Collection of specimens and laboratory diagnosis of foot and mouth disease Dr. Claudiu DIACONU Dr. Mihai TURCITU INSTITUTE FOR DIAGNOSIS AND ANIMAL HEALTH Suceava, september 2009

Stability of FMD viruses - influence for diagnosis  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

Sampling in FMD diagnosis Probang – virus - viral genome Blood – antibodies - virus - viral genome Foot lesions - virus Milk – antibodies - virus - viral genome Mouth lesions - virus

Stadiu evolutiv al bolii – metode de diagnostic Sursa: WRL Pirbright

The samples in FMD diagnosis  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

Conditioning of samples  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 Transport medium

Conditioning of samples  Blood serum  Whole blood (EDTA)  Minimum 4 sample  Use suitable recipients, disposable or sterile

Conditioning of samples  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

Principles of package  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

International shipment of infectious materials 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 – 40 0 to C The packaging - 1,2 m drop test

Laboratory diagnosis of FMD  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

Virus, antigen and ribonucleic acid detection

ELISA antigen detection Stage 5 – adding mixture chromogen - substrate and stop of the reaction OPD  H 2 O 2 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

Viral genome detection  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)

Viral genome detection 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)

Viral genome detection 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

Virus isolation of cell cultures 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

Serological tests  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.

Serology – antibodies to structural proteins 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

Serology – antibodies to nonstructural proteins ∎ 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

“Pen - side” tests “Pen - side” tests Antibody detection in serum or whole blood Detection of viral proteins Detection of viral genome – isothermal amplification with Bst polimerase

Terrestrial Animal Health Code 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!