Veterinary Laboratory Manual, 9th Ed - 2006

Veterinary Laboratory Manual

9th edition Veterinary Laboratory Service

VETERINARY LABORATORY MANUAL II

Edition Year Under the Title

1st edition 1945 Instructions for the Collection and Despatch of Material for Pathological and Bacteriological Examination.

2nd edition 1947 Instructions for the Collection and Despatch of Material for Pathological and Bacteriological Examination.

3rd edition 1950 Instructions for the Collection and Despatch of Material for Laboratory Examination

4th edition 1961 Collection and Despatch of Material for Laboratory Examination

5th edition 1974 Collection and Despatch of Material for Laboratory Examination

6th edition 1981 Collection of Material for Laboratory Examination

7th edition 1985 Collection of Material for Laboratory Examination

8th edition 1995 Laboratory Specimen Submission Manual

9th edition 2006 Veterinary Laboratory Manual

Prepared by staff of Elizabeth Macarthur Agricultural Institute, Menangle and Regional Veterinary Laboratories, Orange and Wollongbar ISBN 0 7305 6656 0

VETERINARY LABORATORY MANUAL III

INTRODUCTION

This 9th edition of the Manual is published for the first time on the Web. Its aim is to provide accessible, up-to-date information to laboratory clients. The general format of the previous edition, edited by Graeme Eamens, has been retained. Many laboratory staff have contributed to this revised Manual. Also, field veterinarians have provided suggestions, which have been incorporated. The Manual includes information about specimen collection and submission in general, as well as for specific diseases. For further information about tests for diseases of commercial livestock, please contact your nearest Regional Veterinary Laboratory or Customer Service at 1800 675 623.

VETERINARY LABORATORY MANUAL IV

TABLE OF CONTENTS

INTRODUCTION.....................................................................................................................................iii VETERINARY LABORATORY DIRECTORY ....................................................................................... ix SPECIMENS (GENERAL) ...................................................................................................................... 1

CONDITIONS FOR ACCEPTANCE ................................................................................................................ 1 LABORATORY CHARGES.............................................................................................................................. 1 SPECIMEN SUBMISSION FORM ................................................................................................................... 1 COLLECTION AND LABELLING OF SPECIMENS......................................................................................... 3 PACKING OF SPECIMENS............................................................................................................................. 4 TRANSPORT OF SPECIMENS....................................................................................................................... 5 DISEASE SURVEILLANCE ............................................................................................................................. 6 SPECIAL REQUIREMENTS FOR TESTING OF STOCK FOR OVERSEAS EXPORT................................... 7 SPECIAL REQUIREMENTS FOR TESTING OF STOCK FOR INTERSTATE MOVEMENT .......................... 8 CHECKLIST OF EQUIPMENT FOR CLINICAL AND NECROPSY EXAMINATIONS ..................................... 9

SPECIMENS (BY DISCIPLINE)............................................................................................................ 11 BACTERIOLOGY.............................................................................................................................................. 11

STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 11 COLLECTION OF SPECIMENS.................................................................................................................... 11 STORAGE OF BACTERIOLOGICAL SPECIMENS PRIOR TO DESPATCH................................................ 13 DESPATCH OF SPECIMENS ....................................................................................................................... 14

BIOCHEMISTRY ............................................................................................................................................... 14 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 14 COLLECTION OF SPECIMENS.................................................................................................................... 14 STORAGE OF SPECIMENS PRIOR TO DESPATCH................................................................................... 15 DESPATCH OF SPECIMENS ....................................................................................................................... 15 GENETICS .................................................................................................................................................... 15 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 15 COLLECTION OF SPECIMENS.................................................................................................................... 15 STORAGE OF SPECIMENS AND DESPATCH OF SPECIMENS ................................................................ 16

GROSS PATHOLOGY ...................................................................................................................................... 16 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 16 COLLECTION OF SPECIMENS FOR GROSS PATHOLOGY ...................................................................... 16 STORAGE OF SPECIMENS PRIOR TO DESPATCH................................................................................... 16

HISTOPATHOLOGY ......................................................................................................................................... 16 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 16 COLLECTION OF SPECIMENS.................................................................................................................... 17

HAEMATOLOGY............................................................................................................................................... 18 BLOOD .......................................................................................................................................................... 18 BLOOD FILMS............................................................................................................................................... 18 PREPARATION OF BLOOD FILMS .............................................................................................................. 18 STORAGE AND DESPATCH OF SPECIMENS ............................................................................................ 18

PARASITOLOGY .............................................................................................................................................. 18 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 18 COLLECTION OF SPECIMENS.................................................................................................................... 19 STORAGE AND DESPATCH OF SPECIMENS ............................................................................................ 19

SEROLOGY....................................................................................................................................................... 19 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 20 COLLECTION OF SPECIMENS.................................................................................................................... 20 STORAGE AND DESPATCH OF SPECIMENS ............................................................................................ 21 TYPES OF SEROLOGICAL TESTS.............................................................................................................. 21

TOXICOLOGY................................................................................................................................................... 24 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 24 COLLECTION OF SPECIMENS.................................................................................................................... 24 STORAGE OF SPECIMENS PRIOR TO DESPATCH................................................................................... 24

VIROLOGY........................................................................................................................................................ 25 DIAGNOSIS OF VIRAL DISEASE ................................................................................................................. 25 STANDARD CONTAINERS AND EQUIPMENT............................................................................................ 27 COLLECTION OF SPECIMENS.................................................................................................................... 27 STORAGE AND DESPATCH OF SPECIMENS ............................................................................................ 28

SPECIMENS (BY DISEASE OR SYNDROME).................................................................................... 31 DISEASES OF LIVESTOCK ............................................................................................................................. 31

ABORTION (GENERAL)................................................................................................................................ 31 ABORTION IN CATTLE................................................................................................................................. 31 ABORTION IN SHEEP AND GOATS ............................................................................................................ 31 ABORTION IN HORSES ............................................................................................................................... 32

VETERINARY LABORATORY MANUAL V

ABORTION IN PIGS...................................................................................................................................... 32 ACETONAEMIA............................................................................................................................................. 32 ACTINOBACILLOSIS AND ACTINOMYCOSIS............................................................................................. 32 ACTINOBACILLUS SEMINIS INFECTION IN RAMS .................................................................................... 32 AFLATOXICOSIS .......................................................................................................................................... 33 AKABANE DISEASE IN SHEEP AND CATTLE............................................................................................. 33 ALGAL POISONING ...................................................................................................................................... 33 ALPHA MANNOSIDOSIS OF CATTLE.......................................................................................................... 33 ANAPLASMOSIS........................................................................................................................................... 33 ANAEMIA....................................................................................................................................................... 33 ANNUAL RYEGRASS TOXICOSIS (ART) .................................................................................................... 33 ANTHELMINTIC RESISTANCE..................................................................................................................... 34 ANTHRAX...................................................................................................................................................... 36 ARSENIC POISONING.................................................................................................................................. 36 ARTHRITIS AND POLYARTHRITIS .............................................................................................................. 37 ARTHROGRYPOSIS AND HYDRANENCEPHALY....................................................................................... 37 ASPERGILLOSIS .......................................................................................................................................... 37 ATAXIA .......................................................................................................................................................... 37 ATROPHIC RHINITIS OF SWINE ................................................................................................................. 38 BABESIOSIS ................................................................................................................................................. 38 BALANO POSTHITIS IN RAMS..................................................................................................................... 38 BENIGN FOOTROT....................................................................................................................................... 38 BETA MANNOSIDOSIS OF GOATS ............................................................................................................. 38 'BIG KNEE' IN GOATS .................................................................................................................................. 38 BLACK DISEASE........................................................................................................................................... 38 BLACKLEG.................................................................................................................................................... 38 BLOAT ........................................................................................................................................................... 38 BLUE-GREEN ALGAL POISONING.............................................................................................................. 39 BORDER DISEASE IN SHEEP ..................................................................................................................... 39 BOTULISM .................................................................................................................................................... 39 BOVINE LEUKOSIS ...................................................................................................................................... 39 BOVINE LEUCOCYTE ADHESION DEFICIENCY (BLAD) ........................................................................... 39 BOVINE MALIGNANT CATARRH ................................................................................................................. 39 BOVINE VIRUS DIARRHOEA ....................................................................................................................... 40 BRACKEN FERN POISONING...................................................................................................................... 40 BRUCELLOSIS BOVINE ............................................................................................................................... 40 BRUCELLOSIS OVINE.................................................................................................................................. 40 BRUCELLOSIS PORCINE ............................................................................................................................ 40 CALCULI........................................................................................................................................................ 41 CAMPYLOBACTER ABORTION OF SHEEP ................................................................................................ 41 CAMPYLOBACTER ENTERITIS ................................................................................................................... 41 CAMPYLOBACTERIOSIS OF CATTLE......................................................................................................... 41 CAMPYLOBACTERIOSIS OF PIGS.............................................................................................................. 42 CAPRINE ARTHRITIS ENCEPHALITIS (CAE).............................................................................................. 42 CARDIOMYOPATHY AND WOOLLY HAIRCOAT (CWH) SYNDROME ....................................................... 42 CASEOUS LYMPHADENITIS (CLA) ............................................................................................................. 42 CHLAMYDIAL INFECTIONS ......................................................................................................................... 43 CITRULLINAEMIA ......................................................................................................................................... 43 CLOVER DISEASE........................................................................................................................................ 43 COBALT DEFICIENCY.................................................................................................................................. 43 COCCIDIOSIS ............................................................................................................................................... 44 COCCIDIOSIS – HEPATIC IN RABBITS....................................................................................................... 44 COLIBACILLOSIS.......................................................................................................................................... 44 COMPLEX VETERBRAL MALFORMATION (CVM) ...................................................................................... 44 CONGENITAL ABNORMALITIES.................................................................................................................. 45 CONTAGIOUS EQUINE METRITIS (CEM) ................................................................................................... 45 CONTAGIOUS OPHTHALMIA....................................................................................................................... 45 CONTAGIOUS PUSTULAR DERMATITIS .................................................................................................... 45 COPPER DEFICIENCY ................................................................................................................................. 45 COPPER POISONING OF SHEEP AND CATTLE ........................................................................................ 46 CORYNEBACTERIUM EQUI IN HORSES OR PIGS .................................................................................... 46 CRYPTOSPORIDIOSIS................................................................................................................................. 46 CYANIDE POISONING.................................................................................................................................. 47 CYSTICERCOSIS.......................................................................................................................................... 47 DEFICIENCY OF URIDINE MONOPHOSPHATE SYNTHETASE (DUMPS) ................................................ 47 DERMATOPHILOSIS .................................................................................................................................... 47 DIARRHOEA.................................................................................................................................................. 47 DRENCHING MORTALITIES ........................................................................................................................ 48 DWARFISM IN DEXTER CATTLE................................................................................................................. 48 ENCEPHALOMYELITIS AND ENCEPHALITIS ............................................................................................. 48

VETERINARY LABORATORY MANUAL VI

ENCEPHALOMYOCARDITIS (EMC) OF PIGS ............................................................................................. 48 ENTEROTOXAEMIA ..................................................................................................................................... 48 ENZOOTIC ATAXIA....................................................................................................................................... 49 ENZOOTIC BOVINE LEUKOSIS (EBL)......................................................................................................... 49 ENZOOTIC HAEMATURIA OF CATTLE ....................................................................................................... 49 ENZOOTIC PNEUMONIA OF PIGS .............................................................................................................. 49 EPERYTHROZOONOSIS OF SHEEP........................................................................................................... 50 EPHEMERAL FEVER.................................................................................................................................... 50 EPIDIDYMITIS............................................................................................................................................... 50 EQUINE BABESIOSIS................................................................................................................................... 50 EQUINE INFECTIOUS ANAEMIA (EIA) ........................................................................................................ 50 EQUINE VIRAL ARTERITIS (EVA)................................................................................................................ 50 ERYSIPELAS................................................................................................................................................. 51 EXOTIC DISEASES....................................................................................................................................... 51 EXUDATIVE EPIDERMITIS IN PIGS............................................................................................................. 51 FACIAL ECZEMA .......................................................................................................................................... 52 FACTOR VIII DEFICIENCY ........................................................................................................................... 52 FACTOR XI DEFICIENCY ............................................................................................................................. 52 FASCIOLIASIS IN SHEEP AND CATTLE ..................................................................................................... 52 FISTULOUS WITHERS ................................................................................................................................. 52 FLEECE ROT ................................................................................................................................................ 52 FOCAL SYMMETRICAL ENCEPHALOMALACIA (FSE) ............................................................................... 53 FOOT ABSCESS IN SHEEP ......................................................................................................................... 53 FOOTROT IN SHEEP.................................................................................................................................... 53 FOOTROT IN GOATS AND CATTLE............................................................................................................ 56 FUNGAL INFECTIONS (OTHER THAN MYCOTOXICOSES)....................................................................... 56 GENERALIZED GLYCOGENOSIS................................................................................................................ 57 GENETIC DISEASES .................................................................................................................................... 57 GOITRE ......................................................................................................................................................... 58 GRAIN POISONING ...................................................................................................................................... 58 GRASS TETANY ........................................................................................................................................... 58 HAEMAGGLUTINATING ENCEPHALOMYELITIS VIRUS (HEV) INFECTION OF PIGS.............................. 59 HAEMATURIA ............................................................................................................................................... 59 HAEMOGLOBINURIA.................................................................................................................................... 59 HAEMOPHILIA .............................................................................................................................................. 59 HEEL ABSCESS - OVINE ............................................................................................................................. 59 HELIOTROPE POISONING .......................................................................................................................... 59 HENDRAVIRUS INFECTION......................................................................................................................... 59 HEPATOSIS DIETETICA............................................................................................................................... 60 HERPESVIRUS VULVOVAGINITIS/BALAN0POSTHITIS IN GOATS........................................................... 60 HISTOPHILUS OVIS/ HAEMOPHILUS SOMNUS INFECTIONS .................................................................. 60 HYPOCALCAEMIA ........................................................................................................................................ 60 HYPOGLYCAEMIA........................................................................................................................................ 61 HYPOMAGNESAEMIA .................................................................................................................................. 61 HYPOPHOSPHATAEMIA.............................................................................................................................. 61 ILL THRIFT .................................................................................................................................................... 61 INFECTIOUS BOVINE RHINOTRACHEITIS (IBR) AND INFECTIOUS PUSTULAR VULVO VAGINITIS (IPV) ............................................................................................................................................ 62 INFERTILITY IN CATTLE, SHEEP, PIGS ..................................................................................................... 62 INHERITED DEFECTS.................................................................................................................................. 62 INHERITED CONGENITAL MYOCLONUS ................................................................................................... 62 IRON TOXICITY SYNDROME IN PIGLETS .................................................................................................. 63 ITCHMITE IN SHEEP .................................................................................................................................... 63 JAUNDICE..................................................................................................................................................... 63 JOHNE'S DISEASE ....................................................................................................................................... 63 KIKUYU POISONING .................................................................................................................................... 66 LEAD POISONING ........................................................................................................................................ 66 LEPTOSPIROSIS .......................................................................................................................................... 66 LICE RESISTANCE TO INSECTICIDES IN SHEEP ..................................................................................... 68 LISTERIOSIS................................................................................................................................................. 68 LIVER FLUKE INFECTION............................................................................................................................ 68 LUPINOSIS.................................................................................................................................................... 69 LYME DISEASE............................................................................................................................................. 69 LYSSAVIRUS ................................................................................................................................................ 70 MALIGNANT OEDEMA ................................................................................................................................. 70 MALNUTRITION............................................................................................................................................ 70 MANGE.......................................................................................................................................................... 70 MANNOSIDOSIS........................................................................................................................................... 71 ALPHA-MANNOSIDOSIS .............................................................................................................................. 71 BETA-MANNOSIDOSIS ................................................................................................................................ 71

VETERINARY LABORATORY MANUAL VII

MAPLE SYRUP URINE DISEASE (MSUD)................................................................................................... 71 MASTITIS (BOVINE) ..................................................................................................................................... 72 MASTITIS (CAPRINE) ................................................................................................................................... 73 MASTITIS (OVINE)........................................................................................................................................ 73 MASTITIS METRITIS AGALACTIA SYNDROME IN SOWS.......................................................................... 73 MENANGLE VIRUS INFECTION................................................................................................................... 73 METABOLIC DISEASES ............................................................................................................................... 74 MUCOSAL DISEASE..................................................................................................................................... 74 MULBERRY HEART DISEASE ..................................................................................................................... 74 MUSCULAR DEGENERATION, NUTRITIONAL ........................................................................................... 74 MYCOPLASMOSIS ....................................................................................................................................... 75 MYCOTIC DERMATITIS................................................................................................................................ 76 MYCOTOXICOSIS......................................................................................................................................... 76 NASAL GRANULOMA OF CATTLE .............................................................................................................. 76 NECROBACILLOSIS ..................................................................................................................................... 76 NEOPLASMS................................................................................................................................................. 77 NEOSPOROSIS ............................................................................................................................................ 77 NERVOUS DISORDERS............................................................................................................................... 77 NITRATE-NITRITE POISONING ................................................................................................................... 78 OEDEMA DISEASE OF PIGS ....................................................................................................................... 78 OPHTHALMIA................................................................................................................................................ 78 ORGANOCHLORINE AND ORGANOPHOSPHATE POISONING................................................................ 78 OSTEOCHONDROSIS IN PIGS.................................................................................................................... 79 OSTEOMALACIA, OSTEOPOROSIS............................................................................................................ 79 OSTERTAGIOSIS.......................................................................................................................................... 79 OVINE BRUCELLOSIS.................................................................................................................................. 79 OXALATE POISONING ................................................................................................................................. 79 PAPULAR STOMATITIS OF CALVES........................................................................................................... 79 PARAKERATOSIS OF SWINE...................................................................................................................... 79 PARAMPHISTOMIASIS................................................................................................................................. 80 PARASITES (EXTERNAL) ............................................................................................................................ 80 PARASITES (INTERNAL).............................................................................................................................. 80 PARVOVIRUS INFECTION IN PIGS ............................................................................................................. 84 PASTEURELLOSIS ....................................................................................................................................... 85 PEPSINOGEN ESTIMATIONS FROM SERUM OR PLASMA....................................................................... 85 PERINATAL MORTALITIES IN CATTLE....................................................................................................... 85 PERINATAL MORTALITIES IN SHEEP AND GOATS .................................................................................. 85 PESTIVIRUS INFECTION ............................................................................................................................. 86 PHALARIS POISONING................................................................................................................................ 87 PHOSPHORUS DEFICIENCY....................................................................................................................... 87 PHOTOSENSITIZATION ............................................................................................................................... 87 PIGLET ANAEMIA......................................................................................................................................... 87 PINKEYE ....................................................................................................................................................... 87 PLANT POISONING ...................................................................................................................................... 87 PNEUMONIA ................................................................................................................................................. 87 POISONING (CHEMICAL)............................................................................................................................. 88 POISONING (PLANT).................................................................................................................................... 88 IDENTIFICATION OF SUSPECT POISONOUS PLANTS ............................................................................. 88 POLIOENCEPHALOMALACIA (PEM) ........................................................................................................... 89 POLYARTHRITIS .......................................................................................................................................... 89 POMPE'S DISEASE ...................................................................................................................................... 89 PORCINE COLITIS........................................................................................................................................ 89 PORCINE ENTEROVIRUS ENCEPHALOMYELITIS .................................................................................... 91 PORCINE ENZOOTIC PNEUMONIA ............................................................................................................ 91 PORCINE PLEUROPNEUMONIA ................................................................................................................. 91 PORCINE INTESTINAL HAEMORRHAGE SYNDROME.............................................................................. 91 PORCINE MYOCARDITIS (PMC) ................................................................................................................. 92 PORCINE PROLIFERATIVE ENTEROPATHY.............................................................................................. 92 PORCINE STRESS SYNDROME (PSS) ....................................................................................................... 92 PREGNANCY TOXAEMIA............................................................................................................................. 93 PSEUDOCOWPOX ....................................................................................................................................... 93 PROTOPORPHYRIA ..................................................................................................................................... 93 PYELONEPHRITIS (BOVINE) ....................................................................................................................... 93 PYOGENIC INFECTIONS ............................................................................................................................. 93 PYRROLIZIDINE ALKALOIDOSIS ................................................................................................................ 93 Q FEVER ....................................................................................................................................................... 94 RED GUT IN SHEEP ..................................................................................................................................... 94 RHODOCOCCUS EQUI PNEUMONIA IN HORSES..................................................................................... 94 RHODOCOCCUS EQUI LYMPHADENITIS IN PIGS .................................................................................... 94 RINGWORM (DERMATOMYCOSIS) ............................................................................................................ 94

VETERINARY LABORATORY MANUAL VIII

ROCK FERN POISONING ............................................................................................................................ 94 ROTAVIRUS INFECTION.............................................................................................................................. 95 RYEGRASS STAGGERS .............................................................................................................................. 95 SALMONELLOSIS......................................................................................................................................... 95 SALT POISONING IN PIGS .......................................................................................................................... 95 SARCOSPORIDIOSIS................................................................................................................................... 95 SCABBY MOUTH .......................................................................................................................................... 95 SCOURING.................................................................................................................................................... 96 SELENIUM DEFICIENCY.............................................................................................................................. 96 SEMEN EXAMINATION ................................................................................................................................ 96 STRYCHNINE POISONING .......................................................................................................................... 98 SWAINSONA POISONING............................................................................................................................ 98 SWINE DYSENTERY .................................................................................................................................... 98 TAPEWORM INFESTATION IN SHEEP ....................................................................................................... 99 TEAT LESIONS ............................................................................................................................................. 99 TETANUS ...................................................................................................................................................... 99 TICK FEVER OF CATTLE ............................................................................................................................. 99 TOXAEMIC JAUNDICE ............................................................................................................................... 100 TOXOPLASMOSIS IN CATS....................................................................................................................... 100 TOXOPLASMOSIS IN SHEEP AND GOATS .............................................................................................. 100 TOXOPLASMOSIS IN OTHER ANIMALS ................................................................................................... 100 TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY (TSE).................................................................. 101 TRICHOMONIASIS OF CATTLE................................................................................................................. 101 TUBERCULOSIS......................................................................................................................................... 101 ULCERATIVE SPIROCHAETOSIS IN PIGS................................................................................................ 102 ULCERS, GASTRIC IN PIGS ...................................................................................................................... 102 UREA POISONING...................................................................................................................................... 102 URINE EXAMINATION ................................................................................................................................ 102

DISEASES OF POULTRY............................................................................................................................... 105 STANDARD CONTAINERS AND EQUIPMENT.......................................................................................... 105 COLLECTION OF SPECIMENS.................................................................................................................. 105 STORAGE AND DESPATCH OF SPECIMENS .......................................................................................... 105 SEROLOGICAL TESTS AVAILABLE FOR POULTRY................................................................................ 105 AVIAN ENCEPHALOMYELITIS (AE)........................................................................................................... 106 BIG LIVER AND SPLEEN DISEASE ........................................................................................................... 106 LEUKOSIS................................................................................................................................................... 107 MAREK'S DISEASE..................................................................................................................................... 107 MYCOPLASMOSIS (AVIAN) ....................................................................................................................... 107 PASTEURELLOSIS (AVIAN)....................................................................................................................... 108 PULLORUM DISEASE ................................................................................................................................ 108 RETICULOENDOTHELIOSIS (RE) ............................................................................................................. 108 TUBERCULOSIS (AVIAN)........................................................................................................................... 108

DISEASES OF CAGE BIRDS, AVIARY BIRDS AND RACING PIGEONS..................................................... 109 STANDARD CONTAINERS AND EQUIPMENT.......................................................................................... 109 COLLECTION OF SPECIMENS.................................................................................................................. 109 STORAGE AND DESPATCH OF SPECIMENS .......................................................................................... 109

DISEASES OF BEES ...................................................................................................................................... 110 DISEASES OF FISH........................................................................................................................................ 111

STANDARD CONTAINERS AND EQUIPMENT.......................................................................................... 111 HISTORY AND DIAGNOSIS........................................................................................................................ 111 COLLECTION, STORAGE AND DESPATCH OF SPECIMENS.................................................................. 112 SOME DISEASES OF FISH, CRUSTACEANS AND SHELLFISH ALREADY ENCOUNTERED IN NSW........................................................................................................................................................ 113

FEEDS AND PASTURE ANALYSIS .................................................................................................. 114 WATER TESTING............................................................................................................................... 116

VETERINARY LABORATORY MANUAL IX

VETERINARY LABORATORY DIRECTORY

Phone ‘Customer Service’ at 1800 675 623 to contact any of the following veterinary laboratories Location Address ContactsMenangle Regional Veterinary Laboratory

Elizabeth Macarthur Agricultural Institute ‘Camden Park’ Woodbridge Road Menangle NSW 2568 Postal address: (PMB 8, Camden 2570) Officer in Charge: Keith Walker Pathologists: Steven Hum Rod Reece Richard Luong Elizabeth Macarthur Agricultural Institute (Main Reception)

Ph: 02 4640 6327Fax: 02 4640 6400

Ph: 02 4640 6333Fax: 02 4640 6300

Orange Regional Veterinary Laboratory Agricultural Research and Veterinary Centre Forest Road Orange NSW 2800 Officer in Charge: Graham Bailey Pathologists: Patrick Staples Erica Bunker

Ph: 02 6391 3858Fax: 02 6391 3899

Wollongbar Regional Veterinary Laboratory Wollongbar Agricultural Institute 1243 Bruxner Highway Wollongbar NSW 2477 Officer in Charge: Graeme Fraser Pathologists: John Boulton Paul Gill

Ph: 02 6626 1103Fax: 02 6626 1276

VETERINARY LABORATORY MANUAL 1

SPECIMENS (GENERAL)

All NSW Department of Primary Industries Veterinary Laboratories operate according to ISO 17025, and are accredited by the National Association of Testing Authorities (NATA). Details on the scope of this accreditation can be found on the NATA website at http://www.nata.asn.au. CONDITIONS FOR ACCEPTANCE

The Veterinary Laboratories examine specimens from all livestock, poultry, native and feral animals and birds, fish and bees. In relation to pets, cage birds, racing dogs and horses, it is policy to examine only specimens where the health of the flock, herd, aviary etc., is involved or where public health is likely to be involved or for import and export purposes. Specimens must be accompanied by a Specimen Submission Form signed by the submitter. The submitter thereby accepts responsibility for payment of laboratory charges for the tests requested. Where the submitter expects the work to be funded (entirely or partially) by NSW Department of Primary Industries, the reason for this request and the source of funds (where known) should be clearly and conspicuously stated. Specimens are generally only accepted on referral from a veterinarian. Exceptions include WormTest kits, which can be submitted by primary producers. The Genetics section, at EMAI also accepts bovine hair samples collected by cattle owners for genetic disease testing where certification is not required. CASES LIKELY TO INVOLVE CRIMINAL INVESTIGATIONS Examination is not undertaken on specimens likely to involve criminal prosecutions (e.g. in cases of suspected malicious poisoning). If a case is likely to involve police matters or matters controlled by other regulatory authorities, those bodies should be contacted by phone and consulted before any action is taken. Often they have special procedures to be followed with respect to sampling/examination, and with respect to sealing, custody, care and transport of exhibits. They may also have analyses performed by the Government Analytical Laboratories. Laboratory Reports on samples submitted by field veterinarians as routine diagnostic

specimens are unlikely to be admissible as evidence in criminal prosecutions and civil actions. INFORMATION ANALYSIS Test results and findings may be provided to other authorised staff and used for statistical, surveillance, extension, certification and regulatory purposes in accordance with Department policies. The information assists disease and residue control programs and underpins marked access for agricultural products. The source of the information will remain confidential unless otherwise required by law or regulatory policies.

LABORATORY CHARGES NSW Department of Primary Industries operates the Veterinary Laboratories on a cost recovery basis. Submitters will normally be invoiced soon after the final report is issued. The laboratory fees for most common tests can be downloaded from http://www.dpi.nsw.gov.au/agriculture/vetmanual/submission/lab-charges#fees. Laboratory fees for tests not listed can be obtained by calling Customer Service at 1800 675 623. Laboratory charges are subsidised for testing in the following circumstances*: • Notifiable disease • Emergency (exotic) disease • National TSE Surveillance Program • Mortality investigations *See ‘Disease Surveillance’ below for more details. Because it is often difficult for submitters to specify the precise tests that will be required, many find it useful to indicate a maximum laboratory fee on the Specimen Advice Form. The Pathologist will consult with the submitter before exceeding this amount.

SPECIMEN SUBMISSION FORM

Field veterinarians should supply all pertinent details of the history, clinical signs and necropsy findings. This information will allow the laboratory veterinarian interpret the laboratory results and suggest additional testing as appropriate. All information supplied will be included in the Laboratory Report.

http://www.dpi.nsw.gov.au/agriculture/vetmanual/submission/lab-charges#fees

http://www.nata.asn.au


NSW Department of Primary Industries' Specimen Submission Form facilitates the orderly collection of this information. Specimen Submission Forms are supplied in pads of 100 pages. Alternatively, the Form can be downloaded for printing from the NSW Department of Primary Industries web site at http://www.agric.nsw.gov.au/reader/das-vettesting. An official Specimen Submission Form should be used with all specimens submitted. Special forms may also be available for special investigations (eg footrot, ovine or bovine Johnes disease,). These forms provide a check list for information required by the laboratory in these investigations. Specimen Submission forms should be completed using black or blue ballpoint pens, as this ensures good photocopies. Be as concise as possible. Long narratives waste your time and ours. The space available on the form indicates the level of detail required. For more complex investigations, a second sheet may be attached. Such detailed advice is best emailed to the laboratory for inclusion in the laboratory report. The following provides guidance on completing each section of the form: OWNER Provide the normal trading name and full address, including the property name of the owners, e.g. A. Brown, A. Brown & Sons, Mia Mia Pastoral Co. Please print clearly. The same name must be used in all specimen submissions from a property to allow previous submissions from the property to be traced. RURAL LANDS PROTECTION BOARD and RLPB Property ID The affected property's RLPB (Rural Lands Protection Board) district and its RLPB property ID is required for statistical and reporting purposes. The Property ID is the Property Identification Code (PIC), tail tag number, or Assessment Number. SUBMITTER The full name and address (including postcode) should be printed. The submitter's telephone and facsimile numbers should also be given to allow

contact between the laboratory and the veterinarian. Provide your email address if you would prefer to receive your results by email. DISEASE SUSPECTED For Diagnostic cases, give the first two disease possibilities you suspect. SPECIMENS SUBMITTED Give a full list of the specimens submitted together with the examinations requested on each. The number of containers, swabs and blood samples, should be clearly listed so that the laboratory staff can check to be sure that all the specimens submitted have been received. Use the Specimen Submission Key List in addition to the Sample Submission Form if there are insufficient lines on the latter. TESTS REQUESTED Be as specific as possible, e.g. Brucella CFT and ELISA. This is especially important for regulatory/ movement/ export testing. It should be clear which test should be applied to each sample. Your request is equivalent to an order for services and you will be charged for all tests you have requested. If you wish the laboratory to hold a specimen for possible testing later, write 'HOLD' beside this specimen. REASON FOR TEST Tick the most appropriate checkbox. SPECIES Include the species, breed, age and sex of affected animals. For species, select from the following: cattle, sheep, pigs, poultry, goats, horses, fish, bees, deer. If others, state clearly. For breed, give breed or predominant breed in crosses. For sex, select from the following: male, female, castrate, spayed, mixed group, unknown. NUMBER OF ANIMALS We use the numbers provided to calculate the epidemiological statistics, 'period prevalence' and 'cumulative mortality'. For the statistics to have meaning, the following conventions should be followed: At risk: The total number of animals or units (eg litters, pens) at the start of the nominated period. Please specify the unit.

http://www.agric.nsw.gov.au/reader/das-vettesting


Affected: The total number of animals or units that have been affected (sick or dead) during the nominated period eg year, month, lifetime. Please specify the period. Dead: The total number of animals or units dead during the same nominated period. Further information eg daily mortality or prevalence can be provided in the 'History' section. HISTORY AND CLINICAL FINDINGS Include details of husbandry, nutrition, clinical signs, treatments, and necropsy findings. PREVIOUS REFERENCES The laboratory reference number (eg. W04/01762), or date of any previous submissions relating to the condition from this owner should b given, This allows the duty pathologist to check previous reports, offer more informed comment, and maintain continuity between individual submissions from the same owner. POSTMORTEM FINDINGS An accurate summary of significant postmortem findings will help us provide interpretation of laboratory test results.

COLLECTION AND LABELLING OF SPECIMENS

SPECIMENS (BY DISCIPLINE) For detailed information on collection and handling of specimens for each laboratory discipline,see Specimens (by discipline) in this Manual. The following is a brief guide: Bacteriology Swabs of tissues eg heart blood, intestinal content, in transport medium. 30 ml of chilled lesion, fluid or tissue eg liver, lung, intestine in a screw-capped container. Biochemistry Full 10ml plain and LiHep blood tube. Genetics 50 tail hairs with roots. Gross Pathology Representative samples of affected tissue with any adjacent normal tissue. Haematology Full 10ml EDTA blood tube and blood smear.

Histopathology Representative samples of affected tissue with adjacent normal tissue. Tissues should be in 1cm thick slices in 10x their volume of buffered formalin solution. Parasitology Approximately 30g of faeces for faecal egg count. Serology Full 10ml plain blood tube.

Toxicology Approximately 50ml of ingesta, faeces or fresh tissue. Virology Full 10ml plain and EDTA blood tubes. 30ml of fresh chilled tissue eg heart, spleen or swabs of lesions or tissues in PBGS. NB All specimens should be clearly labelled with the animal identification. NSW Department of Primary Industries does not supply specimen containers or consumables. SPECIMENS (BY DISEASE OR SYNDROME) For details of the most appropriate specimens and tests for a wide variety of animal diseases, see Specimens (by disease or syndrome) in this Manual, with subsections: • Diseases of livestock • Diseases of poultry • Diseases of caged birds • Diseases of bees • Diseases of fish In general, it is better to send too many rather than too few samples. Specimens will be held in the laboratory under appropriate conditions pending the results of initial tests. The pathologist should be given sufficient history and specimens to allow the application of other examinations which may be suggested from the history or lesions. Such additional testing will be undertaken only after consultation with the submitter. When investigating difficult or unusual disease problems, we encourage field veterinarians to consult with NSW Department of Primary Industries laboratory veterinarians to decide on the most appropriate sampling and testing strategies.


Care must be taken in the collection and despatch of samples to the laboratory to ensure that you, your staff, personnel handling the specimens during transit and the staff at the Laboratory who have to unpack and handle the specimens are not subjected to any risk of infection.

PACKING OF SPECIMENS Submitters are responsible for correctly packing and consigning specimens in compliance with current International Air Transport Association (IATA) regulations, which are updated annually in the IATA publication ‘Dangerous Goods Regulations’. Incorrect packing may result in refusal by a courier to carry the consignment and/or in legal action. NSW Department of Primary Industries does not supply specimen containers or consumables. On request, recyclable specimen containers or eskies will be returned to submitters. There is a small charge for this service. IATA divides infectious substances into Category A (IATA Packing Instruction 602) and Category B (IATA Packing Instruction 650). Packing Instruction 602 is more stringent than Packing Instruction 650. • Within each Category, infectious

substances are given a UN Number and Proper Shipping Name appropriate for their hazard classification.

Category A (IATA Packing Instruction 602): An infectious substance which is transported in a form that, when exposure occurs, is capable of causing permanent disability, life-threatening or fatal disease to humans or animals. • UN 2814 (Infectious substance,

affecting humans*). • UN 2900 (Infectious substance,

affecting animals only).

*Proper Shipping Name. Examples include Hendravirus, Bacillus anthracis (cultures only), and exotic pathogens such as FMD virus and Nipah virus. See IATA indicative list of Category A substances. Category B (IATA Packing Instruction 650): An infectious substance which does not meet the criteria for Category A. • UN 3373 (Diagnostic specimens)

NB Virtually all specimens sent by veterinarians to laboratories are transported as ‘Diagnostic specimens’ and must be packed to comply with IATA Packing Instruction 650.

For postage, IATA Packing Instruction 650 applies, since carriage by air may be involved. For carriage by road, other regulations apply but, in general, are satisfied by packing for carriage by air. IATA PACKING INSTRUCTION 650 (UN 3373 DIAGNOSTIC SPECIMENS) This instruction covers the labelling and packing of Category B infectious substance as defined by IATA. ‘Diagnostic specimens’ is the Proper Shipping Name. NB Virtually all specimens sent by veterinarians to laboratories are transported as ‘Diagnostic specimens’ and must be packed to comply with IATA Packing Instruction 650. See Guidelines for Packing Diagnostic Specimens below for practical advice on packing to comply with IATA Packing Instruction 650. A number of companies sell suitable packing, but by far the most common outer container is the foam esky enclosed within a rigid cardboard box. The Consignment Note* must include the following details: • Proper Shipping Name ie ‘Diagnostic

specimens’ • Net quantity* and • UN number ie ‘UN 3373’

*Maximum 4 kg of solid or 4 L of liquid material (excluding coolant). A maximum of 1 L within each primary container.

A UN 3373 DIAGNOSTIC SPECIMENS diamond-shaped label must be applied next to the Consignment Note. These labels can be obtained from RVLs or from couriers.

*Pre-addressed Consignment Notes carrying the above endorsements, and also the account number of NSW Department of Primary Industries (to charge the cost of freight to the laboratory).are available from your Regional Veterinary Laboratory.

IATA PACKING INSTRUCTION 602 (UN2814 INFECTIOUS SUBSTANCE


AFFECTING HUMANS or UN2900 INFECTIOUS SUBSTANCE AFFECTING ANIMALS ONLY) This instruction covers the labelling and packing of Category A Infectious Substance as defined by IATA (see above). It includes those agents that cause serious communicable disease. Examples include Hendravirus, Bacillus anthracis (cultures only), and exotic pathogens such as FMD virus and Nipah virus. See IATA indicative list of Category A substances. IATA Packing Instruction 602 is more stringent than 650, and requires: • Person packing to have been trained

by an IATA-approved trainer within the past two years.

• Completed Shipper’s Declaration for Dangerous Goods form with the package.

GUIDELINES FOR PACKING DIAGNOSTIC SPECIMENS (IATA PACKING INSTRUCTION 650) ‘Dangerous Goods Regulations’, published annually by the International Air Transport Association (IATA), should be consulted for advice on specimen packing requirements. NB Virtually all specimens sent by veterinarians to laboratories are transported as ‘Diagnostic specimens’ and must be packed to comply with IATA Packing Instruction 650. The following notes are provided to assist submitters. Primary containers

i. Tighten the lids of all primary specimen containers. For histopathology containers, tape the lid to prevent loosening. For glass slides, wrap together between stiff cardboard.

ii. Clearly label each primary specimen container or slide with the animal identification.

iii. Check that the specimens tally with those identified on the Specimen Submission Form.

iv. Estimate the total net weight of specimens and record this on the Consignment Note.

Secondary container(s)

i. Seal all primary containers from each property/problem into a

watertight secondary container with sufficient absorbent material to absorb all fluid from the primary container(s). A sturdy plastic bag will suffice as a secondary container - it must be sealed watertight.

ii. Indelibly label the secondary container with the owner's name.

iii. Put Specimen Submission Form(s) into a separate plastic bag.

Outer container (usually an esky)

i. Put specimen secondary container(s), Specimen Submission Form(s), cooler bricks and tight packing into the outer container (usually an esky). Crumpled newspaper will suffice for tight packing.

ii. If the esky is foam, seal it into a cardboard box.

iii. Complete the Consignment Note and stick it onto the outer container.

iv. Stick a UN 3373 DIAGNOSTIC SPECIMENS diamond-shaped label adjacent to the consignment note. These labels are available from RVLs or from some couriers.

Call Customer Service (1800 675 623 if you need the Account Number of NSW Department of Primary Industries for a particular courier (to charge the cost of freight to the laboratory). Pre-addressed Consignment Notes, available from your Regional Veterinary Laboratory, include this Account Number NB Suspect ANTHRAX submissions must have a clear warning under the lid of the outer packing. This warning must be on top of samples and not packed in with the samples.

TRANSPORT OF SPECIMENS Specimens (other than those for export testing), should be submitted to the Regional Veterinary Laboratory which services your region. Depending on the tests specified, the samples may be sent by that laboratory to another appropriate Departmental testing centre. This may be another RVL or one of the Central Laboratories at EMAI, Menangle. A map showing the location of laboratories is available at http://www.dpi.nsw.gov.au/agriculture/vetmanual/contact . Generally specimens should not be sent by overnight courier on Friday or at the

http://www.dpi.nsw.gov.au/agriculture/vetmanual/contact


weekend. Weekend delivery attracts a heavy surcharge from all courier companies. Before calling your courier to pick up specimens on a Friday, call the laboratory to discuss options. The cost of transport of submissions to the laboratory is included in the test charges. Submitters can therefore charge the cost of freight to the NSW Department of Primary Industries’ Account Number on the Consignment Note. COURIERS Most submissions are sent to our laboratories by overnight courier. Commercial couriers may use either road or air transport and specimens should therefore be packed in accordance with the more stringent IATA requirements for air transport. NSW Department of Primary Industries has contracts or agreements with several large courier companies as well as with local companies. Some companies offer same day delivery. Call the laboratory to discuss transport options before contacting a courier company. FORWARDING LIVE OR DEAD ANIMALS The laboratory should be consulted before live or whole dead animals are forwarded. We will determine the feasibility of the operation and make arrangements for receiving the animals. All costs associated with freight and carcase disposal will be borne by the submitter unless prior arrangements have been made. It is the responsibility of the sender to meet with the requirements not only of the transport authority, but also with the requirements of welfare of the animal during transport. The following should be used as guidelines:

i. Containers must meet the requirements of the transport authority.

ii. The containers should be large enough for the animal to stand, to lie down fully extended and to turn round.

iii. Suitable bedding should be provided.

iv. Animals should be placed in the container and delivered to the terminal or railway station shortly before the estimated time of departure. Animals should not be

left confined for long periods awaiting or during transport.

DISEASE SURVEILLANCE

NSW Department of Primary Industries collects epidemiological data on the prevalence of animal disease by monitoring the presenting problem and result of testing of material submitted to its laboratories. This data is used to satisfy the requirements of our trading partners and to direct research and extension efforts of our staff. This passively-collected data is supplemented by data collected while testing healthy animals for export or interstate movement and by active surveillance programs funded by industry or by the Department. For notifiable diseases (including emergency diseases) and for mortality investigations, NSW Department of Primary Industries encourages veterinarians to submit samples to their laboratories by offering some 'free' testing.

NOTIFIABLE DISEASES A full list of the currently notifiable diseases is available at http://www.dpi.nsw.gov.au/agriculture/vetmanual/submission/disease-surveillance. Veterinarians and livestock owners have an obligation under various Acts of Parliament, to report any suspected occurrence of these diseases to NSW Department of Primary Industries staff. Generally, no fee will be charged for tests conducted at a NSW Department of Primary Industries laboratory to exclude an endemic notifiable disease. For emergency (exotic) disease, all testing to exclude that disease and to establish an alternative diagnosis will be free of charge to the submitter. Full details of the eligibility criteria have been circulated to veterinarians in NSW. For endemic diseases, submission of specimens to a NSW Department of Primary Industries' laboratory accompanied by a fully completed Specimen Submission Form nominating the suspected disease is a suitable form of notification. For emergency (exotic) disease, contact the Animal Disease Watch Hotline 1800 675 888 or the local Senior Field Veterinary Officer.

http://www.dpi.nsw.gov.au/agriculture/vetmanual/submission/disease-surveillance


NATIONAL TSE SURVEILLANCE PROGRAM (NTSESP)

Full details of this program are available at http://www.animalhealthaustralia.com.au/programs/adsp/adsp_home.cfm. See also Transmissible spongiform encephalopathy in this Manual.. Veterinarians are encouraged to submit specimens from adult sheep and cattle with progressive neurological disease. The laboratory charges for this testing are charged to the NTSESP. Veterinarians and owners receive incentive payments.

