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Theses

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2017 - 20197
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Subject: Veterinary Microbiology × End date: 2020 × Start date: 2015 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    DETECTION AND IDENTIFICATION OF EHRLICHIA SPECIES AMONG DOGS, INCONTACT HUMANS AND TICK VECTORS IN HIMACHAL PRADESH
    (CSHHPKV Palampur, 2019-09-30) ANGARIA, SHIVANI; Chahota, Rajesh
    Vector-borne diseases (VBDs) in animals and humans have gained worldwide importance in the climate change scenario. Ehrlichiosis is one of such rickettsial disease. Ehrlichia is an obligate, intra-cytoplasmic, gram-negative bacteria that cause ehrlichiosis in dogs and other vertebrates, transmitted by tick-vectors and occur mainly in the humid and warm regions. These bacteria multiply within the cytoplasm of White Blood Cells (WBCs) of vertebrate hosts and affect various organs. Canine ehrlichiosis is an important tick-borne disease of dogs worldwide and may lead to zoonosis. Till date, not much work has been done on ehrlichiosis, molecular identification and genetic characterization of native strains/species of Ehrlichia prevalent in Himachal Pradesh, therefore, this study was planned to detect the prevalence of Ehrlichia in dogs, tick vectors and humans in different locations of the state. A total of 215 samples were collected including blood samples from dogs (n = 177), blood samples from in-contact humans (n = 24) and ticks collected from the body of the dogs (n = 24). Samples were collected from 105 locations covering 38 tehsils of 9 districts of Himachal Pradesh. All the samples were tested using Ehrlichia genus and three Ehrlichia species (E. canis, E. ewingii and E. chaffeensis) specific PCR tests. 11 (6.2%) of blood samples from dogs were found positive for ehrlichiosis by examination of buffy-coat smears. By PCR tests, out of 177 blood samples of dogs, 107 (60.4%) were found positive for one or more Ehrlichia species. 61 (34.4%) samples were found harboring multiple Ehrlichia species. All the ticks were identified as Rhiphicephalus sanguineus. From ticks, 4.2 per cent samples were found positive, while no human blood sample was found positive. Phylogenetic positions of all the detected Ehrlichia species/strains were determined which were genetically characterized after nucleotide sequencing of partial 16S rRNA gene. It was found that all E. canis, E. platys species/strains were making clusters with earlier reported species/strains from different parts of India and abroad.
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    ThesisItemOpen Access
    DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH
    (CSKHPKV. Palampur, 2019-07-20) THAKUR, DEEPIKA; Dhar, Prasenjit
    Diarrhea is among the most common clinical conditions seen in dogs leading to high morbidity and mortality. It can have various causes, including infectious agents, like viruses, bacteria and parasites. The aim of the study was to isolate enteric DNA viruses from diarrheic dogs of Himachal Pradesh and characterize them at molecular level. A total of 238 fecal/other samples were collected from diarrheic / apparently healthy dogs along with brief clinical history of the animal. The faecal samples were subjected to DNA extraction and PCR for the detection of enteric DNA viruses viz. CPV, CAV-1 and Dog CV with primers targeting the VP2, pⅦ and replicase gene of CPV, CAV-1 and Dog CV respectively of each virus were used in the study. 109 (45.79 %) samples were found positive for CPV-2b and 4 (1.68%) samples were found positive for CAV-1. No samples were found positive for Dog CV. HA along with HI and DotELISA were standardized for virus detection from clinical samples and infected cell culture fluids using hyper immune sera raised in rabbits against CPV and CAV-1. Dot-ELISA was found to be almost similar to in sensitivity HA-HI for detection of viruses. Growth studies of CPV and CAV- 1 were done on MDCK, A-72 and Vero cells. CPV could be adapted to MDCK and A-72 cells but not to Vero cells while adaptation of CAV-1 was not possible on any cell culture. Physicochemical characterization of CPV was done by checking the effect of temperature, pH, chloroform and formalin on the virus. The VP2 (CPV-2) and pⅦ (CAV-1) gene fragment was sequenced directly which revealed close genetic similarity to Indian and Italian isolates of CPV and CAV, respectively. Microbiological studies on fecal samples revealed 37 bacterial isolates that were resistanct to penicillin and streptomycin in antibiogram and were found concurrently associated with clinical cases of viral diarrhea. In conclusion, diarrhea in dogs in H.P are mostly caused by CPV type 2b strain and occasionally by CAV-1 and is frequently associated with other bacterial species.
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    ThesisItemOpen Access
    DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH.
