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ThesisItem Open Access PREVALENCE, HAEMATOLOGICAL, BIOCHEMICAL AND HISTOPATHOLOGICAL OBSERVATIONS OF PARAMPHISTOMOSIS OF DOMESTIC RUMINANTS IN TIRUPATI(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-12) PREETHI, M; VENU, R(MAJOR); SRINIVASA RAO, K; SRILATHA, Ch; VINOD KUMAR, NABSTRACT: The present study on ‘prevalence, haematological, biochemical and histopathological observations of paramphistomosis of domestic ruminants in Tirupati’ was conducted in cattle, sheep and goats. A total of 2133 samples (dung, blood, serum, tissues and amphistome specimens) from slaughtered domestic ruminants was examined. In direct faecal smear examination, an overall prevalence of 24.29 percent of paramphistomosis infection was recorded, whereas in faecal sedimentation method, 32.51 percent was observed. In cattle, the prevalence of infection by direct faecal smear and faecal sedimentation methods were 17.43 and 31.19 percent, respectively. In sheep, the prevalence rates were recorded higher than cattle. In goats, the prevalence of paramphistomosis by direct faecal smear examination was 20.66 percent, whereas by faecal sedimentation method, it was 30.52 percent. Out of 109 cattle carcasses, in 47 cases (43.12) amphistomes were found in rumen, reticulum and bileduct during slaughterhouse examination. In sheep and goat, the prevalence rates were 42.15 and 40.85 percent, respectively by slaughterhouse examination. Age-wise, higher prevalence was recorded in cattle of 2-4 years followed by older animals of above 4 years and young animals of <2 years. Slightly higher prevalence (26.79%) was noticed in >1- 2 years old sheep than <1year age group. In goats, the prevalence of infection was lower in the age group of <1year, when compared to their counterparts in sheep. Based on slaughterhouse study, the sheep of <1 year old were more infected (56.28%) than >1-2 years old sheep (36.85%). In contrast, the higher prevalence was noticed in >1-2 years (63.27%) than < 1 year old goats (21.74%). Sex-wise, the prevalence of infection in male cattle was slightly higher than females by direct faecal smear and slaughterhouse examinations, whereas in sedimentation method, female animals showed higher infection than male. The prevalence of infection in female sheep was higher than male sheep. Overall, statistically no significance difference was observed between male and females, in respect to their diagnostic method. Blood samples were screened for haematological observations such as PCV, Hb and RBC counts. Statistically, significant difference was noted between immature amphistomosis and uninfected groups of cattle, sheep and goats. No significant difference was noted between infected and uninfected animals of cattle and sheep in relation to PCV, Hb and RBC values, but in goats significant difference was observed. In DLC, statistically, no significant difference was observed in immature, infected and uninfected amphistomosis cattle. In immature and uninfected sheep, neutrophil and monocyte values showed significant alterations, but in goats it was in neutrophil and lymphocytes. Serum sample screening revealed, low levels of total proteins and albumins in immature amphistomosis infected cattle, sheep and goats. Gross and histopathological changes of amphistome infected rumen, reticulum and bile duct were observed. The adult amphistome parasites invading the rumen mucosa by sucker and plugging of mucosa of bile duct was noticed in cattle. In the bile duct of cattle, massive and extensive infiltration of plasma cells, lymphocytes and monocytes in between acinar structures was recorded. Hyperplasia of bile duct, degenerative changes and necrotic changes in acinar mucosal lining epithelial cells were appreciated prominently. In immature amphistomosis, duodenum of sheep revealed thickened and edematous; mucosal surface was severely congested, petechial haemorrhages and necrosis were also noticed. Histopathological studies of infected