ThesisItem Open AccessPATHOLOGY AND MOLECULAR DIAGNOSIS OF NECROTIC ENTERITIS IN CHICKEN(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) BEHERA, DEBASISH; Pathak, Debesh ChandraThe present research work was carried out with an aim to study the pathology necrotic enteritis with isolation and molecular detection of C. perfringens and experimental production of the NE in chicken to compare between the C. perfringens type A and C in terms of hematological, biochemical and pathomorphological alterations. Total 320 numbers of samples based on different clinical signs/pathological conditions were collected from 15 districts of Assam. Isolation and identification of C. perfringens was done by cultural and morphological characteristics and confirmation was done by detection of cpa gene of C. perfringens by PCR. In this study 20 (15.03%) intestinal content and 9 (4.81%) cloaca swabs were found to be positive for cpa gene of C. perfringens. Isolation of bacteria from the samples collected during monsoon was found to be highest in comparison to other seasons. Study showed around 80% of the total isolates of C. perfringens was from the birds of 4-6 weeks of age. The C. perfringens isolated from the enteritis samples were found to be highest. Total 29 samples were found to be positive for cpa gene (324 bp) encoding for alpha gene and cpb gene (180 bp) encoding for beta gene was detected in 11 isolates. The additional virulence toxin genes of C. perfringens like TpeL and NetB were also detected. The gross lesions of NE in field condition revealed haemorrhagic, eroded, detached dead mucosal tissues in intestine. Formations of diphtheritic membrane, distention of intestine were also observed in intestine. Liver, kidneys and lungs showed congestion, haemorrhage and focal areas of necrosis. Spleen and Bursa of Fabricious in some birds was found to be moderately enlarged. The gross lesion of brain was found to be limited to mild congestion of meningeal blood vessels. Histopathology of NE in chickens revealed congestion of blood vessels in the lamina propria and submucosa with vacuolation of epithelial cells of intestinal villi along with necrosis making the villi broader and shorter. Different developmental stages of coccidia were also seen in the mucosal epithelial cells. In other organs such as liver, kidneys, heart, lungs, spleen, bursa of Fabricius and brain showed variable nature of histopathological lesions like congestion, haemorrhage and focal areas of coagulative necrosis. Experimental production of NE was done in chicken by infecting C. perfringens isolate Type A and type C with and without coccidia in separate groups. The clinical signs shown by the experimentally infected birds were diarrhoea, dehydration, depression, reluctance to move, loss of appetite, ruffled feathers, drooping of wings and head and huddling. The clinical pathology of experimental birds showed, significant decrease in TEC level, hemoglobin (g/dl) level as well as in PCV (%) and significant increase in TLC in the birds of infected group in comparison to the control. Serum ALT and AST both showed a significant increase (P<0.01) and total protein showed a significantly decreased (P<0.05) level. The gross lesion revealed congestion and haemorrhage and focal areas necrosis of mucosa of intestine. Enlargement, congestion, haemorrhage with focal areas of necrosis of the liver were common gross findings in all the experimentally infected groups. This might be due to damage to RBC in entero-hepatic circulation by α toxin of Clostridium perfringens. Kidneys, heart, lungs, spleen and Bursa of Fabricius revealed moderate degree of congestion and haemorrhage. Variable degrees of vascular changes in terms of gross lesions were observed in all most all the infected groups. The histopathological lesions revealed developmental stages of coccidia (schizonts & merozoites) and infiltration of large no of mononuclear cells and few polymorhonuclear cells in intestine. The intestinal villi have undergone necrosis and necrosed cells were sloughed off from the mucosa. Liver revealed marked fatty change in hepatocytes, congestion in the sinusoids. Kidneys showed focal areas of inter tubular congestion. Heart and lungs revealed focal areas of mononuclear cell infiltration as well as congestion and haemorrhage. Spleen and Bursa of Fabricius showed depletion of follicles and brain showed neuronal degeneration and necrosis with neuronophagia. Based upon the clinical signs, gross and histopathology, the present study revealed the groups infected by both coccidia and clostridial isolate showed distinctly more pronounced qualitatively and quantitatively in terms of clinical signs and pathological lesions. It has been also observed in this study that C. perfringens type A was found to be more virulent in terms of pathogenesis and pathomorphology in comparison to C. perfringens type C. TEM evaluation of experimentally