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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
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 Nair
The 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.
Open Access
PREVALENCE AND PATHOLOGY OF POLIOENCEPHALOMALACIA IN GOATS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 1999) N. DIVAKARAN NAIR; A. Rajan
Polioencephalomalacia is a significant emerging disease problem in goats Although, PEM in goats was disease problem m goats. recognized and reported as early as 1956, its etiology is poorly understood and symptomatolgy, pathological features and therapeutic approach have not been well defined and documented. Hence an investigation was undertaken to assess the prevalence of the disease based on the data available from i -F i-hw citate for a period from 1991 76 Veterinary hospitals of to 1994. This data documented revealed an increasing trend to the occurrence of the disease and significantly high o n 4 Pt +-hP first five months of which incidence was recorded m the first a. amVhcprvpd in the month of April, the peak incidence was observe spontaneous cases of the disease were studied m detail and experiments were conducted taking goat as a model using selected incriminating agents such as Amprolium ;350 mg/kg body weight), Amprolium and rice gruel (350 mo/kg, and ad libitum rice gruel), nee gruel (ad libitum), sodium sulphate (150 mg/kg body weight followed by 500 mg/kg body weight on the 10th day) BHC (2.5 mg/kg followed py 5 mg/kg body weight on the 10th day) and Ficus tsiela mOXb. The experiment was for a period of 45 days. 5V ii /-mptomatology, weight of the animals at fortnightly intervals, weight of the brain, CSF protein concentration. Drain autofluorescence, gross and histopathological alterations of the brain and ultrastructural pathology were the markers utilized for evaluating the disease processes. The sodium sulphate, BHC and Amprolium and rice gruel created group showed symptoms and lesions more or less similar to the spontaneous cases. Only few animals in each group developed the disease such as two in amprolium and rice gruel treated group, four in BHC treated group and chree in sodium sulphate group. This showed that individual idiosyncracy plays an important role in the manifestation of the disease. The symptoms developed at different latent periods were not progressive as compared to the spontaneous cases where Che symptoms were progressive. The symptoms included lethargy, depression, knuckling at the fetlock, frequent cremors, opisthotonos and loss of eye preservation reflex. Blindness was seen in one of the, natural cases. The histological lesions of the brain in all the cases were comparable in different segments of the brain. Mostly ic was characterized by diffuse laminar cortical aeaeneration and necrosis, occasional neuronal swelling, ii i giial cell reaction and white matter vacuolation. Vascular changes predominated in the sodium sulphate group and also in the natural cases. There was glial cell response in the form of nodules in sodium sulphate group and natural cases. A predominant perivascular and neuropil accumulation of lymphocytes, gitter cells and monocytes were .seen in the natural cases. These were considered as secondary deposition following a toxic degenerative neuropathy. The necrotic focicould well be delineated in few of these cases by the bluish or creamy autofluroscence of the affected brain, but was not found to be of any primary diagnostic value as all the affected brain did not show fluorescence. Ultrastructural investigation revealed the basic reaction of the brain tissue to be similar in both the experimental and natural cases except for their intensity. Ultrastructural lesions were characterized by neuronal swelling, membrane lysis, segregation of the filamentous and granular component of nucleolus, cytoplasmic organellar aamage such as fragmentation of RER, partial degranulation of ribosomes, mitochondrial swelling, cristolysis and lornplete disappearance of organelle. Neuropil spongiosis and splitting of myelin at the intraperiod line and firmation of multiple vacuolations of the white matter were • onaracteristic. From this observations it was clearly iv deiineated that the primary insult was a biochemical one which caused much damage to the volume control mechanism of the cell and subsequent cellular damage. The CSF protein evaluation revealed high protein level in the spontaneous cases whereas in the experimental cases, the concentration remained within the normal range indicating that it has no diagnostic value. In this investigation it has not been possible to induce PEM with Amprolium even at a dose rate of 350 mg/kg body weight and it was proved that amprolium is not a cause for PEM. Rice gruel ad libitum was found to be tolerated by the animal except one which showed dullness and abdominal distension towards the end of the experiment. Diffuse neuronal degeneration was observed in the brain of this animal. Based on this observation it was concluded that rice gruel consumption every day as a component of the concentrate feed in goats might not cause any detrimental effects and the problem comes only when it is fed in large quantities on a single day. B'^icus tsiela Roxb. though produced vascular damage and aiifuse neuronal degeneration in one of the experimental animals, goats were found to be highly resistant to Ficus r-iela Roxb. toxicity and the variation in the susceptibility of different species to this toxicity was brought to light. From this investigation it was also clarified that sodium sulphate, BHC, rice ^ and amprolium could be initiating agents of PEM under certain circumstances and no single cause seems to be responsible for PEM.
