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ThesisItem Open Access PATHOLOGICAL AND MOLECULAR STUDIES ON JSRV (JAAGSIEKTE SHEEP RETROVIRUS) IN SHEEP AFFECTED WITH OVINE PULMONARY ADENOCARCINOMA IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) SAMATHA, V; RAMA DEVI, V (MAJOR); SATHEESH, K; SUBRAMANYAM, K.V.; VINOO, ROvine pulmonary adenocarcinoma (OPA) is a naturally occurring transmissible tumor of lungs in sheep and goats caused by an exogenous Jaagsiekte Sheep Retrovirus (exJSRV). A major obstacle in JSRV research is the difficulty to culture the etiological agent in vitro and absence of serological tests for diagnosis. Of late, PCR diagnostic techniques are being used to diagnose OPA at field level. There are reports of OPA from India, but molecular studies on JSRV are lacking. The present work was planned to study the gross and microscopic lesions and to carry out molecular diagnosis along with molecular characterization of JSRV by sequencing U3, gag and Env-TM genes. In addition, exons of 5, 7 and 8 of p53 gene in OPA tumors were sequenced to determine any mutations. The materials for the present study were collected from slaughter houses, private organized farms and from field mortalities. A total of 150 lung samples were collected and of these, 22 samples were found positive for OPA based on gross, histopathology and by molecular diagnosis of JSRV by U3-hnPCR. Grossly, the lung samples from OPA cases revealed diffuse areas of consolidation or tumor nodules and the cut surface had meaty appearance with moist surfaces resembling classical form of OPA. Histologically, sections from consolidated and/or nodular areas of lungs revealed multiple nonencapsulated neoplastic areas of different sizes composed of cuboidal to columnar epithelium lining the alveolar and bronchiolar walls.The neoplastic epithelium was mainly arranged in two typesviz. papillary or acinar growth patterns. In few lungs, bronchioloalveolar growth pattern was noticed. In advanced cases, thickening of alveolar septa was noticed due to connective tissue proliferation and cellular infiltration in the interstitium. Myxomatous nodules were evident in some areas. Immunostaining for JSRV-CA protein was performed on eight OPA lung samples and the positive staining was indicated by the presence of intracytoplasmic fine brown granular staining of the neoplastic cells, alveolar macrophages and desquamated tumor cells. RNA was extracted from the suspected OPA lungs and lymph node tissues and cDNA was prepared and was amplified by U3-hn PCR to detect JSRV transcripts using specific primers. A total of 22 sheep (14.7%) were found positive for OPA out of 150 animals examined. cDNA prepared from 22 OPA lung samples was used for conducting PCR to amplify gag and Env-TM genes of exJSRV. A PCR that was specific for the exons 5,7 and 8 of p53 gene in lung tumor tissues of OPA was performed and an amplified products of 332 bp,210 bp and 220 bp were detected respectively. The PCR amplicons obtained in different reactions were sequenced by Sanger dideoxychain termination method at Bioserve Biotechnologies Pvt. Ltd, Hyderabad. All the sequences were aligned and the deduced sequences were submitted to GenBank. Pair wise divergence and similarity was calculated using MEGA 7. The nucleotide sequence analysis of five JSRV sequences (samples 4-8) from the OPA affected sheep for U3 region of exJSRV showed more similarity (81-98%) with a UK strain (AF105220.1) than (69-73%) with a South African strain (M80216). The phylogenetic analysis of these sequences revealed that they were slightly divergent from other Gannavaram isolates available in the database. The nucleotide sequence analysis of eight JSRV sequences (samples 1-8) from the OPA affected sheep for gag region of exJSRV showed similarity (99-100%) with both enJSRV NTRCVSc_enJSRV 1 to 12) and (100%) a few exJSRV (MG192314, MG192315, MG192316 and DQ838494). The absence of Sca1 site was noticed. The present sequences were similar to exJSRV isolates reported from Delhi and China. The nucleotide sequence analysis of four exJSRVsequences (samples 4-7) from the OPA affected sheep for Env-TM region showed more similarity (94-9%) with UK strain (AF105220.1) and (92-94%) with USA strains (AF357971) than (80-82%) South African strain (M80216). The region of cytoplasmic tail obtained is incomplete in its aminoterminal, but the information revealed conservation of YX part of the YXXM motif of the exJSRV. The nucleotide sequence analysis of p53 gene at exons 5,7 and 8 in OPA tumor samples revealed no mutations that may suggest involvement of other regions of p53 or alternative genes in development of OPA. Based on the pathological and molecular studies carried out on OPA affected lung tissues of sheep, the JSRV sequences obtained were characterized as exogenous JSRV.ThesisItem Open Access DETECTION AND MOLECULAR CHARACTERIZATION OF SOIL TRANSMITTED HELMINTHS OF ZOONOTIC SIGNIFICANCE IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-12) GNANI CHARITHA, V; CHENGALVA RAYULU, V (MAJOR); MALA KONDAIAH, P; ASWANI KUMAR, K; JAGADEESH BABU, AThe present study was carried out to