infected birds showed disruption of intercellular junctional complexes, formation of gaps between enterocytes and delimitation of boundaries of individual enterocytes. Disintegration of nuclear material, dilatation of endoplasmic reticulum, disruption of cristae of mitochondria, increase intracytoplasmic vacuolizations and membrane bound vesicles were also prominent ultrastructural alterations in this study. Data were subjected to statistical analysis and analyzed by SAS System ('Local', X64_7PRO) using one way analysis of variance (ANOVA). Means were presented as mean ± standard error (SE) and were compared by the Duncan test at the 0.05 level of probability. ThesisItem Open AccessPATHOLOGY OF HEPATO-RENAL DYSFUNCTION IN DOGS (Canis lupus familiaris)(College of Veterinary Science, Assam Agricultural University Khanapara, Guwahati-781022, 2016-07) Umesh, Tari Chubita; Tamuli, Sarojini MahantaAn investigation was carried out in naturally occurring hepatic and renal dysfunctions in dogs from in and around Guwahati city; to study the prevalence, clinical symptoms, hemato-biochemical changes, urine pathology, gross and histopathology and histoenzymic activity. During the period from June, 2015 to May, 2016; a total of 9564 dogs were surveyed for incidence of hepato-renal dysfunction; 511(5.34%) were recorded as positive out of which 148 (1.55%) were positive for hepatic, 182 (1.82%) for renal and 181 (1.89%) for hepato-renal dysfunction. This revealed that hepato-renal dysfunction is prevalent in the dogs of Guwahati region. In a detailed examination of 155 dogs, 87 (56.13%) were reported positive for both hepatic and renal dysfunctions, out of which 31 (20%) were positive for hepatic, 35 (22.58%) for renal and 21 (13.54%) were positive for hepato-renal dysfunctions. Hepatic dysfunction showed highest prevalence in pre-monsoon season (21.95%); renal dysfunction showed highest prevalence in winter season (27.02%) and hepato-renal dysfunction showed highest prevalence in post-monsoon (23.08%) season. The age-wise prevalence for hepatic dysfunction was highest in dogs belonging to age group of 1-˂3 years of age (33.33%), for renal dysfunction it was the age group of 9 years and above (37.04%) and in hepato-renal dysfunction highest prevalence was seen in the age group of 6-˂9 years (23.53%). Sex wise prevalence of hepatic dysfunction was higher in males (22.47%) whereas prevalence of renal dysfunction was higher in females (25.76%). But the prevalence of hepato-renal dysfunction was found to be almost equal in both males (13.48%) and females (13.64%). Breed wise prevalence of hepatic dysfunction was highest in German Shepherd breed, for renal dysfunction it was highest in Pug breed and for hepato-renal dysfunction, highest prevalence was also seen in German Shepherd breed. Mortality due to hepatic, renal and hepato-renal dysfunction was found to be 18.06%, with 3.87% mortality for hepatic dysfunction, 3.22% mortality for renal dysfunction and 10.96% mortality for hepato-renal dysfunction. The clinical symptoms observed were elevated body temperature, anorexia, dehydration, dull and depressed appearance, rough body coat, pale mucous membrane, rapid pulse rate, shallow breathing, emaciation, anaemia, vomiting, diarrhoea, edema, melena, dark yellow to coffee colored urine. Some cases showed exclusive hepatic dysfunction signs of jaundice, ascites and abdominal pain. Some cases showed signs of renal dysfunction like stranguria, oliguria, urea breath and ulcers in the mouth. Giemsa’s stain and Modified Knott’s method were used for detecting hemoparasites like Babesia gibsoni, Babesia canis, Ehrlichia canis and Dirofilaria immitis in the blood of affected dogs. Hematological parameters like hemoglobin, packed cell volume, total erythrocyte count, mean corpuscular volume and mean corpuscular hemoglobin concentration revealed that dogs suffered from normocytic and normochromic anaemia. Biochemical markers included in liver and kidney function tests revealed that affected dogs showed mild to severe dysfunction of liver and kidneys. Postmortem study revealed several changes in liver like centrilobular necrosis, bridging necrosis, steatosis, acute hepatitis, portal cirrhosis, Glissonian cirrhosis, biliary cirrhosis, cholangitis, hemosiderosis and glucocorticoid hepatopathy; and also in kidneys like acute interstitial nephritis, acute glomerulonephritis, chronic interstitial nephritis, membranous glomerulonephritis, membrano-prolifereative glomerulonephritis, sclerotic glomerulonephritis, protein losing nephropathy, oxalate nephrosis, amyloidosis and calcification of tubules and glomeruli. Other organs like spleen, lungs, heart, stomach, intestine and brain were also observed to be affected. Three dogs revealed neoplastic changes like hepatocellular carcinoma, renal carcinoma and metastatic cancer of liver, kidneys and lungs. Special stains like Masson’s