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ThesisItem Open 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, ROvine 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.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.ThesisItem Open Access STUDIES ON THE PATHOLOGY OF ENDOPARASITIC DISEASES IN BACKYARD CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) YAMUNA, M; ANNAPURNA, P (MAJOR); RAMA DEVI, V; SREEDEVI, CThe present study is undertaken to know the incidence of endoparasitic diseases in backyard chicken, to describe the lesions associated with the diseases and to carry out morphological identification of parasites in possible cases. A total of 1350 backyard chicken of either sex and of different age groups procured from villages in and around Gannavaram apart from the necropsies conducted in the Department of Veterinary Pathology, NTR C.V.Sc, Gannavaram were examined for endoparasitic diseases, if any. Organs showing parasitic lesions or parasites were collected for further studies. Thin blood smears were prepared for detection of hemoprotozoan infection, if any. Out of 1350 backyard chicken examined, 433 birds (32.07%) revealed definite lesions of endoparasitic diseases in the gastrointestinal tract. Based on gross and histopathological examination, the diseases were broadly grouped under diseases caused by cestodes (4%), nematodes (11.7%), protozoa (16%) and mixed infection (0.37%). No trematode and haemoprotozoa infection were noticed. Morphological identification of various parasites was carried out in the present study. Among cestodes, Cotugnia digonopora (1.48%) and Raillietina spp. (2.52%) were found in the small intestines. Raillietina spp. comprised of R. cesticillus (0.89%), R. echinobothrida (0.89%) and R. tetragona (0.74%). In C. digonopora infection, intestines were pale and numerous tapeworms were seen in the lumen on cut section. Histopathologically, sections of tapeworms in the lumen and within the mucosa, blunting of villi, goblet cell hyperplasia and infiltration of mononuclear cells were noticed. In Raillietina spp. infection, segmented parasites were noticed in the lumen of small intestine in all the three Raillietina spp. Histopathologically, cut sections of parasites within the lumen of intestines and mononuclear cell infiltration were seen in all the three species of Raillietina. In addition, in R. cesticillus infection, villous hyperplasia and lymphoid depletion in the follicles were noticed. Edema and hyperplasia of mucosal epithelium were seen with R. echinobothrida whereas fusion of villi and necrosis were noticed in R. tetragona infection. In the present study, Dispharynx nasuta (0.15%), Ascaridia galli (11.11%), Cheilospirura hamulosa (0.07%), Heterakis gallinarum (0.15%) and Tetrameres mohtedai (0.22%) were the nematodes noticed causing diseases in backyard chicken. In D. nasuta infection, dark, circular areas were visible through the serosa of proventriculus. The mucosa showed, ulcers and slender worms on the surface. Histopathologically, cut sections of worms within the lumen, metaplasia of the surface epithelium into stratified squamous type, infiltration with mononuclear cells and lymphoid hyperplasia were the salient features noticed. In A. galli infection, small intestines showed mild congestion of serosal vessels in heavy infection. Slender roundworms were seen on cut section. Microscopically, cut section of worms in the lumen of intestine, goblet cell hyperplasia, blunting of villi and mononuclear cell infiltration were noticed in the mucosa. In C. hamulosa infection, gizzard revealed erosions in the koilin layer and reddish, coiled worms attached to underlying mucosa. Histopathologically, cut sections of parasite, mucosal necrosis and severe infiltration of mononuclear cells were observed along with lymphoid hyperplasia. H. gallinarum was found freely within the lumen of caecum. Microscopically, cut sections of worms, necrosis of mucosal epithelium along with mononuclear cell infiltration were noticed in the ceacum. T. mohtedai infection was seen in the proventriculus of chicken. Grossly, dark spots were seen through the serosa and on cut section, reddish, subspherical female worms of T. mohtedai were observed. Microscopically, proventricular glands revealed cut section of the parasite and atrophy of epithelium. In the present study, coccidiosis (16%) was the only protozoan disease recorded that occurred as caecal (6.81%) and intestinal (9.19 %) forms. In both the forms, the carcass was pale and emaciated. Mucosal scrapings from the caeca and intestines revealed coccidial oocysts in caecal and intestinal coccidiosis respectively. Based on micrometry and sporulation time, the coccidia were identified as E. tenella in caecal coccidiosis and as E. acervulina, E. necatrix, E. maxima, E. brunetti and E. mitis in