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HISTOMORPHOLOGICAL EVALUATION OF STROMAL COLLAGEN AND IMMUNOHISTOCHEMICAL ANALYSIS OF HEAT SHOCK PROTEIN 47 IN CANINE MAMMARY TUMOURS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-22) KEERTHANA SANTHOSH; Dr. Devi S.S
The present study was conducted to histomorphologically evaluate the stromal collagen and to analyse the immunohistochemical localisation of Heat shock protein 47 (Hsp47) in canine mammary tumours (CMTs). Twenty five CMT samples were collected from Department of Surgery and Radiology, TVCC, Mannuthy and University Veterinary Hospital, Kokkalai. The highest occurrence of mammary tumour was observed in dogs of nine to eleven years age group and the mean age of occurrence of CMTs in the studied population was 9.04±0.54 years. Out of 25 CMTs, the highest occurrence was observed among female dogs. Data on breed-wise occurrence showed that Labrador retriever breeds were mostly affected. Grossly CMTs varied in shape, colour and consistency with a mean size of 10.34±0.91 cm. Based on histological features, 24 tumours were classified as malignant and only one as benign. Twenty four malignant CMTs were subjected to grading on the basis of tubule formation, nuclear pleomorphism and mitotic figures.Grade II tumours were high in number which was followed by Grade III tumours. Tumours were histologically classified into different subtypes and ductal carcinomas were high in number followed by solid carcinomas and tubulopapillary carcinomas. Tumour to stroma ratio (TSR) was assessed in H and E stained sections and immunohistochemically using anti-cytokeratin antibody and subsequently classified tumours into those with high TSR (low stroma), moderate TSR (moderate stroma) and low TSR (high stroma). Tumours with high and moderate stroma were further analysed and based on the extent of fibroplasia, stroma was classified into three: stroma with severe fibroplasia, stroma with moderate fibroplasia and stroma with mild fibroplasia. Nine CMT samples had severe fibroplasia, 11 had moderate fibroplasia and only two had mild fibroplasia. Collagen in the stroma was visualized by employing several staining techniques. Picrosirius red stain imparted red colour to collagen whereas Masson’s trichrome gave blue colour to collagen. Varying grades of collagen deposition could be appreciated in the stroma of cancerous tissue. With Herovici staining newly deposited young and immature collagen in the stroma of CMTs was identified. The sequences of changes occurring in collagen fibres during the progression from moderately differentiated CMTs to poorly differentiated ones were assessed in picrosirius red stained histological sections using polarized microscopy. In moderately differentiated tumours, thick yellow orange birefringence was noticed. In poorly differentiated tumours, a transition from yellow orange to greenish yellow birefringence was seen. Immunohistochemistry was performed to study the expression of Hsp47 in CMTs. Out of 25 tumour cases, 19 (seventy six per cent) were strongly positive, four (sixteen per cent) were moderately positive and two (eight per cent) had weak immunopositivity. Analysis of data revealed that IHC scores of Hsp47 positively correlated with tumour grades and all tumours with high fibroplasia showed strong immunopositivity for the protein indicating a clear association between the stromal fibroplasia and Hsp47. The deposition of collagen type I in CMTs having high and moderate fibroplasia was analysed immunohistochemically using antibodies directed against COL1A1. The results revealed that tumours having high and moderate stromal fibroplasia gave either very strong or strong immunostaining for the protein indicating an increased deposition of collagen type I in neoplasms of canine mammary glands. Statistical analysis revealed a strong correlation between COL1A1 expression and Hsp47 expression. The study proved that Hsp47 directly enhanced fibroplasia and favoured collagen deposition. Increased immunostaining of these two proteins corresponded to increase in grades of CMTs. Further studies in a larger population would help in confirming the potential roles of Hsp47 and COL1A1 as prognostic and therapeutic biomarkers in CMTs.
