Chicken Infectious Anemia (CIA) was first recognized by Yuasa et al. in 1979 as a new disease in young chickens caused by a novel virus (CIAV). Since the first description of this disease and subsequent isolation of the virus in cell culture in Japan, CIAV has been isolated in virtually all countries with a poultry industry. CIA is characterized by aplastic anemia and generalized lymphoid atrophy with concomitant immunosuppression and is frequently complicated by secondary viral, bacterial, or fungal infections. CIAV plays a major role in the etiology of a number of multifactorial diseases associated with hemorrhagic syndrome and/or aplastic anemia. In addition to anemia and associated syndromes, subclinical chicken infectious anemia virus infections, without anemia and increased mortality, are frequently observed in commercial flocks (Schat & van Santen, 2008). In Argentina, CIA was identified in 1991 and the virus was isolated for the first time in 1993 (Buscaglia et. al,. 1994). Since then, other isolates have been reported (Buscaglia, 1996; 1999) and several others have been characterized (Craig et. al, 2009). Buscaglia showed, at the IXth Argentinean Virology Congress, the presence of CIAV antibodies in backyard hens provided to poor families in a social program called Pro Huerta (Buscaglia, 2008). The results of serological tests on sera obtained from backyard chickens from different locations of Buenos Aires Province unrelated to the Pro Huerta program were presented in part and combined with the data from sera from the Pro Huerta program that was presented, but not published in detail yet (Buscaglia, 2010). More sera unrelated to the Pro Huerta program were obtained from small flocks of chickens that were distant from commercial poultry operations. Results from samples obtained from wild birds from the province of Buenos Aires will be presented and compare to both types of backyard chickens. In summary the aim of the present study was to determine the presence of antibodies to CIAV in free living wild birds, from more backyard chickens not related with the Pro Huerta and compare them with the sera from Pro Huerta chickens reported previously.
Materials & Methods
Sera were obtained from two types of backyard poultry populations randomly sampled and wild birds that either arrived to the Fundación Ecológica Pinamar or were obtained by the author in different circumstances from places in the province of Buenos Aires:
a) Four hundred sera were obtained from chickens which were part of a social program for food security (called Pro-Huerta) established by INTA (Instituto Nacional de Technologia Agropecuaria).
b) A second group of 350 sera were collected from backyard chickens from locations such as General Lamadrid, Laprida, General Madariaga, Paraje Juancho, City Bell, Gonnet y La Plata in the province of Buenos Aires. All donor flocks from these samples were far from commercial poultry operations.
c) A third group of 25 sera were obtained from different species of wild birds from different locations of the province of Buenos Aires.
The samples mention in a) and 300 samples of b) were tested at a 1:100 dilution using the commercial IDEXX enzyme linked immunosorbant assay (ELISA) CIAV antibody test kit. Samples of the second group were double checked using the indirect fluorescence antibody (IFA) test reported elsewhere (McNulty et al., 1989; Yuasa et al., 1985) and used essentially as described previously (Buscaglia & Tohya, 1997). Samples from wild birds (group c) were obtained from the district of Pinamar, General Lamadrid, City Bell and Gonnet. They were only tested using IFA as 50 samples from group b).
MDCC-MSB-1 cells were obtained from Dr Takeshi Mikami (at that time at the Department of Veterinary Microbiology, Faculty of Agriculture. The University of Tokyo, Tokyo, Japan) and from the Department of Avian and Aquatic Animal Medicine (DAAAM), Cornell University (CU), U.S.A. Both MSB-1 cell lines were grown at 39o C or 41° C in an atmosphere of 5% CO2 in RPMI 1640 medium supplemented with 5% fetal bovine serum or in LM Hahn medium with 10% fetal bovine serum. The cells were sub cultured at 2- to 3-day intervals and seeded at 0.5 X 106/ml. More MSB-1 cells were cuture as above, but at Dr K. A. Schat's lab at the Department of Microbiology and Immunology, CU during 2010. Smears were prepared with the virus described below and as mention also below.
