Progress 10/01/99 to 12/31/03
Outputs
We have undertaken research on dual infection of chickens with infectious laryngotracheitis virus (ILTV), a highly contagious respiratory of chickens, and fowlpox virus (FPV). Although FPV and ILTV are different viruses belonging to genus avipoxvirus and alpha herpes virus, respectively; these viruses manifest almost similar symptoms and signs in the upper respiratory tract of infected chickens. Severe forms of the diseases could occur in flocks due to either FPV or ILTV infection or both. Clinical respiratory infections or subclinical cases may be misdiagnosed in chickens. Therefore accurate diagnostic methods which differentiate one virus from the other are needed to ensure that infections are identified promptly. We examined tissue samples from chickens clinically diagnosed as FPV infection and employing two techniques; a double-stain immunohistochemistry (IHC), and a sensitive and rapid multiplex PCR technique, using two different primer sets; one specific to ILTV,
and another primer set specific to FPV. Both FPV and ILTV were detected from only (2/7) chicken samples by both PCR and IHC; however, chicken samples (5/7) were positive by IHC and PCR for FPV only. More work is in progress to determine the frequency and extent of dual infection of chickens.
Impacts
The results demonstrate that chickens are simultaneously infected with two differing avian viruses and also emphasize the specificity and sensitivity with which these techniques could be used for differential diagnosis of dual infection.
Publications
- Theodros Tadese and Willie M. Reed. 2003. Detection of specific reticuloendotheliosis virus sequence and protein from REV-integrated fowlpox virus strains. J. virol. Methods 110: 99-104. Simultaneous infection of chickens with fowlpox virus (FPV) and infectious laryngotracheitis virus (ILTV) by immunohistopathology and multiplex PCR technique (Manuscript to be submitted).
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Progress 01/01/02 to 12/31/02
Outputs
Pox has been a continual problem in vaccinated poultry. Variants of field isolates of FPV have been responsible, and are more likely to have been created by the integration of REV in the genome of FPV. We have demonstrated the presence of REV protein in CEFs infected with field isolates of fowl poxvirus. Using polymerase chain reaction (PCR), field isolates (5/6) and vaccine strains (2/7) of FPV were found to have a 291 bp repeat sequence of REV-LTR integrated in their genome. We adopted an immunofluorescence (IF) technique, using a monoclonal antibody (MAb) to the REV envelope protein to confirm the presence of specific REV protein. While the IF indicated the presence and localization of REV protein in defined boundaries within cells infected with field isolates of FPV carrying REV (FPV-REV) the MAb reacted specifically to a REV protein with a relative molecular mass of 62 kD. These data have the potential to advance our current understanding of the integration of
REV in field isolates of FPV which have attributed, in recent years, to considerable economic loss in the poultry industry. Currently, we are attempting to isolate and purify the specific REV protein for further studies and sequence analysis.
Impacts
This work will ultimately lead to the development of more efficacious vaccines to prevent avian pox and will also explain why avian pox infections have increased dramatically in the U.S. in the last few years.
Publications
- Theodros Tadese and Willie M. Reed. 2003. Use of restriction fragment length polymorphism (RFLP), immunoblotting (IB), and polymerase chain reaction (PRC) in the differentiation of avian posviruses. J Vet Diag Invest. (in press).
- Theodros Tadese, E.A. Potter, and W.M. Reed. 2003 Development of a mixed antigen agar gel enzyme assay (AGEA) for the detection of antibodies to poxvirus in chicken and tukey sera. J Vet Med Sci. (in press).
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Progress 01/01/01 to 12/31/01
Outputs
Outbreaks of pox in vaccinated commercial flocks due to variant or field isolates of fowl poxvirus (FPV) are on the increase. Recently, We have been investigating additional samples of FPV from various regions of U.S. All field isolates examined thus far have had integrated reticuloendotheliosis virus FPV-REV in their genome. REV is the most common retrovirus associated with neoplastic disease in poultry. Currently, We are furthering our studies on a limited number of these strains as well as vaccine strains known to carry REV to shed light on the molecular nature of FPV-REV to identify and isolate specific proteins that could have contributed to the antigenic difference among the strains. The specific proteins may serve as immuno-modulatory protein/s causing the strains to become virtually invisible to the immune system of previously vaccinated poultry. As an extension of our previous studies, we have been purifying specific REV protein from FPV-REV infected cell
cultures and the proteins are being purified using monoclonal antibodies specific for envelope polypeptides expressing REV specific and strain common epitopes. The monoclonal antibodies proved capable of recognizing specific proteins from the purified strains. Immunofluorescence (IF) techniques are also being used to identify and localize the magnitude and specificity in which REV proteins are encoded in FPV-REV infected cultures. These proteins will be used as a basis for construction of cDNAs for future studies.
Impacts
This work will ultimately lead to the development of more efficacious vaccines to prevent avian pox and will also explain why avian pox infections have increased dramatically in the U.S. in the last few years.
Publications
- Tadese, T. and Reed, W.M. Reed. 2001. Use of fragment length polymorphism (RFLP), Immunoblotting (IB) and polymerase chain reaction (PCR) in the differentiation of vaccine and field strains of fowlpox virus (FPV) (manuscript in press)
- Tadese,T., Potter,E.A., and Reed, W.M. 2001. Development of a mixed agar gel enzyme assay (AGEA) for the detection of antibodies to poxvirus in chicken and turkey sera. (manuscript ubmitted)
- Tadese, T. and Reed, W.M. Localization and profiling of reticuloendotheliosis virus (REV) protein/s in cell cultures infected with REV integrated fowlpox virus FPV-REV strains. Abstract received to be presented at the American Veterinary Medical Association annual meeting, Nashville, Tennessee, July 13-17, 2002.
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Progress 01/01/00 to 12/31/00
Outputs
Variants of fowl poxvirus (FPV) have been responsible for pox diseases in previously vaccinated commercial flocks. These variants are being isolated in different regions of the U.S. and the molecular nature of these variants is not well understood. Avian poxviruses are known to recombine with other avipoxviruses to generate different strains with a potential to infect previously vaccinated poultry. There is also a possibility that variants of FPV could have evolved as a result of combining with REV, a naturally occurring retrovirus known to cause neoplastic disease conditions in poultry. We have examined 6 field isolates and seven vaccine strains of FPV by enzyme-linked immunoassay (ELISA) and polymerase chain reaction (PCR) technique designed to specifically amplify REV-LTR sequences. The results have revealed that 5 of the field isolates (3 by both PCR and ELISA, and 2 by PCR alone) were positive for REV, but 2 of the vaccine strains were positive by PCR alone.
However, by immunoblotting, 5 of the 6 field isolates and 4 of the 5 vaccine strains showed a 62kD protein known to be specified by REV, suggesting an intimate association between REV and FPV strains. By integration into FPV, REV could alter gene function, enhance pathogenicity to vaccinated poultry. Further work is in progress to map and elucidate the nature of REV integration.
Impacts
This work will lead to the development of more efficacious vaccines to prevent avian pox and will also explain why avian pox infections have increased dramatically in the U.S. in the last several years.
Publications
- Tadese, T. and Reed, W.M. Abstract presented at the American Veterinary Medical Association annual meeting, Salt Lake City, UT, July 22-26, 2000.
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