Trichome, Periodic acid Schiff’s, Von Kossa’s and Prussian blue stains were used to differentiate the specific changes in the affected tissues which did not appear very pronounced by routine staining methods. Histoenzymic study revealed mild to no activity of LDH in degenerated cells and AKP enzyme showed moderate to high activity in the degenerated cells and sinusoidal spaces of the liver and kidneys. ThesisItem Open AccessSTUDIES ON THE PREVALENCE, PATHOLOGY AND MOLECULAR DIAGNOSIS OF DUCK VIRUS ENTERITIS IN ASSAM(Assam Agricultural University, Khanapara,Guwahati, 2016-06) SAHARIAH, PARAG JYOTI; Upadhyaya, T.N.Duck plague or duck viral enteritis is an acute and contagious and economically important viral disease of ducks having high morbidity and mortality rate. A total thirty nos. of duck plague outbreaks occurring in certain district of Assam were attended during the period of February, 2014 to March 2016. Out of a total 5252 ducks at risk, 2956 (56.28%) were affected clinically and 2449 (46.62%) died. The overall morbidity and mortality were 56.28% and 46.62% respectively, however the cause specific mortality for DP in the present study was found to be 82.85%. Highest mortality was recorded in duckling (55.80%) followed by grower (51.24%) and adult ducks (35.43%) respectively. In the present investigation, altogether 445 serum samples were collected from the ducks from affected as well as some other ducks from the surrounding areas of the outbreak from different parts of Assam. All the serum samples were subjected to indirect ELISA test for detection of the duck plague viral antibody. Out of the total 445 serum samples tested for detection of DP viral antibody, 171 (38.42%) serum samples showed positive in ELISA. A total of 131 numbers of duck carcasses were subjected to necropsy examination. Externally, the carcasses were markedly emaciated, the vent was soiled with greenish-white faecal materials and occulo-nasal discharges were also observed. Grossly, the vascular changes were invariably present in all the visceral organs including the brain. The longitudinal folds of the esophagus showed presence of thick yellowish-white patchy diphtheritic membrane. In a few cases, the intestinal annular bands appeared as intensely reddened rings due to haemorrhages and were visible from external and internal surfaces. The liver was moderately enlarged with presence of scattered petechiae and focal greyish-white necrotic areas. The coronary vessels of the heart were engorged. There was presence of petechiael to echymotic haemorrhages in the epicardium particularly in and around the coronary groove, which give the heart a characteristics paint brush appearance. Microscopic lesions were characterized by haemorrhages, congestion, degeneration and necrotic changes of the parenchymatous organs. Liver showed varying degrees of degeneration with multiple areas of focal coagulative necrosis. Intra-nuclear, eosinophilic inclusion bodies with a distinct halo were observed inside the degenerated hepatocytes. Congestion of the blood vessels in the myocardium, haemorrhages between the muscle fibres and epicardium of heart were evident. There were rupture of the blood vascular wall and escape of blood into the surrounding musculature. The intestinal annular band showed congestion, haemorrhages and depletion of the lymphocytic cell populations. Lymphocytic depletion was also observed in the spleenic and bursal follicles. For in-situ demonstration of the DP virus Fluorescence Antibody Test was used. On fluorescent microscopy (FAT) DP virus was demonstrated in the liver, spleen, bursa of Fabricius, brain, thymus and intestinal annular band. A total of 380 numbers of samples were collected from clinically affected (107) and dead ducks (273) for molecular diagnosis of the disease. Out of total 380 samples, 231 (84.61%) post mortem samples and 68 (63.55%) clinical samples showed positive for duck plague virus specific nucleic acid. Highest numbers of tissue samples that showed positive for PCR were liver (91.80%) and spleen (91.53%). In clinical samples 79.10 per cent was positive in whole blood, 40.91 percent was positive in cloacal swabs and 33.34 percent in tracheal swab. In biochemical study, the ALT and AST activities in serum and tissues were significantly higher in DP affected ducks in comparison to the healthy ducks. The virus could be successfully isolated in 9-11 day old duck embryos from the field samples. The infected CAM and the embryos showed extensive haemorrhages throughout the body. Embryopathy was observed within 4-8 days post infection. ThesisItem Open AccessEXPERIMENTAL STUDIES ON ACUTE AND CHRONIC CHLORPYRIPHOS TOXICITY IN INDIGENOUS CHICKEN(Assam Agricultural University, Khanapara, Guwahati, 2013-06) BEGUM, SHAMEEM ARA; Upadhyaya, T. N.The present study was conducted in thirty two numbers of 3 months- old indigenous chickens. The birds were divided into