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Avian leukosis virus sub-group
J (ALV-J) -
Developing laboratory technologies for diagnosis in Australia
by T. Bagust, S. Fenton and M. Reddy
August 2004
RIRDC Publication No 04/116 RIRDC Project No UM-49A
The major overseas broiler breeding companies have recently made significant progress in reducing ALV-J infection in their elite flocks. The collective wisdom of industry and of these scientists suggests however, that it will be more difficult to control ALV-J, than it was to control subgroups A and B in earlier decades. These increased difficulties reflect two key biological features of ALV-J, which are (1) the range of antigenic variation that is being observed amongst J-strains, (2) the much enhanced efficiency of ALV-J for infection via contact transmission in broiler chickens.
During the past decade ALV-J has emerged as a serious cause of mortality and suboptimal performance in commercial broiler breeders. Since its discovery in the United Kingdom in 1991, ALV-J has been diagnosed in many countries, including in Australia since 1998. Significant economic losses can be associated with ALV-J infection. The loss rate in commercial broiler breeders infected with ALV-J and intercurrent stressors can be as high as 1.5% per week in excess of normal mortality from approximately 20 weeks of age onwards. Consequential and replacement costs for these breeders can present a major economic loss for the poultry industry. The ALV-J associated loss rates, which have been reported in breeders worldwide, vary from 3-20%. Progeny broilers infected with ALV-J also tend to show reduced growth, unevenness of growth rates within flocks and a greater susceptibility to developing serious diseases when challenged by immunosuppressive viruses or secondary bacterial invaders.
With this background knowledge of ALV-J and the clear potential of ALV-J to cause significant economic losses through mortalities and reduced productivity in Australia’s chicken meat industry, the current project was undertaken during 2000-2003 with the following experimental and operational objectives:
• Develop the virological and serological test systems required to enable culture and detection of the presence of ALV-J infection in vitro and to undertake comparison of isolates antigenically.
• Develop for Australian application the molecular biological techniques required for ALV-J identification and final diagnosis, with recognition of any genomic variation occurring amongst isolates.
• Develop practical methodologies for diagnosis of ALV-J related field problems in broiler flocks.
• Develop an active ALV-J international information network.
Virological culture and serological investigations were undertaken which included comparative assessments of the effectiveness of commercially available avian leukosis ELISA-based reagents, as well as extensive investigations into the propagation of ALV-J in diploid chicken embryonic fibroblasts (CEF) cell cultures as well as a transformed CEF cell line (DF-1). The DF-1 line was imported as part of the present investigations and is classified as C/E (will propagate all sub-groups of ALV except ALV-E). The most sensitive substrate for the detection of ALV-J has consistently been found to be C/O (chicken embryo fibroblasts which can propagate all ALV subgroups) cultures, which are prepared from a small SPF (specified pathogen free) flock, flock, which is maintained at the University of Melbourne for the purposes of this project. Standard SPF CEF cultures have been found to contain unacceptably high levels of endogenous leukosis viral antigens, while DF-1 cell cultures have been found to lack adequate sensitivity (50 to 1000 - fold less) for isolation of ALV-J compared to C/O SPF cell cultures.
Using the C/O SPF cell culture test systems for detection of ALV-J, samples were examined from the broiler breeder flocks of collaborating commercial organizations in Australia.
In summary the following results were obtained during the project:
(1) ALV-J infection of broiler breeders has been detected in grandparent (GP) and parent (P) flocks being maintained in Qld, NSW, Victoria and South Australia.
(2) Detection of virus: ALV-J has been isolated in cell culture and identified by polymerase chain reaction (PCR) testing in field samples of tumours, whole blood, buffy-coat, albumens, meconium and also in feather follicles. The sample of choice which has been selected for screening flocks is to use buffy-coats from whole blood, while if large numbers of birds are to be tested, serums can be used most effectively.