intestinal coccidiosis. Grossly, in caecal coccidiosis ballooning of caeca with dark petechiae with bloody contents and dried caecal cores were seen on cut scetion. Microscopically, extensive erosion of mucosal epithelium that contained different stages of coccidia was a constant feature noticed. Haemorrhages, fusion of villi and infiltration of mononuclear cells were seen. Out of 124 birds affected with intestinal coccidiosis, concurrent infection with five species of coccidia was seen in 98 birds that showed lesions throughout the small intestines while 26 birds were infected with three species of coccidia in which lesions were restricted to parts of small intestine. Grossly, linear haemorrhages visible through the serosa and ballooning of intestines was seen. On section, mucosa was haemorrhagic and the contents were bloody with clots. Histopathologically, haemorrhages, necrosis, fusion of villi and hyperplastic changes were noticed in the mucosa. Various developing stages of coccidian in epithelial cells were observed along with severe infiltration of macrophages, plasma cells and heterophils. In the present study, mixed parasitic infection (0.37%) that comprised of A. galli in combination with C. digonopora (0.22 %) and with R. Tetragona (0.15%) was noticed. In all the cases, intestines revealed both round worms and tapeworms in the lumen grossly and microscopically. In addition, necrosis (A. galli and C. digonopora) and hyperplasia (A. galli and R. tetragona) were also noticed in the mucosa microscopically. The present study revealed 32.07% of endoparasitic diseases in backyard chicken and recorded the associated pathological changes. Of the various diseases noticed, coccidiosis was the most predominant in the area under study.ThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON UTERINE LESIONS IN SLAUGHTERED SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-03) VIJAYA DURGA, G; ANNAPURNA, P(MAJOR); SATHEESH, K; RAVI KUMAR, PThe present study was undertaken to know the prevalence of uterine lesions in slaughtered sheep, to describe the gross and histopathological changes in different uterine lesions and to isolate various bacterial agents from the uterine lesions in possible cases. A total of 198 uteri of non pregnant sheep aged above one year were collected from various slaughter houses and from animals necropsied in the Department of Pathology, NTR College of Veterinary Science, Gannavaram. On gross and microscopic examination, definitive lesions were noticed in 61 uteri with a prevalence of 30.8%. Based on the predominant pathological lesion noticed, the uterine lesions were grouped under haemorrhages (3.03%), inflammation (18.18%), hydrometra (1.01%), endometrial hyperplasia (2.02%), adenomyosis (1.01%) and miscellaneous conditions (5.55%). Petechial or ecchymotic uterine haemorrhages were noticed grossly and on cut section while focal accumulation of erythrocytes and macrophages laden with haemosiderin were seen within endometrial stroma microscopically. Inflammatory conditions comprised of acute endometritis (3.03%), chronic endometritis (13.13%) and pyometra (2.02%). In acute endometritis, the uteri were either soft and enlarged or hyperemic and showed few focal haemorrhages on endometrial surface. Turbid exudates were present in the lumen. Microscopically, exudate consisting predominantly of polymorphs in the lumen, necrosis of surface epithelium and diffuse neutrophil infiltration in stroma were noticed in the endometrium. The endometrial glands revealed polymorphs within lumen and periglandularly. In chronic endometritis, uterus was hard to touch and showed asymmetrical enlargement of horns or mild annular constrictions in some cases. The contents were turbid whitish to clear fluidy. Microscopically, diffuse mononuclear infiltration especially of plasma cells in the stroma and in the glandular lumen was characteristic. Hyperplasia of endometrium, lymphoid follicle formation beneath the epithelium, extensive periglandualr fibrosis and hyalinisation of blood vessels were the other salient features. In pyometra, uterine horns were enlarged and contained yellow to greenish, creamy contents. Microscopically, exudate with large number of polymorphs in the lumen and endometrial hyperplasia were noticed. The characteristic feature was infiltration of polymorphs along with mononuclear cells in all the layers of uterus. Endometrial glands were dilated and cystic. In hydrometra, there was distension of uterine horns grossly and the lumen contained clear fluid. Microscopically, edematous stroma with plasma cell infiltration, cystic endometrial glands and edematous myometrium were observed. In endometrial hyperplasia, uterus was enlarged, firm and heavy grossly. Multiple cysts of 1-2 mm were embedded in endometrium that contained watery to viscous creamy