Dr. Claudiu DIACONU

Dr. Mihai TURCITU

INSTITUTE FOR DIAGNOSIS AND ANIMAL HEALTH

Suceava, 7 -10 september 2009

Susceptibility of FMD viruses at the environmental factors (pH, temperature)

∎ optimal pH 7,2 – 7,6 ∎ stable 7,0 – 8,5 (at low temperature) ∎ fast inactivation 6,8 and 9 >

∎ storage at refrigeration temperatures during transport (avoid freezing/thawing cycles).

the manner of samples collection

Manipulation and conditioning of samples

Glassware, transport medium, antibiotic mixture

The quality of results and the quality of global diagnosis reflect the quality of samples

Probang – virus - viral genome

Blood – antibodies - virus - viral genome

Foot lesions - virus

Milk – antibodies - virus - viral genome

Mouth lesions - virus

Sursa: WRL Pirbright

Epithelium and vesicular fluid Serum and whole blood

Oesophagopharyngeal (probang) fluid

Organs- heart from young animals - lymphnodes– prescapular (ruminants) - submandibular (swine)

- mezenteric (all species) - spleen, kidneys, liver

- from fatal cases

Epithelium of unruptured or recently ruptured vesicles (min. 1g, 2 cmp)

Vesicular fluid

Phosphate buffer 0,04 M (PBS, MEM)

Glicerol (1/1)

Phenol red solution1%

penicillin– 1000 UI / ml

mycostatin– 100 UI / ml

neomycin – 100 UI / ml

polymyxin – 50 UI / ml

pH 7,2 – 7,4

Tra

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Blood serum Whole blood (EDTA)

Minimum 4 sample Use suitable recipients, disposable or sterile

Oesophagopharyngeal fluid (probang)

◦ Phosphate buffer 0,08 M◦ Bovine serum albumin 0,01%◦ Phenol red◦ Antibiotic mixture

2 ml transport medium 2 ml sample

Check pH Few hours at refrigeration temperature Freezing for a longer time

Safe condition for transport

Not to present a hazard to persons or environment during shipment

It is essential to have no any leakage of contaminated materials

outside the parcel

♦ Primary containers – watertight, identified

♦ Secondary containers – screw cap, rubber washer,

crushproof

♦ Tertiary container – cool packs - submission form attached outside

Alternative methods of packing with similar level of safety

IATA - “Dangerous Good Regulations”

Infectious substance, affecting animals – UN 2900

Technical spcifications:

• secondary container – withstanding without leakage an internal pressure of 95 kPa in the range of – 400 to + 550C

• The packaging - 1,2 m drop test

Accurate and fast diagnosis

Confirmation or not of clinical suspicion

Doesn’t replace the clinical investigation of livestock

The accuracy of results depends on the quality of sampling

Complete epidemiological information for a rationale

interpretation of the results

Stage 5 – adding mixture chromogen - substrate and stop of the reaction

OPD H2O2 Colour reaction

Phase 1 – Coating of microplate with rabbit antiserum – capture antibodies

Phase 2 – Transfer of processed samples

Stage 3 – Transfer of guinea – pig antiserum – detecting antibodies

Stage 4 – adding of conjugate – antiguinea – pig HRP antiserum

Amplification of a segment from FMD genome by RT - PCR

Objectives

Identification of all genotypes – RT - PCR Generic –

amplification of a highly conserved regions among all

genotypes (regions 5`NTR or3D)

Specificity – avoid of false – positive results (ability to

discriminate FMD/SVD)

Interpretation:

∎ Analysis of amplification curves –Real Time RT-PCR technique:

increased sensibility , low risk of intercontamination among

samples (closed system), fast generation of results (cca 4

hours)

∎ Agar gel electrophoresis –Nested PCR technique: increased

sensibility, high risk of intercontamination among samples

(open system), results in a medium interval of time (cca 8-9

hours)

Curves obtained by amplification of genic fragments (regions 3D)

1. (protocol Callahan) 2. (5`NTR - protocol Reid)

Electrophoresis of amplification products - test nested PCR (protocol Bahri)

1. PCR1 2. PCR2

Primary cultures - primary bovine thyroid cells – maximum susceptibility to FMD viruses

- kidney cells (calf, lamb, piglets)

Stabilized cell lines – PK15, IBRS – 2, BHK21- stability

Identification of isolated viral strain by antigen detection ELISA/RT - PCR

to certify individual animals for international trade;

to confirm suspected subclinical cases or retrospective diagnosis;

substantiate of absence of infection – in vaccinated livestock;

prove efficacy of vaccination.

SP seroconversion – after infection

- postvaccination

VNT – “gold standard”

- reference strain, high containment unit

- 2 – 3 days to perform

BFL ELISA – screening

- high sensibility

- reproducibility, robustness

SPC ELISA – similar sensibility

- detection limit

- high specificity, robustness

Serotype – specific tests

∎ Status of persistent infected animal (ruminant) – “carrier”

persistence 28 days infectious viral particles in the oesophagopharyngeal region

∎ Seroconversion of NSP antibodies – specific for infection

- not serotype - specific

- not induced by modern, purified vaccines

Discrimination vaccinated / infected animals

Antibody detection in serum or whole blood

Detection of viral proteins

Detection of viral genome – isothermal amplification with Bst polimerase

An outbreak of FMD

FMDV has been isolated and identified as such from an animal or a product derived from that animal; or

viral antigen or viral ribonucleic acid (RNA) specific to one or more of the serotypes of FMDV has been identified in samples from one or more animals, whether showing clinical signs consistent with FMD or not, or epidemiologically linked to a confirmed or suspected outbreak of FMD, or giving cause for suspicion of previous association or contact with FMDV; or

antibodies to structural or nonstructural proteins of FMDV that are not a consequence of vaccination, have been identified in one or more animals showing clinical signs consistent with FMD, or epidemiologically linked to a confirmed or suspected outbreak of FMD, or giving cause for suspicion of previous association or contact with FMDV.

Thank you!