Superoxide dismutase SodB is a protective antigen against Campylobacter jejuni colonisation in chickens

Abstract

Campylobacter is the leading cause of foodborne diarrhoeal illness in the developed world and consumption or handling of contaminated poultry meat is the principal source of infection. Strategies to control Campylobacter in broilers prior to slaughter are urgently required and are predicted to limit the incidence of human campylobacteriosis. Towards this aim, a purified recombinant subunit vaccine based on the superoxide dismutase (SodB) protein of C. jejuni M1 was developed and tested in White Leghorn birds. Birds were vaccinated on the day of hatch and 14 days later with SodB fused to glutathione S-transferase (GST) or purified GST alone. Birds were challenged with C. jejuni M1 at 28 days of age and caecal Campylobacter counts determined at weekly intervals. Across three independent trials, the vaccine induced a statistically significant 1 log10 reduction in caecal Campylobacter numbers in vaccinated birds compared to age-matched GST-vaccinated controls. Significant induction of antigen-specific serum IgY was detected in all vaccinated birds, however the magnitude and timing of SodB-specific IgY did not correlate with lower numbers of C. jejuni. Antibodies from SodB-vaccinated chickens detected the protein in the periplasm and not membrane fractions or on the bacterial surface, suggesting that the protection observed may not be strictly antibody-mediated. SodB may be useful as a constituent of vaccines for control of C. jejuni infection in broiler birds, however modest protection was observed late relative to the life of broiler birds and further studies are required to potentiate the magnitude and timing of protection.

Keywords: Antibody; Campylobacter jejuni; Chickens; Protection; Superoxide dismutase; Vaccine.

Fig. 1

Purity and immunogenicity of vaccine preparations. (A) SDS–PAGE analysis of GST, GST–CjaA and GST–SodB vaccine preparations, followed by silver staining. (B) Western blot of the vaccine preparations in panel A using anti-GST antibody. (C) Western blot of the vaccine preparations in panel A using sera from Campylobacter-infected but non-vaccinated chickens. For all the lanes presented a total of 1 μg protein was used, in a final volume of 20 μl, mixed 1:1 with denaturing running buffer.

Fig. 2

Purified recombinant SodB-based vaccine, but not a CjaA-based vaccine, was protective against homologousCampylobacterchallenge in chickens. Data represent the arithmetic mean of log transformed caecal counts of C. jejuni from a minimum of 3 independent trials where each antigen was tested concomitantly, each using 3–6 birds per time interval, ± the standard error of the mean (SEM). Data for the GST and GST–CjaA control groups was available from an additional experiment that did not test GST–SodB concomitantly but which had an identical design to the three experiments that tested GST–SodB in parallel. Samples were collected at weekly intervals at post-mortem examination and as a result the lines are inferred to reflect the course of excretion, and are not longitudinal values from the same animals. Values in the table are mean CjaA- and SodB-specific serum IgY and bile secretory IgA levels in vaccinated and control groups measured by ELISA at the time intervals on the x-axis. Changes compared to the GST control group were determined using a generalised linear model (R² = 0.47) and post-hoc Dunnet's tests. Statistically significant differences to the GST control group are denoted with and *** for P ≤ 0.001.

Fig. 3

Serum IgY levels and caecal Campylobacter counts do not correlate in individual birds. The data represent a linear regression in individual birds of caecal Campylobacter counts on fold changes in OD_450 nm values in ELISAs measuring antigen-specific serum IgY levels in the GST–CjaA (panel A) and GST–SodB (panel B) vaccinated birds. Fold changes in serum IgY (indicated on the x axis) were calculated by dividing the OD_450 nm reading for individual birds in the Campylobacter vaccine groups by the mean reactivity of the GST only vaccinated group to the corresponding antigen.

Fig. 4

SodB is absent from the outer membrane and surface of C. jejuni. (A) Immunoblotting of inner membrane, outer membrane and periplasmic subcellular fractions of C. jejuni 11168H with sera from GST–SodB vaccinated birds collected immediately prior to challenge. (B) Immunofluorescence microscopy of C. jejuni 11168H stained with sera from the same birds used in panel A and from GST only vaccinated birds as a negative control.