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ThesisItem Open Access PORCINE DERIVED SCAFFOLD ASSISTED FULL THICKNESS SKIN WOUND HEALING IN RABBIT MODEL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-06-15) SUVANEETH P; SUVANEETH P; N. Divakaran Nair; N. Divakaran NairThe remodeling and regenerative responses of porcine cholecyst on full thickness skin wounds were evaluated in this study using rabbit as an animal model. Comparison of porcine cholecyst, porcine cholecyst seeded with autologous bone marrow cells and open wound control were made for evaluation. Porcine cholecyst was decellularized using a non-enzymatic, non-detergent based protocol and was checked for cellularity prior to in vivo evaluation. The in vivo evaluations were done on full thickness skin wound healing model in New Zealand White rabbits. The inflammatory, remodeling, and regenerative responses were evaluated 7, 14, 21 and 28 days post implantation. The H&E stained sections were evaluated for inflammatory and remodeling responses. Collagenization was evaluated and quantified using Masson’s trichrome and Picrosirius red staining. Differential collagenization was quantified using Herovici staining. Elastin deposition was ascertained using Verhoeff-Van Gieson’s staining. Proliferative response of the fibroblasts was evaluated by argyrophilic nucleolar organizer region (AgNOR) histochemistry and proliferating cell nuclear antigen (PCNA) immunostaining. Dermal cellular proliferation was quantified using PCNA immunohistochemistry. Re-epithelialization and epidermal formation was quantified using cytokeratin based immunohistochemistry. Vimentin immunohistochemistry was used to assess the mesenchymal cell response. ASMA immunohistochemistry was carried out to assess myofibroblast activity and CD 31 for neoangiogenesis. The results of the current study indicated that porcine derived cholecyst scaffolds are very well suited to be used as a bioscaffold material for full thickness skin wound healing, owing to their biochemical, biodegradable, biocompatible and tissue remodeling responses. The use of porcine cholecyst in full thickness skin wounds showed improved epithelialization and faster remodeling devoid of infections or graft rejections in all the animals under study. Scab formation, ulcerations, infections and other complications were also not observed in any of the scaffold assisted wounds. The scaffold enhanced cellular proliferation and keratinocyte activity. Controlled collagenization and faster replacement of immature collagen to mature collagen were also observed in scaffold assisted treatments. Improved neoangiogenesis were seen on scaffold assisted wounds from day seven itself which aided in faster healing of granulation tissue. Complete healing occurred around 14 days in graft assisted treatments, which was significantly early for the size of the wounds. Porcine cholecyst assisted healed wounds showed minimal wound contraction and reduced the chances of scar formation. The presence of autologous marrow cells enhanced proliferating cells, possibly due to the presence of bone marrow derived mesenchymal stem cells, as observed by a higher mesenchymal cell activity in vivo in marrow cells supplied scaffold. Use of autologous marrow cells has improved the time of healing in porcine cholecyst assisted skin wound healing, but not significantly from the application of a non-cell seeded matrix.ThesisItem Open Access EXPRESSION OF MECHANISTIC TARGET OF RAPAMYCIN(mTOR) AND DISHEVELLED, EGL 10 AND PLECKSTRIN DOMAIN CONTAINING MTOR INTERACTING PROTEIN (DEPTOR) IN CANINE SUPERFICIAL AND MAMMARY TUMOURS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-09-30) RAIMON MATHEW; Sajitha I.S.The present work was undertaken to study the expression of two proteins, critical in the cell signaling pathway namely, mechanistic target of Rapamycin (mTOR) and Dishevelled, EGL10 and Pleckstrin domain containing mTOR interacting protein (DEPTOR) in canine mammary and superficial tumours. Excision biopsy samples of mammary and skin tumour suspected growths from dogs presented to University Veterinary hospitals, Mannuthy and Kokkalai were collected for the study during the period from January 2018 to March 2019. Out of the 129 tumour suspected cases, 22 were mammary tumour growths and 17 were superficial tumour growths. Age wise analysis revealed that the mean age of the dogs affected with mammary tumours and superficial