1. ASSESSMENT OF DIAGNOSTIC METHODS AND STANDARD DIAGNOSTIC PROCEDURES
Prof. Dr. Ivaylo Chenchev, PhD, DVSc
National Diagnostic and Research Veterinary Medical Institute, Bulgaria

2. DIAGNOSTIC METHODS AND STANDARD DIAGNOSTIC PROCEDURES
OFFICIAL DOCUMENT FOR LABORATORY METHODS IS
REGULATION (EC) No 882/2004 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL
of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules

3. DIAGNOSTIC METHODS AND STANDARD DIAGNOSTIC PROCEDURES
OFFICIAL DOCUMENT FOR MOVMENT OF EQUINES IN EU IS
REGULATIONS
COMMISSION REGULATION (EU) No 595/2010
of 2 July 2010
amending Annexes VIII, X and XI to Regulation (EC) No 1774/2002 of the European Parliament and of the Council laying down health rules concerning animal by-products not intended for human consumption

4. DIAGNOSTIC METHODS AND STANDARD DIAGNOSTIC PROCEDURES
African Horse Sickness (AHS)
Equine Infectious anemia (EIA)
Equine Viral Arteritis (EVA, EAV)
Equine Herpes type 1 (EHV 1)
Equine Herpes type 4 (EHV 4)
Equine Influenza virus (EIV)
West Nile Fever (WNF)
Other Encephalomyelitis (EEE, JEE.VEE)

5. African Horse Sickness
Mortality
Horses: Mortality 50-95%
Pulmonary form – up to 95%
Cardiac form – 50% or higher
Mixed form – 70-80%
Horsesickness fever - typically recover
Other Equidae
Mules: 50%
European or Asian donkeys: 5-10%
None in African donkeys and zebras

6. African Horse Sickness
Refrigerate but do not freeze the following specimens and send them to the laboratory:
Blood, preferably with heparin as an anticoagulant (other anticoagulants can be used), or blood in an equal volume of OCG Pieces of spleen, mediastinal and mesenteric lymph nodes, lung, and liver At least 5 mL of serum from acute and convalescent animals
In addition, send the following:
Blood smears (at least six) fixed in absolute methanol
Tissues in 10-percent formalin, from spleen, liver, lung, kidney, heart, lymph nodes, and brain

7. African Horse Sickness Laboratory Diagnosis: Virus Isolation
Confirmation of an initial case of AHS in an area normally free of the disease requires isolation and identification of the virus.
AHSV can be isolated from heparinized blood, spleen, lymph node, or lung collected at necropsy using cell culture (BHK21 or Vero cells)
Intracerebral inoculation of mice that are 2 to 3 days old
Intravenous inoculation of embryonated eggs at day 10 to 12.
The incubation period in mice can be 4 to 20 days; then the mice die.
Viral isolates are identified by group-specific tests such as complement fixation
Enzyme-linked immunosorbent assay (ELISA)
Immunofluorescence
Determination of the serotype is done by plaque reduction or plaque inhibition using known antisera.
Real Time PCR

8. African Horse Sickness Laboratory Diagnosis: Serology
The antibody to AHS can be detected starting about 10 days after infection.
Group-specific tests are complement fixation (CF antibody present 4 to 6 months)
Immunofluorescent assay (IFA)
ELISA
Immunodiffusion
Detectable antibodies are present for 1 to 4 years after infection.

9. Equine Infectious anemia (EIA) Laboratory diagnosis
Antemortem specimens
Serum (Serology – AGID, ELISA Ab)
Whole blood (VI, PCR, viral detection not routine)
Postmortem specimens
Whole blood (VI, PCR, viral detection not routine
Clinicopathology – “swamp fever” of equines; typically sub clinical; primary infection febrile respiratory; chronic infection and shedding eventually glomerulonephritis

10. Equine Infectious anemia (EIA) Laboratory diagnosis
AGID Test
Advantages:
Gold Standard (Coggins test)
Detects Antibody to EAV
Easy, inexpensive,
requires few reagents/equipment
Disadvantages:
Semi quantitative
Moderate sensitivity
Subjective interpretation
Requires 72 hours
Further testing of positives
Antibodies not detectable for several days + - AG AS

11. Equine Infectious anemia (EIA) AGID Test

12. Equine Viral Arteritis (EVA, EAV) Laboratory diagnosis
Virus isolation
ELISA for antibodies
Virus Neutralization Test
Real Time PCR

13. Equine Viral Arteritis (EVA, EAV) Laboratory diagnosis
Antemortem speciments
Paired serum (Serology – modified SN, IFA, ELISA)
Nasopharyngeal, conjuctival swab or wash, citrated blood, semen (PCR, VI, viral detection not routine)
Postmortem speciments
Part of lung, trachea, spleen, colon, cecum&associated lymph nodes, small and medium sized arteries (FA, PCR, VI, viral detection not routine)
Clinicopathology – subclinical febrile illness with leucopenia, depression, edema, panvasculitis, abortion storms on breeding farms
Virus culturable only for first 2 weeks post-infection

