DEVELOPMENT OF LATEX AGGLUTINATION TESTS AGAINST PASTEURELLA MULTOCIDA
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Date
2014-07-19
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CSKHPKV, Palampur
Abstract
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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