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ThesisItem Open Access ETIOPATHOLOGY OF LAMB MORTALITY IN RAYALASEEMA REGION OF ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) VENKATA RAGHAVENDRA, SANGUBOTLA; ANAND KUMAR, A(MAJOR); AMARAVATHI, P; CHENGALVA RAYULU, V; MADHAVA RAO, TABSTRACT: Present study was undertaken to study the etiopathology of lamb mortality in Rayalaseema region of Andhra Pradesh state to find out the prevalence and probable causes for lamb mortality. In an epidemiological survey, a total of 1798 lambs were recorded as dead in four districts of Rayalaseema region, of them the mortalty rate was in descending order Anatapur, Chittoor, Kadapa and Kurnool. The mortality was higher in females than males. Maximum mortality was observed in winter season and at 1-3 months age group. The mortality was majorly due to infectious origin 1533 (85.26%) followed by non-infectious origin 223 (12.4%) and miscellaneous causes 42 (2.34%). Majority of deaths were due to respiratory system affections followed by digestive system affections, multiple system involvement, combined digestive & respiratory systems, non specific affections, circulatory system, urinary system and combined respiratory & urinary system in descending order. Out of 100 faecal samples of ailing lambs examined, 68 samples were positive for different parasitic ova and oocysts. In the age group of 4-6 months it was maximum. The positive cases of parasitic ova in ailing lambs were more in females than in males. Various parasitic ova identified were eggs of Strongyle, Moniezia sp, Schistosoma sp, Haemonchus sp, Paramphistome sp, Trichuris sp and oocysts of Coccidia sp. The samples collected during the postmortem examination revealed ova of Trichuris sp, Moniezia sp, Strongyle and cysts of Balantidium sp. Out of 102 blood samples collected from ailing lambs the blood picture was decreased PCV, Hb, TEC, MCHC, neutrophils, monocytes, eosinophils and increased TLC, MCV, MCH, lymphocytes were observed when compared with apparently healthy group (n=10). Males showed increased PCV, neutrophils, eosinophils, MCV and in females increased TLC, lymphocytes, monocytes and MCHC, where as almost similar Mean±SE values of Hb, TEC were noted in both males and females. Out of 50 serum samples from ailing lambs screened, increased TP, albumin, globulin, A/G ratio, creatinine and decreased calcium, ALT were observed when compared with apparently healthy group (n=10). Increased TP, albumin, globulin, AST were reported in males whereas increased A/G ratio, calcium, ALT in females and similar values of creatinine in both sexes were noted. A total of 53 different samples collected from ailing lambs and affected organs were subjected for culture isolation of microorganisms and molecular characterization by using PCR assay. E.coli, Salmonella sp and S.aureus isolates were obtained by culture method. E.coli isolate was positive for stx2 gene, isolates of Salmonella sp for invA gene and isolates of S.aureus for nuc gene. The prevalence of gross and histopathological changes was noted from 179 dead lambs in Rayalaseema region of Andhra Pradesh during the period of study from December, 2015 to June, 2016. Lambs from Chittoor, Kadapa, Anantapur and Kurnool districts were died in descending order. The mortality was more in females than males. The highest mortality was noted in summer followed by winter. Lambs died at 4-6 months age followed by 1-3 months age, 7-9 months, 10-12 months age and <1 month age in descending order. The mortality of lambs in this region more due to multiple system affections and due to inflammatory origin majorly followed by non inflammatory origin i.e. 179 dead lambs. Inflammatory origin conditions included inflammation of multiple organs involvement was recorded to be highest. Gross lesions in respiratory system observed were tracheitis, congestion, consolidation, suppuration, aspiration, nodular growths and haemorrhages of lungs. In circulatory system, haemorrhages, congestion of coronary blood vessels, gelatinization and necrosis of epicardial fat, small pin point to streaks of pale areas on epicardium and hydro pericardium were observed in heart where as in spleen petechial haemorrhages and congestion were noted. Mild to severe enlargement, edema and hemorrhagic mesenteric lymph nodes and enlarged supra scapular