Virus strains and Monoclonal antibodies
The Cux-1 strain of CIAV (von Bulow et al., 1983) was obtained, with permission from the USDA by the DAAAM from Mr. R. Wellenstein (SPAFAS. Inc., Norwich, Connecticut), after an unknown number of passages in MSB-1 cells. The same strain was obtained with permission of the Argentinean Ministerio de Economía y Obras y Servicios Públicos, Secretaria de Agricultura Ganadería y Pesca, Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA) from DAAAM. Cux-1 CIAV was used to infect MSB-1 cells for the IFA test as described by McNulty et al. (1989). Supernatant fluids from hybridoma 51.3 (Chandratilleke et al., 1991) were used to detect the presence of CIAV-specific antigens. This hybridoma produces monoclonal antibodies (MAb) against the Cux-l strain of CIAV and cross-reacts with at least 2 other CIAV isolated in the USA.
Detection of antibodies against CIAV by IFA
Cux-1- infected and uninfected control MSB-1 cells were used as positive - and negative- antigen preparations for detection of antibodies. These were prepared as described by Lucio et al. (1990) with a few modifications (Buscaglia & Tohya, 1997).
Results & Discussion
None of the sera from the third and second group were positive in the ELISA test while 91% of the birds from the first group were positive with high titers. Sera from the second group, which were negative in the ELISA test, were also negative using the IFA.
The main differences between the two types of backyard birds sampled are their genetic background and the vaccines they received. Unlike the backyard flocks in the second group, Pro Huerta chickens were developed at INTA and were vaccinated with herpes virus of turkey (HVT) at the first day of age.
Non commercial birds in Europe had shown the presence of CAIV antibodies (de Wit et al., 2004: Wunderwald & Hoop, 2002). The first report of antibodies detected in non commercial poultry and especially in backyard poultry in the Americas was published in 2006 (Hernandez-Divers et al., 2006) and the second was presented at the IXth Argentinean Virology Congress (Buscaglia, 2008). Although Craig et al. (2009) stated that no further studies on CAIV in Argentina were reported after 1994, information mentioned in the introduction shows the contrary (Buscaglia 1996; 1999. 2008; Buscaglia et al., 2003).
Either the source of chickens used for the Pro Huerta program had CIAV or the environment in which they were kept was contaminated. A third, albeit unlikely, cause could be that the HVT vaccine was contaminated with CIAV. Hernandez- Divers et al. in 2008 pointed out that pathogens of free-ranging chickens create a risk of disease for wild birds. The probable harm done perhaps to the free living birds and chicken population in Argentina is potentially serious because CIAV can be spread among others birds since the chickens given by this social program could be apparently a source of virus. CIAV was probably introduced by commercial poultry operations. However, the role of the Pro Huerta flocks as a reservoir for CIAV should be considered. However, preliminary studies conducted specially on free living wild birds, since they may also be reservoir for the virus transmission and so pose a threat to backyard and commercial poultry did not show positive data. The need for further studies should be considered since CIAV has already been recognized in wild birds in Europe (Campbell, 2001). Means of control and biosecurity measures should be a high priority for poultry farmers and veterinarians. It is interesting that the authors, who recently characterized isolates of CIAV in Argentina (Craig et al., 2009), belong to the same institution that distributed the Pro Huerta chickens among poor families. For that reason it is highly recommended that they participate and apply sanitary measures to eradicate and eliminate the disease from those birds if it exists. None of these chicken flocks are checked for the presence of infectious pathogens with the exception of Avian Influenza (AI) and Newcastle disease (Buscaglia et al., 2003) and thus their health status is unknown. The Pro Huerta flocks that helped to understand the absence of AI in Argentina (Buscaglia et al., 2007) may also be a reservoir for other avian diseases that could cause an economic and environmental threat, not only to the commercial poultry industry but to other backyard birds and potentially free living birds as well.
None of the sera from the second and third group were positive in the ELISA test while 91% of the birds from the first group were positive with high titers. All sera from the second group tested by ELISA or IFA resulted negative. The sera from wild birds up to the moment were negative to the IFA test as well as the sera from backyard poultry unrelated to the Pro Huerta program. Social programs such as the one mentioned in the present work should evaluate consequences before being implemented and the presence of infectious pathogens should be checked.
I wish to thank Dr. Karel A. Schat for allowing me the use of his lab at Cornell University for preparation of the smears used to test the wild birds sera and more backyard chicken sera.
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