    (CSKHPKV, Palampur, 2019-07) Thakur, Deepika; Dhar, Prasenjit
    Diarrhea is among the most common clinical conditions seen in dogs leading to high morbidity and mortality. It can have various causes, including infectious agents, like viruses, bacteria and parasites. The aim of the study was to isolate enteric DNA viruses from diarrheic dogs of Himachal Pradesh and characterize them at molecular level. A total of 238 fecal/other samples were collected from diarrheic / apparently healthy dogs along with brief clinical history of the animal. The faecal samples were subjected to DNA extraction and PCR for the detection of enteric DNA viruses viz. CPV, CAV-1 and Dog CV with primers targeting the VP2, pⅦ and replicase gene of CPV, CAV-1 and Dog CV respectively of each virus were used in the study. 109 (45.79 %) samples were found positive for CPV-2b and 4 (1.68%) samples were found positive for CAV-1. No samples were found positive for Dog CV. HA along with HI and Dot- ELISA were standardized for virus detection from clinical samples and infected cell culture fluids using hyper immune sera raised in rabbits against CPV and CAV-1. Dot-ELISA was found to be almost similar to in sensitivity HA-HI for detection of viruses. Growth studies of CPV and CAV- 1 were done on MDCK, A-72 and Vero cells. CPV could be adapted to MDCK and A-72 cells but not to Vero cells while adaptation of CAV-1 was not possible on any cell culture. Physicochemical characterization of CPV was done by checking the effect of temperature, pH, chloroform and formalin on the virus. The VP2 (CPV-2) and pⅦ (CAV-1) gene fragment was sequenced directly which revealed close genetic similarity to Indian and Italian isolates of CPV and CAV, respectively. Microbiological studies on fecal samples revealed 37 bacterial isolates that were resistanct to penicillin and streptomycin in antibiogram and were found concurrently associated with clinical cases of viral diarrhea. In conclusion, diarrhea in dogs in H.P are mostly caused by CPV type 2b strain and occasionally by CAV-1 and is frequently associated with other bacterial species.
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    ThesisItemOpen Access
    MICROBIOLOGICAL INVESTIGATIONS ON RESPIRATORY INFECTIONS OF SHEEP AND GOATS WITH PARTICULAR EMPHASIS ON MYCOPLASMA
    (CSKHPKV, Palampur, 2019-07) Sunaina; Verma, Subhash
    In this study, a total of 163 of 198 morbid samples (nasal and tracheal swabs, lung tissues and pleural fluid) from sheep and goats showing respiratory disease signs were positive for bacteria belonging to seven genera and ten species. Staphylococcus aureus (34.94%) was major bacteria followed by E. coli (21.50%). Other bacteria included Bacillus spp., Pseudomonas aeruginosa, S. epidermidis etc. Nine (6.2%) samples also yielded growth resembling mollicutes which were confirmed by PCR. Out of 37, 83.8% S. aureus carried at least one virulence gene. The mecA was detected in 64.51% S. aureus followed by coa and lukPV in 14 (45.16%) and arcA in 12 (38.50%) isolates. Most S. aureus were sensitive to chloramphenicol, ceftriaxone and tetracycline. Penicillin was at least effective antibiotic. Out of 40, 55% isolates of E. coli carried at least one virulence gene. The eaeA, stx2 and UAL were detected in six, eleven and four E. coli isolates. Most E. coli were susceptible to chloramphenicol and ciprofloxacin. MAR index > 0.2 was recorded for S. aureus (75.6%) and E. coli (10%). Trueperella pyogenes was detected in 10 and Histophilus somni in one nasal swab out 50 nasal swabs of goats. In total, 64 of 198 samples were positive for Mycoplasma spp. using generic-specific primers targeting 16S rRNA gene. Also, 12.5% samples detected positive both for M. mycoides cluster and M. capricolum subsp. capripneumoniae, all of which emanated from an outbreak CCPP having case fatality of 45.07%. The 16S rRNA sequencing identified S. maltophilia, S. entericus and Enterococcus spp. These findings suggest that S. aureus is a major opportunistic pathogen of respiratory tract of sheep and goats harbouring many virulent and AMR genes with high MAR indices and MDR phenotype. This calls for rational usage of antibiotics in veterinary practice. Species-specific PCR should be first choice for diagnosis of CCPP, since MCCP is difficult to culture. Finally, the role of novel bacterial sequences recovered from respiratory tract of goats should be investigated in health and disease.
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    ThesisItemOpen Access
    EVALUATION OF A CANDIDATE GNP-APRV2 FOOTROT NANOVACCINE IN MICE
    (CSKHPKV, Palampur, 2017) Srivastva, Pratiksha; Verma, Subhash
    Footrot is one of the most important diseases of small ruminants worldwide. It is a chronic debilitating disease causing separation of hoof leading to severe lameness. Dichelobacter nodosus, a multi-serogroup gram negative anaerobic rod is principal causative agent of footrot. Disease causes heavy loss to sheep industries every year in terms of expenditure incurred in treatment programs, however treatment is of limited value so focus is shifted towards prophylactic immunisation. Currently available footrot vaccines provide only serogroup-specific immunity. Acidic serine protease (Aprv2) secreted by virulent D. nodosus can serve as a better vaccine candidate as it is conserved in all the 10 serogroups. Gold nanoparticles have been proved to possess great potential as antigen carrier and adjuvant, therefore in this study, a candidate gold nanoparticle-AprV2 vaccine was evaluated against footrot in mice. Recombinant AprV2 was expressed and purified followed by its adsorbtion over the surface of gold nanoparticles (18-20nm). Five groups consisting of 10 mice in each group were administered with three doses of GNP-AprV2 nanovaccine, GNP-AprV2 nanovaccine with MPLA, AprV2, GNP and PBS, respectively at a fortnight interval to evaluate the immune response by measuring IgG levels. GNP AprV2-nanovaccine induced higher IgG responses in comparison to AprV2 and MPLA adjuvanted GNP-AprV2 nanovaccine when measured at 45 days. However, the level of IgG were higher for MPLA at earlier time points (14 & 28 days).
