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.

Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by Escherichia coli cause systemic vascular damage that manifests as edema disease in swine and hemolytic uremic syndrome in humans. In vitro, Stx inhibit protein synthesis and, depending on circumstances, induce necrosis, apoptosis, or both. The mechanism of in vivo Stx-mediated vascular damage is not known. The ability of Stx to cause apoptosis of vasculature in vivo was studied in pigs with edema disease that was produced by oral inoculation with Stx-producing E. coli. Arterioles of ileum and brain were evaluated by terminal dUTP nick-end labeling (TUNEL) assay for DNA fragmentation in myocytes (10 infected pigs, 5 control pigs) and by transmission electron microscopy for ultrastructural changes characteristic of apoptosis (17 infected pigs, 8 control pigs). In comparison with controls, increased numbers of TUNEL-positive arterioles were detected in 6/10 (60%) subclinically affected pigs 14-15 days after inoculation. Ultrastructurally, lesions in myocytes consisted of lysis (necrosis), with cytoplasmic debris and nuclear fragments contained between intact basement membranes. Endothelial cell changes ranged from acute swelling to necrosis and detachment from basement membrane. Subclinically affected pigs (n = 14) tended to have changes predominantly in myocytes, whereas pigs with clinical illness (n = 3) more commonly had changes in endothelial cells. The arteriolar lesions and clinical signs of edema disease are attributed to the effects of Stx on vasculature. Therefore, our findings suggest that the Stx-induced arteriolar lesions seen in this study were primarily necrotic, not apoptotic. We suspect that necrosis was the principal cause of the DNA fragmentation detected.

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.

Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery.

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmDeltaNOX and pKmDeltaNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.

Ultrastructure and DNA fragmentation analysis of arterioles in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by E. coli are potent cytotoxins that affect the vascular system. In humans, systemic toxemia causes renal glomerular damage manifested as hemolytic uremic syndrome. In swine, Stx-producing E. coli (STEC) cause edema disease that is characterized microscopically by segmental arteriolar smooth muscle cell (SMC) lesions. Our objectives were to characterize ultrastructurally and by TUNEL the type of death (apoptosis or necrosis) that occurs in SMCs during edema disease. Increased DNA fragmentation consistent with apoptosis was detected by TUNEL in arterioles of challenged pigs 14-15 days post inoculation. Ultrastructurally 3 grades of SMC lesions were distinguished: 1) Partial loss of SMCs, intercellular space filled with granular cellular debris admixed with membrane bound vacuoles; 2) Complete loss of SMCs; only granular cellular debris and clear vacuoles remained within basement membrane; 3) Inflammation of media; SMCs replaced by a rim of cellular debris located in the periphery of vessel wall. The most common lesion detected was grade 1 (9 ilea and 4 brains). We did not find apoptotic nuclear changes in SMCs or apoptotic inclusion bodies within resident cells. Our study indicates, that (1) Stx produced during edema disease does not cause SMC apoptosis 14-15 dpi; (2) SMCs undergo an array of changes from degeneration to necrosis.

Edema disease as a model for systemic disease induced by Shiga toxin-producing E. coli.

Edema disease (ED) is a naturally occurring disease of weaned pigs caused by host adapted strains of E. coli that produce Shiga toxin (STEC). We determined the temporal and quantitative relationships between intestinal colonization by STEC, levels of Shiga toxin (Stx2e) in the gut, in the blood, and clinical manifestations of ED. Bacterial colonization (10(8) CFU/cm ileum) was highest 4 days post inoculation (pi) in animals that did not develop clinical disease and 6 days pi in animals with clinical signs of ED. The mean time for the development of clinical signs of ED was 6 days pi (range 4-10). Average peak titers of Stx2e in the ileum were 1:16,384 in asymptomatic animals and 1:32,768 in clinical animals. Titers of Stx2e in the feces reflected the toxin titers in the ileum but were lower. Intestinal titers of Stx2e and the density of bacterial colonization were predictive of clinical ED for a group of animals but not for individuals. Approximately 50% of the pigs that had Stx2e titers of > or = 1:4096 and a bacterial density of > or = 10(6) CFU/cm in their ileum, had clinical ED. Pigs that had intestinal Stx2e titers < 1:4096 were asymptomatic. Stx2e was detected in the red cell fraction of blood from some of the pigs with clinical ED and in some that were asymptomatic. Stx2e was not detected in the serum of any animals. ED may be a useful model for predicting the temporal and quantitative relationships between bacterial colonization, Stx levels in the gut and blood and systemic disease for STEC in other species.

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.

Cloning and expression of porcine recombinant soluble tumor necrosis factor receptor 1.

