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1

PCR-based methods for detection of JSRV in experimentally and naturally infected sheep

100%
Kycko A., Reichert M.
Bulletin of the Veterinary Institute in Puławy
|
2010
|
tom 54
|
nr 4
Application of real-time PCR using Taqman probe was tested for jaagsiekte sheep retrovirus (JSRV) detection. Sensitivity of real-time PCR and hemi-nested PCR methods was compared using plasmid DNA. The methods, along with RT PCR and real-time RT PCR, were tested for the possibility of JSRV genome (LTR region) detection in biological material from experimentally and naturally infected sheep. The experimental group of eight animals was used, including five lambs infected with JSRV by intratracheal inoculation at the age of 2 weeks. The samples collected from the animals ante-mortem included blood and respiratory tract fluid. Lung tissue, mediastinal lymph nodes, spleen, and liver were collected post-mortem. The field studies included blood samples collected from sheep from Polish flocks and lung samples obtained from slaughterhouse. In addition, DNA samples isolated from blood of sheep from the abroad located flocks with history of ovine pulmonary adenomatosis (OPA) were also included. Lung samples were examined histologically for the presence of pulmonary adenocarcinoma. The sensitivity of PCR, hemi-nested PCR, and real-time PCR using Taqman probe was evaluated as 10³, 10², and 10² viral copies, respectively. Both viral RNA and DNA were detected in the lung fluid taken from JSRV infected sheep showing clinical sings of OPA and in all neoplastic tissues. Proviral DNA was found in mediastinal lymph node of one experimental sheep. Five of the 66 DNA samples from the abroad located farms were positive for the presence of JSRV LTR. All blood and lung samples collected from Polish sheep were negative for the presence of JSRV LTR. The characteristic adenocarcinoma lesions were found in all lung sections of experimentally infected sheep. Implementation of the real-time PCR method is a good alternative to traditional PCR and hnPCR in JSRV detection and, apart from histopathological and immunohistochemical examinations, may be used as a confirmatory test in clinically suspected cases, or as a screening method in control or eradication scheme.
2

Two-dimensional electrophoretic analysis of polypeptides expressed by normal and ovine pulmonary adenocarcinoma affected lung tissues

100%
Kycko A., Reichert M.
Bulletin of the Veterinary Institute in Puławy
|
2008
|
tom 52
|
nr 1
Frozen lung tissue sections from 2 healthy and 2 adenocarcinoma affected sheep were lysed in appropriate buffer. The two-dimensional (2D) electrophoresis of the protein lysates was performed. The resulting gels were visualised by silver and Coomassie Blue staining, then scanned and analysed using appropriate software. There was one spot present on the image obtained from the analysis of healthy tissue and no spot was found on cancer 2D gel image. The spot was excised and analysed using mass spectrometry. As a result, cytosolic NADP-isocitrate dehydrogenase was identified. In addition, several other protein spots of different intensity in neoplastic tissues, as compared with healthy ones, were found. The last finding reflects changes in protein expression in neoplastic and healthy tissues. These preliminary results can serve as the basis for more detailed investigations of the neoplastic tissue proteome, e.g.: isoelectric focusing in narrow pH range and analysis of correlation between tissue and serum protein profiles. The analysis of serum proteins from affected sheep can reveal markers of neoplastic process and help in preclinical diagnosis of ovine pulmonary adenocarcinoma.
3

Detection of jaagsiekte sheep retrovirus in respiratory tract fluid and lung tissue of experimentally infected lambs

80%
Kycko A., Jasik A., Reichert M.
Bulletin of the Veterinary Institute in Puławy
|
2008
|
tom 52
|
nr 1
The possibility of jaagsiekte sheep retrovirus (JSRV) genome detection in peripheral blood leukocytes and respiratory tract fluid of ovine pulmonary adenocarcinoma (OPA) affected sheep was tested. Five of six lambs used in the experiment were infected with JSRV by intratracheal inoculation. The blood samples were taken from all the lambs at 10-d intervals for 3 months and the white blood cells were subjected to DNA isolation followed by PCR amplification for the detection of proviral DNA that gave negative results for all the samples. The respiratory tract fluid was collected from the nostrils of lamb No. 6, which developed clinical sign of OPA 2 months after the inoculation. The fluid was examined using PCR and reverse transcriptase PCR (RT-PCR) for the presence of proviral DNA or viral RNA. The presence of OPA in the lamb was subsequently confirmed by histopathological examination and the detection of proviral DNA in the lung tissue. Results of the standard PCR amplification performed on the DNA isolated from the nasal discharge from lamb No. 6 was negative, while the RT-PCR gave positive results confirming the presence of virions in the lung fluid. Results of the study show the RT-PCR technique may be a useful tool in ante-mortem diagnosis of OPA, especially in distinguishing it from other respiratory diseases.
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