lymphnode in one case were recorded. Congested cerebral blood vessels were noted. In the digestive system, rumen and reticulum filled with plenty of food material (phytobezoars), that was hard and dry, Haemonchus sp worms in abomasum, empty stomach and intestines, duodenal haemorrhages, enteritis (catarrhal and haemorrhagic), thickened intestinal mucosa, congested mesenteric blood vessels, volvulus and tape worms in intestinal portions were observed. The liver showed the lesions including congestion, necrotic areas, haemorrhages, calcification, enlarged and infarcted, cirrhotic changes, icteric patches on surface, larva migrans, abscesses and distended gall bladder. In renal system, softened (pulpy) kidneys, congestion, haemorrhages, cysts in medulla, dilatation of renal pelvis of kidneys, urinary bladder distention and urethral stricture were recorded. Three cases of Anthrax suspected lambs (confirmed by blood smear examination), One dog bite case and three abortion cases reported were not subjected to post mortem. A total of 453 tissue samples of different affected organs included lungs, liver, kidney, heart, intestine, lymphnode, spleen and brain were collected from 179 lambs that were died and subjected to histopathology. In respiratory system, pneumonia was major condition encountered and included different types, non specific pneumonia, suppurative pneumonia, bronchopneumonia, Jaagsiekte, interstitial pneumonia, fibrinous pneumonia, verminous pneumonia, aspiration pneumonia and Suppurative pneumonia combined with Jaagsiekte. In circulatory system, the lesions of heart included myocarditis, presence of Sarcocysts and parasites in myocardium. In spleen, congestion, presence of hemosiderin pigment, thickened trabeculae, degeneration and hyperplasia of splenic corpuscle and lymphoid aggregation around splenic corpuscle were recorded. In mesenteric lymphnodes the lesions included congestion, depletion of lymphoid cells, edema, haemorrhages, fibrous tissue proliferation, reactive hyperplasia, section of parasite, cyst in the vessel and abscess. A case of lymphosarcoma was also observed. Brain lesions like neuronal degeneration, vacuolation, apoptosis, neuronophagia, satellitosis and focal to diffused mono nuclear cell infiltration were observed. In kidney samples, congestion, degeneration of tubules, presence of casts, necrosis of tubules, shrunken glomeruli, fibrous tissue proliferation, cystic spaces, tubular edema, fatty change in glomeruli as well as in tubules, cloudy swelling, presence of hemosiderin pigment, infarction were noted. Accordingly the conditions classified as nonspecific nephritis, glomerular nephritis, haemorrhagic glomerular nephritis, haemorrhagic nephritis, parasitic ova and parasite in cystic fluid were recorded. In intestines congestion, haemorrhages, ulcers, fibrous tissue proliferation, degeneration of glands, various villous abnormalities were observed. The conditions were classified as catarrhal enteritis with parasitic load, nonspecific enteritis, haemorrhagic enteritis, diptheritic enteritis, necrotic enteritis. In liver sinusoidal congestion, fibrous tissue proliferation, focal to diffused necrosis, mild to moderate fatty change, degeneration of hepatocytes, bileduct epithelial hyperplasia and proliferation, calcification, presence of hemosiderin pigment were noted. Further the conditions classified as hepatitis, presence of parasitic ova, granuloma formation, abscess, haemorrhages and fibrotic nodule and bacterial colonies in portal triad.ThesisItem Open Access PATHOMORPHOLOGICAL STUDIES ON SODIUM FLUORIDE TOXICITY IN FEMALE RATS WITH SPECIAL REFERENCE TO THE REPRODUCTIVE TOXICITY AND ITS AMELIORATION(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-10) REDDI PRIYANKA, K; SUJATHA, K(MAJOR); SRILATHA, Ch; RANI PRAMEELA, DABSTRACT: Fluorosis is one of the most important public health hazards across the world, resulting from chronic exposure of higher amounts of fluoride. Most of the times, it is characterized by dental mottling (Dental) and crippling (Skeletal) disorders. Livestock exposed to forages and grasses contaminated with fluoride-rich industrial effluents, deep well water, feed supplements and mineral mixtures containg excess fluoride are more susceptible for fluoride toxicity. Apart from the skeletal and dental abnormalities fluoride can able to produce