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2 OIE/FAO and National Reference Laboratory for Avian Influenza and Newcastle Disease, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell’Università 10—35020 Legnaro (Padova), Italy
3 Corresponding author (email: gcattoli{at}izsvenezie.it)
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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According to the revised World Organization for Animal Health (OIE) definition, all avian influenza viruses belonging to H5 and H7 subtypes are defined as notifiable avian influenza (NAI) viruses, regardless of their virulence for birds (OIE, 2005). All avian influenza viruses of low pathogenicity for poultry (LPAI), including low pathogenic H5 and H7 viruses, are maintained in nature in wild birds, mainly in Anseriformes and Charadriformes. Recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in Russia, Central Asia, Europe, and Africa suggest the virus may have been carried by wild birds during their autumn and spring migrations. To gain more information on the presence and persistence of HPAI H5N1, several countries and international organizations developed and applied surveillance programs targeting wild bird populations. However, prior to implementing a surveillance program it is essential that the objectives are defined in order to select the most appropriate diagnostic approach.
Generally speaking, surveillance in wild birds can have two main objectives: to establish the circulation and prevalence of all avian influenza viruses (including H5 and H7 LPAI and HPAI), and to establish the presence or absence of HPAI viruses (e.g., HPAI H5N1) in the selected population at a given point in time. Assessment of NAI viruses in wild birds is relevant to identify areas at higher risk of their introduction into domestic poultry and better understand the ecology of these viruses in their natural reservoirs. The collection of viral strains and knowledge of the most prevalent hemagglutinin (H) and neuraminidase (N) subtype combinations also can be instrumental for development of vaccines for poultry. Surveillance programs focusing on detection of HPAI viruses in the wild could act as an early warning system. This would allow rapid implementation of preventive measures in a country or in a defined area.
The two main objectives mentioned above can be achieved within the same surveillance program. However, because different diagnostic approaches may be more suitable to one or the other purpose, the main objective of a surveillance program should be identified and prioritized. We discuss the use and application of difference diagnostic approaches for wild bird surveillance. In addition, in some cases environmental sampling can be used to determine the persistence of infectious viral particles in the environment, although this type of sampling requires validation and improvement before it can be deemed a reliable means of detecting environmental contamination.
Correct selection of the target population is the first important step of any surveillance program. Although this topic is beyond the scope of our paper and will not be addressed, it is important to bear in mind that among the vast variety of wild birds, the target population might be different on the basis of the two main objectives listed above. In addition, selection of the most appropriate sample, and adequate collection, storage, and shipment is essential for the success of any surveillance program, regardless of the diagnostic approach.
Wild bird surveillance, mainly based on cloacal sampling of captured or hunted waterfowl in Europe and Africa in recent times, revealed active circulation of many LPAI subtypes. However, no HPAI H5N1 viruses were detected, even in countries in which the pathogenic virus was confirmed in ill or dead wild and domesticated birds (SANCO, 2006; Gaidet et al., 2007). This might indicate that the approach used in these surveillance programs is suitable for finding AI viruses but not HPAI viruses.
Environmental detection of AI viruses may be a possible indicator to establish virus persistence following cleaning and disinfection procedures during and after an AI outbreak (Suarez et al., 2003). However, sampling to determine the presence and persistence of AI viruses in the natural environment has not been extensively applied. Although not specifically recommended, collection of fresh faecal dropping can substitute for cloacal swabbing in wild birds (Munster et al., 2005).
Serology offers a relatively inexpensive and practical way to determine circulation and prevalence of influenza viruses in the avian population. In the framework of a surveillance effort in domesticated birds, serological diagnosis is considered a suitable approach to monitor the AI-free status of farms, including backyard and industrial birds. On the contrary, the application of current serological methods in wild bird surveillance will generate information of very limited use. Antibodies to the group antigen of influenza A viruses may be detected by agar gel immunodiffusion (AGID) and immunoenzymatic tests. These tests are unable to give any indication of the virus subtype. Because several species of wild birds are the natural reservoirs of AI viruses, the detection of group-specific rather than subtype-specific antibodies is a common finding of no diagnostic relevance.
The above-mentioned tests have additional limitations for use in wild fowl. Waterfowl usually do not produce serum precipitins (Higgins, 1989) and, therefore in spite of exposure, they rarely yield positive results in the AGID test. Furthermore, most of the current commercial immunoenzymatic tests are based on indirect detection of specific antibodies through antispecies conjugates. These tests were developed for detecting infection in intensively reared poultry, namely chickens and turkeys, and therefore employ antichicken IgG secondary antibodies. These kits have not been validated for wildfowl, and their reliability in these species remains to be established.
