Advanced Laboratory Techniques in Poultry Disease Diagnosis

Version 706 Version 706

Dr. Joseph GiambroneProfessor

U http://www.auburn.edu/~giambjj U /email: [email protected]

201 Department of Poultry Science260 Lem Morrison Drive

Auburn University, AL 36849-5416

Teresa DormitorioResearch Associate III

Email: [email protected]

Preface

ADVANCED LABORATORY TECHNIQUES IN AVIAN MEDICINE MANUAL 2

http://www.auburn.edu/~giambjj/

The purpose of this book is to provide the diagnostic laboratory, which is already experienced and equipped for diagnosis of avian diseases, more advanced and sophisticated techniques for disease diagnosis. The book is divided into two sections. The first section gives credit to the time honored traditional methods. The second provides an introduction to newly developed techniques in molecular biology. Diagnostic methods will be covered for infectious organisms only, which include bacteria, mycoplasma, fungi and viruses. For the molecular diagnosis of DNA containing microorganisms, the Mycoplasma species will be used. The very common avian viruses, infectious bursal disease and avian reoviruses, are used as an example of RNA containing microorganisms. Only the most commonly found organisms in each group will be covered, but the techniques are similar for less important species. The mention of any product or company name does not imply endorsement.

Acknowledgments

This book and CD depended upon many people without whom it could not have been written. Sincere thanks go out to former graduate student, Wayne Duck, who suggested a need for this book and thereby helped in the preparation of some of the initial materials. We would also like to thank Loraine M. Hyde from Poultry Science Department for her help in typing, checking, and typesetting this manuscript. Thanks to the Film Lab of AU for there help in scanning the photos and organizing the Book.

TABLE OF CONTENTS


PageAuthors 2Preface 3Acknowledgments 3TABLE OF CONTENTS 4Introduction 5

I.TRADITIONAL DIAGNOSTIC METHODSIsolation and Identification of Microorganisms 51) Bacteria

a) Salmonella 7b) Escherichia coli 9c) Pasteurella multocida 10d) Staphylococcus aureus 11e) Mycoplasma 12

2) Fungia) Aspergillus 14

3) Viruses 15 a) Cultivation of viruses in chicken embryos 19Routes of inoculation and collection of specimens for avian influenza 19

b) Propagation in chicken tissues 25 c) Propagation in cell culture 26

Chicken kidney cells 27 Chicken embryo fibroblasts 28 Chicken embryo liver cells 29 Tracheal rings 30 Cell lines and Secondary cells 31

d) Application of cell culture techniques in virology 31 e) Virus Identification 32

B. Serological procedures 411) Immunodiffusion 452) Agglutination—Salmonella 473) Hemagglutination Inhibition—ND, MG, IBV 474) Immunofluorescence 495) Virus Neutralization—IBV, AE, IBDV 536) Enzyme Linked Immunoabsorbent Assay 57

C. Immunosuppression1) Introduction 632) Definition 633) Evaluation 63

a) Antibody 63b) CMI 64

4) Causes 645) Prevention 65

II. MOLECULAR BIOLOGICAL TECHNIQUES 105A. Nucleic Acids 105

1) Propagation, purification and quantification of IBDV RNA 1122) Quick IBDV RNA isolation procedure 1293) Restriction fragment length polymorphism 133

a) Mycoplasma gallisepticum 136b) Silver stain 138

4) Hybridization 142a) Radioactive Probes 148b) Non-radioactive Probes 150c) Dot and Slot Blot 152d) Southern Blot 152e) Northern Blot 167f) In situ Hybridization 184g) Tissue Print Hybridization 185h) In situ PCR 188i) Nested PCR 192

5) Polymerase Chain Reaction 195a) Restriction fragment length polymorphism 207b) Real Time PCR for avian influenza 210

b) Sequencing 2256) Microarray Assay 236

Proteins 2371)Electrophoretic Separation 2392)Dot and Western Immunoblots 2443)Monoclonal antibodies—production and uses 258

a) Antigen capture ELISA 267b) Immunoperoxidase test 271

Appendix1.Selected list of suppliers 2742.Procedures for Preparation of Buffers and Reagents 2793.Commonly Used Abbreviations 285

Glossary 287

INTRODUCTION


Much of the rapid development in the poultry industry worldwide has been due to improvements in genetics, nutrition and disease control. Knowledge of the cause of diseases has expanded dramatically over the years. Advances in the diagnosis, treatment and vaccination have contributed to improved disease control. It is extremely important to identify a pathogen before the disease can be adequately controlled. However, the isolation of an organism from a lesion does not always mean that it is directly responsible for the disease. The agent may be a secondary invader or become a primary pathogen after the bird’s immune system was suppressed. This immunodepression could be brought about by a variety of agents and environmental conditions. In addition, birds may be submitted late in the course of the disease and only secondary invaders such as bacteria are readily isolated. Affected birds should be submitted as early as possible to increase the chance of isolation of primary invaders, especially viruses.

Diseases may be caused by one or more agents. Therefore, it is important to undergo a routine battery of tests; otherwise you may miss one or more affecting agents. It may be also necessary to collect serum from live, diseased birds to check for abnormally high or low levels of antibody against a variety of common infectious organisms. Submission of at least 10 birds from a disease flock is usually adequate. A combination of normal, sick and recently dead birds and/or tissues, blood specimens, samples of feed, water and litter, plus a thorough history of the flock should be submitted. The time honored traditional methods of isolation and identification of disease pathogens and/or the antibodies they induce is still the backbone of the diagnostic laboratory. However, more sophisticated techniques using molecular biological techniques such as monoclonal antibodies, nucleic acid probes, polymerase chain reaction, and restriction fragment length polymorphism are now being used routinely in diagnostic laboratories. It is the subject of these advanced techniques, which sets this book apart from its predecessors.

In the chapters on molecular biology, introductory material will explain the basis of each technique after which specific methodology will follow which gives details in step by step fashion. The specific techniques will be centered on one DNA containing organism, e.g. mycoplasma or RNA microbe, e.g. infectious bursal disease virus. These pathogens are featured since they are extremely important pathogens of poultry and because much more is known about their genetic material. However, at the molecular level, genetic manipulations are basically the same and techniques described herein can be adapted for most avian pathogens with little modifications.

I. TRADITIONAL DIAGNOSTIC METHODS

A.Isolation and Identification of Microorganisms

Bacteria

Bacteria, along with blue-green algae, are prokaryotic cells. That is, in contrast to eukaryotic cells, they have no nucleus; rather the genetic material is restricted to an area of the cytoplasm called the nucleoid. Prokaryotic cells also do not have cytoplasmic compartment such as mitochondria and lysosomes that are found in eukaryotes. However, a structure that is found in prokaryotes but not in eukaryotic animal cells is the cell wall which allows bacteria to resist osmotic stress. These cell walls differ in complexity and bacteria are usually divided into two major groups, the gram-positive and gram-negative organisms, which reflect their cell wall structure. The possession of this cell wall, which is not a constituent of animal cells, gives rise to the different antibiotic sensitivities of prokaryotic and eukaryotic cells. Prokaryotes and eukaryotes also differ in some important metabolic pathways, particularly in their energy metabolism and many bacterial species can adopt an anaerobic existence.

In this section, we shall look at the structure of typical bacterial cells and the ways in which they liberate energy from complex organic molecules. Various aspects of bacterial structure and metabolism are the basis of bacterial identification and taxonomy. Bacteria are constantly accumulating mutational changes and their environment imposes a strong selective pressure on them. Thus, they constantly and rapidly evolve. In addition, they exchange genetic information, usually between members of the same species but occasionally between members of different species. We shall see how this occurs.

Bacteria have parasites, the viruses called bacteriophages which are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. Eventually, these lyse the infected bacterial cell liberating new infection phage particles. The interrelationships of bacteria and


the pages will be investigated.

Taxonomy

The basis of bacterial identification is rooted in taxonomy. Taxonomy is concerned with cataloging bacterial species and nowadays generally uses molecular biology (genetic) approaches. It is now recognized that many of the classical (physiology-based) schemes for differentiation of bacteria provide little insight into their genetic relationships and in some instances are scientifically incorrect. New information has resulted in renaming of certain bacterial species and in some instances has required totally re-organizing relationships within and between many bacterial families. Genetic methods provide much more precise identification of bacteria but are more difficult to perform than physiology-based methods.

Family: a group of related genera. Genus: a group of related species. Species: a group of related strains. Type: sets of strain within a species (e.g. biotypes, serotypes). Strain: one line or a single isolate of a particular species.

The most commonly used term is the species name (e.g. Streptococcus pyogenes or Streptococcus pyogenes abbreviation S. pyogenes). There is always two parts to the species name one defining the genus in this case "Streptococcus" and the other the species (in this case "pyogenes"). The genus name is always capitalized but the species name is not. Both species and genus are underlined or in italics.

B. The Diagnostic Laboratory

The diagnostic laboratory uses taxonomic principles to identify bacterial species from birds. When birds are suspected of having a bacterial infection, it is usual to isolate visible colonies of the organism in pure culture (on agar plates) and then speciate the organism. Physiological methods for speciation of bacteria (based on morphological and metabolic characteristics) are simple to perform, reliable and easy to learn and are the backbone of hospital clinical microbiology laboratory. More advanced reference laboratories, or laboratories based in larger medical schools additionally use genetic testing.

Isolation by culture and identification of bacteria from patients, aids treatment since infectious diseases caused by different bacteria has a variety of clinical courses and consequences. Susceptibility testing of isolates (i.e. establishing the minimal inhibitory concentration [MIC]) can help in selection of antibiotics for therapy. Recognizing that certain species (or strains) are being isolated atypically may suggest that an outbreak has occurred e.g. from contaminated hospital supplies or poor aseptic technique on the part of certain personnel.

Steps in diagnostic isolation and identification of bacteria

Step 1. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on culture plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one - several days. It is not uncommon for cultures to contain more than one bacterial species (mixed cultures). If they are not separated from one another, subsequent tests can’t be readily interpreted. Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Whether the organism requires oxygen for growth is another important differentiating characteristic.

Step 2. Colonies are Gram stained and individual bacterial cells observed under the microscope.

Step 3. The bacteria are speciated using these isolated colonies. This often requires an additional 24 hr of growth.

Step 4. Antibiotic susceptibility testing is performed (optional)

The Gram Stain, a colony is dried on a slide and treated as follows: 3

Step 1. Staining with crystal violet.


Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.

Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram negative) and not others (Gram positive).

Step 4. Counterstaining with safranin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink.

Under the microscope the appearance of bacteria are observed including: Are they Gram positive or negative? What is the morphology (rod, coccus, spiral, pleomorphic [variable form] etc)? Do cells occur singly or in chains, pairs etc? How large are the cells? There are other less commonly employed stains available (e.g. for spores and capsules). Another similar colony from the primary isolation plate is then examined for biochemical properties (e.g. will it ferment a sugar such as lactose). In some instances the bacteria are identified (e.g. by aggregation) with commercially available antibodies recognizing defined surface antigens. As noted above genetic tests are now widely used.

Genetic characterization of bacteria

Whole genomes of a representative strain of many of the major human pathogens have been sequenced, and this is referred to as genomics. This huge data-base of sequences is highly useful in helping design diagnostic tests. However, rarely are more than one or two representative genomes sequenced. There is a lot of variability in sequences among individual strains. Thus for practical reasons, genetic comparisons must involve multiple strains. Certain genes have been selected to define common traits among species and then this information is used to develop diagnostic tests.

1. Sequencing of 16S ribosomal RNA molecules (16S rRNA) has become the "gold standard" in bacterial taxonomy. The molecule is approximately sixteen hundred nucleotides in length. The sequence of 16S rRNA differentiates bacterial species.

2. Once the sequence is known, specific genes (e.g. 16S rRNA) are detected by amplification using the polymerase chain reaction, PCR. The amplified product is then detected most simply by fluorescence (“real time” PCR) or by gel electrophoresis (the molecular weight of the product).

3. DNA-DNA homology (or how well two strands of DNA from different bacteria bind [hybridize] together) is employed to compare the genetic relatedness of bacterial strains/species. If the DNA from two bacterial strains display a high degree of homology (i.e. they bind well) the strains are considered to be members of the same species.

4. The guanine (G)+ cytosine (C) content usually expressed as a percentage (% GC) is now only of historical value.

Chemical analysis

Commonly fatty acid profiling is used. The chain length of structural fatty acids present in the membranes of bacteria is determined. Protein profiling is rapidly expanding. Characterization of secreted metabolic products (e.g. volatile alcohols and short chain fatty acids) is also employed.

Rapid diagnosis without prior culture

Certain pathogens either can’t be isolated in the laboratory or grow extremely poorly. Successful isolation can be slow and in some instances currently impossible. Direct detection of bacteria without culture is possible in some cases; some examples are given below. Bacterial DNA sequences can be amplified directly from human body fluids using PCR. For example, great success has been achieved in rapid diagnosis of tuberculosis. A simple approach to rapid diagnosis (as an example of antigen detection) is used in many doctor's offices for the group A streptococcus. The patient's throat is swabbed and streptococcal antigen extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is detected by aggregation (agglutination) of antibody coated latex beads. Direct microscopic observation of certain clinical samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum). However, sensitivity is poor and many false negatives occur. Serologic identification of an antibody response (in


patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred. This is commonly used in

Salmonella

Introduction

Avian salmonellosis is divisible into three diseases: pullorum disease (S. pullorum), fowl typhoid (S. gallinarum), and paratyphoid. Pullorum and typhoid are not often seen in commercial poultry companies, where serologic testing and eradication of positive breeder flocks is practiced, but are common in small backyard flocks. Paratyphoid is common in commercial poultry operations worldwide. Two common paratyphoid organisms are S. enteritidis and S. typhimurium. S. enteritis occurs in commercial layer (2% of US) and S. typhimurium in poultry flocks. They are common causes of gastroenteritis in humans through contaminated poultry products. In the US, S. enteritidis is not a pathogen in poultry, but is an important cause of disease in some parts of Europe.

Salmonella are horizontally and vertically transmitted. Pullorum and paratyphoid diseases primarily affect young poultry, whereas typhoid can occur at any age. Lesions include fibrinopurulent perihepatitis, pericarditis, and necrosis of the intestinal and reproductive tracts.

Sample Collection

Liver, spleen, heart, gall bladder, blood, ovary, yolk sac, joints, eye and brain can be used for isolation on non-selective media. The gut is commonly colonized by salmonellae, with the ceca most often infected. Tissues may be ground and inoculated onto agar or broth. Gut tissues generally require selective media to inhibit common nonpathogenic contaminants. The yolk sac of day-old chicks is a good source for isolation. Feed, water and litter may also be taken from poultry houses. Sterile cotton swabs can be used for isolation. Cotton swabs can be dragged along litter to check for environmental contamination or be used to check breeder nests, laying cages or hatchery machines. Swabs can be stored under refrigeration in a sterile holding media such as 200 gr of Bacto Skim Milk in 1 litter of distilled water.

Culture Media

None-Selective media include beef extract and beef infusion. Selective media include tetrathionate broths, selenite enrichment broths, MacConkey's agar or eosin methylene blue agar (EMG). Selective plating media include brilliant green (BG) agar supplemented with novobiocin (BGN) and XLD agar supplemented with novobiocin (XLDN). Salmonella colonies on BG and BGN agar are transparent pink to deep fuchsia, surrounded by a reddish medium. The HB2BS positive colonies on XLD or XLDN agars are jet black. Pink colonies/to 2mm in diameter are present on MacConkey's agar and dark colonies 1mm in diameter on EMB. A Gram stain reveals negative rods.

Rapid Salmonella Detection Techniques

A variety of rapid detection systems include enzyme immunoassay antigen capture assays, DNA probes, and immunofluorescence. These techniques will be discussed later in the book.

Basic Identification Media

A combination of triple-sugar-iron (TSI) and lysine-iron (LI) agars are sufficient for presumptive identification of salmonella. On TSI agar, salmonellae produce an alkaline (red) slant and acid (yellow) butt, with gas bubbles in the agar and a blackening due to HB2BS production. Salmonellae will show lysine decarboxylation, with a deeper purple (alkaline) slant and alkaline or neutral butt with a slight blackening due to HB2BS production. Before doing serological screening procedures, the culture should be further evaluated using additional identification media (Table 1.0). Commercial kits employing more extensive tests include API—20E (Analy Lab Products, Plainview, NY), or Enterotube I (Roche Diagnostics, Montclair, NJ) are also available.


Table 1.0. Reactions of Salmonella Cultures in Media

Media S. pullorum S. gallinarun Paratyphoid

Dextrose A A AG

Lactose - - -

Sucrose - - -

Mannitol A A AG

Maltose - A AG

Dulcitol - A AG

Malonate broth - - -

Urea broth - - -

Motility media - - +

A = Acid, G = gas produced

Figure 0.1.0 Slide agglutination

Serologic Identification

These methods including slide (figure 1.0) and plate agglutination will be discussed in a later chapter. References

Mallison, E.T. and G.H. Snoeyenbos, 1989. "Salmonellosis." In A Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co. Dubuque, Iowa. pp. 3-11.

Escherichia

Introduction

Escherichia coli cause a common systemic infection in poultry known as colibacillosis. Colibacillosis occurs as an acute septicemia, or chronic airsacculitis, polyserositis, or infectious process in young poultry. Coligranuloma is a chronic infection resulting in lowered egg production, fertility and hatchability in adult birds.

Clinical disease

Clinical signs are not specific and vary with bird age, duration of infection and concurrent disease conditions. In septicaemia in young birds, signs include: anorexia, inactivity and somnolence. Lesions may


be seen as swollen, dark-colored liver and spleen and Ascities. Chronically affected birds may have fibrinopurulent airsacculitis, pericarditis, perihepatitis, dermatitis and lymphoid depletion of the bursa and thymus. Arthritis, osteomyelitis, salpingitis, and granulomatous enteritis, hepatitis and pneumonia may occur in older birds.

Sample Collection

Heart, liver, lungs, spleen, bone marrow, joints and air sacs are all good specimens for isolation using a sterile swab or needle or ground tissue. Cultures may be stored in E. coli broth upon refrigeration.

Culture media

E. coli, a gram – rod (figure 1.2), grows well in meat media, Tryptose blood, blood agar, SI medium, Lysine iron agar (LIA), MacConkey's agar (figure 1.1). Differential biochemical media can be used such as triple iron agar slants or identification kits (API-2OE or Enterotube I). On blue agar E. coli will show white glistening, raised colonies 1-to-3 mm diameter and under the microscope as gram-negative rods. On MacConkey's agar pink, 1-2 mm diameter dry colonies with dimple will be evident. On TSI slant, E. coli will produce a yellow slant and butt with gas but no HB2BS (no black color). On SMI medium the indole reaction is positive, HB2BS negative and motility +/-. In LIA the slant will be alkaline and the butt acid with no HB2BS production.

Figure 1.1 E. coli

Figure 1.2 Gram negative rods

References

Arp, L.H., 1989. "Colibacillosis." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 12-13.

Pasteurella

Introduction


The disease caused by the infection with Pasteurella multocida, a bipolar encapsulated rod (figure 1.3), inpoultry is called fowl cholera. It is common world wide and affects all species of birds including turkeys, chickens, quail and wild water fowl.

Pasteurella

Figure 1.3 Bipolar encapsulated rods

Clinical disease

The disease occurs in birds of any age, but is more common in semi mature to mature birds. It can occur as an acute septicemic disease with high morbidity and mortality, or chronic with low level of performance in adult flocks. Signs include depression, diarrhea, respiratory signs, cyanosis, lameness and/or acute death. Lesions include hyperemia, hemorrhages, swollen liver, focal necrotic areas in the liver and spleen and increased pericardial fluids. Swollen joints and exudate in the wattles, comb and turbinates may be seen in chronic cases.

Sample Collection

The organism may be isolated from the liver, spleen, gall bladder, bone marrow, heart and affected joints with a sterile needle or swab. The organism is fairly stable on short term storage. Preferred Culture Media

Dextrose Starch agar (DSA), blood agar or trypticase soy agar are recommended for primary isolation. On DSA, 24 hour colonies are circular, 1-3 mm in diameter, smooth, translucent, and glistening. Colonies on blood agar are similar to those on DSA, but appear grayish and translucent. P. multocida cells are typically rods of 0.2-0.4 x 0.6-2.5 um occurring singular in pairs of short claims. Cells in tissues or from agar show bipolar staining with Giemsa, Wayson's or Wright's stains. Capsules can be demonstrated by mixing a loop full of India Ink on a slide and the colony and examining it at high magnification.

