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20153
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Subject: Veterinary Pathology × End date: 2015 × Start date: 2015 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    PATHOLOGICAL STUDIES AND RT-PCR DETECTION OF JAAGSIEKTE SHEEP RETROVIRUS (JSRV) IN SHEEP IN NATURAL CASES OF OVINE PULMONARY ADENOCARCINOMA
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P) INDIA, 2015-11) NANDA KISHORE, K V S; RAMA DEVI, V(MAJOR); SATHEESH, K; SATHEESH, K
    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 ymph 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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    ThesisItemOpen Access
    PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF SHEEP POX
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P) INDIA, 2015-08) SAI ASWINI, BONTA; SATHEESH, K; RAMADEVI, V; SUBRAMANYAM, K V
    ABSTRACT: Sheep farming is the major source of livelihood for landless poor in India. Pox infection in sheep causes a huge economic losses in the Indian subcontinent in terms of poor hide quality, decreased productivity, abortions and even mortality in lambs. Hence, the present study was undertaken to describe the gross and histopathological changes in affected organs and to study the ultrastructural changes in skin and lung and also to confirm the disease by PCR. Sheep pox is caused by SPPV, a member of the genus Capripoxvirus and family Pox viridae. In the present study a total of 1240 sheep (Nellore Dora breed) of both sex and different age groups from various slaughter houses and field were screened for clinical signs/ lesions of sheep pox and of these, 77 animals were found affected (including both ailing and dead) with an overall incidence of 6.2%. Among 77 affected sheep, detailed necropsy was carried out in 16 animals and the representative tissue samples were collected for further studies. A few skin/scab samples from ailing animals were also collected at random for PCR studies. Clinically, the ailing animals showed rise of body temperature, occulonasal discharges and characteristic cutaneous pox eruptions at different stages of development. The symptoms were more severe in young lambs. At necropsy, gross lesions of sheep pox were predominantly seen on the skin and lungs followed by other internal organs. The skin lesions were characterized by papules, nodules and scabs mostly on wool-less parts of the body. The papules were greyish white, circular, hard that coalesced to form white and firm nodules with ulcerated centres. Some papules were encrusted to form the scab. The affected lungs were edematous and congested with irregular areas of consolidation besides rib impressions on surface. A few macules and numerous papules/nodules were noticed along with a very few pustules. Round and white papules were seen on the tongue, rumen, and reticulum. Liver, kidney, heart and spleen showed small, white and circular necrotic areas. Lymph nodes were enlarged and edematous. In the present study, microscopic lesions were noticed in the skin, lungs and in other internal organs like tongue, rumen, reticulum, liver, kidney, heart and spleen. Sections of skin showed hyperkeratosis, acanthosis, parakeratosis, hydropic changes with microvesicle formation and presence of eosinophilic intracytoplasmic inclusion bodies in epidermal layer. In the dermis folliculitis, vasculitis, presence of SPC’s and infiltration of mononuclear cells and fibroblasts were seen. In the lungs, congestion, focal areas of necrosis, proliferative alveolitis, foetalization, thickened interalveolar septa with infiltration of mononuclear cells were seen. The alveolar lumen revealed eosinophilic amorphous material and presence of sheep pox cells with eosinophilic intracytoplasmic inclusion bodies. Proliferative bronchiolitis and peribronchiolar lymphoid aggregations were the other changes observed in affected sheep. The sections of tongue revealed congestion, ulcerations and formation of microvesicles. In the rumen, hyperkeratization, necrosis of squamous epithelial cells and presence of characteristic cytoplasmic inclusions in the prickle cells were seen. Reticulum revealed necrosis, sloughing of squamous epithelium with presence of eosinophilic intracytoplasmic inclusion bodies. Sections of liver showed sinusoidal dilatation, presence of inclusion bodies in few hepatocytes and formation of new bile ductules. The kidney sections showed interstitial nephritis, presence of hyaline casts, eosinophilic intracytoplasmic inclusion bodies in the tubular epithelium and necrotic foci. Sections of heart revealed haemorrhages and infiltration of lymphocytes in between the muscle fibers. Depletion of lymphocytes in the lymphoid follicles was seen in the spleen and lymph nodes of pox affected sheep. In the present study, the ultrastructural changes were studied in the skin and lung tissue samples by TEM. The skin revealed hydropic changes and presence of numerous electron dense particles suggestive of immature virions. In the lung, proliferation of type II pneumocytes, presence of the immature virus particles in affected alveolar epithelial cells and macrophages, thickened interalveolar septa along with nuclear and chromatin changes were seen. The virus suspension from skin papule/ nodule was passaged thrice in embryonated eggs through CAM route that revealed death of embryo as early as 48 hours PI. Grossly, congestion, edema, hemorrhages and necrosis and microscopically congestion were noticed in the CAM. In the present study, molecular diagnosis of pox by PCR was carried out with skin and lung samples from affected sheep using B68 and B69 primers specific for P32 gene of Capripoxvirus that yielded an amplified product of 390 bp, confirming the Capripoxvirus infection in the samples tested. The present study revealed the gross, histopathological and ultrastructural lesions in different organs in natural cases of sheep pox. Further, the disease was confirmed based on the pathological and molecular diagnosis of Capripox infection in the affected sheep in the study area.
