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Avian influenza diagnostic techniques

Avian influenza diagnostic techniques. Saliha Hammoumi CIRAD Montpellier, France. AI laboratory diagnosis. Serological diagnosis Useful for epidemiological surveillance or to supplement virological diagnosis Virological diagnosis

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Avian influenza diagnostic techniques

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  1. Avian influenza diagnostic techniques Saliha Hammoumi CIRAD Montpellier, France

  2. 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)

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

  4. Antigen + control Test Serum 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

  5. Enzyme Linked ImmunoSorbent Assay (ELISA) • Commercial kits available for chicken and turkey • Detection of antibody directed against NP • ELISA indirect • Good sensitivity • Rapid results

  6. Haemagglutination Inhibition Assay (HIA) • 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 + + RBC Reference antigen Diluted test serum Serum dilution 1/4096 1/2 + serum : 1/1024 HA+ HA-

  7. 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

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

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

  10. 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

  11. M+25 M-124 Detection of type A influenza: real-time RT-PCR specific of M gene Taqman Technology Primers Spackman: M+25/M-124 Probe M+64 FAM-BHQ1 Q-RT-PCR OIE protocole M+25 M-124 M+64 M gene

  12. Amplicon of 601pb M2 M1 M-AIVsens (H5N2) (230) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTCAATGGGAATGGAGACC IVAMM2E (H1N1) (234) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTAAATGGGAATGGAGACC AB036778 (H3N2) (244) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATC NC_002016 (H1N1) (244) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGATC AY210255 (H1N1) (219) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTCAATGGGAATGGGGATC CY000794 (H3N2) (243) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTCAATGGGAATGGGGATC CY006188 (H1N2) (232) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAATGGGGATC NC_004907 (H9N2) (251) ACTGCAGCGTAGACGATTTGTCCAAAATGCCCTAAATGGGAATGGAGACC (H5N1)/segment 7) (244) ACTGCAGCGTAGACGATTTGTCCAAAATGCCCTAAATGGGAATGGAGACC NC_007363 (H5N1) (244) ACTGCAGCGTAGACGCTTTGTCCAGAATGCCTTAAATGGAAATGGAGATC AY627887 (H5N1) (242) ACTGCAGCGTAGACGCTTTGTCCAGAATGCCCTAAATGGAAATGGAGATC AM040045 (H5N1) (226) ACTGCAGCGTAGACGCTTTGTCCAGAACGCCCTAAATGGAAATGGAGATC AB239319 (H5N1) (223) ACTGCAGCGTAGACGCTTTGTCCAGAATGCCCTAAATGGAAATGGAGATC AB239326 (H5N1) (220) ACTGCAGCGTAGACGCTTTGTCCAGAATGCCCTAAATGGAAATGGAGATC DQ100569 (H5N1) (181) ACTGCAGCGTAGACGCTTTGTCCAGAATGCCCTAAATGGAAATGGAGATC Consensus (251) ACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTAAATGGGAATGGAGATC (780) TGTTGCAGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (784) TACTGCAGCAAGTATCATTGGGATCTTGCACCCGATATTGTGGATTCTTGATCGTC (794) TATTGCCGCAAATATCATTGGGATCTTGCACTTGACATTGTGGATGCTTGATCGTC (794) TATTGCCGCAAATATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (769) TGTTGCTGCGAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (793) TGTTGCTGCGAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (782) TGTTGCTGCGAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (801) TGTTGCAGCAAGTATCATTGGGATATTGCACTTGATATTGTGGATTCTTGATCGTC (794) TGTTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (794) TGTTGCCGCAAGTATCATTGGGATACTGCACTTGATATTGTGGATTCTTGATCGTC (792) TGTTGCCGCAAATATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (775) -------------------------------------------------- (773) TGTTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (770) TGTTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (731) TGTTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC (801) TGTTGCCGCAAGTATCATTGGGATCTTGCACTTGATATTGTGGATTCTTGATCGTC 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 M+25 M-124 M+64 M1 M2 M gene

  13. 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

  14. Detection of H7 subtypes • Real-time RT-PCR  Primers LH6H7 / RH4H7 (VLA, Weybridge, UK)  Taqman probe H7pro11 –5’(FAM ) /…/ (BHQ1)-3’ Probe H7pro11 GK 7.3  GK 7.4 LH6H7 RH4H7 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

  15. 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)

  16. 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 : +4°C for few days or -80°C, dry ice, liquid nitrogen • Process samples in BSL2 or BSL3 • Specific material required • Specific pathogen free eggs • Egg incubators

  17. 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 37°C • 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

  18. Alternative isolation methods • Isolation on primary chicken embryo fibroblasts • Isolation on Madine Darby Canine Kidney cells (MDCK)

  19. AI team in CIRAD Montpellier • Yane Kandassamy • Thierry Lefrançois • Frédéric Petitclerc • Saliha Hammoumi • Patricia Gil • Colette Grillet • Emmanuel Albina

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