OBJECTIVE: To clone, sequence, and express porcine recombinant soluble tumor necrosis factor receptor 1 (sTNFR1). PROCEDURE: A polymerase chain reaction (PCR)-based library enrichment technique was used to isolate a fragment of porcine TNFR1. The mature extracellular domain of porcine TNFR1 was subcloned into an expression vector and expressed in Escherichia coli as a fusion protein. Protein product was purified by immunoaffinity chromatography, using a commercially available affinity gel specific for the marker peptide of the fusion protein. The bioactivity of the purified protein was tested for its ability to inhibit TNF-mediated cytotoxicity in a PK(15) bioassay. RESULTS: A 927-base pair fragment of porcine TNFR1 encoding the entire extracellular and transmembrane domains, as well as 75 amino acids of the cytoplasmic domain, was isolated from a porcine lung cDNA library. The extracellular domain was expressed as a soluble TNFR1 fusion protein with a yield of 120 to 150 microg/L of culture. Affinity-purified porcine sTNFR1 was able to inhibit TNF-mediated cytotoxicity of porcine PK(15) cells in dose-dependent manner. CONCLUSIONS: Porcine recombinant sTNFR1 inhibits TNF bioactivity in vitro. This recombinant protein will be useful for developing TNFR1 antibodies and studying the roles of TNF and TNFR1 in the pathogenesis of infectious diseases in swine.

Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves.

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.

Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves.

A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2. This multiplex PCR was specific and sensitive. It will be useful for identification of E. coli strains which cause diarrhea in calves.

Differentiation of F18ab+ from F18ac+ Escherichia coli by single-strand conformational polymorphism analysis of the major fimbrial subunit gene (fedA).

Toxin-producing Escherichia coli expressing F18 fimbriae colonizes the small intestines of weaned pigs and causes diarrhea, edema disease, or both. The F18 family is composed of two antigenic variants, F18ab and F18ac. Because many strains do not express F18 fimbriae in vitro, identification and differentiation of these two variants are difficult. Single-strand conformational polymorphism (SSCP) analysis is a rapid method for identifying genetic mutations and polymorphisms. The F18 major fimbrial subunit genes (fedA) of 138 strains were amplified by PCR, and genetic differences were detected by SSCP analysis. The SSCP analysis of the fedA gene differentiated F18ab+ strains from F18ac+ strains. Most strains classified as F18ab+ by SSCP analysis contained Shiga toxin 2e and enterotoxin genes. Most strains classified as F18ac+ by SSCP analysis contained only enterotoxin genes. The SSCP analysis was a useful method for predicting the antigenicity of F18+ E. coli and could also be used for analysis of other virulence genes in E. coli and other pathogenic bacteria.

Effect of dietary stress on fecal shedding of Escherichia coli O157:H7 in calves.

Two groups of calves were subjected to dietary stress by withholding of food beginning 1 or 14 days after inoculation with 10(10) CFU of Escherichia coli O157:H7. Following treatment, neither group had a significant increase in fecal shedding of E. coli O157:H7. A third group of calves had food withheld for 48 h prior to inoculation with 10(7) CFU of E. coli O157:H7. These calves were more susceptible to infection and shed significantly more E. coli O157:H7 organisms than calves maintained on a normal diet.

Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs.

The role of Escherichia coli heat-stable enterotoxin B (STb) in neonatal porcine diarrhea caused by enterotoxigenic E. coli was examined by comparing adherent isogenic strains with or without STb. The cloned STb gene (in the plasmid pRAS1) was electroporated into a nonenterotoxigenic strain (226M) which expresses the F41 adhesin. Strain 226M pRAS1 adhered and expressed STb in vivo, causing fluid secretion in ligated ileal loops in neonatal pigs. Although strain 226M pRAS1 caused very mild diarrhea in some orally inoculated neonatal pigs, the weight loss in these pigs was similar to that caused by the parental strain without STb. We conclude that STb does not significantly contribute to diarrhea caused by enterotoxigenic E. coli in neonatal pigs.

Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves.

Cattle are an important reservoir of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains, foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome in humans. EHEC O157:H7 strains are not pathogenic in calves >3 weeks old. Our objective was to determine if EHEC O157:H7 strains are pathogenic in neonatal calves. Calves <36 h old inoculated with EHEC O157:H7 developed diarrhea and enterocolitis with attaching and effacing (A/E) lesions in both the large and small intestines by 18 h postinoculation. The severity of diarrhea and inflammation, and also the frequency and extent of A/E lesions, increased by 3 days postinoculation. We conclude that EHEC O157:H7 strains are pathogenic in neonatal calves. The neonatal calf model is relevant for studying the pathogenesis of EHEC O157:H7 infections in cattle. It should also be useful for identifying ways to reduce EHEC O157:H7 infections in cattle and thus reduce the risk of EHEC O157:H7 disease in humans.

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.

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.