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Enterotoxigenic Escherichia coli (ETEC) strains cause diarrhea in humans and animals by colonizing the small intestine and producing enterotoxins which cause fluid secretion. Adherence is mediated by filamentous surface structures called pili, fimbriae, or adhesins. A variety of antigenic types of adhesins are expressed by ETEC strains that cause diarrhea in pigs, including K88, K99, 987P, and F41. The E. coli enterotoxins are classified based on their thermal stability and are broadly divided into heat-labile enterotoxins (LTI and LTII) and heat-stable enterotoxins (STa and STb). The E. coli STa enterotoxins have been further subdivided into STaP, which is associated with porcine ETEC strains, and STaH, which is found in human ETEC strains. Adherence and enterotoxin expression are both required for ETEC strains to cause diarrhea (18).

The STb enterotoxin is prevalent in E. coli strains isolated from pigs with diarrhea but is rarely found in E. coli strains isolated from humans or cattle (6, 7, 11-13). ETEC strains that express STb isolated from humans have been reported, but these strains were not associated with diarrhea (7). In porcine ETEC strains, STb is the most prevalent enterotoxin identified; however, STb⁺ strains frequently express another enterotoxin, either LT or STaP (13). Additionally, strains that are only STb⁺ rarely hybridized with DNA probes for one of the known adhesins of porcine ETEC strains (3). E. coli strains that contain only STb and no other enterotoxin have been isolated from pigs, but these strains frequently lack known adhesins and are nonpathogenic (1, 8). Thus, confirmation of a specific role for STb in porcine diarrhea caused by ETEC strains has been complicated by the presence of other enterotoxin genes or the lack of a known ETEC adhesin in most isolates that express only STb.

In this study, we investigated the role of STb in ETEC-mediated diarrhea in neonatal pigs. We constructed an adherent strain that expressed only STb and isogenic control strains containing a recombinant plasmid encoding STaP or the cloning vector alone. These strains were examined for adherence and in vivo expression of enterotoxins in ligated ileal loops. The strains were also examined for pathogenicity by oral inoculation of neonatal pigs.

Strain construction. Strain 226M was used as a host for pBR322 or recombinant plasmids encoding STb (pRAS1) or STaP (pCH4). Strain 226M is an adherent, nontoxigenic mutant of ETEC strain 431 that has lost a plasmid encoding K99 and STaP but retained the chromosomal genes for F41 (2). Strain 226M colonizes the ileum but causes very mild diarrhea and minimal weight loss in neonatal pigs (2). Strain 234M was also used as a host for pRAS1. Strain 234M is an independent mutant of strain 431 that has lost the genes encoding K99 (by deletion) but retained the STaP gene and F41 genes (2). Strain 234M, like wild-type strain 431, colonizes the ileum, expresses STaP, and causes severe diarrhea and significant weight loss in neonatal pigs (2). Strain 123 is a nontoxigenic, nonadherent control strain that has been described previously (16, 20). Strain 1790 is a nonadherent strain which expresses only STb (20).

In vivo enterotoxin expression and adherence. Ligated ileal loops in neonatal pigs were used to confirm that derivatives of strain 226M containing cloned genes for STaP and STb would adhere and express enterotoxin in vivo. Caesarian-derived, colostrum-deprived, neonatal pigs less than 1 day old were anesthetized, and a series of ileal loops were created. The loops were each approximately 6 cm long and separated by 3-cm interloops. Each loop was inoculated with 3 × 10⁸ CFU of one of the strains. Soybean trypsin inhibitor (Sigma), which prevents degradation of STb (20, 21), was included in some loops. The pigs were kept at 35°C and given butorphanol tartrate (Torbugesic; Fort Dodge Laboratories, Fort Dodge, Iowa) to relieve postsurgical discomfort. At 6 h postinoculation, the pigs were sacrificed and necropsied. The fluid volume and length of each loop were recorded. The loops were fixed in formalin and processed for histopathology. Adherence was determined by microscopic examination of stained sections for bacterial layers. Figure 1 shows the amount of fluid accumulated in ligated ileal loops in neonatal pigs at 6 h following inoculation with the indicated strains. Strain 1790, which is nonadherent and expresses only STb enterotoxin, was used as a control for STb expression and did not cause fluid secretion in the absence of TI as has been shown previously (20, 21). There was no difference in fluid secretion in loops inoculated with any of the adherent strains with or without trypsin inhibitor; these data were combined to obtain the mean final fluid volumes per centimeter shown in Fig. 1. All of the strains containing either the STaP or STb enterotoxin gene caused significantly more fluid secretion than the nontoxigenic control strain (strain 123) or strain 226M with or without the cloning vector pBR322 (P < 0.05; Student's t test). Strain 226M containing the cloned STb (226M pRAS1) or STaP (226M pCH4) gene adhered as well as the adherent, nontoxigenic (strains 226M and 226M pBR322) and wild-type (strain 431) parents.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Mean fluid accumulation per cm (± standard error of the mean) and bacterial adherence for the indicated strains in ligated ileal loops made in neonatal pigs. Fluid accumulation is expressed as final loop volume per centimeter (Vf/cm). Loop adherence is expressed as number of loops with adherent bacteria per number of loops examined. The number of loops for each strain is the same for fluid accumulation measurements and adherence determinations.

Oral inoculation of neonatal pigs. Caesarean-derived, colostrum-deprived neonatal pigs were orally inoculated to compare the virulence of strain 226M pRAS1 (STb⁺) with that of the nontoxigenic parent strain. Neonatal pigs were housed in isolation at 35°C and given an intraperitoneal (i.p.) injection of serum, but they were not fed or given water. Pigs less than 8 h old were weighed and orally inoculated with 10¹⁰ CFU of one of the strains. At 18 h following inoculation, the pigs were weighed again, examined for diarrhea, and necropsied, and samples of ileum were collected for bacteriology and histopathology as previously described (2). Strains 226M, 226M pCH4 (STaP⁺), and 226M pBR322 were used as controls. The pigs' weight changes and the number of pigs with diarrhea for each of the strains used are shown in Table 1. Strain 226M pCH4 (STaP⁺) caused severe diarrhea and weight loss in all inoculated pigs, similar to the weight loss and diarrhea seen in pigs inoculated with the wild-type parent, strain 431 (2). However, strain 226M pRAS1 (STb⁺) caused only mild diarrhea with weight loss that was not significantly different (P < 0.05; Student's t test) from the weight loss caused by the nontoxigenic strain 226M or by 226M containing the cloning vector, pBR322. Table 1 also shows that strain 226M pRAS1 colonized the ileum, adhered, and grew as well in vivo as strain 226M. Despite the severe diarrhea and weight loss caused by strain 226M pCH4, this strain did not colonize as well as strain 226M or 226M pRAS1.