three different groups viz. Group A, Group B and Group C. Group C was given no treatment and served as control for both the treatment groups. Group A Birds served as acute toxicity group and were administered a single LD50 dose of chlorpyriphos i.e. 32 mg/kg body weight. Blood was collected from wing vein or jugular vein at zero hours and post treatment at every 2 hrs interval up to 6 hrs, then at 12 hours and subsequently at 12 hrs intervals till death. Group B birds served as chronic toxicity group and were administered with 1/90th of LD50, i.e. 0.36 mg/kg body weight of chlorpyriphos WW in acute toxicity group were excitation, bloody diarrhoea and excessive salivation with drooping of wings. The birds sat on their hocks with curled toes, were unable to stand, showed tremor, convulsions and recumbency before death. In chronic toxicity group, birds showed slightly staggering gait, leg weakness, tremor and diarrhoea. Some of the birds developed curled toes with pale mucous membrane and prominent keel bones. There was reduction in body weight gain of the insecticide treated chronic toxicity group of birds. The haematological parameters (Hb, TLC, TEC) were significantly increased due to chlorpyriphos exposure in both the insecticide treated groups compared to control groups. In DLC, heterophil per cent was found to be increased and lymphocyte per cent was found to be decreased in both acute and chronic toxicity group compared to control group. Dose dependent significant increase in serum enzyme activities (alkaline phosphatase, aspertate aminotransferase, alanine aminotransferase) due to administration of chlorpyriphos were observed in both the treated groups. During the experimentation, inhibition of cholinesterase activities indicating neurotoxicity due to administration of chlorpyriphos in both the treated groups was observed. Gross lesions of chlorpyriphos treated chickens showed congestion, patchy areas of pale discolouration in the liver, with distension of the gall bladder. The striking changes in other organs, viz. kidneys, lungs and brain, were mainly congestion and haemorrhages of variable intensity with dose and time of exposure to chlorpyriphos. Histopathological alterations in the liver appeared to be dose and time dependent as evident from the severity of changes in low and high dosed chickens. The changes observed in acute toxicity were congestion, haemorrhages, focal mononuclear cell aggregation, hepatocellular necrosis, dilatation of sinusoids, mild fatty changes and disruption of hepatic cords. In addition, mild to moderate proliferation of the biliary epithelial cells around the portal vein with formation of new bile ducts were observed. Kidneys showed congestion and focal to diffuse haemorrhages in the parenchyma; cellular swelling and mild vacuolar degeneration in the tubules and focal areas of haemorrhage and coagulative necrosis along with dilatation of the Bowman’s space in the glomerulus. Lungs revealed congestion and diffuse haemorrhages with accumulation of edematous fluid and fibrous tissue proliferation in the interalveolar septa. Brain showed neuronal degeneration, satellitosis and neuronophagia in the cerebrum along with mild gliosis, demyelination and congestion. The cerebral neurons showed degeneration and formation of vacuoles in the cytoplasm. There were degenerative changes in the Purkinje cells along with demyelination in the molecular layer of the cerebellum. Intestine revealed mild haemorrhages, mononuclear cell infiltration and sloughing of mucosal epithelial cells from the basement membrane along with hyperplasia and hypertrophy of intestinal glands. The changes in the spleen were congestion and focal haemorrhages with isolated depletion of lymphocytes in the splenic follicles. Changes in the bursa of Fabricius consisted of moderate depletion of lymphocytes with congestion, interfollicular and intrafollicular haemorrhages. The harderian gland revealed mild haemorrhages, depletion of plasma cells and necrosis. Proventriculus showed mild hyperplasia of mucosal epithelium, glandular necrosis, elongation and distension of crypts and infiltration of mononuclear cells in the lamina propria. Heart showed mild haemorrhage in the myocardium. The histopathological changes in chronic toxicity were similar to that of acute toxicity but became prominent towards the end of the experimental period. Additionally, in chronic toxicity, there was mild haemorrhage in the caecal tonsils after the 3rd week of the treatment period, the intensity of which increased subsequently with mild depletion of lymphocytes in the follicles. Gross and histopathological changes in various organs of birds treated with chlorpyriphos were observed with typical organophosphorus dose dependent toxicity signs. Microscopic changes observed in different organs viz. liver, kidney, brain, lungs, spleen, caecal tonsils, bursa of Fabricius were typical to