An investigation into the serological and molecular diagnosis of Jaagsiekte Sheep Retrovirus (JSRV)

Abstract

The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards.