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ThesisItem Open Access Pathomorphological and molecular detection of avian leukosis virus infection in chicken(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Tamuly, Nibedita; Dutta, BiswajitAvian Leukosis being a common neoplastic disease of the commercial poultry farm, causes significant economic losses to the farmers. The present study was undertaken to determine the status of the infection in the poultry population from 7 different locations in Kamrup district of Assam. During the period of study, twenty two (22) outbreaks of avian leucosis were recorded from seven (7) different locations of undivided Kamrup district of Assam. A total 243 numbers of post mortem was conducted from which 65 positive cases were reported on the basis of gross examination, histopathological alteration and molecular detection. The overall mortality percentage was recorded as 4.11%. Among different age groups maximum mortality was reported in adult birds above 20 weeks of age (7.36%). However few cases were also reported below 16 weeks of age. Breed/strain wise study revealed highest mortality was reported in BV-380(6.17%) followed by BV-300 (4.47%) which was further followed by Kamrupa (3.49%) and Daothigir (3.47%). Season wise occurrence of the infection was more during winter (4.86%) followed by pre-monsoon (4.11%) and post monsoon (3.68%). Clinically, affected birds did not exhibit any typical clinical signs, however some of the affected birds showed signs like anaemia with pale comb, emaciation with decrease growth rate and productivity and osteopetrosis. The gross pathological study gives a presumptive diagnosis of the diseases where prominent lesions were found in liver, spleen, kidney and heart. In all the cases hepatomegaly was most commonly seen. The affected liver also showed nodular, eucosis or diffuse form of lesions. Spleen, kidney and heart also showed enlargement, necrosis and the presence of nodular growth. Even though the bursal involvement could not be detected due to its rudimentary form in adult birds but in two cases the lesions in bursa was prominent. Microscopic alterations were severe in liver, spleen, kidney, heart and lung characterized diffuse infiltration of immature lymphoid cells, causing distortion of normal parenchyma. Molecular detection by targeting gp85 env gene produced amplification bands at 229 bp. The phylogenetic analysis of the resultant sequences showed 99-100% homology with the endogenous forms of isolates from China, USA and South Korea. Virus can be isolated on 6th day old embryo where replication of the virus was showed by severe hemorrhages and mortality 48-96 hours of post infection. In field condition presence of other neoplastic diseases like Marek’s disease produces similar lesions which complicates proper diagnosis of avian leucosis. In such situations differential diagnosis can be made on the basis of cell cytology, histopathology and Polymerase chain reaction. In histopathology Marek’s diseases affected tissue showed infiltration of pleomorphic cells and on molecular detection positive samples produced bands at 225bp. Myeloid form and erythroid forms were not found during the study. And the present study reveals that infective form of subgroup E of avian leucosis is circulating in the residential poultry population which might undergo mutation along with exogenous forms and create a more severe form of the disease.ThesisItem Open Access Pathomorphological and molecular studies of respiratory mannheimiosis in goats(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Mazumder, Amdedul Islam; Begum, Shameem AraThe present investigation was conducted to study the pathomorphological and molecular studies of respiratory Mannheimiosis in goats for a period of one year from March 2021 to February 2022. The materials for the present study were collected from various sources such as slaughter houses in and around Guwahati and from postmortem examinations carried out in the Department of Veterinary Pathology, College of Veterinary Science, A.A.U., Khanapara, Guwahati-22. Based on gross observation 30 lungs showing lesions of pneumonia were collected during post mortem examination. Twenty one lung samples showing pneumonic lesions were also collected from slaughter houses. For detailed bacteriological and pathological studies all of the 51 pneumonic lungs were chosen. A total of 43 isolates of bacteria were obtained in the present study out of which seven isolates were morphologically and biochemically positive for Mannheimia haemolytica (16.28%). Apart from this, other bacteria isolated were Pasteurella multocida (23.26%), E. coli (20.93%), Klebsiella spp. (18.60%), Staphylococcus spp. (13.95%) and Streptococcus spp. (6.98%). All the 7 isolates of Mannheimia haemolytica were screened for Lkt and 16s rRNA gene respectively. The Lkt gene with amplicon size 206 bp and the 16s rRNA gene with amplicon size 1500 bp was detected in all the 7 isolates of Mannheimia haemolytica. The Phylogenetic analysis of 16s rRNA gene of Mannheimia haemolytica isolated from goats in the present study showed percent identity above 97 percent with other strains of Mannheimia haemolytica present in the NCBI Gene Bank throughout the world. Different types of pneumonia associated with respiratory Mannheimiosis recorded during the present study were bronchophneumonia (37.25%), interstitial pneumonia (27.45%), haemorrhagic pneumonia (19.61%), suppurative pneumonia (11.76%), and fibrinous pneumonia (3.92%). Patchy areas of consolidation in the cranioventral portion of lungs were the most commonly observed gross lesion in bronchopneumonia. Microscopically, bronchopneumonia was characterized by neutrophils and mono-nuclear cell infiltration with presence of fibrin in the bronchi, bronchiole, alveolar lumen and pleura. Interstitial pneumonia cases were characterized by enlarged and rubbery lungs which do not collapse when the thorax is opened. The interlobular septa were distended with exudate. Microscopically, alveolar wall was thickened due to infiltration of polymorphonuclear cells and lined by cuboidal epithelial cells. Alveolar lumen was also filled with polymorphonuclear cells, macrophages and desquamated epithelial cells. Haemorrhagic pneumonia cases revealed multifocal, patchy to diffuse areas of haemorrhage throughout the lung surface. Microscopically, there was hemorrhage within the alveoli and inter alveolar septa with leukocytic infiltration in the bronchus. The wall of the bronchus also showed the inflammatory changes. Areas of emphysema were also observed. Gross pathological alterations observed in suppurative pneumonia were multiple focal abscess formation on lung surface. Presence of creamy suppuration could also be noticed in tracheal lumen. Microscopically, heavy infiltration of neutrophils could be seen in bronchial and alveolar lumen. In some cases necrotic mass admixed with bacterial colonies surrounded by thick connective tissue capsule were also recorded with infiltration of polymorphonuclear cells, mononuclear cells, plasma cells and macrophages. In fibrinous pneumonia, lungs were covered with stringy net like material. Excess serous fluid was present in the pleural and peritoneal cavities. In few cases the lungs was tightly adhered to the thoracic wall due to deposition of fibrin. The interlobular septa were prominent due to accumulation of fibrin. Microscopically, fibrinous pneumonia was characterized by the presence of intra alveolar fibrin in the form of “fibrin balls” within the alveolar spaces. The traditional ‘oat cells’ and necrotic macrophages were present inside the damaged alveoli.ThesisItem Open Access Pathology and molecular diagnosis of helicobacter infection in pig(College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Dutta, Kongkon Jyoti; Tamuli, Sarojini MahantaHelicobacter is a zoonotic bacterium that has been associated with gastritis and ulcearative lesion in the stomach of pig. Prevalence of Helicobacter infection in pig was determined from four districts of Assam during the period from July, 2021 to June, 2022. The prevalence of Helicobacter infection was found to be 39.9% and 45.67% by RUT and PCR test, respectively. The age wise prevalence of Helicobacter infection revealed highest in adult pigs 42.37% (RUT) and 48% (PCR) in comparison with piglet 23.07% (RUT) and 16.66% (PCR). Among different farms the highest prevalence was recorded in unorganized farms 45.53% (RUT) and 52.17% (PCR) in compared to organized farms 32.96% (RUT) and 37.10% (PCR). The presence of HLOs in different regions of the stomach by RUT was recorded as Parsoesophagea (1.48%), Cardia (2.95%), Fundus (15.27%), and Pylorus (20.27%). The maximum positivity was recorded in pylorus and fundus region with 20.27% and 15.27% respectively. The presence of HLOs in different grades of gastric macroscopic lesions was determined. The positivity of HLOs in different grades of gastric macroscopic lesions recorded as: 1+ (Early or mild) (28.12%), 2+ (Severe) (37.03%), and 3+ (more severe+ ulcers) (56.89%). The maximum positive RUT was detected in Grade 3+ (58.89%). HLOs were detected by brush cytology from the mucosal surface satined with Giemsa and Gram’s stain in 22.66% and 16.74% of the samples respectively. The gross lesions of gastritis were recorded in 203 (58%) stomachs out of 350 pig carcasses examined. The present investigation recorded early or mild gastric