MORTALITY INVESTIGATIONS NSW Department of Primary Industries subsidizes laboratory fees for the investigation of mortalities in livestock. To qualify for this subsidy: • The animals involved must be from a

commercial livestock herd or flock. Horse mortalities are eligible. For poultry more than 100 birds must have died.

• All owner and property details must be provided on a fully completed Specimen Submission Form.

NB Foetuses and neonates up to one day of age are not eligible for this subsidy. When using this subsidy, submitters pay the first $80 of the laboratory fee and NSW Department of Primary Industries up to $140 of the residual. (These values are GST inclusive)

SPECIAL REQUIREMENTS FOR TESTING OF STOCK FOR OVERSEAS EXPORT

The following requirements should be fulfilled:

i. Intending exporters (owners and/or agents) must advise the Animal Quarantine Inspection Service (AQIS) [phone (02) 8334 7434; fax (02) 8334 7430] of their intent to export, at least four (4) weeks prior to the commencement of testing, otherwise there can be no guarantee that testing will be completed and certification provided to allow export to proceed on schedule. Field veterinarians should check to ensure this requirement has been met.

ii. The veterinarian submitting material

is totally responsible for determining the tests required. This information must be included on the Specimen

Submission Form. Specific requirements for testing of stock for each overseas export destination can be obtained from the Animal Quarantine Inspection Service (AQIS) [phone (02) 8334 7434; fax (02) 8334 7430).

iii. The name of the official veterinarian

supplying the information on export testing requirements should be advised and stated on the Specimen Submission Form. This will allow the laboratory to check on any changes in requirements, where necessary, and to prepare quotations for the cost of laboratory services.

iv. The name of the agent or shipping

company involved should be supplied on the Specimen Submission Form with telephone/facsimile numbers.

v. The Officer in Charge of the

Regional Veterinary Laboratory, Menangle or his/her appointed nominee should be advised of the expected dates of sampling and shipment of the animals prior to the submission of the specimens.

vi. The vendor of the stock should be

listed as the owner of the stock rather than the agent or shipping company. This allows different lots to be traced, when animals are being purchased from several properties.

vii. Full legal animal identification details

should be cross-matched to sample identification and documented.

viii. It is particularly important for export

testing that samples of good quality and adequate quantity be submitted and that they be correctly labelled.

ix. For all overseas export testing the

samples should be forwarded directly to:

Officer in Charge Regional Veterinary Laboratory Elizabeth Macarthur Agricultural Institute Woodbridge Road MENANGLE NSW 2568 The Regional Veterinary Laboratory and the Central Laboratories at EMAI, Menangle offer almost all required export tests for livestock. The RVL Menangle co-ordinates

http://www.animalhealthaustralia.com.au/programs/adsp/adsp_home.cfm



most export testing in NSW and can be contacted by all field veterinarians directly for enquiries and assistance. Alternatively, certain tests can be performed within Regional Veterinary Laboratories at Orange, and Wollongbar for export certification.

SPECIAL REQUIREMENTS FOR TESTING OF STOCK FOR INTERSTATE MOVEMENT

i. Advice on requirements for interstate movement should be obtained from the District Veterinarian of the local Rural Lands Protection Board. Nomination of tests to be performed is the responsibility of the authorised official submitting veterinarian.

ii. In cases where large numbers of

animals are to be tested for interstate movement (i.e. greater than 200), the Officer-in-Charge of the local Regional Veterinary Laboratory or his/her appointed nominee should be advised of the expected dates of sampling and

transport of the animals, prior to the submission of the specimens.

iii. The vendor of the stock should be

listed as the owner of the stock, rather than the agent or transport company. This allows different lots to be traced, when animals are being purchased from several properties.

iv. Full legal animal identification must

be documented.

v. It is particularly important that samples of good quality and adequate quantity be submitted, and that they be correctly labelled. This will expedite testing.

vi. For interstate movement, send

specimens to the relevant Regional Veterinary Laboratory for the district of origin of the stock.

Those tests unavailable at Regional Veterinary Laboratories will be undertaken at EMAI following referral of specimens from the Regional Veterinary Laboratories.


CHECKLIST OF EQUIPMENT FOR CLINICAL AND NECROPSY EXAMINATIONS Suggest Checklist for Clinical and Necropsy Examinations 1 Personal • Rubber boots

• Overalls • Gloves

i. disposable ii. post mortem gloves or gauntlets

• Post mortem apron (optional) • Towel and soap • Wet weather gear

2 Animal Husbandry • Halter • Nose grips • Bleeding choke rope • Twitch

Pig handler • Drugs

i. Rompun 2% ii. Xylocaine 2%, etc

3 Clinical • Stethoscope • Thermometers • Obstetrical gloves

4 Clinico-pathological

• Nitrate and cyanide test

5 Clinical Specimens • Vacutainers - plain, heparin and EDTA • Vacutainer needle holders and needles (18G) • Sterile containers (100 ml) • Sterile bottles

• 1 oz Macartney - 25 ml • Bijou Macartney - 5 ml

• Swabs • Transport media

• Amies charcoal transport medium in bottles of commercial swab packs

• Phosphate buffered glycerol saline (PBGS) in bottles • Microscope slides (and spreaders for blood films) • Syringes (1-20 ml) and needles (14-26 G) • Scalpel handle and disposable blades • Scissors • Non-sterile faecal containers (50-100 ml) • Vaginal mucus pipettes • Ethanol/iodine for skin asepsis • Biopsy instruments and small bottles of fixative

6 Euthanasia • Rifle and ammunition • Euthanasia solution and syringe/needle • Killing knife

7 Necropsy • Knives • 20 cm skinning 2.5 cm wide straight blade • 20 cm skinning 2.5 cm wide curved blade • 15 cm boning knife pointed straight

• Steel - 30 cm Butcher's • Footrot secateurs

• 7.5 cm blades • Handsaw 30 cm blade and replacement blade • Scissors

• Mayo straight 16 cm • Mayo straight 14 cm • Double sharp 11 cm fine • Pointed/1 rounded end and knob (for gut running)


Suggest Checklist for Clinical and Necropsy Examinations • Bone forceps • Toothed forceps

• 20 cm • 18 cm • 13 cm

• Scalpel handles • No. 4

• Scalpel blades • No. 24 and/or No. 20

• Rib cutters • Meatsaw/hacksaw • Cleaner/hatchet • Buckets - plastic (2) • Trays

• plastic (large) • plastic (small) • Sterile scissors and forceps for virological tissue samples

8 Pathological Specimens

• Sterile containers • 100 ml • 25 ml • 5 ml

• Plastic bags 0.1 mm thick • large • medium • small

• Rubber bands • Microscope slides and diamond pencils, or markable slides and

pencil • Histopath jars with 10% neutral buffered formalin

• Large 500 ml • Medium 250 ml • Small 125 ml

• Special fixatives (e.g. Bouins) if required • Ball of string

9 Decontamination /Disinfection

• Container and 20 litres water • Disinfectant – one litre Lysol or similar • Nail scrubbing brush (for cleaning instruments) • Long handled scrubbing brush (for cleaning boots) • Roll paper towel • Plastic garbage bags - suitable container for contaminated 'sharps'

10 Clerical • Clip board • Postmortem and Specimen Submission Forms • Specimen collection handbook • Pencil • Marking pens • Black biro • Adhesive labels • String-tie labels

11 Storage • Large metal esky with a blood vacutainer box and small foam esky inside

• Frozen cold bricks, dry ice or portable car fridge • Serum storage plastic disposable tubes - 5 ml


SPECIMENS (BY DISCIPLINE)

BACTERIOLOGY

A bacterial disease can only be diagnosed when a pathogenic organism can be demonstrated, either on smear, culture, using molecular techniques or in tissue sections, in association with pathological changes. Many pathogenic organisms are present in normal animals, e.g. Clostridium perfringens in intestinal contents, so that recovery of the organism alone may not necessarily be significant. In other cases e.g. E coli, there are many serotypes, but only a few are commonly pathogenic. STANDARD CONTAINERS AND EQUIPMENT

Sterile plastic bottles and jars These are available commercially in a range of sizes from about 10 to 100 ml. They are used for fluids, discharges, small lesions, etc. Plastic bags For larger specimens of organs or foetuses. The primary receptacle must be leakproof and not contain more than 500g. Plastic bags may be suitable. A minimum bag thickness of 0.1 mm should be used. All bags should be securely sealed by ziplocking or double folding and application of stout rubber bands or equivalent, the bag should be packed inside a second bag which also contains sufficient absorbent material to absorb fluid should the inner bag leak and securely sealed as above. Plastic bags must not be used if bone, horns or other sharp objects may puncture the bag. Swabs Swab transport systems for the recovery of organisms are available commercially. They consist of plain cotton wool sterile swabs, which are inserted into a semisolid transport medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium) particularly for fastidious bacteria, before submission to the laboratory. There are different transport mediums available commercially, such as swab/transport medium packs for general purposes, swab/charcoal transport medium for more sensitive organisms, swab/transport systems modified for anaerobe recovery. Some testing involving special stains or antigen testing may require dry swabs or specific transport conditions

Microscope slides Use slides of standard size and ensure they are clean. Some commercially available slides are not washed and are not suitable for making satisfactory smears. Slides with frosted ends are preferred for labelling purposes. Ensure slides are packed such that they don’t break in transit eg slide holder, wrapped in tissue/paper with sufficient padding. Milk sample bottles Sterile 30 ml Macartney bottles or any equivalent sterile, wide-mouthed, screw-topped, plastic container. e.g. sterile 30 ml universal containers. Pipettes for collecting vaginal mucus Polystyrene artificial insemination pipettes, approximately 50. cm long x 2mm internal diameter and 2 ml capacity. These are available commercially. Pipettes for collecting preputial scrapings from bulls See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/02-bull-vd-coll.pdf Plastic pipettes made from polystyrene or polypropylene with suggested dimensions of external diameter 9.5 mm, internal diameter 6.4 mm, 61.0 cm in length, straight except for a bend at the end that is held by the operator during collection; the other end, which contacts the preputial surfaces, is bevelled. They should be fitted to 90 ml firm rubber bulbs for suction while scraping.

NB Do not use non sterile containers, gloves or fragile containers when submitting material for bacteriological examination. Aspirates submitted in syringes with needles will not be examined.

COLLECTION OF SPECIMENS

Specimens should be collected aseptically and submitted promptly in individual sterile non-leaking, screw-topped, or plastic jars. If small, the entire lesion or organ should be submitted. If the lesions are large or widespread, submit a portion of the affected tissue containing the lesion and surrounding area. Alternatively an aspirate of the lesion can be taken and transferred to a small sterile container and submitted

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34601/02-bull-vd-coll.pdf


If a septicaemia/bacteraemia is suspected, submit portions of liver, spleen, heart blood and lung, in separate containers. Swabs Are generally inferior to the above. In the case of exudates, a smear(s) should be made and submitted with the swab. for culture should be submitted as soon as possible in transport medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium) is preferred., Stuart Transport Medium is unsuitable for contagious equine metritis (Taylorella equigenitalis) transport if the swabs are to be processed more than 24 hours after collection. Aborted foetuses and foetal membranes For foetuses from smaller animals (e.g. sheep, goats, pigs) and smaller foetuses from the larger animals, the entire foetus together with the foetal membranes should be enclosed in separate plastic bags and submitted chilled. . are generally inferior to the above. In the case of exudates, a smear(s) should be made and submitted with the swab. In the case of the large foetuses which cannot be delivered to a laboratory, a post-mortem examination should be conducted and the appropriate specimens should be collected aseptically into sterile jars (see ‘Abortion’). Impression smears of foetal membranes should be taken prior to despatch of specimens.

Portions of the placenta or cotyledons should also be taken and fixed in buffered formalin prior to despatch. Faecal samples and rectal swabs Collect approximately 30 g faeces direct from the rectum into a sterile, wide-mouthed non-leaking container. Do not overfill the container. (preferred collection method) Rectal swabs, heavily impregnated with faeces, should be submitted in transport medium such as Stuart Transport Medium or Amies Charcoal Transport Medium. For Johne's faecal culture, see the section ‘Johne's disease’. NB. If a parasitological examination is also required, a duplicate sample should be collected.

Smears of intestinal mucosa and pathological lesions Firmly smear suspect area with slide. A number of smears can often be made on each slide. AIR DRY and wrap separately or place in slide protective holders for transport (commercially available). Always leave one (frosted) end of the slide clean for handling and labelling. Smears should be clearly labelled, Intestinal contents Should be expressed into sterile, wide-mouthed, non-leaking containers, avoiding gross contamination of the sample or the container (preferred collection method). Swabs of intestinal contents should be placed in a transport medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium). Milk samples Wash the udder and the teats thoroughly and dry with a paper towel. Swab the teats with 70 per cent alcohol. The first couple of squirts should be discarded unless used for a field test and, about 10 ml milk squirted into a sterile 30 ml Macartney bottle or sterile 30 ml plastic universal container held nearly horizontally. Avoid touching the mouth of the bottle with the teat. Code the samples numerically. The samples must be submitted chilled using either crushed ice or cooling bricks in an insulated container. Milks must be refrigerated if transport is slightly delayed, or frozen if transport is delayed more than 2-3 days.

Preputial material from bulls Preputial scrapings submitted unchilled in selective transport medium for T foetus (InPouch TF) See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/02-in-pouch-guide.pdf Selective transport medium for T foetus (InPouch TF), together with instructions for the collections and despatch of specimens will be forwarded from the laboratory on request. Preputial samples should not be frozen or refrigerated. Keep at temperatures between 18oC and 30oC. Semen samples

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34603/02-in-pouch-guide.pdf


Must be collected aseptically avoiding contamination by preputial material. Samples collected using an artificial vagina are often grossly contaminated unless they are cultured shortly after collection. From 0.5 to 1.0 ml is sufficient for bacteriological examination. Attempts to collect large volumes by repeated ejaculation increase the risk of contamination. Urine samples For routine cultural examinations to be meaningful, urine samples should reach the laboratory within a few hours of collection. If there are likely delays before or during transport, specimens must be chilled, but should be processed by the laboratory within 72 hours of collection. When direct examination for leptospires is required, submit a fresh urine specimen specifically for leptospiras motility examination at the laboratory within 20 minutes of collection. Otherwise, for leptospire morphology submit a separate urine sample of at least 20 ml, preserved with 0.25 ml of undiluted formalin or 1.5 ml of 10% formalin. Vaginal mucus samples Mucus can be collected from the ventral fornix of the vagina and the external os of the cervix by guiding a plastic artificial insemination pipette by hand (per rectum) and applying GENTLE suction to the external end. To diagnose bovine venereal campylobacteriosis (BVC) in abortion or infertility cases:

i. (Preferred method): Vaginal mucus samples are collected for demonstration of C fetus subsp venerealis antibodies by BVC ELISA test. PBST diluent and instructions for collection of mucus samples are available from the laboratory. See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/01-bvc-elisa-guide.pdf. Vaginal mucus is collected using a plain, sterile cotton swab and placing the cotton swab end into phosphate buffered saline plus Tween (PBST) for transport. The swabs, PBST and instructions are provided by the laboratory.

ii. For isolation of Campylobacter, direct inoculation to Campylobacter transport media (CTM) is ideal.

If specimens for Campylobacter culture will not reach the laboratory within 6-8 hours, then sealed pipettes from which air bubbles are excluded should be used (Atmospheric oxygen is lethal to Campylobacter fetus). Plastic pipettes can be sealed adjacent to the mucus using a pair of pliers previously heated in a flame. on dry ice is recommended. Consult with your courier if you intend to use dry ice for submission of samples.

Bacteria with particular requirements Mycoplasma, Spirochaetes (including Leptospira, Brachyspira), Chlamydia, obligate anaerobic bacteria (including Dichelobacter nodosus) have special requirements. If culture is required, consult with your Regional Veterinary Laboratory.

STORAGE OF BACTERIOLOGICAL SPECIMENS PRIOR TO DESPATCH

In general, all samples for bacteriological examination, except smears, should be kept chilled (2-8oC) but not frozen, from the time of collection until they are received in the laboratory. This will ensure the minimum growth of contaminants. Insulated containers with frozen icebricks or plastic bags of crushed ice should be taken in the car to all investigations and specimens placed therein as soon as possible. Delays in transport over weekends and public holidays must be considered. Specimens are better held refrigerated until the sender is sure the transport to the laboratory will not be delayed. Samples of vaginal mucus for bacteriological examination for Campylobacter fetus should be submitted on dry ice in an insulated container. Consult with your courier. Specimens best stored at room temperature (not refrigerated) The following samples should be stored and transported between 18oC and 37oC (ie not refrigerated or frozen): • Samples submitted in Campylobacter

Transport Medium (CETM) • Samples submitted in Trichomonas

Transport Medium (In Pouch) • Swabs submitted in Amies Charcoal

medium for Contagious Equine Metritis (CEM)

• Swabs submitted in Amies Charcoal medium for or Atrophic rhinitis.

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34595/01-bvc-elisa-guide.pdf


• Tissues for fungal culture (temperatures of less than 15oC can be detrimental to fungal survival).

DESPATCH OF SPECIMENS

See section ‘Packaging and sending specimens to the laboratory’. In addition, when forwarding specimens for bacteriological examination the following procedures should be adopted: • All specimens for transport should be

packed in accordance with IATA requirements for diagnostic specimens (Packing Instructions 650) so as to prevent leakage and risk of accidental exposure of personnel handling the container.

• Specimens for transport from cases of suspected tuberculosis or anthrax should always be submitted individually in containers separate from all other specimens and in accordance with IATA requirements for infectious substances (Packing Instructions 602). Pack such that on opening the outer packaging, a note advising ‘Suspect TB’ or ‘Suspect Anthrax’.

• The specimen advice form is best sealed in a plastic bag to avoid contamination or damage from water.

• Check to ensure that specimens are submitted at the temperature appropriate for the disease condition, i.e. chilled, or at room temperature. Check with your courier.

BIOCHEMISTRY

NSW Department of Primary Industries’ laboratories do not carry out any biochemical analysis; all such testing is outsourced to laboratories that are accredited by NATA to conduct biochemical tests. In some cases it is more appropriate to apply a test on fresh samples in the field rather than submit samples to a laboratory, e.g. urine analysis, where commercially available kits will provide a more reliable answer on the spot, rather than the same test applied in the laboratory 24 to 48 hours later. STANDARD CONTAINERS AND EQUIPMENT

Contamination of glassware and other collection equipment by traces of the element of interest has been an issue in the past. Vacuum blood tubes and needles,

and disposable plastic containers have eliminated this problem. Standard containers for Biochemistry are: • Blood vacuum tubes (10 ml),

siliconized, without anticoagulant: For blood and serum samples.

• Blood vacuum tubes (10 ml) with EDTA or heparin: For plasma samples.

• Serum vials: 5 ml vials or bottles with leak-proof closures.

• Bottles, jars, plastic bags - for tissues, organs, etc., as listed for Bacteriology.


Blood and serum samples Blood samples should be collected aseptically into the appropriate 10 ml vacuum tube. Contamination of the sample by soil, faeces, hair, etc., must be avoided. Where whole blood will not arrive at the laboratory within 24 hours of collection, sera should be separated from the clot as soon as possible and frozen or chilled in 5 ml serum vials. Chilling should also continue in transit. Care should be taken to avoid haemolysis (see "Serology - Avoiding Haemolysis of Samples"). Haemolysis will seriously interfere with the level of serum magnesium, phosphate, total protein, albumin and enzyme levels. Sera for Vitamin A or E analysis must be protected from heat and light. Separated sera or plasma must be placed into 5 ml containers wrapped in foil, then forwarded chilled to the laboratory. Plasma samples At least 2 ml of plasma should be submitted frozen in 5 ml screw capped containers. The blood sample should be collected in the appropriate blood vacuum tube. Where whole blood will not arrive at the laboratory within 24 hours of collection, plasma should be separated by centrifugation as soon as possible and preferably within 4 hours of collection. The plasma should be removed, free of all cells and then kept chilled or frozen. Chilling should also continue in transit. Plasma for Vitamin A or E analysis must be protected from heat and light. Separated sera or plasma must be placed into 5 ml


containers wrapped in foil, then forwarded chilled to the laboratory. Tissue samples At least 50 g of tissue should be submitted frozen in a screw capped container. Care must be taken to avoid contamination by soil, faeces, ruminal or intestinal contents when collecting tissue for biochemical analysis. Different organs must be submitted in separate containers. If other examinations are required, e.g. bacteriology, pathology, then duplicate samples as required for the other examinations should be submitted in separate containers. Tissues for vitamin A or E analysis These should be protected from heat and light; wrap in foil or brown paper and submit chilled to the laboratory.

STORAGE OF SPECIMENS PRIOR TO DESPATCH

Samples of tissues must be chilled or frozen as appropriate until despatch.

DESPATCH OF SPECIMENS Specimens should be forwarded in an insulated container with an icebrick. To prevent frozen samples from becoming heated during transit, always ensure there are several ice bricks to ensure samples arrive at least chilled: • Always seal the specimen advice form

in a separate plastic bag.

GUIDE TO INTERPRETATION OF BIOCHEMICAL PARAMETERS IN SHEEP AND CATTLE Normal values are supplied by the laboratories to which we outsource and are included in our reports to submitters. Suggested normal values for some analytes are included under specific diseases in the section ‘Specimens (by disease or syndrome)’ of this manual. Where values supplied by laboratories differ from these suggested values, the former should be used. The ‘Serum enzymology’ section includes information on enzyme changes see in various conditions. Refer to relevant section of the manual. See:

Acetonaemia Copper deficiency Hypomagnesaemia Hypocalcaemia Hypophosphataemia

Muscular degeneration, nutritional Selenium deficiency Serum enzymology Vitamin A and E Vitamin B12

GENETICS

The field veterinarian should check the Specimens (By Disease or Syndrome) section of this manual for details on the specimens required for diagnosis of the specific disease suspected. Where DNA based tests have been developed for genotyping at a defined locus the preferred sample is hair roots. DNA can also be isolated from anticoagulant-treated blood and from semen; however the tests will be significantly more expensive than with those that exploit hair roots as a source of DNA. Only with prior arrangement with the laboratory can other tissue samples be the utilised as the source of DNA for routine tests. Where DNA tests are not available, diagnosis may be possible by histological examination of suitable tissues and findings used to complement historical observations, breeding data and clinical findings.

STANDARD CONTAINERS AND EQUIPMENT

Vacuum blood tubes (5mL or 10mL, with anticoagulant EDTA or heparin) For blood samples. Paper envelope or pre-labelled ‘ziplock’ plastic bags For tail hairs.


Hair Tease out 20 to 25 hairs growing at the distal end of the tail. Select hairs that are free of visible faecal contamination. Tie a knot in the hair shafts approximately one quarter of the distance from their proximal end. Firmly grasp the knotted hairs and pull with a quick action. Ensure roots are present at the proximal end of the hair shafts. If desired, cut off and discard the distal half of the shafts. Place the shafts (with roots) in an envelope labelled with the animals identity, and post to EMAI. See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34605/dpi-samp-coll-prot.pdf


Use of forceps or artery clamps may facilitate collection of suitable samples from newly-born calves. Clearly state the breed of the subject and DNA test required. Blood Fill evacuated blood tubes, to end of draw, from the coccygeal vessels or the jugular vein. Immediately after drawing the sample ensure mixing of anticoagulant and blood by repeated but gentle inversion of the tubes. Label each tube with the identity of the subject. Semen A semen straw or 0.5mL of raw semen in a serum vial.

STORAGE OF SPECIMENS AND DESPATCH OF SPECIMENS

Samples of tissues other than hairs must be chilled until despatch. Do not freeze blood samples. Clean, dry hairs are stable at ambient temperature for an extended period of time. Specimens other than hairs should be forwarded in an insulated container with an icebrick. Use sufficient icebricks to ensure samples arrive at least chilled. Refer to Guidelines for Packaging Specimens for more detail. Always seal the specimen advice form in a separate plastic bag.

GROSS PATHOLOGY

Provided that the field veterinarian can be sure that the specimens will be delivered to the Regional Veterinary Laboratory promptly, there are advantages in submitting the whole animal or whole affected organs. Submission of the animal, live or dead, should be considered when there is a: • High mortality or morbidity from an

unknown cause. • Continuing problem and previous

examinations have not established a diagnosis.

All animals either live or dead, submitted to the laboratory must be accompanied by the appropriate specimen advice form with all relevant details. The laboratory should be advised by phone that the animal is being

delivered. Any dead animal should arrive in time to allow a post mortem examination on the same day. STANDARD CONTAINERS AND EQUIPMENT

Specimens for gross pathology should be submitted in glass or plastic screw-top containers or strong plastic bags. When live or dead animals are submitted, the containers used should prevent contamination of the environment with possible pathogens.

COLLECTION OF SPECIMENS FOR GROSS PATHOLOGY

When submitting whole animals, ensure that they are typical of the syndrome being investigated. When the necropsy is done in the field and a laboratory opinion is sought on gross changes detected, then a large portion of the tissue or organ should be submitted chilled but not frozen, containing the lesion and adjoining normal tissue for comparison. If material is submitted for macroscopic examination, small portions in buffered formalin should also be taken and submitted for histopathological examination.


Tissue samples should be kept chilled from the time of collecting until they are received at the laboratory. NB Do not freeze samples being submitted for either gross or histopathological examination

HISTOPATHOLOGY

In general, the histopathologist will only be able to offer comment on the significance of lesions when an appropriate range of specimens has been submitted, together with a good history and a description of the clinical and necropsy findings. STANDARD CONTAINERS AND EQUIPMENT

(Neutral) Buffered Formalin Buffered formalin solution is the recommended fixative because of its stability, longer life of preserved tissues and


a negligible fixation deposit. It is prepared as follows: Commercial Formalin * 100 mlNaH2PO4.2H2O 4.0 gNa2HPO4 6.5 g

Water to 1 litre* Commercial formalin is Formalin B.P. 34-38% formaldehyde. It should be used neat in this formulation. Buffer salts are available commercially in prepared packs. Other fixatives Other fixatives for special purposes are available from the laboratory. Containers These must have a tightly fitting lid to prevent leakage. Containers must have sufficiently wide mouths to allow tissue to be easily withdrawn after fixation. Don’t squeeze large tissue samples into containers.

COLLECTION OF SPECIMENS Tissues collected at necropsy should be preserved immediately. If fresh organs are being submitted for bacteriological or gross pathological examination, then a small block of the organ (sliced 0.5 cm and no more than 1 cm thick) should be fixed in buffered formalin at the time of collection, for histopathology. Always submit an adequate range of tissue specimens. Preserved tissue should be submitted in at least 10 times its volume of fixative. Decomposing or frozen tissues are not suitable for histopathological examination Organs Organs smaller than 1 cm in thickness can be fixed whole. Organs greater than 1 cm thick must be sectioned. Tissue for fixation should be sliced 0.5 cm (and no more than 1 cm) thick. They should include part of the lesion and adjacent apparently healthy tissue. Gastrointestinal tract and other tubular organs

Ensure that the mucosal surface of tubular organs (eg gut, uterus, bladder) is exposed by excision of the wall before immersion in fixative. Handle only the edges of delicate tissues so that histological detail is not lost. Spinal cords After exposure, the spinal cord should be lifted by grasping the dura with tissue forceps. Each spinal nerve root is then severed as it is exposed. The dura is then opened longitudinally in the dorsal midline and the cord and dura submitted whole in a large container of buffered formalin. To prevent fixation of the cord in a curled position, Transect the cord at 10 to 20 cm intervals, leaving a part of the dura intact at each transection site, to keep the cord segments connected. The cord will then be fixed in short, straight segments. Open the dural to allow better penetration of the fixative. Brain There are several methods of exposing the brain for removal. Convenient methods for large animals (eg longitudinal and transverse craniotomy) are used in the National TSE Surveillance Program (http://www.animalhealthaustralia.com.au/programs/adsp/adsp_home.cfm) It is preferable to submit the brain whole in a wide mouth container with 10 times its volume of buffered formalin solution. After 24 hours fixation, the formalin solution can be changed to hasten fixation. Ideally, bovine and equine brains should be immersed in at least 6 litres of buffered formalin solution (e.g. in plastic buckets) for at least 48 hours, then submitted in a large container of fresh buffered formalin solution. It is essential to avoid distortion of the brain during fixation. Compression of brain surface resting on the base of the container can be avoided by adding neat formalin until the brain floats. If the brain cannot be submitted whole, it may be cut transversely into two or three pieces. Always cut transversely so that all brain sections can be examined (grossly and histologically) for bilateral lesions. Do not cut the brain longitudinally.



HAEMATOLOGY

BLOOD For red and white cell counts, haemoglobin estimation and derived parameters, collect blood in EDTA (purple top) tubes. EDTA blood may not be suitable for differential white cell count and morphology if blood films are not made within 1 hour of blood collection.

BLOOD FILMS Fresh blood films should be made from EDTA blood immediately after collection. They should then be air dried. Leucocytes degenerate when blood stands for a few hours. Use dry slides, free of dirt and grease. Avoid contamination when preparing blood films.

PREPARATION OF BLOOD FILMS i. Use a glass slide as the spreader

The spreading edge must be smooth (i.e. no chips out of it) and clean. Spreaders can be re-used if cleaned and dried after each use.

ii. Place a small drop of blood (thoroughly but gently mixed) in the centre of the slide about 1 cm from the end. Grasp the spreader with thumb and middle finger using the index finger to put light pressure on the spreader.

iii. Place the spreader beyond the drop and at 45o to the slide. Draw it back until it touches the drop and the blood spreads evenly across the slide almost to the edges.

iv. Quickly and smoothly push the spreader right to the end of the slide at the 45o angle.

v. Wave the slide quickly in the air to dry it. Label at the thick end with the owner's name and animal ID (if necessary) using a lead pencil.

STORAGE AND DESPATCH OF SPECIMENS

Blood should be kept chilled, not frozen, whilst being held prior to despatch and during transport to the laboratory. Blood should be submitted to the laboratory chilled in insulated containers. Avoid contact between the blood sample and icebricks.

NB Do not chill blood films or expose them to formalin vapour. Blood films can be submitted in the same container as chilled specimens, provided the blood films are securely packed and well wrapped for insulation against chilling.

PARASITOLOGY

Faecal egg counts and egg type (by larval differentiation) are a guide to the size and type of worm burden. Faecal egg counts are influenced by faecal consistency and bulk, host resistance, stage of pregnancy and effects of lactation. Starvation will increase egg counts and inappetence may cause the count to increase by up to 30 to 40 times. Diarrhoea will reduce egg counts. Faecal egg counts will vary according to the parasite species involved and whether the worm burden consists of sexually mature parasites. The history should provide details of recent anthelmintic treatments including drench resistance status, stocking rate, pasture availability, swampy areas, grazing rotation. STANDARD CONTAINERS AND EQUIPMENT

Jars Plastic (5 ml, 20 ml, 70 ml) screw top containers for insects, parasites, aliquots of gut washings, skin scrapings. Parasite Faecal Collection Kits Collection kits are available from your Regional Veterinary Laboratory: • WormTest kits contain 10 containers for

monitoring a single mob or grazing group in a herd.

• DrenchTest kits include 50 containers for one control and four test drench groups. Use additional WormTest kits for extra drench groups.

• Horse WormTest kits are suitable for 5 horses.

• DrenchRite® kits for bulk faecal collection allow farmers to test for resistance with a single muster.

Vacuum blood tubes (with anticoagulant) For heartworm antigen and microfilaria tests in dogs (generally for export purposes).


Vacuum blood tubes (plain) For serology of liver fluke infection in cattle.

COLLECTION OF SPECIMENS Faecal samples For individual animals at least 30 g of fresh faeces should be collected (preferably from the rectum) and placed directly into a jar. The jar should be filled and the lid closed tightly in order to minimise exposure of worm eggs to air which would allow egg development. Wormtest Kit samples should be collected from 10 animals, including apparently healthy and affected animals in each mob. Complete instructions for collection of samples are provided with each WormTest kit. Gastrointestinal tract The total gastrointestinal tract should be submitted direct to the laboratory when it can be delivered by the submitter, double bagged in strong clear plastic bags. Only submit unpreserved chilled gastrointestinal tracts if delivery to the laboratory is assured within 36- 48 hours. The tract should not be opened and each part should be tied off at the appropriate junction, i.e. between the stomach (abomasum) and small intestines and between the small and large intestines. Alternatively the organs can be washed by the field veterinarian and aliquots (from washings of known volume) submitted to the laboratory. Contact the laboratory for instructions on the procedure. Insects and snails for identification These should be submitted in 70% alcohol in a small leak proof container. Mites Acute skin lesions should be scraped with a scalpel until blood is produced. Moisten the scalpel with liquid paraffin. Scrape particularly at the edge of any visible lesion. Before scraping, hair or wool must be clipped as close to the skin as possible. Scrapings should be sealed in a small, wide mouthed bottle and submitted unpreserved. Parasites for identification Flukes, tapeworms and roundworms should be washed in water and preserved in 5 per cent formalin. Always include the head of the tapeworm.

When identification of parasitic cysts is required, tissues should be submitted chilled in a jar. Blood Collect a full tube of blood. For tubes with anticoagulant ensure blood is gently mixed with anticoagulant. Plain blood tubes should be allowed to clot at room temperature.


Faecal samples should be kept cool, but not frozen. Even prolonged chilling at 5oC kills the eggs of most species and makes samples unsuitable for larval culture for strongyle egg identification. Faecal samples should be submitted as soon after collection as possible, chilled in an insulated container, with icebricks wrapped in newspaper to prevent freezing. Gastrointestinal tract washings should be preserved in 5% formalin. Also see specific parasitic diseases: • Anthelmintic resistance in sheep • Babesiosis • Coccidiosis • Cryptosporidiosis • Cysticercosis • Drenching mortalities • Equine babesiosis • Fasciolosis in sheep and cattle • Ostertagiosis • Paramphistomiasis • Parasites internal/external/resistance • Sarcosporidiosis • Tapeworms • Tick fever • Toxoplasmosis • Trichomoniasis of cattle • Total worm counts (interpretation) • Worm egg counts (interpretation)

SEROLOGY

Serology is available for a range of bacterial and viral diseases, as well as for some chlamydial, mycoplasmal, rickettsial, protozoan and metazoan diseases. Such tests may be available in Regional Veterinary Laboratories, or only in Central Veterinary Laboratories at EMAI, Menangle. A serological test is used to show the presence or absence of antibody to a specific aetiological agent or group of agents. The


presence of antibody indicates exposure to the organism, which may be due to a current clinical condition or to an earlier unrelated infection. In tests where results are expressed in titres, the best evidence of infection is the demonstration of a four-fold titre rise between samples collected early in the clinical episode and those collected 2-3 weeks later. A titre variation of less than four-fold (i.e. one dilution) is within the normal variation of a serological test and is not significant. Since many animals in endemic areas may have antibody to a given organism, a single sample from an affected animal may not allow the serological test to be interpreted. A single serum sample is particularly useful in eliminating a diagnostic possibility. Frequently, presence of antibody in the acute phase or absence in the convalescent phase will eliminate a diagnostic possibility. Haemolysed or contaminated samples often give unreliable results in a complement fixation test (CFT): the serum may be anti complementary or give non-specific low titre positives, particularly in sheep and goat CFT's. Poor quality samples will give poor quality results, necessitating retesting of the animals involved (see Avoiding haemolysis of blood samples). STANDARD CONTAINERS AND EQUIPMENT

Blood vacuum tubes, 10 ml These should be silicone coated, without anticoagulant.

Sterile screw capped containers, 5 ml For serum samples.


Blood samples should be collected aseptically, using a 10 ml blood vacuum tube. At least 5 ml of blood should be collected. When a range of serological tests is required (particularly when viral serology and non-viral serology is to be undertaken), duplicate samples should be collected. Contamination of the container and stopper should be avoided. Blood and faecal material should be removed prior to despatch, to reduce the risk of contamination of laboratory staff handling the specimens.

Use a separate sterile needle to avoid mechanically transmitting infectious agents from one animal to another. Avoiding haemolysis of samples Haemolysis occurs as a result of poor collection techniques, contaminated equipment or poor handling of the sample once it is collected. Common causes of haemolysis include: • Use of non sterile containers for

collection or storage. • Contamination by faecal and other

material due to faulty aseptic techniques.

• Contamination of the sample by water. • A slow flow from the needle, due to

obstruction of the needle, or failure to insert into mid-vein.

• Forcibly expelling blood through a needle.

• Heating of samples, usually in car boots or through back windows of car, or after prolonged exposure to direct sunlight during collection.

• Freezing. NB. Pig blood haemolyses quickly. Serum should always be separated from the clot within 4 hours of collection. Labelling of samples Samples must be labelled serially (e.g. from 1 to 30) with a water proof pen, preferably on an adhesive label. Keep a key list which correlates sample numbers with animal identification. Do not label the stopper, which is removed during testing. The specimen advice form submitted with the samples should list clinical details beside each sample number. This will allow the laboratory to offer an informed comment on the results and perhaps apply other relevant tests. DO NOT label samples with tag numbers, names, etc., as this leads to confusion and errors in reading numbers in the laboratory. It also makes it very difficult for the laboratory to ensure all sample are present or to check on missing or broken samples. DO NOT label containers with water soluble ink. It smudges when wet and may rub off if samples are chilled or frozen. Serum can be frozen, provided there are no blood cells present in it.



Samples should be allowed to clot before transporting them over any distance. Clots may not retract readily in cold weather or if they are chilled too soon after collection. Samples should be held in a warm room until the clot retracts. Once the clot has retracted, blood samples must be held chilled to reduce contamination, haemolysis and autolysis. If the laboratory will not get the samples within 48 hrs of collection, decant the serum into a 5 ml sterile, screw capped plastic container and submit the serum sample only. If virological examination of the clot is also needed (e.g Pestivirus antigen detection), submit clot separately.

TYPES OF SEROLOGICAL TESTS Complement Fixation Test (CFT) The test quantifies complement-fixing antibodies to a range of antigens. Test serum, complement and antigen are incubated then sensitised erythrocytes are added. If there is specific antibody present, an antigen-antibody complex is formed which binds complement and sensitised erythrocytes are not lysed. Results are given in titre form, reflecting a doubling serial dilution of serum from 1:4 upwards. The titre indicates the dilution at which 50% or more of the erythrocytes are not lysed. Enzyme-Linked Immunosorbent Assay (ELISA) The ELISA detects specific antibodies in serum which are allowed to bind to antigen on a solid phase. To detect the bound serum antibodies, the test uses a second antibody system (directed against antibodies of the animal species under test) to which is linked an enzyme. This enzyme catalyses a colour reaction, and the amount of colour which develops reflects the level of original serum antibody. This colour is measured quantitatively and is termed the serum absorbance or optical density. The second antibody is usually species-specific, but may react with closely related animal species at a different concentration. Thus each ELISA is geared towards one test animal species. An absorbed ELISA (e.g. Johne's ELISA) involves the above steps, but also includes treatment of serum to absorb non-specific antibodies before testing.

The level of ELISA-reactive antibodies can be measured in a qualitative and quantitative way. The quantitative measure is relative to control sera. This may be in terms of:

i. Absorbance (Optical Density, OD) of the test serum, or,

ii. ELISA ratio - compares the OD of the test serum to that of a negative control, but taking into account the performance of one or more positive control sera on the plate to give a valid test. Depending on the test, ELISA ratios of 2 or 3 are common cut-off points, or,

iii. ELISA value or ELISA units - depending on the test, this can be used to reflect either:

• the OD of the test sample relevant to a standard curve, originating from a range of positive and negative controls, or,

• a mathematical formula which expresses the OD of the test sample as a percentage of the positive control, with subtraction of the negative control OD from each, ie: (test OD - neg OD) / (pos OD - neg OD) x 100

(Agar Gel Immunodiffusion (AGID) Syn: Gel Diffusion Precipitin Test (GDPT) Specific antibody is detected by placing test serums and control serum in wells in a gel around a central antigen well. Migration of antigen and antibody towards each other results in a visible line of precipitation where an antigen/antibody complex is formed. A GDPT reaction reflects the position of a specific line of reaction relevant to the serum and antigen wells, on a limited scale of trace, 1, 2 or 3. A reading of 2 indicates a line midway between the serum and antibody wells. A trace and 1 reaction are located closer to the serum well, whereas a 3 reaction is located closer to the antigen well. Trace is the weakest reading, and 3 reflects the highest level of antibody detected. Serum Agglutination Test (SAT) Tests for agglutinating antibody in a tube-based test, where clearing of the tube due to agglutination is used to determine an endpoint. Results are expressed as a titre, and the dilution series used (and therefore the titre) reflects the standard protocol for the test required. In some tests, the endpoint readings are converted to international units rather than titres.


Microscopic Agglutination Test (MAT) A test used for Leptospiral antibody which measures the ability of sera at varying dilutions to give an endpoint of 50% agglutination of one of a range of live leptospiral serovars. Indirect Fluorescent Antibody Test (IFAT) A slide-based assay using serum at varying dilutions to show specific binding to the test antigen on the slide. The fluorescent-labelled second antibody provides a measurable signal of bound antibody from the test serum.

Rapid Plate Test (RPT) An agglutination assay used for some poultry pathogens, and performed with diluted serum on a solid base. Latex Agglutination Test (LAT) Antigen coated-latex beads are reacted with diluted sera, with a positive test giving a lattice of latex beads due to agglutination. Rose Bengal Test (RBT) A spot agglutination test performed on a solid base and used as a screening test for Br. abortus. Gives a qualitative (+/-) result only.


SUMMARY OF AVAILABLE SEROLOGICAL TESTS FOR VARIOUS NON-VIRAL DISEASES Host Disease Agent Available Diagnostic Test Cattle Leptospirosis L pomona

L hardjo Other Lepto serovars include: copenhageni, tarassovi, grippotyphosa, canicola, australis, zanoni

MAT (serovar specific) MAT* (serovar specific)

Campylobacteriosis Campylobacter fetus venerealis ELISA (vaginal mucus) Johne's disease M paratuberculosis ELISA, CFT* Salmonellosis S Typhimurium

S Dublin SAT (serogroup specific)

Chlamydiosis/SBE C psittaci CFT Brucellosis B abortus ELISA, RBT*, SAT* Liver fluke F hepatica ELISA Q fever C burnetti CFT* Neosporosis Neospora caninum ELISA Sheep Brucellosis B ovis CFT, ELISA Salmonellosis S Typhimurium

S Dublin SAT (serogroup specific)

A. seminis epididymitis

A seminis CFT (at ARI Yeerongpilly)

Johne's disease M paratuberculosis AGID, CFT* Chlamydiosis C psittaci CFT Liver fluke F hepatica ELISA Toxoplasmosis T gondii LAT Goats Johne's disease M paratuberculosis ELISA, AGID, CFT* Toxoplasmosis T gondii LAT Brucellosis B abortus SAT* Q fever C burnetii CFT* Horses Babesiosis B equi IFAT* Leptospirosis L pomona

L tarassovi MAT*(serovar specific)

Brucellosis/ Fistulous withers

B abortus SAT*

Pigs Mycoplasmosis M hyopneumoniae M hyorhinis

ELISA

Leptospirosis L pomona, L tarassovi, L bratislava

MAT (serovar specific)

Brucellosis B suis RBT, SAT Poultry Pullorum S pullorum RPT Mycoplasmosis M gallinarum

M synoviae RPT

Dogs

Leptospirosis L canicola L copenhageni

MAT (serovar specific)

Key AGID Agar gel immunodiffusion test; CFT Complement fixation test ELISA Enzyme linked immunosorbent assay IFAT Indirect fluorescent antibody test; LAT Latex agglutination test MAT Microscopic agglutination test RPT Rapid plate test RBT Rose Bengal test SAT Serum agglutination test; * Commercial test only


TOXICOLOGY

It is not possible to comprehensively screen samples for 'poisons' or 'toxins'. It is up to the submitter to consider the history, clinical signs and lesions (if any) and identify specific toxins for analysis. Contact your Regional Veterinary Laboratory if you are unsure whether a test is available for a particular toxin. In cases of suspected poisoning, it is important that an effect be demonstrated in the animal. For example, nitrate poisoning is confirmed by demonstrating the presence of nitrate in the serum or blood of the animal, not by demonstrating the presence of nitrate in pasture plants in the paddock. It is important that the history provided includes details of treatment with any suspected toxic compound, particularly in relation to the strength of the preparation and the time since treatment or access to the material. STANDARD CONTAINERS AND EQUIPMENT

Blood vacuum tubes, 10 ml For blood samples

Leakproof 5 ml tubes or bottles For serum samples.