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    ThesisItemOpen Access
    PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF GOATPOX
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P) INDIA, 2015-08) JAYASRI, K; ANNAPURNA, P(MAJOR); RAMA DEVI, V; LAKSHMI KAVITHA, K
    ABSTRACT: Goat farming has an important role in improving the socio-economic conditions of rural poor in India. Goat pox is a highly contagious disease of goats caused by members of the genus Capripoxvirus. In the present study, goat pox was diagnosed in goats based on the clinical, gross, histopathological and ultrastructural examinations and was further confirmed by molecular diagnosis. The samples were collected from field, slaughter houses and animals necropsied at Department of Veterinary Pathology, NTR College of Veterinary Science, Gannavaram. A total of 1060 goats were screened for clinical signs / lesions of goat pox and of these, 119 animals including both ailing and dead animals were found affected, with an overall incidence of 11.22%. Among the 119 affected goats, detailed necropsy was carried out in 23 animals and the representative tissue samples were collected for further studies. A few skin/scab samples from ailing animals were also collected at random for PCR studies. The clinical signs noticed in the ailing animals were papular and nodular lesions of pox on the body, rise in body temperature, occulo-nasal discharges, coughing and dyspnoea that were more severe in the young animals.In the necropsied animals, pox lesions were found consistently in the skin and lungs of goats with varied involvement of other systems/ organs. Grossly, skin revealed papules, nodules and scabs. Papules were predominant, grayish white, hard, firm and circular that varied from 0.5 to 3 cm in diameter. Lungs revealed macules and papules of which the papular stage was predominant. Macules were red, circular areas that revealed congestion on section. Papules appeared circular, grayish white, firm and measured 0.5 to 1.5 cm in diameter that also showed necrotic centers and nodule formation in some cases. A single papule was noticed in the tracheal mucosa in one case. In the digestive system, papules were noticed on the tongue, dental pad, esophagus, rumen, reticulum and abomasum in a few animals. Circular, pale foci of varied sizes on the liver and kidney and edema of bronchial and mesenteric lymph nodes were also observed in some of the necropsied animals. In the present study, microscopic lesions were noticed consistently in the skin and lungs of goat pox affected animals. Other internal organs affected were trachea, tongue, dental pad, esophagus, rumen, reticulum, abomasum, liver,kidney and lymph nodes. In the skin, hyperkeratosis and parakeratosis in the keratin layer, vacuolation, ballooning degeneration, necrosis, microvesiculation and intracytoplasmic, eosinophilic inclusions in the prickle cell layer were noticed. Dermis showed a few distinct cells with vacuolated nuclei and marginated chromatin that resembled sheep pox cells. In the lungs, the predominant lesion was hyperplasia and hypertrophy of type II pneumocytes and characteristic inclusions were observed within the alveolar epithelial cells. The lumen of the alveoli contained fibrinous exudates and large cells similar to sheep pox cells. Bronchial and bronchiolar mucosa revealed hyperplasia and hypertrophy along with the presence of characteristic inclusions within the epithelial cells. Tracheal mucosa revealed ulceration with loss of cilia in the epithelium. In the digestive system, characteristic inclusions were observed in the epithelium of dental pad, rumen and reticulum. The epithelium of tongue showed microvesicles and hyperkeratosis was observed in the rumen. In addition, reticulum revealed acanthosis and abomasum showed vacuolar degeneration. Liver showed severe congestion and mononuclear cell infiltration in the portal areas. The predominant lesion in the kidney was interstitial nephritis. Vacuolar degeneration of tubular epithelium and congestion of glomeruli were also noticed. Bronchial and mesenteric lymph nodes revealed mild depletion of lymphoid cells. In the present investigation, ultrastructural changes were studied in the skin and lung tissue samples of affected goats by TEM. Skin sections showed loss of normal integrity of cells and fibrosis between the epithelial cells. Numerous VLP’s in keratinocytes and immature virions in the cytoplasm of prickle cells along with clumping of chromatin in the nucleus were observed. Lung sections revealed hyperplasia of type II pneumocytes that contained immature or mature virions. The alveolar epithelial cells showed hyperchromatic nucleus, vacuolation of cytoplasm and dilated cisternae of rough endoplamic reticulum.Inoculation of embryonated chicken eggs with the proviral material thrice resulted in embryo mortality as early as 48 hours PI. Grossly, thickened and edematous CAM was noticed. In addition, hemorrhages during 2nd passage and pox like lesions and necrosis during third passage were also found. Microscopically, sections of CAM revealed vacuolation of ectoderm consistently. Hemorrhages in the 2nd passage and necrosis along with a few, intracytoplasmic, eosinophilic inclusions within the ectodermal cells were also evident in the 3rd passage. Molecular diagnosis of pox by PCR upto genus level was carried out in skin and lung tissue samples of necropsied animals and in a few skin/scab samples from ailing animals using B68 and B69 primers specific for P32 gene of Capripoxvirus. An amplified product of 390 bp confirmed the presence of capripox viral DNA in the samples. In the present investigation, the clinical signs and gross, histopathological and ultrastructural lesions in various organs of goats in natural cases of goat pox were described and based on pathological and molecular diagnosis, goat pox was confirmed in the affected animals in the area under study.