determine the prevalence of soil transmitted helminths of zoonotic significance among canine faecal and soil samples of Andhra Pradesh (A.P) along with molecular characterization of Toxocara and Ancylostoma spp. The overall prevalence of gastrointestinal (GI) parasites was 83.5 percent (n=745) out of 892 canine faecal samples screened by conventional microscopy. Significantly (P ≤ 0.01) higher prevalence of GI parasites was recorded in Costal Andhra (87.1%) than Rayalseema (75.2%) region of A.P. Eleven parasitic species including five nematodes, four cestodes and two protozoans were isolated from the faecal samples. The most ubiquitous parasites were Ancylostoma spp. (33.5%) and Toxocara canis (20.7%) followed by Strogyloides stercoralis (5.04%), Cystoisospora spp. (3.69%), Toxascaris leonina (3.2%), Trichuris vulpis (2.9%), Taenia spp. (2.5%), Dipylidium caninum (2.1%), Entamoeba spp. (1.23%), Diphyllobothrium latum (0.45%) and Spirometra spp. (0.34%). Concurrent mixed infection was recorded in 7.7% of dogs. Dog associated risk factors such as sex, age, breed and domestication along with effect of urbanization and seasonal influences on the prevalence of parasites were analyzed. The overall prevalence of soil transmitted parasites was 43.5 percent out of 390 soil samples screened. High contamination index of Toxocara spp. (18.5%) followed by hookworms (12.3%) was recorded. Rural soils (48.7%) were found more contaminated than urban areas (44.0%). More number of soil samples were positive for different parasitic stages collected from the garden soils (75.5%) followed by play/school grounds (65.2%), animal dwelling areas (37.3%), parks (34.0%) and veterinary dispensaries (24.0%). The prevalence of Ancylostoma spp. and Isospora spp. were positively correlated (P <0.05) with rainfall while Toxocara and Toxascaris spp. were independent of temperature influence. Responses retrieved from the questionnaire survey revealed that more than half of the pet owners (63.7%) were unaware of zoonotic soil transmitted helminths. Genomic DNA (gDNA) of 151 faecal samples were screened for Toxocara spp. using PCR-RFLP targeting ITS-2 gene. Upon genotyping of PCR amplicons (n=50) with RsaI enzyme, all the canine faecal isolates were identified as T. Canis (287 bp and 244bp). Amplification of gDNA from soil samples (n=72) yielded different product sizes viz., ~540bp (ITS-2) and ~500bp (ITS-2). Genotyping of randomly selected ~540bp product (n=32) from soil isolates with RsaI enzyme confirmed T. cati (37.5%) with three fragments (286bp, 150bp and 103bp) and T. canis (62.5%) with two fragments (287bp and 244bp). Sequencing and phylogentic analysis of ITS-2 products (~540bp) revealed 100% homozygous within the species and with the other geographical isolates of T. canis and T. cati. However, the other amplified product of ~500bp (n=21) with RsaI enzyme yielded two fragments (263bp and 219bp) and the sequencing result revealed 81.8 percent identity with T. cati. The phylogenetic analysis of ITS-2 sequences of 500bp products of A.P constituted a different sub branch and diverging from T. cati, T. canis, T. malayseinsis and T. vitulorum thus categorizing as a separate group or variant. Randomly selected 169 faecal and 48 soil samples from A.P were screened for detection of Ancylostoma spp. using a two step semi-nested PCR targeting ITS-1, ITS-2 and 5.8S gene. On gel electrophoresis, 132 (78.10%) samples showed primary PCR band at ~450bp size while secondary amplicons were observed at ~410bp size. Twenty one out of 48 soil PCR products were found positive in first round PCR and as well as in semi-nested PCR. Genotyping of semi-nested PCR products with BfaI and AhdI restriction enzymes revealed A. caninum (43/50) and A. ceylanicum (7/50) from faecal and only A. caninum (21/21) from soil isolates. The phylogeny of A.P isolates of A. caninum and A. ceylanicum revealed 100% homology within the species and with the other geographical isolates. Taken together, present data suggest the potential role of pet/stary dogs as being the main sources of contamination and signifies the need of integrated approaches to minimize the risk at different settings.ThesisItem Open Access ROLE OF COMMON EFFLUX PROTEIN INHIBITORS ON PHARMACODYANMICS AND PHARMACOKINETICS OF ENROFLOXACIN IN BROILER CHICKEN(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-11) SRIVIDYA, GULLAPUDI; SRINIVASA RAO, G (MAJOR); RAVIKUMAR, P; RAMA DEVI, V; MURALIDHAR, MEnrofloxacin, an antimicrobial fluoroquinolone is most commonly used against majority of gram negative bacterial and mycoplasma infections in majority of livestock including poultry. Due to indiscriminate usage of enrofloxacin in the poultry farming and clinical practice, resistance had been developed to this quinolne drug. Microbes develop resistance to antibiotics by various pathways. Among them, efflux pump mediated drug resistance is one of the important pathways identified in the recent past. Phytochemicals namely, theobromine, glycyrrhetenic acid and glycyrrhizic acid and capsaicin were identified as efflux pump inhibitors. It was described that phytochemicals which possess efflux