structural and functional alterations in many of the soft tissues like reproductive organs, liver, kidney, brain, gastro intestinal tract, thyroid and endocrine glands by impairing the antioxidant status of the body. In recent years, experimental studies pertaining to toxic effects of fluoride on reproductive organs and developmental toxicity have got great attention. In this aspect, many authors reported effects of fluoride on male reproductive system where as information regarding female reproductive system is inadequate. Provision of nutritional supplements with high antioxidant properties is the farthest objective as remedy to mitigate the complications of fluoride toxicity. Emblica officinalis (Amla) is proposed for its potent antioxidant property. ZnO nanoparticles are well documented for their multidisciplinary biological activities in the field of medicine. Hence, this present study was undertaken to investigate pathomorphological changes in the reproductive system and other soft tissues of female rats induced by fluoride and its amelioration with Emblica officinalis (Amla) and to study the efficacy of Zinc oxide nano particles coated Emblica officinalis. The present study was carried out by procuring 108 female Wister albino rats (weighing around 200 g) that were randomly assigned to 6 Groups with 18 rats in each Group. Group I served as vehicle control and received distilled water whereas animals in Groups II, III, IV, V and VI received sodium fluoride @ of 18 mg/ kg b.wt, Emblica officinalis @ of 100 mg/ rat/ day, ZnO nano particles @ of 10 mg/ kg b.wt, sodium fluoride @ of 18 mg/ kg b.wt + Emblica officinalis @ of 100 mg/ rat/ day, sodium fluoride @ of 18 mg/ kg b.wt + ZnO nano particles coated Emblica officinalis @ of 100 mg/ rat/ day respectively. Six rats from each group were sacrificed at fortnight intervals. Hematologically, significant reduction was recorded in the TEC, Hb and PCV and TLC in Group II. Serum biochemical analysis revealed significant decrease in total protein, SGPT, SGOT, cholesterol, calcium and increased creatinine, alkaline phosphatase and acid phosphatase levels in Group II. Significant increase in the concentration of TBARS, indicator for oxidative damage and decreased levels of antioxidants like catalase, SOD, GPx and GSH levels in liver, kidney, brain and uterus of Group II rats were noticed. Decreased serum Progesterone (P) and Estrogen (E2) concentration were noticed in Group II rats. Whereas, clear hematological, biochemical and hormonal improvement was noticed in Group V and VI. No alternations were noticed in all these parameters in Group III and IV. On histopathological examination, uterus of Group II rats revealed distorted lamina propria with moderate infiltration of MNCs, squeezed endometrial cells revealing large empty spaces, severe degenerative changes in endometrial glandular epithelium, periglandular infiltration of MNCs and fibrous tissue proliferation, variation in size and shape of the glands, severe hyperplastic changes with complete obliteration of glandular lumen and overall reduction in number of glands in the endometrium. Whereas in myometrium, highly distorted smooth muscle architecture with irregularly arranged funicular layers, infiltration of MNCs and eosinophils and edematous changes between muscle fibers were noticed. Uterine lumen was completely obliterated. In Group V and VI, these changes were less intense and by the end of 6th week uterus came to near normal appearance. Ovaries of Group II rats showed highly degenerated germinal epithelium, degenerated and necrosed stromal tissue with engorged blood vessels, distorted basement membrane separating the theca folliculi from the zona granulosa, vacuolated spaces in granulosa cells, severely disintegrated oocytes and nucleus in secondary follicles, increased number of atretic follicles, pyknotic follicular cells, vacuolated corpus luteum and marked reduction in the number of follicles at different developmental stages. These changes were less severe in Group V and VI. In liver, severe congestion of central vein, dilated sinusoidal spaces with congestion, degenerated hepatocytes with karyomegaly and hyperchromatic nuclei, individualization of hepatocytes, focal loss of hepatocytes with MNC aggregates and disorganization of hepatic cords, microgranuloma formation, mild to moderate