Download Slide 1

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
Mouth lesions - virus
Milk – antibodies
- virus
- viral genome
Foot 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

Transport medium

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


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
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
Stage 5 – adding mixture chromogen - substrate and stop of the reaction
OPD  H2O2
Colour reaction

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!