P. multocida can be further identified with biochemical tests. Fructose, galactose, glucose, and sucrose are fermented without gas production. Indole and oxidase are produced and there is no hemolysis of blood or growth on MacConkey's agar.

References

Rhoades, K.R., R.B. Rimler and T.S. Sandhu, 1989. "Pasteurellosis and Pseudotuberculosis." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 14-21.


Staphylococcus

Introduction

Staphylococcus aureus (Figure 1.4) is frequently the cause of arthritis, synovitis, and localized abscesses in joints, foot pads, skin, and over the breast muscle. The organism is ubiquitous in poultry houses and is a common primary and secondary invader.

Figure 1.4 Colonies on blood agar

Clinical Disease

Staphylococcosis appears to be a classical opportunistic infection. Clinical disease is more frequent in birds subjected to poor husbandry conditions such as overcrowding, sharp objects in the house, poor ventilation, wet damp litter and birds that are immunosuppressed. The organism typically occurs following recent viral or mycoplasma infections in the joints. The infection often occurs locally through a wound and then may spread and become septicemic. The liver, spleen, and kidneys, may become swollen. S. aureus may begin as a swelling in the breast area, foot pad or gangrenous dermatitis or as yolk sac infections from a hatchery or breeder flock. Localized lesions may contain a white or yellow cheesy exudate. Septicemic lesions may have necrotic and/or hemorrhage foci and cause swelling and discoloration of the tissues.

Sample Collection

Specimens for culture include blood or exudate from lesions. They can be collected from sterile swabs, loops or by needles and syringes. No special precaution is needed for handling, transportation or storage of materials.

Culture media

Staphylococci grow readily on ordinary media. Blood agar or thioglycollate broth supports the growth of the organism. Selective media include Manitol-salt agar or the similar staphylococcus 110 medium.

Agent Identification

On agar cultures, staphylococci produce 1 to 3 mm diameter, circular, opaque, smooth, raised colonies in 18 to 24 hours. S. aureus are hemolytic on blood agars (figure 1.5). On manitol-salt agar, S. aureus colonies are surrounded by a yellow halo. Colonies are examined microscopically to confirm that they contain gram-positive cocci. A positive coagulase test will confirm they are pathogenic staphylococci. Commercially available desiccated rabbit plasma containing either citrate or EDTA is used for the coagulase test. A commercial coagulase test that uses microtubes (STAPHase, Analylab Products, Plainview, NY) is also available.


Figure 1.5. Isolation of organisms from tissues

References

Jensen, M.M. and J.K. Skeeles, 1989. "Staphylococcosis." In a Laboratory manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing Co., Dubuque, Iowa, pp. 43-44.

Mycoplasma

Introduction

Mycoplasma are tiny prokaryotic organisms characterized by their lack of a cell wall (figure 1.6). There are numerous species of mycoplasma that infect poultry, however, the most common and pathogenic are M. gallisepticum and M. synoviae. They are found in commercial breeder and layer flocks world wide and may cause drops in egg production, fertility and hatchability as well as respiratory and skeletal system disease. They may be transmitted in flocks both vertically and horizontally.

Clinical disease

M. gallisepticum (MG) is a cause of respiratory disease and egg production drops in chickens and turkeys. Severe airsacculitis, swollen sinuses, coughing, rales, depressed weight gain, poor feed conversion, mortality and increased condemnation in the processing plant. M. synoviae causes lesions of synovitis and respiratory disease in chickens and turkeys.

Sample Collection

Cultures may be taken from the trachea, choanal cleft, affected joints, sinuses or air sacs, with sterile swabs. Tissues may be shipped frozen for later isolation. Isolated culture may be shipped in broth medium by overnight carrier.

Culture media

Mycoplasmae are fastidious organisms that require a protein based medium enriched with 10—15% serum. Supplementation with yeast and/or glucose is helpful. M. synoviae requires nicotinamide adenine


dinucleotide (NAD), cysteine hydrochloride is added as a reducing agent for the NAD. A commonly used media is Frey's (Table 1.1).

Figure 1.6. Mycoplasma colonies

Table 1.1. Frey's Media Formulation

Constant Amount

Mycoplasma broth base (BBL, Cockeysville, MD) 22.5 g

Glucose 3 gr

Swine Serum 120 ml

Cysteine hydrochloride 0.1 gr

NAD 0.1 gr

Phenol red 2.5 ml

Thallium acetate (10%) 2.5 to 5 ml

Penicillin G Potassium 10P6P units

Distilled HB2BO 1,000 ml

Adjust pH to 7.8 with 20% NaOH and filter sterilize

Broth cultures incubated at 37 aerobically are generally more sensitive than agar. Cultures are incubated until the phenol red indicator changes to orange, but not yellow. This may take anywhere from 2 to 5 days. Agar plates are examined for colonies under low magnification under regular light or with a dissecting microscope. Colonies are usually evident after 3 to 5 days.


Tiny, smooth colonies 0.1 to 1 mm in diameter with dense, elevated centers are suggestive of mycoplasma (Figure 1.6). Mycoplasma speciation is by serological methods using polyclonal or monoclonal antibody. Serological tests include immunodiffusion, agglutination, enzyme linked immunosorbent assay (ELISA) and immunofluorescence. The problem with polyclonal serum is that there can be cross reactions between MG and MS. Also, the serum may contain antibodies against the serum present in the medium and give false positives. Breeders given inactivated vaccines, especially vaccines against Pasteurella, may have false positive serologic reactions up to 6 weeks post vaccination. Therefore, the use of monoclonal antibodies, to be discussed later in this book, is most desirable.

References

Kleven, S.H. and H.W. Yoder, 1989. "Mycoplasmosis." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens, Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 57-62.


Mycology (Fungi)

Fungi are eukaryotic organisms that do not contain chlorophyll, but have cell walls, filamentous structures, and produce spores. These organisms grow as saprophytes and decompose dead organic matter. There are between 100,000 to 200,000 species depending on how they are classified. About 300 species are presently known to be pathogenic for man.

There are four types of mycotic diseases:

1. Hypersensitivity - an allergic reaction to molds and spores.

2. Mycotoxicoses - poisoning of man and animals by feeds and food products contaminated by fungi which produce toxins from the grain substrate.

3. Mycetismus- the ingestion of preformed toxin (mushroom poisoning).

4. Infection

Aspergillus

Introduction

The most common fungal disease of poultry is Aspergillosis. It is primarily a respiratory disease, but the organism can spread to the brain and eye causing central nervous signs and blindness. The organism is common in warm moist environments, which include hatcheries and poultry houses. Young birds are most susceptible, since their immune system and respiratory tract cilia, responsible for trapping foreign objects, are less developed at that age.

Clinical Disease

Aspergillus fumigatus and A. flavus are common causes of disease in commercial young poultry. The pulmonary system is the initial point of entry, but the agent may spread to the gastrointestinal tract, eye or central nervous system. There are two forms of the disease. The acute form occurs as brooder pneumonia in young animals causing respiratory disease and death. The chronic form occurs in older birds and may result in respiratory signs or torticollis and cloudy eyes. Small, white cheesy nodules may occur in acute disease in the lungs, airsacs or intestinal tract. Plaques (yellow or gray) may occur in chronic cases in the brain or respiratory tract.

Sample Collection

Lesions are the preferred source for culture using sterile swabs or inoculation loops. The samples may be shipped or stored for a short time at refrigeration temperatures.

Culture Media

Initial isolation may be accomplished on blood agar, or Sabouraud's dextrose agar. Specimens can be smeared on plates or minced in a grinder with sterile saline. The plates can be inoculated at 37C for 1 to 3 days. Chloramphenicol (0.5 g/liter) can be added to the media to inhibit bacteria growth.


Figure 2.1 Fungal culture


Small greenish blue colonies with fluffy down (Figure 2.1) can be transferred to Czapek's solution agar (Difco Lab, Detroit, MI) for a definitive diagnosis. Scrapings of a colony or from a lesion can be placed on a microscope slide and stained with 20% KOH. Branching septate hyphae 4 — 6 micron in diameter will be evident. The presence of the conidial head is needed to differentiate the various species of Aspergillus. Lactophenol, a semi-permanent mounting medium, contains 20 gr of phenol, 40 ml of glycerin, 20 ml of lactic acid and 20 ml of distilled HB2BO. For staining hyphae and examination of conidia, 0.05 g of cotton blue can be added to make lactophenol cotton blue. A piece of colony can be teased apart with a needle, stained, marked and mounted with a cover slip. Species identification may be achieved on the basis of morphological criteria upon microscopic examination. References

Richard, J.L. and E.S. Beneke, 1989. "Mycosis and Mycotoxicosis." In a Laboratory Manual for the Isolation and identification of Avian Pathogens. Kendall/Hunt Publishing Co., Dubuque, Iowa, pp. 70-76.

Viruses

Introduction

Viruses are important subcellular pathogens of poultry. Viruses are tiny obligate intracellular organisms. They can only be seen with the electron microscope, and since they don't have cellular organelles or metabolic machinery, they can only be propagated in a living host and are not affected by common antibiotics. Important viruses of poultry include: infectious bronchitis, Newcastle disease, influenza, laryngotracheitis, and pneumo viruses which cause respiratory diseases; Marek's disease and lymphoid leukosis viruses, which cause lymphoid tumors, and immunosuppression; adenoviruses, chicken anemia virus, reoviruses, and infectious bursal disease viruses, which cause morbidity, mortality and/or immunosuppression; and fowl pox virus which causes skin and oral lesions.

Knowledge of viral replication and genetics is necessary for understanding the interaction between the virus and the host cell. The interaction at the cellular level and progression of a particular viral infection determines disease pathogenesis and clinical manifestations. The host immune response to the presence of viruses will be examined later.

Interaction Between Viruses and Host Cells

The interaction between viruses and their host cells is intimately tied to the replication cycle of the virus. Moreover, the interaction of virus with cellular components and structures during the replication process influences how viruses cause disease. Overall, there are four possible primary effects of viral infection on a host cell. Most infections cause no apparent cellular pathology or morphological alteration; however, replication may cause cytopathology (cell rounding, detachment, syncytium formation, etc.), malignant transformation, or cell lysis (death).


Cell Death

Cell death during viral replication can be caused by a variety of factors. The most likely factor is the inhibition of basal cellular synthesis of biomolecules, such as proteins. During the replication cycle, the virus induces the cellular machinery to manufacture largely viral products rather than those the cell would normally make. As a result, the predominant products synthesized by the cell are viral and the cellular products necessary for the survival of the cell are not present or present in too low a quantity to maintain its viability. In addition to the lack of essential cellular products, this event results in accumulation of viral products (RNA, DNA, proteins) in excess, which can be toxic for the cells. In the release phase of the replication cycle of some viruses, apoptosis of the host cell is stimulated. In other instances, inhibition of the synthesis of cellular macromolecules causes damage to lysosomal membranes and subsequent release of hydrolytic enzymes resulting in cell death.

Cellular Effects

Cytopathic effect (CPE) denotes all morphologic changes in cells resulting from virus infection. Infected cells sometimes have an altered cell membrane; as a result the infected cell membrane is capable of fusing with its neighbor cell. It is thought this altered membrane is the result of the insertion of viral proteins during the replication cycle. The result of fusion is the generation of a multinucleate cell or syncythia. The formation of syncythia is characteristic for several enveloped viruses, such as herpesviruses and paramyxoviruses. The altered cell membrane also is altered with regard to its permeability, allowing influxes of various ions, toxins, antibiotics, etc. These multinucleate cells are large and are sometimes called "multinucleate giant cells".

Another aspect of CPE is the disruption of the cytoskeleton, leading to a "rounded up" appearance of the infected cell. The cell in this case will either lyse or form syncythia. CPE occurrence in clinical specimens can indicate viral infection and CPE is used as the basis for the plaque assay used in viral enumeration. Infection of cells with some viruses (e.g., poxviruses and rabies virus) is characterized by the formation of cytoplasmic inclusion bodies. Inclusion bodies are discrete areas containing viral proteins or viral particles. They often have a characteristic location and appearance within an infected cell, depending upon the virus.

Malignant Transformation

In this process, viral infection results in host cells that are characterized by altered morphology, growth control, cellular properties, and/or biochemical properties. Malignant transformation and resulting neoplasia may occur when the viral genome (or a portion) is incorporated into the host genome or when viral products are themselves oncogenic. Viruses causing malignant transformation are referred to as tumor viruses. Viruses from different families have been shown to possess the ability to transform host cells. The tumor viruses have no common property (size, shape, chemical composition) other than the development of malignancy in the host cell. Malignant transformation is often characterized by altered cellular morphology. This includes the loss of their characteristic shape and assumption of a rounded up, refractile appearance as described for CPE. This is the result of the disaggregation of actin filaments and decreased surface adhesion.

Altered cell growth, the hallmark for malignant transformation, is exhibited in viral cells that have lost contact inhibition of growth or movement, have a reduced requirement for serum growth factors, and/or no longer respond to cell cycle signals associated with growth and maturation of the cell (immortality). Some of the altered cellular properties exhibited by malignantly transformed cells include continual induction of DNA synthesis, chromosomal changes, appearance of new or embryonic surface antigens, and increased agglutination by lectins. Commonly altered biochemical properties of malignantly transformed cells include reduced levels of cyclic AMP. Cyclic AMP is a chemical signal associated with the cell cycle and by keeping the levels reduced the cell continually divides. Also involved is the increased secretion of plasminogen activator (clot formation), fermentation for the production of lactic acid (known as the Warburg effect), loss of fibronectin, and changes in the sugar components of glycoproteins and glycolipids.

Oncogenesis

Although cause-and-effect has been difficult to obtain, a number of DNA and RNA viruses have been associated with neoplastic transformation. Viruses implicated in oncogenesis either carry a copy of a gene associated with cell growth and proliferation or alter expression of the host cell’s copy of the gene. Effected genes include those that stimulate and those that inhibit cell growth. Viral genes that transform infected cells


are known as oncogenes (v-onc genes), which stimulate uncontrolled cell growth and proliferation. The discovery of oncogenes led to the finding that all cells contain analogous genes, called proto-oncogenes (c-onc genes), which are normally quiescent in cells as they are active at some point in development. Proto-oncogenes include cellular products such as growth factors, transcription factors, and growth factor receptors.

DNA viruses associated with oncogenesis include the Marek’s disease virus (Herpesviridae). This virus is typically circular episomic (independent of the host chromosome, rather than integrated) nucleic acids. The oncogenes (v-onc) encode proteins associated with the replication cycle of the virus. RNA viruses associated with oncogenesis include members of the family Retroviridae (e.g., avian leukosis virus). These viruses integrate their genomes (or a copy of the genome) into the host chromosome; referred to as proviruses or proviral DNA. Viral integration is mediated by the terminal ends of the genome, known as LTRs (long terminal repeats). LTRs contain promoter/enhancer regions, in addition to sequences involved with integration of the provirus into the host genome. Retroviruses can cause oncogeneses by encoding oncogenes themselves or by altering the expression of cellular oncogenes or proto-oncogenes through insertion of their genomes into the host chromosome close to these genes.

No Morphological or Functional Changes

In some instances, infection with viral production can occur with no discernable change in the host cell. This is referred to as an endosymbiotic infection. This is probably dependent upon the replication needs of the virus. Most likely the virus requires cellular processes to be active in order for viral replication to take place and thus does not alter the features of the cell.

Pathogenesis of Viral Infections

Pathogenesis is defined as the origination and development of a disease. Viral infections can be acute, chronic, latent or persistent. The first step in the disease process is exposure.

Exposure and Transmission

Exposure may occur by direct contact with an infected animal, by indirect contact with secretions / excretions from an infected animal, or by mechanical or biological vectors. Transmission of virus from mother to offspring (transplacental, perinatal, colostrum) is called vertical transmission. Transmission via other than mother to offspring is horizontal transmission. Activation of latent, nonreplicating virus can occur within an individual with no acquisition of the agent from an exogenous source.

Portal of Entry

Viruses enter the host through the respiratory tract (aerosolized droplets), the alimentary tract (oral-fecal contamination), the genitourinary tract (breeding, artificial insemination), the conjunctivae (aerosolized droplets), and through breaches of the skin (abrasions, needles, insect bites, etc.). Whether or not infection ensues following entry depends upon the ability of the virus to encounter and initiate infection in susceptible cells. The susceptibility of cells to a given virus depends largely on their surface receptors, which allow for attachment and subsequent penetration of the virus.

Localized and Disseminated Infections

Following infection, the virus replicates at or near the site of viral entry (primary replication). Some viruses remain confined to this initial site of replication and produce localized infections. An example is the common cold and similar infections in domestic animals caused by rhinovirus. Other viruses cause disseminated (systemic) infections by spreading to additional organs via the bloodstream, lymphatics or nerves. The initial spread of virus to other organs by the blood stream is referred to as primary viremia. Viremia can be either by virus free in the plasma or by virus associated with blood cells. After multiplication in these organs, there may be a secondary viremia with spread to target organs.

The virus is transmitted in a fecal-oral fashion. It initially replicates in the cells of the tonsils, migrates to the intestines and mesenteric lymph nodes. From the mesenteric lymph nodes, the virus enters the central nervous system. Once in the central nervous system, the neurological symptoms of: ataxia, tremors, loss of


coordination, stiffening of the limbs, convulsions, paralysis, and coma are observed. The preference of a particular virus for a specific tissue or cell type is known as tropism.

Mechanisms of Viral Infections

Virus replication occurs in target organs causing cell damage. The number of cells infected and/or the extent of damage may result in tissue/organ dysfunction and in clinical manifestation of disease. The interval between initial infection and the appearances of clinical signs is the incubation period. Incubation periods are short in diseases in which the virus grows rapidly at the site of entry (e.g., influenza) and longer if infections are generalized (e.g., canine distemper). Some viruses infect animals but cause no overt signs of illness. Such infections are termed subclinical (asymptomatic or unapparent). There are numerous factors that may influence the outcome of viral infections. These include preexisting immunity, genetics of the animal, age of the animal, and stress related factors such as nutritional status, housing, etc.

The mechanisms by which viruses cause disease are complex. Disease may result from direct effects of the virus on host cells, such as cell death, CPE, and malignant transformation. Alternatively, disease results from indirect effects caused by the immunologic and physiologic responses of the host. An example of indirect physiologic response is infection with rotavirus, which causes diarrhea in young animals and humans. Diarrhea may be caused by rotavirus-infected erythrocytes that are stimulated to produce cytokines, exciting enteric neurons, and inducing the secretion of excess fluids and electrolytes into the large intestine. The virus spreads from the CNS to peripheral nerves within axons. The host responds to the presence of the virus-infected neurons by inducing a cell-mediated immune response. Macrophages, neutrophils, and specific cytotoxic T lymphocytes are activated to kill bornavirus-infected neurons. The result is chronic inflammation in the CNS that corresponds with the neurological signs associated with the disease.

Two very important terms used in the discussion of microbial diseases are pathogenicity and virulence. Pathogenicity denotes the ability of a virus or other microbial/parasitic agent to cause disease. Virulence is the degree of pathogenicity. An avirulent virus is one lacking the capacity to cause disease. An attenuated virus is one whose capacity to cause disease has been weakened frequently by multiple passages in cell cultures, embryonated eggs or animals.

Virus Shedding

Virus shedding is the mechanism of excretion of the progeny virions to spread to a new host, thus maintaining the virus in a population of hosts. Viruses are typically shed via body openings or surfaces. For localized infections, virus is typically shed via the portal of entry. In disseminated infections, virus may be shed by a variety of routes. Not all viruses are shed from their hosts. These include viruses that replicate in sites such as the nervous system, as in viral encephalitis, and dead-end hosts.

Evasion of Host Defenses

In an effort to ward off the infection, the host initiates an inflammatory response. Principal components of this response include interferons, cytotoxic T lymphocytes, antibody producing B-lymphocytes, a variety of effector molecules, and complement. These various components work in concert and augment one another in an attempt to rid the host of the infecting virus. In this effort to rid itself of the infecting virus, the inflammatory response causes many of the clinical signs and lesions associated with viral infections.

Interferons (α and β) are produced by virus-infected cells. They act to stop further virus replication in the infected and neighboring cells. Interferons also enhance antigen expression on infected cells, thereby making them more recognizable to cytotoxic T cells. Some viruses (e.g., adenovirus) produce RNAs that block the phosphorylation of an initiation factor, that reducing the ability of interferon block viral replication.Cytotoxic T cells kill viral infected cells by releasing perforins, which create pores in the virus-infected cell. Granzymes are then released into the virus-infected cell, which degrade the cell components. Lastly, cytotoxic T cells stimulate apoptosis of the host cell.