lesions (1+) in 31.53%, severe lesion (2+) in 39.90% and more severe lesions and ulceration (3+) in 28.57% of the stomach examined grossly. The recorded lesions were various grades of lesions as thickening of the gastric wall, corrugation of the gastric folds, congestion and haemorrhages, necrosis and sloughing of the gastric mucosa, erosions and ulceration in the parsoesophagea as well as glandular regions of the stomach. The frequency of various histopathological lesions in affected stomach were Hyperkeratosis (17.28%), Parakeratosis (14.81%), Epithelial hyperplasia (44.44%), Glandular degeneration (60.49%), Vacuolation of glandular epithelium (56.79%), Lymhoid follicle (62.96%), Inflamatory cell infiltration (Neutrophil (32.09%), Lymphocyte (45.67%), Eosinophil (23.45%), Macrophage (28.39), Plasma cell (25.92%), Sloughing & erosion (53.08%), Ulcer, Congestion and Haemorrhages (39.50%), Glandular abscess (9.87%), Fibrosis (4.93%), Metaplasia and Dysplasia (3.70%), and Detection of Helicobacter (9.87%). Ultrastructurally, Helicobacters with bacillary morphology, inflammatory cells on the gastric mucosa and biofilm formation by coccoid form of Helicobacters were detected. PCR analysis of the gastric samples showed the presence of Helicobacter spp in 37 (45.67%) and Helicobacter suis in 17 (20.98%) samples. PCR detection rate was found to be higher from the pylorus and fundic region and severe gastric lesions of the stomach. To study the zoonotic potential of Helicobacters, a total of 30 saliva samples from pig farmers were subjected for PCR out of which Helicobacter spp. was detected in 8 numbers of samples. Further, Helicobacter suis (2) and Helicobacter pylori (3). Phylogenetic analysis revealed that Helicobacter suis detected from pig handlers and pig stomach samples shares above 99% identity suggesting zoonotic transmissions of Helicobacters from pig to human.ThesisItem Open Access PATHOMORPHOLOGICAL AND MOLECULAR DIAGNOSIS OF INFECTIOUS BURSAL DISEASE(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2020-12) ISLAM, MUZAHARUL; GOSWAMI, S.The present investigation was carried out to know the pathomorphology of Infectious Bursal Disease in different organized and un-organized poultry farms in and around the Guwahati city, Kamrup district, Assam. Altogether 1650 birds were necropsied, out of which 1279 birds from 29 different outbreaks were diagnosed as suspected of IBD based on gross lesions observed. Clinical signs commonly recorded were vent pecking, dullness, depression, anorexia, ruffled feathers and yellowish white diarrhea. In gross pathological study, bursa of Fabricius were found to be invariably affected with lesions like swelling, edema and hemorrhage. Few bursa of Fabricius showed atrophy. Moderate to severe haemorrhages in the breast and thigh muscle were consistently observed. The kidneys were enlarged and mottle with whitish pale colour appearance. Hemorrhagic lesions were also noticed in extra bursal lymphoid organs like spleen, thymus and caecal tonsils. Histopathologically, there were mild to severe depletion of lymphocytes in the bursa of Fabricius, spleen, thymus and caecal tonsils. Heterophilic infiltrations were also consistently observed in these organs. Complete depletion of lymphocytes with formation of cystic cavity were noticed in some bursal follicle. Marked thickening of the inter-follicular space with proliferated fibrous connective tissue were another characteristic bursal lesion observed. Muscle sections revealed haemorrhages and necrosis. Kidney showed degenerative changes and necrosis in proximal and distal convoluted tubules. There were glomerular shrinkage in few cases. Degenerative changes of the hepatocytes were the commonly observed histopathological changes in liver. In scanning electron microscopic study of bursa of Fabricius, moderate to severe erosion and ulceration of mucosal plica and exposure of reticular fibers were noticed after exuviations of epithelial and other cells were noticed. Button like depressed structures were seen in follicles and some follicles appeared as empty craters due to complete lymphocytic depletion. Spleen and thymus also showed moderate to severe lymphocyte depletion characterized by presence of homogenous mass. In transmission electron microscopic, spherical virus particle without a clear membrane were observed in the cellular debris of the cytoplasm of the follicular cells. Mitochondrial changes like crystolysis were also recorded. ABSTRACT The disease was confirmed by detection of viral antigen and viral nucleic acid in 165 representative samples by Indirect IPT and 612 representative samples by RT-PCR.ThesisItem Open Access PATHOLOGY 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.