fluid. Microscopically, cystic endometrial glands with periglandular fibrosis was found consistently. Proliferation of endometrial glands, infiltration of mononuclear cells in the stroma and hyalinisation of blood vessels were the other features noticed. Uteri with adenomyosis showed thickened uterine wall grossly and presence of endometrial glands between the muscle bundles in the myometrium microscopically. Miscellaneous conditions comprised of pigmentation due to melanin deposition (5.05%) and a cyst on the uterus (0.5%). Melanin was seen mostly in the caruncles and in few cases, in the intercaruncular region. Microscopically, brownish to black melanin pigment was seen within the superficial layer of endometrium and the caruncles without any other accompanying histopathological lesion. A single case of small, fluid filled serosal inclusion cyst was noticed grossly on the left horn of uterus. Microscopically, a thin walled cyst that contained pink stained fluid was noticed in the outer surface of perimetrium. From various uterine lesions, 38 samples yielded 4 bacterial isolates that comprised of E.coli (55.3%), Staphylococcus sp. (28.9%), Klebsiella sp. (13.2%) and Proteus sp. (2.6%). The present study revealed the prevalence and pathomorphological changes of a spectrum of uterine lesions in ewes of which chronic endometritis was the most common lesion recorded. E. coli was found to be the major bacterial pathogen isolated from sheep uterus in the area under study.ThesisItem Open Access CLINICO-PATHOLOGICAL STUDIES ON CANINE PYOMETRA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-12) SOLMON SINGH, B; ANNAPURNA, P(MAJOR); RAMA DEVI, V; RAVI KUMAR, PThe present study was undertaken at NTR College of Veterinary Science, Gannavaram to know the incidence, to carry out hematological, biochemical and bacteriological studies and to describe the lesions in canine pyometra. During the period of study, out of 140 bitches presented to Department of Surgery and Radiology at Teaching Veterinary Clinical Complex (TVCC) for various ailments, 20 bitches (14.3%) were diagnosed with pyometra by clinical examination, radiography and ultrasonography and various samples collected from these were used for further studies carried out in the present study. Highest incidence of pyometra (70%) was found in the age group of 6-10 years and the mean age of occurrence was 8.05 ± 0.61 years. Among the breeds, Pomeranian (35%) was mostly affected followed by Labrador (30%), Spitz (15%), German Shepherd (10%) and Boxer and Pug breeds (5% each). Nulliparous bitches (65%) were mostly affected followed by primiparous (25%) and pleuriparous (10%) animals. Open (85%) and closed (15%) types of pyometra were noticed and all the bitches were in diestrus. Anorexia, lethargy, vomition and normal temperature were observed in most of the cases. Mucopurulent to hemorrhagic vaginal discharges with fetid odour were seen in all open pyometra cases. In the present study, a significant decrease was noticed in the mean values of PCV, Hb and TRBC in pyometric bitches compared to control animals whereas no significant change was seen in MCV, MCH and MCHC that indicated a normocytic, normochromic anemia. Significant leucocytosis with regenerative shift to left was noticed. Significant increase was noted in the mean values of BUN, creatinine and AST and ALT in pyometric bitches compared to control dogs that indicated an altered renal and hepatic function respectively. The lesions noticed in the hysterectomised uteri from 20 pyometric bitches were grouped into types II, III and IV. Type II lesions (75%) consisted of open and closed pyometra. Grossly, horns were uniformly distended and revealed whitish cysts on the surface that exuded creamy pus on incision. Histopathologically, marked infiltration of plasma cells along with cystic glandular hyperplasia was characteristic. Myometrium revealed necrosis, plasma cell infiltration and showed adenomyosis in one case. Type III lesions (20%) consisted of open pyometra cases that showed marked sacculations of uterine horns grossly. Annular constrictions, whitish cysts, focal hemorrhages and ulceration were seen on cut section. Histopathologically, marked infiltration of polymorphs in the endometrial stroma and within glandular lumen was the consistent finding. Endometrial glands showed necrotic debris with bacterial colonies in the lumen and prominent periglandular fibrosis. Myometrium showed fibrosis and atrophy. A single open pyometra case (5%) with type IV lesion was noticed that showed highly distended uterine horns especially at the ovarian end. On cut section, granular endometrial surface and creamy white fluidy contents were noticed. Histopathologically, very thin endometrial layer with few cystic glands, stromal infiltration with mononuclears and atrophy and fibrosis of myometrium were observed. In