insecticide poisoning. Considerable histochemical changes were noted in the hepatocytes and biliary epithelial cells of the liver. Alkaline phosphatase showed increased activity in the proliferated biliary epithelia and moderate to strong in the sinusoids of the degenerated areas in both the treated groups. Cholinesterase activities were inhibited in the degenerated areas of the brain particularly in the grey matter in both the treated groups. Gas chromatography revealed maximum accumulation of CPF in the brain followed by muscle, liver and kidney in descending order. Ultrastructural studies showed degenerative changes in the mitochondria, endoplasmic reticulum, nucleus and cell membrane. ThesisItem Open AccessMOLECULAR BASED DIAGNOSIS ALONG WITH HISTOMORPHOLOGICAL, IMMUNOHISTOCHEMICAL AND ULTRASTRUCTURAL STUDIES ON CLASSICAL SWINE FEVER VIRUS INFECTION IN PIGS(Assam Agricultural University, Khanapara, Guwahati, 2013-07) PEGU, SEEMA RANI; Rahman, T.Classical swine fever (CSF) is a fatal viral disease affecting pigs and wild boars causing severe economic loss mainly in countries with dense pig populations. The disease is prevalent in almost all pig producing states in the country, and more particularly in the north eastern states of India. Besides clinical signs, gross and histopathological examinations, a precise laboratory diagnosis is required to confirm and to take the early decision for control of the disease during large outbreaks. In the present study, a total of six outbreaks were attended from January 2011 to January 2013. One hundred sixty-nine clinical and post mortem samples were collected from CSF suspected animals of which seventy eight samples were tested to detect E2 gene region of CSFV by nested RT-PCR and 5´ NTR in Real time RT-PCR. Nested RT-PCR showed 73% positivity and Real time RT-PCR showed 83% positivity in various clinical and post mortem samples. A total of 25 samples were tested for insitu detection of CSF glycoprotein 55(Gp 55) by Indirect fluorescence antibody tests(I-FAT) in cryosections as well as in paraffin embedded tissue sections and 18(72%) samples showed positivity in cryosections, and only 8(32%) samples showed positive result in paraffin sections. However, a total of 25 samples tested by Indirect immunoperoxidase test ( I-IPT) in paraffin sections, 18(72%)samples were found positive. Haematological study revealed significant decrease in haemoglobin percent, TEC, PCV, TLC and platelet in the CSF affected animals. In DLC, significant decrease in lymphocyte percent was recorded along with relative increase in the granulocyte percent. The clinical signs of classical swine fever were inactiveness, off fed, high rise of temperature, huddling, erythematous skin lesions, conjunctivitis and posterior weakness. The gross changes recorded were turkey egg kidney, enlarged and haemorrhagic lymph nodes and button ulcers in large intestine. Histopathological alteration recorded were interstitial and glomerular nephritis, lymphoid follicular depletion in all the lymphoid organs, perivascular cuffing, degeneration and necrosis of purkinje’s cells in the brain and ulcerative lesions and necrotic enteritis in large intestine. Enzyme histochemical study revealed weak ATPase activity in the lymphoid organs indicating B cell depletion and weak nonspecific esterase activity in the lymphoid organs indicating T cell depletion in CSF affected animals. The distribution of apoptotic cells in different lymphoid tissues were detected by TUNEL staining. There was significant increase in the number of apoptotic cells, mostly lymphocytes and smaller number of macrophages in the lymphoid follicles and interfollicular areas in lymph node, spleen, palatine tonsil and ileum. Transmission electron microscopic study (TEM) revealed, thickening of glomerular basement membrane in the glomeruli, glomerular mesangial cell proliferation, shortening and swelling of foot processes along with spherical electron dense virus particle within the dilated cytoplasmic vesicles of podocytes in the kidney. In the lymph node, TEM revealed proliferation and dilatation of rough endoplasmic reticulum cisternae and loss of cristae in the swollen mitochondria in macrophages. Lymphocyte apoptosis was observed in the lymphoid follicle characterized by condensation and margination of chromatin and fragmentation of lymphocyte nuclei and cytoplasm. In the present study, nucleic acid based technique (RT-PCR) targeting E2 gene of CSFV in various clinical and post mortem samples showed a highly sensitive and satisfactory result indicated that RT PCR is a very reliable diagnostic tool with minimum time requirement for detection of CSFV during CSF outbreaks from antimortem as well as post mortem samples. However, monoclonal antibody based in-situ immunodiagnostic assays like indirect FAT and indirect IPT used in the present study could efficiently demonstrate CSFV antigen in different