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ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF CHICKEN INFECTIOUS ANEMIA IN COMMERCIAL CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY, TIRUPATI - 517 502. (A.P.) INDIA, 2024-02) MOUNIKA PENUBAKULA; SAMATHA .V (MAJOR); SUDHA RANI CHOWDARY .CH; SRIVIDYA .GIn the present study, a total of 217 commercial chicken among 50 chicken flocks were suspected for CIA based on history and clinical signs. CIA was diagnosed in 135 birds including 116 layers (71.6%) and 19 broilers (34.5%) with an overall prevalence of 62.2% based on hematology, gross lesions, histopathology, electron microscopy and molecular studies. Out of 116 CIA cases in layers the highest prevalence was recorded in East Godavari (90.47%), followed by Chittoor (81.81%), Kakinada (80%) and lowest in Eluru (52.38%). The prevalence of CIA in broilers was found highest in East Godavari (39.1%) followed by Chittoor (38.88%) with lowest prevalence in Visakhapatnam (21.42%). In the present study, age wise prevalence of CIA in commercial chicken showed highest prevalence in layer growers of 5-16 weeks of age (79.8%) and the lowest prevalence in layer chicks aged ≤4 weeks age (27.2%). Concurrent infections with other bacterial (36.3%), viral (24.2%), protozoal (28.7%) infections were noticed with overall occurrence of 48.88%. Concurrent infections were confirmed in layer growers (55.17%), layer adults (42.30%) followed by broiler chicken (42.1%) and layer chick (33.33%). The commercial chicken affected with chicken infectious anemia exhibited pale comb and wattles, anorexia, generalised weakness, decline in egg production and ruffled feathers. Blood samples collected randomly from the birds suspected for CIA grossly appeared as watery contents and there was a significant decline in the Hemoglobin concentration (5.56 ± 0.29 g/dl), PCV (17 ± 0.79 %) and TEC (1.46 ± 0.09 x 106/µL) in CIA affected flock when compared to that of healthy flocks. Grossly, CIA affected chicken exhibited small, shrunken thymus and bursa. Pale pink to yellow discoloration of bone marrow of femur along with oily jelly to watery like consistency was observed. Atrophy of spleen was noticed in many cases whereas enlarged pale livers and swollen pale kidneys were observed in few cases. Petechiae to patchy hemorrhages on thigh muscle, breast muscles and in the oral cavity were also noticed. Cytologically, bone marrow collected from affected chicken revealed presence of vacuolar spaces due to replacement of hematopoietic tissue by adipose tissue. Thymus sections revealed multiple hemorrhages along with moderate to severe depletion of lymphocytes in cortex and medullary areas and apoptotic changes in nucleus like pyknosis and karyorrhexis in thymic lymphocytes whereas few lymphocytes exhibited the presence of eosinophilic, intranuclear inclusion bodies. All the sections from bone marrow of femur revealed aplasia of all cell types including granulocytic, agranulocytic and hemopoietic precursors along with complete replacement of bone marrow by adipose tissue in the form of vacuolar spaces. Hemocytoblast cells with more cytoplasm and large nuclei revealed eosinophilic, intranuclear inclusions. Bursa of Fabricius showed moderate atrophy of lymphoid follicles, cystic changes in bursal follicles. Spleen revealed moderate to severe lymphoid depletion in white pulp along with proliferation of macrophages, reticular cells and hemosiderosis. Severe congestion of blood vessels, multiple hemorrhagic foci and degenerative changes were noticed in tissue sections of liver and kidneys. In the present study, different concurrent infections were noticed in 66 CIA affected commercial chicken. Marek’s disease was confirmed in a total of 16 cases revealing neoplastic nodules on liver and heart. Tissue imprints revealed numerous pleomorphic cells and histologically, infiltration of neoplastic lymphoid cells in hepatic parenchyma and myocardium was noticed. Different bacterial diseases included fibrinopurulent serositis (12), gangrenous dermatitis (7) and Fowl Cholera (5). Protozoal infections like intestinal and caecal coccidiosis (19) were also recorded. Fibrinous pericarditis revealed fibrinous exudation along with inflammatory cell infiltration in pericardium and samples collected from these lesions yielded characteristic green metallic sheen colonies on EMB agar confirming E.coli. Gangrenous dermatitis appeared as dark, moist lesions at the base of the wing indicating blue wing. Necrotic and inflammatory changes were evident in subcutaneous tissue and lesions yielded Gram positive cocci suggestive of Staphylococcal infection. Multiple hemorrhagic foci on epicardium and numerous necrotic foci on liver were noticed in Fowl cholera. Microscopically, hemorrhages and necrotic changes were observed in heart and liver. Tissue imprints of heart revealed presence of bipolar organisms and swabs collected revealed non hemolytic colonies on blood agar suggestive of Pasteurellosis. Distended intestinal segments and caeca along with hemorrhagic and necrotic contents were suspected for coccidiosis. Intestinal and caecal mucosal scrapings revealed presence of different stages of coccidia suggestive of Eimeria Spp. Severe hemorrhages and necrosis in mucosa and submucosa along with presence of different stages of coccidia was evident. Five thymus and three bone marrow samples collected from CIA affected birds were subjected to TEM and revealed severe scarcity of cells and presence of apoptotic changes in the nuclei and intranuclear viral inclusions. Apoptotic thymocytes revealed chromatin aggregation as large, dark condensed masses that abut the nuclear membrane. Bone marrow samples revealed large adipose cells replacing the bone marrow whereas few electron