Enterotoxigenic 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 ؊ (⌬eltAB) 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 (Nal r ) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal r parent, 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.

Although heat-stable (ST) and heat-labile (LT) enterotoxins produced by enterotoxigenic Escherichia coli (ETEC) have been documented as important factors associated with diarrheal diseases, investigations assessing the contributions of individual enterotoxins to the pathogenesis of E. coli infection have been limited. To address the individual roles of enterotoxins in the diarrheal؉ astA STb ؉ strain did not, although diarrhea developed in several piglets. The changes in the blood packed-cell volume and plasma total protein of gnotobiotic piglets inoculated with the LT-positive strains were significantly greater than those of pigs inoculated with the K88 astA/pBR322 strain (P ‫؍‬ 0.012, P ‫؍‬ 0.002). Immunochemistry image analysis also suggested that LT enhanced bacterial colonization in a gnotobiotic piglet model. This investigation suggested that LT is a major contributor to the virulence of K88؉ ETEC and that isogenic constructs are a useful tool for studying the pathogenesis of ETEC infection.Escherichia coli strains that colonize the small intestines, invade intestinal epithelial cells, and/or produce one or more toxins are important causes of diarrheal disease in both farm animals and humans. The virulence of enterotoxigenic E. coli (ETEC) is believed to be associated with the production of fimbrial adhesins and enterotoxins (1,19,35,36,51,54). Fimbrial adhesins mediate the attachment of bacteria to the surface of host epithelium cells and allow bacterial colonization. Fimbriae produced by different ETEC strains are quite diverse (21). In swine, ETEC strains that produce K88 (F4) or F18 are the most common currently associated with diarrheal diseases (19). These fimbriae apparently bind to glycoconjugates in the porcine enterocyte brush borders, and the absence of the respective glycoconjugate renders the animal resistant to bacterial colonization and consequent diarrheal diseases (14,15,20,48,49).Enterotoxins, including heat-stable enterotoxins (STa and STb) and heat-labile enterotoxin (LT) (23,25,39,45), have been found to disrupt intestinal fluid homeostasis and to cause hypersecretion of fluid and electrolytes through activation of adenylate cyclase (by LT) or guanylate cyclase (by STa) in small intestinal mucosal cells (26, 34). There are two major serogroups of LT found among E. coli strains: LT-I and LT-II. LT-I is associated with diarrheal diseases of both humans and animals, while LT-II is typically associated with diarrheal disease in animals. STs are small and monomeric molecules and may be associated with either human or animal disease (45, 55). STa and STb are the two classes of STs first recognized and differ from each other in both structure and enzyme activity (11,12). STa is produced by ETEC and other bacteria, while STb is found only associated with ETEC. A third ST, enteroaggregative E. coli (EAEC) EAST1, has been more recently identified. It is a plasmid-mediated enterotoxin, of low molecular weight and is frequently but not exclusively associated with EAEC isolated from children with p...

Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ⌬SPI-1 and ⌬invG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ⌬SPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ⌬SPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ⌬SPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.

In 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. coli heat-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 (⌬estB) did not significantly differ from that of the parent. However, deletion of the LT genes (⌬eltAB) 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.

Salmonella enterica subsp. enterica serovar 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 Salmonella Enteritidis, we constructed chromosomal deletion mutants of the tatB and tatC genes, which are essential components of the Tat translocon. We observed that the tat mutations 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 Salmonella Enteritidis tat mutants by introducing episomal copies of the tatABC genes. When tested in chickens by use of a Salmonella Enteritidis ⌬tatB strain, 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 Salmonella Enteritidis pathogenesis.

Escherichia coli O157:H7 is an important food-borne pathogen and cause of hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are an important reservoir of E. coli O157:H7, in which the organism colonizes the intestinal tract and is shed in the feces. Vaccination of cattle has significant potential as a pre-harvest intervention strategy for E. coli O157:H7; however, basic information about the bovine immune responses to important bacterial colonization factors resulting from infection has not been reported. The serum and fecal IgG and IgA antibody responses of adult cattle to E. coli O157:H7 intimin, translocated intimin receptor (Tir), E. coli-secreted proteins (Esp)A, EspB and O157 lipopolysaccharide (LPS) in response to infection were determined. All animals were seropositive for all five antigens prior to inoculation, with antibody titers to EspB and O157 LPS significantly higher (P<0.05) than those to Tir, intimin and EspA. After inoculation, the cattle became colonized and developed significant increases in their serum antibody titers to intimin, Tir, EspB, EspA and O157 LPS (P<0.05); however, by 42 days post-inoculation the titers to all except EspB were on the decline. In contrast, pre- and post-inoculation fecal IgG and IgA antibodies to these same antigens were not detected (<1:5). These results indicate that cattle respond serologically to E. coli O157:H7 type III secreted proteins, intimin and O157 LPS during the course of infection and the response is correlated with the extent of fecal shedding.

A 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 coli O157: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. coli O157:H7 was isolated and identified from TRM using standard culture methods involving selective enrichment, immunomagnetic separation, and PCR confirmation. The probability to detect E. coli O157: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. coli O157: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. coli O157: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. coli O157:H7 colonization of the terminal rectum of commercially fed cattle at harvest.

Preharvest intervention strategies to reduce Escherichia coli O157:H7 in cattle have been sought as a means to reduce human foodborne illness. A blinded clinical trial was conducted to test the effect of a vaccine product on the probability that feedlot steers, under conditions of natural exposure, shed E. coli O157:H7 in feces, are colonized by this organism in the terminal rectum, or develop a humoral response to the respective antigens. Steers (n = 288) were assigned randomly to 36 pens (eight head per pen), and pens were randomized to vaccination treatment in a balanced fashion within six dietary treatments of an unrelated nutrition study. Treatments included vaccination or placebo (three doses at 3-week intervals). Fecal samples for culture (n = 1,410) were collected from the rectum of each steer on pretreatment day 0 and posttreatment days 14, 28, 42, and 56. Terminal rectum mucosal (TRM) cells were aseptically collected for culture at harvest (day 57 posttreatment) by scraping the mucosa 3.0 to 5.5 cm proximal to the rectoanal junction. E. coli O157:H7 was isolated and identified with selective enrichment, immunomagnetic separation, and PCR confirmation. Vaccinated cattle were 98.3% less likely to be colonized by E. coli O157:H7 in TRM cells (odds ratio = 0.014, P < 0.0001). Diet was also associated with the probability of cattle being colonized (P = 0.04). Vaccinated cattle demonstrated significant humoral responses to Tir and O157 lipopolysaccharide. These results provide evidence that this vaccine product reduces E. coli O157:H7 colonization of the terminal rectum of feedlot beef cattle under conditions of natural exposure, a first step in its evaluation as an effective intervention for food and environmental safety.