Open Access
ASSESSMENT OF THE ROLE OF AFLATOXIN IN THE AETIOLOGY OF CARCINOMA OF THE MUCOSA OF THE ETHMOID
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1995) SURINDER CHAUDHARY, K.; Rajan, A.; SURINDER CHAUDHARY, K.
The present investigations were planned to assess the role of aflatoxin B, (APE.) and/or virus in the aetiology of ethmoid carcinoma using pig as a model in yivQ and bovine ethmoid mucosa culture in vitro. Thirty-two, Large White Yorkshire piglings of two-three months age were procured from the University Pig Breeding farm, Mannuthy and divided at random into four groups of eight each. The pigs in group I and group II were administered aflatoxin B^ (0.070 mg/kg b.wt/inoculum by intravenous route at weekly interval for six months) and/or ethmoid tumour extract (2 ml/pig/inoculum, intranasally, at fortnight interval for three months) . The pigs in group III were administered ethmoid tumour extract alone, while the pigs in group IV were kept as negative controls. During the period of observation of 18 months all the pigs of different groups given AFB, and/or ethmoid tumour extract appeared healthy and no clinical manifestation of the carcinoma of the mucosa of ethmoid was observed. However, there was appreciable reduction in the weight and mild degree of depression. 11 In the AFBj^ treated pigs, sacrificed at 9, 12, 15 and 18 months of investigation, the ethmoid mucosa had greyish white, soft and oedematous appearance along with scattered small pale elevations at necropsy. Histologically, the ethmoid mucosa exhibited hyperaemia, varying degree of mononuclear cell infiltration and fatty degeneration in the initial stages. In the later stages, there was proliferation of mucous glands showing acinar, tubular or papillary arrangements. Occasionally papillary projection of the surface epithelium and focal squamous metaplasia were also observed. Ultrastructural features of the cells of the ethmoid mucosa consisted of both productive and degenerative changes. The cells had sparse cytoplasmic organelles. The poor cytoplasmic contents and irregular nucleus with nucleolar margination were the other electron microscopic features observed in the ethmoid mucosa of AFB^ treated pigs. AFB^ in the range of 43.12-139.43 ppb could be detected in the blood of 52.37 per cent of the ethmoid tumour bearing cattle analysed in the present study. The blood samples from the AFB^ treated pigs were positive for AFB^ (40-160 ppb) upto 10 days after the withdrawal of treatment whereas AFM^ could be detected in blood sample of one pig only upto 3 days after the treatment. The ethmoid mucosa analysed after 3 months and at subsequent specified intervals was consistently negative for AFB and AFM By concerted efforts cells of the mucosa of the ethmoid were established ^ vitro. AFB^^ treatment of long term epithelial cultures initiated from the primary culture of bovine ethmoid mucosa origin resulted in morphological transformation accompanied by increased growth in soft agar and cytochemical positivity of gamma-glutamyl transpeptidase. This confirmed the tumourigenicity of AFB^. The xenotransplantation of these iji vitro transformed epithelial cells in mice was not successful. Electron microscopic studies of the cells of the carcinoma of the ethmoid mucosa in spontaneous cases of cattle revealed varying ultrastructural features. The neoplasticoells were either well differentiated secretory structures or undifferentiated ones. Desmosomes and tight junctions were seen between the epithelial cells. Endoplasmic reticulum and mitochondria varied in their contents and degree of disorganization. Nucleus was highly pleomorphic and predominantly euchromatinic. The retroviral like particles were demonstrated intracellularly and occasionally in extracellular spaces in the neoplastic cells of 7 tumour bearing cattle. Similar particles were also seen in the cell free ethmoid tumour extract in three of 21 tissues examined.
Open Access
IMMUNOPATHOLOGICAL RESPONSE OF THE DUCK /ylaas platyrrhyncos domesticusi TO SUBLETHAL DOSE OF SELECTED AGRO - CHEMICALS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1998) VUAYAN., N.; Valsala, K.V.; VUAYAN., N.