dense viral aggregates in the nucleus of hemocytoblast are also observed. In the present study, molecular diagnosis of CIA disease in commercial chicken was carried out using thymus, bone marrow, spleen and liver tissue samples for amplification of VP1, VP2 and VP3 genes of Chicken infectious anemia virus by specific primers and yielded amplicons of size 1390 bp for VP1 gene, 713 bp and 367 bp for VP2 and VP3 genes respectively confirming the presence of CIA viral DNA in the samples. In conclusion, the present study has defined characteristic gross and microscopic features of CIA in commercial chicken and diagnosis was done based on PCR by amplification of VP1, VP2 and VP3 genes of CIAV.ThesisItem Open Access CYTOLOGICAL, PATHOMORPHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES ON CANINE CUTANEOUS NEOPLASMS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-04) SIRISHA KAVETI; SUDHA RANI CHOWDARY .CH (MAJOR); SAMATHA .V; SRINIVASA RAO .TThe present study was undertaken to know the occurrence of canine cutaneous neoplasms, to classify and study the gross, cytology, histopathology and AgNOR count and to evaluate different tumor markers by IHC. In the present study, 62 canine cutaneous neoplasms were histologically classified into 19 types, of which epithelial neoplasms (59.7%) had the highest occurrence followed by mesenchymal (17.7%), round cell and melanocytic neoplasms (11.3% each). The occurrence of malignant tumors (64.5%) was higher compared to benign tumors (35.5%). In the present study, dogs affected with cutaneous neoplasms ranged from 1 to 15 years of age with highest occurrence in 7 to 9 years age group. Males (71%) were more affected than females (29%). The highest occurrence of canine cutaneous neoplasms was recorded in Non - descript dogs followed by Labrador retriever and Spitz. With respect to location, the occurrence was highest in head (37.1%) followed by extremities (32.3%), trunk (19.4%) and neck (11.3%) regions. Grossly, the tumors were 1 to 10 cm in diameter, round to oval, dome, irregular and cauliflower shaped with soft to firm consistency. Cytology of benign tumors revealed uniform to mild pleomorphic cells, low nuclear to cytoplasmic ratio and condensed chromatin, while, cytology of malignant tumors revealed high cellularity, moderate to severe pleomorphism, anisocytosis, anisokaryosis, high nuclear to cytoplasmic ratio, single to multiple nuclei and nucleoli and emperipolesis. The different histologic types of canine cutaneous neoplasms recorded in the present study were papilloma (4.8%), basal cell carcinoma (12.9%), squamous cell carcinoma (4.8%), basosquamous cell carcinoma (3.2%), trichoblastoma (6.5%), sebaceous adenoma and sebaceous epithelioma (3.2% each), sebaceous carcinoma (8.1%), hepatoid gland adenoma (6.5%), hepatoid gland carcinoma and apocrine gland carcinoma (3.2% each), lipoma (3.2%), fibroma (1.6%), fibrosarcoma (8.1%), haemangiopericytoma (1.6%), haemangioma (3.2%), plasma cell tumor (3.2%), mast cell tumor (8.1%) and malignant melanoma (11.3%). Of all the histologic types, basal cell carcinoma had the highest occurrence. The proliferative cellular activity of canine cutaneous neoplasms was demonstrated by AgNOR staining. The mean AgNOR count ranged from 1.68 to 3.08 and malignant tumors had higher AgNOR count compared to benign tumors. Twenty three canine cutaneous tumors were subjected to immunostaining of different tumor markers viz. cytokeratin, vimentin, S100 and CD117. Basal cell carcinoma, squamous cell carcinoma, sebaceous carcinoma, hepatoid gland carcinoma and apocrine gland carcinoma were positive to cytokeratin, fibrosarcoma and haemangiopericytoma were positive to vimentin, mast cell tumors were positive to CD117 and malignant melanomas were positive to S100 and vimentin. The present study has provided certain insights on the occurrence of canine cutaneous neoplasms in this geographical region of Andhra Pradesh. It has also put on record the cytological, pathomorphological and immunohistochemical findings of different canine cutaneous neoplasms.ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF FOWL POX IN BACKYARD CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2023-03) MOLIKA KOTTAPU; SAMATHA .V (MAJOR); SUDHARANI CHOWDHARY .CH; JYOTHI SREE .CHIn the present study, fowl pox was diagnosed in 119 birds out of 128 suspected backyard chicken with an overall occurrence of 92.96% based on gross lesions, histopathology, ultrastructural and molecular studies. Fowl pox in backyard chicken was recorded in eight coastal districts of Andhra Pradesh and the highest occurrence of fowl pox was found in Krishna district (100%), followed by West Godavari (96.8%), Bapatla (93.75%), Konaseema (92.3%), Kakinada (90%), Prakasam (88.8% ), Anakapalli (75%) and Visakhapatnam (66.6%). The highest occurrence of fowl pox in the present study was in the winter season (47.9%), followed by summer (29.4%), and rainy seasons (22.7%). Occurrence of cutaneous form of fowl pox in backyard chicken was mostly seen in chicks of ≤ 9 weeks of age (97.10%), followed by adults aged > 28 weeks (92.30%) and growers aged 9-28 weeks (80.0%) whereas diphtheritic form of fowl pox was mostly noticed in chicks followed by growers and mortality rate was also highest in chicks when compared to others. Both cutaneous (85.71%) and diphtheritic forms (14.28%), of fowl pox was recorded in the present study. Among 102 cases of cutaneous form, highest occurrence of pock lesions were observed at the eye lids (33.4%), followed by beak (25.5%), corner of the mouth (11.77%), shank region (8.824%), feet (6.87%), comb and wattles (4.95%), at the wings (3.93%), cloacal aperture (2.95%) and ventral aspect of abdomen (1.97%). The highest occurrence of diphtheritic form was observed on mucous