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ThesisItem Open 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, RajeshVector-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.ThesisItem Open Access DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH(CSKHPKV. Palampur, 2019-07-20) THAKUR, DEEPIKA; Dhar, PrasenjitDiarrhea 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.ThesisItem Open Access DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH.(CSKHPKV, Palampur, 2019-07) Thakur, Deepika; Dhar, PrasenjitDiarrhea 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.ThesisItem Open Access MICROBIOLOGICAL INVESTIGATIONS ON RESPIRATORY INFECTIONS OF SHEEP AND GOATS WITH PARTICULAR EMPHASIS ON MYCOPLASMA(CSKHPKV, Palampur, 2019-07) Sunaina; Verma, SubhashIn 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.ThesisItem Open Access STUDIES ON RESPIRATORY PATHOGENS IN POULTRY OF HIMACHAL PRADESH WITH EMPHASIS ON IDENTIFICATION OF HAEMOPHILUS SPP.(CSKHPKV, Palampur, 2008-07) Durairajan, R.; Sharma, MandeepThesisItem Open Access MOLECULAR CHARACTERIZATION OF DIFFERENT CHLAMYDIAE ASSOCIATED WITH VARIOUS INFECTIONS OF RUMINANTS(CSKHPKV, Palampur, 2011-07) Bhardwaj, Brijesh; Chahota, RajeshChlamydial infections have been reported previously among domestic ruminants from various disorders affecting reproductive system, respiratory system and digestive system in Himachal Pradesh, but the molecular characterization of various species/strains of chlamydiae involved has not been done yet, hence we conducted a molecular epidemiological investigation to detect the prevalence of different chlamydial species/strains in domestic ruminants in different parts of state, migratory tracts and in organized farms of Himachal Pradesh. To know the overall chlamydial prevalence in different disease conditions, total 404 samples were screened using Chlamydiales order specific primers based on 23S rRNA and family Chlamydiaceae specific primers based on VD2 region of ompA gene. Result of chlamydial screening showed that 93 (23%) samples, out of 404 samples were positive for chlamydiae, which showed the higher involvement of chlamydiae in various disease conditions. Out of 205 samples of reproductive disorders, 55 (26.82%) samples were showed chlamydial involvement. Among the cases of reproductive disorders, highest i.e. 38 per cent chlamydial association was detected in endometritis followed by 23 per cent in abortions. Out of 147 samples of pneumonia, 23 (15.64%) samples were found positive for chlamydial infection and from 39 samples of enteritis, chlamydial involvement was found in 33.33 per cent samples. Whereas, two samples of conjunctivitis were also found positive for chlamydial involvement. Molecular characterization and genetic variability studies of chlamydial species/strains detected among ruminants by PCR were done either by PCR-RFLP or by study of nucleotide sequence variation of ompA gene in VD2 region. Overall 57 PCR positive samples were characterized and out of which 39 (68.4%) samples were found C. psittaci, 16 (28%) samples were C. abortus, and two (3.5%) samples were found C. pecorum. From PCR positive samples, isolation of chlamydial strains was also attempted using 6 to 8 day old embryonated chicken eggs and isolation of chlamydiae up to 40.74 per cent could be achieved. Besides chlamydiae, involvement of other bacterial agents was also tested in samples from reproductive diseases. Various bacterial isolates like Brucella melitensis 2 (1.1%), Staphylococcus spp. 48 (26.37%), Streptococcus spp. 7 (3.84%), E. coli 47 (25.82%), Bacillus spp. 31 (17.03%), Klebsiella spp. 11 (6.04%), Arcanobacterium spp. 18 (10%), Pseudomonas spp. 13 (7.14%) and other bacterial species. 24 (13.19%) were isolated from different female reproductive disorders of the livestock. The results showed high prevalence of chlamydial infection among ruminants and involvement of multiple chlamydial species was detected in this study.ThesisItem Open Access DEVELOPMENT OF LATEX AGGLUTINATION TESTS AGAINST PASTEURELLA MULTOCIDA(CSKHPKV, Palampur, 2014-07-19) Kour, Amitoz; Verma, SubhashPasteurella multocida is a causative agent of a number of economically important diseases in livestock including Haemorrhagic Septicaemia (HS) in cattle and buffalo. Due to the peracute and fatal nature of the disease, there is urgent need for rapid diagnosis so that appropriate therapeutic and preventive measures could be undertaken. A study was designed to extract different antigens from P. multocida capsular type A and B that included whole cell lysate (WCL), capsular antigen, outer membrane proteins (OMPs) and lipopolysaccharides (LPS) to develop latex agglutination test(s) for pen-site diagnosis of HS. Bacteria were grown on brain heart infusion broth and whole cell antigen was obtained by centrifugation of sonicated P. multocida suspended in HEPES buffer. OMPs were extracted by ultracentrifugation of the supernatant obtained by addition of HEPES Buffer containing Sodium lauryl sarcosinate detergent and the detergent insoluble OMP enriched fractions were obtained, while capsular antigen was separated by fractional precipitation with addition of polar organic solvents which yielded high molecular weight capsular polysaccharide. LPS was extracted by formalinized saline killing of bacteria followed by ultracentrifugation. Total protein concentration was found to be 8.97 mg.ml-1 and 5.67 mg.ml-1 for OMP capsular type A & OMP type B respectively, while for WCL it was 22.38 mg.ml-1 & 26.89 mg.ml-1. Carbohydrate estimation of capsular type A & type B of LPS and capsular polysaccharide concentrations were estimated to be 188.32 μg.ml-1, 330.71 μg.ml-1 and 4.08 mg.ml-1, 2.38 mg.ml-1 respectively. OMPs & whole cell lysate extracted were subjected to discontinuous SDS-PAGE. Nine polypeptides of MW ranging from 15-100 kDa from OMPs preparation & fifteen polypeptides of MW ranging from 25-98 kDa from whole cell lysate were visualised. These antigens were used to raise hyper-immune sera in rabbits that were used to sensitise latex beads (carboxylate modified polystyrene) by covalent coupling using glutaraldehyde. The raised antisera was tested by AGPT and ELISA to detect presence of polyclonal antibodies. The sensitised latex beads (SLB) with polyclonal sera against WCL were tested in agglutination assay against WCL, OMP(s), LPS and capsular antigens of both strains. All these tests showed agglutination within 60 seconds. Cross reaction was seen with WCL and OMP(s) between both types of P. multocida. The specificity of SLB was also checked by agglutinating the particles with prepared antigens of other bacteria like E. coli, S. aureus, A. lignieresii and P. aeruginosa which showed no agglutination with latex beads. SLB were also tested by reacting them with nasal swabs from healthy as well as infected animal giving no agglutination. Nasal swabs of infected animals were then streaked on BA and suspected colonies suspended in PBS when agglutinated with SLB showed agglutination. These colonies were then confirmed by colony PCR. Minimum concentrations of antigen (100 ng.ml-1) were detected by SLB showing its sensitivity. This test therefore can be used as an aid to provide rapid and confirmatory diagnosis for P. multocida infections of livestock particularly HS.ThesisItem Open Access EVALUATION OF A CANDIDATE GNP-APRV2 FOOTROT NANOVACCINE IN MICE(CSKHPKV, Palampur, 2017) Srivastva, Pratiksha; Verma, SubhashFootrot 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).ThesisItem Open Access MOLECULAR AND IMMUNOLOGICAL EVALUATION OF NEOPLASMS IN DOGS WITH PARTICULAR EMPHASIS ON LYMPHOMA(CSKHPKV, Palampur, 2018-07-24) Sanjeev kumar; Verma, SubhashIn 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.