pump inhibitory activity if combined with classical antimicrobial agents reduces the development of resistance and also improves their therapeutic efficacy. In the present study, pharmacokinetic and pharmacodynamic interaction between enrofloxacin, a fluoroquinolone antibacterial agent and efflux protein inhibitors capsaicin, theobromine, glycyrrhetenic acid and glycyrrhizic acid were investigated using chicken as model system. Broiler chickens weighing about 1.5-2.0 kg were randomly divided into six groups with six birds in each group. Group I birds did not receive any treatment and is primarily meant for standardization of HPLC assay for determination of enrofloxacin/ciprofloxacin in chicken plasma. Enrofloxacin alone (10 mg.kg-1, PO) was administered to birds in group II that served as control. Birds in groups III, IV, V and VI were received enrofloxacin (10 mg.kg-1, PO) after one hour of oral administration of efflux protein inhibitors, capsaicin (15 mg.kg-1), theobromine (125 mg.kg-1), glycyrrhetenic acid(100 mg.kg-1) and glycyrrhizic acid(100 mg.kg-1) respectively. Blood samples were collected from tarsal vein into heparinized tubes, at predetermined time intervals prior to and 0.166, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24 and 48 h post-enrofloxacin administration. Plasma was separated and used for HPLC analysis to determine plasma concentrations of enrofloxacin. Based on plasma concentrations of enrofloxacin, pharmacokinetic parameters for enrofloxacin were determined using non-compartmental method. Detectable concentrations of enrofloxacin in birds persisted upto 48h in all groups. The mean Cmax, AUC(0-t), AUMC(0-t) , (Vd/F), MRT and (ClB/F) in enrofloxacin control group were 2.17 μg/ml, 36.28+4.80 μg.h.mL-1, 633.04+85.53 μg.h2.mL-1, 4.47+0.43 L.kg-1, 16.60+0.42 h and 0.28+0.03 L.kg-1.h-1. Results obtained from the study revealed that upon coadministration of theobromine, glycyrrhetenic acid and glycyrrhizic acid, the maximum plasma concentration (Cmax) increased to 2.62+0.14, 2.94+0.03 and 2.96+0.04 μg.mL-1 , area under the plasma drug concentration time profile (AUC0-t) enhanced to 48.93+1.29, 63.35+1.08 and 43.37+0.98 μg.h.mL-1, mean residence time (MRT) were increased significantly to 19.05+0.42, 17.70+0.25 and 17.27+0.69 h suggesting that enrofloxacin residence time was prolonged in birds. The phytochemical, capsaicin co-administration significantly shortened the half life and increased the elimination rate constant, tmax of enrofloxacin in chicken. Synergisitic interaction of efflux protein inhibitory phytochemicals capsaicin, theobromine, glycyrrhetenic acid and glycyrrhizic acid with enrofloxacin were noticed in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against S. aureus ATCC 25923, E.coli ATCC25922, K. pneumoniae ATCC700603 and P.aureginosa ATCC27853 in vitro. MIC (μg/ml) of enrofloxacin against E. coli, S. aureus, K. pneumoniae and P. aureginosa were 0.02, 0.20, 1.65 and 2.43 respectively. The MIC (μg/ml) of enrofloxacin in the presence of capsaicin was decreased to 0.090 (55%) against S.aureus, 0.012 (40%) against E.coli , 0.266 (84%) against K. pneumoniae, 0.404 (83%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of theobromine was reduced to 0.110 (45%) against S.aureus, 0.012 (66%) against E.coli, 0.258 (84%) against K. pneumoniae, 0.450 (81%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of glycyrrhetenic acid was reduced to 0.041(80%) against S. aureus, 0.012 (40%) against E. coli, 0.404 (76%) against K. pneumoniae, 0.450 (81%) against P. aureginosa. The MIC (μg/ml) of enrofloxacin in the presence of glycyrrhizic acid was reduced to 0.110 (45%) against S. aureus, 0.012 (40%) against E. coli, 0.404 (76%) against K. pneumoniae, 0.45 (81%) against P. aureginosa. In vitro results of revealed that efflux protein inhibitors potentiated the antibacterial activity of enrofloxacin against the selected bacterial strains. The PK-PD mathematical integration of pharmacokinetic and pharmacodynamic data obtained in the study was attempted. The surrogate markers commonly applied for optimization of the dose with pharmacokinetic and pharmacodynamic parameters were Cmax/MIC>10, AUC/MIC>125. The Cmax/MIC in group II, group III, group IV, group V and group VI against E.coli ATCC25922 were 86.92, 80.2, 104.72, 117.36, and 118.48 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against S.aureus ATCC25923 were 10.86, 10.02, 13.09, 14.67, and 14.81 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against K. pneumonia ATCC 700603 were 1.31, 1.21, 1.58, 1.77, 1.79 respectively. The Cmax/MIC in group II, group III, group IV, group V and group VI against P.aureginosa ATCC 27853 were 0.89, 0.82, 1.07, 1.20, 1.21 respectively. The ratio of AUC/MIC>125 will prevent the development of resistance against the antibacterial agent. The AUC/MIC in group II, group III, group IV, group V and group VI against E.coli ATCC25922 were 1813.93, 1859.15, 2446.45, 3167.30, and 2165.40 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against S.aureus ATCC25923 were181.39, 185.91, 244.64, 316.73 and 216.54 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against