Some viruses reduce the expression of MHC class I antigens on the surface of the host cell (e.g., cytomegalovirus, bovine herpesvirus type I, adenoviruses). As cytotoxic T cells cannot detect viral antigens that are not complexed with MHC class I antigens, virus-infected cells cannot be destroyed in this manner, allowing "survival" of the virus within the host. However, cells with no or insufficient MHC class I antigen on


their surface are recognized by natural killer cells, which kill the cell in a manner similar to that described for cytotoxic T cells. Antibody producing B-lymphocytes secrete specific antibodies to neutralize the infectious virions when the cell liberates them. Antigen-antibody complexes in turn can activate the complement system. Complement aids in stimulating inflammation and the effective neutralization of virus and in the destruction of viral infected cells.

The various effector molecules (cytokines) that are produced by the cells of the immune system have many roles, including the induction of fever and the attraction of other inflammatory cells, (e.g., neutrophils and macrophages) to the injured site. Some viruses possess receptors for a variety of cytokines (e.g., vaccinia virus has receptors for interleukin-1, which stimulates fever production). When immune cells release the cytokine, it is bound to the virus. This, in turn, reduces the amount of the cytokines available to modulate immune responses. This enhances the "survival" of the virus within the host. An alternate mechanism to evade the immune response is to have many antigenic types (serotypes). An immune response to one serotype does not guarantee protection from another serotype of the same virus. For example, there are over 100 serotypes of rhinovirus and 24 serotypes of bluetongue virus.

Persistent Viral Infections

Some viruses have the ability to abrogate the inflammatory response and cause persistent infections. They accomplish this in a number of ways, including the destruction of T lymphocytes causing immunosuppression, the avoidance of immunologic surveillance by altering antigen expression, and by the inhibition of interferon production.

There are three clinically important types of persistent infections:

Chronic-carrier infections

These are organisms that continually produce and shed large quantities of virus for extended periods of time. As a result they continually spread the virus to others. Some chronic-carriers are asymptomatic or exhibit disease with very mild symptoms. Examples include infections with equine arthritis virus, feline panleukopenia virus, and avian polyoma virus.

Latent infections

A special type of persistent infection is one in which the virus is maintained in the host in a "non-productive" state. Herpesviruses are notorious for causing latent infections. The viral genome is maintained in neurons in a closed circular form, and is periodically reactivated (often during stressful conditions) resulting in a productive infection and viral shedding. Latent infections also occur with retroviruses in which the proviral DNA is incorporated into the host cell genome. Cell transformation and malignancy may result if the integrated transcript causes a disruption of normal cellular control processes.

Slow Virus Infections

This refers to those viral infections in which there is a prolonged period between initial infection and onset of disease. In this case, viral growth is not slow, but rather the incubation and progression of disease are extended.

CULTIVATION OF VIRUSES IN CHICKEN EMBRYOS

Propagation of viruses is done for their initial isolation and detection, passage for stock cultures, chemical analysis, vaccine production, preparation of antigens for serological tests, and for other immunological and molecular needs Since viruses can only be propagated in living hosts, embryonating eggs, tissues and cell cultures have been commonly used for their cultivation. Chicken embryos are used because of their (1) availability, (2) economy, (3) convenient size, (4) freedom from latent infection and extraneous contamination, and (5) lack of production of antibodies against the viral inoculum. Eggs from healthy, disease-free flocks should be used.


Incubation of embryos is usually at 98.8—99.5F (37.1—37.5C) throughout the entire period. Lower temperatures may be required under certain circumstances.

Knowledge of the development of the avian embryo is necessary for utilization of this medium for cultivation of viruses. The embryo commences development as a sheet of cells overlying the upper pole of the yolk. The embryo is recognized with difficulty during the first few days, but at 4- or 5-days of incubation it may be readily detected by candling. From the 10th day the embryo rapidly increases in size and feathers appear. As the embryo increases in size, there is an accompanying decrease in the volume of the extraembryonic fluids. At the time of hatching there is no free fluid in any of the extraembryonic cavities. Throughout incubation there is a steady loss of water by transpiration through the shell.

The amnion and chorion arise by a process of folding and overgrowth of the somatopleure. The amnion develops first over the head and then the caudal region. By fusion of the lateral folds, the amnion completely envelops the embryo, except for the yolk sac, from the 5th day of incubation. From the 6th to 13th days there is an average of about 1 ml of amniotic fluid. By the 10th day, the chorion almost completely surrounds the entire egg contents and is in immediate contact with the shell membrane.

The allantois appears on the 3rd day as a diverticulum from the ventral wall of the hind gut into the extraembryonic cavity and rapidly enlarges up to the 11th or 13th day. During the process of enlargement, the outer layer of the allantois fuses with the outer layer of the amnion and the inner layer of the chorion to form the allantoic cavity. The amount of allantoic fluid varies from about 1 ml on the 6th day to 10 ml on the 13th day. The fused chorion and allantois is known as the chorioallantoic membrane, which is highly vascular and constitutes the respiratory organ of the embryo.

In the early stages of development, the amniotic and allantoic fluids are solutions of physiologic salts. After about the 12th day, the protein content and viscosity of the amniotic fluid increases. The allantoic cavity receives the output of the kidneys, and after the 12th or 13th day the allantoic fluid becomes turbid because of the presence of urates. The yolk sac consists of a steadily enlarging sheet of cells. From the 12th day on, the yolk material becomes progressively drier and the yolk sac more fragile. During the last 24 to 48 hours of incubation, the yolk sac is drawn into the abdominal cavity.

Routes of Inoculation and Collection of Specimens

The various procedures outlined for inoculation of chicken embryos and for collection of specimens are a compilation of methods. Tissues and organs from embryos and birds should be collected aseptically using standard recovery procedures. The CAM, yolk sac and embryo or bird tissues should be ground as a 10% suspension in a sterile diluent with antibiotics and then centrifuged at low speed (1,500 x g for 20 minutes before inoculation).

Some of the factors influencing the growth of viruses in chicken embryos are (1) age of the embryo, (2) route of inoculation, (3) concentration of virus and volume of inoculum, (4) temperature of incubation, and (5) time of incubation following inoculation. The presence of maternal antibody in the yolk of hens immunized against or recovered from certain viral infections, precludes the use of the yolk sac route for initial isolation and subsequent passage of viruses. All fluids from live birds suspected of having virus material should have antibiotics such as gentamicin, penicillin+streptomycin and fungizone added to it before inoculation into embryos.

It may take several “blind” passages (no pathology), before noticeable pathologic changes take place in the embryo, if the virus is in small amount. When working with viral infected material, one should always practice sterile technique and work under a class II microbiological safety cabinet.

Allantoic Cavity inoculation employs embryos of 9- to 12-days incubation. The inoculum is generally 0.1—0.2 cc. Some of the avian viruses which grow well in the allantoic entoderm are those of Newcastle disease, infectious bronchitis, and influenza. This route has the advantage of simplicity of inoculation and collection of specimens when large quantities of virus-infected fluid are to be obtained for use in chemical analysis, vaccine production, and preparation of antigen for serologic tests.

1.Candle the embryos and select an area of the chorioallantoic membrane distant from the embryo and amniotic cavity and free of large blood vessels about 3 mm below the base of the air cell. In this area, make a pencil mark at the point for inoculation.


2.Make a similar mark at the upper extremity of the shell over the air cell.

3.Apply tincture of suitable disinfectant to the holes and allow to dry.

4.Drill a small hole through the shell at each mark but do not pierce the shell membrane.

5.Using a syringe with a 25 gauge 5/8 inch (16 mm) needle, inoculate 0.1 to 0.2 ml inoculum per eggs by inserting the entire length of the needle vertically through the hole and injecting the desired amount.

6.Seal the hole with glue or hot wax and return the eggs to the incubator.

7.Candle the eggs daily for 3 to 7 days for signs of death (absence of blood vessels and a dead embryo at the bottom of the egg).

8.Eggs dying in 24 hours should be discarded, since their death was probably due to bacteria (which can be determined by isolation of fluids in media) or trauma.

9.Embryos dying after that time should be refrigerated for 1 hour then fluids harvested and frozen at -70C for later use.

10.Blind passage of suspected viral inoculum can be accomplished by reinoculating the allantoic fluids every 5 to 7 days into fresh eggs until pathology or death occurs. Initial isolation of some viruses from clinically ill birds including infectious bronchitis virus (IBV) may take as many as 3 passages for embryo death to occur.

11.Always check live or dead embryos after harvesting fluids for evidence of pathologic changes such as curling and stunting for IBV, and stunting and hemorrhages (Figure 3.1) for Newcastle disease virus (NDV) or reoviruses.

Figure 3.1 Embryo pathology induced by virus in embryo on the right

12.All eggs should be disinfected before harvesting.

13.Crack the eggshell over the air cell by tapping the eggshell with the blunt end of sterile forceps. Remove the eggshell which covers the air cell, being careful not to rupture the underlying membranes, and discard pieces of the eggshell in disinfectant. Discard forceps in a beaker of disinfectant.

14.Use forceps to tear the eggshell membrane, the CAM, and the amniotic membrane to release the fluid. Depress the membranes over the yolk sac with the forceps and allow the fluid to collect and pool above the forceps. Using a 5-ml pipette or syringe and needle, aspirate the fluid and place it into a sterile 12 x 75-mm snap-cap tube or other suitable vial. It may be necessary to carefully peel back the eggshell membrane from the CAM to permit a better view of the membranes.

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MERGEFORMATINET

Viral isolation in embryos

15.Clarify the fluid by centrifugation at 1500 x g for 10 minutes and test the fluid for evidence of virus infection using hemagglutination, electron microscopy, or other suitable methods.


16.Store at -70C for passage or other use.

I. Avian Influenza Isolation

Swab preparation

BHIB with Penicillin/Streptomycin (P/S)

Procedure:Add 900 ml ddH2O to 1 L flask.Add 37g BHI powder and dissolve completely by stirring over low heat.Pour half of the BHIB solution into another 1L falsk and cover both with foil.Autoclave flasks for 15 min on a liquid exhaust cycle and cool on bench top.In hood, add 6.02g of Penicillin-G to 150 ml beaker.WEAR GLOVES AND MASK!In hood, add 10g of streptomycin sulfate to 150 ml beaker. WEAR GLOVES AND MASK!In hood, add 50 ml ddH2O to 150 ml beaker and dissolve P/S.Transfer P/S/ solution to 100 ml flask and add 100 ddH2O to 100 ml.Filter P/S solution through a 0.22um filtration device into a sterile 125mL bottle.Label and store in refrigerator until BHIB is cool.Once BHIB is cool, add P/S solution (50 ml to each flask) and stir.Pour into ten sterile 125 mL bottles.Label bottles, put tape around bottle neck/cap, and store in freezer. To prepare sampling vials:Using sterile technique and working under a hood pipette 1.8mL BHIB with P/S into each disposable 5mL capped tubes.Freeze vials in lab freezer until needed for sampling.

II. Sample collection

Collect cloacal swabs, place in tubes and store on ice. Samples can be shipped with cool packs if they will arrive at the laboratory within 48 hours. Upon arrival in the lab, centrifuge fluid at low speed (500-1500 x g) to sediment debris. Supernatant should be kept at 22-25 C for up to 15-60 min, to allow the antibiotics to reduce the level of bacterial contamination. Supplementation with additional antibiotics may be needed. If further reduction in bacterial contamination is required to reduce embryo deaths or nonviral HA activity of egg fluids, the supernatant can be filtered through prewet 0.22-0.45-µm filter. However, filtration can remove low levels of virus from samples and reduce isolation rates. Put samples in 3 vials/case then store at -700C.

III. Processing cloacal swabs – inoculation

Candle 10 day old fertile eggs. Mark the edge of the air sac on viable eggs and discard dead/infertile eggs.Arrange eggs on a plastic flat in six rows of four eggs.Label eggs with sample/bird number and egg letter (A thru D for each sample)Retrieve samples (Swabs in vials of BHIB) from the ultra cold freezer, thaw quickly in a 37 C water bath, and then keep cold for the rest of the procedure, either in the refrigerator or an ice bath.Vortex samples for 1 min, then centrifuge for 15-20 minutes at 1200 rpm.Sanitize eggs by lightly wiping with sterile swab dipped sparingly in 5% iodine in alcohol solution.Sanitize egg punch with 70% alcohol. Use the egg punch to puncture a small hole in the shell just above the air sac line. Do not damage the membrane that lies just below the shell.Inoculate eggs using a 23 gauge needle on a 1cc syringe. Pull up 0.6 ml of sample and inoculate 0.15 mL into each egg through the punched holes, inserting the needle vertically, slightly pointed toward the front of the egg.Cover the holes with a small drop of Duco cement.Incubate eggs for 48-72 hours in a 37 C humidified incubator.Clean out and sanitize hoods with 70% alcohol.

IV. Processing cloacal swabs – harvest

Label a 96-well plate with one half-row per sample number and two sets of A-D columns.


Add 0.025mL PBS to each sample well and 0.50mL PBS to each control well.Transfer eggs into cardboard trays and sterilize shells with a quick squirt of isopropanol.Use an egg cracker and forceps sterilized by flaming to crack and peel away the top of the shell.Using a sterile disposable pipette, place a drop of chorioallantoic fluid from each egg into the appropriate well. Use a new pipet for each row/sample.Cover tray of open eggs with foil and return to refrigerator.After harvesting all the eggs, add 0.05mL 0.5% CRBC to each well of plate.Cover and let sit at room temperature for 45 minutes.Read and record results: pelleted CRBCs that run in a tear-drop shape upon tilting the plate 45 degrees indicate a NEGATIVE well; hemagglutinated CRBCs (formed in a lattice-work, giving a solid pink appearance to the entire well) indicate a POSITIVE well.Harvest all the chorioallantoic fluid from each positive egg separately into a labeled tube.Perform an HA titer on the fluid harvested from each egg.Pool harvests from eggs of the same sample with similar titers.Aliquot into labeled cryovials and freeze in the ultra-cold freezer.

Controls:Cell control – Add 0.05mL PBS and 0.05 mL CRBC to the bottom row of the plate. Each well should pellet and run in a tear-drop upon tilting. (This is a negative control that tests CRBC integrity and insures there is no agglutinating virus present in the blood suspension).

V. Processing cloacal swabs – Inoculating Re-passes

Thaw virus samples in 37 C water bath, then keep cold for the rest of the procedure. Vortex about 1 minute.Dilute samples in BHIB with P/S. Dilute 1:10 if re-passing to increase titer or to check a questionable (+/-) first pass; dilute 1:100 if re-passing to make more stock.Centrifuge diluted samples for 15-20 minutes at 1500 rpm.Filter samples through a 0.22 um filter into a sterile labeled cryovial.Inoculate and harvest samples as outlined above.

VI. Hemagglutination (HA) Test

Add 0.05mL PBS to each well of a 96-well plate labeled with the sample number for every two rows and “2, 4, 8, 16, 32, 64, 128, 256” (virus dilutions) across the top.Add 0.05mL virus isolate to the first well of appropriate rows.Mix and transfer 0.05mL across each row, discarding the final 0.05mL.Add 0.05mL 0.5% CRBC to each well.Cover and let sit at room temperature for 45 minutes.Read and record results: pelleted CRBCs that run in a tear-drop shape upon tilting the plate 45 degrees indicate a NEGATIVE well; hemagglutinated CRBCs (formed in a lattice-work, giving a solid pink appearance to the entire well) indicate a POSITIVE well. Results are read as the inverse of the furthest dilution producing complete agglutination, ie, the last positive well.

Controls:Cell control – Add 0.05mL PBS and 0.05 mL CRBC to the bottom row of the plate. Each well should pellet and run in a tear-drop upon tilting. (This is a negative control that tests CRBC integrity and insures there is no agglutinating virus present in the blood suspension). Chorioallantoic Membrane inoculation employs 10- to 12-day-old embryos and inoculum of 0.1-0.5 cc. This route is effective for primary isolation and cultivation of the viruses of fowl pox, laryngotracheitis, infectious bursal disease virus, reoviruses, which produce easily visible foci or "pocks." The chorioallantoic membrane is a suitable site for study of the development of pathologic alterations and inclusion bodies, and titration of viruses by the pock-counting technique (Figure 3.2).


Figure 3.0.2 Plaque on CAM

1.Candle embryos for viability.

2.Mark an area about 1/4 inch below and parallel to the base of the air cell. Disinfect with 70% alcohol or Betadine® solutions.

3.Drill or punch a hole at this mark being very careful not to tear the shell membrane. Punch a hole directly at the top of the air cell.

4.Holding the embryo in the same position and using a rubber bulb, draw air out of the air cell by placing the bulb over the hole at the top of the embryo. This negative pressure creates the artificial air cell by pulling the CAM down.

5.Using a 25-27 gauge needle, insert it into the artificial air sac about 1/8 inch and release the inoculum. Make sure the embryo is lying horizontally for 24 hours of incubation then return to upright position.

6.The following procedure is most commonly used for harvesting the CAM.

a) Crack the eggshell over the false air cell by tapping the eggshell with the blunt end of sterile forceps. Remove the eggshell as close to the edge of the false air cell as possible and discard pieces of eggshell in disinfectant. Discard the forceps in a beaker of disinfectant.

b) Observe the CAM for signs of thickening (edema) and plaque formation.

c) Harvest the CAM by grasping it with sterile forceps, stripping away excess fluids with a second set of forceps. Place harvested CAM in a sterile petri plate for further examination or in a 12 x 75-mm snap-cap tube or other suitable vial for storage.

d) Freeze and store the vial containing the CAM at -70C.


Yolk Sac inoculation is performed with 5- to 8-day-old embryos and inoculum of 0.2-1.0 cc. This route can be used for initial isolation of reoviruses.

1.Rotate the egg until blood vessels can be seen close to the margin of the air cell.These vessels may appear as nothing more than an array of faint lines, orange in color, extending from a clear halo. The embryo is within the area of the halo.

2.With an egg punch, make a hole in the top of the shell.

3.Use a 25-27 gauge, 1 ½-in. length needle. Insert the needle straight down into the yolk sac until its point is one-half the depth of the egg. Aspirate some yolk material in the egg, and then reinoculate the material with suspect virus material into the embryo.

4.For harvesting the Yolk-Sac Fluid:

a) Open the egg in the same way as described above for harvesting AAF.

b) Rupture the CAM to allow access to the yolk-sac membrane.

c) Grasp the yolk-sac membrane with forceps and carefully lift it to separate it from the embryo and other membranes. Using a second set of forceps, strip off the excess yolk and place the yolk sac in a sterile 12 x 75-mm snap-cap tube or other suitable vial for storage.

d) The fluid can also be taken directly by aspiration through a large (small gauge) needle or pipette (Figure 3.3). Store yolk sac at -70C.


Figure 3.0.3 Harvesting virus from embryos

PROPAGATION IN CHICKEN TISSUES

Many tissues of the chicken can serve to propagate viruses. One of the most common is the bursa of Fabricius (Figure 3.4). This organ is a sac-like organ in the form of a diverticulum at the lower end of the alimentary tract in birds. It produces B lymphocytes which can differentiate into plasma cells upon antigen stimulation. Mature B-cell can then produce immunoglobulins, which are active against pathogenic organisms. Infectious bursal disease virus, reoviruses, adenoviruses, lymphoid leukosis viruses, and Marek’s disease virus will readily propagate in the bursa. Infectious bursal disease virus (IBDV) replicates to a very high titer nearly 10P9P/ml in the bursa. This virus can cause severe morbidity, mortality and/or immunosuppression in susceptible chickens. Specific pathogen free (SPF) chickens between the ages of 3 to 6 weeks are commonly used for the propagation of this virus. Chickens are normally given by eye and nose drop about 10P3P/ml of the virus. The chicks are housed in isolation units maintained with filtered air and sacrificed 3 days post infection. The bursae are placed in NET buffer and stored at -70C until needed. Bursae can be ground with a blender or grinder in buffer at a 10% suspension and then stored in an ultracold freezer.


Figure 3.0.4 Harvesting IBDV from inflamed bursa

References

Senne, D.A., 1989. "Virus Propagation in Embryonating Eggs." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 176-181.

Villegas, P., 1986. "Cultivation of Viruses in Chicken Embryos." In A Laboratory Manual of Avian Diseases. University of Georgia, Athens, GA. pp. 1-5.