the present study, a total of 6 isolates viz. E.coli (30%), Klebsiella sp. (20%), Pseudomonas sp. (15%), Shigella sp. (10%), S.aureus (10%), and Proteus sp. (5%) were obtained in pure cultures from 18 uterine samples whereas a mixed culture of E.coli with Shigella was obtained from two samples (10%) and E.coli was found to be the most common bacteria isolated from pyometra cases. The clinico-pathological studies on canine pyometra were carried out in the present study. The hematological and biochemical changes and the lesions noticed in pyometric bitches were characteristic and E.coli was the most predominant organism isolated from cases of pyometra.ThesisItem Open Access PATHOLOGY AND MOLECULAR DIAGNOSIS OF CANINE PARVO VIRAL INFECTION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-10) RAMYA, Ch.; SATHEESH, K(MAJOR); ANNAPURNA, P; MALAKONDIAH, PThe present work was undertaken to know the incidence, to study haemato-biochemical changes, to describe the lesions and to carry out molecular diagnosis of CPV in dogs by PCR. A total of 85 dogs with suspected clinical signs of CPV infection presented to the TVCC, veterinary hospitals and pet clinics in selected districts (West Godavari, Krishna and Guntur) of A.P during the period from December 2017 to July 2018 were screened and various materials were collected for the present study. Of the 85 dogs screened, 37 were found positive by PCR constituting an overall incidence of 43.52% of CPV in the study area. The incidence was found to be highest in Guntur (65%) followed by Krishna (37.5%) and West Godavari (35.29%) districts. Non-descript, unvaccinated male pups of 0-3 months were found to be at higher risk especially during monsoon season in the area under study. Clinical signs noticed in the affected dogs were vomition and haemorrhagic diarrhoea with eventual death mostly in young pups. The salient haemato-biochemical findings were anaemia, neutropenia, lymphopenia, increased BUN, creatinine, AST, ALT, ALP values and decreased total serum protein and albumin. In nine dogs that died during the course of the disease and confirmed positive for CPV by PCR, necropsy was carried out. The animals were cachectic with blood tinged faeces at perianal region. Lesions were noticed in the intestine, heart, stomach, spleen, lung, liver and kidneys. Intestine was congested and the contents varied from watery to yellow mucoid and often bloody. Microscopically, villous atrophy, fusion, haemorrhages, necrosis and diptheritic membrane formation were observed in the mucosa. Goblet cell hyperplasia, cystic enlargement of crypts and mononuclear infiltration were seen in the lamina propria. Mesenteric lymph nodes were grossly enlarged that showed lymphoid depletion. Heart of CPV affected dogs had a mottled and pale appearance. Microscopically, necrosis of cardiac muscle, interstitial myocarditis and haemorrhages between muscle fibres were noticed. Stomach revealed mucosal congestion and haemorrhagic to mucoid contents. Microscopically, haemorrhages, mononuclear infiltration and necrosis were noticed in the mucosa. Lymphoid depletion was seen in the spleen. Grossly, lung appeared congested that revealed vascular congestion and mild septal thickening microscopically. Liver was pale and showed degenerative changes and focal necrosis of hepatocytes. Kidneys appeared congested grossly that revealed degeneration and necrosis of tubular epithelium. In the present study, molecular diagnosis of CPV in 37 samples using PCR assay by using CPV-2ab primers yielded a product of 681 bp specific for VP-2 gene of capsid protein of CPV. In conclusion, the present study revealed the occurrence of CPV infection in dogs in the study area with an incidence of 43.52% and described the haemato-biochemical and pathological changes apart from molecular diagnosis of the infection by PCR.ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF CLOSTRIDIUM PERFRINGENS TYPE D INFECTION IN SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2018-06) RACHEL DIVYA, MEKATHOTI; SATHEESH, K(MAJOR); ANNAPURNA, P; SUBRAMANYAM, K.V.Enterotoxemia in sheep is a fatal disease and is attributable to a toxigenic type of Clostridium perfringens type D associated with epsilon and alpha toxins. Hence, the present study was undertaken to know the incidence, to elucidate lesions in various organs, to carry out bacterial isolation and identification and molecular confirmation of Clostridium perfringens type D by multiplex PCR. In the present study, a total of 927 indigenous sheep of either sex and of different age groups were screened for clinical signs of enterotoxemia and of these, 157 (16.93%) were found to be clinically affected. A detailed necropsy was carried out in 27 dead animals out of the affected animals and the representative samples were collected for further studies. Urine samples