Brad T. Bosworth

Pathogenesis of Escherichia coli O157:H7 in weaned calves.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) that cause diarrhea, hemorrhagic colitis, and hemorrhagic uremic syndrome in humans. One strategy for reducing human foodborne EHEC infections is to reduce the levels of EHEC in cattle. Bovine O157:H7 infection models will facilitate identification of virulence factors involved in bovine infections. O157:H7 cause severe diarrhea and attaching and effacing (A/E) mucosal lesions in colostrum-deprived neonatal (< 2 h) calves. We hypothesized that O157:H7 also cause A/E lesions in older calves, but these were not detected in earlier studies because intestinal levels of O157:H7 were too low (< 10(6) CFU/g of tissue) for detection of focally distributed microscopic lesions. Weaned 3- to 4-month-old calves were fasted 48 h, inoculated via stomach tube with 10(10) CFU of O157:H7 or nonpathogenic E. coli, necropsied 4 d pi and examined histologically. Calves inoculated with O157:H7 had higher intestinal levels of inoculated E. coli than control animals. The rectum was the major site of colonization. A/E lesions were seen in the rectum and cecum of calves with high levels of O157:H7. Weaned calves, like neonatal calves, are susceptible to intestinal damage induced by EHEC O157:H7. The rectum and cecum may be principal sites of EHEC O157:H7 colonization during the carrier-shedder state in cattle.

Prevalences of Some Virulence Genes among Escherichia Coli Isolates from Swine Presented to a Diagnostic Laboratory in Iowa

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Sequence of the bovine CD44 cDNA: Comparison with human and mouse sequences

CD44 is a cell-surface glycoprotein involved in leukocyte adherence, T-cell activation and lymphocyte homing. We have isolated and sequenced a cDNA clone which encodes for bovine CD44. The predicted amino acid sequence of bovine CD44 has an overall high similarity with that of human and mouse CD44, 79.5 and 73.2%, respectively. In all three species, CD44 has a similar transmembrane region and cytoplasmic tail. In addition, all of the cysteine residues and a majority of the putative N-linked glycosylation sites in the extracytoplasmic domain are conserved between bovine, human and mouse. All three species have an area of low interspecies similarity within the extracytoplasmic domain. This area has a similarity of 34% between bovine and human, 27% between bovine and mouse, and 35% between human and mouse. The location of this area of low similarity is conserved between species.

Pathogenesis of O157:H7 Escherichia Coli Infection in Neonatal Calves

Cattle have been implicated as an important reservoir of Shiga-like toxin-producing Escherichia coli (SLTEC) O157:H7, enterohemorrhagic E. coli (EHEC) that cause hemorrhagic colitis and hemorrhagic uremic syndrome in humans. Naturally- or experimentally-infected cattle can shed low levels of E. coli O157:H7 long-term, but little is known about the pathogenesis of E. coli O157:H7 infection in cattle. E. coli O157:H7 induce characteristic attaching and effacing (A/E) mucosal lesions in ceca and colons of 1-day-old gnotobiotic piglets and this model is used to study the pathogenesis of SLTEC infections. A/E lesions were not detected in histologic sections of the intestines from adult cattle or 3- to 14-week-old calves infected with E. coli O157:H7. Our objective was to determine if E. coli O157:H7 induce A/E lesions in neonatal calves. Colostrum-deprived calves (< 12-h-old) were bottle-fed with antibiotic-free milk replacer containing 10(10) colony forming units (CFU) of O157:H7 (SLT-I+, SLT-II+) or nonpathogenic E. coli, necropsied 18 h postinfection and their intestines examined histologically. Bacterial attachment, effacement of microvillous borders, and destruction of epithelium were observed in the intestines of the neonatal calves inoculated with E. coli O157:H7. No lesions were observed in calves inoculated with nonpathogenic E. coli. The distribution of intestinal lesions in neonatal calves resembled that in gnotobiotic pigs. Neonatal calves are apparently more susceptible to A/E lesions induced by E. coli O157:H7 than are older calves or adult cattle and provide a model for studying the pathogenesis of E. coli O157:H7 infections in cattle.