14. Equine Viral Arteritis (EVA, EAV) ELISA
Advantages
Commercial kits available
Rapid (same day)
Can be semi-automated
Disadvantages
Requires expensive
equipment
False positive reactions
Positives require confirmation

15. Equine Viral Arteritis (EVA, EAV) Laboratory diagnosis
One step RT-PCR for EAV with using only positive control in semen fluid

16. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
Virus isolation
ELISA for antibodies and antigens
Virus Neutralization Test
Real Time PCR

17. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
Methods available for the laboratory diagnosis of equine herpesvirus respiratory infections include:
Virus isolation
Polymerase chain reaction (PCR)
Immunofluorescent detection of viral antigens
Serologic testing (ELISA, CF, SN)
The cytopathic effect of EHV-1 and EHV-4 is characteristic, and seroidentification of the two herpesviruses can be made with type-specific monoclonal antibodies.
Amplification of viral DNA using PCR is a rapid, sensitive and increasing utilized assay for detection of EHV-1 or EHV-4 respiratory tract infection.
When direct antigen detection methods are used for a rapid laboratory diagnosis of EHV-1 or EHV-4, it is important to confirm the direct test results by virus isolation.

18. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
Electro microscopic picture of EHV 1 after virus isolation

19. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
Immunofluorescent detection of viral antigens

20. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
ELISA for distinguishes the antibodies against two serotypes EHV 1 and EHV 4

21. Equine Herpes type 1 (EHV 1) and (EHV 4) DIAGNOSIS
Conventional PCR for EHV 1 and EHV 4

22. Equine Influenza virus (EIV) Laboratory Diagnosis
Presumptive diagnosis
Serologic diagnosis
Clinical signs/lesions
Antigen capture tests
Definitive diagnosis
Isolation and characterization of the virus
Molecular detection with subtyping/pathotyping

23. Equine Influenza virus (EIV) Serologic Tests:
Type-Specific Tests (type A):
Enzyme-linked immunosorbent assay (ELISA) IgG
Detects all subtypes (H3, H7)
Subtype-Specific Tests (H or N subtype):
Hemaggltination-inhibition test
Neuraminidase-inhibition test
Detects only homologous subtype

24. Equine Influenza virus (EIV) Serologic Tests
Limited value because of routine use of vaccine
Hemagglutination-inhibition test (HI)
Enzyme-linked immunosorbent assay (ELISA)

25. Subtype-Specific Tests for EIV HI/NI (antibodies)
Advantages
Gold standard
Quantitative (titer)
Rapid (same day)
Disadvantages
Requires many reagents (antigens/antiserums)
Non-specific (steric) inhibition
Requires pre-treatment of serum to remove normal serum agglutinins (false negatives)

26. WNF - Clinical Signs in Horses
Paralysis of lips, facial muscles, or tongue
Head tilt, difficulty swallowing
Altered mentation
Sound sensitive
Blindness
Troubling righting
Drowsiness
Flu-like, anorexia, depression
Muscle and skin twitching
Hyperesthesia
Propulsive walking
Weakness, ataxia, recumbency
Seizures

27. WNF – Laboratory Diagnosis in Horses
Serology: blood and CSF
ELISA IgM and IgG
Virus isolation
Isolation cell cultures (C6/36 and VERO-6)
RT-PCR

28. ELISA - Principe Enzyme Linked Immunosorbent Assay (ELISA)
Term was coined by Engvall and Pearlmann in 1971
Different Types
Sandwich
Indirect
Competitive
Similar To RIA, Except No Radiolabel
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs

29. ELISA - Principe

30. Sandwich ELISA 2 antibodies required Must recognize different epitopes
1st antibody is referred to as capture Ab
2nd antibody detection Ab
2nd antibody is biotinylated
Enzymes commonly used: HRP (Horse Radish Peroxidase) and AKP (Alkaline Phosphatase)
Substrate is TMB (Chromogen)

31. ELISA Plate
96 well plate
Made of plastic on which protein can be adsorbed (bind) easily
Usually done 4C
Special buffer used that will not denature Ab and maximize binding
Blocking step ensures no empty spaces are left
Blocking reagent is often 10% FBS

32. PCR as diagnostic tool Advantages Fast Highly sensitive
Highly specific
Disadvantages
Highly sensitive (contamination!!!)
Highly specific (mutants may escape detection due to mutations in primer or probe region

33. Steps in PCR Preparation of sample (e.g. tissue homogenate)
Isolation of genetic material
Amplification of the target (PCR)
Detection of amplicons (gel / real-time)

34. Principle of PCR Viral RNAcDNA synthesis(RT-step) Denaturation
Annealing N x extension
Amplicon(s)

35. Principle of PCR Real time detection Advantages
With probes additional spec ificity
Low risk of contamination (closed system!)
Quantification possible
Fast
Robotisation possible
Disadvantages
SYBR Green less specific than probes (Specificity can been hanced through melting curves)
More sophisticated equipment needed

36. Real time detection
Denaturation
Annealing
Elongation
Detection