Open Access
IMMUNOHISTOCHEMICAL EVALUATION OF THE EXPRESSION OF GLUTAMATE AMMONIA LIGASE IN THE BRAIN OF RABIES AFFECTED DOGS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-23) PULLAGURA NIKHITHASREE; Dr. Divya C
The current research work was designed with the objective of studying immunohistochemical localisation of glutamate ammonia ligase (GLUL) protein in different anatomical sites of brain in canine rabies as well as the comparison of immunohistochemical signals in furious and dumb forms of the disease along with gross and histopathological changes in rabid dog brains. The carcasses of 219 rabies suspected dogs that were brought to the Department of Veterinary Pathology, CVAS, Mannuthy, for necropsy between January 2021 and August 2022 formed the source of the samples in this study. One hundred and thirty-threebrain samples tested positive for dFAT were further confirmed by employing polymerase chain reaction targeting N gene of rabies which yielded amplicon size of 533bp. Gross, cytological and histopathological alterations in brain of rabies were recorded. Immunohistochemical staining revealed localisation of GLUL in glial cells in different anatomical sites of brain with different staining intensity. Diffuse strong immunopositivity was noticed in the microglial cells, astrocytic end feet process and axonal fibres in cerebellum, hippocampus, brainstem and cerebrum in furious form. Whereas weak immunopositivity was noticed in all the four parts of brain in dumb form. Comparison of immunofluorescence with clinical forms of rabies revealed that furious form exhibited higher grade of fluorescence where as lower grade of fluorescence was noticed in dumb form. Further, in different clinical forms of rabies, immunohistochemical localisation of GLUL was compared with immunofluorescence of the viral antigen. In furious form of rabies, immunofluorescence was higher and immunopositivity for GLUL was stronger. The dumb form, in contrast, demonstrated low immunofluorescence and weak immunopositivity. Immunofluorescence and immunopositivity were positively correlated in both furious and dumb form. However, the significant assosciation and correlation between immunofluorescence and immunopositivity were greater in hippocampus and cerebellum (p< 0.01) compared to brainstem and cerebrum (p>0.05).Mechanism by which GLUL is increased and decreased in furious and dumb form respectively was substantiated from the present study. It is postulated that increased levels of GLUL leads to excitotoxic conditions manifested as furious form and decreased levels of GLUL leads to neurodegenerative changes manifested as dumb form.
Open Access
IMMUNOHISTOCHEMICAL LOCALISATION OF M2 MACROPHAGES IN CANINE MAMMARY TUMOURS USING CD163 MARKER
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-16) NAVYA ELIZABETH SAJEEV; Dr. Sajitha I. S.
The present study entitled “Immunohistochemical localisation of M2 macrophages in canine mammary tumours using CD163 marker” was undertaken to study the expression of a phenotypical marker of M2 macrophages namely CD163 in CMT. It has been reported that presence of the protumourigenic M2 macrophage phenotype was associated with faster and worse progression in human breast cancer. But the studies in veterinary oncology are sparse in this area. Hence, the differential expression of the marker with respect to the grade of the tumour was also evaluated. Excision biopsy samples of mammary tumour suspected growths from presented to University Veterinary Hospitals, Mannuthy and Kokkalai were collected for the study during the time period of December 2020 to October 2021. The highest incidence of CMT was seen in the age group of seven to eight years. All the mammary tumours recorded were in female dogs. Dogs belonging to the breed Labrador (28%) represented the majority of the cases followed by Cross breed (16%) and Dachshund and Rottweiler (12%). Ductal carcinoma, tubulopapillary carcinoma, solid carcinoma, comedocarcinoma, cribriform carcinoma and mixed type of tumours were the histological types observed. Of the 25 samples collected 44% were grade II tumours whereas 28% were grade III and 28% were grade I tumours. Immunohistochemistry was performed to quantify the presence of M2 macrophages by the expression of CD163 marker protein in all cases of canine mammary tumours. Sixteen per cent (4/25) of the tumours were strongly positive (+++) and fifty-six per cent (14/25) were moderately positive (++). The remaining twenty eight percent (7/25) showed weak immunostaining (+) for the protein. Statistical analysis revealed significant positive correlation between the grade of the tumour and CD163 expression (p=0.01). High grade malignant tumours were shown to have strong to moderate CD163 expression, indicating the higher infiltration of M2 macrophages. Lower grades or benign tumours had weak expression of the marker protein indicating lower infiltration. The present study identified the significance of M2 macrophage marker, CD 163 in CMT, with respect to grades of tumours. Further studies with larger number of samples and analysis of its expression in various stages of the disease will help in confirming its role as a prognostic and therapeutic biomarker in CMT.
