Differential Detection of Avian Oncogenic Viruses in Poultry Layer Farms and Turkeys by Use of Multiplex PCR

Abstract

bAvian oncogenic viruses include Marek's disease virus (MDV), a highly contagious herpesvirus, as well as retroviruses such as avian leukosis virus (ALV) subgroups A to J and reticuloendotheliosis virus (REV). In this study, we examined the incidence of these viruses in suspected samples collected from poultry layer farms of South India, mainly in the Namakkal district of Tamil Nadu, a highly dense poultry-growing area in India. The histopathology-positive tissue sections were identified and further confirmed by immunohistochemistry using virus-specific antibodies. The viruses belonging to all 3 groups (MDV, ALV, and REV) were isolated in a cell culture system and confirmed by immunofluorescence using virus-specific antibodies. PCR appeared to be the method of choice for rapid and accurate diagnosis of these viruses. The multiplex PCR primers specific to MDV, ALV, REV, and chicken DNA were designed for rapid differential diagnosis. The specificity of the primers was checked by amplification of DNA from virus-infected cell culture in comparison with uninfected samples, and sensitivity was evaluated by calculating the minimum copy number at which amplification occurs in the cloned PCR products. The sequences of the amplicons were compared by BLAST analysis. PCR tests demonstrated the presence of single, dual, or triple viruses in some of the samples. Of 169 samples screened by multiplex PCR, 9 samples were positive for MDV, 17 samples were positive for ALV, 12 samples were positive for REV, and 17 samples were positive for both ALV and REV. Three samples were positive for all three viruses. ALV-positive samples were further subjected to subgroup-specific PCR, which gave positive results for subgroups B and D but not for subgroup J. Multiplex PCR appeared to be a useful technique for rapid differential diagnosis of avian oncogenic viruses and detection of multiple infections of avian oncogenic viruses under field conditions.

Pathogenic characteristics of Marek's disease virus field strains prevalent in China and the effectiveness of existing vaccines against them

“…PCR and ELISA (using p27 mAb) methods were used to test Avian leucosis virus (ALV) (Gopal et al, 2012;Yun et al, 2013). The ALV HLJ09SH01 strain, which was isolated from China , was used as positive control for amplification of ALV in this study.…”

“…PCR and IFA (using gp90 mAb) methods were used to test Reticuloendotheliosis Virus (REV) (Gopal et al, 2012;Xue et al, 2012.). The REV HLJR0901 strain was isolated from China (Jiang et al, 2014.…”

Isolation and full-genome sequence of two reticuloendotheliosis virus strains from mixed infections with Marek’s disease virus in China

“…The samples collected from sick chickens of suspected MD were sent to us, and we tested oncogenic viruses [including MDV, REV, and avian leukosis virus (ALV)] and chicken anemia virus (CAV) with PCR assays [10][11][12][13]. In the total of 20 sick chickens from Shandong Province, the testing results showed that 15 were MDV positive, six were REV positive, none was detected with ALV and CAV, and six were mixed infected with MDV and REV.…”

Applying the isothermal DNA amplification method in identifying the Marek’s disease virus in poultry

“…Czułość reakcji PCR w detekcji wirusa MDV w odniesieniu do reakcji LAMP jest ok. 10 × niższa (46,47). Dostępne są także dane literaturowe na temat detekcji wirusów MD z zastosowaniem reakcji real-time PCR (2,16,21,42,47). Pewnych trudności dostarcza porównanie wyników uzyskanych przez autorów badań.…”