Comparison of the Contributions of Heat-Labile Enterotoxin and Heat-Stable Enterotoxin b to the Virulence of Enterotoxigenic \u3ci\u3eEscherichia coli \u3c/i\u3ein F4ac Receptor-Positive Young Pigs

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 study, we compared these two toxins in terms of importance for piglets \u3e1 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

Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides

AbstractThe current control method for contagious bovine pleuropneumonia (CBPP) in Africa is vaccination with a live, attenuated strain of Mycoplasma mycoides subsp. mycoides (Mmm). However, this method is not very efficient and often causes serious adverse reactions. Several studies have attempted to induce protection using inactivated mycoplasma, but with widely contradictory results. Therefore, we compared the protective capacity of the live T1/44 vaccine with two inactivated preparations of Mmm strain Afadé, inoculated with an adjuvant. Protection was measured after a challenge with Afadé. The protection levels were 31%, 80.8% and 74.1% for the formalin-inactivated, heat-inactivated and live attenuated preparations, respectively. These findings indicate that low doses of heat-inactivated Mmm can offer protection to a level similar to the current live attenuated (T1/44) vaccine formulation

Reduced intestinal colonization of adult beef cattle by \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 \u3ci\u3etir\u3c/i\u3e deletion and nalidixic-acid-resistant mutants lacking flagellar expression

The importance of the Escherichia coli O157:H7 translocated intimin receptor (Tir) protein in intestinal colonization and the effect of infection with Tir+ strains on protection against subsequent challenge was studied in adult beef cattle. Cattle were orally inoculated (C1) with a Shiga toxin-2++ E. coli O157:H7 strain that was Tir+ or Tir-, and 42 days later were re-challenged (C2) with the nalidixic acid (Nal) R parent strain to test whether prior infection had any effect on fecal shedding. During the first 14 days post-C1, the NalS wildtype (WT) strain was shed at significantly higher levels and for a longer period than the other strains; however, the mean levels of shedding of the NalR and Δtir complemented strains were not significantly different from that of the Tir- strains. The Δtir, Tir complemented mutant, and Δtir vector control strains inadvertently did not express flagellin, and did not effectively colonize the intestine. We were unable to determine whether Tir exposure at C1 had any effect on protection. Further, those given an initial inoculation with a non-flagellated variant of E. coli O157:H7 were more susceptible to a second challenge with motile E. coli O157:H7 than those originally inoculated with motile strains. The cause of the loss of expression of flagellin was not addressed. We suggest that either the flagellum or a factor that regulates both its production and that of some other effector has a significant function in colonization

Relationship between Heat-Labile Enterotoxin Secretion Capacity and Virulence in Wild Type Porcine-Origin Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e Strains

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system(T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme- linked immunosorbent assay tomeasure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P\u3c0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92%of the variation in time-to-a-moribund-condition (R2 = 0.92, P\u3c0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenicWT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407.MaximumLikelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATPbinding capacity of GspE and LT secretion, and between the latter and virulence

Relative Importance of Heat-Labile Enterotoxin in the Causation of Severe Diarrheal Disease in the Gnotobiotic Piglet Model by a Strain of Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e That Produces Multiple Enterotoxins

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- (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 Nalr parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT- mutant was 67% lower (P \u3c 0.05) and that of those inoculated with the complemented strain was 36% higher (P \u3c 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 \u3c 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

Pathogenicity of an Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e Hemolysin (\u3ci\u3ehlyA\u3c/i\u3e) Mutant in Gnotobiotic Piglets

Pigs infected with hemolytic F4+ strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4+ enterotoxigenic E. coli to 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

Significance of Heat-Stable and Heat-Labile Enterotoxins in Porcine Colibacillosis in an Additive Model for Pathogenicity Studies

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 disease caused by K88-positive ETEC in young pigs, enterotoxin-positive and -negative isogenic E. coli strains were constructed by using pBR322 to clone and express LT and STb. Four strains, K88(+) astA, K88(+) astA/pBR322, K88(+) astA STb(+), and K88(+) astA LT(+), were constructed and subsequently included in gnotobiotic piglet challenge studies, and their pathogenesis was assessed. The results indicated that all K88(+) isogenic strains were able to colonize the small intestines of piglets exhibiting the K88 receptor. However, only LT- and STb-positive strains caused appreciable diarrhea. Piglets inoculated with the K88(+) astA LT(+) strain became dehydrated within 18 h, while those inoculated with the K88(+) 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

Relative Importance of Heat-Labile Enterotoxin in the Causation of Severe Diarrheal Disease in the Gnotobiotic Piglet Model by a Strain of Enterotoxigenic Escherichia coli That Produces Multiple Enterotoxins

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(−) (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 (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

Salmonella enterica Serovar Enteritidis tatB and tatC Mutants Are Impaired in Caco-2 Cell Invasion In Vitro and Show Reduced Systemic Spread in Chickens▿ †

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 ΔtatB 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