A comparison of Shiga-toxin negative Escherichia coli O157 aflagellate and intimin deficient mutants in porcine in vitro and in vivo models of infection

Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, experimental infection studies have demonstrated that Stx-positive E. coli O157:H7 can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months and that persistence was not dependent upon intimin. We have shown that the flagellum of Stx-negative E. coli O157:H7 does not have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on a porcine (IPI-21) cell line, porcine in vitro organ culture (IVOC) and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nal(r) and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cells. In porcine IVOC association assays, E. coli O157:H7 NCTC12900nal(r) was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants significantly adhered in greater numbers to more IVOC gastrointestinal tissues than the parent. Groups of 14-week-old pigs were dosed orally with 10(10)CFU/10ml of either E. coli O157:H7 NCTC12900nal(r) or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon. These data suggest that colonisation and persistence of Stx-negative E. coli O157:H7 in pigs, involves mechanisms that do not require the flagellum or intimin.     

Levamisole synergizes proliferation of intestinal IgA+ cells in weaned pigs immunized with vaccine candidate F4ac+ nonenterotoxigenic Escherichia coli strain

Levamisole (2, 3, 5, 6-tetrahydro-6-phenylimidazole 2,1-b thiazole) is a well-known nonspecific stimulator of host defence mechanisms. In previous investigations, we have found that levamisole acts on cell-mediated immunity in challenge-induced porcine postweaning colibacillosis (PWC). We assume that levamisole could also act synergistically on humoural immune response when applied as an adjuvant with vaccine candidate strains for oral immunization of weaned pigs against PWC. The influence of levamisole in combination with experimental F4ac⁺ nonenterotoxigenic Escherichia coli (non-ETEC) vacinal strain on proliferation of IgA⁺ cells was examined in 4-week-old weaned pigs experimentally infected with ETEC. We have performed identification and morphometric quantification of the plasma cell phenotype within jejunal/ileal mucosa. Plasma cells were identified by immunohistochemistry with monoclonal anti-IgA antibodies and quantifying by use of digital image analysis. Quantification of IgA⁺ cells from levamisole-primed vaccinated and challenge-infected weaned pigs showed significantly increased number ( P  < 0.05 for both jejunum and ileum) compared with those observed in unprimed vaccinated/challenge-infected controls. It is suggested from these results that levamisole may contribute in initiation of local humoural immune response to enteric pathogens, such as enterotoxigenic E. coli .     

Vaccination of calves with EspA, a key colonisation factor of Escherichia coli O157:H7, induces antigen-specific humoral responses but does not confer protection against intestinal colonisation

Enterohaemorrhagic Escherichia coli (EHEC) infections in humans are frequently associated with direct or indirect contact with ruminant faeces and may result in haemorrhagic colitis and severe renal and neurological sequelae. Broadly cross-protective vaccines for control of EHEC do not yet exist and the molecular mechanisms that influence bacterial persistence in the intestines of ruminants are incompletely understood. We sought to determine the role in colonisation and protective efficacy of EspA, which forms a filamentous extension of the locus of enterocyte effacement-encoded type III secretion system that injects EHEC proteins into enterocytes. A non-polar deletion of espA severely impaired the ability of E. coli O157:H7 to colonise the intestines of calves. Vaccination of calves with highly purified recombinant EspA induced high-titre antigen-specific IgG1 (also reactive to native EspA) and salivary IgA responses, however these responses did not protect calves against intestinal colonisation by E. coli O157:H7 upon experimental infection.     

Incidence of common DNA sequences in bovine and porcine Escherichia coli strains causing diarrhoea

Seventeen bovine and 56 porcine Escherichia coli isolates from cases of diarrhoea and from healthy animals were examined for DNA sequences homologous to the genes for verocytotoxins (VT), enterotoxins and human enterohaemorrhagic E coli/enteropathogenic E coli (EHEC EPEC) sequences. VT-1 was the most common toxin among the bovine isolates and VT-2 the most common in the porcine isolates. No isolates had homologous sequences to enteropathogenic adherence factor, but 71.2 per cent hybridised to the DNA probe encoding specific EHEC sequences, and 95.9 per cent showed homology with a 23 kb DNA fragment common to EHEC and EPEC plasmids.     

Colonization of the small intestine of weaned pigs by enterotoxigenic Escherichia coli that lack known colonization factors.