tumours was 7.85±0.41 years and 6.55±0.59 years respectively. The highest incidence of both the tumours was seen in the age group of seven to nine years. All the mammary tumours were recorded in female dogs but in case of superficial tumours male dogs (58.82 per cent) were more affected. Data on breed-wise occurrence showed the highest occurence of both the type of tumours in Labradors. Among the mammary tumours, 90 per cent were malignant and 10 per cent were benign, where as in case of superficial tumours majority (76.47 per cent) were benign in nature. Grossly CMTs varied in shape, colour and consistency with a mean size of 6.70±0.60 cm. The superficial tumours were predominantly round to oval in shape with grayish white colour having a mean size of 4.65±0.37cm. On grading of malignant CMTs, majority of the simple carcinomas, which are the most malignant type, had higher grade of II or III. Immunohistochemistry for mTOR and DEPTOR has revealed that both the proteins were significantly expressed (p0.05) with expression of these proteins. The mean expression of mTOR in mammary tumours and superficial tumours was significantly higher (p<0.01) than that of DEPTOR. Further, it was also found that there was significant positive correlation (p<0.01) between the expression of mTOR and DEPTOR in both these type of tumours. Thus, the present study identified that the two key proteins in cell signaling pathway, the mTOR and DEPTOR, were significantly expressed in canine mammary and superficial tumours. Further it was shown that their expression was positively correlated. Hence the oncogene role of DEPTOR in CMTs and CSTs was established through the study.ThesisItem Open Access EVALUATION OF IN VIVO HOST RESPONSE OF DIFFERENTLY DECELLULARISED OMENTAL SCAFFOLDS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-31) ASHNA SULAIMAN; Dhanush Krishna B.In this study, efficiency of decellularisation protocols as well as host response of differently decellularised bovine omental scaffolds were evaluated in a rat model. Bile treated omental scaffold (BBO), triton X- 100 treated omental scaffolds (TBO) and un-processed omental scaffolds (UBO) were used during the implantation period along with sham control (SCO). Efficiency of different decellularisation protocols was assessed by DNA quantification, 4, 6- diamidino-2-phenylindole (DAPI) staining, scanning electron microscopy and histological examination. Cellular and tissue response was evaluated semi quantitatively using ISO-10993-6: 2016 at 7, 14 and 30 days post- implantation (PI). CD4+ and CD8+ T-cells in retrieved implants and spleen were evaluated immunohistochemically to study the graft acceptance of these implants. Proliferation index was also evaluated by Ki67 immunolabelling. The results of this study showed that bile treatment had better decellularisation efficiency than triton X- 100. BBO showed better remodeling response in terms of neovascularisation, fatty infiltration and neocollagenisation than TBO and UBO. At all time periods studied, BBO implant had significantly (p< 0.05) lesser CD4+ per cent and higher CD8+ per cent than TBO and UBO, respectively. UBO showed significantly higher CD4+ per cent at all time periods than BBO and TBO. The CD4+/CD8+ ratio for BBO was significantly (p< 0.05) lower than TBO and UBO. Besides, BBO showed high Ki67 index (p<0.05) than TBO at 30 days PI. These results document that bile as an effective decellularising agent with less tissue toxicity than chemical detergent, triton X- 100. Apart from these, BBO enhanced remodeling potential through neovascularisation, cellular proliferation and neocollagenisation with significantly lower CD4+/CD8+ ratio and higher Ki67 index. Thus, bile treated omental scaffold may be a promising candidate for applications in human and veterinary medicine for improving patient’s life.ThesisItem Open Access EVALUATION OF IN VIVO HOST RESPONSE OF DIFFERENTLY DECELLULARISED OMENTAL SCAFFOLDS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-08-29) SULAIMAN, ASHNA; Krishna B., DhanushIn this study, efficiency of decellularisation protocols as well as host response of differently decellularised bovine omental scaffolds were evaluated in a rat model. Bile treated omental scaffold (BBO), triton X- 100 treated omental scaffolds (TBO) and un-processed omental scaffolds (UBO) were used during the implantation period along with sham control (SCO). Efficiency of different