lymphoid and non lymphoid organs. ThesisItem Open AccessPREVALENCE AND PATHOLOGY OF FLUOROSIS IN CATTLE IN ASSAM(Assam Agricultural University, Khanapara, Guwahati, 2013-04) Bonia, Rajib; Baruah, G. K.The present investigation was carried out with a view to determine the prevalence and pathology of fluorosis in cattle in Assam. A survey work was undertaken to know the prevalence of fluoride poisoning in cattle in some areas of Karbi Anglong, Nagaon and Guwahati, Assam by visiting door to door to the farmers house in the areas where fluoride content in the water was reported high. The overall prevalence of fluorosis with characteristic clinical symptoms and lesions in cattle in Assam was recorded as 28.63% (268/936) out of which district wise prevalence of fluorosis was 11.85% (111/936) in Karbi Anglong, 9.40% (88/936) in Nagaon and 7.37% (69/936) in Guwahati. Age wise prevalence of fluorosis was recorded as 17.94% (168/936), 5.98% (56/936) and 4.70% (44/936) in calves below one year, 1-3 years and above three years respectively. All the animals affected from fluorosis showed mild to severe dental lesions like yellowish brown pigmentation, irregular wearing and mottling. None of the affected animals showed characteristic signs of osteofluorosis. The values of haemoglobin (Hb) concentration, total erythrocyte count (TEC) and total leucocyte count (TLC) were reduced significantly in all the affected animals compared to healthy animals. The differential leucocyte count (DLC) revealed significant increase of eosinophils in all the affected animals and the neutrophils count though decreased but not significantly. Other cellular counts remained within the normal range. The urine samples of the affected animals showed presence of trace amount of bile pigment and proteins. Glucose, ketone bodies, blood and bile salts were absent. There was significant (P<0.01) variation of fluoride concentration in the serum, urine and milk between healthy and affected animals. Present study also recorded the highest concentration of fluoride in water and soil in Karbi Anglong district whereas the highest concentration of fluoride in forages was recorded in Nagaon district. Out of all the areas surveyed the highest concentration of fluoride in water, forages and soil was recorded in Kheroni areas of Karbi Anglong district. To undertake a systematic study, experimental acute and chronic fluoride toxicity in cattle below one year of age was carried out. Experimental studies on fluoride toxicity revealed variation of symptoms, clinicopathological alterations and the presence of fluoride content in the serum, urine and tissues like liver, kidney, teeth and bone which were in accordance to dose and period of exposure to the fluoride. Gross, histopathological, histochemical and ultrastructural alterations in tissues were also recorded. ThesisItem Open AccessPREVALENCE, PATHOLOGY AND MOLECULAR STUDIES OF PESTE DES PETITS RUMINANTS IN GOATS OF ASSAM(Assam Agricultural University, Khanapara, Guwahati, 2015-07) ISLAM, MUZAHARUL; Pathak, D. C.Peste des petits ruminants is an acute, febrile, emerging and economically important viral disease of goats having high morbidity and mortality rate. In the present investigation, 456 serum samples collected from affected and apparently healthy goats from different places of Assam were screened for seroprevalence of PPR in goats by HI test and c ELISA test. Out of 456 serum samples screened, PPR viral antibody could be detected in 269 samples by HI test (145 serum samples from affected goats and 124 from apparently healthy goats) and 209 samples by c ELISA test (136 from affected goats and 73 from apparently healthy goats). 60 serum samples (9 from affected goats and 51 from apparently healthy goats) showed positive in HI test but were found negative by c ELISA test. In comparative study it was revealed that HI test was more sensitive than c ELISA. The haematological study of 26 affected goats showed significant increase in total erythrocyte count, haemoglobin, packed cell volume and significant decrease in total leucocytes count. Lymphopenia was constant finding in differential leucocytes count. Biochemical study revealed significant decrease in serum protein and significant increase in serum potassium level, with nonsignificant increase of serum sodium level. All the 10 necropsied carcasses showed emaciation and dehydration with soiled hindquarters and sunken eye balls. Ulcerative lesions on gum, lips, dental pad and tongue, enteritis and linear haemorrhages on the crests of the folds of large intestine were invariably observed. The liver was enlarged with engorged gall bladder. Spleen and lymph nodes were enlarged. The lungs showed congestion and consolidation of anterior and cardiac lobes with emphysema in diaphragmatic lobes. On cut section, lung showed large quantities of white frothy exudate particularly in the bronchi. The histopathological