duodenum of sheep revealed, plugging of the mucosa with sucker and extensive infiltration of macrophages and plasma cells deep into the mucosa. Based on morphology, 5 amphistomes were recognized viz., Cotylophoron cotylophorum, Paramphistomum cervi, Gastrothylax crumenifer, Fischoederius elongatus and Gigantocotyle explanatum. Mixed infections were noticed higher at 50, 73.6 and 62 percent, in cattle, sheep and goat, respectively. In immature amphistomosis infected ruminants, haematological and biochemical parameters were estimated and showed a great variation and microscopic examination of duodenal wall scrapings revealed immature amphistomes.ThesisItem Open Access IDENTIFICATION OF SARCOCYSTIS SPECIES IN CATTLE (Bos taurus) BY PCR-RFLP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-10) MOUNIKA, K; SREEDEVI, C(MAJOR); VENU, R; SRINIVASA RAO, TABSTRACT: The present study was carried out to determine the prevalence of bovine sarcocystosis and identify various species of Sarcocystis from cattle in different regions of Chittoor district, Andhra Pradesh by PCR-RFLP. Macroscopic examination and microscopic examination by pepsin-HCl digestion method of 150 slaughtered cattle from Tirupati, Chandragiri, Chittoor, Renigunta and Pakala regions in Chittoor district revealed an overall 91.33 per cent of prevalence of sarcocystosis. The infection was highly prevalent in Tirupati (97.14 %) compared to that of Chandragiri (92.5 %), Renigunta (90.0 %),Chittoor (80.0 %), and Pakala (70.0 %) regions of Chittoor district. The prevalence of macroscopic and microscopic sarcocysts was 6.57 and 93.43 per cent respectively. Macroscopic cysts were exclusively observed in oesophagus. The prevalence of infection increased with advance in age and there was a significant relationship between the prevalence of infection and age groups of cattle (P<0.001). There was no significant difference (P>0.001) between the prevalence of Sarcocystis infection in male (91.76 %) and female (90.76 %) cattle. Genomic DNA was extracted separately from bradyzoites of all 137 cattle that were positive for sarcocystosis and amplified 18S rRNA gene of Sarcocystis that yielded PCR product of 900 bp. Digestion of 18S rRNA gene PCR products was performed with restriction endonuclease BseLI to detect different species of Sarcocystis. On digestion with restriction endonuclease three different patterns were observed on agarose gel electrophoresis, one with 513 bp and 343 bp and other with 525 bp, 241 bp and 141 bp which were referred to Sarcocystis cruzi and S. hirsuta respectively. While another with 532 bp and 335 bp was referred to S. fusiformis. S. cruzi (93.43 %) was significantly more prevalent in Chittoor district in comparison with the S. hirsuta (4.38 %) and S. fusiformis (2.19 %). Infection of cattle with S. hominis was not observed in the study area. These findings provide evidence that Sarcocystis species of cattle and water buffaloes are not strictly intermediate host specific but might occasionally infect water buffaloes and cattle, respectively where both hosts occur and natural cross transmission through dogs are possible. S. fusiformis is able to use the cattle as an intermediate host and is not restricted to buffalo. PCR-RFLP was helpful in studying the molecular epidemiology of sarcocystosis in cattle and was a good method in discriminating between species of Sarcocystis.ThesisItem Open Access CLONING, EXPRESSION AND CHARACTERIZATION OF PARAFLAGELLAR ROD GENE OF TRYPANOSOMA EVANSI(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2016-01) SIVAJOTHI, S; CHENGALVA RAYULU, V(MAJOR); MALA KONDAIAH, P; SREENIVASULU, D; SRILATHA, ChABSTRACT : Present study was undertaken with an objective to isolate, clone, express and characterize the paraflagellar rod gene of Trypanosoma evansi. Local isolate of Trypanosoma evansi collected from naturally infected cow was multiplied in Wistar rats. Total RNA was extracted from DEAE-cellulose column chromatography purified trypanosomes by using