Experimental Infection of Attwater's/Greater Prairie Chicken Hybrids with the Reticuloendotheliosis Virus
Reticuloendotheliosis virus (REV), a common pathogen of poultry, has been associated with runting and neoplasia in an endangered subspecies of grouse, the Attwater's prairie chicken. The pathogenesis of REV infection was examined in experimentally infected prairie chickens. Three groups of four Attwater's/greater prairie chicken hybrids were infected intravenously with varying doses (tissue culture infective dose [TCID50], 200, 1000, and 5000) of a prairie chicken-isolated REV. A fourth group of four birds was not infected. Blood was collected prior to infection, and at various times up to 37 wk following infection. Peripheral blood mononuclear cells were examined for integrated proviral DNA by a single-amplification polymerase chain reaction (PCR) and nested PCR of a region within the pol gene. The nested PCR identified REV proviral DNA in all REV-inoculated birds by 2 wk postinfection and confirmed chronic infection throughout the study. With the exception of a bird that died from bacterial pneumonia 8 wk postinfection, neoplasia, resembling that seen in naturally occurring infections, was observed in all birds, even those receiving as little as 200 TCID50 of virus.
[Show abstract] [Hide abstract] ABSTRACT: A highly sensitive real-time PCR method was developed in this study for reticuloendotheliosis virus (REV) detection and quantitation. The real-time PCR method, with a minimum detection limit of 10 proviral DNA copies, was 100 times more sensitive than the conventional PCR. It was also shown to be highly specific, as no positive signals were detected for other common avian DNA viruses. The coefficients of variation of intra- and inter-assay reproducibility were both less than 2%. The chicken β-actin gene was co-amplified and used as the internal control to monitor the efficiency of DNA extraction and PCR amplification. Specific pathogen free chickens were infected with REV at different ages and the blood was detected with the real-time PCR method. High levels of proviral DNA were detected in the blood of REV-infected chickens during the experiment and chickens infected early had higher proviral loads from 2 weeks post-infection compared with late infected chickens. This study provides an excellent research and diagnostic tool that can be used for REV detection and quantitation.0Comments 7Citations
- "This real-time PCR assay had high reproducibility with relatively small intra-and inter-assay variability . Furthermore, this new method was able to detect as few as 10 target copies of the proviral DNA, which is 100 times more sensitive than the conventional PCR, 60 times more sensitive than the combined method with a single-amplification reaction and a Southern blot (Drew et al., 1998), and 10 times more sensitive than the nested PCR (Ryan et al., 2006 ). Therefore, the real-time PCR method developed in this study can be used to screen large number of birds for REV infection at much earlier stages of infection with lower proviral loads. "
[Show abstract] [Hide abstract] ABSTRACT: Reoccurring infection of reticuloendotheliosis virus (REV), an avian oncogenic retrovirus, has been a major obstacle in attempts to breed and release an endangered grouse, the Attwater's prairie chicken (Tympanicus cupido attwateri). REV infection of these birds in breeding facilities was found to result in significant decreases in the CD4(+) and increases in the CD8(+) lymphocyte populations, although experimental infection of birds resulted in only increases in the CD8(+) lymphocytes. Because our indirect immunofluorescent assay readily detected infection of both CD4(+) and CD8(+) lymphocytes, a triple labeling flow cytometric procedure was developed to quantify the individual lymphocytes infected in vivo with REV. Lymphocytes were gated with a biotinylated pan-leukocyte marker bound to streptavidin R-PE-Cy5. Chicken CD4 or CD8 specific mouse MAb directly labeled with R-PE identified the phenotype and with permeabilizing of cells, infection was indirectly labeled with rabbit IgG specific for the REV gag polypeptide and FITC conjugated goat anti-rabbit antibody. More than 50% of the total lymphocytes and of the total CD4(+) or CD8(+) lymphocytes supported in vivo viral expression in all infected birds examined. Remarkably, this level of infection was detected in the absence of visible clinical signs of illness.0Comments 7Citations
- "Early detection of infection is critical in maintaining flocks free of infected birds that can remain persistently infected for months. The current assay of choice for detection of REV is PCR amplification of either the polymerase gene or the LTR of the integrated provirus (Drew et al. 1998; Bohls et al., 2006a). The ready identification of infection in leukocytes by direct detection of immobilized cells expressing REV antigen, as with IFA or immunoperoxidase staining, may provide the basis for a more sensitive, less expensive assay. "
- [Show abstract] [Hide abstract] ABSTRACT: This study was designed to detect reticuloendotheliosis virus (REV) as a contaminant in fowl pox vaccines. A total of 30 fowl pox vaccine samples were examined for the presence of REV using both in vitro and in vivo methods. In in vitro testing, the fowl pox vaccine samples were inoculated into chicken embryo fibroblast cultures prepared from specific-pathogen-free embryonated chicken eggs, and the cultures were examined using PCR to detect REV. In in vivo testing, each fowl pox vaccine sample was inoculated into 5-d-old specific-pathogen-free chicks, which were kept under observation for up to 12 wk postinoculation; serum samples were collected at 15, 30, and 45 d postinoculation for the detection of REV-specific antibodies using ELISA. Tissue samples were collected at 8 and 12 wk postinoculation for histopathological examination. Of the tested vaccines, only one imported vaccine sample tested positive for REV using PCR. Serum samples collected from chicks infected with the PCR-positive vaccine batch also tested positive for REV-specific antibodies using ELISA. Histopathological examination of the liver, spleen, and bursa of Fabricius demonstrated the presence of tumor cells in these organs, confirming the results obtained using PCR and ELISA, and indicating that the sample was contaminated with REV. These data clearly indicate that the screening of all commercial poultry vaccines for viruses is an important factor in assuring the biosafety of animal vaccines.0Comments 15Citations