K. pneumoniae ATCC 700603 were 21.98, 22.53, 29.65, 38.39 and 26.24 h respectively. The AUC/MIC in group II, group III, group IV, group V and group VI against P.aureginosa ATCC 27853 were14.92, 15.30, 20.13, 26.06 and 17.82 h respectively. Based on the PK-PD integration values obtained in the present study it appears that enrofloxacin at the dose rate of 10 mg.Kg-1 is effective against E. coli and S. aureus but not effective against K. pneumoniae and P. aureginosa. Administration of efflux pump inhibitors namely theobromine, glycyrrhetenic acid and glycyrrhizic acid increases bioavailability, maximum plasma concentration of enrofloxacin whereas capsaicin significantly reduced the tmax and half life and increased the elimination rate constant in broiler chicken. The MIC, MBC which were used as indicators of antibacterial effect of enrofloxacin were significantly increased in the presence of efflux protein inhibitors.ThesisItem Open Access GASTROINTESTINAL PARASITISM IN SHEEP AND GOATS IN CERTAIN DISTRICTS OF HYDERABAD-KARNATAK REGION KARNATAKA STATE WITH SPECIAL REFERENCE TO ANTHELMINTIC RESISTANCE(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-10) ADEPPA, J; SREEDEVI, C(MAJOR); Placid E., D’Souza; Ravi Kumar, P; SrinivasaRao, TThe present study was carried out to determine the prevalence of GI parasites and efficacy of commonly used anthelmintics in sheep and goats in Hyderabad-Karnataka region, Karnataka. Faecal samples were collected from sheep (1876) and goats (1745) in H-K region (Bidar, Kalaburgi and Raichur districts), Karnataka for a period of one year to determine the prevalence of gastrointestinal parasitism. Processing of faecal samples by standard faecal floatation and sedimentation techniques revealed an overall prevalence of 65.78 per cent. The rate of prevalence was significantly higher (P<0.05) in goats (67.9%) when compared to that in sheep (63.8%). The rate of prevalence in Bidar, Kalaburgi and Raichur districts was 68.76, 61.8 and 62.28 per cent respectively, without significant (P>0.05) differences in prevalence of GI parasitic infection between districts. The infected small ruminants were parasitized with one or two of eight different species and genera of parasites, each. Out of 3621 faecal samples examined, infection with single parasite (43.94%) was more commonly observed than multiple species infection (21.84%). The most prevalent species were strongyles (27.64%) followed by Eimeria spp. (7.91%), Strongyloides papillosus (3.87%), Trichuris ovis (2.54%), Moniezia spp. (1.02%), amphistomes (0.8%), Buxtonella sulcata (0.11%) and Schistosoma spindale (0.06%). Coproculture studies indicated predominance of Haemonchus contortus larvae. Statistically there was no significant (P>0.05) difference between prevalence of GI parasitic infection and age of animals. The overall prevalence was high in post-weaning lambs and kids (69.61%) than in pre-weaning lambs and kids (68.60%) and adults (60.12%). With regard to the gender of animals, the overall prevalence of GI parasitic infection was significantly (P<0.01) high in female animals (67.9%) than in males (62.85%). The overall prevalence was significantly (P<0.001) high in rainy season (69.04) followed by winter (65.63 %) and summer (62.52%). To determine the efficacy of thiabendazole and ivermectin against H. contortus of sheep and goats in H-K region, egg hatch assay (EHA), larval development assay (LDA) and PCR-RFLP were employed. The results of the EHA revealed significant (P<0.05) inhibitory effect on egg hatching rates in sheep (LD50 = 0.12 μg/mL) and goats (LD50 = 0.115 μg/mL) with thiabendazole. LDA studies also showed significant (P<0.05) inhibitory effect on larval development in sheep (LD50 = 0.133 μg/mL) and goats (LD50 = 0.131 μg/mL) with thiabendazole. Similarly the results of the LDA exhibited inhibitory effect on larval development with ivermectin (LD50 = 0.0428 μg/mL; LD50 = 0.0431 μg/mL). Analyzing the overall situation the study highlights that the thiabendazole and ivermectin had extremely high LD50 than discriminating dose in EHA and LDA, reflecting benzimidazoles and ivermectin resistance. A total of 450 (250 from sheep and 200 from goats) adult male H. contortus from different areas in H-K region were genotyped at codon 200 in β-tubulin isotype 1 gene for the detection of BZ resistance by PCR-RFLP. The genotypic frequencies of three genotypes (homozygous resistant ‘rr’, heterozygous ‘rS’ and homozygous susceptible ‘SS’) for BZ resistance in sheep and goats varied significantly (P<0.01) in the studied area. Out of 250 parasites genotyped in sheep, 43 (17.2%) were ‘rr’, 179 (71.6%) were ‘rS’ and 28 (11.2%) were ‘SS’ type. Among 200 parasites genotyped in goats, 38 (19.0%) were ‘rr’, 129 (64.5%) were ‘rS’ and 33 (16.5%) were ‘SS’ type. Overall, the proportion of BZ resistant (‘r’) allele was significantly (P<0.01) high when compared to per cent prevalence of susceptible allele (‘S’) in worms collected from sheep and goats in H-K region. Among different areas of H-K region the frequency of resistant allele was significantly higher (P<0.01) in Sedam and Lingasugur regions in