Trizol LS reagent. Complementary DNA (cDNA) was synthesized from the RNA of host cell free T. evansi parasites by reverse transcription using oligo dT primers. RT-PCR was standardized to amplify the cDNA by targeting 1800 bp unique for PFR 2 gene of T. evansi. Amplification of cDNA was confirmed on agarose gel electrophoresis. The concentration of PCR amplicon was found to be 40 ng/μl after extracting from the gel. The gel purified PCR product (PFR 2 gene of T. evansi) was cloned into pTZ57R/T vector system. Transformation of competent Escherichia coli DH5α cells with ligated PFR 2 gene T. evansi was successfully carried out in LB agar with X-Gal and IPTG. Developed recombinants were observed as white colonies and non-recombinants as blue colonies. Presence of inserts was confirmed initially by Colony-PCR and then by Plasmid-PCR. Nucleotide sequence of the PFR 2 gene of T. evansi S.V.V.U. isolate (Accession No. KT277497) of the present study revealed 100 % homology with T. evansi China isolate and 99% homology with T. evansi Izatnagar and Bikaner isolates. Variation in nucleotide mutations at 4 positions with T. evansi Izatnagar and 3 positions with T. evansi Bikaner isolates were observed. The amino acid mutations in the PFR 2 gene of T. evansi S.V.V.U. isolate displayed regularity at 4 positions when compared to T. evansi China, Izatnagar and Bikaner isolates. Tree topology based on the Neighbor-joining (NJ) method of phylogenetic analysis has showed a close homology with other Trypanosomatidae species sequences with 100% bootstrap values. Restriction digestion of insert DNA of PFR 2 gene as well as pET 32a vector was carried out with EcoR I and Hind III enzymes and subjected for ligation by using T4 DNA ligase. The recombinant protein was sub-cloned into pET 32a and expressed in the BL21 (DE3) pLysS expression system. A high level of expression of recombinant protein of PFR 2 gene of T. evansi was noted following four hours of induction with 1 mM IPTG. Molecular weight of the Ni-NTA column chromatography purified recombinant protein of PFR 2 gene of T. evansi was found to be approximately 90 kDa after resolving by SDS-PAGE. PFR 2 gene of T. evansi S.V.V.U. isolate was further characterized by determination of its protein profile with SDS-PAGE analysis and western blotting against hyper immune serum. Indirect ELISA was optimized for detection of specific antibody titre against recombinant protein of PFR 2 gene of T. evansi. Based on the ELISA result, it is evident that PFR 2 gene products are eliciting very good immune response. However, further study is required to know the protective effect of the antibodies in laboratory animal models and to explore the PFR 2 gene of T. evansi as potential candidate for diagnostic and vaccine target against surra. Findings of the present study confirmed the existence of PFR 2 gene in Indian cattle isolate of T. evansi. Cloning, expression and characterization of PFR 2 gene of T. evansi of cattle isolate carried out in the present investigation is probably the first report in India.ThesisItem Open Access STUDIES ON INCIDENCE OF PARASITES OF QUAILS (Coturnix coturnix) AND PIGEONS (Columba livia} IN AND AROUND TIRUPATI. ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1998-04) SRINIVASA RAO, R; HAFEEZ, Md(MAJOR); RAMAKRISHNA REDDY, P; VENKATESWARLU, UABSTRACT: A study was undertaken to record the ecto and endo parasites of pigeons and quails in and around Tirupat~A. ndhra Pradesh. Out of the 162 pigeons examined. 60 were positive for ectoparasites with a percentage incidence of 34.03. Pigeons had lice. ticks and mites. Among lice the species recorded were CdurnMa cofumbae, M?mpon gahae, Menacanthus. sb9rnineus. Gonlbcotes gaiiin8e and tipewus capons. Lipewus capcxls was the least prevalent lice with an incidence of 3 per cent and the most prevalent was Colurnbkuh columbae with 56.6 per cent. The only tick species recorded was Argas pemiws with an incidence rate of 1.6 per cent. Two species of mites were observed viz., Ornithonyssus bursa (6.6%) and Megninia columbae (8.33%). Among the total number of pigeons examined. 