Intestinal colonization of 3-week-old weaned pigs by enterotoxigenic Escherichia coli (ETEC) strains that were originally isolated from weaned pigs with fatal diarrhea and that lacked K88, K99, F41, and 987P adhesins (4P- ETEC) was studied by histologic, immunofluorescent, and electron microscopic techniques. In the first experiment, 16 principal pigs were inoculated orogastrically with ETEC strain 2134 (serogroup O157: H19) or 2171 (serogroup 0141:H4), and eight control pigs were not inoculated. In the second experiment, 24 principals were inoculated with ETEC strain 2134, and 12 controls were inoculated with a nonenterotoxigenic strain of E. coli. Principal and control pigs were necropsied at intervals from 24 to 72 hours after inoculation of principals to provide the tissues used for this report. Results from the two experiments and with both ETEC strains were similar and therefore were combined. Adhesion by 4P- ETEC was demonstrated in ileum but not in cecum or colon in 22/40 principal pigs sampled at 24 to 72 hours after orogastric inoculation. Adherent bacteria were most apparent on the intestinal villi covering Peyer's patches. Only occasional adherent bacteria were detected in ileal sections from a few (4/20) of the control pigs. Adherence by 4P- ETEC was characterized by "patches" of bacteria closely associated with the lateral surfaces and less frequently with the tips and the bases of intact villi. In most cases, the adherent bacteria were separated from epithelial cell microvilli and other bacterial cells by a 50-400-nm space. Filamentous bacterial appendages bridged this space and formed a network among adjacent bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of rotavirus and enterotoxigenic Escherichia coli in conventionally-reared dairy calves

A study was made of the effects of rotavirus and/or enterotoxigenic Escherichia coli (ETEC) on dairy calves born and suckled on the farm and subsequently reared in isolation. Calves were orally inoculated at 6 days old with either rotavirus (5), ETEC (7), rotavirus and ETEC (5) or remained uninoculated controls (4), and their reactions were recorded by clinical, microbiological, and pathological observations. Rotavirus infection consistently produced diarrhoea, while ETEC inoculated alone did not colonise the intestine. In dual infections, both rotavirus and ETEC multiplied, although the severity of diarrhoea was not greater than that caused by rotavirus alone. Some ETEC-inoculated calves developed subsequent naturally-acquired rotavirus infections, but in these no ETEC multiplication occurred. The results suggest that prior or simultaneous rotavirus infection is necessary to enable ETEC colonisation of the intestine in convenstional calves of this age.     

Virulence and fitness gene patterns of Shiga toxin-encoding Escherichia coli isolated from pigs with edema disease or diarrhea in Germany

Fecal Escherichia coli isolates (n = 3,218) from piglets with edema disease or diarrhea were screened for the genes of Stx2 and Stx2 variants. A total of 283 E. coli isolates (8.8%) proved exclusively positive for Stx2e and most of these (85.1%) harbored genes for F18 fimbria. No recognized adhesins were detectable in 14.5% of the isolates. Genes for heat-stable or heat-labile E. coli enterotoxins were found in F18+ as well as F18 isolates (51.9% and 33.3%, respectively). Five isolates also harbored fyuA and irp2 genes which are indicative of a high pathogenicity island in E. coli. All Stx2e+ isolates lacked genes for intimin, EHEC hemolysin, STEC autoagglutinating adhesin, subtilase cytotoxin, serine protease Espl. The majority of Stx2e+ isolates belonged to phylogenetic groups A (59.3%) and D (38.9%) and only few isolates were classified as B1 and B2 (1.8%). The results suggest that Stx2e-producing E. coli strains are highly prevalent in diseased pigs in Germany. Despite their significant diversity, most strains possess all typical features (Stx2e, F18) of porcine edema disease E. coli. However, a considerable portion of porcine strains resembles published human Stx2e+ strains in that they lack any recognized pig-associated adhesin. Thus, a zoonotic potential cannot be excluded for these strains.     

Antibiotic resistance,ESBL genes,integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum β-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1–15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for bla_TEM and bla_CTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.     