Open Access
HISTOMORPHOLOGICAL, IMMUNOHISTOCHEMICAL AND MOLECULAR STUDIES ON DESMOPLASTIC REACTIONS IN CANINE MAMMARY TUMOURS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR,KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-11-10) DEVI S.S; Dr. Ajith Jacob George
The present study entitled ‘Histomorphological, immunohistochemical and molecular studies on desmoplastic reactions in canine mammary tumours’ was conducted to analyse the role of cancer associated stroma in canine mammary tumours. The histopathological features and grades of tumours were examined in relation to stromal types. As part of the study, the stroma was stratified histomorphologically, expressions of relevant genes and localisation of various proteins involved in stromal reprogramming were examined.A total of 50 canine mammary tumour cases presented to the University Veterinary Hospitals at Mannuthy and Kokkalai during the period from March 2019 to March 2021 were considered for the present study. Occurrence of CMTs was found to be more in female dogs between eight to 12 years. Inguinal glands were found to be the most affected glands. Out of the 50 cases, 39 tumours were malignant of which 30 tumours had either rich or moderate quantity of stroma. These 30 tumours were subjected to histological subtyping and malignancy grading. Ductal carcinomas were identified to be the most common histological type and among the observed cases, Grade II tumours formed the majority. Stroma associated with all these tumours were histomorphologically classified into fibrotic, inflammatory and mixed types. Fibrotic stroma was again subdivided into sclerotic, desmoplastic and stroma of intermediate maturity. On analysing the stromal type in relation to various histological types of CMTs, it was observed that inflammatory or desmoplastic stroma were a feature of highly aggressive tumours like micropapillary carcinoma, comedocarcinoma and carcinosarcoma, while majority of the ductal carcinomas and carcinoma arising in benign mixed tumours had sclerotic stroma. Similarly sclerotic stroma was a feature of Grade I carcinomas while higher grade tumours had either desmoplastic or inflammatory stroma. The cytological features of carcinomas in the study were also analysed in relation to stromal types. Cytological grading of tumours based on the evaluation of stained fine needle aspirated from tumours gave 87.5 per cent concordance rate with histological grading system. Sensitivity and specificity of cytological grading with respect to histological grade was assessed and 100 per cent sensitivity was identified for Grade 1 carcinomas, while the greatest specificity was observed for Grade 3 carcinomas. Grade 1 carcinomas were mainly having sclerotic stroma while Grade 3 carcinomas had either desmoplastic or inflammatory stroma. The desmoplastic stromal reactions in CMTs progressed through the sequential stages of TACS-1, 2 and 3 similar to that described in human breast cancers. Scanning electron microscopy and special staining techniques using Masson’s trichrome, Picrosirius red and Herovici were also employed to demonstrate the desmoplastic reactions occurring in the stroma associated with CMTs. The expression of three main genes involved in desmoplasia and stromal reprogramming namely α-SMA, TGF-β1 and MMP-9 were analysed in normal mammary gland, low grade non metastatic carcinomas and high grade metastatic carcinomas. It was observed that all the three genes were significantly upregulated in high grade metastatic tumours. Along with α-SMA, TGF-β1 and MMP-9, two more proteins of significance viz FAP and SDF-1 were subjected to localisation by IHC. Statistical analysis using Fisher’s exact test revealed significant association between IHC scores of these proteins and grades of tumour. Increased immunostaining corresponded to increase in tumour grades. An understanding of cancer associated stroma (CAS) and its cross talk with tumour cells is very much crucial while predicting prognosis and designing therapeutic protocols. With respect to Veterinary oncology, characterization of CAS, identification of diagnostic and prognostic stromal markers and actionable therapeutic stromal targets remain unexplored even today and thus, the present study was a basic and preliminary attempt to characterise the desmoplastic stromal reactions and identify stromal molecules that could aid in diagnosis, prognosis and treatment of CMTs.