61.41% 2 1.59 had one or the other infection. Helminths were present in 57 per cent and protozoa were present in 28 per cent of pigeons. The endoparastic fauna af pigeons recorded was composed of three cestode species, two nematode species and three species of protozoa. The cestodes noted were R. tetragons, R. echinobofhrida and R. Cesticillus. Nematodes observed were Ascandia gall; and Heterakis gallinarum. The protozoa recorded were Eimeria species. Haemoproreus columbae and Trichomonos gallinae. Over all percentage inctdence of ectoparasites on quails was 37.89 2 3.4 per cent. The incidence of ectoparasites in farm quails was far less (1 0.47 2 7.03) than that observed in migratory quails (37.14 2 3.66%). The only type of ectoparasite recorded was lice. The four species of lice recorded were Guntocotes gallinae. Goniodes d~sstmiIisG~ onmdes gigas and Menopc~gl allnae. Examination of both farm and migratory quails did not reveal any helm~nth parasites. But they were posltrve for the oocysts of Enneria and lsospora specles with the percentage ~ncidenceo f 47 and 42 respectivety. Two drugs. one pyrethrod. dettramethrin (Butox) and the other a neem based herbal compound (Nimbitor) were studied for ttmr efficacy against ectoparasites of pigeons. Butox in the concentrations of 0.1. 0.15 and 0.2 per cent produced maximum efficacy after 24, 12 and 6 hours repsectively. Nimbitor when used in 1, 1 .S and 2.0 percent concentration exhibited maximum response after 48, 24 and 12 hours respectively.ThesisItem Open Access STUDIES ON ANTICOCCIDIAL DRUG RESISTANCE OF FIELD ISOLATES OF EIMERIA TENELLA FROM BROILER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1996-08) SURYA KUMARI, BARREY; PADMAVATHI, P(MAJOR); RAMESH, A.J; SATYANARAYANA CHETTY, MThesisItem Open Access EFFECTS OF IRRADIATION ON LARVAL TREMATODES WITH SPECIAL REFERENCE TO AMPHISTOME CERCARIAE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1980) BHASKARA RAO, THOLETI; VENKATESWARA RAO, B(MAJOR); VENKAT RATNAM, A; RAMA RAO, PThesisItem Open Access HELMINTH PARASITES OF RODENTS WITH SPECIAL REFERENCE TO ZOONOTIC POTENTIAL AND HOST - PARASITE ASSOCIATIONS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 1979) BHASKAR RAO, TOPALLE; VENKATESWARA RAO, B(MAJOR); RAMARAO, PThesisItem Open Access DIAGNOSIS OF BOVINE SARCOCYSTOSIS BY IMMUNOFLUORESCENT ANTIBODY TECHNIQUE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2012-03) DASMA BAI, BANOTHU; UDAYA KUMAR, M(MAJOR); CHENGALVA RAYULU, V; NARASIMHA REDDY, Y; ANAND KUMAR, AABSTRACT: Sarcocystis species are widely prevalent in man and animals, causing significant impact on animal and public health throughout the world. A laboratory standardized immunofluorescent antibody technique was used to study the seroprevalence of bovine sarcocystosis and the efficacy of same was compared with the traditional diagnostic methods like macroscopic, microscopic (squash and pepsin HCl acid muscle digestion). Histopathological changes and viability of bradyzoites in the affected esophageal muscles were also studied in the present investigation. The gross examination of oesophagi revealed creamy white colored thin walled macrocysts of Sarcocystis spp appearing in different shapes (fusiform, elliptical, ovoidal and globular etc) and sizes ranging from 2.0-18.0 x 1.0-5.0 mm with an average size of 10.47 + 0.295 x 3.08 + 0.089 mm. None of the organs showed any kind of gross lesions around the macrocysts embedded in the muscles. Microscopic examination of esophagus and diaphragmatic muscles by squash technique revealed the presence of microcysts arranged horizontally in between the muscle fibers of esophagus where as the pepsin HCl acid digestion of muscle samples of esophagi and diaphragm showed live bradyzoites in gliding motion. Histopathological studies suggested two possible etiologic agents of bovine sarcocystosis namely: S. cruzi, characterized by