PROPAGATION IN CELL CULTURE

Introduction

Cell cultures are used in laboratories for the isolation, identification, and propagation of viruses and for the detection of neutralizing antibodies. Cell cultures have advantages over animals or embryonated eggs. Cell cultures are economical, and they are a homogeneous population of cells, free of immunological and hormonal influences that might affect replication of the virus. In addition, many cell lines can be stored in liquid nitrogen and be readily available.

Aseptic technique is important even in the presence of antibiotics. Contamination can be reduced using laminar flow hoods and using alcohol cleaned gloves and wearing a mask.

Laboratory equipment

Culture media and solutions need a large supply of pure water. The water must be deionized, double-distilled, or both, to remove traces of cytotoxic organic and inorganic materials. Reverse osmosis followed by glass distillation is used.

In general, cells are cultured in plastic petri dishes, flasks, or roller bottles specially coated for cell culture. Plastic vessels are sterilized after use by autoclaving and discarded. It is possible, however, to recycle used plastics. Other equipment that is needed include an inverted microscope, and, if cells are cultured in dishes or tubes open to the atmosphere, an incubator in which the atmosphere can be maintained with 85% relative humidity and 5% carbon dioxide. Incubator cleanliness is important to prevent contaminating the cultures. Mouth pipetting is not acceptable. Use either a rubber pipette filler or an electric apparatus. An autoclave is needed to sterilize, clean and disinfect lab ware at 20 lbs of pressure for 20 minutes.

Media and Solutions

The different cell-culture media all have the following composition:1)A balanced salt solution.2)A protein supplement such as serum.3)An antibiotic mixture to control microbial contamination.Cell culture media can be bought nearly complete (except for sera and antibiotics) commercially from many sources in a powdered or liquid format. This medium provides the cells nutrients and conditions for growth. Complex media such as minimum essential medium (MEM), M199 and RPMI-1640 are normally purchased


as 10x liquid concentrate or in powdered form). They are normally prepared with buffered salts such as Hank's or Earle's. Additional vitamins, amino acids (particularly L-glutamine which is not stable at refrigerated temperature) are added to improve growth. A typical avian cell culture growth media contains the following:

Table 3.1. Balanced Salt Solutions (BSS) and Phosphate-Buffered Saline (PBS)

200 mM Glutamine 10ml

7.5% NaHCOB3B 29.30 ml

10x MEM 100 ml

HB2BO 760.7

Serum* (10%) 100ml

Total 1,000 ml

*Maintenance media may contain any where from 0 to 3% Fetal Bovine Serum depending on confluency and age of the cells.

Hanks' and Earle's BSS are frequently used as bases for growth medium. Their function is to maintain a physiological pH (7.2 to 7.6) and osmotic pressure and to provide water, glucose, and inorganic ions needed for normal cell metabolism. They usually contain an indicator of pH, e.g., phenol red. BSS and PBS are also frequently used to wash inocula and dead cells from cultures, to remove serum-containing media before trypsinization, and to dilute trypsin solutions.

The stock solutions can be autoclaved, sterilized by filtration, and stored frozen or at 4C. For use, one part of the 10x solution is added to nine parts of water, and it is finally sterilized either by filtration or by autoclaving.

Trypsin-Versene SolutionTrypsin is needed to digest connective tissue so individual cells suspensions can be made. Stock solutions of up to 2.5% trypsin can be prepared or obtained commercially. Sterilize by filtration. Dispense in 50-to-100-ml quantities and freeze at -20C. Use as 1x (final concentrations: 0.05% trypsin and 0.025% versene (TV)) by mixing 100 ml 10x TV with 900 ml of sterile glass-distilled water. It is recommended that TV be warmed to 37C before use.

Sodium Bicarbonate SolutionFor pH control, sodium bicarbonate is added to the medium just before use. Various concentrations from 1.4% to 10% have been used. Sterilize by filtration and store at room temperature.

Neutral Red SolutionA 1% solution of neutral red (Difco, Detroit, MI) can be prepared in water, sterilized by filtration and stored at room temperature to observe pH of the medium.

Antibiotic SolutionPenicillin G sodium and dihydrostreptomycin sulfate are purchase desiccated and stored in the refrigerator or in solution and stored frozen. A final concentration of 100 IU penicillin and 100 ug of dihydrostreptomycin are normally used to control bacteria. Gentamicin sulfate stock solution at 10 mg/1 ml can be substituted at a use level of 0.1 ml/10 ml of medium. To control fungi Nystatin (Mycostatin, Squibb, New York, NY) or amphotericin B (Fungizone, Squibb) can be added at 40 IU or 2 ug per ml of medium, respectively.

SeraFetal bovine (FBS) or calf serum (CS) are routinely added to media for cell cultures. The sera provide unknown cofactors needed for cell growth. Only purchase sera, which have been tested to be free of mycoplasma.

SterilizationMany items are purchased sterilized. Others including neutral red, versene and water can be prepared by


autoclaving. Media containing thermolabile compounds such as amino acids, (antibiotics, serum or trypsin) must be sterilized by filtration. Pressure filtration through membrane filters (Millipore, Corp., Bedford, MA or Gelman Sciences, Ann Arbor, MI) is routinely used.

Preparation of primary avian cell cultures requires that organs be aseptically removed from embryos or young chicks. Organs must be cut into small pieces and tissues dispersed into a suspension of single cells by enzymatic digestion. They are then allowed to grow into confluence in an incubator.

Chicken Kidney Cells (CEK)

Kidney cells can be prepared from 18-20-day-old embryos or day-old to three-day-old chicks. The amounts indicated here are for preparing kidney cells from 10-15 embryos.

1.Prepare media and trypsin solution and set in 37C water bath.

2.Spray eggs with disinfectant and allow drying under a sterile hood.

3.Using sterile technique (sterile equipment and media) remove embryos with blunt ended curved forceps and put into tray. Wash embryos with 70% alcohol or sterile distilled water.

4.Use regular dissection methods or cut the backbone right above wing joint and separate. This exposes the kidneys without having to touch the intestines and viscera.

5.Remove kidneys and put into glass beaker containing phosphate buffer solution (PBS) or Hank's balanced salt solution (HBSS).

6.Pour off supernatant and clean kidneys. If there are any large chunks, mince lightly with scissors or squeeze gently with forceps. Wash three to four times with PBS or HBSS. Use 75-100 ml PBS total.

7.Drain off the last wash and pour the tissue fragments into a trypsinization flask containing a magnetic stir bar. Add 50-100 ml prewarmed (37C) trypsin-EDTA solution.

8.Put the flask on a stirrer base in 37C incubator and stir very slowly for 15-20 minutes.

9.When the supernatant is cloudy, shake flask, and then set it down for several minutes to let the clumps settle out. Take out one drop of supernatant and put it on a glass slide and observe. If there are many single cells and small clumps (two to 10 cells) with few very large clumps then it is time to pour off the supernatant. Have ready a sterile graduated centrifuge tube with 5 ml of cold heat-inactivated calf serum in it. (Set in a pan of ice.) Pour supernatant through gauze covered funnel into this tube. (The serum stops the trypsin action). With fresh trypsin repeat process one to two times (10 min. ea.) more. Do not extend trypsinization time past 1 hr. Centrifuge at 1000 RPM for 10 minutes.

10.The kidney cells (and RBC's) will pellet. Note the amount of cells obtained. Pour off trypsin solution. Resuspend cells in 3-5 mls of minimal essential medium (MEM) or Hams F-10 with Earl's balanced salt solution (EBSS). Add the cells to the appropriate amount of MEM (EBSS) with 10% heat-inactivated fetal calf serum (growth media). One ml of cell pack can be resuspended in approximately 200 ml of MEM (EBSS). Cells can be counted in a hemocytometer by resuspending in a known amount of media. Make 1 to 10 dilutions of cells in trypan blue. You will want approximately 2.5 x 10P6P cells/ml of media to plate out the cells. The 35 mmP2P plates require 2 ml and 60 mm P2P plates require 5 ml. Do one plate first and observe after the cells are allowed to settle for a few minutes.

The cells should form a monolayer in one to two days. When monolayer is formed, they may be inoculated or if it is desirable, they may be inoculated simultaneously. After the cells have formed a monolayer, the old media can be poured off, the monolayer washed with PBS. The cells are then ready to be infected with virus or given maintenance media with 0-3% serum. The CEKS's are mainly epithelial cells and are used for growth of infectious bronchitis virus, adenoviruses and reoviruses. CEFC's are mainly fibroblasts and are used for growing Newcastle disease, infectious bursal disease, and herpes viruses.

Chicken Embryo Fibroblast (CEF)


1.Use nine-11 day old embryos. The technique described here is for three to five embryos. Spray eggs with a disinfectant (70% alcohol is commonly used). Using sterile technique, open shell and remove embryo with blunt ended curved forceps.

2.Place embryos in petri dish and cut off heads and limbs.

3.Transfer bodies to new petri dish or beaker containing PBS. In the beaker, the bodies can be fragmented by carefully chopping them with sterile scissors. Another procedure that can be used when large number of embryos is to be processed is as follows: attach a cannula to a 35 or 50 cc syringe, remove plunger, and pour tissue chunks into barrel and force through cannula with the plunger into a 30 ml beaker. Keep the cannula and syringe sterile and use it to draw off supernatant from above settled tissue chunks during PBS washes.

4.Wash with PBS 3-4 times to remove red blood cells.

5.Pour tissue fragments into trypsinization flask containing magnetic stirring bar. Add about 50 ml pre-warmed (37C) trypsin solution to flask and put on stir plate at slow speed on 37C incubator for 10-15 minutes. Stop trypsinization by adding 1ml calf serum or by placing the flask on ice for 3-5 min. Another trypsinization may be done on the clumps of tissues after the supernatant with single cells has been decanted.

6.Strain cells through two folds of sterile gauge.

7.Centrifuge at 1000 rpm for 10 min and discard supernatant.

8.Add fresh PBS and vortex to wash and suspend cells.

9.Centrifuge again at 1000 rpm for 10 min and discard supernatant.

10.Note the amount of pelleted cells obtained. Resuspend cells in 1X MEM containing glutamine and 10% FBS. One ml of cells can be diluted in 80 ml of media. Cells can be plated into 5, 25 or 100 ml flasks, or in a roller bottle. Lids of containers kept in a COB2B atmosphere need to be loosened to allow exchanges of gases. CEFC's will grow in a non- COB2B atmosphere and their lids need to be kept tightly closed.

MEM (1X) Glutamine 5ml 5mlNaHCO3 14.65ml 14.64mlH2O 375.35ml 410.35ml FBS 50ml 50ml Pen-Strep 10ml 10ml

NOTE: Secondary cells may be made from a confluent monolayer of CEF. Dilute trypsin solution 1:2 with Hank's Balanced Salt Solution (HBSS). Pour off media on CEF plates, wash plates with 1 ml trypsin solution (for 60 mm size dish) and pour off immediately. Add 2 ml trypsin solution to each plate and incubate in 37C COB2B incubator for two to five minutes. Remove trypsinized cells from dish with a pipette and put into centrifuge tube with 1 ml serum to stop reaction. Centrifuge 10 min. at 1000 rpm. Secondary cells may be plated 1:3 as heavy as the primary culture.

Normal CEFs CPE in CEFS


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Primary Chicken Embryo Liver Cells (CELIC) 1.Use 13-15 day-old embryos and spray eggs with a disinfectant. 2.Using sterile technique, remove embryos from eggs, open embryos to expose livers.

3.Remove the livers with curved, blunt ended forceps and put them into a beaker containing sterile buffer solution. Be sure to cut out the gall bladder before putting into the buffer.

4.Trim off any visible connective tissue or pieces of attached intestine. Mince tissue lightly with scissors or forceps.

5.Allow the liver pieces to settle to bottom of beaker. Decant and discard buffer containing RBC's. Wash three times or until the buffer is clear. (Usually 100 ml of buffer is enough for the collection and washes).

6.Drain off the last wash and pour the tissue fragments into a trypsinization flask, rinsing the beaker out with the trypsin solution. Add 50 ml prewarmed (37C) trypsin solution to the flask, which already has a magnetic stirrer bar in it.

7.Put flask into 37C incubator and stir gently for 15-20 minutes. Check cells as for CEKC step #9.

8.Follow CEKC procedure for remaining steps.

9.Dilute liver cells 1:150 in MEM.

Chick Embryo Tracheal Rings

1.Tracheal ring cultures are organ cultures and do not form single cell monolayers. They are used for primary culturing of many respiratory viruses. Use either embryos (19-20 day-old) or one-day-old chickens. Open disinfected egg shell and remove embryo cutting away the yolk sac.

2.Cut skin until trachea is completely exposed.

3.Carefully remove the trachea with forceps and remove all fatty tissue surrounding it.

4.Place trachea in glass petri dish containing approximately 5 mls of HBSS.


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5.Lay tracheas on sterile filter paper and place on tissue chopper. Use sterile razor blade and cut trachea into rings at medium speed.

6.Wash mucous from inside of rings with a syringe and needle containing HBSS. Place rings in a separate petri dish containing HBSS.

7.With small forceps, place individual rings into sterile test tubes. Cover with 0.5-1.0 mls of media. Be sure rings are immersed in solution.

8.Put tubes in rack and rotate at 37C for 24 hours. Mucous may again need to be washed from inside of the rings with HBSS.

9.At the end of 24 hours, check for ciliary movement under the microscope (use either the 4X or 10 X objectives).

10. Score the ciliary movement as follows:If half the ring has movement, the ring would be assigned a 2.If 3/4 of the ring has movement, the ring would be assigned a 3.If the entire ring has movement, the ring would be assigned a 4.Rings with reading lower than 2 are not used.

11.The rings are now ready to be inoculated. The ciliary movement should be read after 3-5 or 7 days, depending upon the virus being studied.

Tracheal rings can be used to detect the presence of infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and laryngotracheitis virus (LT). They can also be used to run Virus Neutralization Tests for IBV. Tracheal rings can also be used to evaluate ciliary activity after challenge with field isolates. Rings are prepared from adult birds four days after challenge. The ciliary activity is evaluated as described.

Procedure for Inoculating Preformed Monolayers

1.Swirl plate to resuspend as many RBC's and debris as possible and then decant and discard growth medium.

2.Wash monolayer gently with 2-3 mls of prewarmed PBS and discard.

3.Add 0.1 ml sample inoculum to the small 10 x 35 mm plates or 0.2 ml for the larger size (60 mm). Rock each plate gently to distribute inoculum evenly over the cell monolayer.

4.Incubate inoculated cultures in 37C incubator for 45 minutes to allow virus to absorb Rock tray once or twice during incubation if possible.

5.Add 2 ml maintenance medium to each 35 mm plate (or 5 ml for 60 mm plates.)

Note: Maintenance media — 0% - 3% heat-inactivated calf serum.

6.Incubate at 37C. Check plates daily for damage to the cells or cytopathogenic effect (CPE).

7.To harvest samples, freeze plates and then thaw two to three times, shaking flask when media is partially thawed to help dislodge cells and collect. Alternatively, cell monolayers can be removed by removing media and then scraping adherent cells with a sterilized "rubber policeman". Virus will be present both extracellularly in media and intracellular in cells. Freezing and thawing or sonication for five minutes will disrupt cells to remove virus. For some highly intracellular viruses such as herpes viruses it is best not to disrupt the cells.

Cell Lines and Secondary Cultures

A cell line is a population of cells derived from an animal tissue, which can be continually propagated over numerous passage mammalian cell lines which support avian virus growth. They include VERO and BGM—70 cells. VERO cells are derived from kidney tumors of African green monkeys whereas BGM cells are kidney cells derived from a bovine tumor. The tumor cells’ genetic material allows them to grow indefinitely. Avian viruses such as Newcastle disease, infectious bronchitis, infectious bursal disease and reoviruses have been adapted to these cell lines. Cell lines from tumors of ducks and chickens will also support avian


herpesviruses, avian leukosis viruses, Marek's disease virus and the chicken anemia virus. Primary Chicken Embryo Fibroblast Cells (CEFC's) that can be passaged up to four times in cell culture are called secondary cells. The advantages of all lines and secondary cells are that they don't require live animals embryos and can be stored frozen in liquid nitrogen so they are readily available when needed. The passage and use of these lines or cells is as described under CEFC's.

Freezing cells

A low temperature freezer (-70C) or liquid nitrogen container (-196C) is needed. The freezer should be plugged into an electric surge protector and be equipped with an alarm in case of temperature rise, and backed up with a gas powered electric generator in case of long term power failure. The liquid nitrogen tank should be checked monthly with a ruler to measure depth of nitrogen in the tank. The tank normally needs to be refilled every four to six weeks depending on usage.

Procedure for freezing cells

1)Use only actively growing cells (2 to 5 days of age).

2)Prepare the cells as outlined for passage of secondary CEFC's.

3)Centrifuge the cells at 400 g for 5 mins and discard the supernatant fluid.

4)Resuspend cells in cold culture medium or calf serum containing 10% dimethyl sulfoxide.

5)Transfer the cells to prechilled freezing vials and place in an insulation container which allows for a gradual drop in temperature of 1C per minute. Place the container in a -20C freezer for 1 hr. then -70C for 8 hours and, if available, liquid nitrogen. Cells are viable for months at -70C and for years at -196C.

6)For use, cells should be thawed in a water bath at room temperature.

7)Thawed cells should be plated at 2x the density of primary cell lines. Maintenance of these cells is as previously mentioned.

Application of Cell Culture Methods for Virology

Virus multiplication in cell culture can be detected in several ways:

1)Morphologic alternation of the cell, called cytopathic effect (CPE) due to degeneration of cellular organelles. The CPE can be seen as holes in the monolayer (Figure 3.4).


Figure3.4 Viral CPE (hole in monolayer)

Prior to death, cells may round up, become refractile or partially detach from the monolayer.

2)The formation of giant cells or syncythia (fusion of cell membranes).

3)The pH changes in the medium (red to yellow color change) due to changes in cell metabolism.

4)Serologic methods such as fluorescence or immunoperoxidase assays, can detect viral multiplication in cells. As with chicken embryos, viruses upon initial isolation may have to be passaged blindly (no visible CPE) several times before their presence becomes apparent.

VIRUS IDENTIFICATION

Animal viruses are classified based on their physical and chemical characteristics. Viruses are first divided into two groups based on their nucleic acid content. Deoxyribonucleic acid (DNA) viruses are divided into seven families, five of which contain avian pathogens. These families also contain either single or double stranded nucleic acid. Ribonucleic acid (RNA) viruses are divided into 16 families, nine of which cause disease in poultry. Some of the DNA families and representative viruses are 1) Adenovirus — inclusion body hepatitis; 2) Herpes virus — Marek's disease, and 3) Pox virus — Fowl pox. Some of the most important RNA virus families and representative individuals include: 1) orthomyxovirus — Avian influenza; 2) paramyxovirus — Newcastle disease; 3) coronavirus — infectious bronchitis; 4) Retrovirus — leukosis; 5) picorna virus — avian encepholamyelitis; 6) reovirus — viral arthritis; and 7) birna virus — infectious bursal disease. Reoviruses and birna viruses contain double stranded RNA.

Other Criteria for classifying viruses include:

1)Presence of a lipoprotein envelope; 2) diameter of the virion, and 3) symmetry of nucleocapsid.

Knowledge of these criteria will help place the virus in a recognized family, however, to positively identify a virus serologic methods (reacting an unknown virus with a known antibody) are often required (Table 3.2).

Table 3.2. Important Biological, Physico-chemical Properties of Enveloped and Nonenveloped VirionsCharacteristic Nonenveloped Virus Enveloped VirusUltraviolet radiation Sensitive SensitiveGamma radiation Sensitive SensitiveThermostability Thermostable ThermolabileSusceptibility to ice crystal damage Yes ExtensiveInactivation by lipid solventsand detergents

No Yes

Determining type of nucleic acid

The type of nucleic acid (either DNA or RNA, but not both for viruses) can be determined by various specific inhibitors that affect virus replication. Thymidine analogs are a simple method to determine if the virus contains DNA. 5'-iodo-2'- deoxy uridine (IUDR) (Calbiochem Corp, San Diego, CA) is commonly used. A


simple method is as follows.

1.Prepare maintenance medium with 50 ug/ml IUDR. The media must be homogenized and sterilized by filtration (Run sterility check on each concentration). Prepare dilutions of virus with and without IUDR.

Note:IUDR goes into solution faster if the pH of the medium is increased. Tighten the cap of the bottle and place it on a magnetic stirrer preferably at 37C for approximately 15 minutes.

2.Inoculate preformed monolayers with the appropriate dilutions of the virus being tested. Use two to three plates per dilution of IUDR.

3.Absorb virus at 37C for 45 minutes and discard excess fluid into a beaker containing disinfectant solution.