membrane of the laryngeal orifice (29.42%), trachea (29.42%), oral cavity (23.53%) and oesophageal tract (17.65%) in a total of 17 cases. Backyard chicken affected with fowl pox exhibited clinical signs like stunted growth, emaciation and decline in egg production in laying hens. Birds affected with cutaneous form of fowl pox revealed alopecia, ocular discharges, closure of eyes, blepharitis and blindness. Respiratory signs like nasal discharges, gasping and other clinical signs like swelling of infraorbital sinuses swelling of head, difficulty in swallowing and asphyxia were also noticed. Grossly, cutaneous form of fowl pox revealed pock lesions ranging from initial small erosions to large developed nodular, proliferative wart like projections on eyelids, beak, corner of mouth, comb, wattles, wings, shank region and feet, whereas nodular to purulent, cheesy patches at laryngeal orifice, trachea, oral cavity and oesophageal tract were noticed in diphtheritic form. In the present study, pock lesions from both cutaneous and diphtheritic forms of fowl pox cases revealed characteristic histopathological features. Majority of tissue sections from cutaneous form of fowl pox (25.5%) revealed severe congestion of blood vessels and haemorrhages in epidermis and dermis, ballooning degeneration of epidermal cells, hyperplasia of epidermis (acanthosis) in focal areas along with presence of medium to large singular or multiple intracytoplasmic eosinophilic inclusion bodies (Bollinger bodies). Degenerative changes and necrosis of the cells of uropygial glands and feather follicles were also seen in addition to numerous Bollinger bodies in 56 (54.9%) cases whereas liquefactive necrosis of epidermal cells, microabscess development and severe infiltration of heterophils was noticed in 18 (17.64%) cases. Proliferative wart like lesions (1.96%) exhibited finger like projections of epithelium due to severe hyperplasia and connective tissue proliferation. Superficial mineralised and necrotic layers were observed over the hyperplastic epidermal cells in large firm crusted nodules whereas increased fibrous connective tissue, increased collagen was noticed in epidermis and dermis in healing lesions. Microscopically, all the diphtheritic pock lesions revealed characteristic changes like severe congestion of blood vessels, haemorrhages and degeneration of epithelium of mucous membranes of oral cavity, tongue, oesophagus, trachea and laryngeal orifice along with presence of small to medium solid or ring shaped Bollinger bodies. Severe necrosis of mucosal cells was evident and was covered by haemorrhagic, fibrinoheterophilic exudate in purulent pock lesions. Diffuse loss of cilia from lining epithelium and disorientation of ciliated epithelium in focal areas in addition to proliferation of epithelial cells in mucosa of trachea was noticed. Demonstration of characteristic intracytoplasmic inclusion bodies was performed by Lendrum’s phloxine tartrazine stain that revealed both solid and lipid associated ring shaped inclusion bodies in skin and oesophageal sections. Four cutaneous pock samples collected from fowl pox affected birds were subjected to transmission electron microscopy and all the sections revealed medium to large intracytoplasmic inclusions in epidermal cells. A few affected cells revealed filaments and tonofibrils arranged as loose bundles in cytoplasm whereas various stages of viral particles were observed in cytoplasm of epithelial cells in all the sections. Majority of cells revealed degenerative changes in mitochondria like swelling of the organelle, partial loss of cristae and coalesced mitochondria forming large mitochondrion along with incorporated incomplete virions as an earliest evidence of inclusion body formation. Immature viral particles contained eccentric nucleoid surrounded by halo whereas mature viral particles contained typical inner dumbbell shaped core and two lateral bodies. Characteristic large inclusions contained more virions at the periphery than in central zone. Shedding of extra coats of the mature virions as the broken segments of inner membranes of inclusions resulted in rodlets. In the present study, inoculation of 10 day old embryonated chicken eggs with skin pock suspension resulted in death of embryo between 5 to 7 days of post inoculation. Grossly, the CAM showed severe congestion, haemorrhages, edema and necrosis in first, second and third passages respectively. Microscopically, congestion of blood vessels in ectoderm, edema in mesoderm and haemorrhages in mesoderm was observed on the CAM in the first, second and third passages respectively. Molecular diagnosis of fowl pox disease in skin, oesophagus, trachea and virus propagated CAM tissue samples by using primers specific for P4b gene of fowl pox virus obtained a desired amplicon of 578bp. Nucleotide sequencing and phylogenetic analysis of four cutaneous fowl pox samples revealed that they were in close relation with the FPV isolates from Singapore, North America and USA (KY464130, MH175285 and KC017961 with an identity of 97.28%, 97.21% and 97.16% respectively) when compared to FPVs isolated from Portugal, Brazil, Jammu and Kashmir and India (KM974727, KX863706, MF496042 and HM481406 respectively with an identity of 96.66% to 96.86%). In the present study fowl pox was diagnosed based on the gross, histopathology, electron microscopy and molecular studies in backyard chicken with an overall occurrence as 92.96%.ThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON ENTERITIS IN SLAUGHTERED SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-07) MONIKA, K; SUDHA RANI CHOWDARY, CH(MAJOR); RAMA DEVI, V; SREEDEVI, CIn the present study, a total of 395 sheep from slaughter houses in and around Vijayawada and field mortalities were