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Chaudhary Sarwan Kumar Himachal Pradesh Agriculture University, Palampur

Himachal Pradesh Krishi Vishvavidyalaya (renamed as Chaudhary Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya in June, 2001) was established on 1st November, 1978.The College of Agriculture (established in May, 1966) formed the nucleus of the new farm University. It is ICAR accredited and ISO 9001:2015 certified institution. The Indian Council of Agricultural Research has ranked this University at eleventh place among all farm universities of the country. The University has been given the mandate for making provision for imparting education in agriculture and other allied branches of learning, furthering the advancement of learning and prosecution of research and undertaking extension of such sciences, especially to the rural people of Himachal Pradesh. Over the years, this University has contributed significantly in transforming the farm scenario of Himachal Pradesh. It has developed human resources, varieties and technologies and transferred these to farming community enabling the State to receive the “Krishikarman award” of Govt. of India four times in row for food grain production among small states of the country. Today, the State has earned its name for hill agricultural diversification and the farming community has imposed its faith in the University.

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2008 - 200912010 - 20185
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Subject: Veterinary Microbiology × End date: 2018 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION OF DIFFERENT CHLAMYDIAE ASSOCIATED WITH VARIOUS INFECTIONS OF RUMINANTS
    (CSKHPKV, Palampur, 2011-07) Bhardwaj, Brijesh; Chahota, Rajesh
    Chlamydial 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.
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    ThesisItemOpen Access
    DEVELOPMENT OF LATEX AGGLUTINATION TESTS AGAINST PASTEURELLA MULTOCIDA
    (CSKHPKV, Palampur, 2014-07-19) Kour, Amitoz; Verma, Subhash
    Pasteurella 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.
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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.