Open Access
POST MORTEM EVALUATION OF AVIAN PANCREAS AND THYROID WITH SPECIAL REFERENCE TO INFECTIOUS DISEASES
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-09) GEETHANJALI RAMACHANDRAN; Dr. Pradeep M
Poultry farming and pet bird rearing and breeding are booming sectors in the country but diseases affect the growth detrimentally. Even though occurrence of infectious conditions were studied well, affections of exocrine and endocrine glands were less explored. The present study focused on the systematic recording of gross and histopathological changes in pancreas and thyroid of birds and screened for bacterial and viral infectious agents such as Marek’s disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV). A detailed post mortem examination of 116 poultry and pet bird carcasses was carried out, gross lesions were noted and representative samples were collected from pancreas, thyroid and other visceral organs for histopathology and molecular screening for viruses. Culture was done on suitable medium for bacterial isolation and identification. Gross pancreatic lesions observed in 26.72 per cent of carcasses in which parenchymal mottling (14.6 per cent) was the major gross lesion. Microscopically, Atrophic changes (33.6 per cent) and inflammatory cell infiltration (25.9 per cent) were the most frequent lesions observed in exocrine pancreas while degenerative changes were the major lesions in endocrine pancreas. Gross lesions in thyroid were observed in 23.27 per cent carcasses among which mildly enlarged and red-brown coloured thyroid (14.65 per cent) was the major gross change observed. Microscopically, congestion (35.3 per cent) and inflammatory cells infiltration (25.8 per cent) were the most frequent lesion observed in thyroid. Grading for atrophic changes in pancreas was based on shrinkage of acinar cells with depletion of zymogen granules, cytoplasmic vacuolation and interstitial fibrosis. Grading of infiltration and proliferation of lymphoid neoplastic cells in thyroid were also made. Infectious agents such as E. coli, MDV, IBV, NDV or IBDV were detected in 60.34 per cent of the samples among which the occurrence of E. coli infection (22.4 per cent) was highest followed by MDV (13 per cent). It was observed that younger chicken were the most commonly affected age group. Pathology associated with detected infectious conditions were evaluated.