screened for enteritis. Of these, enteritis was recorded in 110 sheep with an occurrence of 27.84%. Based on gross and histopathological examination, enteritis was classified into catarrhal (20.9%), haemorrhagic (7.3%,), necrotic (9.1%) and parasitic enteritis (62.7%). The etiological agents identified were parasites viz. immature amphistomes, Schistosoma spp., Moniezia expansa, Moniezia benedeni, Stilesia globipunctata, Trichuris ovis, Oesophagostomum spp. and Eimeria spp., bacteria viz. E. coli, Salmonella spp., and Clostridium perfringens and a virus, PPRV. Catarrhal enteritis was of acute and chronic types characterized by presence of catarrhal contents in the lumen of intestines, goblet cell hyperplasia and infiltration of neutrophils and lymphocytes in the lamina propria in acute type, and mononuclear cells in chronic type. Haemorrhagic enteritis was characterized by presence of blood mixed contents, severe mucosal congestion and haemorrhage, coagulative necrosis, villus atrophy and fusion, infiltration of lamina propria with a large number of mononuclear cells and neutrophils, and presence of Gram positive bacteria. Necrotic enteritis was characterized by severe coagulative necrosis of the intestinal mucosa, fusion and atrophy of villi, lacteal dilation, severely hyperplastic to necrotic and cystic crypts, infiltration of the lamina propria with a large number of mononuclear cells and presence of Gram positive bacteria. Majority of the cases of necrotic enteritis were associated with PPR virus infection which showed severe congestion to petechial haemorrhages along the mucosal folds of ileo-caecal junction and rectum giving a zebra striped appearance, syncytial cells in the intestinal mucosa and lymphocytolysis in the Peyer’s patches. In the present study, parasitic enteritis included immature amphistomiasis, schistosomosis, monieziosis, stilesiosis, trichuriosis, oesophagostomosis and coccidiosis. Grossly, in cases of immature amphistomiasis, monieziosis, stilesiosis, and trichuriosis, the parasites were observed in the lumen of intestines, while, schistosomosis, oesophagostomosis and coccidiosis were diagnosed on microscopic examination of the intestines. Nodular lesions in the intestines were observed grossly in schistosomosis, stilesiosis and oesophagostomosis which on histopathology were found to be egg granulomas, hyperplastic villi and pseudotubercular granulomas respectively. In cases of coccidiosis, small greyish white nodules were observed in the intestines which microscopically were found to be hyperplastic crypts and villi harbouring different stages of Eimeria spp. In the present study, 73 samples of intestines were subjected to bacteriological studies. E. coli was the major bacteria isolated from cases of catarrhal enteritis, immature amphistomiasis, monieziosis, stilesiosis, trichuriosis and coccidiosis. Clostridium perfringens was isolated from cases of haemorrhagic enteritis, necrotic enteritis, immature amphistomiasis, monieziosis and coccidiosis, while, Salmonella spp. was isolated only from cases of necrotic enteritis. In the present study, intestines and mesenteric lymph node samples of PPR suspected animals were subjected to one step RT-PCR using a set of primers targeting the N gene of PPRV and yielded a product of 350 bp fragment of N gene in seven samples confirming the presence of PPR virus. In conclusion, the results of the present study revealed occurrence of different types of enteritis, their pathomorphologies and associated etiological agents.ThesisItem Open Access PATHOMORPHOLOGICAL AND MOLECULAR STUDIES OF CANINE TRANSMISSIBLE VENEREAL TUMOR(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-06) YASHASWI, YALAVARTHI; SAMATHA, V(MAJOR); RAMA DEVI, V; RAVI KUMAR, PIn the present study, a total of 72 dogs were suspected for canine TVT based on history and clinical signs. Samples from all the suspected cases were collected from the Veterinary Clinical Complex, NTR College of Veterinary Science, Gannavaram, Veterinary polyclinics, private pet clinics and animal birth control centres present in the coastal districts of Andhra Pradesh. Canine TVT was diagnosed in 68 dogs based on cytology, histopathology, histochemistry, IHC, transmission electron microscopy and molecular studies with an overall occurrence of 94.4%. Canine TVT in dogs was recorded in six coastal districts of Andhra Pradesh and the highest occurrence of TVT was found in Guntur district (100%), followed by Krishna (95.4%), East Godavari (91.6%), West Godavari (90.9%), Visakhapatnam (90%) and Prakasam district (87.5%). The occurrence of canine TVT in the present study was highest in the winter (45.6%), followed by the rainy (29.4%) and summer seasons (25%). High occurrence of TVT was seen in dogs aged between 2-4 years (72.1%), dogs aged 4-6 years (16.2%), 6-8 years (8.8%) and >8 years (2.9%). The occurrence of canine TVT was notably higher in female dogs (83.8%) when compared to that of males (16.2%). In the present study, canine TVT was recorded mostly in non-descript dogs when compared to other breeds. The occurrence of tumor location in 57 female dogs was recorded and was highest at the vestibulo-vulval junction (47.4%), vulval lips (19.3%), vulval mucous membrane (17.5%), posterior vagina (15.8%) and six cases revealed tumors on extragenital sites like skin at perineal region in addition to genital tumors. In 11 male dogs, the occurrence of venereal tumor growth was highest at the base of the penis (36.4%), prepuce (36.4%) and on the tip of penis (27.2%). One case showed tumor at extra genital site on the skin in inguinal area in addition to the genital tumor. Most of the TVT suspected dogs showed clinical signs like bloody discharges from genital tracts in both sexes, excessive licking of