4.Add maintenance medium with IUDR to the dishes.

5.Controls should be included using known DNA and RNA viruses. Both samples and known controls should also be inoculated in regular maintenance medium.

6.Harvest dishes when cytopathogenic effect (Figure 3.5) is observed in controls.

Figure 3.0.5 Viral titration in cell culture by counting plaques (holes) in monolayers

7.Freeze-thaw the dishes three times (The cells can also be sonicated to speed up the process).

8.Titrate pooled controls and all plates where IUDR was used.

9.The virus contains DNA if the titer is 1 logB10B lower with the analog than without.

Determining single or double stranded nucleic acid.

The sensitivity of the virus to actinomycin D can be used to differentiate viruses. Actinomycin D will prevent transcription of messenger RNA from double stranded nucleic acid. This technique can be done as follows: 1) Prepare cell culture media with and without Actinomycin D at 1ug/ml; 2) treat the cells with the drug for two hours before adding the virus; 3) after 48 hours, harvest the virus and titrate it in cell culture. Compare the titer of the virus with and without Actinomycin D. A reduction in 1 logB10B of titer indicates sensitivity to Actinomycin D and that the virus contains double stranded nucleic acid.

Determining the presence of lipoprotein envelope.

The sensitivity of the virus to lipid solvents correlates with whether it has a lipoprotein envelope. To determine the presence of the lipoprotein envelope the following procedure can be done with either of two lipid solvents (ether or chloroform):

1.Dilute virus stock or sample to be tested 1:10 and divide into two aliquots.

2.Add 0.2 ml of CHClB3B to 2 ml of one aliquot in a 15 ml centrifuge tube or 20% volume of ether. Dispense 2 ml of the other aliquot into another 15 ml centrifuge tube.


3.Mix both tubes on Vortex for 10 minutes, keeping the tubes in an ice bath between mixes.

4.Allow the solvent to sediment in refrigerator overnight or by centrifuging (1500 rpm for 30 minutes).

5.Set at room temperature undisturbed for one to two minutes. Using a long sterile Pasteur pipette, collect the clear layer on the top being careful not to pick up any solvent which will appear cloudy. The top layer which has been collected may be left in an opened vial under the hood for 10-15 minutes to allow any solvent present to evaporate prior to inoculation. Cap the vial and refrigerate overnight.

6.Make 10P-1P and 10P-2P dilutions and inoculate undiluted and diluted samples (solvent treated and non-treated) in macro or micro dishes or in embryos.

Note: Glass equipment should be used because the solvents may react with plastic.

7.Compare the titer or CPE of the viral stocks with and without solvent.

If there is a significant drop in titer of the virus treated with solvent, then it contains a lipoprotein envelope.

Morphology of the Virus Particle

The size and shape are also important criteria for classifying viruses. The best method for determining the size of a virion is with the electron microscope (Figures 3.6 and 3.7). Determining the size by membrane filtration is not highly accurate due to virus clumping and occlusion of the membrane pores with cellular debris. The electron microscope is a valuable tool in virology and produces much information required to identify and classify a virus: lipoprotein envelope, size, and morphology. The procedure entails concentration of the virus by ultracentrifugation and resuspension in distilled water. A drop of virus is dropped on a plastic-coated grid and mixed with a drop of 2% phosphotungstic acid in distilled water (adjust to pH 6.5 with KOH). Viral concentrations of at least 10P6P/ml is required to detect virions with this method.

Figure 3.0.6 EM pictures of AIV (left) and IBDV (right) virions

Figure 0.7 EM scope

Table 3.3 outlines the characteristics used to differentiate the families of viruses of importance to avians. After a virus has been classified within a family, it has to be further identified. Serologic identification is very specific, and techniques such as virus-neutralization, immunoprecipitation, hemagglutination-inhibition, immunofluorescence, immunoperoxidase, and enzyme-linked immunosorbent assays are used to identify viruses. The techniques will be discussed later in this book.


Table 3.3Differentiating Criteria for DNA and RNA viruses that are important in avian medicine. I.DNA—Sensitive to thymidine analogsA.Lipid solvent sensitive1.More than 200 nm, Herpes virus (Marek's disease virus).2.Less than 200 nm, Pox virus (Fowl pox virus).B.Lipid solvent resistant1.More than 50 nm, Adenovirus (inclusion body hepatitis virus).II.RNA—Resistant to thymidine analogsA.Lipid solvent sensitive1.HA-positiveP1P

a.Sensitive to Actinomycin D,Orthomyxoviridae (influenza virus)b.Resists Actinomycin D,

Paramyxoviridae (Newcastle disease virus)

2.HA-negativea.Sensitive to Actinomycin D,Retroviridae (Avian Leucosis virus)b.Resists Actinomycin D, Coronaviridae (infectious bronchitis virus)

B.Lipid Solvent Resistant

1.Sensitive to Actinomycin D, Reoviridae (viral tenosynovitis)Birnaviridae (infectious bursal disease virus)2.Resists Actinomycin D, Picornaviridae (avian encephalomyelitis virus) P1PHA = Hemagglutination.


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Collection and Submission of Specimens

The laboratory diagnosis of clinical illness depends to a large extent upon the kind and condition of


submitted specimens. It also depends upon the practicing veterinarian and laboratory working in close concert. Because many of the laboratory tests are for specific disease agents, an adequate clinical history must accompany all submissions. This will permit laboratory staff to perform additional tests if indicated.General guidelines for the collection and submission of specimens are presented below. Most laboratories supply a specimen submission form that should be completed with the available pertinent information. In the absence of a form, the veterinarian should supply as complete a history as possible. Veterinarians should contact the diagnostic laboratory if they have any questions.

Animals

Live, sick animals are preferable to dead animals. Whenever possible, animals should be submitted directly to the diagnostic laboratory for complete necropsy examination. If a herd problem exists, more than one animal should be submitted. Bus and courier service may be used to ship small birds, provided they are packaged in leak-proof insulated containers with sufficient ice or cold packs. Do not freeze animals submitted for necropsy.

Tissues

To minimize contamination during necropsy, it is best to collect a routine set of tissues prior to thorough examination. Recommended tissues are lung, kidney, liver, spleen, small intestine, large intestine, and mesenteric lymph nodes. Brain tissue or head should also be collected if central nervous system disease is suspected. Other tissues containing abnormalities noted during the thorough examination should also be collected. A portion of these tissues should be placed in leak proof plastic bags and placed under refrigeration. While it is recommended that each tissue be placed in a separate bag, it is absolutely essential that intestine be separated from other tissues; otherwise bacteriologic examinations will be compromised.

Tissues should be brought directly to the laboratory, or shipped under refrigeration by over-night mail, bus, or courier service. Tissues collected during the latter part of the week should be frozen and shipped on Monday. Since many viruses produce characteristic microscopic lesions, small pieces (1/4 inch thick) of each tissue should be placed in ten percent buffered formalin for histopathologic examination. An entire longitudinal half of the brain should be submitted. These samples should not be frozen.

Feces

Feces should be collected from acutely ill animals and placed in leak proof containers. While well-saturated swabs are adequate for many individual virologic examinations, several milliliters or grams of feces permit a more complete diagnostic work-up including bacteriologic and parasitologic examinations. Samples should be submitted to the laboratory using cold packs as coolant.

Swabs

Nasal and ocular swabs are useful for isolating viruses from animals with upper respiratory-tract infections. Genital infections may also be diagnosed by examining swabs collected from the reproductive tract. These swabs should be collected from acutely ill animals and placed directly into screw-capped tubes containing a viral transport medium. The sampling of several animals in different stages of the illness increases the likelihood of isolating the causative agent. Swabs are also useful for the sampling of vesicular lesions. Fresh vesicles should be ruptured and the swab saturated with the exuding fluid. Two swabs should be collected, one for virus isolation and one for electron microscopy.

The swab for virus isolation should be placed in viral transport medium and the swab for electron microscopy should be placed in a screw-capped tube containing one or two drops of distilled water. Scab material from the more advanced lesions should also be submitted. There are several commercially available viral transport media that help maintain the viability of viruses during shipment to the laboratory. Most of these transport media are balanced salt solutions containing high protein content and antibiotics to prevent bacterial overgrowth. Many diagnostic laboratories provide their own version of transport medium to practicing veterinarians upon request.


Slides

A number of infectious diseases can be diagnosed by examining slides prepared from blood and tissues. Blood smears and conjunctival scrapings are used for diagnosing viral diseases. Conjunctival scrapings are particularly useful for diagnosing herpesvirus and chlamydial infections. Imprints made from liver, spleen, and lungs are especially useful for diagnosing Chlamydia and herpesvirus infections of psittacine birds.Slides should have sufficient cells to allow thorough examination but should not be so thick as to cause difficulty in staining. A conjunctival scraper or some other device (blunt end of scalpel blade) should be used to scrape the conjunctiva; cotton swabs are not adequate. Matted eyes should be cleaned and flushed prior to scraping the conjunctiva. Tissue imprints should be made by lightly touching the microscope slide with fresh cuts of tissue previously blotted with a paper towel to absorb some of the blood. Slides should be air-dried and sent to the laboratory in slide holders to prevent breakage. Several slides permit a more thorough diagnostic work-up, including cytologic examinations.

Serum

Blood samples should be collected in sterile tubes containing no anticoagulants. These should be submitted to the laboratory in specially designed Styrofoam holders to avoid breakage. Blood samples should not be frozen or allowed to overheat. If samples cannot be delivered to the laboratory within a reasonable time, serum should be refrigerated.

Concentration and Purification of Viruses

Once a virus has been adequately propagated, it needs to be recovered from host cells and debris and purified. This is accomplished by a number of processes that involve differential centrifugation (various speeds), dialysis, precipitations, chromatography and density gradients. The initial step of this process is differential centrifugation; a slow speed (~2,000 x g) is used to remove large cellular debris. This is followed by high-speed centrifugation (40K to 80K x g) to concentrate the virus for small volumes; by dialysis and precipitation for larger volumes; and by cold (-70°C) methanol or polyethylene-glycol precipitation, also for large volumes. Purification is achieved through chromatography and centrifugation through density gradients. Enveloped viruses can be purified by velocity sedimentation through sucrose gradients. Nonenveloped viruses can be purified by centrifugation through cesium chloride gradients.

Infectivity and Storage

Infectivity

Infectivity is a virus particle’s ability to infect a host cell. The temperature outside of a host cell readily affects the virus’ ability to retain its infectivity, particularly in the case of enveloped viruses. As viruses have no metabolic activity of their own, infectivity is the best means to evaluate the integrity of the viral particle following exposure at a particular temperature.

The following are important considerations:

• At 60°C, infectivity of the virus will decrease rapidly within seconds.

• At 37°C, infectivity will decrease dramatically within minutes.

• At 20°C, infectivity decreases within hours.

• Infectivity at the above temperatures influences viral spread by direct contact (at 37°C) and by fomites (at 20°C).

• At 4°C, infectivity in tissues is lost over days. Clinicians should keep this in mind regarding clinical specimens.

Temperatures below freezing are often used for long-term storage. The important consideration is keeping ice crystal formation to a minimum.It should be kept in mind that viruses vary greatly in their resistance and lability. Some are able to survive for hours, days, and even months under environmental conditions, while others are inactivated in a few


minutes under similar conditions.

The three principal methods of storing viruses are:

• Freezing at 70°C with or without a cryopreservative.

• For long term storage, freezing in liquid nitrogen (-196°C).

• Lyophilizing or freeze drying with storage in a freezer or at room temperature.

Virus Visualization

The two major methods mainly used to visualize the structure/morphology of viruses are electron microscopy and atomic force microscopy. Other types of microscopy are used to observe changes induced by virus replication in virus-infected cells. Without a means to visualize viruses, it is difficult to obtain information about structure or virus-cell interactions. Furthermore, being able to visualize viral particles allows one to estimate the number of particles present in a suspension directly. There are other methods that allow one to estimate the number of viruses indirectly. In either case, direct or indirect, enumeration quantification is always an estimate of numbers. This estimate is important when preparing vaccines, when determining the minimum number of virions required to produce disease, and in viral research procedures.

Light Microscopy

While the light microscope is not useful for the direct examination of viruses (except poxviruses), it is useful for observing the effects of viral infection on the host cell. The virus-caused cell damage or destruction is referred to as the cytopathic effect (CPE). Observable cytopathic effects include:

1. Cells rounded up and aggregated in grape-like clusters, as with adenoviruses; 2. Cells round up, shrink, and lyse, leaving large amounts of cellular debris, as with enteroviruses; 3. Cells become swollen and round up in focal areas, as with herpesviruses; and 4. Cells fuse producing multinucleate cells (syncythia), as with paramyxoviruses.

Additionally, inclusion bodies, characteristic of some viruses, can be visualized.

Fluorescence Microscopy

Fluorescence microscopy can be used to visualize virus-infected cells or tissues using virus antigen-specific fluorochrome tagged antibody. The antibody binds specifically to virus antigens within the cells or tissues and thus labels them with a fluorescent tag (usually fluorescein). The fluorescent tag is then visualized with a UV microscope that excites the fluorochrome, which one sees as a colored focus with a relatively dark background. Alternatively, visualization can be performed indirectly by using unlabeled antibodies (as found in convalescent serum) followed by fluorochrome labeled antibodies that bind the first antibody. Fluorescent antibody based assays are commonly used in viral diagnosis and research.

Electron Microscopy

Electron microscopy involves the acceleration of electrons to high energy and magnetically focusing them into the sample. The high-energy electrons have very short wavelengths and thus provide better resolution of very small structures. Electron microscopy has enough resolution power to visualize large polymers, such as DNA, RNA, and large proteins.To facilitate visualization, samples may be coated with heavy metals, such as osmium, prior to examination by electron microscopy. The electrons hit the heavy metals, which are then visualized on a fluorescent screen. Electron microscopy yields 3-dimensional images of virions and their localization within the host cell (nuclear or cytoplasmic) at a given point in time following infection. As the samples are treated with heavy metals, observing virions within live cells is not possible.

Atomic Force Microscopy


The atomic force microscope works by measuring a local property (such as height, optical absorption, magnetism, etc.) with a probe placed very close to the sample. This makes it possible to take measurements over a small area of the sample. Electrons are able to "tunnel" between atoms, resulting in a small, but measurable force. The result of these measurements is a detailed contour map of the surface of a structure.The advantages of atomic force microscopy are minimal sample preparation and use on living specimen. This method has been useful for detailed images of capsid structures and virus-cell interactions.

Immunoelectron Microscopy

This technique allows the visualization of antibody/antigen complexes that are specific to a particular virus. In this method, ultra thin sections are cut and incubated with antibody that is specific for the virus. Following a washing step, the section is incubated with Protein A conjugated gold particles (size range is 5 to 20 nm). The Protein A gold particles bind to the Fc portion of the antibody and are detected by electron microscopy.

Radioimmunoassay

Use: To detect antigen or antibody.Nowadays, radioimmunoassay systems are rarely used in veterinary diagnostic laboratories.There are two basic radioimmunoassay (RIA) systems, liquid phase and solid phase. In the liquid phase system, the antigen-antibody complexes are precipitated by subsequent addition of anti-gammaglobulin. The precipitate is collected by centrifugation and dried. The amount of radioactivity in the precipitate compared to the total radioactivity is a quantitative measure of the antigen-antibody reaction. The labeling is done with 125I (see Immunodiffusion), and anyone of the three components can be labeled.

In the solid phase system, the antibody is coated to the inside of a polystyrene tube and then reacted with antigen. Briefly, the specimen is added to a polystyrene tube previously coated with antiviral antibody. If the antigen is present, it attaches to the bound antibody. Following rinsing, 125I labeled antiviral antibody is added, which reacts with the complex giving a "sandwich effect". The tube is washed and the amount of radioactivity is determined. While the aforementioned techniques of antigen detection are used as the first approach to viral diagnosis, in many instances these techniques are not applicable because appropriate specimens are often not obtainable from live animals. Also, rapid antigen detection systems are not available for numerous viral diseases. In these instances, virus isolation is attempted.

Direct Enumeration of Viruses

Estimating the number of viruses has a number of important uses including research and vaccine production. Electron microscopy is used for the enumeration of viral particles in a cell free solution. A known volume of sample is examined and the number of virions counted. This number is then used to estimate the number of viruses. One limitation is that empty capsids, thus non-infective particles, are also counted. In research, the number of infectious particles and the total number are compared and establish a ratio of total particles/infectious particles for a given virus.

Indirect Enumeration of Viruses

Indirect methods of viral enumeration are those that utilize factors associated with infectivity (biological activity). The three principal methods used to indirectly assess viral concentrations are hemagglutination assays, plaque forming assays, and the limiting dilution method.

Hemagglutination

This assay is based upon the property of many enveloped viruses to agglutinate red blood cells (RBCs).The assay is carried out by adding red cells to dilutions of the virus sample in a microtiter plate, then observing for hemagglutination. It takes many viruses to coat RBCs and result in hemagglutination. For example, it takes approximately 104 influenza virions per hemagglutination unit (HA unit). An HA unit is defined as the highest dilution of the viral sample that causes complete hemagglutination.Hemagglutination is useful in the concentration and purification of some viruses, and as a rapid presumptive test for the presence of these viruses in fluids from infected cell cultures and chicken embryos. It is especially


useful for assaying viral activity of cell cultures infected with hemagglutinating viruses that produce little or no discernible cytopathic effect (CPE). Clinical specimens such as feces can also be directly examined for hemagglutinating activity of particular viruses (discussed further in Chapter 7). Similar type assays that test for enzyme activity of a particular virus (such as one producing reverse transcriptase) can be performed in a similar manner.

Plaque Forming Assay

This assay involves the inoculation of susceptible host cells with virus and using their biological activity to estimate the number of virions present. In the procedure, ten-fold serial dilutions of virus sample are used to inoculate monolayers of host cells. Following incubation to allow the virions to adsorb to the surface of the host cells, the monolayer is overlaid with a gel composed of host cell medium and agarose. The presence of the agar prevents viral spread in the culture of host cells on a large scale, but allows localized cell-to-cell spread. With cytopathic viruses, host cell destruction results in the development of clear zones called plaques, which can be visualized within 24 to 72 hours of incubation. A calculation involving the number of plaques observed, the dilution factor of the sample, and the volume of sample dilution used, yields the plaque forming units (PFU) per milliliter of sample.

The Limiting Dilution Method

This titration-based assay measures an effect on cells in vitro, such as CPE, when exposed to various dilutions of a virus-containing solution. If possible, a known concentration of reference virus culture is used as a positive control. Depending upon the virus, either two-fold or ten-fold serial dilutions of the viral material are made and placed with the cells. The infectivity titer (reciprocal of the highest dilution showing 50% CPE of the infected cultures) is expressed as the TCID50/ml (tissue culture infectious dose). This assay may be used with cultured cells, embryonated eggs or even in laboratory animals.

Miscellaneous Methods Used for Characterization

There are some methods used in virology that are helpful in the identification and classification of an unknown virus. Some of the techniques will be briefly mentioned here, but explained in greater detail later if they are used in the laboratory diagnosis of a particular virus.

Sensitivity to Lipid Solvents

The sensitivity of viruses to lipid solvents, such as chloroform and ether, aids in the taxonomy of some viruses. Any viruses that possess a membranous outer envelope are susceptible to lipid solvents. All enveloped animal viruses, except some poxviruses, are ether sensitive.

Identification of Nucleic Acid Type

This is performed by examining nucleic acid synthesis in cell cultures in the presence of DNA synthesis inhibitors, such as 5-bromo-2-deoxyuridine (BRU). If viral synthesis is inhibited, then virus multiplication will likewise be decreased. In the event that virus growth is not inhibited, the virus is presumed to contain RNA.

Restriction Enzyme Analysis

Restriction enzymes (RE) are endonucleases that cut double-stranded DNA at specific recognition sites, ranging from four to eight base pair palindromic sequences. Restriction endonuclease analysis is particularly useful in the "subserotypic" classification of viruses, in the differentiation of modified-live virus of vaccines from virulent virus, and in the epidemiologic tracking of disease outbreaks. Procedurally, the method entails treating viral DNA with one or more REs, and then separating the resulting fragments according to size by polyacrylamide gel electrophoresis. RNA viruses can be similarly analyzed by first making a complementary DNA (cDNA) strand from the RNA using the enzyme reverse transcriptase, and then amplifying this cDNA by the PCR method described later in this publication.