The study was conducted with the objective of assessing the teratologic, embryotoxic and iramunopathologic effects of two agro-chemicals carbofuran and 2,4-D used in agricultural operations, on duck embryos and ducklings. The embryological studies included the observation on the embryonic defects and weight of the lymphoid organs along with the histopathologic and ultrastructural studies of the lymphoid organs like the bursa, spleen and thymus. The results showed that the contamination of the eggs with Che above chemicals caused moderate deleterious effects on Che lymphoid system, though no teratologic effects could be Observed. The histological and ultrastructural changes also confirmied the deleterious effects at the tissue and cellular 1evel. The immune system of the ducks exposed to the above reals for two different durations were assessed using a battery of tests. The humoral immune system was assessed based on the body weight, lymphoid organ weight haematological evaluation, serum biochemical evaluation and the evaluation of HI titre against NDV. The cell-mediated immune system was assessed by the skin reactivity tests to DNCB and PHA. LMIT and GVHR were also employed to assess the cell-mediated immune system. The histological and ultrastructural studies were also conducted after six weeks and ten weeks of the experiment. The assessment of the immunological profile employing these elegant tests revealed that these agro-chemicals caused mild but significant suppression of the cell-mediated and humoral immune response. By this investigation it was clarified that these agro-chemicals have mild to moderate degree of immunotoxic effect when exposed for a short duration, but the changes were severe when exposed for longer duration. From the studies, it was demonstrated that the immunotoxic effect of carbofuran is more compared to 2,4-D (herbicide) and the changes were dose and time dependent.
Open Access
COMPARATIVE STUDY OF THE IMMUNOPATHOLOGICAL RESPONSE IN OCHRATOXIN A, CADMIUM AND MERCURY TOXICITIES IN DUCKS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Thrissur, 1996) VYAS MADHAVRAO SHINGATGERI; Rajan, A.; VYAS MADHAVRAO SHINGATGERI
The present investigation was undertaken for the first time to assess'the level of contamination of mercury (Hg) and Cadmium (Cd) in the soil, water, plants and biosamples in localities where there is significant duck population and to study the pathological effects of these chemicals and ochratoxin A (OA) with special reference to immune response in the ducks and duck embryos. The residues of these chemicals in various tissues of the ducks were also assessed. Hg and Cd were detected in all the samples from all the localities surveyed and its presence especially in the soil, vegetation, fish, liver and kidney of the ducks from these areas indicated its public health significance. After the administration of OA, HgClj and CdClj, there was dose dependant embryo mortality and reduction in hatchability rate. The mortality percentage in the highest dose group of embryos inoculated with HgClj, CdClj and OA was 55s, 51.6s and 48.3% respectively while the hatchability rate was 29.92%, 35% and 47.83% respectively. This clearly indicated the embryotoxic action of these chemicals. The body weight, weight of the thymus, and the bursa of Fabricius was found lowered in the embryos inoculated with these chemicals. The variations noticed in the weights at different periods of observation between the chemicals were also dose dependant. The weight of the spleen of the embryos inoculated with OA and HgClj was comparable to the normal embryo but with CdCl^, it was found significantly lowered in the highest dose group at the 28th day of incubation. These observations, therefore indicated that Cd has an adverse significant biological effect on the lymphoid organs of the developing embryo. Similar effect was observed with Hg and OA but was relatively low. Histologically, depletion of the lymphoid elements consequent to degeneration, necrosis and cytolysis was the characteristic feature in the bursa of Fabricius and thymus, however, it was comparatively less in the spleen. Ultrastructural changes in the lymphoid cells in all these organs showed membrane alterations, mild to severe mitochondrial changes, ribosomal detachment and fragmentation of RER and nucleolar and nuclear changes indicative of cell damage, and marked reduction in differentiation into plasma cells. Epithelial cells were also found affected. The toxic effects of these chemicals were more or less similar in the bursa of Fabricius, intensity and severity being more with CdClj followed by HgClj and OA in that order. In the thymus, also, CdCls caused severe pathological effects while with HgClj intensity was least. In the spleen there was increased erythrophagocytosis in addition to these changes and was more severe with HgClj. In the liver, severe necrobiotic changes were found in the hepatic cells associated with ultrastructural picture of organellar damage and lytic changes in the nucleus. Int6nsity of th6S6 changes was tnore severe in Cd.Cl2 treated eTubryos followed by those treated with OA and