genitalia, deformation of external genitalia and perineal region due to large tumors. Grossly, TVTs appeared as pink fleshy or dark firm tumors. Tumors on genitalia of both sexes at initial stages appeared as small hyperaemic papules and the progressing tumors appeared nodular, papillary or multilobulated as well as caulifower-like or pedunculated tumors. The predominant cytomorphological pattern of TVT in the present study is the plasmacytic type, followed by the lymphocytic and mixed types. Cytological examination of 68 cases out of 72 suspected dogs revealed canine TVT cells. Canine TVTs were classified into three cytomorphological sub types: plasmacytic (53%), lymphocytic (28%) and mixed (19%), based on the type of tumor cells. Plasmacytic type tumor cells were large ovoid cells with abundant cytoplasm and several clear cytoplasmic vacuoles at cell margins. The cartwheel-shaped nucleus was eccentrically placed and showed one or more prominent nucleoli in the majority of cells. Lymphocytic tumors cells were round cells with scanty cytoplasm. A high nuclear to cytoplasm ratio was evident in the majority of cells. A few cells revealed cytoplasmic and nuclear vacuolation. The nucleus was round and hyperchromatic with coarse chromatin and prominent nucleoli. Mixed type of tumor cells revealed both lymphocytic and plasmacytic tumor cells. Binucleated cells, mitotic figures, neutrophils, erythrocytes and macrophages were observed in many smears. The cytological smears collected from extragenital TVTs in the cutaneous region revealed polymorphonuclear cells and keratinized squamous cells along with TVT cells. In the present study, 68 tissue samples from genital tumors revealed characteristic histopathological features of TVT. The tissue sections revealed a higher number of neoplastic plasma cells and lymphocytes. Histologically, TVT sections were categorised into progressive (35), steady (12) and regressive stages (21). Progressing tumors contained densely packed tumor cells with scanty connective tissue stroma and a significantly higher number of mast cells, mitotic figures and microvessels at the invasion of the tumor. Mild proliferation of fibroblastic cells along with loosely packed round tumor cells were observed in the steady stage of the tumor. The tumors in the regression phase had decreased round tumor cells, decreased mitosis, increased infiltration of TILs and apoptotic cells. Increased proliferation of fibroblasts and increased collagen was evident. Tissue sections (7) from the extra genital tumor growth on the skin had numerous round neoplastic cells in the dermis. The proliferative cellular activity in TVT cells was demonstrated by AgNOR staining in 68 TVT sections. The mean values of AgNOR count were high in progressing tumors (> 7 AgNOR dots) and low in steady (3-7 AgNOR dots) and regression tumors (< 3 AgNOR dots). Immunohistochemical studies of TVT for PCNA and vimentin were carried out. Proliferating cell nuclear antigen (PCNA) showed strong to moderate expression in progressive tumors (61.8%) and in regressing tumors (38.2%) respectively. Vimentin is a specific marker of mesenchymal derivation and immunoreactivity to vimentin was intense in the cytoplasm of more than 90% of the tumor cells. Four TVT positive tissues were subjected to transmission electron microscopy and two revealed plasmacytic, one lymphocytic and one mixed type. Loosely distributed tumor cells and closely packed tumor cells with small protoplasmic microvilli and extensive interdigitation with adjacent cells were observed. Neoplastic lymphocytes and plasma cells were observed and the lymphocytes were round cells with central oval to irregularly round nucleus whereas, plasmacytes were large oval shaped cells with vacuolated cytoplasm and prominent RER with numerous ribosomal clusters. Initial phases of tumor growth revealed elongated cells with elongated nuclei and presence of neovascularization. The presence of more nucleoli and big perichromatin granules were observed, indicating mitotic figures in progressing tumor sections. Regressing tumors revealed collagen between the tumor cells, fibroblast like cells, tissue infiltrating leucocytes and apoptotic cells. Genomic amplification of 22 TVT tissue samples for demonstration of rearrangement of the c-myc gene segment using specific MycS-2 and LINE AS-1 primers revealed an expected product of 550 bp. Based on the cytology, histopathology, histochemistry, immunohistochemistry, electron microscopy and molecular studies, tumors from 68 cases were canine transmissible venereal tumors consisting of lymphocytic, plasmacytic and mixed type of cells at different stages of tumor development.ThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON SPONTANEOUS KIDNEY LESIONS IN SLAUGHTERED PIGS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-06) PADMINI, VAKA; HEMANTH, I(MAJOR); RAMA DEVI, V; SRINIVASA RAO, TThe present study was undertaken to know the occurrence of various spontaneous kidney lesions in slaughtered pigs, to describe the pathomorphological features of kidney lesions and to identify the probable etiological agents. A total of 320 slaughtered pigs of either sex were screened for various pathological conditions of kidney, if any, at different locations in Krishna District, Andhra Pradesh. Gross and microscopic kidney lesions of pathological significance were observed in 130 animals (40.6%) that were grouped under circulatory disturbances (13.85%), degenerative changes (6.92%), inflammatory conditions (60%), parasitic (11.54%) and miscellaneous (7.69%) conditions. Circulatory disturbances comprised of congestion (10.77%) and haemorrhages (3.08%). Renal congestion was characterized by dark red coloured kidneys with engorged blood capillaries in the cortex and medulla. Renal