sheep and goats, respectively. Findings of PCR-RFLP corroborated with the results of EHA and LDA.ThesisItem Open Access COMPARISON OF EXCISION ARTHROPLASTY, DENERVATION AND TRANS-FEMOROARTICULAR WIRING FOR HIP DYSPLASIA IN DOGS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-09) NAGARAJU, N; SREENU, MAKKENA(MAJOR); VASANTH, M.S.; SRINIVAS, MANDA; RAJU, N.K.B.The present study was conducted on clinical cases of dogs with hip disorders viz, dysplastic hip, subluxated and luxated hips. Incidence of hip disorders was 13.31% of orthopedic cases and higher incidence of hip disorders was found in male dogs (62.12%) than in female dogs (37.87%) and breed wise higher incidence was noticed in Labrador retriever breed (28.28%) followed by Non-descriptive dogs (17.68%) whereas, the lowest incidence was observed in Chihuahua and Saint Bernard (1.01%) breeds of dogs. Higher incidence of hip disorders was observed in dogs aged above five years (47.47%) followed by dogs of age 0-1 year and between above one year to five years.Hip extended venterodorsal views of radiographs confirmed the dysplastic, osteoarthritic, subluxated and luxated hips. Dogs with hip disorders under the study were grouped into three groupsgroup I,group II and group III. Excision arthroplasty was done in group I dogs where femoral head and neck was excised and joint capsule was sutured to prevent bone contact between femur and acetabulum. Denervation done in group II dogs wherein craniodorsal gluteal nerves on acetabular rim were destroyed using bone curette. Trans-femoroarticular wiring was done in group III dogs for subluxated and luxated hips using nylon wiring. The dogs were followed up to 60 days. Hematobiochemical changes during the study period in all the three groups were non significant. Physiological parameters were non significant in all the three groups. All the dogs in group I were evidenced by grade I lameness by end of 60 days of study period, five dogs showed grade I lameness in group II and two dogs showed grade I lameness in group III dogs. Radiological evaluation in group I revealed clear gap between acetabulum and excised femoral part. In group II dogs, apparently no change in radiographs were observed during study period. In group III, dogs four dogs showed good apposition of acetabulum and head of femur and reluxation in two dogs. Operation procedures were very simple and less time consuming in group II followed by group I and group III dogs. Functional assessment of dogs by getting the owners feed back over period of six months revealed better results in group I and group II dog than group III dog owners.ThesisItem Open Access CHARACTERIZATION AND EVALUATION OF PHYLOGENETIC STATUS OF KENGURI, MOULI AND YALAGA SHEEP OF KARNATAKA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-09) SIDDALINGSWAMY HIREMATH; VINOO, R(MAJOR); APPANNAVAR, M.M.; MURALIDHAR, M; VENKATA SESHAIAH, Ch.; RAMANI PUSHPA, R.N.Karnataka state hosts five well adapted indigenous sheep breeds viz. Bellary, Kenguri, Hassan, Deccani and Mandya. Apart from these recognized breeds, there are two lesser known genetic groups viz., Mouli and Yalaga reared by farmers in North Karnataka in Vijayapura and Bagalkote districts, respectively. The objective of the present research is to document and compare the phenotypic traits of three genetic groups, Kenguri, Mouli and Yalaga sheep of Karnataka and to study the genetic diversity existing in these genetic groups, using mitochondrial and Y-chromosome markers. Data on morphological and morphometric traits were recorded in the three genetic groups from farmer's flocks in their breeding tracts. Data was classified based on their age and sex. It was observed that the Kenguri sheep were predominantly brown with white patch on forehead. Males are horned and ewes are polled in this breed. The animals are usually with black hooves, medium long drooping ears, short tail, straight face/fore-head or nose line in males and females. The Mouli sheep were generally white with brown patches all over the body, long tail, polled in both sexes, long drooping ears and typical convex face or bowed fore head with Roman nose. The Yalaga sheep were white in colour, with males being horned and females were polled. They have long tail, long drooping ears, straight forehead and straight nose line. The morphometric traits namely body length, height at withers, flank width and face length were longer in Mouli sheep compared to Kenguri and Yalaga sheep. Body weight is also larger in Mouli sheep compared to Kenguri and Yalaga sheep. The trend also holds good for age wise analysis among the three breeds. The genetic diversity and divergence among the three breeds were assessed using mitochondrial DNA. Metadata of fourteen Indian, Chinese, Caucasian breeds and wild Mouflon were also used for the analysis. The wild ancestor of sheep i.e. urial mitochondrial D-loop region was used as an outgroup for phylogenetic analysis. Mitochondrial DNA analysis indicated that there is high variation within these genetic groups but low differentiation between the genetic groups. Also there is high rate of gene flow among the three genetic