(3) Detection of antibody: The IDEXX commercial ELISA system for detection of antibody to ALVJ has been validated, with the reservation that low grade reactions and a prevalence rate of 15% or less are not indicative of flocks infected with ALV-J. Above these prevalences this ELISA test for antibody can be useful i.e. valid when used as a general flock test for detection of ALV-J infection.
(4) While some commercial broiler breeder organisations tested in Australia showed apparent freedom from ALV-J in 2002-03, examination of other organizations have shown that some 30% of the flocks (grandparent and parent) tested were positive. ALV-J virus was able to be isolated from most of these positive flocks.
(5) In all, some 110 isolates of ALV-J (as well as 14 isolates of ALV-A) have been obtained during this study in Australia to date but the disease significance of ALV-A in these broiler stocks is not yet clear. However the presence of ALV-A could well be conducive to recombination with ALV-J, hence careful consideration needs to be given to removal of all exogenous (infectious) leukovirus by the elite breeder organisations in future.
(6) Of further concern was the finding that ALV-A was frequently detected along with ALV-J.
Mixtures of these viruses were obtained in some 16 of the isolates of ALV-J made from the field in Australia in 2000-02. The reasons for this very high rate of co-infection detected can only be speculative at present.
(7) Experimental infection studies performed using broiler breeder stocks known to be free of ALV-J contamination using Australian ALV-J strains, UOM-201 and UOM-224, found both, when inoculated into day old chickens, to be capable of producing neoplasm, mortalities and eggtransmission of infection (see Chapter 6).
Molecular biological techniques for ALV-J identification, final diagnosis and investigation of genomic variation in Australian isolates were developed. The results of studies undertaken (see Chapter 5 and 7 for details) have conclusively demonstrated that the molecular biological detection techniques i.e. Polymerase Chain Reaction (PCR) tests required for detection of ALV-J have been successfully established in this project. Primer sets H5/H7b were found to be able to demonstrate the presence of all Australian isolates of ALV-J in tumours, feathers, buffy-coats, albumens and meconiums. Additional primers (H5/env-A) were required to be developed for the discrimination of ALV-A from ALV-J. This was necessitated by the finding in these studies of a much higher prevalence of ALV-A infection than expected in Australian broiler breeder stocks.
Sequencing examinations of the env portion of the genome of four ALV-J isolates from Australia, and their comparison with the UK (HPRS-103) and USA prototypes (ADOL-Hc1) strains of ALV-J, show that the Australian isolates are more closely related to one another than to the overseas strains.
Hence the evolution of ALV-J would appear to be continuing in Australian breeder stocks. A preliminary observation to the effect that the genome sequences of Australian ALV-J strains would appear to be more analogous to those of the UK than the USA strains is still under investigation.
Robust methodologies for the diagnosis of ALV-J involvement in field problems in broiler (breeder) stocks have also been developed in the course of this project. The recommended culture method using a 7-9 day passage period in C/O SPF cell cultures for buffy-coat or serum, followed by ELISA testing for ALV gs antigen p27 and final identification as ALV-J by PCR using primers sets will identify the presence of ALV-J in those birds showing disease but not tumours. While the ALV-J virus will be detected by this system in feather follicles or egg albumen, the diagnostic specimen of preference is whole blood. For breeders in which tumours are apparent eg. located at the sternum, liver or heart, the PCR test can be applied directly and rapidly (within 48 hrs) after extraction of DNA. In Australia, PCR differentiation of ALV-J from ALV-A should also be undertaken using PCR testing for final identification of isolates from field flocks.
International reference networking was able to be established from early in this project with the two major international reference laboratories for ALV-J i.e. the Compton Institute of Animal Health, UK (Dr. Venugopal Nair) and the Avian Diseases and Oncology Laboratory (ADOL) of the USDA (Dr.
Ally Fadly). Positive relations with exchange of information and feedback have also been fostered with two of the major breeding companies in the world which supply breeding stock into Australia i.e. Aviagen (Scotland) and Cobb Vantress (USA).
 
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