haemorrhages revealed diffuse petechial and large ecchymotic haemorrhages on the surface with numerous extravasated RBCs in the interstitium and within the tubules. Nephrosis refers to the degenerative changes in the renal parenchyma, was characterized by enlarged and pale kidneys with cloudy swelling and vacuolar degeneration of tubular lining epithelium along with the formation of protein and cellular casts in the tubules. Nephritis was categorized into glomerulonephritis (26.93%), interstitial nephritis (32.31%) and tubulo-interstitial nephritis (0.77%). Glomerulonephritis was comprised of acute (3.08%), sub-acute (21.54%) and chronic (2.31%) types. Acute lesions were characterized by pale and edematous kidneys with petechiae on the cortex along with glomerular lesions of congestion, swelling, hypercellularity and reduced Bowman’s space with protein accumulation. Sub-acute type revealed enlarged and pale kidneys with epithelial cresents and synechiae formation, sclerotic glomeruli, peri-glomerular mononuclear cells infiltration and perivasculitis. Chronic type revealed smaller sized kidneys with finely granular surface and lesions of diffuse global glomerular shrinkage, atrophy and necrosis with severe fibroplasia and mononuclear cells infiltration. Interstitial nephritis was comprised of sub-acute (29.23%) and chronic (3.07%) types. Sub-acute condition was characterized by variable sized whitish foci on the renal surface with nodular and diffuse aggregation of lymphoplasmacytic cells around the tubules, glomeruli and blood vessels. In chronic cases, kidneys were hard with diffuse pale areas and revealed diffuse and extensive proliferation of connective tissue all over the interstitium along with severe mononuclear cells infiltration. Tubulo-interstitial nephritis (0.77%) revealed kidneys with greyish white foci surrounded by hyperemic rim and lesions of multi-focal tubular necrosis and liquefaction with infiltrating polymorphs and mononuclear cells, and diffuse interstitial mononuclear cells infiltration. Stephanurosis (kidney worm) affections were characterized by numerous parasitic nodules in the perirenal fat, renal pelvis and around the ureter harbouring the adult and larval forms of the parasite. Microscopically, thick fibrous nodules containing cross section of parasites and severe infiltration of plasma cells, lymphocytes, macrophages and giant cells in the nodule wall were noticed. In the present study, 7.69% of renal cysts was recorded under miscellaneous conditions. Renal cysts were characterised by clear cystic cavities in the renal parenchyma with clear watery fluid with thick limiting membrane. Microscopically, empty or fluid filled cavities lined by single layer of flattened epithelial lining surrounded by proliferative connective tissue proliferation were noticed. Etiological studies revealed isolation of pathogenic bacterial species in 20% samples that comprised of Staphylococcus spp., Streptococcus spp., Escherichia coli and Klebsiella spp. in association with renal congestion, nephrosis, nephritis and kidney worm affections. The present study concluded that a significant occurrence of spontaneous renal lesions exist in slaughtered pigs and the most common pathological condition encountered being nephritis with consistent lesion of mononuclear cell infiltration. Further, this study suggests that a detailed investigations by involving large sample size and employing different molecular detection methods for wider range of etiological agents including zoonotic importance if any, so as to implement proper preventive measures in pig farming and to minimise the economic losses and to curtail zoonotic spill over, in any.ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF INFECTIOUS BURSAL DISEASE IN COMMERCIAL CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2022-03) AKHILA, N; SATHEESH, K (MAJOR); SAMATHA, V; SRINIVASA RAO, TThe present study was undertaken to know the occurrence of IBD in commercial chicken in around Vijayawada and to carry out pathological and molecular diagnosis of IBD. In the present study, a total of 922 IBD suspected birds of 4 to 6 weeks age were procured from different places in and around Vijayawada, from private diagnostic laboratories besides postmortems conducted in NTR College of Veterinary Science, Gannavaram. At necropsy, IBD was diagnosed in 252 birds out of 922 screened with an overall occurrence of 27.33% based on pathological lesions and molecular detection of IBDV by RT-PCR. The disease was more prevalent in 4 to 6 weeks aged birds with majority of cases occurring during the 5th week (40.11%). The occurrence was highest in April to September months (31 to 40%). As per the history, the affected birds showed depression, watery diarrhoea (white to yellowish), soiled vents, anorexia, ruffled feathers, reluctance to move, closed eyes and death. Necropsy conducted in 252 birds revealed predominant gross lesions in the bursa of Fabricius, spleen, thymus, caecal tonsils. Petechiae and ecchymoses on the pectoral and thigh muscles, haemorrhages at the proventriculus-gizzard junction. Bursa was swollen and oedematous with creamy or yellowish discoloration, gelatinous exudates in the serosa, congestion and haemorrhages on the serosal and mucosal surfaces and inner surface of longitudinal laminae in almost all the cases. Occasionally, the bursa became atrophied and whitish or creamy, which sometimes contained cheesy mass in the lumen. The spleen was enlarged and mottled. Kidneys were markedly swollen, congested and liver was enlarged, friable and yellowish in few cases. Histopathology revealed the prominent findings in bursa of Fabricius, characterized by congestion and hemorrhages along with lymphoid depletion of various degrees in the follicles resulting