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    ThesisItemOpen Access
    MOLECULAR AND IMMUNOLOGICAL EVALUATION OF NEOPLASMS IN DOGS WITH PARTICULAR EMPHASIS ON LYMPHOMA
    (CSKHPKV, Palampur, 2018-07-24) Sanjeev kumar; Verma, Subhash
    In this study, a total of 56 canine tumors were recorded and prevalence of tumors based on sex, age and site were documented. Females (55.36%) were more susceptible than male dogs (44.64%). Highest prevalence of tumors (35.71%) was recorded in the age group of six to nine years and lowest (5.36%) in the age group of less than three years. Most common site of tumor was genitalia. The TVT has highest prevalence (37.50%) followed by squamous cell carcinoma (12.50%) and then adenocarcimoma (10.79%). One case of oral TVT as a primary tumor was also detected. Neoplastic conditions of dogs like lymphoma, mast cell tumor (MCT) and chronic myeloid leukaemia (CML) were diagnosis using various molecular tools. Polymerase chain reaction for antigen receptor rearrangement (PARR) assay for lymphoma, PCR for detection of mutation at juxtamembrane domain of c-kit gene in mast cell tumors and a two-step nested PCR for detection of bcr-abl fusion gene in CML were used. Out of 123 blood samples, two blood samples were found positive for T cell lymphoma using PARR assay. Out of 21 tissue samples, one tumor tissue sample was found positive for MCT. Out of 10 blood samples, three blood samples were found positive for CML. It is concluded that various diagnostic approaches particularly molecular test like PCR was able to detect hidden neoplastic conditions in dogs which were otherwise hard to diagnose clinically and using imaging or cytology techniques. The study also concludes that neoplastic conditions in dogs are common and should form the part of clinical enquiry by the veterinarians.
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    ThesisItemOpen Access
    SELECTION AND EVALUATION OF PHAGE DISPLAY PEPTIDES AGAINST PASTEURELLA MULTOCIDA
    (CSKHPKV, Palampur, 2018-07-24) Dhial, Kritika; Sharma, Mandeep
    Haemorrhagic septicemia is an acute fatal septicaemic disease of cattle and buffaloes and is caused by P. multocida serotype B: 2 in India. Despite its economic importance, there is no specific field level diagnosis for this disease. The pathogenicity of the organism is associated with various virulence factors such as the capsule, lipopolysaccharides, adhesins, toxins, siderophores, sialides and outer membrane proteins. They facilitate the colonisation and invasion of the host tissue. These surface antigens could be a target for both therapeutics as well as diagnostics. Keeping this in mind, the current study was planned to select ligands in the form of peptides using phage display peptide library against major structural components of the Pasteurella multocida and characterize them by using phage ELISA. Ph.D.-12 phage display library was used to select phage peptides. The library titer was 1.8 × 1011 pfu/ml. This heterogenous mixture of phages carrying diverse peptides as ligands was amplified to a final concentration of 2.1x1013 pfu/ml. These amplified phages were then subjected to the alternate selection/subtraction methodology of panning using suspension method in which alternate rounds of positive selection against P. multocida and negative selection against Haemophilus influenzae and Actinobacillus lignieresii were performed. For round 1 of positive selection, 100μl of 2.1x1013 pfu/ml phages were employed and 5.8 × 106 pfu/ml of eluted phages were recovered. For negative selection of round 1 these recovered eluted phages were amplified and 1013 pfu/ml phages were employed. Around 2.8 × 1011 pfu/ml phages remained unbound which were re-amplified to carry out round 2 of positive selection. 1.3 × 1011 pfu/ml were eluted and amplified for round 2 of negative selection to the concentration 1013 pfu/ml and 1.4 × 1012 phages were left unbound. After completing round 3 of alternate selection/subtraction, 4.8 × 1012 pfu/ml were recovered. This study showed that with every round of selection, the non-binding and non-specific phages reduced and the pool of selectively binding phages to the P. multocida with each amplification increased. Further to analyse the affinity of selectively binding phages indirect phage ELISA was carried out. Out of a total of 48 phages, 16 clonal phages were selected for indirect phage ELISA. Out of these 16, five clonal phages viz. B6, B3, B7, B5 and A5 bound their target with high intensity giving higher OD values at 450 nm. These did not or bound poorly to closely related bacteria as the OD values were close to the negative control. The phages have been submitted for sequencing to further characterize them for their structural and functional attributes. This study concludes that it possible to select peptides against the intended target and that such peptides could be used for specific diagnosis of pathogens or as antimicrobial therapeutics after thorough characterization.