Bottles and jars For tissue samples

Slides These must be clean. Some brands available have not been washed and are not suitable unless they are washed.


Particular care should be taken in collecting and packaging specimens for toxicology, because there is often a possibility that infectious agents are involved and these can create hazards to staff handling the material in a laboratory. Therefore avoid contaminating the outside of any submitted containers with tissues and ensure they are leakproof. Tissue samples At least 100 g of tissue should be collected, taking care to avoid contamination with soil, faeces or intestinal contents.

Separate organs should be placed in separate containers. Body fat is the preferred tissue for insecticide residue testing. For biopsy material, a minimum of 2g is required. Blood samples At least 8 ml of blood in a blood vacuum tube, free from contamination with faeces etc. Serum samples At least 2 ml of serum should be submitted. Blood smears Thick air dried smears should be prepared, taking care to leave one end of the slide clean. They should be dry before being wrapped in paper. Suspected toxic material Suspected material, feedstuffs or plants should NOT be sent unless appropriate specimens from affected animals have also been sent. At least 50 g of material should be forwarded. Plant material Examination for nitrate nitrite and cyanide are best performed in the field. Plants for identification should be pressed and dried. Refer section on "Poisoning - plant" for submission of plants for identification.

Ingesta Nitrate and nitrite disappears rapidly from ingesta, and thus ingesta is of no value in diagnosing nitrate/nitrite poisoning. For other chemical poisons, eg. arsenic, lead, at least 250 g of ingesta should be submitted in an air tight, leakproof container.


Tissues should be frozen. Herbage and ingesta samples for toxin examination should also be frozen. Other animal specimens should be chilled.


INTERPRETATION OF ARSENIC, LEAD AND COPPER CONCENTRATIONS IN CATTLE AND SHEEP

Analyte Sample Units Deficient Normal Toxic Arsenic Liver mg/kg (wet wt) < 0.5 * > 8 Lead EDTA blood

Kidney Faeces

µmol/L mg/kg (wet wt) mg/kg (wet wt)

< 1.2 < 4

< 10

> 1.2 > 25 > 25

Copper Liver Kidney

mg/kg (wet wt) mg/kg(wet wt)

< 4 ** 20-70 4-6

† > 100 >8

* Liver arsenic may be in range 2 to 8 mg/kg if several days elapsed since toxic exposure. ** Typical liver copper for cattle is > 20 and sheep > 40. † In sheep, liver copper may increase up to 200 mg/kg (wet wt) before poisoning occurs NB

i. Interpret concentrations between normal and toxic according to clinical and pathological findings.

ii. Concentrations based on dry wt are approximately 5 times the above (wet wt) values. iii. Conversion from mg/kg to SI units is as follows: • As mg/kg x13.3 = As μmol/kg • Pb mg/kg x 4.8 = Pb μmol/kg • Cu mg/kg x 0.0157 = Cu mmol/kg

VIROLOGY

Virological examinations involve demonstration of a pathogenic virus or detection of antibody to virus. Findings must be interpreted in the light of history, clinical findings, lesions, etc. It is not possible to screen for a wide range of viruses. Submitters should forward specimens to be tested for specific viruses. If there is any doubt about the availability of a test, the laboratory should be contacted for advice. DIAGNOSIS OF VIRAL DISEASE

The diagnosis of a viral disease can be based on Histopathology, Virus isolation, Virus Serology, or Virus or Virus Antigen Detection. HISTOPATHOLOGY Cytological changes can indicate a viral aetiology. VIRUS ISOLATION Cultivation and identification of virus grown in tissue culture or eggs inoculated with specimen. NB Failure to cultivate the virus does not rule out a viral aetiology. Cultivation of a virus does not necessarily mean it caused the disease process.

VIRAL SEROLOGY Detection and quantitation of virus specific antibody in the serum of an infected animal. Plasma is also an acceptable sample for most serological tests.

Presence of antibody can be a result of clinical disease, unapparent infection, passive immunity or vaccination. Tests variously demonstrate group specific antibody, type specific antibody or a cross reaction. Viral serology can be applied with the following limitations:

Single Serum • Useful only to eliminate a diagnostic

possibility. Presence of antibody in the acute phase may eliminate the diagnostic possibility; absence of antibody in the convalescent phase eliminates the diagnostic possibility.

• Accurate interpretation often difficult because time of collection may be critical. A negative test on serum < 3 weeks after a suspected viral condition leaves the possibility that the animal was infected but had not yet produced detectable antibody.

Paired Sera • A change from negative to positive

antibody status is known as seroconversion and indicates infection.

• A four-fold rise in antibody titre between acute and convalescent samples can also indicate infection by the specific pathogen.

However both the above may also be due to cross reaction or booster immunisation. They may also indicate stress induced reactivation of latent infection.


The relationship between virus and antibody varies between diseases. In some, virus does not occur in animals with detectable antibody, e.g., Akabane, Ephemeral Fever whereas in others, virus and antibody can be present in the one animal, e.g., EIA, IBR, CAE, EBL and occasionally Pestivirus. NSW Department of Primary Industries offers the following types of viral serology tests: • Agar Gel Immunodiffusion Test (AGID) • Virus Neutralisation Test (VNT) • Haemagglutination Inhibition (HI) Test • Enzyme Linked Immunosorbent Assay

(ELISA) Agar Gel Immunodiffusion (AGID) Test AGID involves diffusion of viral antigen and antibody towards each other through a gel. When they combine, they precipitate in the gel. This produces a visible line where the concentrations of antigens and antibody are balanced. An excess of antigen or antibody can alter the location and appearance of the precipitin line. Each test sample for viral antibody is tested against a known viral antigen to examine the relationship of any precipitin line formed with an adjacent standard reference line formed between known positive serum and virus antigen. This reference line is optimised to give a strong visible precipitin line centrally located between the antigen and positive serum wells (termed a 3 reaction - see below). For viral serology, a specific precipitin line formed by the tested serum sample is recorded as 1, 2, 3, or >3 to describe its relative position to the serum well and the antigen well: Description / Position of Precipitin Line ResultTurn on end of reference line 1Line, closer to serum well 2Line, midway between serum well and antigen well 3Line, closer to antigen well >3

A 1, 2, 3 or >3 antibody reaction is a positive test for antibody, and indicates that an animal has been infected with the specific virus (or a related virus). Strength of viral antibody levels in the test generally does not reflect the severity and stage of infection with any certainty. Virus Neutralisation Test (VNT)

Serum is usually titrated in two fold serial dilutions beginning at 1/4 or 1/10. The virus neutralising antibody titre is the reciprocal of the serum dilution that will neutralise a standard amount of virus (usually 100TCID50). Two serial dilutions rise (i.e. 4-fold) in titre in the convalescent serum sample compared to the acute serum sample is considered significant. This occasionally occurs as a result of technical or statistical variation, so greater differences can be accepted with greater confidence. Haemagglutination Inhibition (HI) Test HI tests are used to detect antibodies to viruses which agglutinate erythrocytes. HI tests depend on antibody binding a fixed amount of antigen, preventing it from causing haemagglutination. Sera is tested in two-fold dilutions, and results expressed as a titre (negative, 2, 4, 8, .......) etc. More accurate measurement of flock status can be gained by repeat sampling of the flock to determine if a rise in titre (e.g. a rise in titre of two dilutions or more is arbitrarily considered significant) has occurred. Enzyme linked immunosorbent assay (ELISA) ELISA results can express the detection of antibody in a qualitative (+ or -) or a quantitative way. The three common means of expressing a quantitative result are absorbance (optical density), ELISA ratio and ELISA value (unit).

Absorbance (Optical Density) The amount of antibody in a sample is proportional to the absorbance (optical density, OD) given by it. The OD may be standardised against one or more serum controls on the ELISA plate. ELISA ratio The ELISA ratio indicates the strength of the sample OD compared to that of a negative serum control on the same ELISA plate. (E/R = OD test/OD neg control) ELISA value or ELISA units The sample OD is fitted to a curve determined by the performance of control positive and negative samples, and the result given as an ELISA value (eg 0 100). This is often a measurement of how the sample OD compares with that of a high positive control (taken as a value of 100). Percent inhibition The results of blocking or competitive ELISAs are expressed as the reduction in OD (as a %) relative to the OD given


by a reference positive (often monoclonal) antibody.

ELISA ODs, ratios or values may be placed into categories (negative/inconclusive/positive) determined by previous experience of the performance of the test. VIRUS OR VIRUS ANTIGEN DETECTION The following types of test are available for the direct detection of virus or viral antigen: • Electron microscopy (EM) • Agar Gel Immunodiffusion (AGID) Test • Enzyme linked Immunosorbent Assay

(ELISA) • Latex Agglutination Test • Haemagglutination (HA) Test Electron microscopy (EM) Allows detection and identification of virus particles on a morphological basis e.g. Rotavirus, Poxvirus. Agar Gel Immunodiffusion (AGID) Test Antigen detection is useful where virus infected tissues contain soluble virus antigens that visibly precipitate with specific antibody, eg: • Parvovirus in mummified pig foetuses • Pestivirus in gut scrapings of cases of

mucosal disease • Rotavirus in faeces. In such cases, the sample is tested against a known positive serum and the relationship of any precipitin line formed with a standard reference line, originating from an adjacent positive antigen, is examined. A test sample giving a line of similar position and intensity, which is continuous through a turn of identity with the reference line is classed as +++ for viral antigen. The results are recorded in terms of relative strength (+, ++, +++, or XS) against the reference line. Enzyme linked Immunosorbent Assay (ELISA) Can be used to detect either virus antigen or intact virus particles. Results can be expressed qualitatively (+ or ) or quantitatively (OD's or ELISA ratios or ELISA values). eg: • Pestivirus detection in the Pestivirus

antigen capture ELISA (PACE). Latex Agglutination Test

Can be used to detect viral antigen or virus particles. Results are only qualitative (+ or ), eg: • Rotavirus detection Haemagglutination (HA) Test Can be used to screen for haemagglutinating viruses, eg: • Newcastle Disease • Avian Influenza • Egg Drop Syndrome On poultry tissue or allantoic fluid from eggs. Results can be given as a titre derived from a doubling dilution of the sample (e.g. 2, 4, 8, ...etc). To determine which virus is responsible, HA positive samples can be tested in the presence of specific antibody in a direct (or reverse) HI test. The results are qualitative (+ or - ).


Blood vacuum tubes, silicone coated For serum samples and blood clots (for isolation of, or detection of antigen to, some viruses). Please do not use serum separator tubes. Blood vacuum tubes with heparin or EDTA For blood for virus isolation (of mainly the arboviruses) or preparation of buffy coat samples for antigen detection. Sterile bottles for samples of tissues, organs Sterile 5 ml vials: For serum and fluid samples. Swabs and PBGS bottles: For discharges, etc. Plain sterile swabs are used and immediately placed in small bottles of phosphate buffered gelatin saline (PBGS). These are available from the Regional Veterinary Laboratories. Sterile instruments An adequate range of sterile scissors and tissue forceps should be available. The containers and equipment listed are suitable for the routine collection of tissues for virus examination. Containers and equipment for special requirements will normally be supplied by the laboratory, after prior arrangements have been made.

COLLECTION OF SPECIMENS Specimens for virus isolation must be collected by aseptic techniques, using


sterile instruments and sterile containers. Avoid contamination from other tissues as well as that from extraneous sources. Submissions for virus isolation should be accompanied by serum from the affected animal(s) whenever possible. Blood This should be collected using the appropriate blood vacuum tube, avoiding contamination of the sample. A separate needle should be used for each animal. Avoid contamination of the outside of the tube by blood, soil and faeces, as this creates difficulties in handling the specimens within the laboratory. Serum If blood samples cannot arrive at the laboratory within 2 days, the serum should be poured off aseptically into a sterile 5 ml vial. The clot should also be submitted as it may be required for virus isolation or antigen detection. Tissues and organs These must be collected aseptically, using separate sterile instruments and containers for each tissue or organ. Contamination between tissues must also be avoided. Portions of tissue (no greater than 2 cm x 2 cm x 2 cm) should be taken. Scabs Scabs and underlying tissue should be submitted in a sterile bottle. Swabs Swabs from excretions, exudates, mucosal surfaces and orifices should be taken

carefully, avoiding contamination from other sites. Each swab should then be transferred to PBGS at room temperature, then chilled (but never frozen) for transport to the laboratory. Faeces At least 10 g of fresh faeces should be collected into a clean jar.


All virological specimens should be chilled prior to and during transport. If more than 48 hours is to elapse between collection and receipt at the laboratory, specimens except blood samples should generally be frozen. For isolation of Arboviruses (especially Bluetongue) and Herpesviruses (e.g. IBR), specimens should never be frozen. These viruses have extremely poor survival at 20oC with just a single freeze, and tissues for their isolation must be kept chilled. All specimens should be clearly labelled and sent in a leakproof container. Check that screw caps are tight, especially after freezing. All samples should be packed in insulated containers with sufficient icebricks to ensure that they are still cold when received at the laboratory. However, care should be taken to prevent direct contact between coolant bricks and specimens, which may otherwise become frozen.

SUMMARY OF AVAILABLE VIROLOGICAL TESTS FOR VARIOUS DISEASES OR DISEASE SYNDROMES Section A: Recognised Viral Diseases Host Disease Virus Available Diagnostic

Test Cattle Ephemeral fever (EF) Rhabodovirus VNT, (ELISA), (VI) Bovine pestivirus

Mucosal disease (MD) Infertility Abortion Congenital abnormalities

Pestivirus Antigen ELISA, AGID, VI Antibody ELISA, VNT

Bovine malignant catarrh Bovine herpesvirus Ovine herpesvirus 2 (OHV2)

Nil; use histopath PCR at AgWest

Infectious bovine rhinotracheitis (IBR)

Bovine herpesvirus – Type 1

VNT, VI

Infectious pustular vulvovaginitis (IPV)

Bovine herpesvirus – Type 1

VNT, VI

Encephalitis Bovine herpesvirus – Type 1

VNT, VI


Host Disease Virus Available Diagnostic Test

Mammilitis Bovine herpesvirus – Type 2

EM

Bovine papular stomatitis (pseudocowpox)

Parapox virus EM

Congenital arthrogryposis/hydranencephaly (AG/HE)

Akabane virus Aino virus Palyam virus Pestivirus also causes AG

AGID, VNT, (VI)

Calf scours Rotavirus Coronavirus

LA, EM EM

Enzootic bovine leucosis (EBL) EBL virus (Retrovirus) ELISA, AGID Bluetongue Bluetongue virus (BTV)

(various types) AGID, ELISA, VNT, VI NB. AGID is group specific; VNT is type specific

Abortion congenital abnormalities

Palyam virus (various types) (see also Pestivirus)

AGID, (VNT, VI)

Enzootic haemorrhagic disease of deer (EHD) virus (EHD1 EHD5)

AGID, (VNT, VI)

Ibaraki disease EHD2 AGID, (VNT, VI) Sheep Contagious pustular dermatitis

(Orf, scabby mouth) Parapox virus EM, Histopath

Arthrogryposis/hydranencephaly (AG/HE)

Akabane virus AGID, VNT, ELISA

Ovine pestivirus (Border disease)

Pestivirus Antigen ELISA, AGID, VI, Antibody ELISA, VNT

Bluetongue Bluetongue virus (BTV) (various types)

AGID, ELISA, VNT, VI NB. AGID is group specific; VNT is type specific

Pigs Encephalomyocarditis virus (EMC)

Cardiovirus VI, (VNT)

Swine Pox Poxvirus EM, Histopath Haemagglutinating encephalitis

virus infection (HEV) (Vomiting and wasting disease)

Coronavirus (HI)

Piglet scours Rotavirus LA, EM Parvovirus disease Parvovirus AGID

(for antigen and antibody)

Goats Caprine arthritis encephalitis (CAE, big knees)

Retrovirus ELISA, (VI, AGID)

Vaginitis Caprine herpesvirus VI, (VNT) Horses Equine infectious anaemia (EIA) Retrovirus AGID Equine rhinopneumonitis

Viral abortion Equine herpesvirus –Type 1

VI, (VNT)

Poultry Avian encephalomyelitis (AE) Picornavirus ELISA, Histopath Avian influenza

Fowl plague/ Duck influenza

Orthomyxovirus AGID, ELISA, HA, VI, Histopath

Avian leukosis Retrovirus Histopath Egg drop syndrome (EDS 76) Haemagglutinating

adenovirus 127 (group II) HI, (VNT, HA, VI)

Fowl adenovirus (FAV) acute inclusion body hepatitis (IBH) of chickens < 14 d

Adenovirus (group I) (1 serotype of 12)

AGID, VNT, Histopath AGID is group specific; VNT is serotype specific


Host Disease Virus Available Diagnostic Test

Fowl Pox (FP) Avipoxvirus EM, (VI), Histopath Turkey haemorrhagic enteritis

(THE) Haemorrhagic enteritis (HE) of turkeys

Adenovirus (group III) AGID, Histopath

Herpesvirus of turkeys Herpesvirus (VI), (HVT) Inclusion body hepatitis (IBH) Adenovirus (group I)

(Many serotypes) VI, VNT, Histopath

Infectious bronchitis (IB) Coronavirus (VI), Histopath Infectious bursal disease (IBD) Birnavirus ELISA, (AGID),

Histopath Infectious laryngotracheitis (ILT) Herpesvirus Antigen ELISA,

Histopath, (VI) Marek's disease Herpesvirus (AGID, VI), Histopath Newcastle disease (ND) Paramyxovirus HI, ELISA, HA, VI,

Histopath Reticuloendotheliosis (RE) Retrovirus VNT, (VI), Histopath Fish Epizootic haematopoietic

necrosis (EHN)

Iridovirus VI

Crustacea White spot syndrome (WSS) White spot syndrome virus PCR Section B: Syndromes with Possible Viral Involvement Host Disease Possible Virus Available Diagnostic Test Cattle Respiratory disease Parainfluenza 3 (PI3) ELISA, (VNT) Respiratory syncytial (RSV) ELISA, (VNT) Pestivirus AGID, (ELISA, VI, VNT) Rhinovirus * Adenovirus AGID Sheep Respiratory disease PI3 ELISA, (VNT) Poultry Big liver spleen syndrome

(BLS)

? AGID, Histopath

Key AGID Agar gel immunodiffusion test ELISA Enzyme linked immunosorbent assay EM Electron microscopy HA Haemagglutination test HI Haemagglutination inhibition test LA Latex agglutination test VI Virus isolation VNT Virus neutralisation test * available by arrangement


SPECIMENS (BY DISEASE OR SYNDROME)

DISEASES OF LIVESTOCK

ABORTION (GENERAL) Notes on the individual species should be consulted. Whenever possible, the aborted foetus together with the foetal membranes should be submitted to the laboratory. Every opportunity should be taken to emphasise to owners the importance of collecting the foetus and foetal membranes. Where it is not feasible to submit the foetus, the autopsy must be complete, and a range of tissues and body fluids submitted to eliminate cause of perinatal death other than infectious agents e.g. dystocia. Diagnosis Based on bacteriology, virology, serology and histopathology findings together with the previous clinical history of the dam. Infectious abortions can be distinguished from non-infectious abortion by measuring elevated levels of species-specific IgG in foetal fluid (blood, peritoneal, pleural or pericardial fluid). This test is available for cattle and pigs.

ABORTION IN CATTLE Common infectious causes of abortion in New South Wales are leptospirosis, and campylobacteriosis. Other infectious causes include listeriosis, fungal and viral infections of the dam and foetus. Pestivirus, Akabane and Palyam virus possibly cause abortion. Pestivirus may be associated with abortion at any stage of gestation; akabane and palyam are predominantly associated with abortions of the last trimester of pregnancy. These three viral infections may cause congenital abnormalities. In some regions (e.g. North Coast, Illawarra and South Coast), the most commonly diagnosed infectious cause of abortion is neosporosis. Characteristic lesions are seen in sections of fixed brain and myocardium. Neospora caninum, a sporozoan with a canid-bovine transmission cycle, has recovered from tissues of affected foetuses. Non-infectious causes include plant poisonings and congenital abnormalities.

If a full term calf is examined in the field, and dystocia is indicated, there is little value in laboratory investigation. Specimens required

Aborted foetuses

i. The whole foetus and foetal membranes submitted chilled, or:

ii. Portions of foetal stomach contents, placenta, liver, lung, spleen;

iii. Clear serous body fluid (pericardial, thoracic or peritoneal) or heart blood submitted chilled in separate sterile containers, and,

iv. Sections of cotyledons, brain, myocardium, lung, liver, and kidney in buffered formalin for histopathology.

Cows i. Sera from the aborting animals and

10-15 animals from the same group. Indicate on the Specimen Submission Form which animals have aborted.

ABORTION IN SHEEP AND GOATS

Common causes in New South Wales include toxoplasmosis, campylobacteriosis, listeriosis, brucellosis, Border disease, Akabane virus and pasteurellosis. Other causes include chlamydiosis and conditions causing clinical illness in the ewe or doe, e.g. salmonellosis, hypocalcaemia, pregnancy toxaemia, septicaemia. Habitual abortion can occur in Angora does. Specimens required

Aborted foetuses

i. Foetus and foetal membranes submitted whole in an insulated container. They should be chilled, not frozen. or

ii. Portions of foetal stomach contents, placenta, liver, lung, spleen

iii. Clear serous body fluid (pericardial, thoracic or peritoneal) or heart blood submitted chilled in separate sterile containers, and,

iv. Sections of cotyledons, brain, myocardium, lung, liver, and kidney in buffered formalin for histopathology.

Ewes and does i. Sera from the aborting animals and

up to 10 flock mates submitted chilled. Indicate on the Specimen


Submission Form which animals have aborted.

See Abortion in cattle re specimens for virology

ABORTION IN HORSES In the past, it has frequently been impossible to reach a diagnosis because of an inadequate history and unsuitable specimens submitted. Common infectious agents responsible for equine abortion include Streptococcus spp., Staphylococcus spp., Escherichia coli, Salmonella spp., Klebsiella spp., leptospirosis, equine herpesvirus and fungi.

Non-infectious causes may include maternal pyrexia and malnutrition, cord compression abnormalities, endocrine dysfunction, endometrial incompetence and immunological factors. Specimens required

Aborted foetuses

i. Foetus and foetal membranes submitted whole and chilled in an insulated container, or,

ii. Portions of placenta, liver, lung, kidney and spleen together with foetal stomach contents submitted chilled.

iii. Portions of placenta, liver, adrenal gland, lung, kidney, brain and spleen submitted in buffered formalin for histopathology.

ABORTION IN PIGS

Common causes of abortion in swine in New South Wales include leptospirosis, streptococcal infection and toxaemic conditions in the sow. Other possible causes include genetic factors, oestrogenic feeds, erysipelas, encephalomyocarditis (EMC) virus and vitamin A deficiency. Abortion should be differentiated from parturient and immediate post parturient deaths due to hypoxia and managerial factors such as injury or stress, smothering and chilling. NB Parvovirus infection causes foetal mummification and parturient deaths; it does not normally cause abortion. Specimens required

i. Aborted foetuses and membranes submitted chilled.

ii. Serum sample from the aborting sow and up to 10 herd mates, submitted chilled.

ACETONAEMIA

Syn: Ketosis See also: Pregnancy toxaemia in sheep Occurs in both beef and dairy cattle Diagnosis History, particularly starvation and/or stress in late pregnancy and early lactation; clinical signs, serum biochemistry (beta hydroxy butyrate), demonstration of ketones in urine and milk. Specimens required

i. Serum sample submitted chilled (for beta hydroxybutyrate analysis).

NB Examinations of urine and milk for ketones and glucose should be carried out in the field.

ACTINOBACILLOSIS AND ACTINOMYCOSIS

Diagnosis Demonstration of Actinomyces or Actinobacillus organisms in pus or tissue sections from early lesions; typical histological changes. Granulomatous lesions containing granules in the pus may also be caused by staphylococci and Arcanobacter (syn Corynebacterium) pyogenes. Specimens required

i. At least four pus smears from deep parts of the lesion. Smears from the surface are not satisfactory.

ii. Affected tissue and specimens of pus submitted chilled.

iii. Sections of recent lesions in buffered formalin for histopathology.

ACTINOBACILLUS SEMINIS INFECTION IN RAMS

Diagnosis History, clinical examination (particularly for orchitis in young rams). Demonstration of A seminis on culture of semen. Specimens required

i. Semen sample collected aseptically, submitted chilled.

ii. Serum sample, submitted chilled (to eliminate Brucella ovis as a cause).

iii. Testes, epididymes and accessory sex glands including ampullae and seminal vesicles submitted chilled.


NB The CF test on serum is not a reliable test for A. seminis.

AFLATOXICOSIS Aflatoxins can cause a range of clinical signs including nervous signs, diarrhoea, hepatic disorders and abortions. Diagnosis Based on clinical findings, histopathology, serum enzymology, and aflatoxin analysis where appropriate. Specimens required

i. Serum for enzymology. ii. Suspected foodstuff for aflatoxin

analysis and mycology. iii. Specimens appropriate to the

particular clinical syndrome should be submitted. Irrespective of the syndrome, sections of liver and kidney in buffered formalin should be submitted to check for toxic changes.

AKABANE DISEASE IN SHEEP AND CATTLE

See also: Congenital abnormalities, abortion, arthrogryposis and hydranencephaly' Diagnosis The diagnosis of Akabane disease relies on history, pathological findings and the demonstration of antibodies to Akabane virus in foetal fluids or serum samples taken from calves or lambs which have not fed from the mother. Specimens required

i. At least 2 ml of foetal fluid (preferably pericardial or pleural rather than heart blood or peritoneal fluid which are more contaminated) for virus serology,

or, from a neonate, serum if the animal has not sucked.

ii. Serum sample from the dam, submitted chilled. (This is only useful in eliminating a diagnosis of Akabane or Aino infection)

iii. Brain, spinal cord and muscle in buffered formalin for histopathology.

NB Specimens should also be submitted to exclude pestivirus infection, which can cause arthrogryposis (AG) and CNS lesions.

ALGAL POISONING See: Blue-green algal poisoning'

ALPHA MANNOSIDOSIS OF CATTLE

See: Mannosidosis

ANAPLASMOSIS See: Tick fever

ANAEMIA This can arise from acute or chronic blood loss, by increased erythrocyte destruction or by impaired erythrocyte production. Diagnosis The diagnosis of clinical anaemia should be made in the field. The diagnosis of the cause requires the submission of the appropriate range of specimens. Many conditions, including haemonchosis, fascioliasis and enzootic haematuria are best diagnosed in the field. The following disease conditions should be considered. • Parasitism, including fascioliasis and

other helminthiases. • Enzootic haematura in cattle. • Babesiosis, anaplasmosis and

eperythrozoonosis. • Plant poisonings, including bracken

fern, pyrrolizidine alkaloids, rape. • Copper and cobalt deficiency in

ruminants. • Iron deficiency anaemia in piglets. • Coccidiosis, sarcosporidiosis. Specimens required Depending on clinical and post mortem findings, appropriate tissues should be sent. These should include:

i. Liver, kidney, spleen, skeletal and cardiac muscle and red bone marrow from the femur in buffered formalin.

ii. Blood films for haematology. iii. At least 5 ml EDTA blood for

haematology. iv. Faeces for parasitology v. Serum chilled for copper etc.

ANNUAL RYEGRASS TOXICOSIS (ART)

Toxicity of Wimmera ryegrass to cattle and sheep in WA and SA is associated with a plant toxin of the tunicaminyluracil antibiotic complex produced in galls in the rye grass seed head by parasitic bacteria (Clavibacter formerly Corynebacterium rathayi)


transported by invading plant nematodes (Anguina agrostis). This disease has not yet been recorded in NSW, but a similar disease involving the same toxin and Clavibacter bacterium has been seen in cattle and some sheep in late 1990 along the Darling flood plains, originating from infected "blowaway grass" (Agrostis avenacea). Clinical signs of ART include neurological disturbances characterised by ataxia and collapse with tetanic and clonic convulsions, ending in death, and abortions in ewes. Diagnosis History, clinical findings, gross pathology, histopathology. Evidence of yellow bacterial galls on annual ryegrass plant heads/seeds; identification of Clavibacter sp in feed. Elimination of other diseases associated with neurological signs and CNS oedema (e.g. enterotoxaemia). Specimens required

i. Fixed liver and brain in buffered formalin

ii. Suspect grass or grass seed (e.g. showing bacterial galls).

ANTHELMINTIC RESISTANCE

Repeated treatment of populations of parasites with anthelmintics selects individuals that have innate or acquired resistance to the drugs. Ultimately treatment becomes ineffective because a large proportion of the parasites in the population are resistant. It is now common for sheep to harbour at least one parasite species that is resistant to one of the major drench groups. It is becoming disturbingly common for worms to be resistant to anthelmintics in all of the major drench groups. Drench resistance is common and often highly developed in goats. Only a limited range of anthelmintics are registered for use in goats. Due to their altered drug metabolism goats may require higher dose rates than recommended for sheep. For more information see: http://www.agric.nsw.gov.au/reader/das-vettesting/2566 Diagnosis History, failure to respond to treatment, in vitro larval development assay (LDA, DrenchRite™), or on-farm trial using a faecal egg count reduction test (FECRT).

IN VITRO LARVAL DEVELOPMENT ASSAY (LDA) In vitro larval development assay (LDA, DrenchRite™) is especially useful for initial drench resistance testing on a property as it requires little on-farm effort. However, it is only able to quantify resistance to benzimidazole (BZ), levamisole (LEV), combination (BZ/LEV) and indications for macrocyclic lactones (ML, ie avermectin/milbemycins). It can not be used for increased dose levels or combinations. FAECAL EGG COUNT REDUCTION TEST (FECRT) Faecal egg count reduction test (FECRT) requires considerable on-farm effort. FECRT can be performed using either:

i. A DrenchTest kit, or, ii. Several WormTest kits (one for each

anthelmintic and one for a control group).

FECRT anthelmintics Where nematode resistance is suspected, the trial should contain groups treated with a range of anthelmintics. Traditionally 5 groups (BZ, LEV, BZ/LEVcombination, ivermectin (IVM) and an untreated control group) were used. However, with recent extensive spread of resistance in these chemical groups, other drenches including naphthalophos (NAP, Rametin ™), and its combinations with BZ and LEV, closantel (CLS), and other MLs such as abamectin, and moxidectin should be considered. Since 2002 half-dose ivermectin and 1/3-dose CLS have been recommended for the early detection of emerging resistance. Use of special formulations of triple or quadruple combinations (Triton™ and Q-drench™, respectively) should be included if possible. Groups may be included where treatments are given at greater than the recommended dose rates to fully evaluate the available anthelmintics. In practice, only double dose LEV or double dose LEV plus single BZ have been shown to be effective where the single dose is not effective. However, it is emphasized that the use of double dose rate or combinations of anthelmintics are not general recommendations; they are used to obtain a prompt answer to possibly a serious and immediate problem on specific farms. FECRT pre-trial check sampling (Day -10)

http://www.agric.nsw.gov.au/reader/sheep-internal


To avoid wasted effort, it is of value to arrange for a pre-trial collection of faeces to obtain an estimate of the egg count of the flock. If this is not possible, faecal samples may be collected from the control group at day 0 to assess if egg content in faeces is sufficient to continue the trial. Objective assessment of the efficacy of an anthelmintic is not reliable where the mean faecal egg count of the control group is less than 200 epg. Some authorities even suggest 300 epg. Subjective evaluation of the data may however be possible below this figure. FECRT treatment (Day 0) Selection of trial animals Trial sheep should be preferably be young sheep at weaning, 3-6 months of age, of even body weight and bred on the property. Undrenched weaners are preferred, but if not possible they should NOT have received a drench during the previous 6 weeks or 8-12 weeks (for persistent ML anthelmintics) to avoid testing an already ‘selected’ worm population. Number of trial animals Each group should contain 15 sheep allowing that faeces may not be collected from all animals at the subsequent visit. (A minimum of 10 sheep per group from which collections are made is necessary). Randomisation of trial animals Sheep should be allocated to treatment and control groups randomly. The best method of randomisation is based on pretreatment egg counts; however, as these are usually not available, the sheep should be drafted into as many groups as there are anthelmintics to be tested plus one untreated control group. For example, if 4 drenches are to be tested, the first 5 sheep in the race are allocated at random to 5 groups; the next 5 are similarly allocated until each group contains 15 sheep. Each group should be identified with a different colour marker (coloured ear tag is often the most convenient). Anthelmintic treatment of trial animals Anthelmintics should be administered accurately by either a calibrated syringe or a drench gun previously calibrated. In most situations, sheep should be orally dosed according to the weight of the heaviest animal in the group. If the group is an even line of sheep the dose received on a mg/kg basis will not vary greatly. FECRT sampling (Day 10-14) Collection of faeces

All trial sheep should be sampled between 10 and 14 days after treatment. Faecal samples should be collected from the rectum of sheep within the first hour after yarding and before much handling has occurred. A minimum of 5 g (10 to 15 pellets) should be collected from each sheep. Transportation of faeces Samples in individually sealed containers filled as close to the top as possible to exclude air, should be transported to the laboratory as soon as possible. The samples should be kept cool in an esky with freezer bricks wrapped in newspaper to avoid freezing. Storage or transport of faeces at or below 4oC may affect the hatching of H. contortus eggs in a faecal culture. FECRT laboratory testing Larval differentiation Pooled faecal cultures and larval differentiations should always be carried out on the control group and preferably each of the test groups. This will allow determination of the species composition and species resistance levels. This is important as some species have different susceptibility to different drenches (eg Levamisole may still be effective as a narrow spectrum against Haemonchus, but not as a broad spectrum against other species). Calculation of FECR results The arithmetic mean (‘average’) on epg of samples collected 10-14 days after treatment, together with a 95% confidence interval, most accurately describes the range of in efficacy of an anthelmintic. The arithmetic mean is the most stringent test of a percentage reduction in faecal egg output and therefore is a more conservative measure of an anthelmintic's performance. Geometric means are sometimes requested by trial sponsors, but arithmetic calculations should always be conducted as well. Each set of results will include the following information per group for each genus present (provided a larval differentiation has been conducted): • Arithmetic mean (‘Average’) • Range (Highest and lowest counts) • Standard deviation • Mean percent reduction in epg (i.e. =

FECR) • 95% confidence interval of FECR

(Upper and lower limit of percent reduction for 95% confidence interval)


Interpretation of FECRT results Resistance should be regarded as being present if:

i. the FECR is < 95%, and,

ii. the lower 95% confidence limit of the % reduction in egg count is less than or equal to 90%.

An example of simple test groups without larval differentiation: Control BZ Levam BZ+LevamArithmetic mean 155 76 190 23Highest 520 160 800 80Lowest 40 0 0 0Std Dev 168 72 287 31% Reduction * 51 * 0 * 85Upper 95% conf. 82 69 96Lower 95% conf. * 0 * 0 * 43

Comment: Interpret these results with caution due to low egg counts in the controls. However it appears that:

i. BZ resistance(*) is present. ii. LEV resistance (*) is present. iii. BZ + LEV resistance (*) is present.

ANTHRAX

Anthrax is a notifiable disease. Fees for tests undertaken to confirm or exclude a diagnosis of anthrax are paid by NSW Department of Primary Industries. Caution Anthrax is a zoonotic disease, causing serious illness and sometimes death in humans. Where anthrax is suspected, a post mortem should not be undertaken as it will cause the vegetative forms of Bacillus anthracis to sporulate. These spores will remain a source of infection for long periods. A diagnosis of anthrax can be made from smears of peripheral blood obtained by cutting the ear of the unopened carcase. Where anthrax is suspected as a result of changes seen at necropsy then impression smears of tissue should be submitted in addition to a range of fresh and fixed tissue to establish an alternative diagnosis if necessary. Diagnosis Demonstration of Bacillus anthracis in blood and tissues. Specimens required

i. Blood smears from peripheral blood vessels. Smears should be sent from more than one animal.

• Make thick, air-dried blood smears, leaving one end of the slide clean.

• Fix blood smears with methanol prior to submission.

• Do NOT fix smears by heat or other agents.

ii. From animals other than sheep and cattle, smears from any affected organs, e.g. swellings in throat and lymph nodes of pigs.

NB Always clearly label specimens as ‘Suspect Anthrax’ Pack securely and forward separately from other specimens Advise the laboratory that you are submitting the samples and place a warning under the lid of the outer packaging. This will ensure special biosecurity precautions are undertaken at the laboratory

ARSENIC POISONING Diagnosis History; clinical and post mortem findings, with gastritis or enteritis; detection of a significant concentration of arsenic in the ingesta or tissues of the dead animal. In chronically exposed animals, detection of arsenic in milk or urine. Specimens required Each specimen should be packed in a separate container, clearly labelled and submitted chilled.

i. 250 g ruminal or stomach contents submitted chilled for toxicology (Reinsch test).

ii. 50 g of liver from the dead animal, submitted chilled for toxicology.

NB Arsenic analysis of tissue is not routinely undertaken, due to the costs. In most cases, a positive Reinsch test (indicates >1mg arsenic/kg) on rumen contents from animals with a suggestive history and


lesions is adequate qualitative confirmation.

iii. 50 ml of milk or urine for arsenic analysis (only where chronic exposure is suspected).

iv. 50 g of suspect source material for toxicology, to be examined only if

arsenic poisoning has been confirmed in the animal.

v. Specimens required to allow for a differential diagnosis if arsenic is not the cause.

Interpretation of tissue and secreta arsenic concentrations

Analyte Sample Units Normal Possibly toxic Toxic Arsenic Liver, kidney mg/kg (wet wt) < 0.5 0.5-8 > 8* Arsenic Milk mg/kg (wet wt) < 0.25 0.25-1.5 > 1.5 Arsenic Urine mg/kg (wet wt) < 0.25 2-100

Interpretation depends on the interval between exposure and sampling. Maximum concentrations of arsenic in tissues occur about eight hours after ingestion, and animals that survive for 2 to 3 days may have liver levels as low as 2 mg/kg. Conversely, clinically normal stock, chronically exposed to arsenic (eg via arsenic contaminated feed over a long period or, in the past stock regularly treated in arsenical dips) may have liver levels up to 8mg/kg and milk levels to 1.5 mg/kg.

ARTHRITIS AND POLYARTHRITIS Diagnosis Based on clinical signs, bacteriological, pathological and serological examination. It is often difficult to demonstrate the presence of organisms in chronic lesions. Specimens required

i. Aseptically collected joint fluid, chilled.

ii. Affected, unopened joints, with the joint capsule intact, submitted chilled.

iii. Acute and convalescent serum samples from affected sheep or cattle, submitted chilled for chlamydial serology.

NB There is no routine serological test available for Erysipelas.

ARTHROGRYPOSIS AND HYDRANENCEPHALY

Causes include infections of the dam with Akabane virus, Aino virus, Palyam virus and Pestivirus (Mucosal disease and Border disease), as well as inherited defects. Diagnosis Based on clinical signs and gross pathology. Diagnosis of the cause depends on history and samples from the dam and affected animals. Specimens required

i. Sample of foetal pericardial or pleural fluid submitted chilled for virus isolation and serology, or, a blood sample from the neonatal animal if it has not sucked.

ii. Serum sample from the dam, submitted chilled for serology.

iii. Brain and spinal cord in buffered formalin for histopathology.

NB. Arthrogrypotic animals may have CNS lesions only in the spinal cord.

iv. Sections of extensor muscles in buffered formalin for histopathology.

ASPERGILLOSIS

See: Fungal infections'

ATAXIA Diagnosis of causes of 'ataxia' requires a careful clinical examination to determine whether the condition is due to a nervous, muscular, skeletal, circulatory or respiratory defect. Diagnosis Based on clinical examination supported by history and laboratory examination to define the specific cause. Specimens required

i. Brain, spinal cord and peripheral nerves in buffered formalin for histopathology.

ii. Sections of skeletal and heart muscle in buffered formalin for histopathology.

iii. Sections of lung and other organs with lesions, in buffered formalin for histopathology.

iv. At least 3 ml of serum, free of cells and haemolysis, submitted chilled for serum enzymology and biochemistry.


v. 30 g of liver and kidney, submitted chilled in separate copper free containers for copper analysis.

ATROPHIC RHINITIS OF SWINE

Syn: Enzootic/progressive atrophic rhinitis Diagnosis Clinical signs, gross pathology, bacteriology. Can be diagnosed clinically when large numbers of pigs with snout deformities are present. If bacteriological confirmation of toxigenic P multocida is needed, collect nasal swabs from up to 10 clinically affected pigs (preferably weaners and growers). Despatch swabs in transport media to reach the laboratory within 24 hours of collection. If possible: • Swab nares with alcohol to reduce

contamination. • Use mini tipped, aluminium-shafted

swabs, especially when sampling weaner and grower pigs.

• Advise RVL of intended sampling, so fresh selective media can be available in the laboratory.

Specimens required

i. Snout swabs transported at ambient temperature in Amies Charcoal transport medium.

BABESIOSIS

See: Tick fever

BALANO POSTHITIS IN RAMS Occurs more commonly in the Border Leicester breed, but also reported in Dorset Horn and other breeds. Usually associated with vulvitis in ewes. Diagnosis Clinical findings, bacteriology in early cases to assist in attempting to determine aetiology. Specimens required

i. Swabs from the lesion submitted in Amies transport medium for bacteriology.

ii. Scabs from the lesion submitted chilled for bacteriology.

BENIGN FOOTROT

See: Footrot

BETA MANNOSIDOSIS OF GOATS See: Mannosidosis

'BIG KNEE' IN GOATS

See: Caprine arthritis-encephalitis (CAE)'

BLACK DISEASE Clostridium novyi is present soon after death in a large proportion of livers of normal sheep and cattle, so that demonstration of the organism is not always diagnostic. Diagnosis Demonstration and recovery of Cl novyi from necrotic foci; histopathology. Often associated with the presence of immature liver fluke, Fasciola hepatica. Specimens required

i. At least three (3) impression smears from cut surface of lesion for staining with specific fluorescent antisera.

ii. Portion of liver, submitted chilled for bacteriology, taken from a freshly dead animal.

iii. Portion of liver containing lesions, in buffered formalin for histopathology.

BLACKLEG

Diagnosis Must be based on a careful post mortem examination with demonstration of either Clostridium chauvoei or Clostridium septicum in the lesions of haemorrhagic myositis. Specimens required These must be taken from a freshly dead animals:

i. At least three (3) smears of haemorrhagic muscle exudate for bacteriology.

ii. Portion of affected muscle, collected aseptically, and submitted chilled for bacteriology.

iii. Portion of affected muscle in buffered formalin for histopathology.

BLOAT

Diagnosis Clinical examination, post mortem findings, including injection of eyes, protrusion of tongue and numerous large haematomata in the frontal sinuses. Specimens required Bloat must be diagnosed on history and gross pathology.


Specimens can be taken to eliminate other causes of sudden death, e.g. enterotoxaemia, hypomagnesaemia.

BLUE-GREEN ALGAL POISONING The toxic blue green algae, Microcystis cyanea and Anabaena circinalis are mainly a problem in late summer and autumn. Diagnosis Presence of algae as a green scum on the water. The clinical syndrome includes sudden death or jaundice and photosensitisation. Evidence of a hepatotoxin and gastrointestinal irritation. Specimens required

i. 500 ml of scum and water for examination for toxic algae.

ii. Serum from affected animals for serum chemistry/enzymology

iii. Sections of liver and kidney in buffered formalin.

BORDER DISEASE IN SHEEP

Diagnosis Clinical signs including abortion and the birth of lambs with abnormally hairy birth coats, which may or may not show nervous signs. Demonstration of Pestivirus in affected lambs and antibody in the ewe. Specimens required

i. Affected lambs submitted whole foetal membranes are also required if it is an anteparturient or parturient death.

ii. Blood sample, submitted chilled on the clot, from the affected lamb and its mother for serology.

iii. Heparinised blood sample for antigen ELISA.

iv. Portions of fresh lung, spleen and mesenteric lymph node collected aseptically and kept chilled for antigen ELISA or virus isolation.

v. Brain and spinal cord, fixed in buffered formalin for histopathology.