groups, particularly between Mouli and Yalaga breeds. The observation is contrasting to the phenotypic differences between the two breeds. The phylogenetic analysis indicated that three genetic groups were predominantly clustered in clade A except one Kenguri sheep which is clustered in clade B. To understand paternal origin of the three breeds, the 611 bp promoter region of SRY gene was amplified and sequenced in the three genetic groups. Orthologous region for a total of 17 sheep representing domestic and wild sheep were obtained from the NCBI database. The three sheep breeds under study didn’t reveal any variation in the promoter region of the SRY chromosome, suggesting they are likely to have same paternal origin. Phylogenetic analysis of sequences indicated a plausible paternal lineage from European or Armenian Mouflon for the three genetic groups. The molecular clock analysis based on mitochondrial control region indicated that the Karnataka breeds must have diverged in the lineage A about 20000 yrs ago. A similar number of years ago the Kenguri individual in clade B also might have diverged. This estimate is slightly higher than the time of sheep domestication suggesting possibility of multiple domestication events in the Indian subcontinent. The results in the present study indicate that phenotypically the three genetic groups are different. At molecular level the Mouli and Yalaga sheep are not differentiated based on the mitochondrial DNA analysis. The paternal source for the three genetic groups is likely from the European or Armenian Mouflon.ThesisItem Open Access MOLECULAR EPIDEMIOLOGY OF CANINE PARVOVIRUS IN ANDHRA PRADESH(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) DEEPIKA KUMARI, G; Ramani Pushpa, R.N(MAJOR); Subramanyam, K.V.; Srinivasa Rao, T; Satheesh, KCanine parvovirus-2 (CPV-2) is one of the most pathogenic viral etiologic agents causing hemorrhagic enteritis in dogs. CPV is a small, non-enveloped, single-stranded DNA virus of approximately 5 kb genome and belongs to the genus Protoparvovirus, in the family Parvoviridae.The objective of the present work is to detect, isolate and characterize circulating CPV in different locations of Andhra Pradesh from fecal samples of diarrhoeic dogs and its phylogenetic characterization. A total of 342 fecal samples were obtained from diarrhoeic dogs and preliminary screening of the samples was carried out by haemagglutination assay (HA) using 0.8 % swine RBC, out of which 71 were positive with a titre ranging from 1 in 32 to 1 in 512 (20.76%). The results of HA was confirmed by haemagglutination inhibition assay (HI) using hyperimmune serum raised in rabbits. All 342 fecal samples were screened by PCR using CPV-2ab primers and 233 samples produced an amplicon size of 681 bp. Genotyping of CPV was done by employing multiplex PCR using CPV-2ab and CPV-2b primer pairs. Out of 233 positive samples, 216 (92.70%) samples produced an amplicon size of 681 bp characteristic of CPV-2a and 17 samples (7.29%) yielded two specific amplicons of 681 bp and 427 bp and thus categorized as CPV-2b. The fecal samples not reacted with CPV-2ab and CPV-2b primers were further analysed by PCR-RFLP using restriction enzyme MboII for the detection of CPV-2c strain. Only one sample reacted with 555 primers produced an amplicon size of 583 bp but remain undigested with restriction enzyme. The overall prevalence rate of CPV in Andhra Pradesh was found to be 68.42%. Virus isolation studies from the PCR positive fecal samples were carried out using CRFK and MDCK cell lines. Successful isolation was observed in both the cell lines with varied cytopathic effects. Sixteen out of thirty four processed fecal samples in CRFK cell lines could produce a mild CPE after 72h post infection (PI) at fifth passage level which include increased granularity, rounding of cells and degenerative changes. Five out of nine CPV positive samples produced marked CPE like increased granularity, rounding of cells and complete detachment of cells in MDCK cell lines after 72h PI at third passage. The presence of virus at each passage level was confirmed by PCR assay using H primers. Transmission electron microscopic studies revealed spherical virus like particles of approximately 20 nm in diameter. The Partial VP2 gene sequencing of eighteen CPV field isolates along with the vaccine strain was performed in AB13730 DNA Analyzer and with Chromas lite software. The VP2 gene sequences were compared with CPV reference strains available in the GenBank by BLASTn analysis and were found to be highly specific revealing a maximum identity of 99.84% to 100% with CPV-2a and 99.35 with CPV-2b. One sample which only reacted with 555 primer on sequence analysis, exhibited a nucleotide homology of 99.66 % with CPV-2a. Multiple sequence alignment for the 18 CPV field isolates along with the vaccine strain was done using Clustal W 1.8 program and compared with the reference strains of CPV. The Partial VP2 nucleotide sequences identity of the field isolates when compared with the reference strains was 99.99 % to 100% and a similar identity was also observed among the field isolates. The