PATHOLOGICAL STUDIES AND RT-PCR DETECTION OF JAAGSIEKTE SHEEP RETROVIRUS (JSRV) IN SHEEP IN NATURAL CASES OF OVINE PULMONARY ADENOCARCINOMA
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Date
2015-11
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SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502 (A.P) INDIA.
Abstract
ABSTRACT :
Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring
transmissible adenocarcinoma of lungs in sheep caused by an exogenous
Jaagsiekte Sheep Retrovirus (exJSRV). The main constraint in the diagnosis of
OPA is the absence of JSRV-specific antibodies in infected sheep that has
precluded the use of serological tests and greatly hindered the development of
diagnostic assays. A definite diagnosis of OPA in an individual animal is
possible only by identifying the characteristic clinical signs and gross and
histopathological findings during necropsy. Ultrastructural studies of pulmonary
lesions in OPA are also useful for obtaining further information. However, for
final confirmation, molecular techniques are required. Only a few studies were
conducted on the PCR detection of JSRV and its application at field levels in
India. Hence, keeping this in view the present work was planned to study the
gross, histopathological and ultrastructural changes in lungs and to detect the
presence of JSRV by RT-PCR technique in natural cases of OPA in sheep.
The materials for the present study were collected from slaughter houses,
private organized farms and from field mortalities. The duration of the study
lasted from August 2014 to July 2015. A total of 1150 sheep of either sex, aged
between 6 - 60 months were examined in detail and the representative samples
were collected from 127 suspected animals. Of these, 20 were found positive
(15.74%) for OPA based on gross and histopathology and ultrastructural findings
of lungs. The OPA was further confirmed by the detection of JSRV by RT-PCR
technique.
Grossly, the lung samples from 20 OPA cases revealed diffuse areas of
consolidation or tumor nodules that were solid, hard and light gray in colour and
clearly demarcated from the surrounding parenchyma. The OPA lungs failed to
collapse, and were enlarged, edematous and heavier than the normal. On cut
surface, the consolidated areas or tumor nodules had a granular elevated foci and
showed meaty appearance with moist surfaces and usually associated with frothy
fluid in the air ways resembling classical form of OPA. In some cases, lungs also
revealed abscessation with oozing of purulent material on sectioning. Mediastinal
lymph nodes were enlarged and edematous and there were no metastatic lesions.
Histologically, all the lungs revealed almost similar histopathological
changes with slight variation in early and advanced cases. Sections from
consolidated and/or nodular areas 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 types viz. papillary or acinar growth patterns. Papillary pattern was
characterized by the papillary projections protruding above the epithelial layers
into the alveolar spaces which were supported by a thin connective tissue stalk.
The acinar or glandular type of growth pattern was composed of structures
resembling acini and the lining cells of the acini were cuboidal to columnar with
vacuolated cytoplasm and the nucleus was round or oval, vesicular and located
centrally or basally. Bronchioloalveolar growth pattern, characterized by the
expansion of the neoplastic cells following the alveolar septa, without destruction
of alveolar architecture was noticed in some cases.
Hyperplasia of bronchiolar epithelium and papillary ingrowths into the
lumen of the bronchiole were noticed along with lymphoid cell proliferation
around the bronchioles. In advanced cases, thickening of alveolar septa was
noticed due to connective tissue proliferation and cellular infiltration in the
interstitium. Fibrous connective tissue divided the neoplastic areas into lobules.
Myxomatous nodules were also evident in some areas.
Ultrastructurally, lung sections from neoplastic areas revealed three
groups of cells viz., alveolar type II pneumocytes, Clara cells and
undifferentiated cells on transmission electron microscopy. Type II cells showed
numerous cytoplasmic vacuolations, lamellar bodies, microvilli, well developed
rough endoplasmic reticulum (RER), surfactant bodies, intracellular
microtubules and swollen mitochondria. The nuclei of these cells were oval to
round or mostly distorted showing pyknosis and anisokaryosis and often with
deep indentations and the heterochromatin was dispersed in peripheral and
central clumps. At places, the cells lost their distinct cell boundaries and had
loose junctions. In the early lesions, the type II cells were characterized by the
presence of numerous microtubules and mitochondria. In advanced cases, the
type II cells contained excessive glycogen granules and were separated by
collagen fibres. The Clara cells were characterized by very well-developed
smooth endoplasmic reticulum (SER), swollen mitochondria, electron dense
bodies, perinuclear tubular structures and loose junctions in between the cells.
The nuclei were round or having prominent indentations and with irregular
distribution of chromatin. There were some undifferentiated tumor cells that
were devoid of either lamellar bodies or electron dense granules in the affected
alveoli and bronchioles. Immature intracytoplasmic aggregation of viral particles
of 55-70 nm diameter were noticed in the type II cells and Clara cells.
In the present study, the detection of JSRV by RT-PCR was carried out to
diagnose and confirm OPA in sheep. Total RNA was extracted from lung and
lymph node samples of OPA suspected animals and cDNA was synthesized and
amplification of the resultant cDNA was carried out by PCR. JSRV transcripts
were found consistently in all the 20 OPA lung tumor tissues and in mediastinal
lymph node samples.
In the present investigation, the gross, histopathological and ultrastructural
lesions of OPA were studied. RT-PCR was used for the molecular detection of
JSRV in naturally infected OPA sheep. Further, it was evident that OPA is a
contagious tumor caused by JSRV and has a potential to cause economic losses
in sheep.
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Keywords
JAAGSIEKTE SHEEP RETROVIRUS; SHEEP; OVINE PULMONARY ADENOCARCINOMA; RT-PCR