BOTULISM

There are five (5) major types of botulism (A to E). Types C and D are the usual causes of botulism in cattle; type C in horses and wild birds. Diagnosis Usually based on clinical signs, which include flaccid paralysis, and the absence of gross and histopathological lesions. Important cases may be confirmed by the

demonstration of the toxin in serum, liver and gut contents. Specimens required

i. Frozen serum, liver, stomach contents, small intestinal contents and large intestinal contents for toxin ELISA.

ii. Suspect food material (approx 30g) for toxin ELISA.

BOVINE LEUKOSIS

See Enzootic bovine leukosis'

BOVINE LEUCOCYTE ADHESION DEFICIENCY (BLAD)

(Holstein/Friesian calves) This defect was disseminated throughout the international Holstein herd from a founder bull in the USA whose descendants have been used extensively in the international Holstein herd. The disease is characterised by progressive leucocytosis emanating from a deficiency of an adhesion factor on the surface of leucocytes that is essential for leucocytes to migrate from blood vessels to sites of infection. Consequently, affected calves suffer with repeated bacterial infections. AI centres select against BLAD heterozygotes when screening bulls for entry to their centres. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

For diagnosis of the disease and for heterozygote detection:

i. 20 to 25 hairs, with roots attached, from the distal end of the tail.

BOVINE MALIGNANT CATARRH

Syn: Malignant catarrhal fever Diagnosis Sporadic incidence, clinical signs, histopathology, PCR for ovine herpesvirus-2 (OHV-2). Specimens required

i. Brain and sections of sections of liver, kidney, adrenal gland, bladder and nasal mucosa in buffered formalin for histopathology.



ii. 5 ml of EDTA blood for PCR testing for ovine herpesvirus-2 (OHV-2)

iii. 5 ml of blood on clot submitted chilled for examination for pestivirus (mucosal disease). In dead animals, alternatively submit spleen and kidney chilled for pestivirus.

iv. Ocular and nasal swabs submitted in phosphate buffered gelatin saline (PBGS) for examination for infectious bovine rhinotracheitis (IBR).

BOVINE VIRUS DIARRHOEA

See: Pestivirus

BRACKEN FERN POISONING See also: Enzootic haematuria of cattle In ruminants, principally a disease of cattle, affecting the haematopoietic system and bladder wall. In adult sheep, long term intake of bracken fern by adult sheep can induce "bright blindness", a progressive retinal degeneration. In horses, pigs and other monogastrics rarely encountered, but associated with nervous signs from thiamine deficiency attributed to a thiaminase in the plant. Diagnosis Clinical signs, haematology, histopathology. There is no specific diagnostic test in the live animal for bracken fern poisoning. Specimens should be submitted to eliminate other diseases. Specimens required (for cattle) Live animal

i. 5 ml of blood in EDTA for haematology.

ii. Blood films for haematology. iii. Serum sample, chilled for

phosphorus estimation (see Hyophosphataemia).

Dead animal

i. Sections of liver, kidney, red femoral shaft bone marrow and affected tissues in buffered formalin for histopathology.

ii. From cases of haematuria, sections of bladder wall with lesions, submitted in buffered formalin, for histopathology.

BRUCELLOSIS BOVINE

NSW reached Bovine Brucellosis free status in 1992.

Specimens required Cows

i. Uterine discharges or vaginal mucus, submitted chilled for bacteriology.

ii. Milk samples from the lactating cow, submitted chilled for bacteriology and serology.

iii. 2 ml of serum for serology. Samples can be submitted on the clot if they will be tested within 48 hours of collection.

Bulls

i. 0.5 to 1 ml of semen collected aseptically, and submitted chilled for bacteriology.

ii. 2 ml of serum, submitted chilled for serology.

Aborted foetuses

iii. Specimens as required for diagnosis of abortion in cattle (see Abortion in cattle).

BRUCELLOSIS OVINE

Diagnosis Characterised by infertility in rams due to epididymitis. Abortion in ewes is not a common finding. The diagnosis in rams must be based on the result of a clinical examination and serology, supported in some cases by bacteriological examination of semen. Specimens required

i. Serum samples submitted chilled for serological testing. Samples can be submitted on the clot if they will be tested within 48 hours of collection.

ii. Semen samples collected aseptically and submitted chilled for bacteriology.

iii. Testes, epididymes and accessory sex glands, including ampullae and seminal vesicles, submitted chilled for bacteriology and histopathology.

BRUCELLOSIS PORCINE

Brucella suis infection was last diagnosed in NSW in 1968. Diagnosis History and serology. The agglutination test is of use mainly as a herd test, as there is a tendency for infected pigs to cease to react although they still remain infected and false positives can also occur. Specimens required


i. Serum samples (not blood) submitted chilled for serology (SAT and RBT).

ii. Lesions, submitted chilled for bacteriology.

iii. Aborted foetuses submitted whole for bacteriology

CALCULI

Diagnosis Evidence of uraemia/urinary retention, cystitis and/or swelling and cavitation of kidney pelvis with pressure effects on cortex and medulla. Demonstration of calculus at critical location. Chemical analysis of the calculus may indicate the cause of the calculus formation. NSW Department of Primary Industries outsources this testing. Specimens required i. Diagnosis should be made on clinical and post mortem findings. ii. Calculi for chemical analysis.

CAMPYLOBACTER ABORTION OF SHEEP

Usually attributed to Campylobacter fetus subsp fetus (formerly subsp intestinalis); occasionally Campylobacter jejuni implicated. Diagnosis Late term abortions or birth of weak or dead full term lambs. Typical liver lesions in some full term lambs. Bacteriology. Specimens required Specimens as required for the diagnosis of abortion in sheep.

CAMPYLOBACTER ENTERITIS Campylobacter jejuni and occasionally Campylobacter coli have been associated with enteritis and diarrhoea in calves, lambs, pigs, dogs, poultry and man. However, both may be found in otherwise normal intestinal tracts of sheep and cattle (C jejuni) or pigs, poultry and man (C coli). The role of other Campylobacters such as C sputorum and C hyointestinalis in the aetiology of diarrhoea and intestinal pathology in pigs is uncertain (see Porcine proliferative enteropathy). Diagnosis Bacteriology, histopathology. Specimens required

i. Fresh, chilled small intestine for bacteriology.

ii. Formalin-fixed small intestine for histopathology.

CAMPYLOBACTERIOSIS OF CATTLE

Syn: Bovine venereal campylobacteriosis (BVC); formerly bovine vibriosis Bovine venereal campylobacteriosis (BVC) is caused by Campylobacter fetus subsp venerealis and is characterised by infertility and early embryonic death. Abortion occurs in a small percentage of infected cows. Campylobacter fetus subsp fetus (formerly subsp intestinalis) also causes abortion in cattle. Diagnosis History of reduced breeding efficiency in herds where natural breeding is practised. Clinical signs commonly observed are repeated returns to service, with prolonged interservice intervals and sporadic occurrence of mid to late term abortions. Abortion and infertility due to C fetus subsp venerealis can be confirmed by demonstration of specific antibodies in the vaginal mucus of affected cows by ELISA. C fetus subsp venerealis can also be isolated from the prepuce of infected bulls. In a herd situation, demonstration of antibody in vaginal mucus is the preferred method of diagnosis as it can be difficult to recover the organism from the prepuce. C fetus subsp venerealis and C fetus subsp fetus can be isolated from aborted foetuses. Specimens required Cows

i. Vaginal mucus samples collected for demonstration of C fetus subsp venerealis IgA antibodies by ELISA test. PBST diluent and instructions for collection of vaginal mucus samples for BVC ELISA test are available from the laboratory. See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/01-bvc-elisa-guide.pdf

ii. Abortion investigation: animals

which have aborted can be sampled from 1 week to 3 months after the abortion.

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34595/01-bvc-elisa-guide.pdf


iii. Infertility investigation: representative sampling of herd; at least 10 vaginal mucus samples collected from infertile heifers or cows. This can be done when pregnancy testing reveals infertility.

Bulls

i. Preputial scrapings submitted in Campylobacter transport enrichment medium (TEM) for bacteriology. TEM is supplied from the laboratory together with instructions for collection of samples. See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/02-bull-vd-coll.pdf

Aborted foetuses

i. Specimens as required for diagnosis of abortion in cattle (see Abortion in cattle).

CAMPYLOBACTERIOSIS OF PIGS

See: Porcine proliferative enteropathy; Campylobacter enteritis'

CAPRINE ARTHRITIS ENCEPHALITIS (CAE)

Syn: ‘Big knees’, Caprine retrovirus infection Diagnosis Viral serology; clinical history of nervous signs in young kids, big knees in adults associated with lameness, ill thrift, weight loss, chronic progressive pneumonia, hard udder and mastitis, histopathology of affected tissues. Specimens required

i. Serum samples chilled for viral serology.

ii. Portions of affected tissues in buffered formalin for histopathology (carpal joint, brain, spinal cord, lung).

CARDIOMYOPATHY AND WOOLLY HAIRCOAT (CWH) SYNDROME

(Poll Hereford calves) Dd: Cardiac white muscle disease (nutritional myopathy, selenium deficiency). CWH is a congenital genetic disease, with an autosomal recessive mode of inheritance presumed to involve defective structural protein(s) within desmosomal complexes.

Affected calves can be identified at birth by their distinctive woolly haircoat, (also described as ‘fuzzy’, ‘wirey’ or ‘curly’). Some CWH calves have protruding eyes and keratoconjunctivitis (‘pinkeye’). Cardiac arrhythmias are usually detectible. Death from heart failure occurs between birth and 12 weeks of age. Most calves with CWH die suddenly. Owners occasionally see an affected calf collapse and die, usually following exertion Some CWH calves show clinical signs of progressive heart failure. Diagnosis Clinical signs, history. and gross cardiac ventricular fibrosis, with or without mineralisation. Microscopic examination is necessary to accurately demonstrate the extent of the heart muscle changes. CWH cases can be misdiagnosed as selenium deficiency (cardiac white muscle disease, nutritional myopathy), particularly if the woolly haircoat is overlooked. Calves with CWH are not selenium-deficient. Any woolly-coated Poll Hereford calf that dies during the first few months of life should have a postmortem examination to check for evidence of cardiomyopathy. It is not sufficient to diagnose CWH in a woolly-coated calf without postmortem confirmation of the cardiomyopathy. Specimens required

i. Whole heart, eyes, liver, kidney and skeletal muscle (including diaphragm, tongue, psoas muscle) in buffered formalin.

ii. Fresh liver and kidney for selenium estimation.

iii. 2 x 10 ml of EDTA blood (1.5 mg EDTA/ml of blood) for DNA isolation (from affected calf, dam and sire). These DNA samples will be held pending development of a diagnostic DNA test (not yet available).

iv. 1 x 10 ml lithium heparin blood for glutathione peroxidase (GSHPx).

CASEOUS LYMPHADENITIS (CLA)

Diagnosis Recovery of Corynebacterium pseudotuberculosis from lesions. Specimens required

i. Smears from early lesions or from the periphery of active lesions for bacteriology.



ii. Portions of tissues containing early lesions or from the periphery of active lesions, submitted chilled for bacteriology.

iii. Portions of tissue containing early lesions or from the periphery of active lesions, in buffered formalin for histopathology.

CHLAMYDIAL INFECTIONS

Syn: Chlamydiosis, sporadic bovine encephalomyelitis (SBE), chlamydial arthritis, chlamydial abortion of sheep, avian chlamydiosis Caution Chlamydiosis is a zoonotic disease, causing serious illness and sometimes death in humans. Diagnosis Based on clinical findings with demonstration of group specific antibody rise between 'acute' and 'convalescent' serum samples in association with disease. Demonstration of Chlamydia by FAT on smears of fresh tissues or fluids. Specimens required

i. Serum samples taken in the acute and convalescent stages of disease (3 to 4 weeks apart) from individually identified and observed animals, submitted chilled for chlamydial serology.

ii. Depending on the clinical condition, specimens as required for the investigation of nervous disorders, arthritis or abortion.

Interpretation of Titres The demonstration of a four fold rise in CFT antibody titres, within 3 to 4 weeks of the clinical episode, is considered significant.

CITRULLINAEMIA (Holstein/Friesian calves) Affected calves are clinically normal at birth, become depressed by 24 hours, develop signs of neurological dysfunction by 48 hours. Typical clinical signs are seemingly aimless wandering, tongue protrusion, chewing on fixed objects, head pressing and ultimately collapse and death between the third and fifth day of life. The mutation responsible for this defect was imported into Australia by a US-born bull whose descendants were selected for high EBVs for butter fat content of milk. The mutation is now relatively rare in the Australian Holstein herd due to selection against heterozygotes by AI centres.

Diagnosis Clinical signs, histopathology, confirmed by DNA analysis, Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

(In order of preference) Diagnosis of the disease


ii. Whole brain in buffered formalin for histopathology.

Heterozygote detection


CLOVER DISEASE

Diagnosis Based on flock history, reproductive performance of older ewes in the flock and demonstration of cystic endometritis. Specimens required

i. Reproductive tracts including ovaries from 15 to 20 ewes which did not lamb at the previous lambing. Submit chilled, as it is often difficult to determine endometrial cysts grossly in fixed tissue.

COBALT DEFICIENCY

In NSW, cobalt deficiency is confined to restricted geographical areas. Signs have been seen in sheep and cattle. Diagnosis History of ill thrift, in young animals particularly after elimination of other likely causes, e.g. parasitism, deficiency of other minerals, malnutrition. Response to cobalt supplements or injections of vitamin B12, preferably in young, weaned animals. Evidence of a mild to moderate macrocytic anaemia associated with weepy eyes. Specimens required

ii. 10 ml of blood, clotted in vacuum tubes, from each of 3 to 5 animals in the affected group, submitted chilled for biochemistry.

iii. Specimens should be submitted to eliminate other causes of ill thrift.



COCCIDIOSIS Occurs mainly in young sheep, cattle, goats and pigs. Lesions occur in the mucosa of the intestinal tract. Characterised in ruminants by scouring, anaemia and ill thrift. In pigs, it has been shown to be an important cause of scouring in 10 21 day old suckers in some herds. Diagnosis Clinical signs, impression smears of intestine (acute), demonstration of large numbers of coccidia in faeces (chronic), and histopathology. In acute coccidiosis, oocysts are often not yet present in faeces, and impression smears of affected intestine and histopathology are recommended. In pigs, oocysts may be difficult to detect in faeces during the diarrhoeal phase, and a definitive diagnosis can be made by examination of small intestinal smears of a typically affected pig. Specimens required

i. Faecal sample for oocyst count. ii. In piglets, and other animals with

acute coccidiosis, obtain 5-10 impression smears of ileum and jejunum and air dry.

iii. Portions of intestines (e.g. ileum, jejunum) showing lesions in buffered formalin for histopathology.

COCCIDIOSIS – HEPATIC IN RABBITS

Characterised by mortalities with gross pathology typically revealing an enlarged

liver with multiple yellow or white nodular lesions. Diagnosis Gross pathology, histopathology. Demonstration of Eimeria stiedae. Specimens required

i. Portion of affected liver in buffered formalin for histopathology.

ii. Oocysts of E stiedae may be demonstrable in the exudate of dilated bile ducts and gall bladder

COLIBACILLOSIS

See also: Oedema disease, scouring Diagnosis Bacterial evidence of Escherichia coli enteritis, enterotoxaemia or septicaemia. Demonstration of pathogenic strains of E coli. Specimens required

i. Preferably the whole animal submitted chilled.

ii. Sections of liver, kidney, jejunum, colon and mesenteric lymph node, submitted chilled, for bacteriology.

iii. Sections of lung, liver, kidney, small intestine and mesenteric lymph node submitted in buffered formalin for histopathology.

iv. In animals showing nervous signs, brain submitted in buffered formalin for histopathology.

E coli serotypes commonly associated with colibacillosis in pigs in NSW

0149:K91,K88ac:H10

0141:K85ac:H4 or HNM

0141:K85ab:H4 or HNM

08:K87,K88ac:H19 or HNM

Neonatal mortality + + Neonatal diarrhoea + + Weaner mortality + + + + Weaner diarrhoea + + + + Weaner nervous signs + + Weaner oedema disease + +

POULTRY There is no real information on the prevalence of pathogenic serogroups in poultry in Australia. Typing of poultry clinical isolates is not being undertaken at this time.

COMPLEX VETERBRAL MALFORMATION (CVM)

Affected calves are generally aborted, but in rare instances may be born alive. The

disease is characterised by shortening of the cervical and thoracic vertebra and symmetrical arthrogryposis in the fore and sometimes rear legs. The mutation responsible for this defect has been traced to a founder US Holstein sire whose descendants have been used extensively worldwide. AI centres select against CVM heterozygotes when screening bulls for entry to their centres.


Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

Diagnosis of the disease and heterozygote detection

i. 20 to 25 hairs with roots attached. From the distal end of the tail

CONGENITAL ABNORMALITIES

See also Akabane, abortion, arthrogryposis and hydranencephaly, pestivirus, genetic diseases. In general, the occasional abnormality or freak is of little interest and there is no benefit in submitting such cases. However, increased incidence of congenital abnormalities or repeated occurrence over a number of years warrant further investigation. Advice should be sought from the laboratory in these cases. Diagnosis Clinical signs. It is important to eliminate infectious causes before incriminating environmental, chemical or other teratogenic causes or inherited defects. A detailed history, particularly in relation to events during the first half of pregnancy may be helpful. The breeding history of the animals may be important in some cases. Specimens required

i. Affected animals submitted whole, together with foetal membranes if available.

ii. Serum sample from mother of affected animal, submitted chilled for serology.

iii. Body fluid (preferably pericardial or pleural) from the foetus or serum sample from the neonate prior to sucking, submitted chilled for serology and examination for immunoglobulins.

CONTAGIOUS EQUINE METRITIS (CEM)

Diagnosis Recovery of Taylorella (formerly Haemophilus) equigenitalis from the genital tract. Specimens required Swabs should be taken from each of the following sites and submitted without

refrigeration in Amies charcoal transport medium for bacteriology:

i. Stallions 3 swabs per animal, from the following:

• prepuce and base of penis (smegma)

• urethral fossa and urethral sinus • terminal urethra

ii. Non-pregnant mares - 3 swabs per

animal, from the following sites: • cervix • clitoral sinus • clitoral fossa

iii. Pregnant mares - 2 swabs per

animal from the following sites: • clitoral sinus • clitoral fossa

CONTAGIOUS OPHTHALMIA

See: Ophthalmia

CONTAGIOUS PUSTULAR DERMATITIS

Syn: Scabby mouth, contagious ecthyma, orf Diagnosis Clinical signs, demonstration of virus particles on electron microscopy. Differentiate from Staphylococcal infections and Dermatophilosis. Specimens required

i. Scabs with underlying tissue scrapings, submitted in a sealed container, e.g. a Macartney bottle, for electron microscopy.

COPPER DEFICIENCY

Diagnosis History, clinical signs, serum and liver copper levels, response to therapy. Specimens required Sample collection equipment must be free of copper. Vacuum blood collection tubes are satisfactory for blood testing. NB In all cases of suspected copper deficiency, clotted blood is preferred to liver or kidney.

i. 10 ml of blood, clotted in vacuum tubes, from each of 3 to 5 animals in the affected group, submitted chilled for biochemistry.

ii. Brain and spinal cord, in buffered formalin, from cases of suspected enzootic ataxia.



Interpretation of copper concentrations (cattle and sheep)

Analyte Sample Minimum amount

Special requirement

Units Species Deficient Normal

Copper Serum or hep plasma

1 ml OK on clot µmol/L Cattle Sheep

< 7.5 < 7.5

7,5-16 7.5-20

COPPER POISONING OF SHEEP AND CATTLE

See also: Pyrrolizidine alkaloidosis' Chronic copper poisoning in ruminants of the hepatogenous and/or phytogenous form is quite common. The terminal acute phase is characterised by liver necrosis, haemolytic anaemia, haemoglobinuria and jaundice. Sheep of all ages and preruminant calves accumulate copper in the liver when dietary copper is in excess of 30 mg copper /kg dry weight of feed consumed (adult cattle require and therefore tolerate 100 mg copper /kg of feed). Calf milk replacers should contain <20 mg copper / kg.

Diagnosis Clinical signs, typical post mortem findings, histopathology, analysis of tissues. NB. The diagnosis can be confirmed histologically. Histochemical methods for staining for copper in liver sections although not quantitative, are quicker, easier and cheaper to perform than tissue analysis. Specimens required

i. Sections of liver & kidney, in buffered formalin for histopathology.

ii. 30 g kidney and liver, in separate copper free jars, submitted chilled and unpreserved for copper estimation.

Interpretation of copper concentrations (cattle and sheep) Analyte Sample Minimum

amount Special requirement

Units Species Deficient Normal Elevated

Copper Liver 30g Fresh mg/kg (wet wt)

Cattle Sheep

< 4 < 4

20-70 40-70

> 100 > 200

Copper Kidney 30g Fresh mg/kg (wet wt)

Cattle, sheep

4-6 > 8

Toxicological results must be interpreted in relation to history, clinical findings and histopathology. See Toxicology for suggested normal and toxic values. Kidney is considered superior to liver for biochemistry in acute and chronic copper poisoning: Kidney copper concentrations rise faster in acute toxicosis. Liver concentrations of 200 mg/kg (wet wt) can be found in sheep dying from other causes, especially sheep affected with pyrrolizidine alkaloidosis, which promotes liver copper accumulation.

CORYNEBACTERIUM EQUI IN HORSES OR PIGS

See: Rhodococcus equi pneumonia in horses See: Rhodococcus equi lymphadenitis in pigs'

CRYPTOSPORIDIOSIS Cryptosporidium spp. are coccidial parasites that are not host specific, are not susceptible to conventional anti-coccidial treatment and to date have no available efficacious specific chemotherapy. They are

associated with gastrointestinal or respiratory disease, affecting particularly young animals. Ruminants, pigs, horses, mice, birds, and man are common hosts. Occasionally detected in young cats, dogs, rabbits, guinea pigs and other mammals, as well as in reptiles and fish. Signs most commonly seen are anorexia and diarrhoea in calves, lambs, kids, piglets, foals and deer calves. Disease is spread by infectious oocysts, which are shed in faeces or respiratory secretions, and have high environmental resistance. Host debilitation and/or concurrent viral or bacterial infection may predispose to infection. Immuno-compromised or immuno-incompetent animals are at high risk of infection. Diagnosis Identification of oocysts in faeces, by examination of faecal smears with acid-fast stains or phase contrast microscopy of faecal flotations. Identification of organisms in the brush border of mucosal surfaces on histopathology. Antigen capture ELISA on faeces of calves (where available), although


this technique may be less sensitive than microscopic examination. Specimens required

i. Faecal sample for preparation of smears and examination by MZN or similar stain, or by floatation and examination by phase contrast microscopy.

ii. Formalin-fixed abomasum/stomach (peptic gland epithelium) and small intestine from freshly-killed mammals, reptiles or fish.

iii. For birds, formalin-fixed small intestine, caecum, colon and bursa, or epithelium of the upper respiratory tract and lung from freshly-killed cases.

CYANIDE POISONING Syn: Prussic acid poisoning. See Poisoning (plants) for information on collection of specimens for plant identification.

Diagnosis Clinical signs, post mortem appearance of blood and mucosae, odour of bitter almonds, access to suspected materials or fodders, chemical demonstration of cyanide in plant material or rumen ingesta. Specimens required Plant specimens can often be identified by the local District Agronomist. Cyanogenetic glycosides in plants hydrolyse during transit so that laboratory analysis is often misleadingly negative. Herbage and ingesta should be kept frozen from the time of collection to the time of analysis; chilling is not sufficient. Immersion in 1.3% mercuric chloride also stops hydrolysis.

i. Sample of rumen ingesta in an airtight container, submitted frozen for toxicology.

ii. Blood smears to check for nitrate poisoning.

iii. Samples of suspected plant material for identification and check for cyanide and nitrate. Submit frozen for cyanide toxicology.

iv. Fresh and formalin-fixed specimens to establish an alternative diagnosis when necessary

CYSTICERCOSIS

Diagnosis

Gross pathology and dissection of a viable scolex from the fluid filled cyst, or histopathology of degenerate cysts. Specimens required

i. Fresh tissue with lesions, submitted chilled for parasitology.

ii. Lesions, in buffered formalin, for histopathology

DEFICIENCY OF URIDINE MONOPHOSPHATE SYNTHETASE (DUMPS)

(Holstein/Friesian cattle) Deficiency of uridine monophosphate synthetase results in foetal death during the first trimester of pregnancy. The mutation responsible for the defect has been recorded in North American Holsteins, in a sire line that, fortuitously, was not used widely in the international Holstein herd. It is not known if exists in the Australian Holstein/Friesian herd. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf



DERMATOPHILOSIS Syn: Mycotic dermatitis, ‘lumpy wool’

Diagnosis Gross appearance of the lesions and demonstration of Dermatophilus congolensis microscopically. The causal organism may be isolated from scrapings or a biopsy specimen. Specimens required

i. Sample of wool or hair showing lesions or scabs for gross examination and demonstration of D congolensis in impression smears.

ii. Skin biopsy from the area with lesions, submitted chilled for bacteriology.

iii. Skin biopsy from the area with lesions, in buffered formalin for histopathology.

DIARRHOEA

See Scouring



DRENCHING MORTALITIES Diagnosis Based on history, clinical signs and in some cases, histopathology. Specimens required Product information sheets from the manufacturer should be checked to ensure the appropriate specimens are submitted.

i. Sections of liver, kidney and lung, in buffered formalin for histopathology. Brain and/or spinal cord if any neurological signs evident.

ii. Other samples as required from the product information sheet of the manufacturer

DWARFISM IN DEXTER CATTLE

Syn: Dexter chondrodysplasia, congenital lethal chondrodysplasia (Dexter bulldog). Congenital lethal chondrodysplasia (Dexter bulldog) is the homozygous form of Dexter chondrodysplasia. Affected calves are usually aborted before the seventh month of gestation, with extreme shortening of limbs and vertebral column, gross craniofacial defects (relatively large head with retruded muzzle, cleft palate and protruding tongue), and a large abdominal hernia. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf



Please provide pedigree of the subject with the hair sample.

ENCEPHALOMYELITIS AND ENCEPHALITIS

See also Haemagglutinating encephalitis virus (HEV) of pigs, porcine enterovirus encephalomyelitis, transmissible spongiform encephalopathy (TSE)

For adult sheep and cattle with progressive neurological disease, please submit specimens as required by the National TSE Surveillance Program (http://www.animalhealthaustralia.com.au/programs/adsp/adsp_home.cfm), accompanied by a completed NTSESP

Clinical History and Post-mortem Report form.

Diagnosis Clinical signs, histopathology. Virology and bacteriology to establish an aetiological diagnosis. Specimens required

ii. Brain and portion of spinal cord, in buffered formalin for histopathology.

iii. Sample of cerebrospinal fluid collected aseptically, submitted chilled for bacteriology.

iv. Section of brain and spinal cord collected aseptically, submitted chilled for virology.

v. Serum samples for virus serology. vi. Specimens as required for the

differential diagnosis of nervous disorders.

ENCEPHALOMYOCARDITIS (EMC) OF PIGS

Encephalomyocarditis is caused by a picornavirus virus. The disease is a zoonosis affecting a wide range of mammals including mice, wombats and elephants. Diagnosis History of sudden death, typical white areas on heart and throughout myocardium, virus isolation. Associated with abortion in some instances. Specimens required

i. Fresh heart and spleen collected separately and aseptically submitted chilled for virology.

ii. Sections of heart, liver, kidney, lung, spleen and whole brain in buffered formalin for histopathology.

iii. Section of liver, kidney, spleen, lung and heart blood submitted chilled for bacteriology.

ENTEROTOXAEMIA

Difficulty is often encountered in establishing a diagnosis of enterotoxaemia in sheep, cattle, goats and deer. The following points must be considered: • Clostridium perfringens is a normal

bowel inhabitant and toxin may be present in the gut content of healthy animals.

• Cl perfringens is a common postmortem invader of parenchymal tissues and will be present by 8 hours after death.




• Toxin may diffuse from the gut if the autopsy is delayed. In chronic cases, e.g. in focal symmetrical encephalomalacia, toxin is usually no longer present.

• Typical kidney lesions develop in sheep within a few hours of death, they are not present at the time of death.

• Type D toxin is stable in gut contents in vitro without preservative, but other toxins are very unstable and have disappeared within 12 hours regardless of preservation, cooling, etc.

Diagnosis A positive diagnosis rests on several of the following being present: • History of sudden death • Necropsy findings:

• glycosuria • pulmonary oedema and congestion • fibrin clot in pericardial sac • epicardial haemorrhages • yellow creamy intestinal contents NB: some animals may show minimal gross changes e.g. sudden death with some small intestinal inflammation. • histopathological lesions in brain

and kidney (e.g. demonstration of protein leakage from blood vessels into perivascular spaces of cerebrum).

• abnormal numbers of Cl perfringens like organisms in smears of intestinal mucosa taken from multiple sites from abomasum to colon, with particular attention to the ileum.

• demonstration of epsilon toxin in gut contents by counter-immunoelectrophoresis (CIEP).

Specimens required

i. At least 20 smears from the intestinal mucosa at sites from the abomasum to the colon with particular attention to the ileum. Sites selected should be from areas showing haemorrhages and also from nearby non inflamed areas which may show a heavier flora. 4-5 sites can be smeared on one slide.

ii. Samples of small intestinal content at least 10 ml from sheep and 40 ml from cattle should be collected from several sites in the small intestine, particularly from areas where the contents are a yellow colour and creamy consistency. Submit chilled for toxicology.

iii. Sections of liver and kidney, and whole brain in buffered formalin for histopathology. Urine examination

for glycosuria should be carried out in the field.

NB Indicate vaccination history with all submissions of suspected enterotoxaemia..

ENZOOTIC ATAXIA Syn Copper deficiency'

ENZOOTIC BOVINE LEUKOSIS (EBL)

A retrovirus infection of cattle which can be spread by blood transmission from rectal palpation, vaccination, tattooing or gouge dehorning where repeatedly-used equipment is employed. Congenital transmission also occurs but is less important. The virus has been eliminated from dairy herds in NSW but beef cattle remain infected at a low prevalence. Like other retrovirus infections (AIDS, CAE, EIA) antibody indicates current (not just previous) infection. Affected animals usually develop antibody within 2-3 months of spread. Diagnosis Clinical signs, confirmed by virus serology, histopathology and haematology. Specimens required

i. Serum or preserved milk samples (bromopol preservation as used for milk cell counts) for virus serology (ELISA or GDPT)

ii. Formalin fixed sections of affected lymph nodes, spleen, thymus, intestine or other organs showing gross lesions for histopathology.

ENZOOTIC HAEMATURIA OF CATTLE

See also Bracken fern poisoning Diagnosis Chronic intermittent haematuria and anaemia. Gross pathology and histopathology of the urinary bladder. Specimens required

i. Sections of urinary bladder with suspect lesions, submitted in buffered formalin for histopathology.

ENZOOTIC PNEUMONIA OF PIGS

See Porcine enzootic pneumonia'


EPERYTHROZOONOSIS OF SHEEP The taxonomy of the causative organism has been revised and it is now known as Mycoplasma ovis. Diagnosis Demonstration of Mycoplasma ovis and anaemia in sheep. The presence of the organism is insufficient to establish the diagnosis as it can be found in normal sheep. Other causes of anaemia must be excluded. Specimens required

i. Blood films from 10 affected and at least 20 apparently normal animals in the flock for examination for M ovis (See Specimens for Haematology - preparation of blood films').

ii. Blood samples from affected animals in EDTA vacuum tubes for haematology.

EPHEMERAL FEVER

Diagnosis General symptomatology and occurrence in the herd and district. Virus antibody rise between acute and convalescent serum samples. Specimens required

i. Paired serum samples (with a 2 to 3 week interval) from individually identified animals collected in the very early acute and convalescent phases submitted chilled or frozen for serology.

NB. Generally virus serology is not carried out unless paired serum samples are submitted.

EPIDIDYMITIS See also Brucellosis-ovine, Actinobacillus seminis, Histophilus ovis Diagnosis Clinical signs and presence of lesions. Recovery of causal organism from semen or reproductive organs. Evidence of infection with specific pathogens, e.g. Brucella ovis, Actinobacillus seminis and Histophilus ovis. Specimens required

i. Serum sample, submitted chilled for serology.

ii. Semen sample collected aseptically, submitted chilled for bacteriology.

iii. Genital organs, including the accessory sex glands, submitted chilled for bacteriology and histopathology.

EQUINE BABESIOSIS This disease caused by the protozoon Babesia equi was introduced to Australia in infected imported horses and transmitted locally by poor injection technique. The disease probably no longer exists in Australia.

Diagnosis Clinical signs of depression and anorexia with intermittent fever. Anaemia, haemoglobinuria and subcutaneous oedema may be present. In chronic cases there may be loss of condition and mild anaemia. Demonstration of the organism in thick and thin blood smears, serology. Specimens required

i. Thick blood smears dried under cover (not by heat or exposure to sunlight) for parasite examination.

ii. Blood films for haematology. iii. 5 ml of blood in EDTA vacuum tube

for haematology. iv. At least 2 ml of serum for serology

(B equi IFAT).

EQUINE INFECTIOUS ANAEMIA (EIA)

Diagnosis Clinical signs of recurrent febrile attacks associated with anaemia over a period. Haematology, serology (AGID - Coggins test) and histopathology. A positive AGID is conclusive evidence of EIA; however, the AGID may occasionally be negative early in the disease course. Specimens required

i. At least 2 ml of serum for viral serology (AGID - Coggins test).

ii. 5 ml of blood in EDTA vacuum tube and blood smears for haematology.

iii. Sections of liver, kidney, spleen and lymph nodes in buffered formalin for histopathology.

EQUINE VIRAL ARTERITIS (EVA)

See also Exotic diseases' Recent identification of seropositive horses and a semen-culture positive stallion in Australia suggest this organism has been established in the horse population at a low level. Clinical disease has not yet been recorded.


EVA is usually subclinical, but clinical disease overseas is characterised by pyrexia lasting 1-5 d, subcutaneous oedema of limbs and ventral abdomen, rhinitis and conjunctivitis with nasal and ocular discharge and photophobia, a skin rash, anorexia, depression, dyspnoea and diarrhoea. Up to 70% of pregnant mares abort during the febrile stage. Affected animals usually recover. Virus is spread by respiratory and venereal transmission. Virus is present in semen, respiratory secretions and also in urine, faeces and aborted foetuses. Diagnosis Serology, virus isolation, histopathology. Specimens required

i. Serum ii. Blood containing anticoagulant,

nasal swabs or washings in PBGS, tissues of aborted foetuses (lung, liver, spleen, thymus, lymph node), semen for virus isolation. From adult horses at necropsy, fresh lung, spleen and lymph nodes draining oedematous areas.

Send chilled unless delays of more than 24 h are anticipated in transit; if so, forward frozen on dry ice.

iii. Tissues from foetuses or adult horses as above, fixed in neutral-buffered formalin for histopathology.

ERYSIPELAS

Erysipelothrix rhusiopathiae causes arthritis (often post-marking) and post-dipping lameness (due to laminitis) in sheep and arthritis, urticaria, valvular endocarditis and fatal septicaemia in pigs. The resultant pyrexia in sows can result in abortion. Diagnosis Clinical signs and history. Gross pathology. Isolation of E rhusiopathiae from affected tissues. There is no routine serological test at present available for Erysipelas. Specimens required Arthritis

i. Whole joint unopened and chilled. If this is not possible, forward aseptically-collected synovial membrane (most desirable) and synovial fluid (of lesser value) in separate sterile containers.

Urticaria and endocarditis

i. Portion of lesion(s) collected aseptically and forwarded in sterile containers for bacteriology.

ii. Portion of lesion(s) in buffered formalin for histopathology.

Septicaemia

i. Sections of liver, kidney, lung, spleen, collected aseptically and forwarded in separate sterile containers for bacteriology.

ii. Portions of above tissues in buffered formalin for histopathology.

iii. From acute clinical cases, sample of citrated blood collected aseptically, submitted chilled for bacteriology.

Abortion As this is associated with maternal pyrexia rather than foetal or placental infection, bacteriological examination of aborted foetuses is usually of little assistance in the diagnosis of Erysipelas abortion.

EXOTIC DISEASES Guidelines on the collection and submission of specimens for exotic diseases can be found in 'Exotic Diseases of Animals: A field Guide for Australian Veterinarians'. Geering WA, Forman AJ and Nunn MJ (1999). This comprehensive book was published by CSIRO Publishing and distributed to all Australian veterinarians. On suspicion of an exotic disease, veterinarians must immediately notify NSW Department of Primary Industries; the exotic disease hotline phone number is 1800 675 888. The Officer manning this phone will advise on what actions need to be taken. In some circumstances, veterinarians on a suspect property will be asked to remain until appropriate arrangements can be made to collect samples and disinfect equipment and samples off the property. Ausvetplan is a comprehensive set of manuals dealing with all aspects of exotic disease management in Australia. The manuals are revised regularly and the current versions can be downloaded from the Animal Health Australia website at http://www.animalhealthaustralia.com.au/.

EXUDATIVE EPIDERMITIS IN PIGS Syn: Greasy pig disease Diagnosis Typical skin lesions on face and body. Isolation of Staphylococcus hyicus.

http://www.animalhealthaustralia.com.au


Specimens required

i. Live or fresh chilled dead piglet for bacteriology and histopathology.

ii. Smears and swabs from skin lesions, submitted in Amies charcoal transport medium.

FACIAL ECZEMA

Diagnosis History of moist, humid weather conditions; abundant, high humus containing pasture; clinical signs, gross pathology and histopathology. Demonstration of numbers of spores of Pithomyces chartarum on pasture. Specimens required

i. Sections of liver and kidney in buffered formalin for histopathology.

ii. At least 1 kg of pasture taken from the lower 5 to 7.5 cm of pasture growth and including the dead leaves and leaf residue, submitted chilled for fungal examination.

FACTOR VIII DEFICIENCY

(Hereford cattle) Syn: Haemophilia A. A sex-linked inherited defect that usually presents as a fatal hemorrhage event following castration. In one herd when a “bleeder” bull was used over heterozygous cows, some daughters were observed to bleed excessively when ear marked. It would be reasonable to expect dehorning and parturition may also present life-threatening challenges to homozygous heifers. Dehorning would also present a life-threatening challenge to bulls that inherited the mutation from their clinically normal heterozygous dam. The condition has been recorded in Hereford cattle in Australia, but may arise in any breed due to new mutations. Diagnosis A history of severe haemorrhages in grandsons of a sire should raise a suspicion of haemophilia A.

FACTOR XI DEFICIENCY (Holstein cattle) An autosomal recessive defect reported in Holsteins in North America and Europe. The mutation responsible for the defect has been defined. The mutation has been

detected in Holstein bulls in Australian artificial breeding centres. Diagnosis History of extended bleeding following dehorning, at parturition, or into milk at the onset of lactation. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

i. 20 to 25 hairs, with roots attached,

from the distal end of the tail.

FASCIOLIASIS IN SHEEP AND CATTLE

See Liver fluke infection

FISTULOUS WITHERS The aetiology is not fully known, but it has often been associated with Brucella abortus infection. Staphylococci and Streptococci are often present in the lesion. Diagnosis Clinical signs, serology. Specimens required

i. Serum sample from affected animal for Brucella abortus serology (RBT and CFT).

ii. Material from the depths of the lesion submitted chilled for bacteriology.

FLEECE ROT

A dermatitis of the skin, caused either by prolonged wetting of the skin or other skin damage. The serous exudate results in matting of the wool. The wool may be discoloured due to the growth of chromogenic bacteria. Diagnosis Clinical signs, bacteriological and macroscopic examination of affected wool. Specimens required

i. Sample of affected wool, submitted for gross examination

ii. Skin biopsy from affected area submitted in buffered formalin for histopathology.



FOCAL SYMMETRICAL ENCEPHALOMALACIA (FSE)

A chronic form of enterotoxaemia. Diagnosis Clinical signs, histopathology of the brain. Specimens required

i. Brain in buffered formalin for histopathology.

ii. Specimens as required to investigate nervous disorders in sheep.

FOOT ABSCESS IN SHEEP

Including: Toe abscess, heel abscess (infectious bulbar necrosis) TOE ABSCESS Caused by a range of pyogenic bacteria, usually involving the front feet and often associated with shelly toe. More common in sheep that have been run in wet conditions for some time. The lesion involves soft tissues of the toe region, often tracking up to break out at the coronet in front of the foot. Toe abscess can also cause underrunning and break out near the heel, but the granulation associated with the initial infection will be found near the toe. HEEL ABSCESS Caused by infection with Fusobacterium necrophorum in association with other bacteria. Occurs in fat, heavy sheep especially ewes in late pregnancy and rams. Outbreaks often seen 7-10 days after susceptible sheep have been run in wet, muddy conditions (wet muddy yards, grazing cereal crops sown in ploughed ground, wet pastures). Can occur in younger sheep if they are put in wet, muddy areas. Usually affects a hind foot, but if severely challenged can affect 2 or 3 feet. Lesions seen as swelling and tenderness in the affected claw with severe lameness, and commonly affect the interphalangeal joint. Suppurative lesions break out in the heel area with discharging sinuses and granulation tissue. Affected foot may be deformed. Diagnosis History and clinical findings. Specimens required None; best diagnosed in the field.

FOOTROT IN SHEEP Includes virulent and benign footrot For the purposes of the Stock Diseases Act and the NSW Footrot Strategic Plan, all forms of non-benign footrot are treated as virulent footrot. For full discussion of footrot classification (including colour photographs) refer to AgFact - Footrot in sheep and goats. (http://www.agric.nsw.gov.au/reader/sheep-footrot/a0956.pdf?). This may take a little while to download. Further information (Policy, AgFacts): http://www.agric.nsw.gov.au/reader/sheep-footrot A. VIRULENT FOOTROT IN SHEEP Caused by strains of Dichelobacter nodosus (formerly Bacteroides nodosus) with a range of virulence. Outbreaks of severe lameness can occur under wet pasture conditions when the mean daily temperature exceeds 9-10oC. Early lesions resemble benign footrot, but progress to severe interdigital dermatitis and underrunning of the sole and hard horn of axial and abaxial wall within 3-4 weeks. Little or no pus present even in severe lesions. The lesions will persist under dry environmental conditions and can affect a high percentage of the mob. Usually more than one foot affected. Not all chronically-infected sheep show lameness. Diagnosis History, clinical examination of a sufficient number of sheep in the flock (to detect underrunning of the hard horn of the hoof and to be confident of the range of lesions present). Bacteriology (clinical diagnosis may be reinforced by demonstration of D nodosus-like organisms in smears). Specimens required

i. Smears of affected feet lesions for D nodosus detection

Smears of affected feet can be used to detect the presence of D nodosus, but are unable to distinguish between virulent and benign strains. In addition, false negative smears can occur. Smears are taken from an active site in the lesion this is at the junction of healthy and diseased tissue. Where underrunning is evident, the horn of the hoof may have to be cut away to reveal the active sites. When the diseased tissue is pared away, a

http://www.agric.nsw.gov.au/reader/sheep-footrot/a0956.pdf


http://www.agric.nsw.gov.au/reader/sheep-footrot

http://www.agric.nsw.gov.au/reader/sheep-footrot


distinct band of active necrosis can be observed along the healthy/ infected border. This area is usually a couple of millimetres wide and is characterised by a greyish to yellow grey exudate. This exudate should abound in D. nodosus and is the material of choice for diagnostic purposes. Smears should be prepared by spreading a small amount of exudate thinly on a microscope slide (using a swab stick, match or point of a knife). Smears may be prepared from the moist necrotic interdigital skin if there is no underrunning of the heel or sole. When preparing the smear, avoid drawing blood, as this renders examination of the smear more difficult. NB • Smears should be made from 5-10

affected animals. • All slides should be numbered clearly

and serially, with a corresponding description of the animal/lesion on the specimen advice sheet. Use of a diamond pencil for numbering is recommended, since legal action may result from the examination findings. If slides with frosted glass ends are available, a dark lead pencil to identify smears is also satisfactory.