decellularisation protocols was assessed by DNA quantification, 4, 6- diamidino-2-phenylindole (DAPI) staining, scanning electron microscopy and histological examination. Cellular and tissue response was evaluated semi- quantitatively using ISO-10993-6: 2016 at 7, 14 and 30 days post- implantation (PI). CD4+ and CD8+ T-cells in retrieved implants and spleen were evaluated immunohistochemically to study the graft acceptance of these implants. Proliferation index was also evaluated by Ki67 immunolabelling. The results of this study showed that bile treatment had better decellularisation efficiency than triton X- 100. BBO showed better remodeling response in terms of neovascularisation, fatty infiltration and neocollagenisation than TBO and UBO. At all time periods studied, BBO implant had significantly (p< 0.05) lesser CD4+ per cent and higher CD8+ per cent than TBO and UBO, respectively. UBO showed significantly higher CD4+ per cent at all time periods than BBO and TBO. The CD4+/CD8+ ratio for BBO was significantly (p< 0.05) lower than TBO and UBO. Besides, BBO showed high Ki67 index (p<0.05) than TBO at 30 days PI. These results document that bile as an effective decellularising agent with less tissue toxicity than chemical detergent, triton X- 100. Apart from these, BBO enhanced remodeling potential through neovascularisation, cellular proliferation and neocollagenisation with significantly lower CD4+/CD8+ ratio and higher Ki67 index. Thus, bile treated omental scaffold may be a promising candidate for applications in human and veterinary medicine for improving patient’s life.ThesisItem Open Access IMMUNOHISTOCHEMICAL EXPRESSION OF EPIDERMAL GROWTH RECEPTOR AND PATHOLOGY OF ORAL AND PHARYNGEAL NEOPLASMS IN DOGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-03) NAIK, MADHURA PRASHANT; R., AnooprajThe present study entitled “Immunohistochemical expression of epidermal growth factor receptor and pathology of oral and pharyngeal neoplasms in dogs” was conducted for obtaining the data on the occurrence of oral and pharyngeal neoplasms in dogs, classification and grading and to compare the expression of epidermal growth factor receptor expression in tumour and normal tissues. A total of 23 oral and pharyngeal lesions were recorded. Out of the 23 lesions, 20 cases were diagnosed as neoplasms and 3 cases were of non-neoplastic origin. Age groups of seven to nine years showed highest incidence of the tumour (45 per cent), 85 per cent of the affected animals were males, while Labrador was the most affected breed (35 per cent). Gingiva formed the common site affected with tumours (60 per cent). The occurrence of malignant and mesenchymal tumours was 55 per cent and 80 per cent respectively. The malignant tumours encountered were histiocytic sarcomas, squamous cell carcinoma, fibrosarcoma, hemangiosarcoma, telangiectatic osteosarcoma and melanoma. Benign tumours included peripheral odontogenic fibroma, osteoma, oral papilloma and fibroma. Malignant tumours were graded based on specific histopathological parameters. Three cases of gingival hyperplasia were also studied. Expression of epidermal growth factor receptor (EGFR) was assessed in tumour specimens and the corresponding normal tissues by immunohistochemistry. Archived samples such as basal cell carcinoma, squamous cell carcinoma, acanthomatous ameloblastoma, desmoplastic ameloblastoma, papilloma and melanoma were also included in the study. When compared to normal tissues, significant strong over expression of EGFR was observed in squamous cell carcinoma, basal cell carcinoma, histiocytic neoplasms, hemangiosarcoma and melanoma while fibrosarcoma and ameloblastoma showed moderate over expression. No significant difference in EGFR expression was observed in papilloma and bone tumours.ThesisItem Open Access PATHOLOGICAL STUDIES AND MOLECULAR DETECTION OF Neospora caninum, Ehrlichia canis and Borrelia burgdorferi INFECTION IN DOGS WITH NERVOUS DISORDERS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-03) H. B, VIDYARANI.; K. S, PrasannaNeurological disorders in dogs with brain affections have multiple aetiology and often confused as rabies. In the present research work we studied gross and histopathological lesions in the rabies negative brain samples and screened them for the presence of Borrelia burgdorferi (B. burgdorferi), Ehrlichia canis (E. canis) and Neospora caninum (N. caninum). Out of the 84 dog carcasses presented to the Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Pookode during January 2018 to June 2019, 36 brain samples were found negative for rabies (42.9 per cent) by using direct Fluorescent Antibody test. The prominent histopathological lesions in the cerebrum, cerebellum and meninges were classified as vascular (86 per cent) and cellular changes (41.66 per cent). The vascular changes were congestion, haemorrhage and vasculitis (90. 