Transcript ASSESMENT OF METHODS

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
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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%
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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
– Serum (Serology – AGID, ELISA Ab)
– 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:
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AS
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Semi quantitative
AG
Moderate sensitivity
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Subjective interpretation
Requires 72 hours
Further testing of positives
Antibodies not detectable for several days
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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
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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 EHV4 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
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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
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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
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Viral RNAcDNA synthesis(RT-step)
Denaturation
Annealing N x extension
Amplicon(s)
Principle of PCR
Real time detection
Advantages
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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