vesicular fatty change, moderate fibrous tissue proliferation in portal areas, marked sinusoidal dilatation with severe congestion were observed in Group II. These changes were less intense in Group V and VI. In kidneys, extensive degenerative changes in tubular epithelial cells, severe intertubular hemorrhages and edema and proteinacious casts in tubules were noticed. Vacuolation in the tuft of glomerulus and atrophied and cystic glomeruli were evident in majority of Group II treated animals. In Group V and VI ameliorated rats, these lesions were less severe. Microscopical examination of Group II lungs showed perivascular and peribronchial infiltration of MNCs and eosinophils, moderate hemorrhages in and around the bronchiole, desquamated bronchiolar epithelium, peribronchial lymphoid aggregation and interstitial edema. These changes were less in intense in Group V and VI. Brains of Group II rats revealed shrinkage of neurons along with focal areas of demyelinating changes and spongiosis, doubling and rounding of Purkinje cells, microgranuloma formation and nerve cell with central chromatolysis and focal areas of gliosis in cerebrum. In Group V and VI ameliorated rats, these lesions were less severe. Microscopic examination of heart revealed mild to moderate hemorrhages, sarcolytic changes and infiltration of MNCs in between cardiac muscle fibers in most of the group II rats. All these changes were mild in Group V and VI. In pancreas, peripancreatic ductular infiltration of MNCs, focal loss of acinar structures exocrine duct hyperplasia and complete degeneration with atrophy of islets of Langerhans were observed in Group II rats. These changes were mild in Group V and VI. Microscopically spleens of Group II animals revealed, depletion of lymphocytes around the central artery and from germinal centers and severe engorgement of red pulp. Group V and VI animals appeared almost similar to control. No specific gross and microscopic alternations were observed in soft tissues of Group III and IV. Increased fluoride concentration was noticed in tissues like liver, kidney, brain and uterus of Group II rats whereas, significant decrease in fluoride concentration was noticed in Group V and VI. In Group II, immunohistochemistry showed increased expression of BAX marker in lining epithelium and glandular epithelium of endometrium of uterus compared to control. Decreased number PCNA positive ovarian follicles were observed in Group II compared to control. Whereas, increased number of ovarian follicles were noticed in Group V and VI. On scanning electron microscopic examination, abraded surface epithelium with distorted cuboidal shape and clumping of surface epithelial cells with decreased number of blebs and microvilli were observed in ovaries of Group II whereas, in Group V and VI, cuboidal cells of the germinal epithelium normal with significant increase in number of blebs and microvilli.ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF MAREK’S DISEASE IN COMMERCIAL LAYER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-11) SRIKANTH, K V; RAMA DEVI, V(MAJOR); ANNAPURNA, P; LAKSHMI KAVITHA, KABSTRACT: Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by Marek’s disease virus (MDV-1). Of late, there are incidences of MD in vaccinated commercial layer chicken in India, causing severe economic losses. Only a few reports are available about the status of the disease in Andhra Pradesh. Hence, keeping this in view the present work was undertaken to study the incidence and gross and histopathological lesions in various organs along with diagnosis of MD by PCR technique. The materials for the present study were procured from Nuzividu, Tanuku and Anaparthy towns, private diagnostic laboratories,Vijayawada and from the post mortems conducted at Department of Veterinary Pathology, NTR College of Veterinary Science, Gannavaram. The duration of the study lasted from December 2015 to October 2016. A total of 520 dead layer birds that were vaccinated previously against the disease were examined in detail and representative samples from suspected MD cases were collected. Of these, 58 birds (11.15%) of 20-52 weeks of age were found positive for MD based on clinical signs, gross and histopathological findings in different organs and by PCR diagnosis. The MD affected chicken exhibited clinical signs of emaciation, weight