sybr-green-i and Avian-Leukosis

Avian leukosis virus subgroup K (ALV-K) is an emerging ALV tumor virus of chickens. We developed a SYBR green-based real-time polymerase chain reaction (PCR) assay for the rapid and economical detection of ALV-K in chicken flocks. The assay was specific for ALV-K and did not cross-react with other ALV subgroup or avian influenza virus, Newcastle disease virus, or Marek's Disease virus. The method was 100 times more sensitive than conventional PCR and 10 times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for the P27 antigen. The assay was also more sensitive than conventional PCR in tests of 86 clinical plasma samples. DF-1 tissue culture cells infected with 1 TCID50 ALV-K particle were identified as negative using ELISA but tested positive with the real-time PCR method. The viral loads in organs and tissues in infected chickens were highest in kidney, lungs, and glandular stomach, and these results matched ELISA findings.

Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Benzothiazoles; Chickens; Diamines; Enzyme-Linked Immunosorbent Assay; Organic Chemicals; Poultry Diseases; Quinolines; Real-Time Polymerase Chain Reaction; Viral Load

Subgroup A, B, and J ALVs are the most prevalent avian leukosis virus (ALV). Our study attempted to develop two SYBR Green I-based real-time PCR (RT-PCR) assays for specific detection of ALV subgroup J (ALV-J) and multiplex detection of ALV subgroups A and B (ALV-A/B), respectively.. The two assays showed high specificity for ALV-J and ALV-A/B and the sensitivity of the two assays was at least 100 times higher than that of the routine PCR assay. The minimum virus detection limit of virus culture, routine PCR and real-time PCR for detection of ALV-A strain was 10(3) TCID50 units, 10(2) TCID50 units and fewer than 10 TCID50 units, respectively. In addition, the coefficients of variation for intra- and inter-assay were both less than 5%. Forty clinical plasma samples were evaluated by real-time PCR, routine PCR, and virus culture with positive rates of 80% (32/40), 72.5% (29/40) and 62.5% (25/40), respectively. When the assay for detection of ALV-J was used to quantify the viral load of various organ tissues in chicken inoculated by ALV-J strains CHN06 and NX0101, the results exhibited that ALV-J genes could be detected in all organ tissues examined and the highest copies of ALV-J were mainly in heart and kidney samples at 30 weeks post-infection. Except in lung, the virus copies of CHN06 group were higher than that of NX0101 group in various organ tissues.. The SYBR Green I-based real-time RT-PCR assay provides a powerful tool for the detection of ALV and study of virus replication and infection.

Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Benzothiazoles; Chickens; Diamines; Genes, Viral; Multiplex Polymerase Chain Reaction; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Viral Load

Studies were performed to determine the effects of Bcell suppression on the pathogenesis of Subgroup J avian leukosis virus (ALV-J) in broiler chickens. Neonatal chickens were treated with cyclophosphamide (CY) or PBS, and then infected with ALV-J (ADOL-7501) at 2 weeks of age. CY treatment induced B cell specific immunosuppression throughout the experiment confirmed by decreased bursal weight, intact lymphocyte mitogenetic activity stimulated by Con A and increased relative subpopulation of CD3-positive cells as measured by flow cytometry. Chickens in this experiment had Mareks disease virus exposure prior to three weeks of age as determined by the presence of lymphocytic infiltration and antibody. Virus neutralizing antibody against ALV-J was first observed at 6 weeks post-infection in some of the infected chickens in the PBS group. As expected, none of the chickens from the CY group and uninfected chickens developed virus-neutralizing antibody. The viremic status was measured by real time RT-PCR using SYBR green I dye. The percentage of viremic chickens was significantly higher, and more chickens had high titered viremia, in the CY treated group. No neoplastic foci consistent with ALVJ infection were observed in any of the experimental chickens. The frequency and intensity of viral antigen expression determined by immunohistochemistry was significantly higher in tissues from CY treated birds than those of PBS treated chickens at 3 weeks post-infection. This study showed that B cell specific immunosuppression with CY treatment in chickens resulted in increase in viremia and viral antigen load in tissues.

Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Benzothiazoles; Body Weight; Bursa of Fabricius; Chickens; Concanavalin A; Cyclophosphamide; Diamines; Flow Cytometry; Immunocompromised Host; Immunohistochemistry; Immunophenotyping; Immunosuppressive Agents; Lymphocyte Activation; Organic Chemicals; Poultry Diseases; Quinolines; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Spleen; Statistics, Nonparametric; Viremia