in atrophy of follicles and formation of cystic spaces within the follicles. Presence of eosinophilic necrotic material in the bursal follicles along with degenerating heterophils, atrophy of plicae, widening of interfollicular spaces with proliferation of fibrous connective tissue and sloughing of bursal epithelium was observed in few cases. Microscopic examination of spleen depicted congestion of splenic vessels and sinusoids, depletion of lymphocytes of varying degrees in white pulp and germinal centers. Thymus revealed decrease in thymocyte density in the cortex and congestion in most of the cases. However, in some cases disruption of normal architecture of cortex and medulla was also evident. Lymphoid depletion in lymphoid nodules, mucosal necrosis along with infiltration of mononuclear cells in the lamina propria were noticed in caecal tonsils in majority of the cases. Kidneys revealed interstitial haemorrhages and congestion in peritubular capillaries, severe infiltration of inflammatory cells, degeneration and individualization of tubular epithelial cells with detachment from the basement membrane and glomerular shrinkage. Liver revealed haemorrhage and severe necrosis of hepatocytes, congestion in the central vein, fatty change in hepatic parenchyma, diffuse coagulative necrosis of hepatocytes, along with severe infiltration of mononuclear cells in the portal areas of the liver. In the present study, pooled bursal samples were collected from 33 birds and utilized for molecular diagnosis of by RT-PCR using specific primers targeting VP2 gene of IBDV. Electrophoretic analysis revealed an amplified product of 480 bp fragment of VP2 gene in 9 samples thus confirming the presence of IBDV. The nucleotide sequencing and phylogenetic analysis of present IBDV sample (OM2024620) revealed it’s close relation with the strains of Kerala (MK 861022.1), Gujarat (AB605259.1), Karnataka (EU788042.1) and Haryana (AY739668.1). In the present study, the pathological and molecular diagnosis of IBD was carried out and the occurrence of IBD in commercial chicken in and around Vijayawada was recorded. It was evident that IBD continues to be a major threat causing economic losses in chicken in the study areaThesisItem Open Access PATHOLOGICAL STUDIES ON NECROTIC ENTERITIS ASSOCIATED WITH CLOSTRIDIUM PERFRINGENS IN COMMERCIAL CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-04) SAI CHARITHA, P.V; ANNAPURNA, P (MAJOR); RAMA DEVI, V; SRINIVASA RAO, TNecrotic enteritis is caused by infection with and toxin production by Clostridium perfringens (type A and C), a gram positive, spore forming anaerobe. Perusal of available literature revealed a very few reports on necrotic enteritis in chicken in the area under study. Hence, the present study was undertaken to know the occurrence of necrotic enteritis associated with C. perfringens in commercial chicken in natural cases, to study the gross and histopathological lesions, to carry out isolation and identification of C. perfringens by conventional bacteriological techniques and confirmation by PCR. In the present study, a total of 180 commercial chicken were collected from private poultry diagnostic centres showing lesions suggestive of necrotic enteritis at necropsy. Based on the pathological, bacteriological and molecular studies, the overall occurrence of necrotic enteritis associated with C. perfringens was found to be 37.8% in commercial chicken that comprised of 26.7 % in broilers and 11.1% in layers. Among the C. perfringens positive birds, concurrent infection with coccidia was noticed in 41.17% of birds. The clinical signs commonly reported in the necrotic enteritis affected birds were loss of appetite, bloody or whitish diarrhoea and sudden death. On bacteriological isolation and identification, a total of 68 isolates of C. perfringens were obtained and were confirmed by PCR with primers targeting the 16S rRNA of C. perfringens. Further, the isolates were also found positive for the alpha toxin gene of C. perfringens by PCR. At necropsy, the birds were found dehydrated and had foetid odour. The lesions were limited to intestines particularly jejunum while duodenum and ileum were affected in few cases. Extraintestinal lesions were seen only in liver in few cases. Grossly, jejunum showed ballooning with gas and blackish discolouration. Whitish foci visible through serosa were seen in some cases. Cut section revealed brownish, foul smelling fluid and necrotic debris along with sloughing of mucosa. An adherent brownish / greyish white diphtheritic membrane covered the mucosa. Histopathologically, in less severe cases, jejunum showed coagulation necrosis at apices of villi with viable deeper layers of mucosa. In more severe cases, the lumen contained mixture of erythrocytes, fibrin and desquamated epithelial cells. Extensive necrosis of villus epithelium, an adherent necrotic diphtheritic membrane over the mucosa and infiltration of mononuclear cells were the other changes noticed. The common finding in all the birds was presence of gram positive rod shaped bacteria along the tips of villi, on exposed lamina propria and in necrotic areas. Sections of duodenum and ileum showed mild coagulative necrosis. Hepatic lesions consisted of congestion and multifocal, greyish white, coalescing foci scattered on the surface of liver grossly. Coagulative necrosis, congestion and mononuclear cell infiltration were seen microscopically. In birds with concurrent coccidiosis, three species of Eimeria viz. E. acervulina, E. necatrix and E. maxima were identified. The intestinal lesions were more severe and consisted of ballooning and haemorrhages grossly. Haemorrhages and necrosis were found on the mucosa. Lumen contained blood clots and necrotic debris. Histopathologically, developmental