Avian influenza diagnostic techniques

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Avian influenza diagnostic techniques

• Saliha Hammoumi
• CIRAD Montpellier, France

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AI laboratory diagnosis

• Serological diagnosis
• Useful for epidemiological surveillance or to
  supplement virological diagnosis
• Virological diagnosis
• Detection of the virus responsible of the
  disease, or its genes or antigenes
• Necessitate early sampling after first symptoms
• Samples Tracheal or cloacal swabs, organs (dead
  birds)

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Serological diagnosis

• Antibody detection
• Agar gel immunodiffusion (AGID)
• Enzyme Linked ImmunoSorbent Assay (ELISA)
• Haemagglutination Inhibition Assay (HIA)

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Agar gel immunodiffusion (AGID)

• detection of antibody directed against NP and M1
• good specificity
• Sensitivity depends on the species
• Can be use with all bird species

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Enzyme Linked ImmunoSorbent Assay (ELISA)

• Commercial kits available for chicken and turkey
• Detection of antibody directed against NP
• ELISA indirect
• Good sensitivity
• Rapid results

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Haemagglutination Inhibition Assay (HIA)


RBC
Reference antigen
Diluted test serum

• Reference antigen necessary
• Red blood cells (RBC) from AIV and NDV free
  chicken
• Serum is positive when the reference antigen does
  not haemagglutinate red blood cells

Serum dilution
1/4096
1/2
serum 1/1024
HA
HA-
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Virological diagnosis

• Detection of virus, its genes or antigens
• Detection of antigen by ELISA
• Detection of viral RNA by RT-PCR
• Detection of infectious virus by isolation on
  embryonated chicken eggs

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Detection of antigen

• Enzyme Linked Immuno-Sorbent Assay (ELISA)
• Samples to be kept at 4C
• Commercial kits Directigen Flu A B Kit
  Becton Dickinson Microbiology Systems
• Rapid results (about 15 min)
• False negative possible if there is
• bacterial contamination

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Detection of viral RNA

• Samples
• Tracheal or cloacal swabs in transport medium
• Organs (dead birds)
• Conservation at low temperature 4C for few
  days or -80C, dry ice, liquid nitrogen
• Process samples in BSL2 or 3
• Specific material required
• Thermocycler for qualitative RT-PCR
• And/or Real-time PCR machine

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Detection of viral RNA

• RNA extraction from samples (manual or automatic)
• Detection of type A influenza
• Matrix (M) gene by (real-time) RT-PCR
• Detection of H5 or H7 subtypes by (real-time)
  RT-PCR on AIV A positive samples
• RT-PCR on the cleavage site of HA for H5 or H7
  positive samples
• Sequencing of the amplified cleavage site for
  determination of pathogenicity

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Detection of type A influenza real-time RT-PCR
specific of M gene
Taqman Technology Primers Spackman
M25/M-124 Probe M64 FAM-BHQ1 Q-RT-PCR OIE
protocole
M25
M-124
M64
M gene
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Detection of type A influenza conventional
RT-PCR specific of M gene

• Detection of the Matrix gene in a region
  conserved among the different influenza A
  subtypes primer M1/M2 (E.Starick et al., 2000)
• Suitable for Influenza detection in birds as well
  as pig, horse and human

M25
M-124
M1
M64
M2
M gene
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Detection of H5 subtypes

• Real-time RT-PCR
• ? Primers H5LH1 / H5RH1 (VLA, Weybridge, UK)
• ? Taqman probe H5PRO 5(FAM ) // (BHQ1)-3

Probe H5PRO
H5-Kha-1
H5-Kha-3
H5LH1
H5RH1
HA-875F
HA-1114R
J3
B2a
Site de clivage
HA Gene

• Conventional RT-PCR primers H5 from VLA,
  Weybridge, UK
• ? HA-875F / HA-1114R
• ? J3 / B2a
• ? H5-Kha1 / H5-Kha3

Different sensitivity and specificity
Complementary RT-PCR
Sequencing of the amplification product
pathotyping
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Detection of H7 subtypes

• Real-time RT-PCR
• ? Primers LH6H7 / RH4H7 (VLA, Weybridge, UK)
• ? Taqman probe H7pro11 5(FAM ) // (BHQ1)-3

Probe H7pro11
LH6H7
RH4H7
GK 7.3 
GK 7.4
Site de clivage
HA Gene

• Conventional RT-PCR primers H7 from VLA,
  Weybridge, UK
• ? Primer GK 7.3 / GK 7.4 

Sequencing of the amplification product
pathotyping
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Detection of H5 or H7 subtypes

• High variation of haemagglutinin gene

Necessary to verify the sequences of the
circulating strains to design new primers so that
false negative can be avoided
ex design of H5 real-time RT-PCR primers by
Spackman et al. (2002)
Observation of differences on HA gene between
North American Strain and Eurasian strains
Modification of H5 primers used in Europe and
Africa (Slomka et al. 2007)
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Isolation in embryonated chicken eggs

• Samples
• Tracheal or cloacal swabs in transport medium
• Organs (dead birds) heart, trachea, spleen,
  brain, liver
• Conservation at low temperature 4C for few
  days or -80C, dry ice, liquid nitrogen
• Process samples in BSL2 or BSL3
• Specific material required
• Specific pathogen free eggs
• Egg incubators

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Isolation in embryonated chicken eggs

• Inoculation of sample in allantoic cavity of
  embryo of 9 to 11 days
• Confirmation of isolation after 2 to 7 days of
  incubation at 37C
• Detection of haemagglutinating activity of
  allantoic fluid
• Confirmation of the presence of Influenza A virus
  by Agar gel immunodiffusion test
• Determination of the subtype by inhibition
  haemagglutination assay with specific antiserums

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Alternative isolation methods

• Isolation on primary chicken embryo fibroblasts
• Isolation on Madine Darby Canine Kidney cells
  (MDCK)

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AI team in CIRAD Montpellier

• Yane Kandassamy
• Thierry Lefrançois
• Frédéric Petitclerc

• Saliha Hammoumi
• Patricia Gil
• Colette Grillet
• Emmanuel Albina