loss, paleness of comb, marked reduction in egg production, ruffled feathers, diarrhoea, lameness and prostration before death. Out of 58 MD cases, visceral tumors of acute form of MD were noticed frequently in the liver (93.1%), kidney (79.3%), spleen (53.5%) and ovaries (20.7%) and less frequently in the proventriculus (17.2%), intestines (13.8%), skin (10.3%), heart (5.2%) and lungs (5.2%). Grossly, the livers were enlarged and had multiple greyish-yellow tumor nodules of either diffuse, nodular or of miliary form. The kidneys were enlarged, irregular and pale and had pinpoint to a few mm greyish white nodules or masses in many cases. The spleen was enlarged and had miliary greyish white lymphomas. The ovaries were enlarged, irregular, pale or greyish white and revealed firm tumorous mass with cauliflower like appearance. The mucosa of proventriculus and the intestinal wall were thickened and nodular tumors were visible through serosa of intestine. Greyish white feather follicular nodules in the skin, greyish white nodular growths in the myocardium and in the lungs were found. Histologically, in the MD affected organs lymphoproliferative lesions were noticed with infiltration of pleomorphic lymphoid cells and the severity of visceral lymphoid lesions were graded into mild, moderate and severe. The sections of liver in severe cases, revealed diffuse or massive proliferation of pleomorphic lymphoid cells or nodular lymphomas compressing the surrounding parenchyma. In severe cases, diffuse proliferation and infiltration of pleomorphic lymphoid cells, plasma cells and a few MD cells or discrete large lymphomas were noticed compressing the renal parenchyma in the kidneys. In severe cases, spleen revealed proliferation and infiltration of lymphoblasts, small and large lymphocytes, macrophages and reticular cells or multiple or solitary lymphomas in the parenchyma. The lesions in the ovaries were characterized by diffuse pleomorphic lymphoid cell infiltration around the ovarian follicles with distortion of ovarian structure in severe cases. In the proventriculus, diffuse infiltration of pleomorphic lymphoid cells in the mucosa, submucosa and within and in between the glands and in the serosa were evident in severe cases. Marked pleomorphic lymphoid cell proliferation was frequently observed in the mucosa, submucosa, muscle layers and subserosa of intestine and lymphomas with nodular aggregation of large and small lymphocytes were clearly evident in the serosa. The lesions in the skin in moderate cases were characterized by nodular infiltration of pleomorphic lymphoid cells below the epidermis, in the dermis and around the feather follicles. In the myocardium, focal degeneration of muscle fibres, proliferation and infiltration of pleomorphic lymphoid cells and rupture of cardiac muscle fibre bundles were seen. The lung sections showed congestion and marked infiltration of pleomorphic lymphoid cells in the wall of the air capillaries and around the secondary bronchus and parabronchus. The sciatic nerve revealed type A lesions in two birds. In the present study, PCR was carried out for the detection of 132 bp repeats and meq gene of serotype 1 MDV in the tumor tissue samples of MD suspected birds that produced two tandem repeats of 132 bp and the primers specific to meq gene produced an expected size of amplicon in MD positive birds. In the present investigation, the pathological and molecular diagnosis of MD was carried out in commercial layer chicken. It was evident that in spite of vaccination, MD continues to be a major threat causing significant losses in commercial poultry flocks.ThesisItem Open Access PATHOLOGICAL AND MOLECULAR DIAGNOSIS OF MAREK’S DISEASE IN COMMERCIAL LAYER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2016-11) SRIKANTH, K.V; RAMA DEVI, V (Major); ANNAPURNA, P; LAKSHMI KAVITHA, KABSTRACT: Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by Marek’s disease virus (MDV-1). Of late, there are incidences of MD in vaccinated commercial layer chicken in India, causing severe economic losses. Only a few reports are available about the status of the disease in Andhra Pradesh. Hence, keeping this in view the present work was undertaken to study the incidence and gross and histopathological lesions in various organs along with diagnosis of MD by PCR technique. The materials for