Open Access
GROSS AND HISTOMORPHOLOGICAL EVALUATION OF THYROID GLAND IN DOGS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-09) NEETHU B; Dr. Prasanna K.S
Thyroid gland is one of the important endocrine gland required for the functioning of almost all the organs of the body and its impairment can adversely affect the body functions. The study of the morphological characters and pathophysiology of the thyroid in dogs is scanty. The present study entitled “Gross and histomorphological evaluation of thyroid gland in dogs” was conducted to find out the pathological findings of the thyroid gland and to correlate it with various infectious diseases affecting the dogs. Sixty-one pair of thyroid glands were collected from dog carcasses brought to the Department of Veterinary Pathology, College of Veterinary and Animal Sciences University, Pookode from various northern districts of Kerala formed the materials of the study. The tissue samples were examined as per the standard operating procedures to screen various gross lesions and further collected in 10 % neutral buffered formalin for histopathological examination. Pooled tissue samples were also collected and stored at -20 ̊c for DNA and RNA extraction for various pathogens. Gross morphological examination of the gland was performed to examine the morphometrical parameters like colour, consistency, length, weight and shape of the gland. Gross pathological lesions were found in 54 dogs out the 61 examined, which constitute an incidence of 88.52 % and histopathological lesions were observed in 58 dogs out of 61 (95 %) which shows high incidence of thyroid pathology in dogs. The gross lesions encountered were paleness of thyroid (31.5 per cent), dark brownish discolouration (51.85 per cent), cystic changes (1.8 per cent), shrinkage of thyroid (44.44 per cent) nodular growth on the dorsal surface (18.52 per cent) as well as nodule embedded deeply in the thyroid parenchyma (27.78 per cent).The histopathological evaluation revealed predominance of non-neoplastic lesions and the lesions encountered were classified as vascular (68.96 per cent), hyperplastic (15.51 per cent), degenerative (82.75 per cent), cystic changes (10.37 per cent), calcification (3.45 per cent), colloidal goiter (27.59 per cent), hyperplastic goiter (15.51 per cent) and neoplastic lesions (4.9 per cent). Molecular detection was also carried out after DNA or RNA extraction. PCR revealed Rabies (22/61), Canine parvovirus (9/61), Canine babesiosis (2/61), Canine herpes (1/61) cases. The present study revealed interaction between thyroid pathology and infectious diseases.
Open Access
PATHOLOGY OF AVIAN REPRODUCTIVE ORGANS AND MOLECULAR DETECTION OF ASSOCIATED VIRUSES
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-12-17) ALIN MOIDEEN T K; Dr. Pradeep M
Economic viability of layer farms and avian breeding houses depends on good egg production which in turn depend on maintenance of healthy reproductive system. The present study entitled “ Pathology of avian reproductive organs and molecular detection of associated viruses” focused on systematic recording and classification of various gross and histopathological changes in ovary and oviduct of female and testis of male bird carcasses and molecular detection of associated viruses such as Marek’s disease virus (MDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), fowl adenovirus (FAdV), avian leukosis virus (ALV), reticuloendotheliosis virus (REV), duck virus enteritis virus (DVEV) and duck Tembusu virus (DTMUV). Three hundred bird carcasses of various species were examined, gross lesions recorded and samples of ovary, oviduct and testis were collected for histopathological and molecular detection. The bird carcasses included different species such as chicken (155), duck (17), turkey (2), guinea fowl (1), quail (5), silver pheasant (1) and parrots (2) and were further grouped into chick/duckling, grower, and adult age groups. Gross pathological changes in reproductive system were observed in 41 percent birds of which 88.62 percent were females. In males, congestive changes in the testicular tunic was the major lesion (8.87 percent) followed by testicular atrophy, lithiasis and neoplasm. In ovary congestion of ovarian follicles (27.27 percent) was the predominant lesion followed by atrophy, egg yolk peritonitis, follicular rupture and hemorrhage, neoplasia and abnormal follicular atresia. The major gross lesions in the oviduct was serosal congestion (25.57 percent) followed by atrophy, salpingitis, egg bound condition, cystic right oviduct and neoplasia. Various histopathological lesions were observed in testicular tunic, seminiferous tubules, epididymis and interstitium of testes including a testicular teratoma. Ovary and oviduct showed vascular changes, ovarian granulosa cell degeneration, atrophic and degenerative changes of mucosal and glandular epithelium of oviduct, ovaritis, salpingitis, and neoplasia. Molecular detection revealed the presence of MDV, REV, IBV, IBDV, FAdV, DVEV, and NDV with or without coinfection in the carcasses while only MDV and REV were detected from the reproductive organs. The study revealed various affections that were not prominent grossly and pressed the need of histological and molecular methods for identifying underlying reproductive disease conditions in birds.
Open Access
PATHOLOGY OF GASTROINTESTINAL LESIONS AND SCREENING FOR ASSOCIATED ENTEROPATHOGENIC VIRAL INFECTIONS IN PIGS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, 2022-06-17) ELIZABETH VARGHESE; Anoopraj R.