• The differential diagnosis of footrot and foot abscess in sheep is best made on clinical examination, with laboratory confirmation where necessary.

ii. Hoof samples for culture (using

special footrot kit in field) Culture of lesions and subsequent testing of D nodosus isolates may assist the field veterinarian to differentiate between benign and early virulent footrot. Under certain conditions isolation and demonstration of virulent strains of D nodosus in the laboratory can be performed using a special footrot kit for sample collection. In such cases, the Regional Veterinary Laboratory should be contacted. B. BENIGN FOOTROT IN SHEEP Caused by infection with benign strains of D nodosus. Seen in sheep grazing wet pastures under mild weather conditions.

Usually affects a number of sheep in the flock, of all age groups. Can cause marked lameness, particularly in rams and heavy sheep. Affected sheep show necrosis and inflammation of the interdigital skin, involving part or all of the soft horn of the axial wall. A small percentage may have some underrunning of the soft horn of the heel. The lesion resolves quickly if sheep are moved to dry pasture or if footbathed in formalin or ZnSO4. Cannot be differentiated from early virulent footrot, but does not progress to underrunning of the hard horn of the hoof. Diagnosis History, clinical examination of a sufficient number of sheep in the flock. In some flocks, a second examination 10 to 14 days later will be necessary to check for progression of the disease, and differentiate from virulent footrot. Bacteriology. The disease is best diagnosed in the field, but in some cases, laboratory culture for virulence testing may be necessary. Specimens required

i. See Virulent Footrot. ii. Smears of affected feet lesions for D

nodosus detection iii. Hoof samples for culture (using

special footrot kit in field) CLASSIFICATION OF DISEASE BASED ON CLINICAL EXAMINATION For full discussion of footrot classification (including colour photographs) refer to AgFact - Footrot in sheep and goats. (http://www.agric.nsw.gov.au/reader/sheep-footrot/a0956.pdf?). This may take a little while to download. In ovine footrot, a distinction between virulent and benign can be made provided there are no recent treatments or environmental conditions which would mask the full expression of disease due to the infecting strain of D nodosus. Examination of sheep after a period of spread (wet pasture conditions/mild temperature) is more reliable to assess flock status. However, additional factors will also influence disease spread and expression, including host resistance and stocking rate.




FOOTROT SCORING SYSTEM Score Description 0 normal 1 non-specific inflammation and/or necrosis of the interdigital skin (IDS) 2 inflammation of the IDS which is due to infection with footrot 3 any lesion in any claw which results in underrunning of the soft horn of the heels or

sole 4 underrunning of any hard horn of the claw

In all circumstances where lameness is occurring, it is recommended that sufficient sheep be examined to ensure that virulent footrot is not present. In most mobs, this will involve the examination of at least 100 sheep. In some mobs, it may require examination of more sheep to detect a

sufficient number of affected sheep (score 2 or greater) to assist in the diagnosis. Based on the examination of at least 100 sheep, the prevalence of score 4 lesions relative to the number of footrot-affected sheep can then be used to make the following classifications:

% of affected sheep with score 4 lesions Disease Classification 1% or more Virulent footrot < 1% May be virulent or benign; refer (i) to (iv) below

i. Disease is also considered

virulent if: • Chronic cases of severe

underrunning (score 4) are present irrespective of prevalence, particularly in areas of the State where the microclimate is unfavourable for full disease expression, in individual mobs subsequent to eradication or in recently purchased mobs of sheep, or,

• Score 4 lesions are present in obviously recent cases, or,

• The veterinarian is confident of a diagnosis of virulent footrot and can justify this (with adequate records).

ii. Caution in footrot diagnosis is

indicated and reinspection necessary if a proportion have score 3 lesions, irrespective of the flock prevalence. This may be:

• Partial expression due to recent virulent infection

• Inadequate environment • Over expression of benign footrot in

a very favourable environment • On reinspection 2-3 weeks

later, recent virulent infections will have progressed under favourable conditions

• If the environment is inadequate, virulent infections will persist at or about the same level but benign cases will have regressed without treatment

• In these circumstances a negative gelatin gel test may be used to support a field diagnosis of benign footrot

iii. Disease is probably benign but

reinspection is advisable if: • After eradication programs on

properties a very low prevalence of underrunning is seen in affected sheep the following spring, where conditions have been conducive for footrot and no treatments undertaken since the eradication program.

A negative gelatin gel test may be used to support a field diagnosis of benign footrot.

iv. Virulent footrot can be excluded

if: • Other identifiable causes of

lameness are demonstrated and there is no evidence of D. nodosus in any sheep examined (minimum of 100)

• After the mob has been through conditions favourable for the expression of footrot and - no recent treatments have

been undertaken and - at least 100 adult sheep are

examined and - 25% or more sheep have D.

nodosus infections and - none have foot scores greater

than score 2.


A negative gelatin gel test may be used to support a field diagnosis of benign footrot.

ROLE OF THE LABORATORY IN FOOTROT DIAGNOSIS A laboratory test must not be used on its own to establish a diagnosis of virulent footrot. Where concern exists on the status of the disease on the property, the producer may elect, at the producer's cost, to utilise laboratory testing as an aid to the flock diagnosis. Culture and virulence testing of D. nodosus is expensive and labour intensive, with cultures requiring 8-18 days to isolate the organism in sufficient purity for characterisation. The gelatin gel test is currently used as the standard laboratory test to evaluate isolates as being benign or virulent (non-benign). The elastase test is available but is not routinely used for estimating isolate virulence. As a minimum, it is anticipated that at least 100 sheep would have been examined in the mob and the lesions in individual sheep recorded, before specimens are collected for laboratory testing. Suitable samples from a minimum of 5 representative affected animals should be forwarded together with an adequate description of lesions in the sampled group. This will allow a minimum of 5 (up to a maximum of 20) isolates to be examined in the laboratory. The following information must be included in the specimen advice form: • number of sheep examined • foot scores of affected sheep • pasture conditions and rainfall over the

past 3-4 weeks • previous footrot history of the flock • history of recent introductions • history of recent treatments • age, breed and sex of the sheep Laboratory-based tests should be interpreted in relation to clinical findings. In general, a positive test result is meaningful, but a negative test result is not a guarantee of footrot freedom. A negative gelatin gel test may be used only to support a field diagnosis of benign footrot. Refer to the booklets:

• ‘Footrot - technical information manual’, published by Coopers Animal Health Australia (for use with the NSW Footrot Strategic Plan) 2nd edn, 1990, edited by R.Walker, SFVO Wagga.

• ‘Footrot Strategic Plan 1991’, published by NSW Farmers, NSW Department of Primary Industries, Rural Lands Protection Boards, Stock and Station Agents Association and University of Sydney.

• ‘Proceedings of Dept of Animal Health Footrot Workshop - Gundagai March 1992’, by J.R. Egerton, University of Sydney.

FOOTROT IN GOATS AND CATTLE

In goats, the clinical signs are not necessarily indicative of the virulence of the organism. Benign sheep strains of D nodosus can cause severe underrunning and lameness in goats; conversely virulent sheep strains may only cause interdigital dermatitis in some goats. Where sheep and goats are run together and spread periods have occurred, then presence of footrot in the goats only is suggestive of a benign strain. Virulence of D nodosus isolates in goats will otherwise require laboratory testing of those isolates. With rare exceptions, cattle isolates of D nodosus to date have proven to be benign for sheep. However it has been shown that a virulent strain for sheep can be harboured by cattle with lesions of foot underrunning and/or discharge. Diagnosis See’Virulent footrot in sheep. Specimens required See’Virulent footrot in sheep.

FUNGAL INFECTIONS (OTHER THAN MYCOTOXICOSES)

Care should be taken in handling specimens from suspect cases as a number of fungal infections are communicable to man. Diagnosis Clinical findings, mycology and histopathology. Specimens required Skin conditions

i. Skin biopsy taken from the margin of an active lesion submitted for mycology* and histopathology is the best specimen.


ii. Hair and deep skin scrapings from the peripheral areas of active lesions, submitted dry, for mycology*.

Systemic conditions

i. Sections of any organs showing lesions submitted for mycology*.

ii. Sections of liver, spleen and lung submitted for mycology*.

iii. Sections of affected organs and liver, kidney and spleen in buffered formalin for histopathology.

*Samples for fungal culture should not be chilled or frozen, as temperatures of less than 15oC can be detrimental to fungal survival. Culture of fungi may take up to 3 weeks.

GENERALIZED GLYCOGENOSIS Syn: Pompe’s disease (Beef Shorthorn and Brahman cattle) The most common clinical presentation is of progressive muscular weakness leading to collapse and inability to rise, usually, but not exclusively, after weaning. There are no pathognomonic gross necropsy findings. PAS positive cytoplasmic vacuolation of peripheral lymphocytes is indicative of the disease. Similarly, light microscopic findings of PAS positive cytoplasmic vacuolation of lymphoid tissue, liver, kidney or neurons are indicative of generalised glycogenosis. There are three known “lethal” mutations in the acidic α-glucosidase gene in cattle, two in Brahmans (commonly known as E7 and E13) and a third in Shorthorns (commonly known as E18). The E7 mutation is the most common, with the E13 mutation restricted to descendants of one American import. Submitters are requested to specify breed of the subject. Submitters are advised to determine (via the Australian Brahman Breeders’ Association) if a Brahman subject is a descendant of the founder for the rare E13 mutation. Diagnosis Clinical signs and history, histopathology, confirm by DNA analysis. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-

syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

(in order of preference) Diagnosis of the disease


ii. Lymphoid tissue and/or whole brain in buffered formalin for histopathology.



GENETIC DISEASES

The majority of inherited diseases recognized in cattle are autosomal recessive. Due to the practice of “line-breeding”, some recessive defects may become relatively common. In addition, undue emphasis upon estimated breeding values (EBVs) to maximize production may lead to high levels of inbreeding and an inevitable increase in prevalence of recessive defects. In contrast, inherited defects that are autosomal dominant are most likely limited to a single herd, or rarely, to a few associated herds. Haemophilia A in Herefords is an example of an X-linked defect, where the clinical disease is most common in males. Potentially there will also be Y-linked defects and also those resulting from mutations in mitochondrial DNA. Autosomal recessive defects are expressed in male and female progeny of clinically-normal, heterozygous parents. One quarter of the progeny of heterozygotes will be affected, half will be heterozygotes like the parents, and the remaining quarter will be homozygous wild-type (normal) - that is, they did not inherit the disease-causing mutation. Registered animals of most cattle breeds have been derived from a limited gene pool. Within modern breeds, herd books are generally closed. Consequently, most recessive defects are usually “breed-specific” and are caused by founder effects relating to a single mutation. There are exceptions where multiple mutations cause the same clinical disease within and across breeds. An affected individual may be heterozygous for two distinct mutations. The term ‘compound heterozygote’ is used to describe these animals. An example would be a Brahman calf affected with generalised glycogenosis if it inherited the exon7 mutation from one parent and the




exon13 mutation from the other parent. Similarly, different mutations in the α-mannosidase gene cause α-mannosidosis in Angus and Galloways. Consequently, an affected Angus/Galloway calf could be heterozygous for both of the mutations. In like manner, a Poll Hereford cross Poll Shorthorn calf may be affected with maple syrup urine disease if it inherits the “breed-specific” mutations from the parents. DNA tests are available for diagnosis of the following genetic diseases, or for detection of heterozygotes. With CVM in Holsteins

and Dwarfism in Dexters, samples should be submitted direct to an external reference laboratory – addresses provided. With DNA-based assays, hair root samples are preferred as they avoid potential problems created by haemopoietic chimerism, which can occur when using tests based on analysis of DNA from cellular elements of blood. Bovine co-twins commonly share haemopoietic stem cells, resulting in DNA in cellular elements of blood containing nucleotide variants from both twins that may be different to the DNA variants found in gametes from an individual co-twin.

Breed of Cattle Disease Angus α-Mannosidosis Murray Grey α-Mannosidosis Galloway α-Mannosidosis Salers ß-Mannosidosis Holstein/Friesian Citrullinaemia

Factor XI deficiency DUMPS Bovine Leucocyte Adhesion Deficiency (BLAD) Complex Vertebral Malformation (CVM)*

Poll Hereford Maple Syrup Urine Disease Inherited Congenital Myoclonus

Limousin Protoporphyria Beef Shorthorn Generalised glycogenosis (Pompes Disease) Brahman Generalised glycogenosis (Pompe's Disease) Dexter Chondroplasia (dwarfism) / Congenital lethal chondrodysplasia (Dexter

bulldog syndrome)**

* Consign samples direct to Dr. Van Haeringen, Laboratorium B.V. PO Box 408, 6700 AK, Wageningen, The Netherlands. ** Consign samples direct to Reprogen, Faculty of Veterinary Science, University of Sydney, PMB 3, Camden, New South Wales 2570 See Specimens (by disease) for details of specimen collection for diagnosis of a specific disease.

GOITRE Diagnosis Clinical findings, histopathology. Specimens required Thyroid glands submitted in buffered formalin for histopathology.

GRAIN POISONING Diagnosis

History of access to grain and demonstration of excess grain in ingesta with a pH of the ruminal content of less than 5. The rumen pH must be taken in the field and is only reliable if taken shortly after death. Elimination of bloat, enterotoxaemia as possible causes. Specimens required

i. Serum or eye fluid for D-lactate. ii. Sections of wall of forestomachs in

buffered formalin for histopathology.

Interpretation of D-lactate concentration Analyte Sample Units Normal Acidosis D-lactate Serum, aqueous humour mM/L < 0.4 >1.0

There is no specific diagnostic test available for grain poisoning. Specimens should be taken to eliminate other possible causes, e.g. enterotoxaemia.

GRASS TETANY See Hypomagnesaemia


HAEMAGGLUTINATING ENCEPHALOMYELITIS VIRUS (HEV) INFECTION OF PIGS

Syn: Vomiting and wasting disease HEV is responsible for two overlapping diseases of young pigs: • Encephalomyelitis (with nervous signs)

of pigs < 10 days old • Vomiting and wasting disease (with

wasting and failure to thrive) of pigs from 7-21 days old.

Diagnosis Histopathology; paired virus serology; virus isolation. Differentiate from enterovirus encephalomyelitis, other neonatal diseases e.g. hypoglycaemia, bacterial meningoencephalitis; differentiate from other causes of runting (bacterial, parasitic, nutritional, toxic). Consider exotic diseases in differential diagnosis of nervous disease: Aujeszky's disease, Teschen disease, Japanese encephalitis. Specimens required

i. Fixed brain, cervical spinal cord, gasserian ganglion (trigeminal nerve ganglion), samples of other visceral organs for histopathology.

ii. Fresh, chilled samples of brain, cervical spinal cord, SI content and a composite of visceral organs (liver, kidney, lung, spleen) collected aseptically into sterile Macartney bottles for virus isolation.

iii. Serum samples from acute and convalescent phases (14 days apart), where possible.

iv. Range of samples for bacteriology, for differential diagnosis e.g. brain, liver, kidney, small intestine.

v. Blood in sodium fluoride for elimination of hypoglycaemia.

HAEMATURIA

See also: Enzootic haematuria of cattle, bracken fern poisoning, poisoning-plant'

HAEMOGLOBINURIA See also: Hypophosphataemia, leptospirosis, tick fever, copper poisoning'

HAEMOPHILIA See: Factor VIII deficiency (Hereford cattle) and Factor XI deficiency (Friesian cattle)'

HEEL ABSCESS - OVINE See: Foot abscess in sheep'

HELIOTROPE POISONING

See: Pyrrolizidine alkaloidosis

HENDRAVIRUS INFECTION Syn: Equine morbillivirus infection Hendravirus infection was first recognised in 1994 in Australia, when it caused an outbreak of acute, fatal respiratory disease that killed 14 horses. During this outbreak the horse-trainer also died and there was a non-fatal infection of another person closely involved with the sick horses. To December 2004, four more sporadic outbreaks reported have involved the death of six horses and one human, and one non-fatal human infection. One human death occurred 14 months after exposure at post mortem examination of an affected horse. Flying foxes (fruit bats) are the subclinical reservoir of Hendravirus. All known cases have occurred in Queensland and coincided with the flying fox breeding season in the later months of the year. Hendravirus is a member of the paramyxovirus family of viruses, and is closely related to Nipahvirus, which caused a major outbreak of acute fatal respiratory disease in pigs and humans (and dogs) in Malaysia in 1999. Like Hendravirus, Nipah virus has flying foxes as its natural reservoir. Hendravirus infection is notifiable. Fees for tests undertaken to confirm or exclude a diagnosis of Hendravirus infection and to establish an alternative diagnosis are paid by NSW Department of Primary Industries. Caution Hendravirus is a zoonotic agent, causing serious illness and sometimes death in humans. Veterinarians should take biosecurity precautions when performing post mortem examinations of horses. Suitable personal protective equipment includes gloves, overalls, and mask to prevent inhalation of aerosols. Call customer service (1800 675 623) for advice. Clinical signs include pyrexia, respiratory distress (dyspnoea, blood-tinged foamy discharge from mouth and nares), sometimes neurological changes (head-pressing, muscle fasciculations) and death. Colic was the initial sign in two recent cases.


Post mortem findings include massive pulmonary congestion and oedema with airways filled with foamy, sometimes blood-stained fluid. Further information at the Queensland Department of Primary Industries' website at http://www2.dpi.qld.gov.au/health/3892.html.

Diagnosis Clinical signs, post mortem findings, histopathology, virology. Specimens required

i. Chilled lung for virology. ii. Fixed lung for histopathology. iii. 10 ml vacuum tubes of clotted and

LiHep blood for virology.

HEPATOSIS DIETETICA A disease of pigs, responsive to vitamin E/selenium therapy. Diagnosis Clinical signs of dullness, anorexia, staggering and possibly mild ascites before sudden death. Specimens required

i. Liver, skeletal muscle, portion of small intestine with mesentery attached, and myocardium in buffered formalin for histopathology.

HERPESVIRUS VULVOVAGINITIS/BALAN0POSTHITIS IN GOATS

See also: Infectious bovine rhinotracheitis (IBR), infectious pustular vulvo-vaginitis (IPV) Diagnosis Clinical signs and history of mating season, straining, dysuria, preputial/ vaginal swelling with papules, vesicles and ulcers which heal in 7-10 days (similar to cows with IPV); Virus isolation and virus serology. Specimens required

i. Swabs of vaginal/preputial lesion secretion in PBGS for virus isolation. Best at papule/vesicle stage, as virus disappears with ulcer formation.

ii. Serum sample submitted chilled for virus serology.

HISTOPHILUS OVIS/ HAEMOPHILUS SOMNUS INFECTIONS

Histophilus ovis and Haemophilus somnus are closely related organisms sometimes associated with the following conditions: Sheep Epididymitis; gangrenous mastitis; abortion; septicaemia, polyarthritis and abscessation in lambs; pneumonia and nephritis. Cattle (in Australia) Purulent vaginitis and endometritis; pneumonia, fibrinopurulent leptomeningitis and ependymitis. (Overseas septicaemia, thromboembolic meningoencephalitis, polyarthritis, tendonitis, and less commonly, mastitis). Diagnosis Clinical findings and pathology, including histopathology. Isolation of H. ovis/ H. somnus from affected tissues NB The organism may be a commensal, carried in the urogenital or upper respiratory tract of normal animals. Specimens required

i. Samples of affected tissues and swabs in Amies charcoal transport medium for bacteriology.

e.g. milk and affected gland (mastitis), liver, lung, kidney, spleen (septicaemia), whole affected joint unopened (arthritis) or synovial fluid and synovial membrane (arthritis), vaginal/uterine swabs and exudate (reproductive), affected epididymis (epididymitis), whole aborted foetus and membranes (abortion), brain (CNS), abscess swab and/or abscess content (abscessation), lung (pneumonia).

ii. Portions of affected tissues fixed in buffered formalin for histopathology.

HYPOCALCAEMIA

Diagnosis Clinical signs, response to therapy, serum or eye fluid calcium level - complication or differential diagnosis of hypomagnesaemia, ketosis, hypophosphataemia. Specimens required

i. At least 2 ml of serum, free of cells and haemolysis, submitted chilled for calcium and magnesium estimation.

http://www.dpi.qld.gov.au/health/3892.html

http://www2.dpi.qld.gov.au/health/3892.html


ii. From cadavers, 0.5-1 ml of aqueous humor, free of blood and cells for calcium estimation. Ideally, filter

aqueous humour (0.45μ membrane filter) to minimise cellular contamination.

Interpretation in cattle and sheep CalciumNormal Serum

Aqueous > 2.25 mmol/L

> 1.4 mmol/LDeficient

Serum Aqueous

< 2.1 mmol/L < 1.1 mmol/L

HYPOGLYCAEMIA

Diagnosis History, clinical signs. Specimens required There is no routine laboratory test for blood glucose concentrations.

HYPOMAGNESAEMIA Syn: Grass tetany, lactation tetany, milk tetany of calves Diagnosis

History, clinical signs, response to therapy, serum, eye fluid or urinary magnesium concentration; complications of hypocalcaemia, differentiation from ketosis. Specimens required

i. At least 2 ml of serum, free of cells and haemolysis, submitted chilled for calcium and magnesium estimation.

ii. From cadavers, 0.5 1 ml of aqueous humor, free of blood and cells, for magnesium estimation. Ideally, filter aqueous humour (0.45μ membrane filter) to minimise cellular contamination.

iii. Alternatively, 1 ml of urine for magnesium estimation.

Interpretation in cattle and sheep MagnesiumNormal Serum > 0.7 mmol/L Aqueous > 0.6 mmol/L Urine > 2 μmol/mosmol

HYPOPHOSPHATAEMIA Diagnosis History, area information, clinical signs, blood or serum inorganic phosphate concentration, response to therapy. Specimens required

i. At least 2 ml of serum free from cells, submitted chilled or frozen for phosphorus estimation.

NB Serum phosphorus levels are of little value if the sample is haemolysed or contaminated. Do not send serum on the clot.

Interpretation in cattle and sheep PhosphorusNormal Serum > 1.3 mmol/LDeficient Serum < 1.0 mmol/L

ILL THRIFT

See also: Anaemia', scouring' Ill thrift is a vaguely defined condition with a variety of causes. Cases of ill thrift associated with anaemia or scouring should be investigated under the syndromes of 'anaemia' or 'scouring'. Other causes of ill thrift not necessarily associated with anaemia and/or scouring include: • Malnutrition, the commonest cause. • Nutritional deficiencies of copper

cobalt, phosphorus or selenium.

• Parasitism, including helminthiasis, fascioliasis and coccidiosis.

• Eperythrozoonosis. • After effects of systemic or chronic

conditions including pyrrolizidine alkaloidosis, sarcosporidiosis and chronic infectious diseases

• Viral: EIA in horses, pestivirus infections in ruminants, EBL in cattle, CAE in goats.

• Specific bacterial diseases e.g. Johne's disease, caseous lymphadenitis.

Diagnosis


Based on clinical findings, laboratory examination to eliminate specific causes and response to treatment. Specimens required Specimens should be submitted to eliminate some of the possible causes only after malnutrition has been eliminated as a contributing factor. The conditions listed should operate on a flock basis so that specimens should be submitted from a number of animals in the flock. The range of specimens to be submitted could include:

i. Faecal samples from 10 to 20 animals for parasitology.

ii. Blood samples as required to eliminate specific deficiencies of selenium, phosphorus and copper.

iii. Sections fixed in buffered formalin, of liver, kidney, cardiac and skeletal muscle, small and large intestine or other organs depending on gross pathological indication of aetiology at field necropsy.

iv. If indicated, sera and/or portions of body organs including the above, submitted fresh for bacteriological, virological and/or parasitological examination.

v. Live unthrifty animal submitted for post-mortem examination.

INFECTIOUS BOVINE RHINOTRACHEITIS (IBR) AND INFECTIOUS PUSTULAR VULVO VAGINITIS (IPV)

Diagnosis Virus isolation and antibody rise between acute and convalescent serum samples. Specimens required Respiratory and ocular disease

i. Swabs from nasal cavities and conjunctivae in phosphate buffered gelatin saline (PBGS).

Vulvo-vaginitis:

i. Acute and convalescent serum samples (collected 3 6 weeks apart), submitted chilled for virus serology.

ii. Swabs of vaginal mucus in PBGS. Abortion

i. Specimens as required for bovine abortion.

Encephalitic form

i. Acute and convalescent serum samples (collected 3 6 weeks apart), submitted chilled for virus serology.

ii. Specimens as required for encephalitis, including brain for virus isolation and histopathology.

INFERTILITY IN CATTLE, SHEEP, PIGS

See also: Abortion (general), congenital abnormalities, copper deficiency, epididymitis, semen examination, trichomoniasis of cattle

Diagnosis Must be based on field investigations supported by the laboratory where necessary. The first step is to establish the stage in the reproductive cycle where the problem occurs, i.e. at joining due to fertilisation failure (male or female infertility), embryonic loss, abortion or perinatal loss. Specimens required Those appropriate to the condition or conditions suspected. A detailed history should be provided to allow consideration of other causes. Detailed investigations should involve prior consultation with the laboratory.

INHERITED DEFECTS See congenital abnormalities, genetic diseases'

INHERITED CONGENITAL MYOCLONUS

(Poll Herefords) Stimulus-responsive myoclonic spasms in bright and alert calves is the striking clinical feature of this disease. The majority of affected calves have hip lesions, most frequent being fractures of the acetabulum, and less commonly, fractures of the head of the femur. No consistent histological findings. Diagnosis History, clinical signs, confirmed by DNA analysis. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf





IRON TOXICITY SYNDROME IN PIGLETS

Diagnosis Clinical history of sudden death of 0-7 day piglets within 1 12 hours of giving iron injection, or vomiting/ diarrhoea/ dyspnoea/ coma and death up to 3 days; selenium deficiency in sow. Specimens required

i. 10 ml of heparinized blood from sow(s), submitted chilled for selenium analysis.

ii. Piglet for post mortem examination and differential diagnosis.

ITCHMITE IN SHEEP

Diagnosis Demonstration of Psorergates ovis mites in deep skin scrapings from suspected cases, examined in the field. Differentiation from other causes of fleece derangement e.g. lice, tender fleeces. Specimens required In cases of dermatitis or skin irritation where mites cannot be seen at field examination, the following samples may be sent to the laboratory:

i. Skin scrapings collected using a scalpel blade dipped in paraffin oil, submitted with the blade enclosed in a secure sample container.

ii. A skin biopsy from the affected area, submitted in buffered formalin (may assist in a differential diagnosis).

NB Wool must be clipped as close to the skin as possible before taking a scraping (see Parasites - External).

JAUNDICE Jaundice is a clinical sign which may arise as a result of a haemolytic crisis or hepatic failure. Common causes include plant poisonings, chronic copper poisoning, leptospirosis and eperythrozoonosis. Diagnosis Clinical signs, history. Specific causes diagnosed on the basis of laboratory examination. Specimens required

i. Sections of liver, kidney and spleen, submitted in buffered formalin for histopathology.

ii. Other specimens appropriate to the disease condition suspected see Copper poisoning,

Eperythrozoonosis, Leptospirosis, Plant poisoning, Pyrrolizidine alkaloidosis.

iii. In the live animal, serum chilled for enzymology.

JOHNE'S DISEASE

Syn: Paratuberculosis Diagnosis History, clinical signs, gross pathology and histopathology. Demonstration of typical organisms in faecal smears and tissues. Isolation of Mycobacterium paratuberculosis from faeces or tissues. Serology. Absorbed ELISA Absorbed ELISA are routinely available for Johne's Disease testing of cattle, goat, alpaca and deer sera. Absorbed ELISA testing is the most sensitive serological method for diagnosis and certification for M. paratuberculosis infection and disease. It also has a very high specificity. AGID An AGID is available for goats and sheep - this has a low sensitivity in identifying infected animals, but its specificity is extremely high. It is of value only in those animals with moderate histological lesions (including all clinical cases); a positive test is indicative of current histological lesions of the disease. ELISA positive goats can be further tested by AGID to identify those infected animals with most advanced lesions. Smears of faeces and tissues Smears of faeces and tissues are of limited sensitivity in subclinical animals, and should be restricted to clinical cases. Faecal and tissue culture Faecal and tissue culture procedures depend on whether the cattle (C) or sheep (S) strain is likely to be involved. The majority of cattle, goat, deer and alpaca infections are attributed to C strains, while sheep infections are predominantly due to S strains. Cattle or goats reared with infected sheep may be infected with S strain; C strain infection in sheep is relatively uncommon. The C strain can be cultured by either conventional (solid media) culture or by radiometric broth (Bactec) culture. For the C strain, conventional culture is usually slower than Bactec culture, but is less expensive. The S strain can only be reliably cultured by radiometric (Bactec) culture, and a longer


incubation period is used compared to the C strain. The current default procedures (based on consumer demand) apply to culture submissions from cattle, goats, alpaca and deer:

Species Method Cattle, deer: Bactec Goats, alpacas: Conventional

If the default procedure is not suitable (i.e. the alternate procedure is required), the required procedure must be advised on the Specimen Submission Form. In addition, where OJD is suspected and a non-ovine species is being tested by culture, this must

be clear on the specimen advice form so that procedures specific to the S strain (i.e. longer incubation; different subculture medium) can be undertaken. The culture process is undertaken in batches, and requires an initial decontamination phase over 5 working days, followed by an incubation phase, and, if growth occurs, a confirmatory phase. The amount of M. paratuberculosis in the sample correlates with the rate of growth (higher numbers reduce the time delay until growth is detected). This in turn modifies the time for culture to be completed. The following table outlines expected time differences between the two strains in current cultural procedures:

C strain S strain

Method Conventional Bactec Bactec Decontamination 5-7 d 5-7 d 5-7 d Incubation 20 wks 8 wks* 12 wks Confirmation by PCR/REA 1-4 wks (if reqd) 1-4 wks 1-4 wks Confirmation by subculture (mycobactin dependency test) Up to 10 wks Up to 10 wks Up to 10 wks

Total time if no growth detected 21 wks 9 wks 13 wks Total time if growth detected and M. paratuberculosis confirmed 12-26 wks 10-21 wks 10-23 wks Total time if growth detected and not M. paratuberculosis 12-24 wks 12-21 wks 12-23 wks

* If growth is detected late (e.g. at week 8), incubation to week 10 may be required to reach a satisfactory level for confirmation. Culture confirmation of M. paratuberculosis Confirmation of M. paratuberculosis from Bactec growth is undertaken when growth reaches a high level, as this optimizes the confirmation rate. Two separate characteristics of M. paratuberculosis are targeted in the confirmation procedure for maximum specificity, to reach a conclusion of culture positive for M. paratuberculosis. When multiple tissues are cultured from an individual, both characteristics need be met on only one tissue for a conclusion of culture positive status for that animal. The first characteristic is measured in a molecular assay involving a polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) that target the IS900 gene sequence. Together, these can identify the presence of M. paratuberculosis DNA with very high specificity. Samples of Bactec growth are collected and then processed by PCR and REA in batches, for greatest efficiency.

Examination for the second characteristic of M. paratuberculosis as part of the confirmatory process from Bactec media is also commenced when growth reaches a high level. This normally requires a sample of growth to be subcultured to solid medium for evidence of typical colonies with mycobactin dependency, and takes up to 10 weeks. Different solid media are used for subculture of the C and S strain. While solid media that supports the S strain can be used to cultivate the C strain, it does so less efficiently than the preferred C strain medium. In contrast, S strain does not grow on the solid C strain medium. As an alternative to subculture to demonstrate mycobactin dependency, testing for a second specific molecular target (e.g. IS1311 PCR and REA) can reduce the time to complete confirmatory testing procedures, and yet retain maximal test specificity. Confirmation of M. paratuberculosis from conventional culture for C strains is equivalent to the second part of the Bactec confirmatory procedure i.e. subculture for


mycobactin dependency. However, additional IS900 PCR and REA testing will be undertaken on growth on solid media typical of M. paratuberculosis in herds where Johne's disease has not previously been confirmed. This should be borne in mind when completing the Specimen Submission Form for samples from herds without prior confirmation of Johne's disease and where conventional culture is requested. Direct PCR Direct PCR on pooled or individual faeces is available for sheep in limited circumstances. It is restricted to use for risk assessment purposes to determine if high shedder animals are present. Strain typing Strain typing can be undertaken on Bactec growth if requested. This relies on PCR and REA targeting the IS1311 gene sequence. NB: Allergic tests and serological tests such as the CFT are unreliable in the diagnosis of the disease, with false positives and false negatives occurring frequently. Individual culling on the grounds of such tests is often impractical. The Johne's CFT is not routinely available. It is restricted for export/certification testing only, and not for routine diagnostic purposes. Specimens required In the live animal:

i. Faeces, submitted chilled for bacteriology

• Individual faecal culture - volume of > 5 g per animal

• Pooled faecal culture - in sheep*, one pellet from each of (up to) 50 sheep per pool. In flock sampling, choose additional sheep to allow for animals from which no sample can be collected (allow 55 sheep for each pool of 50 pellets).

Notes in regard to PFC submissions: • Do not take more than one pellet

per animal • Samples must not overfill a 60 mL

sterile plastic universal container. If pellets are l large, submit only sufficient per pool to fill the 60 mL container.

• Serial faecal culture - in sheep - one pellet from each of (up to) 10 sheep and submit from these same animals on each of three occasions 10-14 days apart. This will create a single pool of up to 30 samples.

ii. Serum sample for ELISA test (cattle, goats) and/or AGID (goats, sheep).

*Note: Pooled faecal culture procedures for cattle and goats are currently under development. In slaughtered animals:

i. Fresh, chilled portions of intestine and intestinal lymph nodes in separate sterile containers for bacteriology. The best samples are usually those with obvious lesions (gut thickening, lymph node enlargement). Samples from the following sites per animal are considered ideal, and are typical of those required for MAP certification. In addition, faeces at slaughter can be useful to assess the risk of shedding.

Cattle: Intestinal portions of 5 cm x 10 cm • proximal ileum (30-45 cm anterior

to ICV) • terminal ileum (11-20 cm anterior to

ICV) • ileocaecal valve (ICV) and attached

segment of ileum • proximal colon • ileal LN • ileocaecal LN

Goats and alpaca: Intestinal portions of 5 cm in length • jejunum • proximal and terminal ileum (1 m

apart) • ileocaecal valve and attached

segment of ileum • proximal colon • ileal LN • ileocaecal LN

Sheep: • terminal ileum • ileal LN (caudal jejunal LN)

ii. Sections of the above, in buffered

formalin for histopathology. In cattle and goats, include fixed caecum. In goats and sheep, can include a section of fixed liver (microgranulomata). NB It is preferable that the above samples be taken in the field. Where this is not possible, do not forward entire intestinal tract for laboratory examination without prior consultation. Unless such samples are received soon after collection, autolytic changes can prevent useful examination,


particularly in serological reactor animals with limited lesions.

KIKUYU POISONING A disease of cattle causing salivation, dehydration, depression, abdominal pain and death associated with grazing on lush kikuyu pastures. The cause is unknown, although mycotoxins have been suggested in some cases. Diagnosis History, clinical signs and histopathology (particularly of omasum and reticulum). Specimens required

i. Sections of omasum, reticulum, rumen abomasum and kidney, in buffered formalin for histopathology.

ii. Sections of liver, kidney and small intestine, submitted in buffered formalin for histopathology (for differential diagnosis).

There is no value at present in examining samples of kikuyu for toxins, fungi, etc.

LEAD POISONING Diagnosis History of illness, clinical signs of central nervous system disturbance, biochemical analysis of tissues. In calves, thymic haemorrhages are a frequent finding at necropsy. Specimens required Live animal

i. 100 g of faeces. ii. 10 mL EDTA blood tube.

Dead animal

i. 50 g of kidney, submitted chilled for toxicology.

ii. Sections of liver and kidney, and whole brain in buffered formalin for histopathology.

Interpretation of lead concentrations (cattle and sheep) Analyte Sample Minimum

amount Units Normal Possibly

toxic Toxic

Lead EDTA blood 5ml µmol/L < 1.2 > 1.2 Lead Kidney (Liver) 50g mg/kg (wet wt) < 4 4-25 > 25 Lead Faeces 100g mg/kg (wet wt) < 10 10-25 > 25

Kidney lead concentrations below 4 mg/kg are considered non-significant. Higher concentrations should be interpreted on the basis of clinical findings and histopathology. Faecal lead concentrations below 10 mg/kg are considered non-significant. Higher levels could be significant, depending on the source of lead.

LEPTOSPIROSIS Leptospirosis is transmissible to man and represents an occupational hazard to veterinarians, abattoir workers, dairy and pig farmers. The most commonly encountered serovars associated with clinical disease are: • Cattle L hardjo, L pomona • Pigs L pomona, L tarassovi • Horses L pomona • Dogs L copenhageni Other serovars do affect domestic animals in Australia, particularly Northern Australia, but such infections are rare in NSW. Zoonotic infections are usually acquired from the maintenance host for each serovar:

Serovar Maintenance Host L. hardjo Cattle L. pomona, L. tarassovi

Pigs

L. copenhageni Rats

In the maintenance host, infections by these specific serovars are likely to be endemic, whereas serovar infections in non maintenance hosts are usually associated with sporadic outbreaks of disease. The most common manifestations of leptospirosis in animals are:

• Abortion (all species) • Milk drop syndrome/ mastitis

(cattle) • Pyrexia/Septicaemia (all species) • Icterus and/or haemoglobinuria

(mainly dogs and calves) • Kidney lesions found at routine

slaughter (pigs, cattle)


• Mortalities following pyrexia and/or icterus/haemoglobinuria

Diagnosis Serology (Microscopic Agglutination Test). Confirmed by demonstration of leptospires via direct microscopic examination of urine or foetal fluids (thoracic, peritoneal, pericardial); and/or by demonstration of organisms in kidney by histopathology. In general culture of tissues, foetal fluids, urine or milk is not undertaken, except under specific arrangements with the receiving laboratory. Specimens required Abortion Samples as outlined in 'Abortion in Cattle' or 'Abortion in Swine'. Whenever possible, submit serum from dam, including 10 15 sera from cohorts, and fresh, chilled entire foetus with membranes (or chilled and fixed foetal tissues, including serous fluids) for bacteriology and histopathology. Milk Drop Syndrome

i. Serum samples from 10 15 affected and recovered cases and cohorts, submitted chilled for serology.

• A convalescent serum sample collected 10 days or more after agalactia often shows a rise in titre to L. hardjo.

ii. Sample of urine preserved with formalin (approx. 1 ml of 10% neutral buffered formalin to 99 mls urine) added immediately after collection, from both affected and recovered cases, for dark ground microscopy. It is worthwhile using a diuretic to obtain a sample with large numbers of organisms and minimal contaminants.

iii. Samples of milk for differential diagnosis of mastitis, submitted as outlined in 'Mastitis'.

iv. In certain cases, following consultation with the Regional Veterinary Laboratory, arrangements may be made to sample cows for culture. This involves field-inoculation into selective liquid media of mid-stream urine collected after diuretic treatment.

Pyrexia

i. Serum samples from 10 15 affected and recovered cases and cohorts, submitted chilled for serology.

ii. Sample of urine preserved with formalin (approx. 1 ml of 10%

neutral buffered formalin to 99 mL urine) added immediately after collection, from both affected and recovered cases.

Icterus/haemoglobinuria Samples as for Pyrexia, but do not collect urine for microscopic examination from animals affected with haemoglobinuria. Urine shedding of leptospires occurs after the haemoglobinuric phase. Kidney lesions in slaughter animals

i. Portions of affected kidneys in buffered formalin for histopathology.

Mortalities following pyrexia and/or icterus/haemoglobinuria

i. Sections of liver and kidney, in buffered formalin for histopathology.

Interpretation of Leptospiral titres For the MAT, titres are given which reflect the standard Leptospiral dilution series for sera. For leptospiral MATs, this is a doubling dilution series starting at 1:25 (L hardjo) or 1:50 (other Leptospires). In cattle, an MAT titre to L hardjo of 25 or greater may be significant. Indication of current infection can only be gained by demonstrating at least a four-fold (i.e. 2 dilution) increase. Titres following vaccination are generally low (less than 400) and do not persist. In cases of abortions in cattle due to L hardjo, titres at the time of abortion may be equivocal and falling. In L pomona infections and L hardjo milk drop syndrome, titres generally rise within 1 3 weeks of the appearance of clinical signs. L pomona is more immunogenic than L hardjo, so titres to L pomona are often 800 or more, whereas in L. hardjo milk drop syndrome, they may be less than 800. L hardjo titres of 200 or more are generally significant. Submission of 10 15 sera from animals in the same group as the affected individuals will give a more accurate assessment of the leptospirosis status of the herd. To determine recent infection, 10 15 paired sera (taken 2 4 weeks apart) should be submitted to detect rising titres (along with the clinical history of the tested animals). In an endemically infected herd at any given time, some titres will be rising, some will be static, and others will be falling.


LICE RESISTANCE TO INSECTICIDES IN SHEEP

Resistance of sheep lice to pyrethroid insecticides is widespread in NSW. No functional organophosphate resistance has been detected. Resistance may be suspected on a history of treatment failure after correct application of the correct amount of lousicide at the correct time, either: • As breakdowns within 3-4 months of

application of backline treatment in sheep treated off-shears or in short wool, or,

• Where sheep in long wool have been thoroughly treated with a pyrethroid and failure to control infestation is obvious within 6 weeks.

NB Long wool application of pyrethroids may leave undesirable residues at shearing. Faulty application of treatment should be considered where infestations are localised to one side or part of the sheep, or where one mob or line is infested and other mobs are free of lice. Diagnosis Examination of sites of, and degree of, infestation in 4-5 affected sheep by counting lice in 10 partings on each side of the infested sheep (each side examined in 2 lines of 5 partings). Demonstration of resistance using an in-vitro laboratory test. NB. It is more productive to inspect a larger number of sheep rather than search a smaller number of sheep more thoroughly. A thorough examination of the dip operation and thoroughness of wetting is recommended, particularly in the case of shower dipping failures with insecticides. Specimens required Testing for OP resistance is not routinely available; it requires controlled temperature and humidity and is a laboratory based test at the Elizabeth Macarthur Agricultural Institute (contact Dr Garry Levot). Submission of live affected sheep would be required.

LISTERIOSIS Listeriosis can be transmitted to man. Diagnosis

Meningoencephalitis (circling disease) Clinical signs and brain pathology. Abortion Post mortem findings and bacteriology. Septicaemia Bacteriology and histopathology. Specimens required Meningoencephalitis

i. Cerebrospinal fluid and sections of brain, liver, spleen and kidney, submitted chilled for bacteriology.

ii. Brain in buffered formalin for histopathology.

Abortion

i. Specimens as required for abortion in the particular species (see Abortion).

Septicaemia

i. Heart blood and sections of liver, kidney and spleen, submitted chilled for bacteriology.

ii. Sections of liver, kidney, spleen and lung, in buffered formalin for histopathology.

LIVER FLUKE INFECTION

See also Parasites Internal Diagnosis Grazing history, clinical examination, serology, faecal egg count, response to treatment. Necropsy findings. ELISA serology is available at EMAI for the detection of infection with both immature and adult fluke. Using the Institut Pourquier (France) antibody ELISA recently adopted (2004) by NSW DPI laboratories, titres appear 2-4 weeks (cattle) after infection and remain high for at least 20 weeks after tretment. The false negative rate is low in cattle (<2%). The test has not been validated for sheep under Australian conditions. Faecal egg counts are only of value in chronic fascioliasis where eggs are being excreted (patent infection); they are of no value in acute and subacute infections. ELISA serology is more sensitive than faecal egg counts for the detection of chronic fascioliasis in both cattle (30% more infected animals detected) and sheep (15-20% more infected animals detected). ELISA serology is the only antemortem diagnostic method for the detection of acute and subacute fascioliasis (before fluke eggs are excreted), which typically occurs in


NSW from autumn to early winter and in late spring. The ELISA test is reported by the manufacturer to be suitable for individual and bulk vat milk samples. The test is currently (2004) undergoing validation by NSW DPI. The milk ELISA is a herd test and for statistical significance a minimum of 12-15 samples should be submitted. Specimens required At necropsy, fascioliasis should be diagnosed in the field and laboratory specimens will not be required.

i. Serum samples from affected cattle

and their cohorts for ELISA serology ii. Faeces for fluke egg counts in the

following situations: • Where serum collection in cattle is

not feasible, or, • In animals where liver fluke has

been detected in the herd or flock and treatment has been given in the previous 5 months, or

• Where paired faeces and serology is specifically sought for comparative study.

