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

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

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

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)

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

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

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

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.

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

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

Equine Infectious anemia (EIA) AGID Test

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

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

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

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

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

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.

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

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

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

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

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

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

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

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)

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

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

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

ELISA - Principe

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)

ELISA Plate 96 well plate Made of plastic on which protein can be adsorbed (bind) easily Usually done overnight @ 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

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

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)

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

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

Real time detection Denaturation Annealing Elongation Detection