were also collected from dead animals to test for glycosuria. Clinically, the affected sheep showed peracute signs with sudden death mostly in young animals. In few acute cases, there was diarrhoea and convulsions. At necropsy, bloated carcass and presence of fluid in the abdominal cavity were noticed on external examination. All the animals revealed predominant gross lesions in intestine, kidney, lung, heart and brain with occasional involvement of other organs. Gas filled, distended intestines were seen grossly that revealed haemorrhages and necrosis of the mucosal epithelium microscopically. Fibrinous pseudomembrane formation and blunting of the villi were the other salient features. Kidneys were swollen and congested that appeared soft, pulpy and had jam like consistency grossly. Microscopic lesions included cloudy swelling and necrosis of the tubular epithelium and haemorrhages. Haemorrhages and necrosis were seen in glomerulus and the interstitium showed infiltration of inflammatory cells and severe vascular congestion. The lungs were edematous and congested grossly and the microscopic lesions were characterized by severe congestion, haemorrhages, alveolar edema, emphysema and thickened alveolar septa. In the heart, straw colored fluid in the pericardial sac and haemorrhages on the epicardium and endocardium were noticed. Microscopic lesions comprised of mild congestion and inflammatory cell infiltration in between the cardiac muscle fibres. In the brain, congestion of meninges, softening and bilaterally symmetric focal areas of hemorrhages were seen grossly. Perivascular edema, neuronophagia, vacuolation were the microscopic lesions seen in cerebrum whereas degeneration of purkinjee cell layer and axonal swelling were noticed in cerebellum. Grossly, liver appeared congested. Microscopically, congestion, cloudy swelling, fatty change and multi focal necrosis were observed. In some cases, hepatic sinusoids were dilated and contained fibrin threads and many bacterial rods. In the present study, intestinal contents collected from necropsied sheep was inoculated in Robertson’s cooked meat medium prepared with BHI broth and thioglycollate broth for bacterial isolation and the culture smears were stained by Gram’s method that revealed Gram positive, short and stumpy rods with blunt ends. Further identification of the bacteria was done by litmus milk test that resulted in the stormy clot fermentation indicating the presence of Clostridium perfringens. The urine samples collected showed glycosuria by Benedict’s test. In the present study, the Clostridium perfringens culture obtained from intestinal contents was used for genomic DNA extraction and the obtained DNA samples were subjected to multiplex PCR for diagnosis of Clostridium perfringens type D that produces alpha and epsilon toxins. Multiplex PCR was carried out in 27 samples by using specific primers for toxin genes viz. alpha toxin gene (cpa) and epsilon toxin gene (etx) for Clostridium perfringens type D and also beta toxin gene (cpb) to differentiate Clostridium perfringens type D from Clostridium perfringens type B. On electrophoretic analysis, an amplicon of 324bp for alpha (cpa) and 655bp for epsilon (etx) were obtained in all the samples confirming the presence of Clostridium perfringens type D in the study area.ThesisItem Open Access STUDIES ON PATHOLOGY OF ENDOSULFAN TOXICITY IN BROILERS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2006-02) SUNEETHA, D; SRILATHA, Ch(MAJOR); SUJATHA, K; CHANDRA SEKHARA RAO, T.SThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON LEAD TOXICITY IN FEMALE RATS WITH SPECIAL REFERENCE TO THE REPRODUCTIVE SYSTEM AND ITS AMELIORATION WITH Emblica officinalis (AMLA) AND Linum usitatissimum (FLAXSEED)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2017-10) AMITAVA PAUL; SUJATHA, K(MAJOR); SRILATHA, Ch; VINOD KUMAR, NABSTRACT: Heavy metal Lead is one of mankind’s oldest environmental and occupational toxins found in almost all phases of biological systems. It is known to cause many adverse effects in man and animals. The major organs affected in the body in lead toxicity include nervous system, renal system, hematologic, cardiovascular and immune system. The deleterious effects of lead on reproductive system both in males and females are also a well-known fact. Animal models are widely used to characterize lead toxicity and to study the ameliorating effects of many herbs. In the current investigation, Emblica officinalis (AMLA) and Linum usitatissimum (FLAXSEED) are being investigated for their protective effect on lead induced toxicity with a major focus on reproductive system of female wistar rats. The present study was carried out by procuring 108 female adult wistar albino rats that were randomly assigned to 6 groups with 18 rats in each group. Group I served as vehicle