Submit separate faecal samples (at least 30 g of fresh faeces each) from at least 10 animals in the flock or herd, in a screw capped jar. Faecal samples may be tested individually, or pooled in groups of two (designed for sheep) or five samples (cattle). Pooling reduces costs but sensitivity is compromised. Submitters should nominate how they wish the samples to be tested. NB For interstate testing for import of ruminants to Western Australia, which is fluke-free, individual samples from up to 30 animals in each consignment are required (further details are available at Department of Agriculture, Western Australia web site under Livestock movement at http://www.agric.wa.gov.au).

For more information, refer to the Agfact A0.9.57 (second edition 1999 revised 2003) ‘Liver fluke disease in sheep and cattle’: http://www.agric.nsw.gov.au/reader/an-diseases/a0-9-57web.pdf

LUPINOSIS A mycotoxicosis mainly associated with grazing on lupin stubble infected with Phomopsis leptostromiformis. Diagnosis History, clinical signs and histopathology. Specimens required

i. Sections of liver and kidney in buffered formalin for histopathology.

ii. If nervous symptoms are seen, specimens as required for nervous disorders.

LYME DISEASE

Lyme Disease is a multisystem disease caused by the spirochaetal bacterium Borrelia burgdorferi. It can affect the joints, heart and nervous system of man, domestic animals and wildlife and is known to occur in Europe, Asia and North America, where it is primarily transmitted by Ixodid ticks. There is clinical, serological and pathological evidence of a Lyme Disease-like in cattle and man in eastern coastal Australia but the organism has not yet been isolated. Likely vectors here are Ixodes holocyclus or other Ixodes spp. In animals, Lyme disease usually presents as lameness associated with polyarthritis; joints are painful and swollen and there may be a low grade pyrexia. There may also be lethargy, stiffness and myalgia, with inability to rise in severe cases. Chronic weight loss, abortion and laminitis like signs can be shown by affected cattle and horses; some horses may have skin hypersensitivity, uveitis and CNS signs resembling encephalitis. Diagnosis Clinical signs, histopathology (typically reveals a fibrinoid polyarthritis). Borrelia burgdorferi is usually present in only low numbers in affected joints, and requires special media for isolation. The organism may be more readily isolated from blood during the early stages of disease. Serology can be undertaken for some animal species outside of the Department's laboratories, by arrangement. Differential diagnosis: • Other causes of arthritis:

Chlamydiosis, mycoplasmal arthritis (See Arthritis, Chlamydial Infections, Mycoplasmosis).

• Ephemeral Fever.

http://www.agric.wa.gov.au/



http://www.agric.nsw.gov.au/reader/an-diseases/a0-9-57web.pdf


• Septicaemic and toxaemic conditions caused by other bacteria.

Specimens required

i. Chilled acute and convalescent serum, especially for alternative diagnosis (Chlamydia)

ii. Fixed tissues for histopathology: joint capsule, brain, heart, peripheral nerve, kidney, liver, skin from tick bite site (in neutral buffered formalin).

iii. Culture may be arranged by consulting RVL staff at EMAI. In cases with polyarthritis, chilled joint fluid and (in PM material) joint capsule is sought. In acute cases, heparinised blood is suggested.

LYSSAVIRUS

Syn: Australian bat lyssavirus, pteropid lyssavirus

Australian bat lyssavirus is closely related to classical rabies virus and causes similar progressive neurological disease in naturally-infected bats and humans. Flying foxes (fruit bats) and insectivorous bats are affected. Subclinical infection apparently occurs in bats, which are the reservoir of the virus. Australian bat lyssavirus infection is notifiable. Fees for tests undertaken to confirm or exclude a diagnosis of lyssavirus infection are paid by NSW Department of Primary Industries.

Caution Lyssavirus is a zoonotic agent, causing fatal infections in humans. Clinical signs in bats include behavioural changes (aggression/docility, shivering, salivation) and paralysis. Where disease is suspected, bats should be euthanased and sent to the laboratory for necropsy. All bats should be handled with care to avoid bites and scratches. The general public should be discouraged from rescuing or handling bats (paralysed, aggressive or unusually-docile bats are at high risk of being clinically-affected). People bitten or scratched by bats should consult a General Practitioner immediately and seek prophylactic vaccination. Individuals regularly handling potentially-affected bats (wildlife carers, veterinary pathologists) should be vaccinated against rabies.

Further advice can be obtained from the Australian Government’s Department of Health and Aging website at http://www.health.gov.au/internet/wcms/Publishing.nsf/Content/cda-pubs-other-bat_lyssa.htm.

Further information on this disease can be obtained from the CSIRO website at http://www.csiro.au/. Diagnosis Clinical signs, histopathology, virology. Specimens required

i. Whole bat (chilled)

MALIGNANT OEDEMA Diagnosis History of mortality, post mortem findings and recovery of pathogenic clostridia from lesions. Best diagnosed in the field because of rapid autolysis of samples. Specimens required

i. Affected tissues from affected animal or freshly dead animal, submitted chilled for bacteriology.

ii. At least three impression smears from lesions for bacteriology.

iii. Sections of affected tissues, in buffered formalin for histopathology.

Material must be taken from affected or freshly dead animals, otherwise examination is worthless.

MALNUTRITION See also: Feeds and pasture analyses Malnutrition is the most common cause of ill thrift. The differential diagnosis should include parasitism, infectious diseases, chronic conditions causing organ dysfunction (e.g. pyrrolizidine alkaloidosis) and deficiencies of particular nutrients (e.g. selenium, copper, phosphorus). Malnutrition should also be considered as a predisposing cause of many conditions, e.g. pregnancy toxaemia, plant poisoning in drought situations.

MANGE Diagnosis Clinical signs, demonstration of parasites in deep skin scrapings or skin biopsy, histopathology.

http://www.health.gov.au/internet/wcms/Publishing.nsf/Content/cda-pubs-other-bat_lyssa.htm

http://www.csiro.au/


Cases are best diagnosed in the field from direct examination of multiple deep skin scrapings. Specimens required

i. Skin scrapings. ii. Skin biopsy from a recently affected

area, in buffered formalin for histopathology (in cases where the disease is suspected but cannot be confirmed from skin scrapings).

MANNOSIDOSIS

ALPHA-MANNOSIDOSIS

(Angus, Murray Grey and Galloway cattle) Affected Angus and Murray Grey calves either fail to survive the immediate postnatal period, or if they do, they show severe, progressive neurological disease characterised by tremors of the head, ataxia, and aggression. The phenotype in Galloways is more severe, with most affected cases aborted or stillborn. Diagnosis History, clinical signs, confirmed by DNA analysis or histopathology. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

(in order of preference, breed of subject must be recorded) Diagnosis of the disease





BETA-MANNOSIDOSIS

(Salers cattle) Affected calves are usually born alive, but exhibit severe neurological abnormalities. These include weakness, incoordination, head-swaying, splaying of the front legs, and a poor sucking reflex. Diagnosis

History, clinical signs, confirmed by DNA analysis or histopathology . Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf

(in order of preference) Diagnosis of the disease





MAPLE SYRUP URINE DISEASE (MSUD)

(Poll Hereford, Hereford and Poll Shorthorn calves) An inborn error in branched chain keto acid metabolism that results in a progressive neurological disease that culminates in death before 5 days of age. At birth, affected calves are clinically normal, but within 24 hours they develop progressive neurological dysfunction characterised by uncoordinated limb movements that prevent the calves from rising. Between 48 and 72 hours after birth the calves collapse and typically are often found in lateral recumbency with opisthotonos. Death intervenes within 5 days of birth. There are no pathognomonic gross findings at necropsy. Widespread vacuolation of white matter tracts in the brain and spinal cord is a common histological manifestation of MSUD in Hereford and Shorthorn calves. Diagnosis History, clinical signs, histopathology, confirm by DNA analysis. Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf (in order of preference, breed of the subject must be recorded) Diagnosis of the disease






ii. Whole brain in buffered formalin for histopathology.



MASTITIS (BOVINE)

Diagnosis Clinical mastitis History, clinical signs and bacteriology Subclinical mastitis Bacteriology, Rapid Mastitis Test, Mixed quarter milk cell counts (individual cow cell counts) in excess of 250,000 averaged over a lactation, elevated milk NAGase activity, increased milk electrical conductivity. Herd mastitis problem Evidence of subclinical mastitis (see above), problems in milking parlour management or milking machine operation, environmental factors such as contaminated water supply or muddy conditions. Bacteriology and antibiotic sensitivity testing is performed by the Regional Veterinary Laboratory. Individual cow cell counts are available through Herd Recording. Cost of cell counts depends upon whether dairy farmer is a herd recording member or not. History required All mastitis submissions should be accompanied by full history of the mastitis case. For clinical cases of mastitis this should include breed, age, previous antibiotic treatment (both for previous clinicals and dry cow treatment), stage of lactation, previous mastitis history. If a herd problem of clinical mastitis is occurring, additional information required is number of cows involved, description of clinical signs and any recent changes in farm management or milking parlour management. Subclinical mastitis problems require an intensive investigation of milking parlour operation. Regional veterinary or dairy livestock officers may provide assistance in these investigations. Specimens required

i. Approximately 20 ml of foremilk collected aseptically from affected quarter into a sterile container.

Mixed quarter samples are not recommended as excessive dilution of the pathogen can occur.

• All samples should be taken prior to antibiotic treatment. If antibiotic treatment has been given, resample case at next occurrence of clinical signs or at least three weeks post treatment.

• Before sample is taken, teat must be washed and dried thoroughly and teat end sterilised with 70% alcohol or undiluted teat dip solution.

• Discard the first few squirts before collecting sample. Samples should be kept on ice until arrival at the laboratory.

Interpretation of results Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus spp (environmental) and are the most common pathogens found in both clinical and subclinical mastitis. In mastitis cases occurring at or near calving, Escherichia coli, Enterobacter spp and other coliforms may be present. Serratia spp and Klebsiella spp can cause persistent mastitis symptoms. Recovery of milk quality in quarters affected by these pathogens may not occur. Incurable mastitis can be caused by Pseudomonas aeruginosa. Nocardia spp and Arcanobacterium (Actinomyces) pyogenes. Recovery of the former two pathogens from mastitis cases should indicate cull for slaughter of the affected cow especially if Nocardia sp are involved as these pathogens pose a potential human health risk. Mastitis due to Arcanobacterium pyogenes usually necessitates drying off affected quarter after treatment and reassessing damage to the quarter after subsequent calving. Recurrent cases of mastitis are usually due to chronic staphylococcal infection. All recurrent cases should be cultured to check cause. Antibiotic sensitivity necessary for recurrent cases. Of the current S4 antibiotics registered for clinical and subclinical mastitis, cloxacillin products are effective for all staphylococcal and streptococcal infections and neomycin-based products effective for staphylococcal and enviromental mastitis pathogens such as coliforms. If other antibiotic products are used, antibiotic sensitivity will be necessary.


MASTITIS (CAPRINE) Diagnosis Bacteriology is the most effective diagnostic method. Rapid Mastitis Test can be used. Cell counts and NAGase are unreliable in herds with caprine arthritis encephalitis (CAE)virus infection. History and specimens required As for bovine samples. Interpretation of results Coagulase-negative staphylococci (Staphylococcus epidermidis) are the most common cause of subclinical mastitis in dairy goats. Clinical mastitis can be caused by coagulase negative staphylococci, Staphylococcus aureus, Streptococcus spp. and enterobacteria such as Escherichia coli, Serratia spp and Klebsiella spp. Care must be taken with treatment. Intramammary antibiotics will have extended witholding periods in goats compared to cows. The blue dye in these antibiotics can be excreted in the milk of treated goats for extended periods of time. Parenteral antibiotics may be used.

MASTITIS (OVINE) Ovine mastitis may be acute/severe, gangrenous, chronic or subclinical. The most frequently isolated bacteria are Staphylococcus aureus, Mannheimia (Pasteurella) haemolytica, Arcanobacterium pyogenes, and Streptococcus spp. Coagulase-negative Staphylococci, Bacillus spp, Micrococcus spp and E coli may also be incriminated in subclinical disease. Less commonly Corynebacterium pseudotuberculosis, Histophilus ovis, Actinobacillus seminis, Pseudomonas spp, Proteus spp and Klebsiella spp are involved. Clostridium perfringens is associated with gangrenous mastitis (also S aureus and E coli). Mycoplasma spp can also cause ovine mastitis. Diagnosis Bacteriology. Other tests including Somatic cell count (high variability makes interpretation difficult; also epithelial cell shedding as a normal phenomenon increases SCC thresholds for identifying mastitic milk), Rapid mastitis test, and NAGase can be applied on a herd basis. History and specimens required As for bovine samples.

MASTITIS METRITIS AGALACTIA SYNDROME IN SOWS

Occurs from 12 to 48 hours after parturition with a rapid fall off in milk production, toxaemia, constipation, fever and vaginal discharges in the sow. Diagnosis The cause or causes of the syndrome are not clearly understood. Management factors are implicated. Specimens required Samples of vaginal discharges are of limited value because of the wide range of organisms normally present in the vagina of the sow. At present, there is little that can be offered in the laboratory in relation to this condition.

MENANGLE VIRUS INFECTION Menangle virus infection was first recognised when it caused a single outbreak of reproductive disease in pigs in a large piggery near Sydney in 1997. A control program, successfully managed by NSW Agriculture, subsequently eradicated the virus from the piggery. A large serological survey failed to find any evidence of infection in other Australian pigs. No further outbreaks of the disease have occurred to date. Flying foxes (fruit bats) near the piggery had antibodies to the Menangle virus. It is believed that flying foxes are the natural host of the virus, and that it was a relatively rare event that led to the virus 'jumping' into pigs. Menangle virus is a member of the paramyxovirus family of viruses, and is closely related to Tioman virus, isolated in 1999 from fruit bats in Malaysia, but which has not yet been associated with disease in any animal species. Menangle virus infection is notifiable. Fees for tests undertaken to confirm or exclude a diagnosis of Menangle virus infection are paid by NSW Department of Primary Industries. Clinical signs include a severe decline in farrowing rate, a high incidence of delayed returns to service and a marked increase in the number of mummified and stillborn piglets, some with severe deformities. Stillborn piglets had a severe non-suppurative encephalomyelitis. Although many pigs were infected, the virus was not highly contagious and thus probably spread


by excretion in faeces and urine, rather than by respiratory aerosols. There was also a severe influenza-like illness in two of the workers at the piggery. The two workers recovered from the illness. Diagnosis History, clinical signs, histopathology, virology. Specimens required

i. Chilled stillborn piglets for necropsy, virology and histopathology.

ii. Fresh heart, lung, spleen, brain and body fluids collected separately and aseptically, submitted chilled for virology.

iii. Sections of heart, liver, kidney, lung, spleen and whole brain in buffered formalin for histopathology.

METABOLIC DISEASES

See 'hypocalcaemia, hypomagnesaemia, hyophosphataemia, biochemistry: guide to biochemical parameters in sheep and cattle

MUCOSAL DISEASE See Pestivirus, abortion, arthrogryposis and hydranencephaly'

MULBERRY HEART DISEASE A cause of sporadic mortalities in rapidly growing pigs, characterised by pronounced myocardial haemorrhage. Many cases are associated with vitamin E and/or selenium deficiency, but these are not the sole cause. More cases tend to occur under conditions of heat stress. Diagnosis History and characteristic necropsy findings of petechial and ecchymotic haemorrhages in the heart, and distension of the pericardium by a serofibrinous transudate. Liver necrosis/ rupture/ haemorrhage is a feature in many cases. Specimens required

i. Heart, liver and brain, in buffered formalin for histopathology.

ii. Kidney and liver submitted chilled or frozen for selenium estimation.

iii. 10 ml of heparinised blood, submitted chilled (not frozen) for biochemistry from 5 to 10 animals in the same pen.

MUSCULAR DEGENERATION, NUTRITIONAL

Syn: Selenium/vitamin E deficiency, white muscle disease, nutritional myopathy, ‘muscular dystrophy’’ This disease complex usually occurs as defined clinical and pathological syndromes which bear a several names. They are thought to have a nutritional pathogenesis involving a dietary deficiency of selenium and/or vitamin E. Diagnosis History, clinical signs, post mortem examination, histopathology, biochemistry, response to therapy. Specimens required

i. 10 ml of heparinised blood submitted chilled (not frozen) for glutathione peroxidase (GSHPx) concentrations, from at least 5 animals in the group.

Selenium status can be measured directly by analysis of blood or tissue selenium concentrations, or indirectly by analysis of blood glutathione peroxidase concentration. The latter, as in most enzyme tests, requires heparinized blood (EDTA is unsuitable). The correlation between glutathione peroxidase (GSHPx) and selenium concentrations is very high in non-deficient animals. In deficient animals, this high mathematical correlation is not present, but of little diagnostic consequence, i.e. in deficient animals, both GSHPx and Se concentrations are low but there is not necessarily a close mathematical correlation between values.

ii. Sections of affected and unaffected skeletal muscle and cardiac muscle, in buffered formalin for histopathology.

iii. At least 2 ml of serum, free of cells and haemolysis, submitted frozen for serum enzymology.

iv. 20 g liver, submitted chilled and protected from light immediately following collection. (See Collection of specimens for biochemistry and Vitamin deficiency).

NB. Whenever possible, submit heparinised blood from 3 5 peer animals rather than tissues of dead cases


Interpretation of blood glutathione peroxidase (GSHPx) concentrations The following values should be taken only as a guide: GSHPx U/g Hb Interpretation Cattle Sheep Disease < 10 <10 Production response < 25 <25 Normal > 100 >50

Note that current blood concentration of GSHPx reflect the selenium intake of the animal at the time the present circulating erythrocytes were formed. An ‘average’ time would be the half life of the erythrocyte for the species and age of animals to be examined (e.g. adult cattle approx. 80 days, adult sheep approx. 60 days, calves and lambs approx. 50 days). The interpretation of selenium status and prognosis is further complicated by factors which include: • Season (winter and summer peaks,

autumn and spring troughs of Se availability).

• Soil type (acid soils < pH 5.5 have 50% reduction in Se availability to plants).

• Pasture type (legumes and other leafy plants lower in Se than grasses).

• Fertilizer history (competitive interaction from sulphur results in reduced selenium in pastures on superphosphate fertilized soils).

• Age of animals preruminant animals more prone to deficiency than ruminants.

For diagnosis of selenium deficiency in sheep, it is recommended that weaners be sampled after access to good feed (e.g. clover). Older sheep normally have lower levels. Diagnosis should be considered on a property basis.

MYCOPLASMOSIS See also Porcine enzootic pneumonia Mycoplasma infections cause a wide range of clinical conditions, particularly among pigs, cattle, sheep, goats, and poultry. Certain mycoplasmas are part of normal mucosal flora, and can outgrow pathogenic mycoplasmas in broth culture procedures. In general, special media is required for growth of mycoplasmas. Some mycoplasmas are slow growing, taking up to 3 weeks to appear in primary culture or in subcultures. Avoid submission of swabs for mycoplasmal culture. Swab materials can

be toxic to mycoplasmas if left in contact for extended periods. Diagnosis Pigs Culture of some pathogenic mycoplasma species can be undertaken using media specific for pig mycoplasmas. Identification of M. hyopneumoniae in lung tissues or nasal swabs by PCR. Identification of M. hyopneumoniae herd infection by ELISA serology. NB: M. hyorhinis is a common inhabitant of the URT and ears (Eustachian tube), and is a common co-inhabitant of pneumonic lungs. It has no pneumonic potential and can rapidly overgrow M. hyopneumoniae cultures. It can cause a serofibrinous polyserositis, or fibrinous arthritis in suckers or weaners less than 10 weeks old. It produces a milder, more sporadic disease than Glasser's disease, and can occasionally affect young adults. (DD: Glasser's disease, Streptococcus suis, Erysipelas). M. hyosynoviae causes synovitis and arthritis (DD: Erysipelas; M. hyorhinis polyserositis of young pigs; Glasser's disease polyserositis or respiratory disease; Pasteurella spp synovitis; Streptococcus suis purulent arthritis or polyserositis; other Streptococci causing purulent arthritis with percutaneous infections/abrasions; leg weakness/osteochondrosis) Cattle, sheep and goats Culture of affected tissue/s. Mycoplasma spp bovine Group 7 is a frequent cause of polyarthritis in calves. It can also cause outbreaks of bovine mastitis and abortion. This organism is readily cultured, and is occasionally cultivable on blood agar cultures without special mycoplasmal media. Other Mycoplasma spp are associated with bovine mastitis, eye lesions, reproductive tract infections, and caprine and ovine arthritis and mastitis.


Specimens required Pigs

i. Serum (or heparin plasma) samples for M. hyopneumoniae ELISA..

ii. Fresh chilled or frozen lung for M. hyopneumoniae PCR (1 cm³).

iii. Nasal swab material collected into sterile PBS for M. hyopneumoniae PCR.

iv. Fresh chilled joint fluid or membrane for M. hyosynoviae culture.

v. Fresh chilled tissues or joints affected with polyserositis/arthritis for M. hyorhinis culture (suckers, weaners).

vi. Fixed lung for histopathology.

Cattle, sheep, goats i. Depending on the pathogen, fresh

tissues, joint fluid, joint membrane, ocular fluid, milk for mycoplasmal culture.

ii. Sections of affected organs in buffered formalin for histopathology.

Poultry

i. Serum for Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) antibody detection by rapid plate test.

NB Culture for avian mycoplasmas is not routinely undertaken. Specialised services are available at some other laboratories.

MYCOTIC DERMATITIS See Dermatophilosis

MYCOTOXICOSIS The effects of mycotoxins vary according to species and age of the animal, the quantity of mycotoxin ingested and the period of exposure. Mycotoxicoses include aflatoxicosis, ergotism, facial eczema, Fusarium spp toxicosis, paspalum staggers, tremorgen intoxication, lupinosis and mouldy corn poisoning. Diagnosis History, clinical signs, histopathology and demonstration of toxic concentrations of mycotoxin in feed. Demonstration of a toxigenic species of fungus in the feed is not enough, the fungi are ubiquitous and only some strains are toxigenic. Mycotoxicosis can only be confirmed by demonstrating biologically effective

concentrations of toxins in the feed the animals ingested. Often, because of the expense of mycotoxin analysis, the diagnosis is made by elimination of other causes. Mycotoxin analysis in feed is a specialised area of chemistry not offered by NSW Department of Primary Industries. Our Laboratories will outsource this testing on request. Specimens required

i. Sections of liver and kidney, in buffered formalin for histopathology to demonstrate toxic effects.

ii. 500g of feed for mycotoxin analysis. iii. Specimens for differential diagnosis,

particularly where nervous system disease is seen clinically, e.g. Fusarium spp. (see Nervous disorders).

NASAL GRANULOMA OF CATTLE

Diagnosis Clinical signs, histopathology. Specimens required

i. Section of affected nasal mucosa, in buffered formalin for histopathology.

ii. Section of affected nasal mucosa, submitted fresh for bacteriology and mycology*.

*Tissues for fungal culture should not be refrigerated or frozen (temperatures of less than 15oC can be detrimental to fungal survival); such specimens should be stored and transported between 18oC and 37oC.

NECROBACILLOSIS Syn: Calf diphtheria, necrotic stomatitis of calves, 'necrobacillosis of lambs' Diagnosis Clinical signs, necropsy findings and demonstration of Fusobacterium necrophorum in lesions. In lambs, multiple large creamy/caseous necrotic liver and lung lesions (and occasionally diaphragmatic lesions) are associated with entry of Fusobacterium necrophorum via primary lesions in the rumen and/or abomasum. Specimens required

i. Smears taken from the depth of the lesions.

ii. Portion of lesion submitted chilled for bacteriology.


iii. Sections of affected organs, submitted in buffered formalin for histopathology.

NEOPLASMS

In cases of biopsies from the live animal, a detailed description is necessary, especially in those cases where the field veterinarian is seeking a prognosis. Diagnosis History, clinical signs, gross pathology and histopathology. The history should include species, age, sex, breed and full description of the tumour giving its location and the rate of development of the tumour. Specimens required

i. Sections of affected organs, in buffered formalin for histopathology.

The sections should be taken both from the centre of the lesion and also from around the margin, at the junction of normal and abnormal tissue.

NEOSPOROSIS Neospora caninum is a coccidian parasite related to Toxoplasma gondii. The dog is the definitive host, with sexual stages in the gut resulting in oocysts shed in faeces. Cattle (an intermediate host species) are infected by ingesting feed contaminated by sporulated N caninum oocysts shed by dogs or by infected cattle tissues (eg infected foetal membranes), or are infected transplacentally. The asexual proliferative stages of Neospora caninum (including tissue cysts), cause:

• Abortion in cattle (world-wide, including coastal NSW) and congenital neonatal neurological disease in cattle (rarely). Neospora abortion is reported in deer (rarely).

• Neurological disease in the dog (rarely)

Diagnosis Cattle History, clinical signs (abortion), histopathology, serology.

Distinctive histological changes are seen in brain (necrogranulomatous encephalitis) and myocardium (non-suppurative myocarditis) of aborted foetuses.

Dog Clinical signs (neurological disease), histopathology, serology.

Specimens required Cattle Aborted foetuses

i. Specimens as required for diagnosis of abortion in cattle (see Abortion in cattle), particularly:

• serous foetal body fluid (pericardial, thoracic or peritoneal) or heart blood for serology

• brain and myocardium (including from autolyzed foetuses) formalin-fixed for histopathology

Cows i. 10 ml of clotted blood in vacuum

tube for serology. A group of aborted and non-aborted animal should be tested. In view of the widespread prevalence of subclinical neosporosis in cattle in coastal NSW areas, seropositivity in an aborted cow does not confirm Neospora abortion.

Dogs i. Spinal cord and brain and other

tissue formalin-fixed for histopathology.

ii. Clotted blood in vacuum tube for serology.

NERVOUS DISORDERS

See also Ataxia', encephalomyelitis, and specific disease syndromes, including transmissible spongiform encephalopathy (TSE). Nervous signs can arise from a wide range of causes, including bacterial, viral, parasitic and protozoon infections, chemical and plant poisonings and metabolic and genetic conditions. Laboratory diagnosis of the cause or causes will require a range of specimens to be submitted, together with a detailed history and clinical examination. For adult sheep and cattle with progressive neurological disease, please submit specimens as required by the National TSE Surveillance Program (http://www.animalhealthaustralia.com.au/aahc/programs/adsp/tsefap/tsefap_home.cfm), accompanied by a completed NTSESP Clinical History and Post-mortem Report form. Diagnosis

http://www.animalhealthaustralia.com.au/aahc/programs/adsp/tsefap/tsefap_home.cfm


History, clinical signs, histopathology and biochemistry. Specimens required

i. Portion of brain, spinal cord and cerebrospinal fluid, collected aseptically and submitted chilled for bacteriology and virology.

ii. Brain and spinal cord, in buffered formalin for histopathology.

iii. Serum sample from the affected animal, submitted chilled for serology.

iv. At least 3 ml of serum, free of cells and haemolysis, submitted chilled or frozen for enzymology and biochemistry.

v. Other specimens as required for specific diseases.

NITRATE-NITRITE POISONING

Diagnosis History, clinical signs, particularly methaemoglobinaemia, chemical confirmation of nitrate or nitrite in the animal. Specimens required Either,

i. 1 ml of serum submitted chilled for toxicology, or,

ii. Thick, air dried blood smears for toxicology.

NB. Testing of plant material, serum or blood is best performed in the field. Appropriate specimens should also be taken to eliminate other causes of sudden death.

OEDEMA DISEASE OF PIGS Diagnosis Clinical signs, necropsy findings and the recovery of enteropathogenic Escherichia coli. Specimens required

i. Segment of ileum or swab in transport medium for bacteriology.

ii. Brain and sections of jejunum and ileum in buffered formalin for histopathology.

iii. Range of fresh and fixed tissues for differential diagnosis.

iv. Moribund or freshly dead animal for necropsy and bacteriological examination.

OPHTHALMIA Syn: Pink-Eye

Diagnosis Clinical signs, identification of causal organisms (Moraxella bovis or IBR in cattle, Branhamella spp or Chlamydia in sheep). Specimens required

i. Smears and swabs of conjunctival surface, submitted chilled for bacteriology.

ii. Swabs of conjunctival sac in phosphate buffered gelatin saline (PBGS), together with paired sera, submitted chilled for virology.

Moraxella bovis can be recovered consistently from the conjunctival sac only when blood agar plates are inoculated immediately swabs are taken. Arrangements can be made with the laboratory for the supply of blood agar plates in special circumstances where the isolation of Moraxella bovis is desirable.

ORGANOCHLORINE AND ORGANOPHOSPHATE POISONING

There may be some delay (approximately 2 weeks) in testing samples for organochlorines and organophosphates. Testing is undertaken at NSW Department of Primary Industries' laboratory at Wollongbar for a range of OC and OP compounds. Currently analytical methods for pesticides in ingesta or tissues are available for the following chemicals: Organochlorines Aldrin, Dieldrin, Endrin, Lindane, DDE, DDD, DDT, HCB, Heptachlor, Heptachlor Epoxide, Alpha Chlordane, Gamma Chlordane, Alpha BHC and Beta BHC. Organophosphates Bromophos Ethyl, Chlorfenvinphos, Chlorpyrifos, Chlorpyrifos Methyl, Diazinon, Dichlorvos, Ethion, Fenitrothion, Fenthion, Malathion, Methacrifos, Methyl Parathion, Parathion, Pirimiphos Methyl, Profenophos, Sulprofos, and others. Diagnosis Based on clinical findings, response to treatment and toxicology. There are no pathological changes observed at necropsy. The history should include details of the specific insecticides to which the animals may have had access (see also 'Specimens for Toxicology'). Specimens required


Acute exposures i. At least 20 g of stomach contents,

liver and brain submitted frozen for toxicology.

ii. Sections of liver, kidney, lung, brain and any other organs with gross lesions, should be submitted in buffered formalin for differential diagnosis on histopathology.

Chronic exposures

i. At least 20 g of fat submitted frozen for toxicology

ii. Sections of liver, kidney, lung, brain and any other organs with gross lesions, should be submitted in buffered formalin for differential diagnosis on histopathology.

OSTEOCHONDROSIS IN PIGS

A condition characterised by joint abnormalities in rapidly growing pigs, usually seen in baconers. Diagnosis History, clinical signs and gross pathology. Specimens required Affected limbs, with unopened joints, submitted chilled for bacteriology, gross pathology and histopathology.

OSTEOMALACIA, OSTEOPOROSIS Disease associated with transient and shifting lameness, bone fractures, perverted appetite and tendency to bone chewing. Affected bones are light, porous and soft, and often the seat of healing fractures.

Diagnosis Clinical signs, gross pathology, histopathology. Most cases are best diagnosed on clinical signs and gross pathology. Histological examination is performed only in certain cases, following laboratory consultation Specimens required

i. Sections of affected bone, submitted

in buffered formalin for histopathology.

OSTERTAGIOSIS

See also Parasites internal Diagnosis Grazing history, age of affected animals, clinical examination, faecal egg count and larval culture, total worm counts. In cattle,

serum or plasma pepsinogen levels (see Pepsinogen estimations from serum or plasma'). Specimens required Live animal

i. At least 30 g of fresh faeces in a screwtop jar filled to capacity, submitted chilled for egg count and larval culture.

ii. In cattle, serum or plasma pepsinogen may be useful, particularly in growing animals.

Dead animal

i. At least 30 g of fresh faeces as above.

ii. The abomasum and abomasal contents whole, with both ends tied off (also tied at the abomaso-duodenal and ileocaecal junctions), submitted chilled or frozen for total worm count and abomasal digest for recovery of histotrophic larvae.

OVINE BRUCELLOSIS

See brucellosis ovine'

OXALATE POISONING Diagnosis History of access to plants containing oxalate, clinical findings, hypocalcaemia, histopathology. Specimens required

i. Sections of liver and kidney, submitted in buffered formalin for histopathology.

ii. At least 2 ml of serum, free of haemolysis and cells, submitted chilled for calcium estimation. (see also Hypocalcaemia).

PAPULAR STOMATITIS OF CALVES

Diagnosis Clinical signs, electron microscopy. Specimens required

i. Digestive tract lesions fixed in buffered formalin for histopathology, if required for differential diagnosis.

ii. Scabs or necrotic debris in a sealed container, submitted chilled for electron microscopy.

PARAKERATOSIS OF SWINE

Diagnosis Clinical signs differentiation sarcoptic mange and exudative epidermitis; histopathology.


Specimens required

i. Portions of oesophagus, tongue and affected skin, submitted in buffered formalin for histopathology.

ii. 10 mL clotted blood tube or 2 mL serum for zinc analysis.

PARAMPHISTOMIASIS

See also Parasites internal' Diagnosis Grazing history and associated seasonal conditions. Clinical findings, demonstration of immature paramphistomes in the duodenum or adult flukes in the rumen and reticulum. Specimens required Dead animal

i. The abomasum and small intestine, unopened and tied off at each end, submitted chilled or frozen for parasitology.

NB Faecal egg counts are of no value in diagnosing clinical paramphistomiasis. The presence of paramphistome eggs indicates non-pathogenic adult stomach flukes are present. Adult paramphistomes may confer some immunity. PARASITES (EXTERNAL)

See also Itchmite, mange' Diagnosis Based on clinical findings and demonstration and identification of parasite on skin scraping or skin biopsy. Specimens required for diagnosis

i. Skin scrapings, submitted unpreserved for parasitological examination.

ii. In cases where parasites cannot be demonstrated in skin scrapings in the field, a skin biopsy sample from an affected area, submitted in buffered formalin for histopathology.

Specimens required for parasite identification

i. Flies, lice, fleas, ticks, spiders, etc. - submit preserved in alcohol/ glycerine (9 parts of 80 per cent alcohol to 1 part glycerine) in a small leak proof container.

ii. Insect larvae - where live larvae are required for insecticide tests, consign about fifty in a ventilated container with about 3 cm damp vermiculite in the bottom. Larvae

can be collected from "struck" wool by placing the wool on a sheet of paper in sunlight.

iii. Mange mites - scrape recent lesions, using a scalpel moistened with liquid paraffin. Transfer scrapings to a small wide mouthed bottle and submit unpreserved.

PARASITES (INTERNAL)

See also Ostertagiosis, liver fluke infection, paramphistomiasis' Faecal egg counts and differential larval counts are a guide to the size and type of worm burden. The faecal egg count depends on a number of factors including faecal consistency and bulk, host resistance, stage of pregnancy and effects of lactation, as well as the parasite involved and whether the parasites are sexually mature. The history should provide details of all recent anthelmintic treatments and flock management, e.g. stocking rates, pasture availability, swampy areas, paddock movement, etc. Further information on internal parasites of sheep or cattle is available on: http://www.agric.nsw.gov.au/reader/sheep-internal http://www.agric.nsw.gov.au/reader/cattlehealth Diagnosis Clinical signs, faecal egg count, larval cultures and total worm count. Specimens required Live animal

i. At least 30 g of faeces collected from the rectum, submitted in a screw top jar filled to capacity for egg count and larval culture. In flock or herd investigations, samples should be collected from 10 to 20 animals showing evidence of parasitism.

WormTest is a convenient system for collecting and submitting faecal samples to the laboratory.

ii. For F hepatica in cattle, serum

sample chilled for ELISA serology. Milk ELISA testing for liver fluke is currently (2004) under validation; check with your veterinary laboratory for availability.

Dead animal



http://www.agric.nsw.gov.au/reader/cattlehealth

http://www.agric.nsw.gov.au/reader/cattlehealth


Preferably: i. The whole gastrointestinal tract

(double bagged in strong clear plastic) submitted unopened, but tied off, either chilled or frozen for parasitological examination.

Alternatively

i. Aliquot samples of 1 L from the stomach and small intestines, submitted in a 5% formalin solution for parasitological examination.

ii. The abomasum and intestine, submitted chilled or frozen for examination for immature parasites.

FAECAL EGG COUNTS (INTERPRETATION) Faecal worm egg counts and differential larval counts are a guide to the parasite burden. The number of helminth ova passed per gram of faeces depends on such factors as faecal consistency and bulk, host resistance, stage of pregnancy, effects of lactation and whether the worm burden consists of sexually mature parasites.

Faecal egg counts are generally lower from cattle than from sheep. If sheep are starved for 24 hours, the count may be increased. Inappetence may cause the count to multiply 30 to 40 times. Diarrhoea depresses the egg count. Any statement of 'significant figures' for ova counts will be only a rough guide. Interpretation must be considered in relation to the specific parasite responsible (as indicated by larval cultures) and the age, origin, state of nutrition and clinical history of the infected animals. See AgNote: WormTest for livestock and guide to egg counts. Sheep An egg count of 500 eggs per gram (epg) is generally considered high enough to require treatment in order to limit pasture contamination and subclinical disease. The following numbers of epg may indicate clinical disease due to that parasite.

Guide to faecal egg counts in sheep (indicating pathogenic burdens)

Species Eggs per gram of faeces Young Sheep Older Sheep Haemonchus contortus 2,000 2,000Ostertagia spp. 500 500Trichostrongylus spp. 500 1,000Nematodirus spp. >200 500Oesophagostomum columbianum 300 1,000Chabertia ovina 500 1,000Fasciola hepatica 100 >100Paramphistomes >500

*Compiled from various reference sources: Cole VG (1986) Appendix 1, pp 233-239; Love SCJ, Hutchinson GW (2003) Table 4, pp 329-333, Skerman KD, Hillard JJ (1966) p7. The egg laying capacity of Ostertagia spp and Nematodirus spp is poor and severe clinical signs may be seen before appreciable numbers of eggs are present in the faeces. Low and medium egg counts will be more significant where the stocking rate is high, when weather conditions are conducive to epidemics (warmth, rain, humidity) and where the biotic potential is high, e.g. Haemonchus contortus. Infections with one parasite only are rarely seen and the additive effects of mixed infections will require assessment. The pathogenicity of immature stages not indicated by egg count should always be

considered. This is of particular significance with Nematodirus, Ostertagia, Chabertia, Fasciola hepatica and paramphistomes. Dictyocaulus filaria is often associated with mixed gastrointestinal infections on the tablelands and slopes. Cattle The clinical history and knowledge of the seasonal pattern of worm parasites in different areas of the State will assist in interpretation of faecal egg counts. In cattle greater than 18 months of age, the egg count gives little indication of the level of the parasite burden.


Guide to faecal egg counts in cattle (indicating possible pathogenic burdens in 6-18 month old cattle)*

Species Eggs per gram of faeces Ostertagia spp. 300->1,500

(<300 possibly significant)Trichostrongylus axei 500-1,000

(medium infections)Haemonchus placei 700->1,500Cooperia spp (in 3-4 month old calves) 1,000-5,000

(10,000-30,000 in acute disease)Oesophagostomum radiatum. >500Bunostomum spp 500-800

(heavy infections)Fasciola hepatica Any egg count is significant

Heavy infections may be indicated by >25 epg. However, there is little relation between egg count and

fluke burden.Paramphistomes >500

(heavy adult infection)*Compiled from various reference sources: Cole VG (1986) Appendix 2, pp240-245; Love SCJ, Hutchinson GW (2003) Table 5, pp 334-338 ; Skerman KD, Hillard JJ (1966) p 8; Smeal MG (1995) pp 358. Horses 600 epg or more may represent a pathogenic cyathostomin (small strongyle) burden. Colic induced by verminous arteritis from migrating Strongylus vulgaris larvae (without eggs in faeces), is not as common as in previous times. Pigs Any positive count of Ascaris suum ova in pigs up to 5 months of age is significant. Young pigs with heavy immature worm burdens may not be passing any eggs in the faeces. In infected pigs, eggs generally appear after the age of 9 to 10 weeks. TOTAL WORM COUNTS (INTERPRETATION) Interpretation of total and differential worm counts is not absolute, but depends on clinical history and several interacting factors such as age, sex and nutritional status of the host. Opinions vary on the significance of counts for various worm species. Sheep Rumen and Reticulum

Paramphistomes. The immature forms occur in the small intestine. Heavy infections of adults, e.g. 5000 paramphistomes may be associated with ill health.

Abomasum

Haemonchus contortus. In young sheep 500 worms (light), 3,000 (heavy) and in adults 1,000 (light), 9,000 (heavy). Ostertagia spp. 3,000 5,000 worms is a heavy infection in young sheep; 5,000-10,000 worms in adult animals may cause mortality. Usually there is a concurrent infection with Trichostrongylus spp. and other intestinal worms. Trichostrongylus axei. Heavy infections (>5,000 worms) may cause unthriftiness and gastritis.

Small intestine Trichostrongylus spp. In young poorly grown weaners, 5,000 worms may cause mortality, while in older weaners, 20,000 worms can be considered a heavy infection. A similar number of worms in adult crossbred sheep can be responsible for ill thrift and scouring but probably 30,000 40,000 worms are needed to cause mortalities. Nematodirus spp. In young sheep 1,000 2,000 worms is considered a light burden, but heavier infections of 5,000-10,000 worms may be an important cause of diarrhoea, ill thrift and sometimes mortality. Usually observed as a concurrent infection with Trichostrongylus spp. In the absence of worm egg counts, the importance of immature stages of Nematodirus spp. should be considered in the diagnosis of clinical parasitism.


Strongyloides spp. Not considered of pathogenic importance under field conditions except in young lambs when infection is heavy (10,000-15,000). May be a problem under penned conditions. Cooperia spp. Heavy infections in young lambs may be of importance. Paramphistomes. Infections with > 5,000 immature stomach fluke cause clinical disease. (80% of immatures are in duodenum). Moniezia spp. Questionable importance.

Large intestine Oesophagostomum spp. There may be a heavy infection with larval forms, or very marked nodule formation in previously exposed older sheep due to Oesophagostomum columbianum, when adults may be rare and egg count consequently low. Oesophagostomum columbianum is usually restricted to northern and western district pastoral areas of NSW. Serious chronic effects are caused by severe nodule formation. In young sheep 100 worms is a serious infection and in adult animals 100-200 worms may be significant. Chabertia ovina. Both immature and adult worms can cause ill effects. In both young and adult sheep, 100-200 worms is a heavy infection. Trichuris ovis. Heavy infections have been observed in sheep during prolonged drought periods. Inflammatory lesions in the caecum resulting from large numbers of parasites probably cause scouring and ill thrift.

Liver Fasciola hepatica. Mortalities from the acute disease may be associated with 700 or more immature liver fluke and from the chronic disease with 50 or more adult fluke.

Lungs Lung worms (Dictyocaulus filaria, Muellerius capillaris) are relatively rare in sheep. Greater than 50-100 worms may be considered a serious burden.

Cattle Rumen and Reticulum

Paramphistomes. The immature forms occur in the small intestine. Heavy

infections of adults, e.g. 5,000 paramphistomes, may be associated with ill health.

Abomasum Haemonchus placei. In 6-month-old calves, 5,000 adult worms may be pathogenic causing anaemia, sub-mandibular oedema, and sometimes diarrhoea. Less important in cattle than H contortus is in sheep. Ostertagia ostertagi. An important pathogenic parasite of both young and adult cattle as 30,000-50,000 adult worms may cause diarrhoea, weight loss, anaemia and harsh coat. The number of immature stages in the histotrophic form in the abomasal wall should be considered when planning anthelmintic treatments against outbreaks of ostertagiosis. Following a single anthelmintic treatment which removes adult worms, the ensuing development of up to, 50,000-90,000 immature worms could easily cause a second serious wave of adult infection (Type II ostertagiosis). Ostertagiosis should be considered in the differential diagnosis of ill thrift and loss of condition in adult cattle, including bulls. Trichostrongylus axei. Usually found in association with Ostertagia ostertagi, but in lower numbers.