The present study entitled “Pathological study of gastrointestinal lesions and screening for associated enteropathogenic viral infections in pigs” focused on systematic recording of gross and histopathological changes in stomach, intestine and mesenteric lymph nodes and screening for Group A rotavirus (GARV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhoea virus (PEDV) and porcine sapovirus (PSaV). A detailed necropsy of 57 pig carcasses were conducted, gross lesions were recorded and samples of stomach, intestine and mesenteric lymph nodes were collected for histopathology and molecular screening. Gross and histopathological changes in either stomach, intestine or mesenteric lymph nodes were evident in all the carcasses presented. Major gross lesions in stomach varied from mucosal erosions to ulcerations, hyperkeratinised pars oesophagea and diphtheritic membrane. Microscopically, the important lesions observed were mucosal necrosis, erosions and ulcers, cellular infiltration in lamina propria, hyperkeratinisation and fibrino-necrotic membrane. Salient gross lesions in intestines were catarrhal to haemorrhagic enteritis, mucosal ulcers in caecum and colon, fibrino-necrotic membranes in ileum, caecum and colon, flaccid and thin-walled small intestine and mesocolonic edema. Principal microscopic changes included Brunner gland hyperplasia, fibrino-necrotic membrane in ileum, caecum and colon, hyperplastic crypts, villous atrophy and fusion, crypt abscess, neutrophilic colitis, hyperplasia and necrosis in Peyer’s patches in ileum and granulomatous infiltration in ileum and colon. Gross lesions in mesenteric lymph nodes were enlargement, congestion and haemorrhage. Major histological changes were varying degrees of lymphocytic depletion, histiocytic and granulomatous infiltration. GARV, TGEV, PEDV and PSaV were not detected in any of the samples screened. However, 80.70 per cent were infected with either PCV2, Salmonella or E. coli among which the incidence of Salmonella isolates were the highest (50.88%). It was observed that weaners were the most commonly affected age groups. Coinfections with at least two of these pathogens were observed in 49.12 per cent cases and severe lesions were encountered in coinfections.
Open Access
PATHOLOGY OF CARDIO-PULMONARY AND RENAL AFFECTIONS IN DUCKS AND SCREENING FOR ASSOCIATED VIRUSES
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD, 2022-02-25) ANAGHA A M; Pradeep M
The present study entitled “Pathology of cardio-pulmonary and renal affections in ducks and screening for associated viruses focused on systematic recording of gross and histopathological changes in heart lungs and kidneys and screening for MDPV, DAdV-1 and ANV. A detailed post mortem examination of 119 duck carcasses was carried out, gross lesions noted and representative samples were collected from heart, lungs and kidneys for histopathology and molecular screening. Gross lung/airsac lesions were observed in 31.09 per cent carcasses and the lesion varied from cloudy airsacs, nodules in air sacs, congestion, consolidation and nodules in lungs. Microscopically lung showed varying degrees of mononuclear cell infiltration and vascular changes. Gross lesions in heart were observed in 20.16 per cent of duck carcasses and the lesion varied from deposition of white material on pericardium, pale discolouration of cardiac muscles, rounding of heart and ventricular hypertrophy. Microscopic lesions included fibrinous pericarditis, myocardial degeneration, epicardial thickening, myocardial fibrosis, microcapillary dilatation and myocardial hemorrhage. Gross lesions in kidneys were congestion, deposition white material, atrophy and enlargement of kidneys. Microscopic lesions in kidneys were associated with glomeruli, tubules, interstitium and vascular changes. Sclerotic changes in kidneys were graded based on extend of fibrous tissue proliferation in Masson trichrome stained sections.A grading system for chronic changes in kidneys based on Masson trichrome staining was carried out for the first time in birds. Grading of lung inflammatory lesions were also made. Adult ducks reared under intensive system showed more chronic changes compared to ducks reared in backyard system which showed more of acute lesions.

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