study showed degeneration, necrosis, ulceration and sloughing off lining epithelium in lips, tongue, small intestine and large intestine. Below the ulcerated areas severe infiltration of mononuclear and polymorphonuclear cells were observed. Some cells of stratum granulosum showed presence of intracytoplasmic eosinophilic inclusion bodies. Hepatocytes showed coagulative necrosis with cytoplasmic and nuclear degeneration. The lungs showed broncho-interstitial pneumonia and presence of intracytoplasmic eosinophilic inclusion bodies. Renal tubular degeneration with coagulative necrosis and atrophy of glomeruli were observed. Edema in both the cortical and medullary areas with severe depletion lymphocytes was observed in the lymph node. Syncytial giant cells were also found in the lymph nodes. Spleen showed depletion of lymphoid population. Some lymphoid follicles were completely destroyed, leaving cystic cavities. In RT-PCR, out of 79 post mortem samples, 58 showed amplification of PPR viral nucleic acid at 463 bp for N gene using N gene specific primers. ThesisItem Open AccessPREVALENCE, PATHOLOGY AND MOLECULAR DIAGNOSIS OF POX IN DOMESTIC BIRDS(AAU, 2016) Pathak, Nayanjyoti; Baruah, G.K.In the present investigation a total of 29 nos. of fowlpox (FP), 13 nos. of pigeonpox (PP) and three nos. of duckpox (DP) outbreaks were recorded, where highest morbidity and cause specific mortality in case of FP was recorded at 0-8 weeks (19.63 % & 23.60 %) followed by 9-20 weeks (5.57 % & 14.43 %) and above 20 weeks (1.39 % & 11.11 %), respectively. Likewise, in PP highest morbidity was recorded at above 20 weeks (35.08 %) followed by 9-20 weeks (30.68 %) and 0-8 weeks (11.11 %). Highest cause specific mortality was recorded at 9-20 weeks (37.03 %) followed by above 20 weeks (30.00 %) and no mortality was recorded at 0-8 weeks. In case of DP outbreaks, highest morbidity was recorded at 0-8 weeks (10.00 %) followed by above 20 weeks (6.66 %), where no mortality was recorded among the ducks. During sero-surveillance study of FP, 27.88 % positive cases were detected by AGID, whereas 77.88 % positive cases were detected by ELISA. Similarly in case of pigeonpox and duckpox 26.82 % and 26.41 % positive cases were detected by ELISA, respectively but no positive cases were detected by AGID. Gross lesions during external examination revealed erosions, crusts and several small, multifocal to coalescing wart-like nodules on various parts of the affected fowls and pigeons, whereas in case of ducks lesions were mostly confined to bill and eye regions. During post-mortem examination of dead birds no any remarkable gross lesions were seen in various visceral organs where in few birds showed fibronecrotic lesions on mucous membrane of the oral cavity and upper respiratory tract. Microscopic lesions revealed varying degrees of hyperplasia and ballooning degeneration of the epithelial cells of the epidermis where most of the cells contained large eosinophilic intracytoplasmic inclusion bodies. During ultrastructural study, intracytoplasmic inclusion bodies were seen in the skin epithelium, which consist of numerous, dumbbell-shaped bodies typical of pox virions. During molecular diagnosis, out of 29 FP, nine PP and three DP suspected samples 86.20 %, 77.77 % and 100 %, respectively were found positive by polymerase chain reaction. Inoculation of fowlpox virus (FPV) in embryonated chicken eggs and inoculation of duckpox virus (DPV) in embryonated duck eggs for isolation showed positive results during the first passage itself , where as pigeonpox and duckpox viruses in embryonated chicken eggs required 2-3 initial passages to get the positive results. Out of the various field isolates, 10 nos. of FPV and six nos. of pigeonpox virus (PPV) were adapted on chorioallantoic membrane (CAM) of developing embryonated chicken eggs, whereas the virus isolated from ducks (n=3) were adapted on CAM of embryonated duck eggs. All total eight nos. of APV isolates were molecular biologically characterized. On phylogenetic analysis it was observed that all the isolates of APV of the present study were clustered along with other APVs corresponding to their species reported from different parts of the world. However, one isolate from duck was clustered along with the isolates of FP, which indicates natural adaptation of FPV in ducks. From the experimental study it was observed that all the chicks inoculated with FPV developed characteristic pox lesions (pustules) within 7-9 days post infection. Similarly, when pigeons were inoculated with PPV then all the pigeons developed pox lesions within 5-9 days. Again in case of ducklings all the ducklings inoculated with DPV developed pox lesions (nodular lesion) within 5-6 days. For comparative study when FPV, PPV and DPV were inoculated into heterologous hosts no lesions were developed. In seroconversion study of experimental birds by ELISA, all the