Small intestine Cooperia spp. The most common small intestinal worm. Always occurs in mixed infections with Ostertagia ostertagi. Heavy burdens of more than 200,000 worms may occur in dairy calves, but clinical signs are due primarily to ostertagiosis. Trichostrongylus spp. Rarely found in large numbers. Bunostomum phlebotomum. In young calves 500 worms may be significant. This hookworm causes anaemia, haemorrhagic enteritis and dark, foetid scour. Moniezia spp. Common in calves but relatively unimportant. Nematodirus spp. Rarely found in large numbers. Heavy infections may be seen in dairy calves (10,000 worms). Low numbers (2,000-3,000) may be found in young cattle in concurrent


infections with other intestinal trichostrongyles. Paramphistomes. Infections with >10,000 immature stomach fluke cause clinical disease. (80% of immature worms are in duodenum).

Large intestine Oesophagostomum radiatum. Found in calves 4-12 months old. A burden of 500-800 worms is a light to medium one, while 1,000 worms may produce clinical signs of parasitism - anaemia, haemorrhagic enteritis, and a watery, mucoid scour. The prepatent stages of this worm in the intestinal wall may also cause ill effects. Trichuris spp. Usually found only in low numbers and is of little pathogenic importance.

Liver Fasciola hepatica. An important cause of liver condemnations at abattoirs. Light infection up to 50; medium infection 50-100 and heavy infection, over 100.

Lungs Dictyocaulus viviparus. Common in dairy calves in cooler areas, but seldom seen in beef cattle. Often seen in conjunction with gastrointestinal parasite burdens.

Pigs Small intestine

Ascaris suum. Very pathogenic. Any number is significant in young pigs. Macracanthorhynchus spp. Not common. Heavy infection is 200 worms. Strongyloides spp. Heavy infections may affect young suckers.

Large intestine Oesophagostomum spp. Widespread and common cause of scouring in pigs. Trichuris spp. Heavy infections can cause typhlitis in young pigs.

Lungs, liver and kidneys Metastrongylus spp. Prevalent lungworm. Immature forms of Ascaris, Stephanurus and Strongyloides may be found wandering in the lung. Stephanurus dentatus (kidney worm) very rare parasite in modern pig husbandry, but still found in feral pigs in some locations. May be re-introduced if

trend to free-range pigs continues. Heavy and pathogenic burdens of 50- 100 larvae.

References Cole VG (1986). Animal Health in Australia Volume 8, Helminth Parasites of Sheep and Cattle. Australian Agricultural Health and Quarantine Service, Department of Primary Industries, AGPS, Canberra. Love SCJ, Hutchinson GW (2003). Pathology and diagnosis of internal parasites in ruminants. In Gross Pathology of Ruminants, Proceedings 350, Post Graduate Foundation in Veterinary Science, University of Sydney, Sydney;Chapter 16:309-338. Skerman KD, Hillard JJ (1966). A Handbook for Studies of Helminth Parasites of Ruminants. Near East Animal Health Institute, Iran Unit, UNDP, FAO, Rome. Smeal MG (1995). Parasites of Cattle, Veterinary Review No. 32, Post Graduate Foundation in Veterinary Science, University of Sydney, Sydney.

PARVOVIRUS INFECTION IN PIGS Infection before 35 days of gestation causes foetal resorption and small litter size, with returns to service. Infection from 35 to 55 days of gestation results in foetal death and mummification, stillborn and weak piglets. Infection after 55 days usually has no effect, but occasionally causes stillbirths if infection takes place before 70 days gestation. Between 80 days and term, the virus does not cross the placenta. Abortion is extremely rare in parvovirus infection. Diagnosis Herd history, clinical findings, histopathology and virology (demonstration of virus antigen or antibody in foetal tissue). Specimens required

i. Whole mummified foetuses and stillborn foetuses submitted chilled for bacteriology, virology and histopathology.

ii. Serology on individual samples from

sows is of little value in diagnosing parvovirus infection. Parvovirus serology should be used as a herd


test or to assess the immune status of individuals.

PASTEURELLOSIS

Infection by Pasteurella spp.and Mannheimia haemolytica (syn: Pasteurella haemolytica) can cause a septicaemic or pneumonic disease. It can also cause abortion and mastitis in sheep. In pigs, toxigenic strains of P. multocida are associated with enzootic atrophic rhinitis (see Atrophic rhinitis of swine). Diagnosis Clinical signs and necropsy findings. Recovery of Pasteurella spp. Histopathology. Toxigenicity testing of snout isolates from pigs. Specimens required

i. Sections of liver, lung, spleen, heart blood and any other organ showing lesions, submitted chilled for bacteriology.

ii. Sections of liver, lung, spleen and kidney, submitted in buffered formalin for histopathology.

NB. In cases of abortion, see specimens required for Abortion.

PEPSINOGEN ESTIMATIONS FROM SERUM OR PLASMA

In adult cattle, estimations of serum or plasma pepsinogen concentrations are considered a useful indicator of abomasal damage and of value in the diagnosis of type II ostertagiosis (caused by the synchronised emergence of histotrophic larvae). Pepsinogen is produced in the gastric mucosa as the inactive precursor of pepsin. Abomasal damage results in increased blood pepsinogen concentration. The assay measures the presence of pepsinogen through the ability of the test serum or plasma to breakdown a protein substrate to peptide fragments. Results are compared with a tryrosine standard, and expressed in terms of IU/L of tyrosine. Specimens required

i. Serum, heparinised or EDTA plasma.

Activity is stable for several days at 4oC for several days and several months at -20oC. Haemolysis has little effect on activity. Interpretation of results

Pepsinogen values should be interpreted on a herd rather than an individual basis. False negatives may occur.

Level (IU/L) Interpretation < 5 No significant abomasal

damage 5-10 Minor damage*

10-15 Moderate damage > 15 Major abomasal damage

* Levels of 5 or greater are considered indicative of damage sufficient to cause production losses.

PERINATAL MORTALITIES IN CATTLE

Bovine stillbirths and neonatal deaths are often related to causes of abortion, and can be part of a foetal neonatal mortality syndrome. The following conditions should be considered, and specimens collected according to the disease entity where appropriate: Dystocia Congenital defects Genetic diseases • Maple syrup urine disease (after first

feed) • Inherited congenital myoclonus (signs

present at birth) • Citrullinaemia (born normal; develops

within 5 days of birth) • Mannosidosis (progressive from birth) • Generalised glycogenosis (progressive

from birth) • Cardiomyopathy and woolly haircoat

syndrome (progressive from birth) Environmental diseases • Mineral deficiencies • Selenium deficiency (WMD; stillbirths

occasionally) Infectious diseases

• Bacterial Leptospirosis, Campylobacter

• Viral Pestivirus, Akabane, Palyam • Chlamydial

PERINATAL MORTALITIES IN SHEEP AND GOATS

Investigations should be based on a field necropsy with laboratory assistance to confirm or make a specific diagnosis


In any detailed investigation, there must be prior arrangement with the laboratory, regarding the number & nature of specimens to be examined. Conditions to be considered should include: • Abortion, including congenital infections • Dystocia • Starvation, mismothering or exposure • Low birth weight from maternal

malnutrition during last 2-3 months of pregnancy, with resultant inability of lamb to gain sufficient early feed.

• Delayed colostrum production in ewes suffering nutritional stress in the last month of pregnancy

• Predation • Postparturient infections, including

necrobacillosis, pneumonia and enteritis.

• Nutritional factors, e.g. selenium deficiency, maternal hypocalcaemia, maternal malnutrition (with foetal anoxia from prolonged parturition), overfat ewes

• Clinical conditions in the ewe likely to result in a prolonged parturition (uterine inertia) and resultant foetal hypoxia or anoxia e.g. clover disease, foot abscess, pregnancy toxaemia, poor body condition due to other causes

• Diagnosis Flock history, field investigation, supported by the laboratory. Specimens required Abortion and parturient death

i. Specimens as required for Abortion in sheep.

ii. iii. Post-parturient death iv. Whole lambs or kids submitted

chilled for bacteriological and pathological examination.

PESTIVIRUS INFECTION

Syn: Mucosal disease; mucosal disease virus infection; bovine viral diarrhoea virus Pestivirus infection is associated with embryonic or foetal death, congenital malformations, perinatal mortality, poor growth rates, respiratory disease, non specific immunosuppression and disease with mortality in calfhood or early adult life. Life long carriers of the virus including cases of mucosal disease [MD]) are a consequence of infection in early foetal life (usually before 90 days gestation).

Foetal infection which persists into and through post natal life will have resulted from one of two circumstances: • Infection of a susceptible dam, probably

through contact with a virus carrier, in early (usually 30 100 days) pregnancy. Immune tolerance to the virus is induced; or,

• The dam herself is a persistently viraemic and immuno-tolerant animal.

Diagnosis Clinical signs and pathology. Detection of antigen or isolation of virus from blood clot or tissue. Detection of antibody in precolostral serum of deformed calves. Specimens required Live animal

i. 10 ml of unclotted blood (heparin blood is preferred), submitted chilled for antigen detection. A clot (from 10 ml of blood collected into a plain tube) may be used for antigen detection but offers lower sensitivity.

ii. Serum from clotted blood is preferred for antibody detection.

iii. In cases of respiratory disease, nasal swabs in PBGS for virus isolation.

Notes: • In cases of embryonic mortality or

abortion, serum samples from 10-15 cows may provide evidence of herd infection and allow more selective investigation to confirm pestivirus involvement.

• A newborn calf with congenital

deformities, the product of infection at 120-180 days gestation, may be seropositive. Some stillborn, apparently-normal calves can also be seropositive. Other affected [=viraemic] animals are usually antibody negative. Demonstration of an antibody titre in a live animal usually indicates that the animal is not persistently infected, assuming it is old enough to have lost maternal antibody (> 6 months). However, around 5% of viraemic animals may have antibody to pestivirus.

Dead animal

i. In cases of mucosal disease, perinatal mortality, abortion or respiratory disease, demonstration of virus antigen by ELISA on fresh tissues provides a rapid diagnosis. Preferred specimens are (in order): spleen, lung, mesenteric lymph


node, bowel and parotid salivary gland submitted chilled. One or several of these should be submitted.

ii. Fresh lung, spleen, and kidney may be submitted chilled for virus isolation, especially from cases of respiratory disease where infections are transient and may not be detected by ELISA.

iii. Foetal fluids (pericardial, pleural or peritoneal, in order of preference) should also be collected to test for IgG and for pestivirus antibody of the IgG level is elevated.

Where pestivirus infection and/or mucosal disease has been confirmed, further testing of maternal relatives and enquiry into the history and management of the herd may indicate how or when infection occurred, and what steps can be taken to eliminate the problem. Options available include removal of carrier families and management to ensure the exposure of breeders to carriers before joining.

PHALARIS POISONING Can cause staggers or sudden death. Diagnosis Flock history, clinical findings, histopathology. Specimens required

i. In cases of staggers, specimens as required for ataxia.

ii. In cases of sudden death, there is no specific diagnostic test available. Specimens should be submitted to exclude other possible causes of death.

PHOSPHORUS DEFICIENCY

See Hypophosphataemia'

PHOTOSENSITIZATION This condition is characterised by the occurrence of pruritus, oedema, frequently with subsequent necrosis, jaundice being an inconstant feature. Plants often causing photosensitization include Hypericum perforatum, medics (Medicago spp.), clovers (Trifolium spp.), and Tribulus spp. A number of other plants are incriminated. Other causes of photosensitization are phenothiazine, facial eczema, algae and conditions giving rise to liver damage. See also Protoporphyria (Limousin calves).

Diagnosis Mainly on clinical examination. Specimens required

i. Sections of liver and kidney, submitted in buffered formalin for histopathology.

ii. Plant material for identification. District Agronomists may be able to identify suspect plant material.

PIGLET ANAEMIA Iron deficiency causes a microcytic hypochromic anaemia in sucking piglets. Diagnosis History, clinical signs, haematology, gross pathology. Specimens required Live animal

i. An EDTA blood sample for haematology.

Dead animal

i. The cadaver submitted chilled for bacteriology and histopathology to investigate a differential diagnosis.

PINKEYE

See Ophthalmia'

PLANT POISONING See Poisoning plant

PNEUMONIA Diagnosis Clinical and post mortem examination. Bacteriological, virological, parasitological, histopathological examination. Specimens required Live animal Especially in feedlot or other intensively reared cattle.

i. Nasal swabs in transport medium for virus isolation.

ii. Paired (acute and convalescent) sera for virus serology (for IBR, Pestivirus, PI3 and RSV).

Dead or slaughter animals

i. Portions of lung, pleural fluid and mediastinal lymph nodes, submitted chilled for microbiology.

ii. Portion of affected lung, trachea, etc submitted chilled for virology.


iii. Sections of lung, submitted in buffered formalin for histopathology

iv. In pigs, see porcine pleuropneumonia and porcine enzootic pneumonia (PEP').

v. In goats where retrovirus infection is suspected, serum sample from affected animal submitted chilled for virology. (see Caprine arthritis encephalitis').

POISONING (CHEMICAL)

See also Arsenic, lead, organochlorine and organophosphate, sodium fluoroacetate, strychnine and urea poisoning NB Examination is not undertaken on specimens likely to involve criminal prosecutions (e.g. in cases of suspected malicious poisoning). In such cases, the police or other relevant regulatory authority should be consulted before any action is taken (see Conditions for acceptance of specimens). Most chemical analyses are specific for at best a narrow group of chemicals eg organophosphates. As the analyses tend to be expensive, clients should carefully consider history, clinical findings and pathological findings so that only the most likely chemical is nominated when completing a request for chemical analysis. Where NSW Department of Primary Industries Laboratories are not capable of conducting the requested analysis, samples will be referred at cost to an outside provider selected on the basis of NATA accreditation and value. Diagnosis History, clinical signs, and in some cases, laboratory examination. Confirmation of chemical poisoning depends upon the demonstration of the poison in body tissues and organs. NB • Irritant poisons will cause a

gastroenteritis. • When specific drugs (e.g. drenches,

dips, feeds, additives) are suspected, then material should be submitted as recommended by the manufacturer.

• A limited range of analyses are available on a routine basis.

Specimens required

i. At least 100 g of stomach content, submitted chilled for toxicology.

NB The Specimen Advice Form should request a particular analysis.

ii. Approximately 500 g of suspect material eg feed, soil for chemical analysis.

NB Analysis of this material is usually only requested after poisoning is confirmed and submitters wish to identify the source of the poison.

iii. Sections of liver, kidney and other organs showing lesions, submitted in buffered formalin for histopathology.

iv. Specimens as required to allow a differential diagnosis, e.g. salmonellosis

POISONING (PLANT)

See also Cyanide, nitrate and oxalate poisoning and pyrrolizidine alkaloidosis Diagnosis History (sudden high morbidity/mortality, evidence that suspected toxic plant has been eaten), clinical signs, necropsy findings, If plant poisoning is suspected, the paddock should be inspected to determine the availability of possible toxic plants. A check can then be made at necropsy to determine if these plants are present in the ingesta. Specimens required

i. Sections of liver, kidney and any other organs showing lesions, submitted in buffered formalin for histopathology.

ii. If nervous signs are seen, specimens as required for Nervous disorders.

NB Examination of ingesta is usually difficult and of limited value. It will only be performed for specific plants to which the animals have had access, and provided that specimens of those plants accompany the specimens of ingesta.

IDENTIFICATION OF SUSPECT POISONOUS PLANTS

Advice in the first instance should be sought from the local District Agronomist. When this is not possible, specimens should be submitted as follows Collection of plant specimens All specimens sent should consist of a small branch or portion of the stem, 20 30 cm long, showing the leaves in position together with flowers and/or fruits. Owing to


the tremendous number of plant species, it is difficult, and sometimes impossible to determine specimens from leaves alone.

For small plants and grasses, the whole of the plant should be sent, with the exception of the terrestrial or ground orchids when only the parts above ground should be collected. With small ferns and fern allies, the rhizome (root like structure) is required, while in the tree ferns the scales or hairs at the base of the stalk of the frond are essential for identification. Eucalypts can be difficult to identify without samples of the buds, fruit and juvenile leaves, in addition to the adult foliage. Information on the bark type, the extent of rough bark when present, habit and the habitat are very important.

Labelling of plant specimens Each plant specimens should be numbered and a second set of specimens with corresponding numbers retained. The data and place of collection should be given, as well details of the habit of growth, height, flower colour, habitat, type of soil, and, in the case of trees, a description of the bark. Specimens from other states, which may be unfamiliar to local botanists, particularly require adequate specimen preparation, precise locality details, and notes on the main features of the plants and their habitats. A list of identifying names or information corresponding to the numbers will be reported. Specimens will only be returned on special request. Not more than 12 specimens will be named in one collection, except under special circumstances. The cooperation of inquirers is appreciated in supplying further information or material when rare specimens, or specimens of special interest, have been identified. Storage and despatch of plant specimens Press and dry specimens between newspaper sheets. Fresh specimens should not be forwarded unless particularly requested.

Specimens forwarded for identification should be addressed to: The Director (Attention: Botanical Inquiry Section) National Herbarium Royal Botanic Gardens SYDNEY 2000

POLIOENCEPHALOMALACIA (PEM) Syn: Cerebrocortical necrosis (CCN) See also: Nervous disorders A sporadically occurring, nervous disease principally of sheep and goats, less commonly of cattle and occasionally pigs, with characteristic brain lesions in advanced cases of cerebrocortical necrosis. The disease in ruminants is considered due to either: • production of thiaminases in the rumen

following changes in the ruminal flora, particularly when indigestion occurs after sudden dietary change, or,

• ingestion of thiaminases in plants (e.g. Nardoo, rock fern) or in chemicals (eg. amprolium in poultry feed/ manure).

Diagnosis Clinical signs (typically progressive higher CNS dysfunction with aimless wandering, blindness, head pressing, recumbency, opisthotonus, convulsions and death). Gross pathology and histopathology of brain. Response to 6 10 mg/kg thiamine IV in live affected animals (preferably those not yet recumbent). Differentiate from tetanus, focal symmetrical encephalomalacia/ enterotoxaemia, lead poisoning, listeriosis, salt poisoning, CAE (goats) and copper deficiency induced leukomalacia. Specimens required

i. Whole brain, submitted in buffered formalin for histopathology.

ii. Specimens as required for diagnosis of other nervous disorders.

POLYARTHRITIS

See Arthritis

POMPE'S DISEASE See Generalised glycogenosis

PORCINE COLITIS Syn: Porcine intestinal spirochaetosis (IS), spirochaetal diarrhoea, non-specific colitis


Dd: Trichuriasis, swine dysentery, salmonellosis, yersiniosis, post-weaning colibacillosis, gastric ulcers, porcine proliferative enteropathy (Lawsonia intracellularis infection) Porcine colitis has been attributed to various non-Brachyspira hyodysenteriae spirochaetes and Campylobacter coli. Porcine intestinal spirochaetosis (spirochaetal diarrhoea) Porcine intestinal spirochaetosis appears to be a distinct entity associated with infection by Brachyspira (formerly Serpulina) pilosicoli. It mainly affects weaners 2-6 weeks post weaning but can affect growers, and leads to loose (’wet cement) faeces, which may vary from watery to mucoid. The disease is expressed as fluctuating and cyclical in a herd, persisting a few days, resolving in 7-10 days, or becoming chronic. Lesions are often mild, patchy and focal superficial colitis and possibly typhlitis. There may be no gross lesions, or mild thickening and oedema of the colonic mucosa with localised mucosal haemorrhages. In resolving lesions, conical scales of fibrinonecrotic material may be dislodged from the mucosal surface by rinsing. Histological changes include mucosal oedema, with shallow, scattered mucosal erosions, crypt dilation, and submucosal congestion; spirochaetes may cover an intact epithelium as a false ‘brush border’, and B pilosicoli may be demonstrated in affected mucosa by PCR or culture. Dietary colitis syndrome (non-specific colitis) Dietary colitis syndrome is a recently described condition of rapidly growing pigs, especially those 8-14 weeks of age. It is a diarrhoeal disease of unknown aetiology, in which large-intestinal histological, and sometimes macroscopic, lesions are seen. Diarrhoea may progress from ‘wet cement’ or ‘cow pat’ faeces to watery diarrhoea with dehydration. Lesions include proprial leucocytosis, with variable epithelial necrosis and erosion. It is likely that this syndrome has a variety of causes; in one associated with a pelleted diet, the only change detected is frothy large intestinal contents. Dietary change, poor quality oils and carbohydrates, ad-lib feeding and high density diets may be associated with this condition. Short chain fatty acids (SCFA) can cause reversible changes to colonic epithelium, comprising epithelial cell loss, reduction of goblet cell numbers, and

replacement of lost epithelium by low cuboidal enterocytes. It is possible that high energy grower diets may escape complete digestion in the small intestine, to be fermented in the large intestine, producing abnormally high levels of SCFA. Many cases reported previously may have been undiagnosed infections by B pilosicoli or possibly other weakly beta-haemolytic spirochaetes, or Lawsonia intracellularis. Trichuriasis Trichuriasis can produce an identical macroscopic mucohaemorrhagic colitis and typhlitis to that of swine dysentery. Disease outbreaks can be caused by massive infestations of immature T suis (no T suis ova in faeces), which are not as easily detected at necropsy as the adult worms. Dietary niacin deficiency Dietary niacin deficiency produces severe, persistent diarrhoea, marked weight loss, and a thickening of the colonic mucosa. Histological changes comprise hypertrophy of the colonic mucosa, associated with a mixed leucocytic, mainly lymphocytic, infiltration of the lamina propria. Diagnosis is confirmed by assessment of the diet, which is usually home-mixed, with corn as the major component. Diagnosis Clinical signs (weaner ill-thrift associated with diarrhoea or mucoid faeces), post-mortem findings, bacteriology, ,parasitology, histopathology, , Confirmation of intestinal spirochaetosis due to B pilosicoli; diagnosis of non-specific colitis or niacin deficiency by dietary history and elimination of infectious agents; confirmation of T suis by parasitological examination; elimination of infectious agents associated with other syndromes or diseases with similar presenting signs (L intracellularis; B hyodysenteriae, Salmonella spp, E coli, Yersinia spp); elimination of other possible causes of ill thrift (e.g. gastric ulceration) Specimens required

i. Sections of affected intestine, submitted in buffered formalin for histopathology.

ii. Impression smears of affected intestinal mucosa for bacteriology.

iii. Fresh colon or caecum or faeces (live animals) for detection of Brachyspira pilosicoli by PCR.

iv. Sections of affected intestine (especially ileum and colon), submitted chilled for bacteriology and PCR for differential diagnosis.


NB. PCR tests are available for three enteric pathogens: Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis.

PORCINE ENTEROVIRUS ENCEPHALOMYELITIS

Syn: Talfan disease A sporadic encephalomyelitis usually affecting pigs from 14 days to weaning, but also seen in older pigs. Low morbidity and mortality. Typically causes posterior paresis and ataxia; may progress to all legs, with recumbency and paddling. Tremors and excitement may occur. Diagnosis Histopathology, virus isolation. Specimens required

i. Brain fixed in neutral buffered formalin for histopathology.

ii. Sample of fresh, chilled brain for virus isolation.

iii. Other samples to eliminate possible differential diagnoses: e.g. bacterial meningoencephalitis; oedema disease; spinal cord abscess or other lesions - including brain and small intestine for bacteriology; fixed spinal cord for histopathology.

PORCINE ENZOOTIC PNEUMONIA

Syn: Mycoplasmal pneumonia Diagnosis Identification of Mycoplasma hyopneumoniae infection in a herd by ELISA serology, or in individual animals by PCR on affected lung. ELISA serology can provide a profile of the M. hyopneumoniae infection status of the herd. Blood samples may be collected from the abattoir or from a subpopulation of animals in the piggery. Seroconversion after natural challenge can take 9 weeks, so targeting older pigs for serological profiling is more cost-efficient. Carriage in the live animal can be detected by PCR from nasal swabs. However, nasal excretion is variable, so a negative swab PCR on a single occasion is of limited diagnostic value. Specimens required Live pigs:

i. Serum (or heparin plasma) samples from affected pigs and their cohorts for ELISA. Since this is a herd disease, the results should be

looked at in this light rather than on an individual animal basis. For this reason, it is recommended that about 30-40 sera be submitted on each occasion.

ii. Nasal swab material collected into sterile PBS for PCR (if required; may be cost prohibitive)

Slaughter pigs:

i. Fresh lung samples submitted chilled or frozen for PCR. For PCR, samples taken from the affected site and measuring 1 cm³ is sufficient.

ii. Fixed affected lung for histopathology

iii. Culture is expensive and not routinely available. For samples where an isolate is required (e.g. for intensive investigation or research), these must be fresh and reach the laboratory within a few hrs of slaughter, and preferably come from well developed lesions.

PORCINE PLEUROPNEUMONIA

Actinobacillus pleuropneumoniae (formerly Haemophilus pleuropneumoniae) causes a fatal pleuropneumonia or non fatal chronic respiratory disease. At least 7 serotypes (1, 2, 3, 5, 7, 11, 12) occur in Australia isolates are currently being serotyped at the Animal Research Institute, Yeerongpilly, Qld. Diagnosis Gross pathology, bacteriology and histopathology. Focal and diffuse gross lesions of reddish black colour with or without pleurisy are suggestive. Concurrent infection with Pasteurella multocida is not uncommon. Specimens required

i. Fresh affected lung and bronchial lymph node for culture.

ii. Fixed affected lung in buffered formalin for histopathology.

PORCINE INTESTINAL HAEMORRHAGE SYNDROME

Syn; Intestinal volvulus, torsion of mesentery, whey bloat, colonic bloat, redgut, intestinal venous infarction. Diagnosis History, clinical signs, post-mortem findings. Predisposing factors include transportation, whey feeding. Clinical signs include abdominal distension and pain, and dyspnoea, but most cases are simply found


dead. Usually but not always associated with palpable torsion of the root of the mesentery that causes intestinal venous infarction. The proximal duodenum, which receives its blood supply from the gastroduodenal vessels, is unaffected.

PORCINE MYOCARDITIS (PMC) Porcine myocarditis (PMC) is a recently recognised condition of uncertain aetiology apparently affecting pigs in only two piggeries in NSW. . It is believed that the syndrome is due to a viral infection predominantly, if not exclusively, occurring in utero. PMC is a notifiable disease. Fees for tests undertaken to confirm or exclude a diagnosis of porcine myocarditis are paid by NSW Department of Primary Industries. PMC causes an increase in stillbirths and pre-weaning mortalities. There may be a variable increase in the incidence of mummified foetuses. The gross pathological changes consist of small pale areas in the myocardium. There is often evidence of cardiac enlargement and an increase in the volume of body fluids, consistent with congestive heart failure. Histologically there is a non-suppurative myocarditis Further information can be obtained from the NSW Department of Primary Industries’ website at http://www.agric.nsw.gov.au/reader/an-health/pmc-qa.htm. Diagnosis History, clinical signs, postmortem findings, histopathology. Specimens required

iii. Chilled stillborn piglets for necropsy, virology and histopathology.

iv. Fresh heart, lung and body fluid collected separately and aseptically, submitted chilled for virology.

v. Sections of heart, liver, kidney, lung, spleen and whole brain in buffered formalin for histopathology.

vi. Sections of liver, kidney, spleen, lung and heart blood submitted chilled for bacteriology.

PORCINE PROLIFERATIVE ENTEROPATHY Porcine Proliferative Enteropathy

Syn: Porcine intestinal adenomatosis (PIA), proliferative haemorrhagic enteropathy (PHE), ileitis, regional ileitis (RI), necrotic enteritis Diagnosis Clinical signs, necropsy findings, histopathological examination of affected intestine, and bacteriological examination, Lawsonia intracellularis is the aetiological agent. It is an intracellular organism and requires rat enterocyte cell lines for maintenance. The uncomplicated proliferation of mucosa due to L. intracellularis is referred to as PIA, while additional changes that may be superimposed on this lesion may result in regional ileitis (muscular hypertrophy), necrotic enteritis (coagulative necrosis) or PHE (haemorrhage). It is likely that necrotic enteritis represents secondary bacterial infection superimposed on a primary L.intracellularis lesion. PIA and RI are associated with weaner ill thrift, but PIA can occur in all ages. PHE is associated with sudden death in finisher and breeder pigs, while necrotic enteritis can cause mortalities in grower pigs. Specimens required

i. Sections of affected intestine (especially ileum and colon), submitted chilled for bacteriology for differential diagnosis.

ii. Sections of affected intestine, submitted in buffered formalin for histopathology

iii. Impression smears of affected intestinal mucosa for bacteriology (MZN stain)

iv. Fresh intestinal contents or faeces for PCR

NB. PCR tests are available for three enteric pathogens: Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis.

PORCINE STRESS SYNDROME (PSS)

Syn: Pale soft exudative pork [PSE], malignant hyperthermia An inherited neuromuscular disease of grower pigs, characterised by a susceptibility to stress-induced collapse and death, particularly of Landrace and Pietrain breeds.

http://www.agric.nsw.gov.au/reader/an-health/pmc-qa.htm


It is caused by a recessive mutation of the HAL gene on chromosome 6 (susceptibility to PSS was initially determined by reaction to the anaesthetic halothane). Animals can be genotyped as stress-resistant (NN), stress-carrier (Nn), or stress-positive (PSS-positive) (nn). A large proportion of the stress-carrier and PSS-positive animals produce carcases with inferior muscle quality. Diagnosis Clinical history and signs, post mortem findings (60-70% of pigs may show gross muscle lesions). Laboratory examination of necropsy specimens cannot assist the field diagnosis. A DNA test for PSS genotyping patented by the University of Toronto is available at commercial laboratories in North America. It is not routinely available in Australia.

PREGNANCY TOXAEMIA

Diagnosis Generally on clinical grounds and demonstration of ketones in urine. Differentiate from hypocalcaemia, metabolic or starvation ketosis, secondary ketosis due to anorexia from other causes and polioencephalomalacia. Specimens required Samples are only to eliminate other possible causes:

i. Sections of liver, kidney, adrenal gland and brain submitted in buffered formalin for histopathology.

ii. At least 2 ml of serum, free of cells and haemolysis, submitted chilled for calcium, magnesium and beta hydroxybutyrate (ketone) estimation.

NB Urine examination for ketones should be carried out in the field.

PSEUDOCOWPOX See Teat lesions

PROTOPORPHYRIA (Limousin and Blonde d'Aquitaine cattle) An acute photosensitivity evident from birth and characterised by alopecia, ulceration and scarring of the pinnae, nares and possibly along the midline. Diagnosis Clinical signs and history; confirmed by DNA analysis.

Specimens required See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-disease-syndrome/diseases_of_livestock/dpi-samp-coll-prot.pdf



PYELONEPHRITIS (BOVINE)

Diagnosis Clinical signs mainly of abnormal urine and recovery of Corynebacterium renale from urine or kidney tissue. Specimens required

i. Sample of urine collected into a sterile container, submitted chilled for bacteriology.

ii. Sections of kidney lesion submitted chilled for bacteriology.

iii. Sections of kidney and other parts of urogenital tract with lesions, submitted in buffered formalin for histopathology.

PYOGENIC INFECTIONS

Diagnosis Recovery of pyogenic organisms from abscesses and infected organs. Specimens required

i. At least four (4) smears from periphery of the lesion for bacteriology.

ii. Portions of affected tissue containing lesions, submitted chilled for bacteriology.

iii. Section of lesion, including the margin, submitted in buffered formalin for histopathology.

PYRROLIZIDINE ALKALOIDOSIS

See also Poisoning, plant' An intoxication of grazing sheep, cattle, pigs and horses when hepatotoxic pyrrolizidine alkaloids are ingested from plants of the genera Senecio, Crotalaria, Heliotropium, Amsinckia, Echium and others. In sheep, the condition may be complicated by chronic copper poisoning. Diagnosis History of access to hepatotoxic plants, clinical and post mortem findings, histopathology.



Specimens required i. Sections of liver and kidney,

submitted in buffered formalin for histopathology.

ii. In cases of suspected copper poisoning, at least 30 g of liver and kidney, in separate copper free jars, submitted chilled for copper estimations.

Q FEVER

Q Fever is usually an asymptomatic infection of animals caused by Coxiella burnetii, but it can cause abortion in sheep and goats and infections in man. Diagnosis The complement fixation test is not a reliable test for Q Fever. At present there is no routine test available for diagnosis. The CFT is used only for export certification. Specimens required

i. For abortions, those required for abortion in sheep and goats.

ii. For export certification, serum sample forwarded chilled for CFT.

RED GUT IN SHEEP

Syn: Intestinal volvulus, torsion of mesentery, colonic bloat, intestinal venous infarction An acute haemorrhagic enterocolitis occurring in sheep grazing some lucerne or clover pastures, or other fresh, young green feed. Some cases show severe abdominal distension, with rapid death. Post mortem findings can include distension of the small and large intestine, severe haemorrhage and congestion of intestinal mucosa and blood stained intestinal contents. There may be rapid autolysis of the carcase. Diagnosis Post mortem findings, especially displacement and/or torsion of the caecum and colon. Differential diagnosis from enterotoxaemia. NB. The ventral necropsy approach is essential for the displacement and/or torsion to be seen. Specimens required

i. As for enterotoxaemia, together with ii. Sections of affected intestine,


RHODOCOCCUS EQUI PNEUMONIA IN HORSES

Syn: Rattles Diagnosis History, clinical signs, and post mortem findings. Recovery of Rhodococcus equi from lungs and pulmonary lymph nodes. Specimens required

i. Portions of affected lung and lymph nodes submitted chilled for bacteriology.

ii. Swabs of lung abscesses in Amies Charcoal Transport medium, submitted chilled for bacteriology.

iii. Portion of affected lung submitted fixed in buffered formalin for histopathology.

RHODOCOCCUS EQUI LYMPHADENITIS IN PIGS

Characterised by oval or spherical lesions in lymph nodes which are encapsulated and easily enucleated. It is often difficult to differentiate from tuberculosis in swine. Diagnosis Recovery of Rhodococcus equi from lesions, histopathology. Specimens required

i. Affected lymph nodes, submitted chilled for bacteriology.

ii. Affected lymph nodes, in buffered formalin for histopathology.

RINGWORM (DERMATOMYCOSIS)

See fungal infections

ROCK FERN POISONING Cheilanthes tenuifolia (rock fern) can cause haemorrhages through out the body with hepatic necrosis. Also associated with staggers or ataxia in sheep. Diagnosis History, clinical signs, post mortem findings, histopathology. Specimens required Live animal

i. 5 ml of blood in EDTA for haematology.

ii. Blood films for haematology (refer to Haematology in first section of this manual).

Dead animal


i. Sections of liver, spleen and kidney submitted in buffered formalin for histopathology.

ii. Specimens to differentiate from other causes of haemorrhage and septicaemia, based on post mortem findings.

Ataxic animal Specimens as required for Ataxia.

ROTAVIRUS INFECTION See also Scouring Causes diarrhoea in young animals. Diagnosis Demonstration of either virus particles in faeces by electron microscopy or soluble antigen by immunodiffusion, latex agglutination or ELISA. Specimens required

i. 20 g of faeces or intestinal contents, submitted chilled for electron microscopy or antigen detection.

ii. Specimens as required for the differential diagnosis of causes of scouring.

RYEGRASS STAGGERS

Syn; Perennial ryegrass staggers Associated with fungal neurotoxins produced by Acremonium loliae in the leaf, stem and seed of perennial ryegrass, which are toxic to sheep and cattle. Outbreaks more common in summer and autumn, and with close grazing of limited available feed. Diagnosis History, clinical signs, histopathology. This syndrome can be diagnosed by clinical signs in the field. Specimens required

i. Brain, submitted in buffered formalin for histopathology.

SALMONELLOSIS

Usually occurs as one of three major syndromes: • Peracute septicaemia seen mostly in

young animals. • Acute enteric form most commonly

seen in adult animals. • Chronic enteric form is common in

pigs, and occurs in cattle. There is persistent diarrhoea, severe emaciation and intermittent fever.

Diagnosis Clinical signs, necropsy findings, recovery of Salmonella from heart blood, spleen, liver, bile, mesenteric lymph nodes and intestinal contents in both septicaemic and acute forms. Serology. In the chronic form, the bacteria may be recovered from the intestinal lesions and less frequently from other viscera. Specimens required Live animal

i. Serum for Salmonella Typhimurium and Salmonella Dublin SAT serology in cattle and sheep.

ii. Faecal sample submitted chilled for bacteriology.

Dead animal:

i. Portion of liver, kidney, lung, bile, spleen, mesenteric lymph nodes and small intestinal contents in separate sterile containers, submitted chilled for bacteriology.

ii. Sections of liver, kidney, lung, spleen and small and large intestine, submitted in buffered formalin for histopathology.

SALT POISONING IN PIGS

Diagnosis History, clinical signs, histopathology. Specimens required

i. Brain, submitted in buffered formalin for histopathology.

ii. Specimens as required for the differential diagnosis of causes of nervous disorders.

SARCOSPORIDIOSIS

Infection with protozoa of the genus Sarcocystis can cause anaemia, ill thrift and myopathy in experimental infections in sheep and an acute febrile disease in calves. Diagnosis Histopathology. Specimens required

i. Sections of cardiac and skeletal muscle, submitted in buffered formalin for histopathology.

SCABBY MOUTH

See Contagious pustular dermatitis'


SCOURING Investigations into causes of scouring in animals should take into consideration: • Age of affected animal9s) • Whether sudden or gradual in onset • Condition of the animal • Numbers affected • Intensity of grazing or housing Diagnosis History, clinical examination, bacteriology, parasitology, histopathology, virology. Specimens required Depending on the age of the animal and the results of the clinical examination, specimens should be forwarded as required for the following diseases: • Colibacillosis, especially in neonates • Rotavirus infection in young animals • Cryptosporidiosis in young animals • Salmonellosis • Pestivirus (Mucosal disease) in cattle, especially < 2 yrs • Parasitism • Johne's disease in cattle, sheep and goats • Yersiniosis • Poisoning, plant or chemical • Proliferative enteropathy in pigs • Swine dysentery in pigs • Enteritis in pigs, calves, dogs associated with Campylobacter jejuni • Copper or selenium deficiency in ruminants • Coccidiosis • Hypomagnesaemia in milk fed calves In general, the following specimens should be submitted. Live animal

i. In cases of suspected enteric or systemic infections at least 30 g of faeces, submitted chilled for bacteriology or virology.

ii. At least 30 g of faeces from 10 to 20 animals in the flock, submitted chilled for parasitology.

iii. Blood or serum samples as required for virology.

Dead animal:

i. Sections of small and large intestine, especially areas showing lesions, submitted chilled for bacteriology.

ii. Sections of liver, kidney, lung and spleen submitted chilled for bacteriology in cases of suspected enteric or systemic infection.

iii. Sections of liver, kidney, spleen, selected sites in the gastrointestinal

tract, cardiac and skeletal muscle in buffered formalin for histopathology.

iv. Specimens as required for specific diseases, e.g. Johne's disease, Pestivirus, arsenic poisoning, if these are suspected.

v. Abomasum and small intestine submitted chilled for total worm count.

SELENIUM DEFICIENCY

See Muscular degeneration, nutritional'

SEMEN EXAMINATION In investigations of infertility, semen examination should be considered after a careful clinical examination. Laboratory examination can be considered: • When the field veterinarian is confident

the semen sample is typical of the semen to be obtained, and

• There is an obvious deficiency with regard to density, motility, wave motion, or

• There is evidence of inflammatory products in the sample, i.e. polymorphs, clotted semen.

Veterinarians should conduct the initial examination in the field. Specimens required

i. For morphology, air dried semen smears.

ii. For live/dead sperm examination, smears stained with freshly prepared nigrosin eosin.

iii. For bacteriology, semen collected aseptically and submitted in sterile containers.

NB. Semen collected using an artificial vagina is not suitable for bacteriology.

SERUM ENZYMOLOGY NSW Department of Primary Industries refers all clinical chemistry testing to a specialist laboratory. Test results are incorporated into the laboratory report which includes normal ranges supplied by the testing laboratory. Serum enzymology is usually undertaken as part of a package of tests eg liver profile. renal profile, ruminant metabolic profile. The most frequently requested serum enzymes are: • Aspartate aminotransferase (AST)

also known as Glutamic oxaloacetic transaminase (GOT)


• Creatine kinase (CK), also known as creatine phosphokinase (CPK)

• Gamma glutamyltransferase (GGT) • Glutamate dehydrogenase (GLDH) Specimens required i. 10 ml of blood, clotted in vacuum

tubes.

NB If blood cannot be sent to the laboratory overnight, it should be allowed to clot and the serum poured off; it should be frozen if the delay will be greater than 24 hrs. Approximately 2ml of serum per animal should be provided. It must be free of haemolysis.

Note that the stability of these enzymes is variable, as is their half life in the blood: Stability in serum sample Half life in blood after insultAST Intermediate WeeksCK Unstable DaysGGT Very stable -GLDH

Intermediate Weeks

Applications and Interpretation of Results in Sheep and Cattle Enzyme Major source Suggested normal serum levels (IU/L)AST Liver, muscle < 100CK Muscle, CNS < 300GGT Liver < 80 sheep < 50 cattleGLDH Liver < 20 sheep < 50 cattle

Typical disease syndromes with elevation of serum enzymes Enzyme Disease process AST Sustained necrosis of hepatic and/or muscular tissue (skeletal or cardiac) CK Sustained necrosis of muscular tissue (eg. WMD) and/or CNS (eg. PE) GGT Bile duct epithelium damage, cholestasis incl cases with biliary hyperplasia (eg.

chronic fascioliasis; pyrrolizidine alkaloidosis) or acute hepatic necrosis GLDH Hepatocellular damage (rises rapidly in acute hepatic necrosis)

SODIUM FLUOROACETATE (1080) POISONING

Causes neurological signs in canines and cardiac signs in ruminants. Continued exposure to low doses can cause myocardial degeneration in cattle and sheep. Diagnosis History, elimination of other causes, detection of poisoned material, e.g. carrot in the stomach contents; chemical analysis confirms fluoroacetate in stomach contents. NSW Department of Primary Industries does not analyse for fluoroacetate. Specimens will be sent to a specialist laboratory on request. Specimens required

i. Approximately 100ml of stomach contents..

ii. In cases of suspected poisoning in sheep and cattle, sections of myocardium, liver and lung


SPORADIC BOVINE ENCEPHALOMYELITIS (SBE)

See also Chlamydial infections' Diagnosis History, clinical signs, histopathology, serology. Specimens required

i. Paired serum samples from individually identified and observed animals collected in the clinical and convalescent phase (2 to 3 weeks apart), submitted chilled or frozen for chlamydial serology.

ii. Portions of liver and spleen collected aseptically, submitted chilled for bacteriology.

iii. Brain and sections of liver, spleen, kidney and other organs with serositis, submitted in buffered formalin for histopathology.


iv. Heparinised or citrated blood from acute cases for chlamydial isolation and FAT.

ST GEORGE DISEASE

Caused by ingestion of desert riceflower (Pimelea simplex). Diagnosis History of access to P simplex, clinical signs. Specimens required

i. Sections of liver, kidney, spleen, heart and lung, submitted in buffered formalin for histopathology.

STRANGLES IN HORSES

Strangles is a notifiable diseases in NSW. Submission of appropriate specimens accompanied by a Specimen Submission Form requesting exclusion of strangles is suitable notification. There are no laboratory fees for tests taken to exclude strangles.

Diagnosis Clinical findings, isolation of Streptococcus equi from lesions. Specimens required

i. Pus samples or swabs taken from lesions and upper respiratory tract, submitted chilled in Amies charcoal transport medium for bacteriology.

STREPTOCOCCUS SUIS INFECTION IN PIGS

Streptococcus suis is associated with meningitis, septicaemia, polyarthritis, endocarditis and bronchopneumonia in young pigs. At least 28 serotypes have been identified. Disease in Australian pigs has been associated with serotypes 1,2,3,7 and 9. Diagnosis Isolation of S suis from affected tissues (brain, joints, lung, liver, kidney, spleen). Specimens required

i. Fresh chilled portions of brain and/or lung, liver, kidney, spleen

ii. Sections of brain, liver, kidney in buffered formalin for histopathology

Serotyping of S suis isolates is not routinely available.