the present study were procured from Nuzividu, Tanuku and Anaparthy towns, private diagnostic laboratories,Vijayawada and from the post mortems conducted at Department of Veterinary Pathology, NTR College of Veterinary Science, Gannavaram. The duration of the study lasted from December 2015 to October 2016. A total of 520 dead layer birds that were vaccinated previously against the disease were examined in detail and representative samples from suspected MD cases were collected. Of these, 58 birds (11.15%) of 20-52 weeks of age were found positive for MD based on clinical signs, gross and histopathological findings in different organs and by PCR diagnosis.The MD affected chicken exhibited clinical signs of emaciation, weight loss, paleness of comb, marked reduction in egg production, ruffled feathers, diarrhoea, lameness and prostration before death.Out of 58 MD cases, visceral tumors of acute form of MD were noticed frequently in the liver (93.1%), kidney (79.3%), spleen (53.5%) and ovaries (20.7%) and less frequently in the proventriculus (17.2%), intestines (13.8%), skin (10.3%), heart (5.2%) and lungs (5.2%).Grossly, the livers were enlarged and had multiple greyish-yellow tumor nodules of either diffuse, nodular or of miliary form. The kidneys were enlarged, irregular and pale and had pinpoint to a few mm greyish white nodules or masses in many cases. The spleen was enlarged and had miliary greyish white lymphomas. The ovaries were enlarged, irregular, pale or greyish white and revealed firm tumorous mass with cauliflower like appearance. The mucosa of proventriculus and the intestinal wall were thickened and nodular tumors were visible through serosa of intestine. Greyish white feather follicular nodules in the skin, greyish white nodular growths in the myocardium and in the lungs were found.Histologically, in the MD affected organs lymphoproliferative lesions were noticed with infiltration of pleomorphic lymphoid cells and the severity of visceral lymphoid lesions were graded into mild, moderate and severe. The sections of liver in severe cases, revealed diffuse or massive proliferation of pleomorphic lymphoid cells or nodular lymphomas compressing the surrounding parenchyma.In severe cases, diffuse proliferation and infiltration of pleomorphic lymphoid cells, plasma cells and a few MD cells or discrete large lymphomas were noticed compressing the renal parenchyma in the kidneys. In severe cases, spleen revealed proliferation and infiltration of lymphoblasts, small and large lymphocytes, macrophages and reticular cells or multiple or solitary lymphomas in the parenchyma. The lesions in the ovaries were characterized by diffuse pleomorphic lymphoid cell infiltration around the ovarian follicles with distortion of ovarian structure in severe cases.In the proventriculus, diffuse infiltration of pleomorphic lymphoid cells in the mucosa, submucosa and within and in between the glands and in the serosa were evident in severe cases. Marked pleomorphic lymphoid cell proliferation was frequently observed in the mucosa, submucosa, muscle layers and subserosa of intestine and lymphomas with nodular aggregation of large and small lymphocytes were clearly evident in the serosa. The lesions in the skin in moderate cases were characterized by nodular infiltration of pleomorphic lymphoid cells below the epidermis, in the dermis and around the feather follicles. In the myocardium, focal degeneration of muscle fibres, proliferation and infiltration of pleomorphic lymphoid cells and rupture of cardiac muscle fibre bundles were seen. The lung sections showed congestion and marked infiltration of pleomorphic lymphoid cells in the wall of the air capillaries and around the secondary bronchus and parabronchus. The sciatic nerve revealed type A lesions in two birds. In the present study, PCR was carried out for the detection of 132 bp repeats and meq gene of serotype 1 MDV in the tumor tissue samples of MD suspected birds that produced two tandem repeats of 132 bp and the primers specific to meq gene produced an expected size of amplicon in MD positive birds. In the present investigation, the pathological and molecular diagnosis of MD was carried out in commercial layer chicken. It was evident that in spite of vaccination, MD continues to be a major threat causing significant losses in commercial poultry flocks.