Design and Evaluation of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Identification of Genes for Nine Different Virulence Factors Associated with Escherichia Coli that Cause Diarrhea and Edema Disease in Swine

A multiplex polymerase chain reaction (mPCR) assay was developed for detection and characterization of pathogenic Escherichia coli that cause diarrhea and edema disease in swine. The mPCR assay was designed as a single reaction for detecting 5 different adhesins (K88, K99, 987P, F41, and F18), 3 enterotoxins (LT, STaP, and STb), and the Shiga toxin (Stx2e) associated with porcine pathogenic E. coli. The specificity of the mPCR assay was evaluated by comparison with results from previous analysis of 100 porcine isolates characterized by colony blot hybridization with DNA probes for the 5 adhesins and 4 toxin genes. There was complete agreement between the 2 methods. The mPCR assay for E. coli pathogens isolated from swine was further evaluated by examination of strains containing virulence factors that are known to have different antigenic subtypes or DNA sequence variations. It was found that the mPCR assays targeting genes encoding for K88 and F18 amplified products with the appropriate sizes from strains containing genes for different K88 and F18 antigenic subtypes; mPCR assays targeting the gene encoding for STaP amplified product from only STaP-positive but not STaH-positive isolates; and mPCR assays targeting the gene encoding for the Stx2 amplified products from only Stx2-positive and not Stx1-positive isolates. Similarly, mPCR assays targeting the gene encoding for LTI did not produce the appropriate product from strains containing genes for LTII. The mPCR assays are simple to perform, and they should be useful for diagnosis of porcine colibacillosis, including the genotypic characterization of E. coli isolates from pigs with diarrhea or edema disease.

Shiga Toxin-Producing Escherichia coli Infection: Temporal and Quantitative Relationships among Colonization, Toxin Production, and Systemic Disease

Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

Intervention with Shiga Toxin (Stx) Antibody after Infection by Stx-Producing Escherichia coli

Shiga toxins (Stxs) produced by Escherichia coli (STEC) cause systemic vascular damage, manifested as hemolytic uremic syndrome in humans and as edema disease in pigs. Edema disease, a naturally occurring disease of pigs, was used to determine whether Stx antibodies, administered after infection and after the onset of Stx production, could prevent the systemic vascular damage and clinical disease caused by Stxs. A total of 119 STEC-infected pigs were treated with low, medium, or high doses of Stx antibody or with placebo. After inoculation with STEC, antibodies or placebo was injected intraperitoneally at 2 days postinoculation (DPI; low dose) or 4 DPI (medium and high doses). Edema disease was prevented with the low- and high-dose Stx antibody treatments administered at 2 and 4 DPI, respectively. High-dose antibody treatment also reduced the incidence and extent of vascular lesions. The degree of protection depended on the dose of antibody and the time of administration.

Early Attachment Sites for Shiga-Toxigenic Escherichia coli O157:H7 in Experimentally Inoculated Weaned Calves

ABSTRACT Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 1010 CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx− O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157+ bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157+ bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157+ bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx− O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle.

Presence of F18ac (2134P) Fimbriae on 4P− Escherichia Coli Isolates from Weaned Pigs with Diarrhea

To cause disease, enterotoxigenic Escherichia coli (ETEC) must produce 1 or more enterotoxins (LT, STa, STb) and colonize the small intestine. Bacterial adhesins, known as fimbriae or pili, promote attachment to and colonization of the small intestine. Four classical fimbrial types, K88, K99, 987P, and F41, are associated with ETEC infections in neonatal pigs. Of these, K88 fimbriae are also associated with ETEC diarrhea in postweaning pigs. With the exception of K88, the fimbrial adhesins associated with ETEC diarrhea in postweaning pigs are less well characterized. ETEC strains are negative for the 4 classical pilus types and are referred to as 4P ETEC. immunologic methods may be lower than estimates based on genetic testing.

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3).

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.