control and they received distilled water, whereas animals in Group II, III, IV, V and VI received lead acetate @ 60 mg/ kg b.wt., Emblica officinalis @ 100 mg/ rat/ day, Linum usitatissimum @ 300 mg/ kg b.wt, lead acetate @ 60 mg/ kg b.wt + Emblica officinalis @ 100 mg/ rat/ day, lead acetate @ 60 mg/ kg b.wt + Linum usitatissimum @ 300 mg/ kg b.wt. respectively for 45 days. Six rats from each group were sacrificed at fortnight intervals. In the present study no mortality was observed in any of the treated group. Across the groups, lead related clinical signs attributable to neurotoxicity were seen. Significant (P<0.05) reduction was recorded in TEC, PCV, Hb and Percent lymphocyte count and significant increase in Percent neutrophil count and TLC was observed in lead treated group (II) in relation to group I. These counts were significantly improved in ameliorated groups (V and VI), when compared to lead treated group (II). Serum biochemical analysis revealed significant decrease (P<0.05) in total protein and increase in SGPT, SGOT, cholesterol, creatinine, ALP and LDH levels in Group II. Significant (P<0.05) increase in the concentration of TBARS and decreased levels of antioxidants like catalase, SOD and GPx levels in liver, kidney, brain and uterus of Group II were noticed. The levels of these parameters were significantly improved in the ameliorated group (Group V and VI). Significant (P<0.05) decrease in serum Progesterone (P) and Estrogen (E2) concentration was noticed in Group II rats. In Group V and VI, significant improvement in hormonal values was noticed compared to Group II. No significant alterations were noticed in all above mentioned parameters in Emblica and Flaxseed treated rats (Group III and IV). In the present study, conspicuous gross lesion was noticed in uterus, liver followed by kidney and lung. Gross lesion includes reduction in size of uterus, paleness of liver, congestion of kidney and lung in lead treated rats (Group II). These changes were less severe in Group V and VI. No significant changes were observed in Emblica and Flaxseed treated rats (Group III and IV). Microscopically uterus of the rats of Group II revealed vacuolar degeneration of lining epithelium of endometrium, narrowing of lumen, degenerated endometrial glandular epithelium, disruption of endometrial glandular structure with, reduced number of endometrial glands, variation in shape and size of the glands, severely degenerated and distorted glands, cystic lumen, complete obliteration of glandular lumen and hyperplastic changes in endometrial gland. In Group V and VI, these changes were less intense. Ovaries of lead acetate treated rats showed, medullary congestion, thickened tunica albuginea, severely degenerated granulosa cell with completely disintegrated oocyte and its nucleus in primary and secondary follicles, lysis of oocyte, cumulus oophorus and clumping of granulosa cells in secondary follicle and significant reduction in the number of follicles at different developmental stages were observed. All these changes were less severe in groups (V and VI). Microscopically, the panel of vital organs comprising liver, kidney, heart, spleen, intestine and lungs were evaluated. The major changes in liver included focal loss of hepatocytes with infiltration of MNCs, bile duct hyperplasia, moderate to severe dilatation and congestion of sinusoids in group II animals. Kidney section showed degenerated and desquamated renal tubular epithelium, congested glumerulus, atrophied and cystic glomeruli. The lead acetate treated group also showed mild to severe thickening of interstitial space, thickening of blood vessels, and severe lymphoid hyperplasia in peribronchial area in lungs. The lesions in brain section included submeningeal hemorrhages, mild to moderate capillary proliferation in cerebral cortex, rounding and loss of purkinje cells in cerebellum, shrinkage of neurons, demyelinating changes and spongiosis and gliosis. Heart showed focal sarcolytic changes, thickening and congested blood vessels. Changes in spleen included thickened blood vessels, thickened capsule and mild to moderate depletion of lymphocyte in white pulp and engorgement of red pulp. Intestine revealed desquamated epithelial cells with increased number of goblet cells, mild to moderate eosinophilic infiltration in sub mucosa and necrosis. The severity of these lesions was of lesser magnitude in Group V and VI. No significant changes were noticed in soft tissues of Emblica (Group III) and Flaxseed (Group IV) treated rats. In rats of group II, immunohistochemistry showed increased expression of BAX marker in both uterus and ovarian tissue compared to Group I. Whereas, in rats of Group V and VI decreased expression of BAX marker was observed compared to Group II. No significant changes were noticed in rats of Group III and IV.