having elongate and septate cysts and S. hirsuta or S. hominis, characterized by having spherical or rounded cysts with thick radially striated cyst wall. The muscle degeneration with focal or diffused mononuclear cells viz: leukocytic infiltration, eosinophils, lymphocytes and macrophages observed in the tissues under study were attributed to the pathogenic effects of S. cruzi. The ruptured or degenerated state of some of the mature sarcocysts surrounded by eosinophils indicated the advanced age of the cyst. Immunofluorescent antibody technique was standardized in the laboratory for the diagnosis of Sarcocyst infection in bovines. Purified, host cell free bradyzoites collected from macrocysts of Sarcocystis spp, in aliquots of 6-8 applied to glass slides and fixed in chilled acetone over night followed by preservation at -200C worked well with 1:16 dilution of positive and negative control sera and 1:40 dilution of rabbit anti-bovine FITC conjugate. The positive sera did not show any cross reaction with T. gondii RH strain and non specific reactions were absent with negative sera. The serosurveillance of bovine sarcocystosis by laboratory standardized IFAT showed 80.14% (323) of cattle and 78.59% (246) of buffaloes positive for anti sarcocystis antibodies out of 403 cattle and 313 buffalo sera tested, respectively showing an overall prevalence of 79.46% out of 716 animals screened. The antibody titers of 6 randomly selected positive samples from different age groups of <2 years, 2-5 years, 5-10 years and >10 years old bovines ranged from 16-64, 32-256, 32-128 and 16-64 with an average titer of 32 + 2.92, 106.6 + 34, 74.6 + 17 and 34.6 + 9, respectively. The age wise prevalence of sarcocystosis in cattle indicated low rate of infection in the age group below 2 years (60%) and an ascending rate of infection in the age groups of 2-5 years (81.33%), 5-10 years (80.52%) and above 10 years (90.9%). Similarly, the incidence was significantly low in the buffaloes of below 2 years (64%) and high percentage of infection (86.51%) in 5-10 years followed by 78.94% in 2-5 years and 77.27% in above 10 years of age groups. No significant difference of infection was observed between male (81.87%) and female (75.19%) animals as well as between non-descriptive (79.63%) and cross bred (77.58%) animals. Esophageal and diaphragmatic muscle samples collected from 100 animals slaughtered at Chengicherla slaughter house, Hyderabad were subjected to visual examination, squash and pepsin HCl acid muscle digestion techniques which revealed the presence of macrocysts in 16% and 0%, microscopic sarcocysts in 8% and 0% and bradyzoites in 76% and 52% esophageal and diaphragmatic muscles, respectively. The sera collected simultaneously from corresponding animals were screened for anti Sarcocystis antibodies by laboratory standardized IFAT and the results were compared with those of visual examination, squash and pepsin HCl acid muscle digestion techniques. The IFAT was found superior in diagnosing sarcocystosis with positivity of 82%, followed by muscle digestion, gross examination and squash techniques with positive rates of 52%, 16% and 8%, respectively. The present study indicated that the visual and microscopic examination of bovine carcass is by no means a satisfactory diagnostic tool and recommends Immunoflourescent antibody technique for the antemortem diagnosis of animals waiting for slaughter at abattoirs in large scale. Experiments were also undertaken to determine whether Sarcocystis would survive storage at different refrigeration temperatures for a period of 9 days. The number of live and dead bradyzoites in one gram of pepsin HCl acid digested bovine esophageal muscle samples previously stored at room temperature, 40C, 00C, and -200C for a period of 48thhr, 8 days, 24thhr, and 24hr were 2x104 and 4 x104, 1x104 and 1 x104, nil and no bradyzoite, respectively when compared to those stored at 0th hr (10x104 and 0 x104).ThesisItem Open Access CERTAIN ASPECTS OF SPIROCERCOSIS WITH REFERENCE TO ITS PREVALENCE AND IMMUNODIAGNOSIS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2006-12) JYOTHISREE, CHITITHOTI; HAFEEZ, Md.