Hemagglutination inhibition assay (HI) provides information on the presence of antibodies directed against a specific subtype and can be applied on a variety of bird species. The sera of certain avian species contain nonspecific hemagglutinins that interfere with the HI test. Such sera are therefore treated prior to the test. In any case, given that wild birds are the reservoir of AI viruses, detection of antibodies against NAI only represents evidence of an exposure to H5 or H7 antigens and no information on whether exposure was recent or not, or on the H and N combination of the viruses or the pathotype.
Traditionally, laboratory protocols for detection and identification of avian influenza viruses were based on virus isolation (VI) in specific-pathogen-free eggs or in cell cultures. This methodology still represents the gold standard and the official method for detection of AI viruses (OIE, 2005). It can be used on all types of clinical, laboratory, and environmental specimens including swabs, organs, faeces, and other organic and inorganic materials. Virus isolation is the only method that can determine the persistence of viable virus in the environment. In extensive surveillance efforts in wild birds, however, its use is limited mainly because it is laborious, time consuming, and cannot be performed on large numbers of samples. In particular, limiting factors appear to be the availability of staff, equipment (e.g., bio-safety cabinets, incubators), eggs, and reagents. Virus isolation can yield positive results only if the samples contain viable virus particles. In some instances, wildlife sample collection occurs in remote areas, where it is not always possible to ensure proper storage and/or prompt shipment of samples to the laboratory. In these cases, VI attempts might lead to false-negative results.
The available immunoenzymatic (antigen capture) methods for influenza virus antigen detection generally are easy to perform and can be applied in the laboratory and directly in the field. They are directed to detection of one of the major viral proteins (nucleoprotein or hemagglutinin) and do not require viable virus to yield a positive result. The methodology is now easily accessible from many companies on the international market. Some products developed for influenza virus detection in humans can be applied to other animal species, such as poultry (Cattoli et al., 2004; Bai et al., 2005; Woolcock and Cardona, 2005). Recently, several kits for the rapid detection of AI antigens in poultry also were developed and marketed. Most of these kits can detect influenza type A antigens (i.e., viral matrix protein or nucleoprotein); thus they cannot provide any information about the H or N subtype or the pathotype. It is not possible to express an overall opinion on the performances of these kits, as full field validation currently is unavailable. However, generally speaking, published data (Cattoli et al., 2004; Woolcock and Cardona, 2005) and laboratory experience in poultry testing indicate that antigen capture methods are useful in some situations but have limited sensitivity and specificity compared to other methods. Analytical sensitivity is generally in a threshold range between 104 and 103 EID50, and this should be kept in mind when issuing a diagnosis based on these methods. Certain types of specimens, such as faeces or internal organs, sometimes lead to false positive results, thus limiting the specificity of the test.
Several protocols based on different molecular methodologies, such as RT-PCR, real-time RT-PCR (rRT-PCR) and NASBA (nucleic acid sequence-based amplification), were developed in the last decade for detection of influenza viral RNA in clinical and laboratory specimens. They are generally very sensitive, specific, and adaptable to high throughputs. PCR-based and sequencing protocols are available to detect subtype and pathotype of the virus directly on clinical materials, thus allowing a rapid turnaround time and faster characterization. Careful evaluation of the method employed to extract the RNA is necessary to avoid false-negative results due to inefficient extraction or presence of contaminants that can inhibit the molecular reactions. On the other hand, molecular methods based on enzymatic amplification of nucleic acid are at risk of generating false-positive results due to cross-contamination of samples, particularly when high numbers of samples are processed at the same time, as might happen during extensive surveillance programs.
Unpublished observations on the performance of rRT-PCR for detection of HPAI H5N1 in carcasses of wild swans (Cygnus olor) at the Italian Reference Laboratory showed that a higher amplification signal and lower cycle threshold (Ct) values could be obtained in tracheal and lung tissues of infected birds. Comparisons of samples from the intestinal contents and pooled lungs and trachea showed differences ranging from 3 to 9 Ct. Whether this was due to a different viral load in the tissues or to the presence of PCR inhibitors in those particular specimens is not known, but sampling the respiratory tract of wild birds may be more suitable for molecular detection of HPAI H5N1 viruses.
