Development of a Pen - Avian Influenza Site Test Kit for the Rapid Diagnosis of H 7 Highly Pathogenic

As well as H5 highly pathogenic avian infiuenza viruses (HPAIV), H7 HPfUV strains have caused serious damages in poultry industries worldwidc. Cascs of bird-to-human transmission of H7 HPAIV have also been reported rl11, On the outbreak of avian influcnza, rapid diagnesis is critica] not on]y for the control of HPAI but also for human health. In the present study, a rapid diagnesis kit based on immunochromatography for the detection of H7 hemagglutinin (HA) antigen of inflaenza A virus was developed using 2 moneclonal antibodies that recognize diff'erent epitopes on the H7 HAs, The kit detected each of the tested [5 H7 influenza virus strains and did not react with influenza A viruses of the other subtypcs than H7 or other avian viral and bacteritd pathogens. Tlie kit deteeted H7 HA antigen in the swabs and tissue homegenates ef the chickens experimentally infected with HPAIV strain AtchickenlNetherlandst 2586103 (H7N7). The results indicatc that the present kit is specific and sensitive enough for the diagnosis of HPAI caused by H7 viruses, thus, recommcnded for the field application as a pen-site test kit. KEy woRDs: avian i]ifluenza yirus, diagnosis kit, H7 HA subtype, iininunochromatography. J, Vet. Mkd. sci. 70(6)/ 5s7-562, 2008 Avian infiuenza viruses belong to genus Influenza A virLLs of family Orthomyxoviridae. Infiuenza A viruses are divided into 16 HA and 9 neuraminidase (NA) subtypes on the basis of their antigenic specificity [1Ol, In addition, they are grouped on the basis of their pathogenicity fbr chicken into HPAIV. in which rnortality rnay be as high as 100%, and low pathogenic avian infiuenza viruses (LPAIV) causing much miLder respiratory disease [1]. Since they are antigenically and genetical]y stasis in their natural hosts, feral water birds that do not show clinical signs, they cari be designated as apathogenic ayian influenza viruses (APAIV) [15, 16]. HPAIV have been restricted to H5 and H7 HA subtypes, although not all viruses of these subtypes are highly pathogenic for chickens. Feral water birds are endemically infected with the APAIV strains of H5 and H7 HA subtypes. The available evidence indicates that these APAIV strains are first ransmitted to domestic water fowls, quails or turkeys, and then in live bird markets to chickens. During multiple infections in chicken population, they may acquire pathogenicity for this species to become HPAIV. Consequently outbreaks of HPAI in poultry are difficult to prevent Ll, 22]. Besides causing havoc in poultry, infrequent chicken-to-human and hurnan-to-human transmissions of H7 HPAIV have been reported [8, 1 1]. Rapid diagnosis of HPAI on the outbreak is critical for its control. WHO, OIE and FAO have also emphasized the need fOr early detection, reponing and introduction of mea*CoRREspoNDENcE To: KmA, H., Laberatory of Microbiology, Department of Disease Control, Graduate School of Vcterinary Medicine, Hokkaido University, Kita 18 Nishj 9, Kita-Ku, Sapporo.Hoklcaido,060-0818,Japan. e-mail:[email protected] sures to control this menace. Although virus isolation is the standard test for diagnosis of avian influenza [18], it requires tirne and bio-safety level 3 (BSL-3) facilities E21]. To supplernent this method other rapid diagnostic tests have been developed which detect either the viral nucleic acids or viral antigens. Nucleic acid based molecular diagnostic techniques share the principle of amplifying the nucleic acid to detection levels, Most common techniques are reverse transcriptase polymerase chain reaction (RT-PCR) [17], RT-PCR with enzyme linked irnmunosorbant assay (ELISA) [9], real-tirne reverse transcriptase polymerase chain reaction (RRT-PCR) [20], nucleic acid sequencebased amplification (NASBA) [6, 71 and loop mediated isothermal amplification (LAMP) [13, 14]. Some of the common techniques used for the detection of viral antigens are indirect fluoTescence antibody test (IFA) and ELISA [12]. Although these techniques provide results in shorter time than the classic method of yirus isolation, they require extensive laboratory set up, special equipment and skilled personne]. To overcome these defects, an antigen capture method based on immunochromatography was used in the present study. The main advantagc ofusing this test is that no extensive laboratory set up, special equipment or ski]led personnel are required and the result can be obtained within 15-30 min [2-5, 23]. Most of the commercially available rapid diagnosis kits detect the nucleoprotein (NP) antigen, whose antigenicity is common among type A influenza viruses [2-4]. Therefbre, availability of a rapid diagnosis kit for the HA antigen subtypc dctermination particularly of HPAIV will be a usefu1 adjunct to the rapid diagnosis of HPAI and consequent implementation of rapid control measures. Rapid diagnosis Japanese Society of Veterinary Science NII-Electronic Library Service apaneseSociety fVeterinary cience 558 R. MANZOOR ET AL. kit fOr the HPAI caused by H5 avian influenza viruses has been described [23]. Here we report an immunochromatography test kit for the rapid diagnosis of H7 avian influenza