3 per cent) and perivascular cuffing with mononuclear cells infiltration (22.6 per cent). The prominent neuronal changes were chromatolysis, shrinkage of neurons, perineuronal vacuolations, degeneration of neurons and neuronophagia. The common glial changes observed were satellitosis, peri glial vacuolations and gliosis. We did other diagnostic methods of blood smear examination for detection of morulae of E. canis and rapid immunochromatographic test for detection of antibodies against the B. burgdorferi in blood samples. Faecal sample examination was carried out for detection of N. caninum oocysts. Stained brain and tissue impression smears (Sellers and Giemsa stain) were examined for the detection of parasitic stages of N. caninum. All these tests were found negative for any of the specific pathogen targeted. Molecular tests were carried out for B. burgdorferi detection by nested PCR targeting 16SrRNA gene and conventional PCR for N. caninum detection by targeting Nc5 gene and none of the samples were found positive. For E. canis detection, the conventional PCR targeting 16SrRNA gene was carried out and one sample (2.78 per cent) was found positive for both genus and species-specific primer sets used. The specific lesions in the brain sample positive for E. canis were congestion, haemorrhage, diffuse vasculitis and perivascular cuffing with mononuclear cell infiltration in cerebrum and cerebellum.ThesisItem Open Access PATHOLOGICAL STUDIES OF ENTERO-HEPATIC AFFECTIONS IN DUCKS AND SCREENING FOR SPECIFIC VIRAL ETIOLOGY(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2019-10-03) SANJAY RAO, NIKHIL; M., PradeepDuck production holds an important position in the Indian poultry scenario, being the second most domesticated species of poultry in the country after chicken. Ducks are reared for both meat and egg production and a healthy liver and intestine capacitate the bird for its best performance and help the farmers for better profitability. Reports on systematic studies on the liver and intestinal lesions of ducks are scanty in India or have only been studied in relation to particular disease aetiology. Even though ducks show less susceptibility to diseases compared to the chicken, a few infectious and non-infectious diseases that affects liver and intestine were reported in ducks from many duck rearing countries. Among the viral diseases, the most important infections reported are duck enteritis virus (DEV) and duck virus hepatitis (DVH). Duck virus hepatitis (DVH) is an acute and contagious disease affecting liver but so far has not been reported in India. The present study was aimed at systematic post mortem recording of gross and histopathological lesions in the liver and intestine of duck carcasses and screening for DVH viruses and DEV by Polymerase chain reaction (PCR) using species specific primers. A total of 112 duck carcasses were examined in the study. Liver showed enlargement with characteristic deep yellow discoloration and firmness in 43.75 per cent cases. Pale yellow or dark brown friable liver recorded in 21.43 per cent cases. Amyloidosis of varying degrees were observed in 61.61 per cent cases of duck livers examined. A correlation between the severity of amyloidosis and gross lesions could also be derived. The enlarged, yellow and firm liver was observed to have moderate to severe amyloidosis histologically, while pale pink liver had moderate amyloidosis. The intestine also showed amyloid deposits in such cases without appreciable gross lesions. Ducks farmed under intensive system of rearing had a higher incidence of amyloidosis particularly in White Pekin ducks. Friable consistency of liver was either due to severe congestion or fatty change. Hepatocellular carcinoma with cirrhosis was identified in one case. DVE and DHV were not detected in any of the cases. Further screening for DVH viruses in a larger population of ducks is warranted. The absence of DEV in the current study may be due to effective control of the