Enteric infections caused by non-enterotoxigenic Escherichia coli in animals: occurrence and pathogenicity mechanisms. A review

Not all E. coli that cause diarrhoea in farm animals act by elaborating the classical heat-labile or heat-stable enterotoxins. These newly recognised animal enteropathogenic E. coli (EPEC) attach to and efface the microvilli of the gut epithelium and resemble the well known human EPEC. In rabbits, only this type of E. coli enteritis is known to be important and a similar disease has been shown to occur in cattle also. There is no doubt that adherence factors are important in the pathogenesis of animal EPEC, but they are not well understood when compared with the adhesion of human EPEC, or to the adhesion of animal ETEC. The enteropathogenic effect is probably due to Shiga-like toxin, a cytotoxin that is active on Vero cells and has also been called Verotoxin. A different type of Verotoxin is produced by most serotypes that are associated with post-weaning diarrhoea and oedema disease in pigs. This toxin is suspected to play a role in the pathogenesis of the latter.     

Rapid identification of virulence genes in enterotoxigenic Escherichia coli isolates associated with diarrhoea in Queensland piggeries

OBJECTIVE: To identify virulence genes in enterotoxigenic E coli (ETEC) isolates associated with diarrhoea in neonatal, 1 to 3 week-old and weaned pigs in southeast Queensland. DESIGN: Multiplex PCR and serotyping were applied to E coli isolates obtained over a 5-year period (1998-2002) from cases diagnosed at Toowoomba Veterinary Laboratory. PROCEDURE: A total of 126 isolates from 25 different Queensland piggeries were tested for haemolytic activity on 5% sheep blood agar and by multiplex PCR for the presence of five commonly recognised fimbrial (F4, F5, F6, F41 and F18) and three enterotoxin genes (STa, STb, LT). A subset of 62 representative isolates were serotyped by slide agglutination. For comparative purposes, multiplex PCR was also performed on the DNA of 31 ETEC isolates from 9 serotypes originating from piggeries in southern New South Wales. RESULTS: A total of 113 (89.7%) of the isolates from Queensland possessed ETEC virulence genes, including 14 of 15 isolates from neonatal pigs (93.3%), 18 of 23 isolates from 1 to 3 week old pigs (78.3%) and 81 of 88 isolates from weaned pigs (92.1%). F4:STa:STb:LT (serotype O149) was the most prevalent pathotype in neonatal and 1-3 week old pigs and F4:STa:STb:LT (serotype O149) and F18:STa:STb:LT (serotype O141) were most prevalent in weaned pigs. In comparison, isolates obtained from neonatal pigs from New South Wales belonged to a more diverse range of pathotypes and serotypes. CONCLUSION: Multiplex PCR was a rapid and specific method for detecting the presence of ETEC virulence genes in porcine E coli isolates. For isolates obtained from cases of suspected colibacillosis in Queensland, growth of a heavy pure culture of haemolytic E coli was a sensitive prognostic indicator of the presence of ETEC virulence genes in the isolate. ETEC pathotypes and serotypes remained stable in Queensland piggeries over the five-year study period and appear to have changed little over the last three decades.     

Longitudinal prevalence study of diarrheagenic Escherichia coli in dairy calves

A longitudinal study (cohort study) elaborating 1,224 rectal swabs from 221 calves aging between 1 and 12 weeks was conducted on 11 dairy farms (i) to ascertain associations between diarrhea and shedding of diarrheagenic E. coli and (ii) to facilitate the zoonotic potential assessment of E. coli strains shed by young calves. Calves were screened weekly by PCR of swab cultures for shedding of enterotoxigenic E coli [ETEC; by detection of heat stable (est) and heat labile enterotoxin genes (elt)], diffusely adhering E. coli [DAEC; diffuse adhesion (daa)], typical enteropathogenic E. coli [EPEC; bundle-forming pili (bfpA) and intimin (eae)] as well as enterohemorrhagic E. coli [EHEC, intimin (eae) and Shiga toxin (stx)]. In addition, EHEC-hemolysin- (Hly(EHEC)) and alpha-hemolysin- (alpha-Hly) producing E. coli were detected by inoculation of blood agar plates. Within the 221 calves, prevalences were 69.7% (25.2% of the 1,224 samples) for Hly(EHEC)-producing E. coli, 55.3% (19.3%) for eae, and 18.2% (4.5%) for stx. E. coli strains exhibiting an alpha-Hly phenotype were detected in 66.5% of the calves and 21.9% of fecal samples. The est gene was detectable in 31.7% of the calves from only 9 of 11 herds and in 7.8% of the samples. Calves shedding DAEC or typical EPEC were not identified. The detection frequency of virulence traits significantly depended on the calves' age and shedding dynamics differed between the traits. A significant correlation between calf diarrhea and shedding of EHEC virulence traits was determined for several postnatal periods (1 week: Hly(EHEC); 1st & 10th week: eae; 4th week stx). Shedding of ETEC (est) was associated with diarrhea in newborn calves (1st week) only. Hly(EHEC)- and alpha-Hly-producing E. coli were shed significantly more frequently by diarrheic calves in 1st and 8th week of life, respectively. The knowledge gained in this study highlights the high prevalence of zoonotic E. coli already in calves.The age-dependent shedding dynamic of the various E. coli pathovars has to be considered regarding prophylaxis as well as planning intervention studies, both for calves and humans.     

