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Subject: Veterinary Pathology × Author: SAMATHA, V × Start date: 2010 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    PATHOLOGICAL AND MOLECULAR STUDIES ON JSRV (JAAGSIEKTE SHEEP RETROVIRUS) IN SHEEP AFFECTED WITH OVINE PULMONARY ADENOCARCINOMA IN ANDHRA PRADESH
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) SAMATHA, V; RAMA DEVI, V (MAJOR); SATHEESH, K; SUBRAMANYAM, K.V.; VINOO, R
    Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring transmissible tumor of lungs in sheep and goats caused by an exogenous Jaagsiekte Sheep Retrovirus (exJSRV). A major obstacle in JSRV research is the difficulty to culture the etiological agent in vitro and absence of serological tests for diagnosis. Of late, PCR diagnostic techniques are being used to diagnose OPA at field level. There are reports of OPA from India, but molecular studies on JSRV are lacking. The present work was planned to study the gross and microscopic lesions and to carry out molecular diagnosis along with molecular characterization of JSRV by sequencing U3, gag and Env-TM genes. In addition, exons of 5, 7 and 8 of p53 gene in OPA tumors were sequenced to determine any mutations. The materials for the present study were collected from slaughter houses, private organized farms and from field mortalities. A total of 150 lung samples were collected and of these, 22 samples were found positive for OPA based on gross, histopathology and by molecular diagnosis of JSRV by U3-hnPCR. Grossly, the lung samples from OPA cases revealed diffuse areas of consolidation or tumor nodules and the cut surface had meaty appearance with moist surfaces resembling classical form of OPA. Histologically, sections from consolidated and/or nodular areas of lungs revealed multiple nonencapsulated neoplastic areas of different sizes composed of cuboidal to columnar epithelium lining the alveolar and bronchiolar walls.The neoplastic epithelium was mainly arranged in two typesviz. papillary or acinar growth patterns. In few lungs, bronchioloalveolar growth pattern was noticed. In advanced cases, thickening of alveolar septa was noticed due to connective tissue proliferation and cellular infiltration in the interstitium. Myxomatous nodules were evident in some areas. Immunostaining for JSRV-CA protein was performed on eight OPA lung samples and the positive staining was indicated by the presence of intracytoplasmic fine brown granular staining of the neoplastic cells, alveolar macrophages and desquamated tumor cells. RNA was extracted from the suspected OPA lungs and lymph node tissues and cDNA was prepared and was amplified by U3-hn PCR to detect JSRV transcripts using specific primers. A total of 22 sheep (14.7%) were found positive for OPA out of 150 animals examined. cDNA prepared from 22 OPA lung samples was used for conducting PCR to amplify gag and Env-TM genes of exJSRV. A PCR that was specific for the exons 5,7 and 8 of p53 gene in lung tumor tissues of OPA was performed and an amplified products of 332 bp,210 bp and 220 bp were detected respectively. The PCR amplicons obtained in different reactions were sequenced by Sanger dideoxychain termination method at Bioserve Biotechnologies Pvt. Ltd, Hyderabad. All the sequences were aligned and the deduced sequences were submitted to GenBank. Pair wise divergence and similarity was calculated using MEGA 7. The nucleotide sequence analysis of five JSRV sequences (samples 4-8) from the OPA affected sheep for U3 region of exJSRV showed more similarity (81-98%) with a UK strain (AF105220.1) than (69-73%) with a South African strain (M80216). The phylogenetic analysis of these sequences revealed that they were slightly divergent from other Gannavaram isolates available in the database. The nucleotide sequence analysis of eight JSRV sequences (samples 1-8) from the OPA affected sheep for gag region of exJSRV showed similarity (99-100%) with both enJSRV NTRCVSc_enJSRV 1 to 12) and (100%) a few exJSRV (MG192314, MG192315, MG192316 and DQ838494). The absence of Sca1 site was noticed. The present sequences were similar to exJSRV isolates reported from Delhi and China. The nucleotide sequence analysis of four exJSRVsequences (samples 4-7) from the OPA affected sheep for Env-TM region showed more similarity (94-9%) with UK strain (AF105220.1) and (92-94%) with USA strains (AF357971) than (80-82%) South African strain (M80216). The region of cytoplasmic tail obtained is incomplete in its aminoterminal, but the information revealed conservation of YX part of the YXXM motif of the exJSRV. The nucleotide sequence analysis of p53 gene at exons 5,7 and 8 in OPA tumor samples revealed no mutations that may suggest involvement of other regions of p53 or alternative genes in development of OPA. Based on the pathological and molecular studies carried out on OPA affected lung tissues of sheep, the JSRV sequences obtained were characterized as exogenous JSRV.