STRYCHNINE POISONING Diagnosis

Clinical signs, confirmation by detection of strychnine in ingesta by chemical analysis. NSW Department of Primary Industries does not analyse for strychnine. Specimens will be sent to a specialist laboratory on request. Specimens required

i. Approximately 100ml of stomach contents.

SWAINSONA POISONING

See also Poisoning, plant' Diagnosis History, clinical signs, histopathology. Specimens required

i. Brain and spinal cord, submitted in buffered formalin for histopathology.

ii. Sections of liver, kidney and lymph nodes, submitted in buffered formalin for histopathology.

SWINE DYSENTERY

Syn: Brachyspira hyodysenteriae infectionDD: Lawsonia intracellularis (proliferative enteritis/ileitis), Brachyspira pilosicoli (intestinal spirochaetosis; spirochaetal colitis), Non-specific (dietary) colitis; less commonly: Salmonella, Whipworm (Trichuriasis), Gastric ulcers. Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) is a relatively common pathogen with a severe impact on production. Swine dysentery can occur in any age (suckers - breeders) but is most common in 15-70 kg grower pigs (5-20 weeks). It may be less common in weaners if medication for respiratory diseases is used. Clinical expression is normally low mortality/ high morbidity, and affected pigs can vary from the classical mucohaemorrhagic diarrhoea - various amounts blood and mucus, or faeces varying from soft, grey to light brown to dark. Severe cases can show watery faeces, with blood and mucus, and stained hindquarters. Swine dysentery may be difficult to detect in straw-based shelters. A less severe, chronic form of loose, dark faeces ("black scour") is associated with intermittent medication. History is typically one of depressed growth rate, and partial loss of appetite that further decreases growth. There is gradual spread in group, and increased incidence of "slab


sided" pigs (hollow in flanks). Abattoir inspection may reveal an index case through evidence of an accumulation of blood-tinged and jelly-like material in the large intestinal wall and between the coils, linked to a history of ‘ordinary’ growth rate. Pathology affects proximal large intestine, causing a fibrinohaemorrhagic colitis and typhlitis. This is often severe and extensive, with erosions, fresh blood and mucus on necropsy. Epithelial hyperplasia is a feature. Diagnosis Gross and histopathology. Smears of affected intestinal mucosa. Identification of Brachyspira hyodysenteriae infection in individual animals by PCR on affected intestine or faeces. Culture of affected intestine or faeces. Specimens required Live pigs

i. Faeces for PCR ii. Faeces for culture if isolation is

required (e.g. for antibiotic sensitivity). It is preferable to advise the laboratory so special selective media can be available.

NB: PCR are available for Lawsonia intracellularis and Brachyspira pilosicoli. Serology is available to exclude Lawsonia, if clinical signs have been evident for over one week. Dead pigs

i. Fresh, chilled portion of affected gut for smears, PCR and possible culture.

ii. Fixed affected intestinal section in buffered formalin for histopathology

TAPEWORM INFESTATION IN SHEEP

Occasionally suspected of causing ill thrift and scouring in sheep under poor nutritional conditions. Diagnosis Response to treatment, must be differentiated from other causes of scouring. Specimens required The condition should be diagnosed in the field.

TEAT LESIONS Includes Pseudocowpox, herpes mammilitis virus'

Can be caused by infection by paravaccinia virus (pseudocowpox), bovine herpes mammilitis virus, bacterial infections, chapping and photosensitisation. Diagnosis Clinical signs, demonstration of virus by electron microscopy, bacteriology. Specimens required

i. Scabs with underlying tissue, and necrotic debris chilled for virology (electron microscopy examination) and bacteriology.

ii. Biopsy of lesion in buffered formalin for histopathology

TETANUS

Diagnosis History, clinical signs, , elimination of other causes including polioencephalomalacia, focal symmetrical encephalomalacia, strychnine poisoning. Specimens required There is no routine diagnostic test for tetanus. Specimens should be forwarded to eliminate other causes.

TICK FEVER OF CATTLE Includes Anaplasmosis, babesiosis Tick fevers are notifiable diseases in NSW. Submission of appropriate specimens accompanied by a Specimen Submission Form requesting exclusion of tick fever is suitable notification. There are no laboratory fees for tests taken to exclude tick fever. Diagnosis Clinical signs, demonstration of organisms in blood or organ smears. In the Tick Quarantine Area, tick fever should be suspected in animals showing haemoglobinuria, elevated temperature, jaundice and anaemia. Babesia bovis (argentina) infections often cause nervous symptoms, followed by coma and death. Specimens required Live animal

i. Thick blood films air dried, avoiding exposure to heat or direct sunlight, for parasitology.

ii. Conventional unstained thin blood smears for haematology. (see Specimens for Haematology).


iii. Blood collected into EDTA, submitted chilled for parasitology and haematology.

iv. At least 2 ml of serum, submitted chilled for serology.

v. Urine sample, submitted chilled for bacteriology and parasitology.

Dead animal

i. Blood films, air dried for parasitology.

ii. Impression smears from the kidney, heart, liver and brain for parasitology.

iii. Brain squash preparation from the grey matter of the cerebral cortex, prepared by crushing a section of the cerebral grey matter the size of a match head between 2 slides and spreading lightly.

iv. Sections of kidney, heart, liver, brain and spleen, submitted chilled for bacteriology and parasitology.

v. Sections of kidney, heart, liver, spleen and brain, submitted in buffered formalin for histopathology.

TOXAEMIC JAUNDICE

See Copper poisoning

TOXOPLASMOSIS IN CATS The cat is the definitive host of the coccidian parasite Toxoplasma gondii, with sexual stages in the gut resulting in oocysts shed in faeces. Clinical disease is not common in cats. Except initially, oocyst excretion is sporadic, and in small numbers. The asexual proliferative stages of Toxoplasma gondii with formation of tissue cysts, cause disease in a wide range of intermediate animal hosts (including humans) that are infected by

• Consuming sporulated T gondii oocysts shed by cats,

• Consuming tissues of other intermediate hosts containing T gondii cysts, or

• Transplacentally. Oocysts of Toxoplasma gondii are highly infectious to humans and animals. There is no routine serological test available. Serological tests are not reliable in cats. Faecal examination will only be performed in special circumstances after prior arrangements have been made with the laboratory. Such examinations pose a high

risk to laboratory workers. In instances where clinical disease requires confirmation in the dead animal, fixed sections in buffered formalin of small intestine, liver, kidney, heart, muscle, spleen, and brain should be submitted for histopathology.

TOXOPLASMOSIS IN SHEEP AND GOATS

Abortion and perinatal loss in both sheep and goats is caused by the asexual proliferative stages of Toxoplasma gondii, with formation of tissue cysts. Infection of the dam can be by sporulated T gondii oocysts from cats, or by T gondii cysts in infected intermediate-host tissues (eg infected placenta). Diagnosis Typical lesions in placenta and foetal brain (necrogranulomas in brain). Serology by Latex test. Specimens required

i. Those required for abortion in sheep (see 'Abortion in Sheep').

NB Foetal membranes must be submitted if toxoplasmosis is to be diagnosed. Serological titres of 4 or more in the Latex test are considered positive.

TOXOPLASMOSIS IN OTHER ANIMALS

The asexual proliferative stages of Toxoplasma gondii with formation of tissue cysts, cause disease in a wide range of intermediate animal hosts (including humans) that are infected by

• Consuming sporulated T gondii oocysts shed by cats,

• Consuming tissues of other intermediate hosts containing T gondii cysts, or

• Transplacentally. Diagnosis Clinical signs, histopathology. Specimens required

i. Sections of liver, kidney, heart, spleen, muscle and brain, submitted in buffered formalin for histopathology.

There are no routine serological tests available for animals other than sheep and goats.


TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY (TSE)

Syn: Prion disease, bovine spongiform encephalopathy (BSE), scrapie in sheep and goats The National TSE Surveillance Program (NTSESP) (http://www.animalhealthaustralia.com.au/aahc/programs/adsp/tsefap/tsefap_home.cfm) is a national program jointly funded by government and industry to demonstrate Australia’s ongoing freedom from BSE and scrapie, and to provide early detection of those diseases should they occur. The NTSESP provides payments for producers, private veterinarians and government agencies for the submission of specimens for laboratory testing from adult cattle and sheep with progressive neurological disease. NTSESP submissions must be accompanied by: • A completed NTSESP Clinical History

and Post-mortem Report Form. • A completed Specimen Submission

Form Diagnosis Clinical signs of progressive neurological disease, histopathology. Immunochemistry is used to specifically identify accumulated abnormal prion protein (PrP), usually in the brain and cord. Specimens required

i. Brain and cord in buffered formalin for histopathology.

ii. Fresh, chilled cervical spinal cord* for detection of prion protein.

*It is prudent to routinely collect at least a sample of fresh, unfixed cervical spinal from all cases of progressive neurological disease in any animal species, for testing for PrP if required.

TRICHOMONIASIS OF CATTLE A contagious venereal disease of cattle causing infertility, abortion and pyometron, caused by the protozoon Tritrichomonas foetus. Trichomoniasis is a notifiable disease in NSW. Submission of appropriate specimens accompanied by a Specimen Submission Form requesting exclusion of trichomoniasis is suitable notification. There are no laboratory fees for tests taken to exclude trichomoniasis.

Diagnosis Herd history of infertility and abortion, demonstration of T foetus. Specimens required Abortion

i. Specimens as required for abortion in cattle. See 'Abortion in Cattle'.

Herd infertility

i. From cows, vaginal or uterine exudates of vaginal mucus from selected females taken 10 to 20 days after service submitted in selective transport medium for T. foetus.

ii. From bulls, preputial scrapings submitted unchilled in selective transport medium for T foetus (InPouch TF). See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/02-in-pouch-guide.pdf

NB Selective transport medium for T. foetus (InPouch TF) together with instructions for the collection and despatch of specimens will be forwarded from the laboratory upon request. See http://www.dpi.nsw.gov.au/agriculture/vetmanual/specimens-by-discipline/bacteriology/02-bull-vd-coll.pdf

TUBERCULOSIS Diagnosis Clinical examination, tuberculin test, necropsy findings, recognition and/or recovery of pathogenic mycobacteria from tissues, histopathology. Tuberculosis is a notifiable disease in NSW. Submission of appropriate specimens accompanied by a Specimen Submission Form requesting exclusion of tuberculosis is suitable notification. There are no laboratory fees for tests taken to exclude tuberculosis. When required, specimens for culture of mycobacteria will be submitted to the Australian Reference Laboratory in Western Australia. Specimens required Suspect TB lesions

i. Portions of lesions unpreserved in sterile sealed jars, submitted chilled for bacteriology.

If considerable delays (> 3 d) will occur before culture, freeze fresh tissues (and prevent from thawing during transport).

http://www.animalhealthaustralia.com.au/aahc/programs/adsp/tsefap/tsefap_home.cfm

http://www.dpi.nsw.gov.au/__data/assets/pdf_file/34603/02-in-pouch-guide.pdf



ii. Portions of lesions, submitted in buffered formalin for histopathology.

Reactor cattle

i. In the case of tuberculin reactor cattle, the following lymph nodes should be collected as aseptically as possible in separate sterile containers, and submitted chilled for culture:

Essential • L & R medial retropharyngeal LN (also

known as suprapharyngeal LN) • L & R bronchial LN • Cranial and caudal mediastinal LN • L & R Supramammary LN (in cows) Desirable • L & R lateral retropharyngeal LN (also

known as atlantal LN) • L & R mandibular LN • L & R parotid LN • Mesenteric LN • L & R internal iliac LN It is important that the property of origin be clearly identified, either by name or tail tag, or tattoo in the case of pigs. NB. Suspect tuberculosis specimens must be packed and despatched separately from other specimens. As organisms responsible for tuberculosis in livestock are pathogenic for man, care should be taken in collecting samples. Specimens should be forwarded in such a manner that they will offer no risk of infection to persons handling the containers in transit or on arrival at the laboratory. NB. Tissues are cultured for 12 weeks before being classed as negative. Positive cultures may appear at approx 8 weeks (possibly earlier in lesioned tissues with massive infection), and can be confirmed either as Mycobacterium bovis or not Mycobacterium bovis by monoclonal dot blot in a matter of days, providing there are sufficient colonies for testing.

ULCERATIVE SPIROCHAETOSIS IN PIGS

Diagnosis Clinical signs, bacteriology, histopathology. Specimens required

i. Smears from skin lesions.

ii. A biopsy from the affected area, in buffered formalin for histopathology.

ULCERS, GASTRIC IN PIGS

Haemorrhage from an ulcerated of the pars oesophagea with haemorrhage is an important cause of anaemia and sudden death in grower and finisher pigs. Ad lib feeding, stress and finely ground grains increase the prevalence of lesions. Diagnosis Gross pathology is usually sufficient to confirm the diagnosis.

UREA POISONING Diagnosis History of access to urea, clinical signs of ataxia, respiratory distress, coma. Specimens required There are no routine laboratory procedures available to diagnose urea poisoning.

URINE EXAMINATION Urine examinations should be performed in the field using the commercially available dip stick kits. The results obtained should be interpreted according to the instructions with the kit. The field veterinarian should then consider diagnostic possibilities according to the results of the urine examination and make a field diagnosis or send the appropriate specimens. Proteinuria • following violent exercise or stress • nephritis • haematuria from any cause • nephrosis, especially if due to chemical

poisoning • inflammation of the postrenal urinary

tract • chronic passive congestion of the

kidney due to a number of conditions including cardiac and hepatic diseases and fevers.

Glycosuria Large quantities of ketone bodies may depress the colour development and result in a false negative reaction. • diabetes • brain lesions including tumours,

haemorrhage, encephalitis, • enterotoxaemia in sheep, also

sometimes in listeriosis and botulism

Acetonuria (Ketonuria)


Occurs in any clinical condition associated with deficient carbohydrate metabolism, including: • ketosis and pregnancy toxaemia in

ruminants • starvation in pregnancy, lactation,

anorexia • diabetes mellitus • acidosis • prolonged vomiting and diarrhoea • febrile and cachexic diseases • after ether or chloroform anaesthesia

Haematuria, haemoglobinuria • neoplasia of the urinary tract • urolithiasis • infectious diseases, including

babesiosis, leptospirosis • chronic copper poisoning • postparturient haemoglobinuria • sulphonamide treatment • plant poisoning, including ranunculas,

turnips, privet, broom • azoturia (myoglobinuria) • acute nephritis, nephrosis, renal

infarcts. Bilirubinuria • hepatocellular disease including

infections and toxins • bile duct obstructions • jaundice due to haemolysis, after liver

damage occurs.

• VIBRIOSIS

See Campylobacteriosis'

VITAMIN DEFICIENCY Diagnosis Nutritional history, clinical findings, response to treatment, biochemistry. NSW Department of Primary Industries laboratories do not analyse for vitamin A or vitamin E. Specimens will be tested at a specialist laboratory when these tests are requested. Specimens required As vitamins A and E are light sensitive and degraded in tissues after collection, all samples for analysis should be protected from light by being wrapped in foil or brown paper and kept chilled during transport to the laboratory.

i. 5 ml of serum, heparin plasma or EDTA plasma

NB Ensure you obtain at least a full vacutainer of blood before separating off the serum/plasma. If inadequate volumes are presented, pools may be prepared where possible.

ii. 20 g liver

Interpretation of vitamin A and E concentrations in blood and liver (cattle, sheep, pigs) Species Sample Units Deficient Deficient Vitamin A Vitamin E Sheep Serum or

plasma µmol/L < 0.9 < 2.3

Cattle Serum or plasma

µmol/L < 0.9 < 4.6

Sheep Liver µmol/kg < 700 < 4.6 Cattle Liver µmol/kg < 11.6 Pig (piglet) Liver µmol/kg < 11 < 7 Pig (adult) Liver µmol/kg < 40 < 7

VOMITING AND WASTING DISEASE OF PIGS

See Haemagglutinating encephalomyelitis virus (HEV) infection'

WHITE MUSCLE DISEASE See Muscular degeneration nutritional

YERSINIOSIS Yersiniosis occurs in a variety of domestic animals. Disease is usually associated with Yersinia . pseudotuberculosis or Yersinia enterocolitica. However, Y intermedia, Y kristenseni and Y frederickseni occasionally cause disease. Yersiniosis can be manifest as one or more of the following conditions:

• Enterocolitis and diarrhoea • Systemic abscesses and mortality • Placentitis, abortion and perinatal

mortality • Pneumonia • Mastitis • Epididymo-orchitis In addition, specific entities have been associated with Y pestis in domestic carnivores (plague) and Y ruckeri in fish (enteric redmouth). Syndromes associated with yersiniosis Y pseudotuberculosis and Y enterocolitica in domestic animals:


Cattle Y. pseudotuberculosis: • Enterocolitis, diarrhoea, ‘flood mud

scours’, mortality • Placentitis, abortion, perinatal mortality • Pneumonia • Mastitis Y. enterocolitica • Abortion Sheep Y. pseudotuberculosis • Enterocolitis, diarrhoea, ill thrift,

mortality • Systemic abscessation, ‘pyaemic

hepatitis’ • Placentitis, abortion, perinatal mortality • Epididymo orchitis Y. enterocolitica • Enterocolitis, diarrhoea, ill thrift Goats Y. pseudotuberculosis • Enterocolitis, mortality • Placentitis, abortion, perinatal mortality,

endometritis, infertility • Systemic abscessation • Mastitis Y. enterocolitica • Enterocolitis, mesenteric lymphadenitis,

diarrhoea, ill thrift, mortality Deer Y. pseudotuberculosis and Y. enterocolitica • Enterocolitis, diarrhoea, mortality • Systemic abscessation Pigs Y. pseudotuberculosis • Mild enterocolitis, mild diarrhoea • Mild systemic abscessation Y. enterocolitica • Mild enterocolitis, mild diarrhoea Poultry (domestic fowl, turkeys, ducks, geese)

Y. enterocolitica • Acute septicaemia, enteritis,

splenomegaly, diarrhoea, mortality • Chronic systemic abscessation, chronic

enteritis, diarrhoea, ill thrift • Systemic myositis (turkeys) Diagnosis Clinical signs, bacteriology, histopathology. Recovery of Yersinia spp from the alimentary tract and internal organs using routine and selective media.

NB Many Yersinia spp inhabit the intestinal

tract of clinically healthy animals and a diagnosis of enteric yersiniosis is confirmed by finding typical histological lesions in sections of the intestine and internal tissues. Recovery of Y pestis is important in view of the zoonotic potential of this bacterium. Y ruckeri is of regulatory importance in fish.

Specimens required i. Samples of faeces, intestinal

contents (particularly ileal contents) and fresh tissue submitted chilled for bacteriology.

ii. Portions of tissue fixed in neutral buffered formalin for histopathology. Samples of fixed internal organs with lesions should be taken if systemic disease is suspected. Segments from all levels of the intestinal tract, particularly ileum and caecum, as well as mesenteric lymph nodes should be taken in cases of enterocolitis.

iii. Samples suitable for diagnosis of other causes of diarrhoea, pyaemia, abortion, pneumonia, mastitis and epididymo-orchitis should be collected as appropriate to the syndrome under investigation.


DISEASES OF POULTRY

Many poultry diseases can be diagnosed in the field. Before specimens are submitted, the field veterinarian should obtain a complete history, examine the flock and affected birds, and perform several necropsies. If a diagnosis cannot be made or if laboratory confirmation is required, then appropriate specimens should be submitted with a full history and description of clinical and necropsy findings. Appropriate tests for specific diseases should be requested.

STANDARD CONTAINERS AND EQUIPMENT See Specimens (General). COLLECTION OF SPECIMENS

Specimens required i. For the investigation of most

diseases, submit at least 5 affected live or freshly dead birds which are, or were, showing typical signs.

ii. Tissues from necropsied birds, fixed in formalin and/or fresh, are also acceptable.

iii. For serology, 1 to 5 ml of blood should be collected from each of 10 to 25 birds. The jugular and wing veins are the best vessels from which to collect blood. Blood should be collected into suitable containers and allowed to clot. If the clotted blood will not reach the laboratory within 48 hours, the serum should be poured off into a suitable clean container before submission to the laboratory. The blood must not be allowed to haemolyse.

iv. For bacterial or fungal culture, whole birds or fresh tissues are required. In general, swabs (with or without transport media) are not suitable, due to loss of viability of fragile organisms and/or overgrowth of contaminants. For the culture of Mycoplasma spp. or Haemophilus paragallinarum, live birds must be submitted.

v. For virology, whole birds, fresh or frozen tissues are acceptable. For virus isolation particularly for NDV and AI- swabs in PBSG (which is available in 5-ml plastic screw-top containers from the RVL Menangle /EMAI Virology Laboratory. If unfamiliar with the correct specimens, please contact the

laboratory for advice before proceeding.


Live birds should be submitted in pet transport boxes or the like, with adequate air holes and absorbent lining. The birds must not be overcrowded. The birds must reach the laboratory overnight (summer) or within 24 hours (winter), unless arrangements have been made for them to receive water and food in transit. Sick birds must be able to withstand transport. If they are not likely to survive, they should be killed humanely and submitted chilled in an insulated container with an icebrick or ice in a sealed container. They must not be frozen. They must reach the laboratory within 24 hours, preferably overnight. Fresh tissues and serum should also be transported chilled, but not frozen, and must reach the laboratory within 24 to 48 hours, preferably overnight. Formalin fixed tissues must be in leak proof containers and packaged securely to prevent breakage in transit. If chlamydiosis or tuberculosis is suspected, particular care must be taken to avoid infection of couriers and laboratory staff. Live birds must not be sent. Dead birds should be sealed inside two plastic bags and submitted chilled inside a strong outer container. The Specimen Submission Form should be attached to the outside of the outer container and should prominently warn that chlamydiosis or tuberculosis is suspected.

SEROLOGICAL TESTS AVAILABLE FOR POULTRY

Serological tests are currently available at EMAI for the following diseases: • Avian encephalomyelitis (AE) • Avian Influenza (AI) • Big Liver and Spleen Disease (BLS) • Egg Drop Syndrome 76 (EDS)

(Haemagglutinating avian adenovirus; adenovirus 127)

• Fowl adenovirus (FAV) • Haemorrhagic enteritis of turkeys (HE;

THE) • Infectious Bursal Disease (IBD) • Marek's Disease (MD)


• Mycoplasma gallisepticum infection (Mg)

• Mycoplasma synoviae infection (Ms) • Newcastle Disease (ND) • Reticuloendotheliosis (REV) • Salmonella pullorum (Sp) Most serological tests are flock tests. Positive results indicate that the flock has been exposed at some time to the infectious agent concerned, either by natural infection (subclinical infection or clinical disease) or due to vaccination. To confirm recent infection, paired serums are required to demonstrate flock seroconversion or rising flock titres. The tests are of limited value for individual birds.

AVIAN ENCEPHALOMYELITIS (AE) Diagnosis Young chickens and turkeys Clinical signs (neurological disease), histopathology, serology. Laying chickens and turkeys Clinical signs (egg production drop), serology. Serology is also used in monitoring efficacy of vaccination. Specimens required

i. Whole live birds, or, ii. Brain, spinal cord, skeletal muscle,

myocardium, proventriculus, gizzard in buffered formalin, for histopathology.

iii. Fresh or frozen sera, collected early during the problem (within 7 days of onset), and again during convalescence (3-4 weeks later). Unless the birds are vaccinated, the titre range could indicate recent exposure and the paired sera may not be required in all circumstances.

BIG LIVER AND SPLEEN DISEASE

Diagnosis Gross lesions, histopathology, serology. Specimens required

i. Whole birds, or: ii. Liver and spleen in buffered

formalin, for histopathology. iii. Sera, for serology.

CHLAMYDIOSIS (AVIAN)

Syn: Psittacosis; ornithosis

Caution Chlamydiosis is a zoonotic disease, causing serious illness and sometimes death in humans. Diagnosis Gross pathology, examination of tissue impression smears for acid fast organisms, antigen detection in tissues by immunofluorescence (IFAT), histopathology, serology (pigeons only), isolation. Specimens required

i. Whole dead birds, for pathology, acid fast and FAT stains.

ii. Liver, spleen submitted fresh, chilled for acid-fast stain and IFAT on fresh smears (made at testing laboratory).

Where (i) or (ii) are not feasible, air dried impression smears from the serosal (not cut) surface of liver and spleen, and from conjunctiva. Submit duplicate smears, for acid fast stain and IFAT.

iii. Liver, spleen, conjunctiva and other representative tissues in buffered formalin, for histopathology.

iv. Sera, for serology. Ideally, paired sera should be submitted to demonstrate rising titres.

NB Serology is unreliable for most avian species other than pigeons.

CHOLERA See Pasteurellosis'

COCCIDIOSIS (AVIAN) Diagnosis Gross lesions, wet intestinal smears, histopathology. Specimens required

i. Whole birds, or: ii. Fresh intestinal scrapings (ie

mucosa plus contents), for microscopic examination for oocysts and schizonts.

iii. Portions of affected gut in buffered formalin, for histopathology.

CORYZA

(Haemophilus paragallinarum infection) Diagnosis Clinical signs, gross lesions, bacteriology. Specimens required

i. Whole live birds, for pathology and bacteriology. (Do not submit dead birds, tissues or swabs for bacteriology, as Haemophilus spp quickly lose viability)


EGG DROP SYNDROME (EDS 76)

A drop in egg production due to EDS in layer flocks is usually associated with the appearance of shell-less eggs. A drop in egg production without shell-less eggs can be due to a variety of causes. These include nutritional, management, environmental and disease factors. Many causes can be identified in the field and specimens should only be submitted to confirm the diagnosis of disease caused by infectious agents. Diagnosis Clinical signs, serology. Specimens required

i. Fresh or frozen sera, collected early during the problem (within 7 days of onset of egg drop), and again during convalescence (3 4 weeks later). If the flock is not vaccinated, the range of titres in conjunction with clinical signs would lead to diagnosis, without the need for paired sera.

FOWL POX

Diagnosis Gross pathology, histopathology, electron microscopy. Specimens required

i. Whole birds, for gross and histopathology.

ii. Lesions fixed in buffered formalin. iii. Fresh tissues for electron

microscopy.

INFECTIOUS BRONCHITIS (IB) Diagnosis Histopathology, virus isolation. Specimens required

i. Whole birds, or: ii. Trachea, lung, kidney, oviduct in

buffered formalin, for histopathology. iii. Fresh trachea, lung, kidney for virus

isolation (and serotyping).

INFECTIOUS BURSAL DISEASE (IBD) Diagnosis Gross pathology, histopathology for clinical or subclinical disease. Serology to monitor exposure or vaccination efficacy. Specimens required

i. Whole birds, or, ii. Bursa of Fabricius in buffered

formalin, for histopathology. iii. Fresh or frozen sera, for serology.

INFECTIOUS LARYNGOTRACHEITIS (ILT) Diagnosis Gross pathology, histopathology, antigen detection in tissues, virus isolation. Specimens required

i. Whole birds, or, ii. Fresh or frozen tracheas (including

larynx) in sterile jars, for antigen ELISA and virus isolation.

iii. Tracheas (including larynx) in buffered formalin, for histopathology. Because of relatively poor sensitivity of the antigen ELISA test, 5-10 tracheas are required.

LEUKOSIS

Diagnosis Gross pathology, histopathology. Specimens required


ii. The following tissues fixed in buffered formalin: liver, spleen, kidney, heart, ovary/testicle, proventriculus, gizzard, sciatic nerves, brain, bursa (if present), tumours (if present).

MAREK'S DISEASE

Diagnosis Clinical signs, gross pathology, histopathology. Specimens required


ii. The following tissues fixed in buffered formalin: liver, spleen, kidney, heart, ovary/testicle, proventriculus, gizzard, pancreas, sciatic nerves, brain, bursa (if present), tumours (if present).

MYCOPLASMOSIS (AVIAN)

(Mycoplasma gallisepticum, M synoviae infections) Diagnosis Gross pathology, histopathology and bacteriology for clinical disease. Bacteriology for subclinical disease. Serology to monitor exposure. Specimens required

i. Whole live birds, for pathology and bacteriology. (Do not submit dead birds, tissues or swabs for bacteriology, as mycoplasmas quickly lose viability).


ii. Fresh sera, for serology. Preferably off the clot and free of red blood cells.

NB Do not submit frozen sera, as false positives may occur.

PASTEURELLOSIS (AVIAN) (Pasteurella multocida, P anatipestifer infections) Diagnosis Clinical signs, gross pathology, bacteriology. Specimens required

i. Whole birds, for gross pathology and bacteriology, or,

ii. Fresh tissues (liver, heart, lung and (from ducks) brain), for bacteriology.

PULLORUM DISEASE

(Salmonella pullorum infection) Diagnosis Serology, bacteriology for detection of carriers. Specimens required

i. For an initial flock test, submit fresh (not frozen) sera from every bird, individually identified.

ii. To check reactors to the field test, submit fresh sera clearly identified to individual birds.

iii. To confirm reactors, submit live or freshly humanely killed birds (clearly identified) for bacteriology.

RETICULOENDOTHELIOSIS (RE)

Diagnosis Gross pathology, histopathology, serology. Specimens required


ii. The following tissues fixed in buffered formalin: liver, spleen, kidney, heart, ovary/testicle, proventriculus, gizzard, sciatic nerves, brain, bursa (if present), tumours (if present).

iii. Sera, for serology.

TUBERCULOSIS (AVIAN) Diagnosis Necropsy, histopathology, isolation of mycobacteria. Specimens required

i. Fresh lesions in sterile sealed jars, for bacteriology.

ii. Portions of lesions in buffered formalin for histopathology.


DISEASES OF CAGE BIRDS, AVIARY BIRDS AND RACING PIGEONS

Isolated mortalities or sporadic disease problems in cage and aviary birds and racing pigeons are NOT examined at Regional Veterinary Laboratories, unless a notifiable or exotic disease is suspected. However, ongoing problems involving a number of birds in larger aviaries or pigeon lofts may be investigated, depending on the circumstances. Before submitting specimens, please phone the RVL to discuss the case. You should have conducted a thorough investigation of the problem and be able to provide a detailed history, clinical and necropsy findings. STANDARD CONTAINERS AND EQUIPMENT See Specimens (General).

COLLECTION OF SPECIMENS Specimens required

i. At least two affected live or freshly dead birds which are typical of the flock problem.

If chlamydiosis is suspected, do not send live birds.

ii. Tissues from necropsied birds, fixed in formalin. Small birds may, after necropsy, be submitted in toto in 10 times their volume of buffered formalin.


As for Diseases of Poultry.


DISEASES OF BEES

Bee diseases are investigated at the Regional Veterinary Laboratories. Diseases seen include American Foul Brood, European Foul Brood and many viral diseases affecting both adults and larvae.

Diagnosis Clinical signs and demonstration of causal agent. Specimens required ADULT BEE DISEASES

i. Preferably 30 adult bees submitted live at ambient temperature for microbiology. Bees should be submitted in aerated containers with a small amount of a candied mixture of sugar and honey.

ii. If only dead specimens are obtainable, submit chilled for microbiology.

LARVAL DISEASES

i. Four (4) air dried larval smears. ii. A brood comb sample containing

diseased larvae, submitted chilled for microbiology.


DISEASES OF FISH

The Special Veterinary Officer (Fish Health), located at the Regional Veterinary Laboratory, Wollongbar can be contacted for advice on the investigation of fish health problems.(Phone: 066 240261) Expertise in the diagnosis of fish diseases is currently available at other Regional Veterinary Laboratories, and the assistance of officers at these locations can also be sought in the first instance.


Water sample containers The types of containers recommended for collection of water samples include: • Polyethylene or glass for ammonia,

nitrate/nitrite, pH, cyanide and algae; • Glass stoppered glass for oxygen, filled

to exclude air; • Nitric acid washed polyethylene for

metals; • Hexane washed glass for pesticides; • Sterile 200 ml glass bottles for

bacteriology, chilled at 4oC, and submitted within 24 hours of collection.

Fixatives mlBOUIN'S FIXATIVE Saturated aqueous picric acid 75Formalin (Commercial solution; 38% formaldehyde w/v) 25Acetic acid 5 DAVIDSON'S SOLUTION Glycerine 10Formalin (38% formaldehyde) 2095% Alcohol 303.5% NaCl solution (or Filtered seawater) 30Glacial acetic acid 10

Fixation time: 24 hours (minimum)

HISTORY AND DIAGNOSIS History taking

i. A comprehensive history assists the investigation. Estimates of morbidity and mortality, based on stocking density on farms or the abundance of the species in natural waterways should be provided. Include measurements of length and weight, with an estimate of the age or stage of growth. Larger fish are more likely to suffer oxygen deprivation, whereas smaller fish tend to be more susceptible to toxic insults. If other inhabitants of the aquatic environment are affected, poor water quality or exposure to toxins should be suspected.

ii. A description of the locality of the

fish kill should include the type of habitat, whether natural or man made, river, stream, lake, pond, dam, tank, raceway, cage or aquarium. On fish farms the design and construction of facilities and the source of water should be related to the number and distribution of ponds affected. Freshwater, brackish,

estuarine or ocean environments are important because of their differences in salinity. A general description of the land use in the local drainage basin or watershed may be appropriate.

iii. Describe the condition of the water

in terms of colour, turbidity, odour and flow. Is the water level high, low or normal? Are algae, organic debris, froth or oil floating at the surface? Does the ecosystem appear "healthy"?

iv. Record the temperature of the water

at several sites and depths. Submit the results of any onsite water quality tests performed. These measurements, especially O2 and pH, could be taken at various sites (inlet, middle and outlet) and depths (surface, mid level and bottom) in the body of water.

v. Details of composition of diet, history

of dietary changes, source or manufacturer of feed, rate of feeding, storage conditions and duration of storage could allow


nutritional deficiencies or toxicities to be identified.

vi. List any treatments, including

chemicals and antibiotics, which have been administered to the fish. On many occasions malachite green or formalin will have been used; although these treatments may have eliminated the causative agent, rendering subsequent examinations negative, they may have caused further direct or indirect injury to the fish and these potential complications should be considered. What dosage regime (time of treatment, duration of treatment and concentration or dose rate of chemical) has been applied?

Diagnosis History, clinical signs, field assessment of water quality*, field evaluation of environmental and ectoparasitic causes of disease**, histopathology, parasitology, bacteriology, virology, toxicology. *Water quality assessment is best performed in the field using commercial test kits for ammonia, nitrite/nitrate, acidity, hardness and copper. **Microscopic examination of wet preparations of gills, skin and fins should allow common parasites to be identified. Diagnostic hints

i. Mortality patterns can be fitted to the time of year, day or season. For example, deaths due to oxygen deprivation are most likely to be clustered in the early hours of the morning when water oxygen levels are depressed. Some diseases, such as infestations with the gill protozoan Chilodonella in many species or epizootic haematopoietic necrosis (EHN) virus in redfin perch, are recurrent seasonal problems.

ii. The signs exhibited by diseased fish

are readily observed in glass sided aquaria, but may be difficult to detect in open water, unless fish are surfacing. Diseased fish may change the depth which they normally occupy in the water column or their spatial relation to inlet or outlet points to a pond. Feeding patterns may be altered. Behavioural abnormalities such as gasping at the surface of the water, flashing or turning upside down may

be displayed. Skin colour may change.

• Superficial lesions affecting

the skin, fins, gills, eyes, mouth or anus may be visualised in the live fish. The results of any clinical examination should be detailed.

iii. Any lesions noted at autopsy or

during handling should be recorded. If several fish are available for examination, an onsite autopsy should be performed on at least one. Collect fresh and preserved specimens from any autopsies and submit these along with any live affected or freshly dead fish which are available.

COLLECTION, STORAGE AND DESPATCH OF SPECIMENS

Specimens required i. Live moribund or affected fish

(preferred specimens). Fish to be submitted are placed in a strong plastic bag inside another plastic bag, together with a half volume of water from the source environment. If likely to be in transit for any length of time, the plastic bag should be inflated with oxygen before twisting, double folding and sealing with strong rubber bands. The plastic bag is then placed inside an esky and sent to the laboratory.

Fish should never be fed before transport; this increases metabolic rate which increases oxygen consumption and may lead to fouling of the water. Sick fish should also be protected from changes in temperature. Despatch of several survivors and/or several healthy control fish is often beneficial in reaching a diagnosis, as this allows the pathologist to compare affected and unaffected/recovered animals in greater detail.

ii. Dead fish (rarely of much diagnostic value, unless they are examined on site) Dead fish are virtually useless after transport, even if chilled, because decomposition in fish occurs rapidly and many ectoparasites will detach from the host soon after death. If only


dead fish are available for submission to the laboratory, submit: • Fixed tissues for histopathology

(must be fixed on site) • Frozen tissues for

toxicology/virology. If only dead fish are submitted, bacteriology is precluded (culture must be undertaken within 1 hour of death to be of value). Frozen fish are unsuitable for histopathology.

iii. Tissues in fixative (submitted in

addition to live fish). This ensures that serviceable specimens will be available should there be transportation delays that result in death or deterioration of other specimens. Small fish may be immersed in fixative whole, with a section of abdomen removed to allow penetration of fixative to internal organs. A range of tissues could be taken from larger fish at autopsy, concentrating especially on those tissues which exhibit gross pathology. In addition to internal organs such as heart, liver, kidney, spleen and intestine, consider submitting brain, skin, muscle and gonad. As autolysis is rapid in the gills, sections of the gill arches should be placed in fixative at the start of the examination. Ten percent neutral buffered formalin or 5% formol saline are suitable fixatives for fish tissues. Bouin's fixative (see ‘Fixatives’ above) can be used if the specimens are small, for rapid penetration and optimal preservation. The tendency of some ectoparasites to detach from gills and skin, even in the presence of formalin, may be overcome by the use of Davidson's solution (see ‘Fixatives’ above).

iv. Fresh tissues (for bacteriological examinations, virology and toxicology). Tissues from large fish (which have been freshly killed) may be removed and transported chilled in sterile

containers. A pool of fresh chilled brain, liver, kidney and spleen is often suitable for isolation of viruses. Swabs are less suitable.

v. Smears of blood and body fluids (for haematology and bacteriology). Many examinations for ectoparasites are best carried out in the field using wet preparations. Rapid blood stains can also be used for smears from fish.

In special cases and after consultation specimens can be forwarded to the Australian Fish Health Reference Laboratory, Geelong, Victoria.

SOME DISEASES OF FISH, CRUSTACEANS AND SHELLFISH ALREADY ENCOUNTERED IN NSW

Fish Kills Attributed to: • Anoxia • Environmental toxins e.g.

superphosphate • Infectious diseases e.g. Epizootic

Haematopoietic Necrosis (EHN) in redfin perch.

Skin Lesions in Fish Often multifactorial; may involve nutritional, parasitic, bacterial and viral pathogens. Histopathology of lesions has been of value. Causative agents include: • Bacterial infection with Aeromonas

hydrophila in hatcheries, seen in stressed adult fish.

• Yellow spot • Red spot in estuarine fish on the North

Coast • Goldfish ulcer disease due to

Aeromonas salmonicida or Vibrio infections. The former is an important pathogen of Salmonids

Shellfish Diseases • Discolouration in Abalone Crustacean Diseases • Protozoal myositis with ataxia in

Yabbies


FEEDS AND PASTURE ANALYSIS

NSW Department of Primary Industries provides an independent Feed Quality Service to sheep, beef and dairy producers. A limited range of analyses is available for pig and poultry feeds. The Feed Quality Service accepts most types of feeds for analysis e.g. grain, hay, pasture, silage, mixed rations, crop residues, browse and leaves. Charges are made for evaluating feed samples, in accordance with Departmental policy. The charges are based on which analyses are requested. For information on analyses and charges, contact the Customer Service Unit on 02 6763 1187.

SAMPLE COLLECTION A standard procedure for the collection of samples should result in a representative sample and minimal sample deterioration. Detailed instructions for the collection of samples are available from Livestock Advisory Officers or directly from the Feed Quality Customer Service Unit on 02 6763 1187. The main points to be considered are: • Samples collected must be

representative of the feed and not taken from one bale or bag.

• Sampling Hay: 10-15 core or grab samples from at least 10 small or 5 large bales selected at random need to be sampled. Mix the sample thoroughly and send approximately 500g in a paper bag to the laboratory.

• Sampling Silage: 10-20 grab samples from fresh silage or from at least 10 small or 5 large selected at random need to be sampled. Mix the sample thoroughly and freeze approximately 500g in a plastic bag and send to the laboratory.

• Sampling pasture: Sample at random 15-20 locations across the paddock. The sample must reflect the pasture consumed by the animal. Mix the sample thoroughly and prepare approximately 500g by either: microwave the sample on high for no longer then 30 seconds, or dry at 600C for 24hrs, or freeze the sample and send to the laboratory.

• Grain and feed mixes: 10-15 grab samples from different locations or bags within the supply. Mix thoroughly and send approximately 500g to the laboratory.

TESTS AVAILABLE Individual and package tests are available from the following list: • Dry Matter • Nitrogen (crude protein) • Digestibility • Organic matter • Metabolizable energy • Acid detergent fibre • Neutral detergent fibre • Lignin • Soluble carbohydrate • Fat • Nutrient minerals (B, Ca, Cu, Fe, K, Mg,

Mn, Na, P, S, Zn) • pH (silage only) • Ammonia-Nitrogen (silage only) Other specialised tests are available. SUBMISSION OF SAMPLES All samples must be accompanied by a Feed Quality Service submission form The form is available from the Feed Quality Customer Service Unit and can be sent to you by fax, e-mail or by post. NO SAMPLE WILL BE TESTED WITHOUT A CORRECTLY COMPLETED SUBMISSION FORM. Each sample should be sealed in a unique, strong paper or plastic bag (not freezer bags etc.) with as much air squeezed out as possible. DO NOT SEND SAMPLES IN GLASS. The outside of the bag should be labelled with: • Your name • Sample type (hay, pasture, silage etc) • A unique sample number or name

Wet samples such as silage, fresh crop (green leaf) and fresh pasture must be sealed in a plastic bag (air tight) and frozen immediately. These samples should then be packed in an esky and sent to the Feed Quality Service. NB. DO NOT DESPATCH FROZEN SAMPLES ON THURSDAYS OR FRIDAYS All samples should be sent to: Feed Quality Service NSW Department of Primary Industries RMB 944 Calala Lane Tamworth NSW 2340


NOTIFICATION TO LABORATORY The Feed Quality Service should be notified by telephone (02) 6763 1187 when samples are sent for analysis. The work load can then be anticipated and a potential turn around time can be given to the officer involved. Any special conditions needed for sample transport can be discussed at the time of notification; e.g. silage and other fresh material (as noted above) needs to be cooled or frozen.

CHARGES FOR FEED QUALITY SERVICES DO NOT SEND ANY MONEY with the sample. An invoice will be issued with the laboratory report. Discounts for large number of samples may be available. Contact the Customer Service Unit (02 6763 1187) for more information.


WATER TESTING

The quality of water from bores, wells, creeks, rivers and farm dams can vary widely. Common problems include hardness, iron and salinity. Water quality can impact on plant growth and animal production, and can affect irrigation equipment. NSW Department of Primary Industries' Environmental Laboratories provide a water testing service for farmers and graziers wishing to determine the suitability of their water for agricultural and domestic applications. Sampling kits are available from all Departmental offices. Standard laboratory reports provide detailed information on pH, salinity, chloride, alkalinity, turbidity, hardness, saturation index, sodium absorption ratio and electrical conductivity. Testing for nutrients, pesticide residues and blue-green algae is also available.

Further information on water testing is available from the NSW Department of Primary Industries website at http://www.agric.nsw.gov.au/reader/das-laboratory/das-farm-water.htm Water for human consumption NSW Department of Primary Industries does not undertake testing of water for human consumption. Contact either the Health Inspector at the Local Council, or the NSW Department of Health's Water Laboratory, Lidcombe regarding any testing for human consumption: NSW Health Division of Analytical Laboratories Weerona Road LIDCOMBE NSW 2141

http://www.agric.nsw.gov.au/reader/das-laboratory/das-farm-water.htm

Documents

Veterinary Laboratory Manual, 9th Ed - 2006