Serotypes and intimin types of intestinal and faecal strains of eae+ Escherichia coli from weaned pigs

Enteropathogenic Escherichia coli (EPEC) that are known to cause severe diarrhoea in children and young rabbits are well characterized, but there are few reports on the serotypes and intimin (eae) types of EPEC in weaned pigs. Based on detection of the eae gene by PCR and by DNA-hybridisation with LEE specific gene probes, 20 intestinal and 17 faecal eae(+) strains from diarrhoeal (164) and non-diarrhoeal (57) weaned pigs from 13 Hungarian farms, representing 12.8% of diarrhoeal and 14.0% of non-diarrhoeal pigs, were identified. The dominant serotype was O123:H11 (40%) among intestinal, and O108:H9 (23%) among faecal strains. The majority (85%) of the intestinal strains possessed eae-beta and 10% carried eae-gamma gene. In contrast, significantly (p<0.025) fewer faecal strains (53%) harboured the eae-beta gene, and 23% were eae-gamma positive. In vitro adhesion tests of intestinal and faecal eae(+) strains indicated adhesion of 20/37 of the strains to PK15 (porcine kidney) cells while only 3/37 strains adhered to HeLa cells. The ultrastructure of intimate bacterial attachment of representative porcine eae(+) strains to PK15 cells showed no pedestal formation, in contrast to the human EPEC (O127:H5, eae-alpha) strain. In conclusion, the data do not demonstrate a significant role for the eae(+)E. coli in porcine post-weaning diarrhoea, but provide new information on a dominant porcine serotype (O123:H11, eae-beta), and on differences of serotypes and intimin types of porcine eae(+) strains according to their site of isolation. Furthermore there was an indication that the PK15 cell line could be used as a model to study in vitro adherence of eae(+)E. coli of some human and porcine origin.     

Persistence of Escherichia coli O157:H7 in experimentally infected swine

These experiments determined the ability of Escherichia coli O157:H7 to colonize and persist in pigs simultaneously inoculated with other pathogenic E. coli strains. Three-months-old pigs were inoculated with a mixture of five E. coli strains. The mixture included two Shiga toxigenic E. coli (STEC) O157:H7 strains, two enterotoxigenic E. coli (ETEC) strains and one enteropathogenic E. coli (EPEC) strain. A high dose mixture with all five strains at 10(10)CFU/animal (CFU: colony forming units) and a low dose mixture with the STEC strains at 10(7)CFU and the EPEC and ETEC strains remaining at 10(10)CFU were used. The STEC strains persisted in the alimentary tracts of some pigs at 2 months post-inoculation, following inoculation with both the high and low dose mixtures. When all strains were given at 10(10)CFU (high dose) the STEC strains persisted in greater numbers and in more pigs than did the other E. coli strains. The results demonstrated that persistent colonization (> or =2 months) by E. coli O157:H7 can occur in pigs. These findings were similar to those reported from sheep inoculated with the same mixture of E. coli strains. The results are consistent with reports suggesting that pigs have the potential to be reservoir hosts for STEC O157:H7.     