stages of coccidia and extensive haemorrhages were seen in the lumen and in the mucosa, along with rod shaped bacteria in the surrounding necrotising debris. The present study revealed an overall occurrence of 37.8% of necrotic enteritis associated with C. perfringens in commercial chicken in the area under study indicating the impact of necrotic enteritis on poultry production leading to economic losses.ThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON LUNG LESIONS IN SLAUGHTERED GOATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-12) SIREESHA, VADDIPALLI; ANNAPURNA, P (MAJOR); RAMA DEVI, V; SRINIVASA RAO, TThe present study on lung lesions in slaughtered goats (Capra hircus) was undertaken to know the incidence, to describe the gross and histopathological changes associated with the lesions and to isolate various bacterial agents in possible cases. A total of 610 non-descriptive goats of either sex and of different age groups from various slaughter houses located in and around Vijayawada and from animals necropsied in the Department of Veterinary Pathology, N.T.R. College of Veterinary Science, Gannavaram were screened for the presence of gross abnormalities of lungs and representative samples were collected from lung lesions for further studies. Out of 610 lungs screened, 130 (21.31%) lungs revealed definite lesions on gross and histopathological examination, that were broadly grouped into abnormalities of inflation 24 (18.46%), circulatory disturbances 27 (20.77%), inflammatory changes 71 (54.61%), neoplastic conditions 05 (3.85%) and miscellaneous conditions 03 (2.31%). Abnormalities of inflation consisted of emphysema (11.54%) and atelectasis (6.92%). Circulatory disturbances comprised of congestion and hemorrhage (14.62%) and edema (6.15%). Inflammatory changes comprised of pneumonia (53.84%) and bronchitis (0.77%). Pneumonia was categorized into bronchopneumonia (46.92%), interstitial pneumonia (6.15%) and granulomatous pneumonia (0.77%). Bronchopneumonia was subdivided into suppurative (30.77%) and fibrinous (16.15%) types. Suppurative bronchopneumonia occurred as acute and chronic types. Acute type was characterized by cranioventral consolidation and mucopurulent exudates or mucoid plugs in airways on cut section. Histopathologically, predominantly neutrophilic exudate in alveoli, bronchi and bronchioles was noticed. Chronic type revealed cranioventral consolidation and multiple purulent foci grossly and on cut section. Microscopically, mononuclear cell infiltration within alveoli and in airways, abscesses and hyperplasia of BALT were noticed. In fibrinous bronchopneumonia, whitish fibrinous deposits were seen on the lungs apart from cranioventral consolidation. Adhesions were seen in some cases. Microscopically, fibrinocellular exudates within alveoli and in airways and fibrinous pleuritis were observed. In interstitial pneumonia, lungs were pale and showed rib impressions on the surface grossly. Cut section showed meaty appearance. Histopathologically, distortion of alveoli and thickened alveolar septa with mononuclear cell infiltration were seen. Granulomatous pneumonia was seen in one case, that showed a single firm nodule grossly and yellowish grey, fleshy mass on cut section of the nodule. Microscopically, a collection of mononuclear cells, epithelioid cells and a few Langhan’s giant cells encircled by fibrous connective tissue was observed. No bacterial, fungal, parasitic or foreign body etiology was noticed. A single case of bronchitis was seen, that showed creamy white nodules on right apical lobe grossly. Microscopically, chronic bronchitis that showed dilated bronchi with hyperplastic mucosa and mononuclear cell infiltration was seen. The neoplastic conditions consisted of jaagsiekte or ovine pulmonary adenocarcinoma (3.85%) that revealed either firm nodules of varying sizes on the lung or extensive involvement of most of the lobe and greyish moist surface and meaty appearance on cut section. Histopathologically, hypertrophy and hyperplasia of alveolar epithelium was noticed resembling jaagsiekte/ ovine pulmonary adenocarcinoma of sheep. Miscellaneous conditions comprised of pulmonary fasciolosis (0.77%) and goat pox (1.54%). Fasciolosis due to an aberrant migration of fluke was seen in a single case that revealed dark linear hemorrhagic tracts on the lungs grossly and on cut section. Histopathologically, fibrino-hemorrhagic tracts that contained cut section of a degenerated trematode surrounded by necrotic cellular debris, erythrocytes and fibrinocellular exudate were noticed. In goat pox, characteristic stages of pox were noticed on the skin, all over the body but mostly around the oral cavity, on the ears and scrotum. Histopathologically, skin lesions consisted of large intracytoplasmic eosinophilic inclusion bodies within epithelial cells and a few cells that resembled sheep pox cells in the dermis. Lung showed few macules, numerous papules and few pustules subpleurally. The microscopic findings were interstitial pneumonia and characteristic cells with vacuolated nuclei and marginated chromatin suggestive of sheep pox cells within the alveoli. Bacteriological studies on 57 lung samples of goats yielded pathogenic bacteria from 53 samples. Of these, 5 types of bacteria were obtained in pure culture from 45 samples viz. Staphylococcus spp. (37.73%), E. coli (30.19%), Pasteurella spp. (7.55%), Klebsiella spp. (7.55%) and Pseudomonas spp. (1.89%) while mixed infections were obtained in 8 samples in a combination of Staphylococcus spp. with E. coli (11.32%) and with Pasteurella spp. (3.77%). In the present study, pneumonia was the major pathological condition affecting goats in the area under study and Staphylococcus spp. was the most predominant bacteria isolated.