At the time of writing, rRT-PCR can probably be considered the preferred molecular method for wild bird surveillance, as it allows high throughput in combination with high sensitivity and specificity. However, no official methods exist for the molecular detection of AI viruses in domestic and wild birds. This results in different protocols used among diagnostic laboratories and can make the comparison and interpretation of results difficult. Recently, a selection of molecular protocols, including conventional RT-PCR and rRT-PCR, were evaluated in inter-laboratory trials (Slomka et al., 2007) and will be included among the recommended protocols in the diagnostic manual for AI published by the European Union.
Based on the characteristics of the different methods and taking into account the main objectives of a surveillance program, guidelines for an appropriate diagnostic approach can be summarized as follows:
To determine circulation and prevalence of AI viruses (including H5 and H7 LPAI and HPAI viruses):
To determine presence or absence of HPAI viruses in the population of a certain area:
To make results comparable and useful to the international scientific community, it is essential that harmonized diagnostic strategies aiming at the detection of influenza viruses, and specifically to HPAI and HPAI H5N1 viruses, are developed and followed worldwide. Regardless of approach used, virus isolation techniques can be delayed but never abandoned, as they still remain the only diagnostic tools that enable complete and genetic characterization of the virus. Collecting virus isolates is of fundamental importance to increase our knowledge of AI, including selection of candidate vaccine strains, as well as pathogenesis and evolutionary studies on influenza viruses.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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CATTOLI, G., A. DRAGO, S. MANIERO, A. TOFFAN, E. BERTOLI, S. FASSINA, C. TERREGINO, C. ROBBI, G. VICENZONI, AND I. CAPUA. 2004. Comparison of three rapid detection systems for type A influenza virus on tracheal swabs of experimentally and naturally infected birds. Avian Pathology 33: 432–437.[Medline]
GAIDET, N., T. DODMAN, A. CARON, G. BALANC A, S. DESVAUX, F. GOUTARD, G. CATTOLI, V. MARTIN, A. TRIPODI, F. LAMARQUE, W. HAGEMEIJER, AND F. MONICAT. 2007. Influenza surveillance in wild birds in Eastern Europe, the Middle East, and Africa: Preliminary results from an ongoing FAO-led survey. In Proceedings of the FAO and OIE International Scientific Conference on Avian Influenza and Wild Birds, 30 and 31 May 2006. Rome, Italy. Supplement of The Journal of Wildlife Diseases, In press.
HIGGINS, D. A. 1989. Precipitating antibodies of the duck (Anas platyrhynchos). Comparative Biochemistry and Physiology 93: 135–144.[Medline]
MUNSTER, V., A. WALLENSTEN, B. OLSEN, G. F. RIMMELZWAAN, A. D. M. E. OSTERHAUS, AND R. A. M. FOUCHIER. 2005. Influenza A virus surveillance in wild birds. In Avian influenza: Prevention and control, R. S. Schrijver and G. Koch (eds.). Wageningen UR Frontis Series. Springer, Dordrecht, NL.
OIE. 2005. Manual of diagnostic tests and vaccines for terrestrial animals—Version adopted May 2005. http://www.oie.int/eng/normes/MMANUAL/A_00037.htm.
SANCO, D.G. 2006. Preliminary results on member states avian influenza surveillance in wild birds, July to December 2005. Working group meeting on surveillance against avian influenza, 9 January 2006, Brussels, Belgium.
SLOMKA, M. J., V. J. COWARD, J. BANKS, B. Z. LONDT, I. H. BROWN, J. VOERMANS, G. KOCH, K. J. HANDBERG, P. H. JORGENSEN, M. CHERBONNEL-PANSART, V. JESTIN, G. CATTOLI, I. CAPUA, A. EJDERSUND, P. THOREN, AND G. CZIFRA. 2007. Identification of sensitive and specific avian influenza PCR methods through blind ring trials organized in the European Union. Avian Diseases 51: 227–234.[Medline]
SUAREZ, D. L., E. SPACKMAN, D. A. SENNE, L. BULAGA, A. C. WELSCH, AND K. FROBERG. 2003. The effect of various disinfectants on detection of avian influenza virus by real time PCR. Avian Diseases 47: 1091–1095.[Medline]
WOOLCOCK, P. R., AND C. J. CARDONA. 2005. Commercial immunoassay kits for the detection of influenza virus type A: Evaluation of their use in poultry. Avian Diseases 49: 477–481.[Medline]
Received for publication 15 December 2006.
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