nucleotide sequences for local CPV-2a isolates exhibited 99.99 % of homology with those of CPV-2b isolates. The nucleotide homology levels between the vaccine strain (CPV-2) and the analysed CPV-2a, CPV-2b isolates was 99.98 and 99.97 %, respectively. The antigenic types CPV-2a and CPV-2b differed from the original CPV-2 in atleast four amino acid positions of the VP2 capsid protein. Amino acid sequence analysis was done using MEGA 7.0 and revealed that thirteen out of 18 found to be new CPV-2a, one as a variant of new CPV-2a and four were characterised as new CPV-2b. Based on the typing systems using key amino acid positions, 77.77% were new CPV-2a and 22.22% were new CPV-2b with no CPV-2c or original CPV-2 found. The phylogenetic analysis revealed that the 14 new CPV-2a isolates were having close ancestral relationship with the Indian and Chinese strains of CPV-2a whereas CPV-2b strains formed a separate clade with the Indian CPV-2b strains. Seroprevalence of CPV was done by Indirect ELISA and HI for a total of 542 sera samples. Indirect ELISA recorded 94.65% in vaccinated and 72.50% in unvaccinated dogs whereas HI detected 56.10% in vaccinated and 16.42% in unvaccinated dogs. The variation in the detection of CPV antibodies by Indirect ELISA and HI from vaccinated and unvaccinated dogs was statistically significant (P < 0.05).ThesisItem Open Access STUDY ON AGEING CHANGES IN THE CORNEA AND RETINA OF BUFFALOES (Bubalus bubalis)(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) PRASANTH BABU, A; JAGAPATHI RAMAYYA, P (MAJOR); NAGAMALLESWARI, Y; SREENU, MAKKENA; LAKSHMI KAVITHA, KThe present study was conducted on age related changes in the cornea and retina of buffaloes. A total of 63 samples from three age groups of buffaloes irrespective of their sex were used in this study. The corneal epithelium of buffaloes was nonkeratinized stratified squamous epithelium with 6 to 7 layers at early age and 12-14 layers of cells in adult animals. There were three types of cells noted in the cornea without any distinct boundaries viz., basal cells, intermediate or wing cells and superficial cells. The Bowman’s membrane was a homogenous and acellular layer and its thickness was increased with advancement of age. The stroma of cornea consisted of uniform collagen fibrils and they were loosely arranged in young animals and tightly packed in aged animals. The keratocytes were interposed between the collagen fibers, which were elongated and thick in young animals and thin in old animals. The Descemet’s membrane was a homogenous non cellular fibrous band with dark anterior band zone and light posterior band zone. The corneal endothelial cells were small and firmly adherent to each other in young buffaloes, but they were enlarged and increased in size in old buffaloes. The age related pigmentation was noticed in epithelium and stroma in aged animals. Numerous sub basal nerve plexuses observed in stroma i.e. between the basal cells, wing cells and superficial cells in all age group of animals. All layers of cornea showed moderate to strong reaction for neutral and acid mucopolysaccharides in buffaloes. In scanning electron microscopy study, no apical microvilli on superficial epithelial cells were noted. The Bowman’s layer was consisted of anterior clear zone lamina lucida and posterior dense zone lamina densa. The immunohistochemical reactivity of cytokeratin 3 was noted in the basal cells of corneal epithelium. The reactivity of CD 31 was increased with age in corneal endothelial cells. In the peripheral portion of retina the retinal pigment epithelium was cuboidal and they were packed with melanin pigment. The thickness of RPE was increased and quantity of melanin pigment was decreased with age advancement. In the photoreceptor layer two types of cells were noted i.e. rod and cone cells. They were tightly packed in young age, whereas loosely arranged in older animals. The number of cones progressively increased in number with advancement of age. The nuclei of outer nuclear layer were displaced into outer plexiform layer in the retina of old animals. The outer limiting membrane was continuous throughout the life in the present study. The thickness of outer plexiform layer was increased with advancement of age due to enhancement of synaptic fibers density. The number and density of horizontal, bipolar and amacrine cells were decreased from young to old buffaloes. The thickness of inner plexiform layer was increased with advancement of age due to increased cystoid spaces and thickening of retinal blood vessels between the synaptic fibers of bipolar, amacrine and ganglion cells. The large α and small β ganglion cells were observed in the retina of buffaloes. The number of α ganglion cells were progressively increased with advancement of age. The corpora amylacea and thickened blood vessels were observed in the nerve fiber layer of retina of old buffaloes. The inner limiting membrane was loose and interrupted in young animals whereas, thick and uninterrupted in old buffaloes. The total thickness of retina was decreased form young to adult animals in the present study. The increased number