disease through appropriate vaccination strategies. The high incidence of amyloidosis in this study and the possible risk of dietary consumption of amyloid livers and its zoonotic potential needs to be investigated.ThesisItem Open Access HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES OF INFECTIOUS BURSAL DISEASE IN CHICKS(College of Veterinary and animal Science,Mannuthy, 2019) RAHMAAN S.B.; Sajitha I. S.The study was conducted to detect the presence of Infectious Bursal Disease (IBD) virus in the bursa of Fabricius of unvaccinated chicks below three weeks of age by polymerase chain reaction (PCR), study the pathology induced by the virus in the lymphoid organs of affected chicks and to evaluate the lymphocyte subpopulation in the bursa of affected chicks using immunohistochemistry (IHC). One hundred samples were collected from dead birds brought for postmortem examination to the Department of Veterinary Pathology, College of Veterinary and Animal Sciences, Mannuthy as well as from egger nurseries and private farms in and around Thrissur. The detailed history and gross lesions were recorded and tissue samples such as bursa of Fabricius, spleen, thymus and other organs were collected for histopathology and PCR. Out of one hundred samples, eight samples were positive for IBD virus by PCR. Gross lesions such as splenomegaly, haemorrhagic typhilitis, swollen and congested kidneys were the common findings in the affected birds. Histopathologically, lymphoid depletion and infiltration of heterophils in bursa of Fabricius, moderate to diffuse lymphoid depletion in spleen and decrease in thymocyte density with congestion in thymus were noticed. Viral antigen was demonstrated in bursa, spleen and liver of affected birds and influx of CD4+ and CD8+ lymphocytes were seen in the bursa of affected chicks by IHC. The present study confirms the occurrence of subclinical IBD in poultry in and around Thrissur and the organ of choice for demonstrating IBD viral antigen by IHC was found to be bursa of Fabricius when compared to other lymphoid organs.ThesisItem Open Access MOLECULAR DIAGNOSIS AND PATHOLOGY OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND PORCINE PARVO VIRUS INFECTIONS(College of Veterinary and animal Science,Mannuthy, 2019) SAIRAM R; Dhanush Krishna B.Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvo virus (PPV) are important emerging viral diseases having significant impact on global swine industries. Hence, the present research work was designed with the objective of detecting PRRSV and PPV from naturally occurring cases in swine population. Gross and histopathological lesions due to PRRSV and PPV infections were studied and these infections were further confirmed with immuno-histochemistry (IHC). 56 samples from pig carcasses suspected for PRRSV and PPV infections were collected during the study period from June 2018 to June 2019 from different swine farms across Kerala. Reverse transcriptase polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) identified eight cases positive for PRRSV and one case positive for PPV, respectively. Common gross lesions of PRRSV positive cases were non-collapsed voluminous lungs, enlarged and congested lymph nodes, splenomegaly, hemopericardium with multifocal whitish areas of necrotic spots in the liver. Gross lesions in PPV positive cases were non-collapsed voluminous lungs, fibrinous pericarditis, splenomegaly and multifocal whitish necrotic foci in kidneys. Histopathologically, PRRSV cases revealed interstitial pneumonia and severe lymphoid depletion in the tonsils of the soft palate, spleen and lymph nodes. PPV positive case displayed interstitial pneumonia, moderate lymphoid depletion in various lymphoid organs and interstitial nephritis. With IHC, viral antigens in positive cases of PRRSV and PPV were demonstrated in lungs and lymphoid organs. In addition, phylogenetic analysis of PRRSV isolate in this study was genetically related to other Indian isolates but forms a separate clade whereas PPV showed close relationship with Korean and Pookode strains. Therefore, this study establishes the presence of PRRSV and PPV in the swine population of Kerala. Routine screening of these viruses in swine farms needs to be practised as an effective measure to control these diseases. So, this study will serve as an evidence for the importance of vaccination against PRRSV and PPV infections of swine populations in near future.
