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ABSTRACTSalmonella entericasubsp. entericaserovar Enteritidis is a leading causative agent of gastroenteritis in humans. This pathogen also colonizes the intestinal tracts of poultry and can spread systemically in chickens. Transfer to humans usually occurs through undercooked or improperly handled poultry meat or eggs. The bacterial twin-arginine transport (Tat) pathway is responsible for the translocation of folded proteins across the cytoplasmic membrane. In order to study the role of the Tat system in the infection and colonization of chickens by SalmonellaEnteritidis, we constructed chromosomal deletion mutants of the tatBand tatCgenes, which are essential components of the Tat translocon. We observed that the tatmutations affected bacterial cell morphology, motility, and sensitivity to albomycin, sodium dodecyl sulfate (SDS), and EDTA. In addition, the mutant strains showed reduced invasion of polarized Caco-2 cells. The wild-type phenotype was restored in all our SalmonellaEnteritidis tatmutants by introducing episomal copies of the tatABCgenes. When tested in chickens by use of a SalmonellaEnteritidis ΔtatBstrain, the Tat system inactivation did not substantially affect cecal colonization, but it delayed systemic infection. Taken together, our data demonstrated that the Tat system plays a role in SalmonellaEnteritidis pathogenesis.

AbstractA large-scale clinical vaccine trial of commercially fed cattle was conducted to test the efficacy of a two-dose regimen of a vaccine product against type III secreted proteins of enterohemorrhagic Escherichia coliO157:H7 on the probability to detect the same organism from terminal rectal mucosa (TRM) as a measure of gut colonization. Vaccine was administered to all cattle within treated pens at arrival processing and at reimplant processing. At harvest, TRM was collected from a sample of cattle from within vaccinated and nonvaccinated pens. The TRM were collected by scraping the mucosa of the terminal rectum 3–5 cm proximal to the rectoanal juncture. E. coliO157:H7 was isolated and identified from TRM using standard culture methods involving selective enrichment, immunomagnetic separation, and PCR confirmation. The probability to detect E. coliO157:H7 from TRM was modeled using a generalized linear mixed model with a logit link function and accounting for random effects of pen within feedlot. Seven hundred eighteen cattle were tested from within 21 pens of cattle (11 vaccinated and 10 not vaccinated) representing 3683 cattle. E. coliO157:H7 was cultured from 68 of 718 (9.5%) TRM samples. Eleven of 382 (2.9%) vaccinated cattle and 57 of 336 (17.0%) nonvaccinated cattle were TRM culture positive. From the multilevel logistic model, vaccinated cattle were 92% less likely to be colonized with E. coliO157:H7 than nonvaccinated cattle (odds ratio [OR] = 0.07, p= 0.0008). Additional explanatory variables were region of the state (OR = 7.4, p= 0.04), and pens with fewer cattle (OR = 0.22, p= 0.05). We concluded that the two-dose vaccine regimen effectively reduced the probability for E. coliO157:H7 colonization of the terminal rectum of commercially fed cattle at harvest.

ABSTRACTIn swine, the most common and severe enterotoxigenic Escherichia coli(ETEC) infections are caused by strains that express K88 (F4)+fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coliheat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets <1 week old and also enhances bacterial colonization of the intestine. In the present study, we compared these two toxins in terms of importance for piglets >1 week old with a study design that involved construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb−mutant (ΔestB) did not significantly differ from that of the parent. However, deletion of the LT genes (ΔeltAB) in the STb−mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4+ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old. However, in contrast to previous studies with gnotobiotic piglets, there was no evidence that the expression of LT enhanced the ability of the F4+ETEC strain to colonize the small intestine.

ABSTRACTEnterotoxigenic Escherichia coli(ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT−(ΔeltAB) and complemented mutants of an F4+LT+STb+EAST1+ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4+fimbria. Sixty percent of the piglets inoculated with the LT−mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nalr) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nalrparent, the mean rate of weight loss (percent per hour) of those inoculated with the LT−mutant was 67% lower (P< 0.05) and that of those inoculated with the complemented strain was 36% higher (P< 0.001). Similarly, piglets inoculated with the LT−mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (P< 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.

ABSTRACTPigs infected with hemolytic F4+strains of enterotoxigenic Escherichia colioften develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4+enterotoxigenic E. colito cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.