of cone cells, formation of cystoids spaces in the inner plexiform layer, increased size of ganglion cells, hyalinization and thickening of blood vessels in inner plexiform and nerve fiber layers were noticed with advancement of age in buffaloes. In the present study SEM revealed that RPE were tightly packed with rod to spherical and elongated melanin pigment granules in young animals and loosely packed in old buffaloes. Numerous capillaries were observed in the inner nuclear layer. Numerous cystoid spaces were also observed in outer plexiform and nerve fiber layers of retina in old animals. Immunohistochemical studies revealed that PAX6 activity on RPE was decreased with advancement of age in buffaloes. The activity of recoverin on photoreceptor cells and their nuclei and binucleate cells of retina was decreased in old animals. The activity of calbindin was strong to moderate in horizontal and amacrine cells and it’s activity decreased with advancement of age in buffaloes. Retina macula was appeared as strip like and pale in color located dorsal to the optic disc in buffaloes. In the present study macula contained large RPE cells with tightly packed melanin pigment, relatively more number of cone cells, cells of inner nuclear and less cystoid degeneration in inner plexiform and nerve fiber layers was observed when compared to peripheral retina. Further, there was less degenerating changes in the macula when compared to peripheral retina with advancement of age in buffaloes. The ora serrata was a nonsensory portion of the retina which includes pars ciliaris retina and pars iridica retina that cover ciliary body and iris. This region consisted of less number of rod cells, more number of horizontal and amacrine cells and few large ganglion cells were observed when compared to the peripheral retina in the present study. Optic disc was round to oval in shape and in this all retinal layers were gradually disappeared near the optic disc. At the junction of pars optica retina and optic disc RPE cells were small and devoid of melanin pigment. The blood vessels were large near optic papilla and they were small and tiny away from the papilla in buffaloes.ThesisItem Open Access IN VITRO ANTICANCER ACTIVITY OF 6–THIOGUANINE AND 6-THIOGUANINE LOADED CHITOSAN NANOPARTICLES WITH OR WITHOUT CURCUMIN AND PHARMACOKINETIC STUDIES IN RATS(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA, 2019-08) RASHMI, R; RAVI KUMAR, P(MAJOR); PRAKASH, N; SRINIVASA RAO, G; RAMA DEVI, V; MURALIDHAR, MCancer is the second leading cause of mortality in the world. Cancer nanotherapeutics are rapidly progressing and being implemented to overcome several limitations of conventional drug delivery systems. 6 thioguanine encapsulated chitosan nanoparticles ( 6 – TG – CNPs ) were formulated by ionic - gelation method. Morphologically , the 6 – TG - CNPs were spherical in shape and showed mean size, PDI, z eta potential and e ntrapment eff iciency of 261.63±6.01nm, 0.3 4 ±0.10, + 15.97±0.46mV and 44.27 per cent , respectively. IR spectra confirmed 6 - TG complex with chitosan. In vitro drug release profile of 6 - TG - CNPs revealed slow but increased ( 91.40 ± 1.08 per cent at 48h) release at pH 4.8 compared to less and sustained ( 73.96 ± 1.12 per cent at 48h) release at pH 7.4. MTT assay was conducted on MCF - 7 and PA - 1 cell lines at 48h incubation to determine per cent cell viability . IC 50 values of 6- TG, 6 - TG - CNPs and curcumin for MCF - 7 were 23.09, 17.82 and 15.73μM , respectively. Likewise, IC 50 values of 6 - TG, 6 - TG - CNPs and c urcumin for PA - 1 were 5.81, 3.92 and 12.89μM , respectively. Combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed percent cell viability of 43.67±0.02 and 49.77±0.05, respectively. The in vitro cytotoxicity potential in terms of per cent cell viability, early apoptosis, G2/M phase arrest and global DNA demethylating activity of 6-TG-CNPs alone and in combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells. The Cmax of 6-TG in 6-TG-CNPs alone and 6-TG-CNPs in curcumin pre-treated groups was found to increased by 1.6-fold and 2-fold, respectively when compared to those observed with 6-TG treated group. The rate and extent of 6-TG absorption was increased by 2.5-fold following 6-TG-CNPs alone and 3.2-fold in curcumin followed by 6-TG-CNPs treated group compared to those observed with 6-TG treated group. The enhanced Cmax and AUC for 6-TG were in the order of curcumin pre-treatment + 6-TG-CNPs>6-TG-CNPs>6-TG groups. The apparent volume of distribution was substantially lower for 6-TG in 6-TG-CNPs alone and 6-TG-CNPs in curcumin pre-treated groups when compared to those observed with 6-TG treated group. Apart from this, volume of distribution at steady-state was also found lower for 6-TG in both the 6-TG-CNPs treated groups. These PK parameters suggests the better tumour targeting efficiency of 6-TG nanoformulations with less toxicity in off-target tissues. Thus, the 6-TG encapsulated chitosan nanoparticles prepared in the present study used either alone or in combination with curcumin present a wide scope for efficient delivery of 6-TG at the target sites.