(Major); UDAY KUMAR, M; ALAHA SINGARI, NABSTRACT : The research work on “Certain aspects of Spirocercosis with reference to its prevalence and immunodiagnosis” was under taken to record the prevalence of spirocercosis in different regions of Andhra Pradesh besides the study of the tissue reactions, haematological and biochemical changes of the disease and the efficacy of certain chemotherapeutic drugs against the spirocercosis and also to prepare an antigen of Spirocerca lupi to detect spirocercosis by immuno diagnostic techniques. A total of 562 dogs were examined in five (5) different places of Adhra Pradesh. Based on faecal sample examination, the over all per cent prevalence of spirocercosis in dogs was 3.02. Among the different (5) places of Adhra Pradesh under this study the prevalence was more in Hyderabad (5.45%) followed by Tirupati(3.6%), Ongole(2%), Kurnool (1.75%) and Guntur (0%). The prevalence of spirocercosis based on post mortem examination was 10 per cent at Tirupati. The prevalence of spirocercosis was more in 1-5 years age group (3.83%) followed by above 5 years age group (2.64%). The prevalence was more in males (3.30%) than females (2.70%). The prevalence was more in non discripts (5.92%) followed by German shepherd (4.09%) and in Pomeranians (1.57%). The prevalence was more in Winter season (5.91%) followed by Rainy season (2.12%) and Summer (1.06%). Radiographic examination showed 47.05 per cent confirmity in suspected cases. The gross pathological lesions like pale lungs, putrid material in the thoracic cavity, exostosis in 7th, 8th and 9th thoracic vertebrae, 5-6 thick hard nodular masses in thoracic portion of aorta and hen egg size nodule in the terminal portion of oesophagus and proximal portion of stomach were generally observed in natural cases of spirocercosis. Histopathological lesions in oesophagus were cut section of parasite and parasitic ova surrounded by inflammatory cells and moderate edema in the serosal layer along with giant cells. Aorta showed infiltration of inflammatory cells in tunica intima, cut section of parasite along with parasitic ova. Severe infiltration of inflammatory cells neutrophils and plasma cells along with fibrous tissue in tunica media. Lung showed focal interstitial pneumonia. Liver showed inflammatory cells between the hepatic cards. Haemorrhages between the cardiac muscle fibres were also observed in the heart. Haematological and biochemical changes in blood and sera samples of naturally infected spirocercosis were low haemoglobin concentration, leukocytosis and Neutrophilia. Serum samples showed low level of Total protein, Albumin and Glucose and increased concentration of Alkaline phosphotase, Amylase and Creatinine. The efficacy of the drugs Ivermectin, Ivermectin plus Prednisalone and Doramectin was tested against naturally infected Canine spirocercosis. The therapeutic efficacy of the above drugs was found to be 100 per cent. Crude antigen of Spirocerca lupi revealed protein concentration as 11.38 mg/ml. Attempts were made to diagnose S.lupi infection in naturally infected dogs by conducting four serodiagnostic tests viz., Agar-Gel immuno diffusion test (AGID), Immuno Electrophoresis (IEP), Counter Immuno Electrophoresis (CIEP) and Passive Haemagglutination Test (PHA). 17 sera samples collected from dogs naturally infected with S.lupi infection were tested against crude S.lupi antigen using AGID, IEP, CIEP and PHA tests. These tests showed a sensitivity of 88.2, 82.3, 88.2 and 76.4 percent in detecting S.lupi infection in naturally infected dogs, respectively The whole homogenate of S.lupi antigen was subjected to 10 per cent SDS-PAGE for the analysis of polypeptide profile. SDS-PAGE revealed a total number of 7, 8 polypeptides, respectively. The apparent molecular weight of polypeptides of these ranged from < 25 KDa to 151 KDa.