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Detection of avian leukosis virus in albumen of chicken eggs.pdf

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Detection of avian leukosis virus in albumen of chicken eggs

Journal of Virological Methods 78 (1999) 1–11 Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction Thuy D. Pham a,*, J. Lloyd Spencer b, Eric S. Johnson a a Department of Biostatistics and Epidemiology, SL 18, Tulane Uni6ersity Medical Center, 1501 Canal Street, New Orleans, LA 70112, USA b Canadian Food Inspection Agency, Ottawa, Ontario, Canada Received 7 July 1998; received in revised form 30 October 1998; accepted 30 October 1998 Abstract A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry. ? 1999 Elsevier Sc