Hemadsorption


Membrane-bound viruses such as orthomyxoviruses and paramyxoviruses obtain their outer envelope by budding through the cell membrane. Prior to budding, viral coded proteins (hemagglutinins) are incorporated into the cell membrane. Such cells will adsorb erythrocytes to their surfaces, and the resulting foci of hemadsorption can be detected microscopically.

Immunological Methods

Animals infected with viruses respond by producing specific antibodies. Detection and measurement of these antibodies, which reflect disease status, are useful in planning herd health programs and studying the epidemiology of disease outbreaks.

While detection of antibodies is also useful in disease diagnosis, it is often a time-consuming process requiring the comparative measurements of antibody in acute and convalescent sera, usually collected 10 to 14 days apart. A more rapid approach is to use specific antiviral antibodies to detect viral antigens directly in clinical specimens. These antibodies are usually obtained by hyperimmunizing rabbits or goats with a specific virus. Alternatively, monoclonal antibodies may be used, if available.

Monoclonal antibodies (mAbs) are prepared in mice by first exposing the mouse to the viral antigen, which sensitizes B cells of the spleen. These cells are collected and chemically fused with a mouse plasmocytoma cell line that secretes IgG. These hybrid cells are then cloned and the resulting hybridomas, which are derived from a single cell, are analyzed for secretion of the specific antiviral IgG. Selected hybridoma cells are injected back into mice intraperitoneally, where the cells grow rapidly, and cause an accumulation of ascitic fluid containing a high concentration of mAb. Monoclonal antibodies are particularly useful in typing and subtyping viruses. When coupled to a fluorochrome, mAbs are widely used for the detection of viruses in tissues. They are also used in a number of commercial ELISAs for identification of viruses.

ReferencesLukert, P.D., 1989. "Virus Identification and Classification." In A Laboratory Manual for the isolation and

Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 182-185.

Schatt, K.A. and H.G. Purchase, 1989. "Cell-Culture Methods." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 167-177.

Senne, D.A., 1989. "Virus Propagation in Embryonating Eggs." In a Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 176-181.

Villegas, P., 1986. "Cultivation of Viruses in Chicken Embryos." In A Laboratory Manualof Avian Diseases. University of Georgia, Athens, GA. pp. 1-5.

B. Serologic Procedures

Serology is the science of using or detecting antibody in the fluids of animals. Antibodies are made up of immunoglobulins (Ig). Various serologic tests may utilize IgG, IgM or IgA in such fluids as tracheal washes, egg yolk, or serum. Serological monitoring is an important tool for detecting the presence of disease agents in poultry flocks. It is also useful for determining the immune status of poultry flocks. A serological test procedure offers a rapid and economical method for disease diagnosis.

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The presence of antibodies in the serum of birds, following a disease outbreak or vaccination, can give assurance that a certain disease did occur or that a vaccination procedure did have an effect. The presence of maternal antibody usually is desirable, and its detection and quantification are important in determining the timing of early vaccination procedures — another reason for serological testing.

Techniques for detection of antibody have evolved from procedures used primarily in the research laboratory. These have been developed to the point where many now are high automated and performed routinely in laboratories. Veterinarians, production managers, servicemen and growers have come to depend heavily on serological test procedures for important decisions on clean-up, depopulation and vaccine use.

Many such testing procedures are slow and time-consuming. The increased demand for serological testing, because of the valuable information it provides, has caused considerable interest in the development of new testing procedures which are reliable, rapid and economical.

Serological procedures are either qualitative or quantitative. The agar gel precipitin (AGP) test, for all practical purposes, is an example of a qualitative technique. There are several disease agents for which AGP test procedures are available. This type of test, however, only confirms the presence or absence of antibody. A quantitative AGP test can be done by preparing dilutions of the antibody in question.

Most serological procedures involve the determination of a titer, and are quantitative in nature. In titer methods, serum is serially diluted to a point (titer) where antibody no longer can be detected. Titers usually are expressed either as the dilution (expressed as a ratio, i.e. 1:64), or as the reciprocal of dilution (i.e. 64).

The concept of titer, or end point dilution, is easily and widely understood by veterinarians and poultry disease experts alike. A high titer is synonymous with disease or possibly with immunity, depending on the type and method of vaccination. Spray vaccination and inactivated (killed virus) vaccines usually produce



high titers.

Examples of quantitative serological tests include virus neutralization (VN), using eggs or cell culture; hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA). Virus neutralization is a good technique, but it takes three to seven days to obtain results, and bacterial contamination frequently is a problem when serum samples are not collected aseptically.

Bacterial and viral agents that hemagglutinate lend themselves to the HI test. This is an excellent procedure that can be performed quickly and economically, but standardize reagents are not available and therefore, results may vary widely from the same sample done by different laboratories. Another problem is that all agents do not hemagglutinate. Agglutination, and specifically micro-agglutination tests, can be performed for many bacteria, but the test is not very sensitive. Viruses are not large enough to be used in agglutination procedures. Antigens must be large enough to be visible in order to detect agglutination when and if it appears.

Regardless of the type of serologic test employed, large-scale testing programs present a series of problems associated with serum samples. These problems relate to the need for trained personnel plus materials and equipment for collection, processing, field storage, and transport of large numbers of sera. The result is increased cost of programs. Two alternative sources for obtaining antibody include egg yolk and whole chicken blood dried on filter paper for certain routine serotests. Problems associated with conventional serum samples can be avoided with these methods. For egg yolk, the yolk can be diluted 1:10 In PBS. For whole blood a more detailed method is described.

Filter paper from Scleicher and Schuall, Inc. Keene, NH, #740 is cut in strips approximately ½ X 8 inches, and three strips are overlapped in the middle and stapled together. The cluster of three strips can be used to collect six samples because blood can be collected on both ends of each strip. Individual birds or flocks can be identified on each strip. Whole blood sample is collected on the filter paper from a small pool of blood formed on the wing surface after puncture of a wing vein with a sharp object. The end (½ to 3/4 inch) of each strip is saturated with blood and complete saturation evidenced by equal blood staining on both surfaces. Partially clotted blood should not be collected.

Samples should then be allowed to dry for at least 30 minutes at room temperature or with a suitable warm air source. Dried blood samples should then be sealed in plastic bags. If the serum samples are to be examined by the ELISA test, then they need not be refrigerated and can be sent unrefrigerated by regular mail to a lab. If sera are to be tested for neutralizing antibodies, then the paper samples should be refrigerated. A portable cooler is ideally suited for this purpose when in the field.

Once samples reach a lab, a standard 4.8 mm-diameter paper punch (Gem Paper Punch, McGill Metal Products C., Marengo, IL) can be used to cut two disks from each sample. These disks are punched directly into one well of a 96-well microplate. After the disks are punched, phosphate buffered saline for the ELISA or cell culture media and antibiotics for the VN test are added to individual samples with a 200-ul pipette or (Medical Laboratory Automation, Inc., Mount Vernon, N.Y.) to rows of 12 samples with a multispenser (Dynatech Lab., Inc., Alexandria, VA.). Samples plus diluent are then agitated for about one hour on a shaker and then stored overnight in a refrigerator for complete elution of the sera from the disks. Disk color is the criterion used to assure complete elution. The color of completely eluted disks is uniformly light, whereas the color of incompletely eluted disks is darker in the center than at the periphery.

After complete elution, the samples can have complement inactivated by incubation at 56C for 30 minutes. The samples are then ready to be transferred into the first well for ELISA or neutralization testing. Comparing results from testing of serum from whole bloods taken with a syringe versus collected by elution from dried blood on paper strips, will be similar. Generally titers obtained from dry blood will be about 1:10 that of the conventional method.

The simplicity, economy, and reproductability of the dried blood collection and processing method make large scale serotesting more feasible. In addition, samples may be collected by persons with little training and experience and can be held for long periods of time before testing. Poultry servicemen can collect samples from flocks and eliminate the need for blood-collection crews. Arrangement of paper strips in clusters eliminates the need for a sample carrier or support, and sample identification can be written directly on the strip.

The one limitation of this technique is the initial dilution factor (1:10) introduced by elution of the sample. This becomes a problem for the NDV-Hl test when using eight or 10 HA units. The lowest Hl titer that can be tested would be 1:10 x 8 HA units or 1:80. This would be a problem especially for young poultry vaccinated


for the first time. It is not uncommon for young vaccinated birds to have titers of less than 1:80, yet they are resistant to challenge. In such cases, testing of circulating antibodies would be of limited value since their immunity probably resides in local sites (i.e. Harderian gland or upper respiratory tract) or is cell-mediated.

Latex Agglutination (LA)

Use: To detect antigen or antibody.LA tests are similar in principle to bacterial agglutination in that latex particles coated with antibody will agglutinate when mixed with the corresponding antigen and thus identifying it. Conversely, the latex particles can be coated with antigens and used to detect antibody. These tests are easy to perform and provide results within minutes. Commercial kits for "in office" use are available for the detection of antibody to some diseases and for detection of some viruses.

Immunoelectron Microscopy

Use: To demonstrate and identify viruses.The negative staining technique of electron microscopy referred to earlier for the demonstration of viruses is also useful for identification. The virus is reacted with immune serum, resulting in clumping that can be seen when viewed under the electron microscope.

Complement Fixation Test

Use: To detect and measure antibody.Complement fixation (CF) tests are most useful as an aid in the diagnosis of acute or recent viral infections, because they primarily detect IgM, the first immunoglobulin class to respond to infection. The test entails the use of viral antigens, guinea pig complement, and an indicator system of "sensitized" sheep RBCs. Reacting with antibody directed against them sensitizes the sheep RBCs. This anti-sheep RBC antibody is referred to as hemolysin and is prepared in rabbits. The antigen and complement are each titrated and diluted. If no specific antibodies are present in the serum being tested, the complement is free to react with the sensitized RBCs, causing lysis. If sufficient antibody is present, the specific antigen-antibody complexes will have bound the complement and no lysis of the RBCs will occur.


ReferencesSkeeles, K., 1985. ELISA's Role in Serological Monitoring of Poultry Flocks. Vineland Update, No. 12, March.

Vineland, N.J.

Immunodiffusion

These tests are routinely used to demonstrate the presence of antibodies against adenovirus or avian influenza in sera, or the presence of viral antigens such as infectious bursal disease, or reoviruses in concentrated cell culture or embryo fluids. The most common technique is the two-dimensional double diffusion procedure, in which both antigen and antibodies diffuse toward each other from separate wells in agar in a petri dish or glass slide (Figure 1.0). This double-diffusion test is known as agar gel precipitin (AGP) or double immunodiffusion (DID) tests.

Figure 1.0 AGPT

Figure 1.0. AGPT test for mycoplasma

A typical AGP test is as follows.

1.Prepare agar gel plates:

NaCl8% (8 gm)Noble Agar*0.7% (0.7 gm)0.1 M PBS pH 7.210 ml1% Thimerosal1 mlPolyethylene Glycol (6000 MW)2 gmDistilled Water89 ml

Autoclave for 10 minutes and pour into small (35 mm) tissue culture plates (2 ml per plate). After the agar has cooled, punch holes with a commercial puncher.

2.Place antigen in the center well and serum samples in outer wells or vice versa. Always include a known positive control. Do not overfill the wells.


3.Place in moisture chamber at room temperature and check for precipitation daily.

4.The antigen and specific antiserum should form a band of precipitation (tiny white line). If the unknown sera samples contain antibodies specific for the antigen in center well, a band should also be present.

Note: The test can also be run using regular glass slides. The agar is poured on the slide and the holes are punched. Humidity must be high in the chamber to avoid desiccation of the agar.

*The agar can also be prepared using purified agar or ionagar #2.

ReferencesVillegas, P., 1986. "Immunodiffusion." In Laboratory Manual, Avian Virus Diseases. University of Georgia

Press, Athens, GA. pp. 13.

Agglutination

The clumping or agglutination of bacteria by antibody is a relatively simple and older technique. The tests are routinely done on breeder or layer flocks for Salmonella and Mycoplasma. The serum plate or slide test is used as an initial screening test. If the serum agglutinates the stained antigen, the bird had been exposed to the organism. Standardized agglutination reagents are available for the previously mentioned organisms. These tests can be done as rapid whole-blood, standard macroscopic tube, microagglutination or microantiglobulin tests.

The rapid whole-blood test is the most widely used for detecting S. pullorum and S. gallinarum infected chickens. In this test a drop of crystal-violet-stained pullorum polyvalent K antigen is mixed on a glass or plastic plate with a loopful of whole blood. The plate is rocked for 2 minutes and results read. If there is visible clumping of the antigen, the sample is positive. If the antigen-blood mixture remains clear the sample is negative.

ReferencesMallison, E.T. and G.H. Snoeyenbos, 1989. "Salmonellosis." In A Laboratory Manual for the Isolation and

Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 3-11.

Hemagglutination-inhibition (HI)

This test is a rapid, sensitive serologic method for determining antibody against Newcastle disease (ND), infectious bronchitis (IB), avian influence (AI) and adenoviruses, as well as Mycoplasma gallisepticum and M. synoviae. In order for this test to be used the organism in question must have the ability to agglutinate red blood cells (hemagglutination). The inhibition or blocking of hemagglutination (HA) of red blood cells by antibody specific for an organism is the basis for this test. The simplicity, ease and the fact that it doesn't require expensive equipment are its main advantages. The major disadvantage is that the preparation and quantitation of HA antigen and procedures for running the HI test vary among laboratories, resulting in nonuniformity of results for the same sample obtained from different laboratories.

The basic reagents in the test are the HA antigen (microorganism), serum diluted two-fold in a saline solution and erythrocyte suspension. The test is usually done in 96 well microtiter plates with micro diluters and pipettors. The constant-antigen, diluted-serum (Beta technique) is most often used. The antigen is first prepared and titrated in saline and incubated with erythrocytes for HA activity. The antigen is then serially diluted in saline and used as a constant amount in the HI test. The amount of HA units depends on the microorganism. In the HI test, the serum, after having the complement activity destroyed, is serially diluted in saline and incubated with a constant amount of antigen. The erythrocytes are then added and the test incubated. Some viruses such as NDV and AI produce an enzyme, neuraminidase, which can cleave the hemagglutinin causing the virus and red blood cells to detach after one hour. Therefore, both the HA and HI test must be read within one hour or false results will occur.

Collection and preparation of chicken red blood cells (RBC's)


1.Use a sterile syringe and needle. For chickens older than 5 weeks, it is advisable to use a 20 gauge, 1 ½" needle.

2.Use Alsever's solution as anticoagulant. Draw the Alsevier’s solution to approximately half the total syringe volume.

3.Using 3-5 known SPF birds which have never been exposed to or vaccinated with the organism you are testing for, draw blood to fill the syringe. Mix well but gently.

4.Dispense into clean graduated centrifuge tube. Centrifuge at about 1,500 RPM's (revolutions per minute) for 5 minutes. Remove and discard the supernatant.

5.Resuspend the RBC's to the original volume using phosphate buffer saline (PBS) solution. Mix well and centrifuge again. Repeat this procedure approximately 3 times.

6.After the last washing, resuspend the RBC's in buffer solution to make a 5% stock solution.

7.Refrigerated RBC's should be good for approximately one week.

Preparation of Newcastle disease virus (NDV) antigen for HI tests

1.Inoculate 9-12-day-old embryonating chicken eggs via the allantoic cavity with 10P3P to 10P5P ELDB50B's of NDV and incubate at 37C.

2.Discard embryos dying within 24 hours post-inoculation.

3.Collect dead eggs and refrigerator for 4 hours when embryo mortality reaches 10 to 30%.

4.Harvest and pool the allantoic fluids (AF), but discard any fluids inadvertently contaminated with RBC.

5.Clarify AF by low speed centrifugation for 10 minutes.

Antigen production for the hemagglutination inhibition (HI) test for infectious bronchitis

1.Dilute the strain of infectious bronchitis virus (IBV) appropriately (10P3P to 10P5P) according to its titer in Hanks balance salt solution.

2.Inoculate 9-11 day chick embryos with 0.1 ml diluted virus.

3.Incubate inoculated embryos, candle and discard dead at 24 hours.

4.Remove embryos from incubator 30-48 hours post-inoculation and place in refrigerator (4C) overnight for a minimum of 3 hours.

From this point on, handle allantoic fluid or antigen preparation on ice. 5.Collect allantoic fluid (AF) as free from red blood cells as possible.

6.Centrifuge AF at 1500 rpm for 30 minutes to remove red blood cells and debris, decant and save supernatant. Note how many ml of AF you have.

7.Concentrate virus by pelleting at 30,000 G for 90 minutes and discard supernatant.

8.Resuspend virus pellet in HEPES buffer at pH 6.5 by adding 1 ml per 100 ml of AF prior to centrifugation.

9.Add an equal volume of phospholipase C (Sigma Chemical) with 1 unit/ml diluted from concentrate in HEPES buffer. The enzyme exposes the hemagglutinin on the viral surface.

10.Mix until the fluid is in a uniform suspension.

11.Incubate 2 hours at 37C, vortexing every 30 min.

12.Storage: The antigen is stable at 4C for 2 months. Repeated freeze-thawing adversely affects antigen


titer.

The antigen must be titrated before use.

Hemagglutination (HA) Determination

1.Add 50ul of PBS to each well of 96 well microtiter plates.

2.Add 50ul of antigen to first well and test antigen undiluted and at a 1:5 dilution.

3.Make serial two fold dilutions of the antigen.

4.Fill each well with 50ul of 0.5% RBC.

5.Seal each plate with saran wrap and incubate at room temperature for 30-40 min. 6.The titer of the antigen is the last dilution of antigen showing agglutination of red blood cells (lacy circular

pattern).

7.The HA titer is equal to 1 HA unit.

Hemagglutination Inhibition Test

1.Make dilution of antigen to yield 8 HA units.

2.Add 50ul of working antigen to all wells except rows A and B. Add PBS in these rows instead.

3.Add 50ul PBS in the 1PstP wells of rows A and B, and working antigen on 1PstP well of C and D.

4.Add 50ul of positive test sera in 1PstP wells of rows E, F and G.

5.Add 50ul of negative test sera in 1PstP well of row H.

6.Make serial two fold dilutions in all wells. 7.Cover plates with saran wrap.

8.Incubate at 37C for one hour.

9.Fill each well with 50ul of 0.5% RBC.

10.Incubate at room temperature for 30-40 min.

11.The HI titer of the serum is computed by multiplying the reciprocal of the serum titer, last serum dilution showing no hemagglutination (button formation), by the number of HA units used in the test (8 HA units) (Figure 3.0).

Figure 3.0 HI test for NDV (wells A-E are positive in rows 8 + 9)

ReferencesBeard, C.W., 1989. "Serologic Procedures." In A Laboratory Manual for the Isolation and Identification of


Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 192-200.

Villegas, P., 1986. "Collection and Preparation of Chicken Red Blood Cells." Preparation of Newcastle disease virus antigen for HI tests." Antigen Production for the Hemagglutination Inhibition (HI) test in Infectious Bronchitis." In Laboratory Manual: Avian Diseases. University of Georgia, Athens, GA. pp. 14-19.

Immunofluorescence

Antigens or antibodies can be labeled with fluorochromes. These labeled reagents, when exposed to a specific wavelength of light, will give off a detectable color. Immunofluorescence is most often used to detect the presence of microbes in tissues or cell cultures, but can be used to detect antibody in serum or antibody secreting cell culture fluids.

Detecting fluorochrome-labeled reagents

To detect fluorochrome-labeled reagents, a specially equipped microscope is required. To detect low levels of fluorescence commonly produced in cell staining experiments, the microscope must have epifluorescence in which the exciting radiation is transmitted through the objective lens onto the surface of the specimen. Absorbing radiation of the appropriate wavelength causes electrons of the fluorochrome to be raised to a higher energy level. As these electrons return to their ground state, light of a characteristic wavelength is emitted. This emitted light produces the fluorescent image seen in the microscope. Individual fluorochromes have characteristic excitation and emission spectra. Filters are used to ensure that the specimen is irradiated only with light at the correct wavelength for excitation. By placing a second set of filters in the viewing light path that only transmit light of the wavelength emitted by the fluorochrome, images are formed only by the emitted light. This produces a black background and a high-resolution image.

Because some fluorochromes have emission spectra that do not overlap, two fluorochromes can be observed on the same sample. This allows the study of two different antigens in the same specimen even when they have identical subcellular distributions.

The most commonly used fluorochromes are fluorescein and rhodamine. They are normally available as isothiocynate derivatives. They can be conjugated to anti-immunoglobulin antibodies, protein A, protein G, avidin, or streptavidin. These conjugates are available from many commercial sources or can be prepared in your laboratory. Filter sets are commercially available that will permit independent observation of these two fluorochromes in the same sample from Sigma Chemical Co. Rhodamine requires an excitation wavelength of 552 and emission wavelength of 5% and produces a red color. Fluorescein needs an excitation wavelength of 495 and produces an emission wavelength of 525 and a green color.

