Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle

Abstract

The induction of secretory and inflammatory responses in calves by Salmonella typhimurium and Salmonella dublin strains was compared, and the effects of mutations in the invH and stn genes were assessed. S. typhimurium induced greater secretory and inflammatory responses than S. dublin in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P. R. Watson, S. M. Paulin, A. P. Bland, P. W. Jones, and T. S. Wallis, Infect. Immun. 63:2743-2754, 1995). These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis. The secretory and inflammatory responses induced by S. typhimurium and S. dublin in bovine ligated ileal loops were not significantly altered by mutation of stn, which suggests that stn does not have a major role in Salmonella-induced enteritis. The invH mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves. The attenuation associated with the invH mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of S. typhimurium to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered. However, lysis of macrophages following infection with S. typhimurium was significantly reduced by the invH mutation. The invH mutation prevented the normal secretion of several proteins, including SipC, by S. typhimurium, indicating that the function of the inv-spa-encoded type III protein secretion system was disrupted. Taken together, these observations implicate inv-spa-dependent effectors in mediation of Salmonella-induced enteritis in cattle. Clearly, however, other undefined serotype-specific virulence factors are also involved in Salmonella-induced enteritis.

FIG. 1

Procedure for the preparation of a genetic construct consisting of a Km^r cassette flanked on each side by approximately 300 bp of DNA complementary to stn. STN1, STN2, STN3, and STN4 are oligonucleotide primers designed to be complementary to the DNA sequence of stn. A more detailed description of this procedure is given in Materials and Methods. The oligonucleotide primers KMR1 and KMR2 were used to check the stn mutation by PCR.

FIG. 2

PCR products generated with primers designed to be complementary to the sequence of stn flanking the predicted site of insertion of Km^r (STN1 and STN4) and with template DNA from wild-type Salmonella strains (lanes 2, 4, 6, and 8) or from stn mutants (lanes 3, 5, 7, and 9). Lane 1, standard markers; lanes 2 and 3, S. typhimurium ST4/74; lanes 4 and 5, S. typhimurium ST12/75; lanes 6 and 7, S. dublin SD2229; lanes 8 and 9, S. dublin SD3246; lane 10, negative control. Sizes of standard markers are shown on the left.

FIG. 3

Secretory and inflammatory responses in bovine ileal loops 12 h after inoculation with S. typhimurium ST4/74 or ST12/75 or with S. dublin SD2229 or SD3246. The secretory response is expressed as the volume of fluid within a loop/length of the loop. The PMN influx ratio is expressed as the PMN influx within a test loop/PMN influx in the control loops. The results from six calves are presented; each strain was tested in triplicate in each animal. Symbols: ▪, wild type; □, stn mutant; ░⃞, invH mutant.

FIG. 4

Mean rectal temperatures of calves following oral inoculation with either S. typhimurium ST4/74 (wild type) (solid line) or its derivative invH mutant (dotted line). Each datum point up to and including that for 48 h is derived from three calves for the wild-type strain and four calves for the invH mutant. After this time, the data for the wild-type strain are from only one calf and the data for the invH mutant are from two calves.

FIG. 5

Mean scour scores of calves following oral inoculation with either S. typhimurium ST4/74 (wild type) (solid line) or its derivative invH mutant (dotted line). Each datum point up to and including that for 48 h is derived from three calves for the wild-type strain and four calves for the invH mutant. After this time, the data for the wild-type strain are from only one calf and the data for the invH mutant are from two calves.

FIG. 6

Recovery of salmonellas from systemic sites, intestinal lymph nodes, and intestinal walls of calves at 18 (a), 54 (b), and 96 (c) h after oral inoculation with S. typhimurium ST4/74 (wild type) (▪) or its derivative invH mutant (□). Each bar represents the mean of triplicate samples from two animals, with the exception of that for the wild-type strain at 96 h, which is the mean from only one animal.

FIG. 7

Recovery of bacteria following incubation with PMNs in the presence or either normal or heat-inactivated bovine serum and after incubation with normal serum alone. Each bar represents the mean from three experiments, each performed in triplicate. Symbols: ▪, wild type S. typhimurium ST4/74; □, invH mutant of S. typhimurium ST4/74; ░⃞, E. coli K-12.

FIG. 8

Lysis of macrophages following incubation with S. typhimurium ST4/74 (▪), S. typhimurium ST12/75 (□), or E. coli K-12 (○) for 1, 2, and 3 h after infection. This is a representative experiment from a total of three experiments and was performed in triplicate. Solid lines, wild type; dotted lines, invH mutant.

FIG. 9

Analysis of proteins secreted by wild-type S. typhimurium ST12/75 (lanes 1) and its derivative invH mutant (lanes 2) by electrophoresis (A) and by Western blotting (B). Proteins from culture supernatants were concentrated and separated on an SDS-polyacrylamide gel and stained with Coomassie brilliant blue. Three proteins present only in the supernatant of the wild-type strain were of molecular sizes similar to those of SipA, SipC, and SipD (87, 42, and 36 kDa, respectively). A band corresponding to the 42-kDa protein was identified by Western blotting with an anti-SipC monoclonal antibody. The sizes of the molecular mass standards are given on the left.