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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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Subject: Veterinary Microbiology × Author: Kour, Amitoz × End date: 2014 × Start date: 2014 × Thesis Type: M.V.Sc. ×
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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.