with HgCl2. In the kidney, the cellular alterations were more in the proximal convoluted tubules and the intensity of these alterations was less in OA and CdCl2 treated embryos compared to those treated with HgClj. The embryos inoculated with OA, HgClj and CdClz, 2-evealed degenerative changes in the lymphoid and other organs. The adverse immunobiological effects of OA, Hg and Cd on the developing embryo were established by histological and ultrastructural studies. In the ducks, OA, HgCls and CdClj were fed orally (at the rate of 300 ug, 10 mg and 15 mg/kg body weight respectively) for 90 days. Clinically ducks showed dullness, restlessness and unusual hyperresponsiveness and inco-ordination of movement, in OA, HgClj and CdClj fed ducks respectively at the later stage of the experiment. Progressive reduction in the gain of the body weight, of the bursa of Fabricius was noticed in all the groups (OA, HgCl2 and CdCl2 treated) while that of the thymus was noticed mainly in CdCl2 and OA treated ducks. There was highly significant reduction in the total erythrocyte count (TEC), Haemoglobin (Hb) concentration and Packed Cell Volume (PCV) in the groups fed OA, Hgcl2 and CdCl2. Highly significant leucopaenia with relative heterophilia and lymphocytopenia was observed in OA and HgCl2 fed ducks, while highly significant leucocytosis with heterophilia and lymphocytopenia was seen with CdClj fed ducks. These alterations revealed the toxic action of these chemicals on the haemopoietic cells and the formed elements of the blood. Estimation of the haemagglutination inhibition (HI) antibody titre, total serum protein (TSP), serum globulin (SG) and serum IgG and IgM levels clarified the adverse effect of OA, Hg and Cd on the humoral immune system, which was evident at an early stage in Cd and Hg treated ducks while at a later stage in OA fed ducks. There was marked reduction in the serum IgM and IgG level in response to New Castle disease Virus (NDV) in ducks fed HgClj and CdCl^ respectively. The highly significant reduction in the T-lymphocyte count in the peripheral blood, increase in the migration indices in response to NDV in leucocyte migration inhibition test (LMIT), reduction in cutaneous response to phytohaemagglutinin-M (PHA-M), 2,4-dinitrochlorobenzene (DNCB) and the decreased spleen indices in Graft versus host reaction (GVHR) indicated the toxicity of OA and Cd on the cell-mediated immunity, which was more pronounced and evident at an early stage in Cd treated ducks than compared to OA treated ducks. However, the results of the migration indices, PHA-M cutaneous response and GVHR demonstrated that the CMI response escape the toxic insult of the Hg and is dependant on the duration of exposure, which was clarified by T-cell dependant lymphocytopaenia and reduction in cutaneous response to DNCB at the last week of the experiment. Significant reduction in the phagocytic ability in all the ducks treated either with OA, HgClj or CdClj was observed by carbon clearance assay. There was reduction in the weight of the bursa of Fabricius and spleen. The thymus showed focal areas of congestion and petechiae in Cd induced toxicity, and was found appreciably reduced and atrophied with Cd and at the later stages with OA. Degenerative and retrograde changes indicative of necrobiosis were observed histologically. Ultrastructural changes were more severe with CdClj followed by HgCl2 and OA in that order. In the spleen of ducks fed CdClj there were proliferative changes initially followed by degenerative changes at later stages. In the thymus, effect of the Cd was more severe on the lymphocytes followed by OA and was least with HgCla. Histological and ultrastructural studies revealed degenerative nephropathic changes in the kidney associated with Hg, OA and Cd toxicity. Intensity of alterations was more with Cd. The hepatic lesions characterised by moderate to severe hepatosis were more pronounced in the case of Hg and Cd in an increasing order. Histological and electron microscopic studies elucidated the neuropathological changes associated with Hg, Cd and OA toxicity. Estimation of OA, Hg and Cd residues indicated a steady increase in the accumulation of these substances in the various tissues depending on the dose and duration of exposure. Highest concentration of OA, Hg and Cd was detected in the kidney, followed by liver. Residues of these substances at varying concentrations were also detected in the muscles, brain, spleen, bursa of Fabricius and thymus. This study brought to light the immunosuppressive effect of OA, Hg and Cd which would compromise the dichotomous defence mechanism of the ducks. Also adverse effects on hepatic, haemopoietic, renal and nervous system were documented. It was clarified by this investigation that immunotoxic effect of these chemicals could lead to breakdown of immunity and outbreak of diseases when the ducks are maintained in a polluted environment. The need for the systematic monitoring of the environment and taking appropriate steps to prevent environmental pollution were stressed. The public health importance of residues of these chemicals was brought to light.