Fluorescence detection is not compatible with histochemical stains, because the components of these stain autofluoresce. Fluorescence detection is not compatible with enzyme detection systems, because the deposition of insoluble compounds after enzyme detection will block the emission of light from the fluorochrome.

Isothiocyanate labeling

Both fluorescein and rhodamine isothiocyanate derivatives are available for coupling reactions. The major problem encountered is either over- or undercoupling, but the level of conjugation can be determined by absorbance readings.

1.Prior to the coupling, prepare a gel filtration column to separate the labeled antibody from the free fluorochrome after the completion of the reaction. Use a gel matrix with an exclusion limit of 20,000-50,000 for globular proteins. Use fine-sized beads (approximately 50 m in diameter).

To determine the size of the column, multiply the volume of the reaction by 20. Prepare a column of this size according to the manufacturer's instructions (swelling, et.).

Equilibrate the column in PBS. Allow the column to run until the buffer level drops below the top of the bed resin. Stop the flow of the column by either using a valve at the bottom of the column or by plugging the end with modeling clay.

2.Prepare an antibody solution of at least 2 mg/ml in 0.1 M sodium carbonate (pH 9.0).


3.Dissolve the fluorescein isothiocyanate (FITC) or tetraethyl-rhodamine isothiocyanate (TRITC) in dimethyl sulfoxide at 1 mg/ml. Prepare fresh for each labeling reaction.

4.For each 1 ml of protein solution, add 50 l of the dye. The dye should be added slowly in 5-l aliquots, and the protein solution should be gently, but continuously, stirred during the addition.

5.Leave the reaction in the dark for 8 hours at 4C.

6.Add NHB4BCl to 50 mm. Incubate for 2 hr at 4C. Add xylene cylanol to 0.1% and glycerol to 5%.

7.Separate the unbound dye from the conjugate by gel filtration. Carefully layer the coupling reaction on the top of the column. Open the block to the column and allow the antibody solution to flow into the column until it just enters the column bed. Carefully add PBS to the top of the column and connect to a buffer supply. The conjugated antibody elutes first and can be seen under room light.

8.Store the conjugate at 4C in the column buffer in a lightproof container. If appropriate, add sodium azide to 0.02% to inhibit microbial contamination. When using low concentrations of antibody (i.e., < 1 mg/ml), it is advantageous to add bovine serum albumin to a final concentration of 1%.

9.For fluorescein coupling, the ratio of fluorescein to protein can be estimated by measuring the absorbance at 495 nm and 280 nm. For rhodamine, measure at 575 nm and 280 nm. The ratio of absorbance for fluorescein (495—280 nm) should be between 0.3 and 1.0; for rhodamine (575—280 nm), between 03. and 0.7. Ratios below these yield low signals, while higher ratios show high backgrounds.

If the ratios are too low, repeat the conjugation using lower levels of antibody and higher levels of dye. If higher levels are found, repeat the labeling with higher levels of antibody and lower levels of dye. Equilibrate and load the column with 10 mM potassium phosphate (pH 8.0). Elute with increasing salt concentrations. Measure the ratios (495/280 or 575/280) of each fraction and select and pool the appropriate fractions.

There are two methods of the fluorescent antibody (FA) test that can be performed, the direct and indirect. The direct method requires only 1 antibody and it must be conjugated. This antibody is called the primary antibody and must be directed against the microorganism. This method is less sensitive than the indirect, but yields less nonspecific fluorescence. It also requires conjugation of numerous antibodies against each organism to be detected.

The indirect method requires two antibodies, the primary and secondary. The primary antibody is directed against the microorganism and is unconjugated. The secondary antibody is conjugated and is directed against the primary antibody. It is usually an anti-immunoglobulin IgG. Commercial conjugated anti-chicken IgG immunoglobulin reagents are available prepared in goats and rabbits. For reasons of increased sensitivity and beyond, only one commercially available conjugate is needed; the indirect test is most often used.

Indirect fluorescence antibody test for viral antigen detection

I. 96-well plate method

1.Aspirate medium from wells of plate containing cells infected with the virus.

2.Wash once with PBS.

3.Immediately aspirate buffer, tap plate on towel to remove excess buffer.

4.Fix for 15 min w/ 1:1 acetone-methanol (Do not let this go more than a couple of minutes more before proceeding to the next step).

5.Pour off acetone-methanol, tap on a towel. Immediately put buffer in wells, this can sit until all plates are fixed.

6.Once all plates are fixed and contain buffer, wait 10 min, and then do one more 10 min wash and pour off.


7.Rinse with HB2BO and pour off. Tap on towel and allow plates to sit upside down (drain). Tap again and then add primary antisera (antimicroorganism).

8.Incubate 1 hour at 37C and rinse with HB2BO.

9.Wash twice (10 min each) with buffer and repeat step 7 (rinse).

10.Add conjugated (secondary) antibody (antichicken IgG) and incubate 1 hr at 37C.

11.Rinse with HB2BO and add buffer to wells and keep in dark until examination under an ultraviolet microscope.

II. Slide Method

1.Scrape the cells where the virus was propagated to 90% CPE from the flask.

2.Centrifuge at 1500 rpm for 10 min.

3.Decant the media.

4.Wash cells with PBS.

5.Centrifuge at 1500 rpm for 5 min.

6.Decant supernatant.

7.Put a drop of the packed cells on a slide and air-dry.

8.Immerse slide in acetone-methanol solution (1:1) for 5 min and wipe dry.

9.Flood fixed samples with primary antisera.

10.Incubate at 37PoPC for 30 min.

11.Wash with PBS 2x thoroughly (5 min/wash).

12.Wipe sides and back of slides.

13.Flood with conjugated (FITC) secondary antibody.

14.Incubate at 37PoPC for 30 min.

15. Wash with PBS. Wipe.

16.Put a drop of mounting liquid (1 part PBS/9 parts glycerol)

17. Cover with cover slip and view with fluorescent microscope (Figure 4.0).


Figure 4.0 FA positive cells

III. Fresh or Frozen Tissue Imprints Method

1.Cut a cross-section of the tissue and imprint the cut side on a slide. Allow to dry.

2.Dip the slide in 1:1 acetone-methanol.

3.Incubate for 2 min at room temperature.

4.Rinse twice in PBS.

5.Flood fixed samples with primary antisera.

6.Incubate at 37C for 30 min.

7.Wash with PBS (2x) thoroughly (5 min/wash)

8.Wipe sides and back of slides

9.Flood with conjugated (FITC) secondary antibody

10.Incubate at 37C for 30 min

11.Wash with PBS and wipe.

12.Put mounting liquid & cover with cover slip and observe microscopically.

IV. Formalin Fixed Paraffin Embedded Tissue Sections Method.

1.Prepare unstained formalin fixed paraffin embedded tissue sections as you would for standard Hematoxylin and Eosin pathologic slides.

2..Deparafinize and rehydrate sections through xylene and graded alcohol series.

a.place slide in xylene for 3 mins and again for 3 min in a fresh solution.

b.place slide in 100% ethanol for 2 mins and then for 2 mins in a fresh solution.

c.place slide in 95% ethanol for 2 mins.

d.rinse slide for 5 min in distilled water.

3.Repeat staining procedures with primary and secondary antibody listed under Indirect FA test using fresh or frozen tissue imprints.

References


Harlow, E. and Lane David, 1989. "Cell Staining." In Antibodies, A Laboratory Manual, Cold Spring Harbor Publishing, Cold Spring Harbor, NY. pp. 353-354, 409.

Virus neutralization (VN) test

The VN test employs the ability of specific antibody to bind to the virus and neutralize its infectivity. Antibody has cell surface receptors, which can attach to receptors on the virus envelope. The antibody, through a variety of mechanisms, can inhibit virus attachment and/or penetration into the host cells or viral uncoating within the cell. Some antibody also has the ability to destroy virus particles prior to attachment or penetration into the cell.

The VN test can be used to determine the amount of antibody in bodily fluids. In this procedure (Beta) a constant amount of virus is used and the antibody is serially diluted. The test can also be used to identify unknown viruses and differentiate between serologic types of subtypes using an Alpha procedure. This technique employs a constant amount of known sera and serially diluting known or unknown virus. In either assay, the virus-serum mixture is allowed to incubate for 30 to 60 minutes usually at 37C and then inoculated into susceptible embryonating eggs, cell cultures or live animals. The VN titer is calculated with an equation, which utilizes the highest dilution of virus or antisera which causes or neutralizes infectivity in the assay system. Infectivity can be measured by morbidity, mortality, gases or microscopic lesions of cytopathic changes in the assay system (susceptible host or cells).

The following procedures are routinely done for common avian viral pathogens. Calculation of Neutralizing Titer

Beta Method: A 50% end point of neutralization is calculated by the method of Reed and Muench. The neutralizing titer is calculated from the endpoint. Table 1.4 shows typical results of a VN test.

Table 5.0 Results for VN Test

Dilution of Serum Infectivity ratio Percent infected

1/16 *1/5 20

1/64 3/5 60

1/128 5/5 100

*Number positive over total number

Method: The Neutralization index (NI) of the serum is the difference between the log titer of the virus control (negative serum) and the log titer of the serum—virus mixture. Tables 5.0 and 5.1 show typical results and the data are calculated as follows:

Titer of virus control (negative serum + virus) = 3.5Titer of positive serum + virus= U-1.5UDifference (NI)= 2.0


P b P Titer calculated by Method of Reed and Muench.

UMean infective dose (IDUBU50UBU) represents the amount of organism capable of infecting 50% of the animal

The equation to use from these data is calculated as follows:=

(0.75 x 0.6) + (1.2)0.6 =log of the serum dilution factor1.2=log of the lower dilution used to calculate proportionate distance

= 1.65 antilog of 10P1.65P = 4950% neutralization endpoint = 1.49

I. Alpha Method (Constant-Serum Diluted Virus)

This procedure is used to quantify antibodies against avian infectious bronchitis virus and avian encephalomyelitis. The serum-virus mixtures are incubated and then assayed for residual virus in chicken embryos by quantal response.

1.Heat inactivate serum sample for about 30 minutes at 56C in water bath. Make 1:2 dilution if quantity is insufficient for the test. You will need at least 2.0 ml of serum for test. Use phosphate buffered saline (PBS) for diluting. Make the lowest dilution possible.

2.Make virus dilutions. Use MASS 42 Infectious Bronchitis Virus (IBV) (Beaudette Strain) or AE virus (Van Roekel Strain). Make dilutions from 10P-1P to 10P-6P in tryptose phosphate broth (TPB) with antibiotics.

3.Virus dilutions are added to the serum tubes:

Tube 1 = 0.4 ml serum + 0.4 ml virus 10P-6P





Incubate for 1 hour at Room Temperature.

4.Place 0.4 ml TPB and 0.4 ml of the virus dilutions 10P-6P through 10P-10P into each separate tube. This is the virus titration.


Proportionate distance =U {50% - (% infected at dilution below 50%)} (% infected at dilution above 50%) - (% infected at dilution below 50%)} = U {50 - 20}_U _ U30_ {60 - 20} {40} = 0.75

5.Incubate for one hour at Room Temperature

6.Leave at least five embryos uninoculated for controls.

7.Seal eggs with Duco cement and place in the incubator at 37C.

8.Candle eggs daily. Discard and disregard all embryonated eggs dead at 24 hours.

9.Record deaths each day for 7 days. On the 7PthP day, open live embryos by refrigeration for 4 hours and check for stunting of embryo which is characteristic of IBV virus. Infected or positive embryos show mortality (gross lesions, hemorrhages, curling, stunting, clumped down and/or kidney urates. Embryos can also be weighed. Embryos weighing less than 80% of the uninoculated control are considered infected (non-neutralized). All eggs are discarded and incinerated.

10.Calculate neutralization index according to previously listed equation.

II.(Beta Procedure)

This procedure is used for infectious bronchitis (in CEKC) and for reovirus and infectious bursal disease virus in CEF.

1.Dilute virus to obtain the appropriate amount of virus to be used in the test. One hundred TCIDB50B is frequently used. The virus dilution is determined from previous titration of the virus by the same methods (i.e. quantal response in cell culture).

2.Add 100 l of virus to well 1 from A to H and 50 l to all other wells except to wells in column 12, which will be the CELL CONTROL.

3.In the first well (column 1, A) add 25 l of the heat-inactivated (56C for 30 min) serum sample using a 25 l microdiluter. All samples are placed in well 1 from A to H (8 samples can be tested per plate).

4.Using the multimicrodiluter, transfer 50l of virus-serum mixture from well 1 to well 2 and continue to well #10. Discard the content of the microdiluters by using sterile blotted paper. Add 50 ul of a known normal serum or PBS to column 11 which will serve as VIRUS CONTROL.

5.Incubate the plates for approximately 30-45 minutes at 37C.

6.Add 0.2 ml of freshly prepared chicken embryo kidney cells, or CEF diluted to contain approximately 5 x 10P4P cells per well, to all wells. Add 50 ul of PBS to all no. 12 wells..

7.Cover the plates with sterile polystyrene covers or with sterile tape.

8.Incubate for approximately 72 to 96 hours.

9.Fix and stain (see technique for staining cell culture monolayers that is listed at the end of this section).

10.The end-point of any serum sample will be the dilution where the virus has been neutralized by the diluted serum. It should look like the cell control and contain no virus specific CPE.

11.Positive and negative controls should ALWAYS be included.

Calculate neutralization index according to previously listed equation.MICRONEUTRALIZATION TEST

1.Prepare the chicken embryo fibroblast (CEF) adapted strain of virus so that approximately 100 infectious units (IU) will be present in 0.05 ml (50 ul).

2.Using the 50 ul pipette, add one drop of the diluted virus to all wells (from 1 to 11) except in well #12. This well will be the cell control.

3.Add 50 ul of the serum sample (Figure 5.1) in the first well (well 1, row A). Follow the same procedure for each one of the serum samples (sample 2 will be located in well 1, row B; sample 3 in well 1, row C;


and so on).

4.Using the 50 ul microdiluter fitted with the handle, dilute all samples in the first well and transfer 50 ul to the second well. Repeat the same procedure up to well #10 and discard the 50 l left.

5.Allow the preparation to incubate at room temperature for 30-45 minutes.

6.Add 0.2 ml of CEF that has been prepared and diluted to be used in microtiter plates (each well should receive 0.2 ml of cells).

7.Add 50 ul of Hank's balanced salt solution to all number 12 wells.

8.Cover the plates with sterile polystyrene covers or with sterile tape.

9.Incubate for approximately 72-96 hours.

10.Fix and stain (Figure 5.0).

Controls:

1.Always include a known positive and negative antiserum.

2.Wells 12 will be cell control.

3.Wells 11 will be virus control.

11.Calculate neutralization index.

Staining CKC Monolayers in Microtiter Dishes

1.Pour the media out of the wells of the microtiter dish. If the wells contain virus, pour the media into disinfectant solution.

2.Wash the cells once with PBS and pour into disinfectant.

3.Fill the wells with 95% ethanol and allow the cells to fix for three to five minutes.

4.Pour off the ethanol.

5.Add 1% crystal violet staining solution (see Appendix for preparation) to the wells and allow the cells to stain for three to five minutes.

6.Pour off the stain and wash the dish with tap water until all excess stain has been removed from the wells (generally three to four washes).

7.Allow the wells to drain and dry the dish.

8.Read the plates as follows:

Virus Control

The wells where the virus has produced cytopathogenic effect (CPE) will be clear. The titer of the virus will be the reciprocal of the highest dilution where there is CPE.

Cell Control

The cell control should be stained dark (purple or blue) since there is no CPE.

Positive Serum Control

Should appear similar to the cell control.

Negative Serum Control


Should look like the virus control.

CRYSTAL VIOLET SOLUTION

Stock Solutions

Solution A:Crystal violet (90% dye-content) 2 gEthanol (95%) 20 ml

Solution B:Ammonium oxalate0.8 g

Distilled water 80 ml

Figure 5.0. VN Test in cell culture

Staining Solution

Mix 1 part of solution A and 9 parts of solution B.

Figure 5.1. Heart BleedingReferences

Beard, C.W., 1989. "Serologic Procedures." In A Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publications, Co., Dubuque, Iowa. pp. 201-207.

Reed, L.J. and H. Meunch, 1938. A simple method for estimating fifty percent endpoints. Am. J. Hyg. 27:493-497.

Villegas, P., 1986. "Virus Neutralization Test in Embryos." "Neutralization Test." Microneutralization Test for Infectious Bursal Disease Virus." Staining CKC Monolayer in Microtiter Dishes." In Laboratory Manual for Avian Viral Diseases." University of Georgia, Athens, GA. pp. 10, 26, 27, 42.

ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)


The ELISA has received considerable attention since its introduction in 1972. A mass of publications has been generated in almost every research area with this immunological assay. Techniques have been developed for almost every pathogen of both man and animal. This includes ELISA techniques for a variety of poultry pathogens. ELISA is convenient, reliable, fast, highly sensitive, and results have compared favorably with other assay procedures such as virus neutralization.

Simplicity and economy are dependent on the availability of the reagents needed for the test, the type of laboratory equipment available and the type and training of laboratory personnel involved in performing the procedure. The indirect ELISA for detection of antibody consists of the following basic steps:

1.Absorption of antigen to a solid phase.

2.Wash

3.Addition of antibody

4.Wash

5.Addition of an enzyme-linked antiglobulin

6.Incubation

7.Wash

8.Addition of substrate (responsible for color reaction)

9.Determination and expression of tests results.

ELISA is simple, but there are more steps in this procedure than in other serological tests commonly used in diagnostic laboratories. Several companies have developed, or are in the process of developing, ELISA kits

for common poultry pathogens. These companies (IDEXX, Portland, ME; Affiniteck, LTD. of Bentonville, Ark; and Kirkegaard and Perry Lab, Gaithersburg, MD.), have developed and marketed world wide commercial kits for determining antibody against a variety of common poultry pathogens. The ever growing list includes Pasteurella multocida, Bordetella avian, Hemorrhagic enteritis virus, infectious bursal disease virus, infectious bronchitis, Newcastle disease, Avian

encephalomyelitis, reovirus, mycoplasma, infectious laryngotracheitis, infectious anemia and avian leukosis viral antigens. These kits are sold in 4-96 well plates for an individual organism. Each kit can test 360 samples in about 4 hours time, costing about 50¢ per sample depending on number of kits purchased, shipping and custom charges.


Figure 6.0. Automated ELISA systems

ELISA is the immunological test of choice for the present — one of the major reasons being the rapid turn-around time once a test is established, and the ability to incorporate computer-assisted analysis and data management as part of the actual test procedure.Most laboratory equipment, including the spectrophotometers used for determining raw ELISA test results, has the capability of being interfaced with a microcomputer (Figure 6.0). Once into the computer, it can be easily analyzed, interpreted, stored, retrieved and reported. The ability to report results graphically, in the form of a histogram or bar graph, is done with relative ease by the computer. Numbers that are difficult to understand by all but the highly trained can be replaced by graphs or pictures that are easy to interpret. The ability to manage, maintain and retrieve records on tests performed at different time intervals can be done by pressing a few keys. The ability of one computer to access another via telephone linkup makes the movement of data from the laboratory back to the submitter, a very rapid process.

The real strength of the ELISA procedure is that results can be recorded photometrically. Specialized spectrophotometers are available which can read test results right in the well in which the test is performed. Results from the spectrophotometer reading (absorbance) can be reported in a number of different ways:

1.As "positive" or "negative." A certain optical density (O.D.) can be predetermined using known negative and positive serum samples that would correlate as being "positive" or "negative."

2.As an O.D. unit or absorbance value. When the quantity of antibody in the sample increases, the O.D. value increases.

3.As a positive/negative ratio (P/N). The O.D. of a positive sample over the O.D. of a negative sample at one set dilution (1:50 or 1:100). The higher the P/N ratio, the more antibody is present.

4.Titer can be determined by carrying out serial dilutions of serum until the P/N ratio is less than or equal to (U<U) 1, An end-point dilution or titer can be determined as with a VN or HI test.

5.Use of a standard curve made from a group of positive serum samples of varying antibody titer. Using one dilution of serum and appropriate standards, one can determine the titer of an unknown serum sample.

6.Combinations of the above.