infected birds (chicks, pigeons and ducklings) inoculated with species specific avipox virus showed presence of antibody. Antibody was also detected in 40 % pigeons and 60 % ducklings inoculated with FPV, 60 % chicks inoculated with PPV and 40 % chicks inoculated with DPV. The scab samples of experimentally infected chicks, pigeons and ducklings showed positive results by PCR but the biopsy samples collected from the inoculated site of the heterologous hosts showed negative results by PCR. Haematological studies in case of avipox infected fowl, pigeon and ducks from both field and experiment cases revealed low levels of haemoglobin, total erythrocyte count and total leucocyte count. In differential leucocyte count, lymphocyte and monocyte percentages were increased, whereas heterophil percentage was decreased in pox infected birds. From the experimental study it could be concluded that though the FPV, PPV and DPV field isolates of this study were host specific but if several passage done on heterologous host then it may adapt the virus and produce characteristic lesions in it. Further study will require to confirm host specificity of APVs. ThesisItem Open AccessMOLECULAR PATHOMORPHOLOGY AND IMMUNODIAGNOSIS OF PPR IN GOATS IN ASSAM(AAU, 2016) Malik, Mahmuda; Rahman, TaiburPeste des petits ruminants is an acute viral disease of small ruminants caused by genus Morbillivirus and family Paramyxoviridae. The disease is characterized by fever, oculo-nasal discharges, stomatitis, diarrhoea and pneumonia. The present study was undertaken to observe the detailed pathology of the disease in natural cases with application of different diagnostic techniques viz., gross pathology, histopathology, ultrastructural study, histoenzymic study, cell culture study, indirect immunofluorescence antibody test and PCR technique . An experimental study was also conducted to study the progression of the disease in goats. In the present study a total of four outbreaks of PPR was attended and the experiment of the diseases was conducted in twelve animals, of which six were kept as control and the rest were treated with PPR antigen. Animals suspected of PPR in field condition showed various clinical signs, like oculo nasal discharge, erosive lesion at the muco-cutaneous junction, dyspnea, greenish diarrhoea and heavy mortality. A thorough necropsy examination revealed gross erosive lesions in the oral cavity, congestion and consolidation of the lungs, linear haemorrhage in the intestine, petechial haemorrhage on the spleen, swollen edematous lymph nodes and congestion in the brain. Histopathological study revealed necrosis, desquamation and ulcer formation on the epithelium lining the tongue and lips. Degeneration and depletion of lymphoid cells in the follicles of lymph node and spleen with presence of syncytia formation. Lungs showed broncho-interstitial pneumonia characterized by thickening of inter alveolar septa with infiltration by macrophages, lymphocytes, fibrosis and a few giant cells. Hyperplasia of the bronchial epithial epithelium with presence of both eosiniphilic intracytoplasmic and intranuclear inclusion bodies in the hyperplastic cells and in the desquamated bronchial and alveolar epithelium. Denudation of the intestinal villi with presence of infiltrating cells, glandular necrosis and depletion of peyer’s patches were present. Ultra structural study showed loss of the normal cell architecture with margination of chromatin in the nucleus. At higher magnification a large number of virion-like particles which appeared as electron dense bodies with a clear halo surrounding them were seen in the cytoplasm of cell. Histoenzymic study revealed mild lactate dehydrogenase activity in the degenerated cells of lymph node, spleen and in the tip of the hyperplastic bronchial epithelium. While intense alkaline phosphatase activity was seen in the affected lung and follicles of lymph node. Cell culture study showed development of cytopathic effects viz., rounding of cell, syncytia formation and elongation in a few cells with presence of intracytoplasmic inclusion bodies. Indirect immunofluorescence antibody test showed emission of apple green fluorescence emitting from the cytoplasm of the affected cells. Experimental studies of PPR infection in goats showed development of clinical sign viz., oculo-nasal discharge, fever, coughing, diarrhea, emaciation and eventually death. Gross and histopathological changes were almost similar to the lesions observed in natural cases. The ultra structural, histoenzymic, cell culture and indirect immunofluorescence studies were performed and the results observed were also similar to that of the natural cases. The haemato-biochemical study of the experimental animals revealed a lower level of total serum protein, with higher level of serum ALT and AST. Haematological study showed an increase in TEC, Hb and PCV, and a decrease in TLC and DLC.