Open Access
OCHRATOXICOSIS IN QUAILS (COTURNIX COTURNIX JAPONICA) WITH SPECIAL REFERENCE TO IMMUNOPATHOLOGICAL RESPONSE
(COLLEGE OF VETERINARY AND ANIMAL SCIENCE,MANNUTHY, 1992) AMIR ABBAS FARSHID; Rajan, A
In this investigation the pathological effects of OA in quail embryo and adult quail were studied with special reference to the immune system. Ochratoxin A (OA) was inoculated into the quail embryos, at the dose rate of 0.02 ^g per embryo. There was 68.66 per cent mortality which clearly indicated the direct action of OA. The embryos which were sacrificed on the 13th and 16th day of incubation showed significant reduction in the body weight, weight of bursa of Fabricius, spleen and thymus. Histological changes in these lymphoid organs were, atrophy, necrosis and depletion of lymphoid elements. Ultrastructurally the changes were time dependent. There were changes in the shape of the nucleus with condensation of granular and fibrillar components of the nucleoli. The adverse immunobiological effect of OA on the developing lymphoid organs was established by histological and ultrastructural studies. OA at the dose rate of 50 /ag/bird/day for a period of 60 days was administered through oesophageal intubation. Pathological changes were sequentially documented with special reference to the immune system. 2 In the OA fed quails, the clinical signs included emaciation and hyperexcitation at the later part of the experiment. There was highly significant reduction in the body weight, weight of bursa of Fabricius, spleen and thymus. The reduction in the weight of lymphoid organs gave evidence to the adverse effects of OA on the immune system. There was reduction in the total erythrocyte count (TEC), haemoglobin (Hb) concentration and packed cell volume in OA fed birds indicating the significant damaging effect on the haemopoietic sytem. Estimation of total serum protein (TSP), serum globulin (SGI) and serum immunoglobulin fractions clarified the biological adverse effect of OA on the humoral immune system. Highly significant reduction in total leukocyte count (TLC) and T cell dependent lymphopenia proved the immunosuppressive effect of OA. There was reduction in T cells, increase migration indices in response to Fowl pox antigen in leukocyte migration inhibition test (LMIT), the reduction in the cutaneous response to 2,4-dinitrochlorobenzene (DNCB) and phytohaemagglutinin- M (PHA-M) and spleen indices in the graft versus host reaction (GVHR), clarified the immunotoxicity of OA on the cell-mediated immune system. Grossly the OA fed birds had pale, friable liver, congested and haemorrhagic kidney, mild enteritis, atrophied 3 bursa of Fabricius, spleen and thymus. Thymus showed petechial haemorrhages and brain revealed cerebral congestion. The lymphoid organs showed degenerative changes and loss of lymphoid elements. Ultrastructurally the bursa of Fabricius showed alteration in lymphoid and epithelial components, lytic cytoplasm and nuclear condensation. The transformation of lymphoid cells into plasmacytoid series of cells was lacking. Similar cellular changes were also seen in the spleen and thymus. These changes gave evidence for the clinical manifestation of defective humoral and cell-mediated response. Kidney tubules were dilated and necrosis of the proximal convoluted tubules was seen along with congestion and focal haemorrhages. Thickening of the glomerular basement membrane was evident. Ultrastructurally fragmentation and lysis of plasma membrane at the luminal surface, numerous cytoplasmic vacuoles and fusion of podocyte foot processes were seen. Liver showed varying degree of fatty change. Ultrastructurally mitochondrial swelling and abnormal mitochondria to severe cytolytic changes we're observed. Brain showed, focal oedema, pyknosis, karyorrhexis, satellitosis of microglial cells and swollen vascular endothelium. This was supported by electron microscopic observations of separation of neuronal elements, fragmentation and vacuolation of perineuronal elements. No gross or histological changes were seen in the heart. Immunostimulation with levamisole at the dose rate of 0.1 mg/quail two doses at 4 day interval, resulted in 4 lymphocytic leukocytosis. TSP and SGI levels were increased significantly associated with increase in IgM and IgG. T cell associated lymphocytosis, decreased migration indices in response to Fowl pox antigen in LMIT, increased cutaneous response to DNCB and PHA-M, were also recorded. There was also elevation of TEC, Hb concentration and PCV. The histological examination of lymphoid organs of immunostimulated quails revealed hyperplasia and blastoid transformation in the bursa of Fabricius, spleen and thymus. This study brought to light the immunotoxicity of OA on the humoral and cell-mediated immune system. ' In addition to this,adverse effects on hepatic, haemopoietic and nervous systems were also documented. Levamisole was demonstrated to regulate the immunodeficiency induced by OA in quails.
Open Access
EMBRYO MORTALITY IN CHICKEN
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Trichur, 1987-10-25) LALITHAKUNJAMMA, C. R; Krlshnan Nalr, M

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