The method of performing the ELISA test, and the details of calculating the end result, may appear somewhat confusing. With appropriate automated equipment and computer-assisted analysis, however, tests can be performed and results obtained with great speed. Hundreds of samples can be tested in a matter of a few hours.The ELISA assay is an antigen-antibody reaction system which uses an antibody-coupled enzyme as the indicator. Color development after reaction of the enzyme with the substrate is directly proportional to the amount of antibody (or antigen) present in the sample being tested (figure 6.1). The commercial assays are bought as kits and set up in microtiter plates by the manufacture for ease of testing large numbers of


samples. The wells of the microtiter plates are coated with a standardized amount of antigen (for the detection of antibodies) or antibody (for the detection of antigen). The sample(s) is incubated in the test well during which time the antigen-antibody complex forms. Excess or unbound material is washed from the well and anti-chicken IgG-horseradish peroxidase conjugate (or anti-antigen conjugate) is added. This binds to the antibody (antigen) bound to the fixed antigen (antibody). This is incubated and the excess is washed away. Lastly a chromogen (orthophenylenediamine or OPD) and enzyme substrate (hydrogen peroxide) are added to the wells. Subsequent color development is proportional to the amount of antibody (antigen) in the original sample.

I.Preparation of Dilution Plates from Commercial Kits. Each Kit contains all necessary materials, reagents and buffers to run the test.

1.Add 200 l of dilution buffer to each well of an uncoated low protein binding 96 well microtiter plate. This plate serves as the serum dilution plate.

2.Add 4 l of unknown serum per well (producing a 1:50 dilution). Start with well A4 and end with well H9 (moving left to right, row by row of wells). For example, wells one through 10 contain the diluted sera of flock one, wells 11-20 contain the diluted sera of flock two, etc.

3.Aspirate and remove any liquid in dilution plate wells A1, A2, A3, H10, H11 and H12.

4.Allow all diluted sera to equilibrate in dilution buffer for 5 min before transferring to an antigen coated ELISA plate.

5.Diluted serum should be tested within 24 hours.

II.Preparation of Reagents

1.1X Wash SolutionDilute 1 ml of concentrated Wash solution in 19 ml of distilled water. Approximately 400 ml of Wash Solution is needed for each 96 well plate.

2.1X Stop Solution

Dilute 1 ml of concentrated Stop Solution in 4 ml of distilled water. Approximately 15 ml is needed per plate.

3.Controls

Dilute an aliquot of each positive control and normal control sera with dilution buffer (1:100) in separate 5 ml test tubes, e.g. 10l/1ml buffer. 300l is needed per plate.

4.Conjugate Solution

Dilute 100 l of horseradish peroxidase conjugated anti-chicken IgG in 10 ml of dilution buffer.

5.Substrate SolutionEach plate will require approximately 10 ml of substrate solution.

III.ELISA Test Procedure

1.Label antigen test plate according to dilution plate identification.

2.Add 50 ul of dilution buffer to each test well (with exception of wells A1, A2, A3, H10, H11 and H12).

3.Add 10 ul of diluted Normal Control Serum to wells A2, H10 and H12.

4.Add 100 l of diluted Positive Control Serum to wells A1, A3 and H11.

5.Transfer 50ul/well of each of the unknowns from the dilution plate to the corresponding wells of the


coated test plate. Do the transfer as quickly as possible.

6.Incubate plate for 30 min at room temperature.

7.Tap out liquid from each well onto an appropriate decontamination vessel.

8.Wash each well with 300 ul of 1X wash solution. Allow to soak for 3 min and tap into waste container. Repeat this procedure two more times.

9.Dispense 100 ul of diluted conjugate into each assay well.

10.Incubate for 30 min at room temperature.

11.Wash as in steps 7 and 8 above.

12.Dispense 100 ul of the Substrate Solution into each test well.

13.Incubate 15 min at room temperature.

14.Add 100 ul Stop Solution to each test well.

15.Allow bubbles to dissipate before reading plate.

Figure 6.1. ELISA Diagram

IV.Processing of Data

Measure and record the absorbance at 490 nm wavelength using a microplate spectrophotometer. If the spectrophotometer is interfaced to a computer and the computer contains software furnished by one of the ELISA kit manufacturers, all calculations, analysis and tabulating of data into bar graphs or histograms will be automatic. If no software or kits are available for your needs or the laboratory is making their own reagents, the calculations can be done manually as shown below. CONTROLS


Negative controls (2) must have a mean ODB490B of less than 0.150.Positive controls must have a mean ODB490B of at least 0.200 greater than the negative controls.

Ex. If NCx= 0.1200.200 minimum differencethen PCx=0.320

INTERPRETATION

The relative levels of antibody are calculated using sample to positive ratios (S/P). Sample to positive ratios of less than 0.2 are considered negative. Samples with S/P ratios greater than 0.2 indicate the presence of antibody and a history of exposure to the agent in question.

CALCULATIONS Provided are examples of the calculations used for S/P ratio and titer determinations.

3. Sample to Positive Ratio (S/P)

sample mean –NCx = S/PPCx- NCx

1.100 - 0.060 = 2.080.560 - 0.060

4.Calculation of Titer at 1:500 DilutionLogB10B Titer = 1.09 (logB10B S/P) + 3.36 " = 1.09 (logB10B 2.08) + 3.36 " = 1.09 (0.318) + 3.36 " = 3.70 Titer = Antilog 3.70 = 5012Samples with ODB490B > 2.001 may be diluted further and retested for more accurate titer determination.

The final titer is determined by multiplying the titer of the sample by the dilution factor in proportion to the 1:500 standard.

i.e., for a 1:4000 dilution,So multiply titer by a factor of 8 for final titer determination.

TECHNICAL TIP

Avoiding Contamination of Test Kit Reagents


1. Negative control mean NCx=

{0.580+0.062} 2 = 0.060

2. Positive Control Mean PCx= 0.580 + 540 = 0.560

Proper handling of test kit reagents is important to get optimal test kit performance. A sudden drop in optical densities could indicate a problem such as contamination of a reagent. Contamination, especially of the test kit conjugate, can happen in many ways—either through introduction of serum or other kit reagents, dirty reagent reservoirs or pouring back unused reagent into the bottle. To avoid contamination and obtain optimal test kit performance until the expiration of the test kit, follow some simple laboratory guidelines.

• Measure all reagents using sterile or clean vessels. Be careful to measure only what is needed for the number of plates being run. This will help to maintain the integrity of the reagents.

• Do not return reagents to the original stock bottles.

• We strongly recommend using disposable pipettes and reservoirs when handling reagents to minimize the risk of contamination. However, if you choose to reuse any disposable device, use a separate reservoir for each reagent and be sure to label them. Also, wash and thoroughly rinse the wells with deionized or distilled water after each use.

• Never use the same reservoir for conjugate and substrate, even if it has been washed.

• Change and discard the disposable reservoirs as frequently as possible.

References

Skeeles, J.K., 1985. ELISA's role in the serological monitoring of poultry flocks. Vineland Update, Vineland, N.J. No. 12, March.

Snyder, D.B., W.W. Marquardt, E.T. Mallison, D.A. Allen, and P.K. Savage. An Enzyme-Linked Immunosorbent Assay Method for the Simultaneous Measurement of Antibody Titer to Multiple Viral, Bacterial or Protein Antigens. Veterinary Immunology and Immunopathology 9:303-317, 1985.

Snyder, D.B., W.W. Marquardt, E.T. Mallinson, P.K. Savage, and D.C. Allen. Rapid Serological Profiling by Enzyme-Linked Immunosorbent Assay. III. Simultaneous Measurements of Antibody Titers to Infectious Bronchitis, Infectious Bursal Disease, and Newcastle Disease Viruses in a Single Serum Dilution. Avian Dis. 28:12-24. 1985.

Thayer, S.G., 1986. "Enzyme-Linked Immunosorbent Assay (ELISA) For Avian Serum Antibody or for Antigen." In a Laboratory Manual for Avian Virus Diseases. P. Villegas, Ed. University of Georgia, Athens, GA. pp. 48-51.

C:Immunosuppression in Poultry

Introduction

Control of infectious diseases depends on flock immunity. Reduced immune responsiveness leading to increased disease losses can seriously damage the poultry industry. Often a laboratory is called on to determine if a poor performing flock has experienced some form of immunosuppression. Immunosuppression describes a variety of disease problems. Vaccine failures and disease outbreaks are blamed on immunosuppression. This chapter will define immunosuppression, describe assays to detect immunosuppression, review immunosuppressive agents and their effects on components of the immune system, and give methods to prevent immunosuppression. Definition

Immunosuppression is a state of temporary or permanent dysfunction of the immune response resulting from damage to the immune system. This leads to increased susceptibility to disease agents and decreased responsiveness to vaccination. Immunosuppressive agents damage the immune system. Immunodysfunction may be caused by infectious and non-infectious agents. Infectious causes include bacteria, viruses and internal parasites. Non-infectious causes include chemicals, hormones, antibiotics, toxins, environmental stresses and lack of dietary ingredients.

Evaluation of immunosuppression


The location, development and function of the immune system are necessary to understand the relationship between immunosuppressive agents and their effects or economic loss. The immune system is dependent on lymphoid tissues and is divided into central and peripheral. Central lymphoid tissues are the bursa and the thymus. Peripheral lymphoid tissue includes the spleen, cecal tonsil, bone marrow and gland of Harder. The central lymphoid tissues become invaded by stem cells derived from the bone marrow or yolk sac, which undergo differentiation and migration as cells destined to form bursal lymphocytes (B-cells) or thymal lymphocytes (T-cells). As chickens age, there is "seeding" of the peripheral lymphoid tissues with centrally derived B and T-cells. When the chicken is mature, the bursa and thymus become vestigial and immunocompetence is dependent on the peripheral immune system. B-cells, when exposed to antigen, divide to produce plasma cells, which secrete antibody (AB), and "memory" cells. Antibodies neutralize antigen; whereas, memory cells retain recognition code to the antigen. On subsequent exposure to the same antigen, memory cells divide to produce more plasma cells, which make antibody during the anamnestic response. T-cells do not produce antibodies. They are involved in cell-mediated immunity (CMI) (effector T-cells), in destruction of infected cells (cytotoxic T-cells), and cells which promote the production of antibody by B-cells (helper T-cells). Memory cells are also produced by T-cells. Macrophages are present early in the development of the immune system and survive for a long time. They become activated during an inflammatory process and replicate under the influence of growth factors produced by lymphocytes. Macrophages are involved in chemotaxis, phagocytosis, microbial killing, intracellular digestion, extracellular killing, and secretion of monokines, interferon, interleukins and hormones. Macrophages also process and present antigen to B- and T-cells.

Immunosuppressive agents can affect all types of immunity, thereby, nonspecifically increasing susceptibility to pathogens. Diseases more severe following immunosuppression are inclusion body hepatitis, gangrenous dermatitis, aplastic anemia, coccidiosis, MD, E. coli septicaemia, reovirus "related diseases," Newcastle Disease virus (NDV), IBV and the "swollen head syndrome." Criteria to evaluate immune functions are: gross and microscopic changes in the morphology of central or peripheral lymphoid tissues, changes in antibody levels, reduction in the CMI response, interference with vaccination, and exacerbation in the course of disease caused by other agents. Atrophy of lymphoid organs and depletion of lymphoid follicles often result from immunosuppressive agents. Changes in lymphoid organs such as the thymus and bursa of Fabricius, therefore, are indicative of immunosuppression. To detect gross changes, weights of lymphoid organs and body weights from infected and control groups can be determined and analyzed statistically. Bursa weight to body weight ratios (B/B) (weight of bursa divided by weight of body of 20 birds per flock x 1,000) can be correlated with immunosuppression. Birds between three-to-six-weeks-of-age normally have B/B ratios from two to four. B/B ratios of one or less are indicative of immunosuppression and are seen in clinically ill birds and birds condemned at processing. Thymus diameter to shank diameter ratios x 10 (T/S ratios) can also be done. T/S ratios of less than one are indicative of atrophy and immunosuppression. Histological changes in lymphoid tissues can be made by microscopic comparisons between infected and control birds.

Antibody response

Most commercial poultry in the world are vaccinated from one to two times against NDV. It is one of the most common and costly diseases of poultry. Therefore, producers are continually monitoring antibody responses in birds as one determination of their immune status against NDV as well as their functioning capability of the immune system.

The HI response has traditionally been a rapid easy test for measuring antibody against NDV. Normal immunized chickens should produce HI titers of at least 2P6P or 2P9P by ELISA. Antibody responses against NDV vaccines can be evaluated using the HI or ELISA as described in previous chapters. Non immunized birds can be either injected with sheep red blood cells (S-RBC's) or tested for natural agglutinins against rabbit red blood cells (R-RBC's). For S-RBC's, poultry are injected twice intramuscularly with 1.0 ml/injection of a 10% volume/volume suspension of S-RBC's at 2 week intervals. At 2 weeks after the injection, birds are bled and serum collected and complement inactivated as stated prior in this book. Sera are tested using a microdilution technique. Sera are diluted in twofold dilutions with PBS in a 96 round bottom well plate for 10 dilutions. An equal volume of a 0.5% suspension of S-RBC's is added to each well and the plates are incubated at room temperature for 60 minutes. The endpoint titer is the last dilution in which the S-RBC's are agglutinated (form a lacy pattern at the bottom of the well). A solid button at the bottom is considered negative. S-RBC's are considered a T-cell antigen, since T-cells are needed as helper cells for B-cells to produced antibody. Normal control chickens should produce titers of around 2P8P after two injections of S-RBC's.


The contrast to the S-RBC test, the R-RBC's are B-cell antigens, since only B-cells are needed to make antibody against them. The basis for this test is that an antigen on the surface of the R-RBC is similar to a bacterial antigen in which all none gnotobiotic birds are exposed. This nonpathogenic bacterium is probably in the air, water and/or feed of all commercially reared poultry. Therefore, with this test, birds do not have to be immunized with the R-RBC's hence the name natural agglutinins. This test is run and evaluated the identical way as the S-RBC's. The R-RBC's are mixed with an equal volume of the bird’s serum which has been diluted in twofold dilutions with PBS. After incubation the wells are checked for hemagglutination. With either of the two tests, a reduction in titer of 10 fold is considered to indicate immunosuppression, when compared to normal control birds. However, it is best to compare the results of multiple animals (suspect and known non-immunosuppressed birds) and analyze the results statistically using a students T-test or Analysis of Variance Assay. Normal control chickens should produce titers of around 2P6P.

CMI responseA number of tests can be used for measuring CMI. The easiest are a measure of the delayed-type hypersensitivity (DTH) or cutaneous basophile hypersensitivity (CBH) reactions. In the DTH test an antigen such as mycobacterium is injected intradermally into the wattle or between the toes of a previously sensitized bird. The increase in thickness after 24 hours as compared to skin injected with saline or antigen injected in an unsensitized bird as measured with micrometer is the DTH response. This DTH is correlated with the T-cell response. Birds are given tuberculin by an intramuscular injection at two sites (in the breast area) with a total of 0.3 ml of tuberculin from Jensen-Salsberry Laboratories, Inc. emulsified in 0.7 ml of Freund's complete adjuvant from Difco Laboratories, Inc., Detroit, Mich. At 14 days after sensitization each chick is tested for DTH by injection of 0.1 ml containing 50 ug of tuberculin into the skin. The interdigital (toe) or wing web skin is used in birds less than four-weeks-of-age, because their wattles are too small to be injected intradermally. The opposite wattle, wing or between the toes on the other foot are injected with 0.1 ml of 0.15 NaCl. The difference in thickness between antigen injected and saline injected skin as measured by calibers is the measure of DTH. Another control would be to inject antigen into unsensitized birds that did not receive tuberculin previously. Sensitized nonimmununosuppressed birds will have a skin which doubles in thickness after 24 hours after receiving tuberculin. Again, it is more accurate to have a number of samples to do a statistical analysis comparing suspect birds with that of normal non-suppressed birds.

A more simplified test than DTH is the CBH test. It is done with phytohemagglutinin-M (PHA-M), a mitogen from Difco laboratories, Detroit, MI. This will cause a cellular reaction in normal unsensitized, nonimmunosuppressed birds and is also correlated with T-cells and CMI. The test requires no previous sensitization and is run and calculated the same way as for tuberculin. The PHA-M is reconstituted and injected in one wattle, wing web, or between the toes. Twenty four hours after injection, the skin thickness is measured and compared to saline injected skin. Again, nonimmunosuppressed birds will show a twofold increase in mitogen injected skin compare to saline injected.

Causes of Immunosuppression

Antibiotics

Antibiotics are capable of depressing the immune response. Therefore, caution must be observed when prescribing compounds at high levels for extended periods. Chlorotetracycline can cause adverse effects on the development of gut-associated lymphoid tissue. Immunosuppression can occur in chicks hatched from eggs dipped in tylosin and gentamycin.

Mycotoxins

Aflatoxins increase susceptibility of poultry to Salmonella, Aspergillosis, coccidiosis, MD, E. coli and IBDV. Effects are dependent of the level of toxin and duration of toxin consumption and age and genetic strain of bird. Ochratoxins, T-2 toxin and fumonisins cause depression in Ig producing cells in the lymphoid organs and a decrease in the size of the bursa and thymus.

DietSelenium deficiency results in immunosuppression. Diets deficient in valine decrease antibody to NDV. Diets devoid in B complex, C and E vitamins cause atrophy of the bursa of Fabricius, thymus and spleen. Consumption of lead, cadmium, mercury and iodine can be immunosuppressive.

Stress

Stress can increase levels of steroids. Steroids decrease lymphoid cell synthesis. Heat stress causes


immunosuppression. Stressed chickens are more susceptible to viral than bacterial infections. Stress of high egg production induces reactivation of adenovirus infections. Stress of force molting decreases antibody responses to NDV and IBDV in broiler breeders.

Bacteria

Bacteria such as E. coli and Mycoplasmae produce factors that depress phagocytosis of neutrophils.

Virus (Table 1.0)Virus-induced immunosuppression causes alterations in the function of a variety of cells, especially lymphocytes and mononuclear phagocytes.

Direct immunosuppression occurs by virus attack on lymphoid organs and cells; whereas, indirect immunosuppression may be by the release of mediators such as hormones, complement and prostaglandins that have direct immunosuppressive activity or that can activate certain species of suppressor cells leading to immune defects.

Table 1.0. Immunosuppressive Viruses_____________________________________________________________________________VirusTypeTargetSuppression_______________________________________________________________________________

IBDBirnavirusB- and T-cellsAB and CMIAL OncovirusB- and T-cells AB and CMIMD HerpesT-cells CMIND ParamyxovirusT-cells CMICAV CircovirusB- and T-cellsAB and CMI IBDV causes atrophy of the bursa of Fabricius, and B-cell lymphoid tissue in the cecal tonsil, gland of Harder and thymus. B/B ratios of IBDV infected broilers may be 0.5 or less at processing. Infections of IBDV during the first week of age cause a permanent suppression in antibody production. IBDV renders chickens more susceptible to MD. Avian Leucosis viruses target B-cells and cause antibody suppression. MDV target T-cells and depress CMI. A consequence of MDV immunosuppression is increased susceptibility to coccidia and reduced antibody response. CAV causes immunodepression of B- and T-cells. The virus causes anaemia (paleness of the comb, shanks and bone marrow). There is degeneration of the bursa and thymus. Hemorrhage of the musculature and internal organs is also seen. Prevention of immunosuppression

IProduce high quality chicks free of mycoplasma, ALV, and E. coli, having high maternal antibody against serologic standard and variant IBDV, and CAV.

IIRear chicks in a clean sanitized environment, with chlorinated water using nipple drinkers, and an adequate diet fortified with vitamins and minerals and free of mycotoxins, pesticides and toxic metals.

IIIUse only approved levels of antibiotics.

IVReduce stress by using adequate heating and cooling and do not over crowd birds.

VUse effective vaccination methods to control IBDV, MDV, and NDV.

Many agents may damage lymphoid organs, and cause immunosuppression. Poor immune responses in birds and atrophy of lymphoid organs are seen in many morbid, dead or condemned poultry. Immunodepression may be permanent or temporary and can interfere with vaccine efficacy and increase susceptibility to other agents. Humoral responses can be evaluated by measurement of serum antibody. CMI responses can be evaluated by DTH or CBH reactions in the wattle, wing web or interdigital skin test. Macrophages can be measured for their ability to phagocytose bacteria or latex beads. Immunosuppression may be apparent as an increase in respiratory or enteric disease, high morbidity or mortality, atrophy of lymphoid organs, high condemnations, poor feed conversion, or low egg production.


Health & Medicine

Advanced Laboratory Techniques in Poultry Disease Diagnosis