Prevalence of the lpfO113 gene cluster among Escherichia coli O157 isolates from different sources

Domestic farm animals represent an important reservoir of infection for Shiga toxin-producing Escherichia coli (STEC). Nevertheless the bacterial factors required to colonise these hosts are poorly defined. In this study, the prevalence of a recently described fimbrial gene cluster, lpfO113, among human and animal isolates of STEC was investigated. lpfO113 has been shown to play a role in the adherence of STEC O113:H21 to epithelial cells. Here the presence of the lpfAO113 gene (predicted to encode a major fimbrial subunit) was examined by PCR in E. coli of serogroups O157 and O26 isolated from pigs (n=38), cattle (n=10), and humans (n=9). In addition, we tested for several other genetic virulence markers including Shiga toxin (stx), intimin (eae), the translocated intimin receptor (tir), EHEC-hemolysin (ehx) and F18 fimbriae (fedA). Overall 45 of the 57 strains (79%) possessed the lpfAO113 gene as determined by the presence of a 573 bp PCR product. Moreover, there was a close correlation between the presence of the lpfAO113 marker and the absence of the eae gene. lpfAO113 was found in all of pig isolates, suggesting a possible role in colonisation of the porcine host. In addition, several E. coli strains isolated from pigs had two fimbrial gene markers, fedA and lpfAO113. lpfAO113 was not present in strains of E. coli O157:H7 as described previously. Overall these results show that lpfAO113 is widely distributed among eae-negative E. coli isolates and thus may represent an important adherence factor in this group of pathogens.     

Analysis of the AIDA-I gene sequence and prevalence in Escherichia coli isolates from pigs with post-weaning diarrhoea and oedema disease

Three hundred and twenty-four strains of Escherichia coli isolated from weaned pigs with diarrhoea or oedema disease in Eastern China were screened by multiplex PCR for the presence of the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). Two AIDA-I positive strains were subjected to analysis of the nucleotide sequence of the complete orfA and orfB of the AIDA gene. The AIDA-I positive E. coli isolates were also assessed for five fimbriae (F4, F5, F6, F18 and F41) by monoclonal antibodies and for toxin genes (STa, STb, LT, EAST1, Stx2e) by PCR. Twenty-one (6.5%) of the isolates possessed AIDA-I genes. Of these isolates, two carried AIDA-I genes as the only demonstrated virulence factors, and the remaining isolates carried other virulence factor genes. Comparing the AIDA-I sequence from porcine and human sources, a high homology of orfA both in porcine E. coli and human E. coli was observed. However, each orfB of the two porcine E. coli isolates was 3864 nucleotides long compared with 3861 for the E. coli 2787 orfB, and showed 96.5% homology to E. coli 2787. The data indicated (1) that AIDA-I may be an occasional virulence factor in post-weaning diarrhoea and oedema disease in pigs, (2) that it has the potential to transfer between porcine and human E. coli, and (3) that there is a genetic diversity in orfB between human and porcine E. coli.     

Association between the porcine Escherichia coli F18 receptor genotype and phenotype and susceptibility to colonisation and postweaning diarrhoea caused by E. coli O138:F18

Porcine postweaning Escherichia coli enteritis is a cause of significant morbidity and mortality in pigs worldwide, and effective prevention remains an unsolved problem. This study examined the correlation between susceptibility of pigs to experimental infection with an E. coli F18 strain and the porcine intestinal F18 receptor genotypes. Thirty-one pigs classified as either belonging to the susceptible or the resistant genotype were inoculated with cultures of an E. coli O138:F18 isolated from a pig with postweaning diarrhoea. Susceptibility to colonisation and diarrhoea was assessed by clinical observations, faecal shedding of the challenge strain, histopathology and microscopic adhesion tests. Ten of 14 (71.4%) genetically susceptible pigs and one of 17 (5.9%) resistant pigs developed diarrhoea attributable to the challenge strain. There was no difference in susceptibility between homozygotic and heterozygotic susceptible pigs. Faecal shedding of the challenge strain correlated with the genetic receptor profile. Twenty pigs examined immunohistochemically revealed focal to extensive small intestinal mucosal colonisation by E. coli O138:F18 in nine of 10 susceptible and three of 10 resistant pigs. Results of in vitro adhesion assays performed with F18 cells on enterocyte preparations from 24 pigs, showed complete concordance with the F18 genotypes. In conclusion, this study showed a high correlation between the porcine intestinal F18 receptor genotypes and susceptibility to disease. However, pigs of the resistant F18 receptor genotype were not entirely protected against intestinal colonisation by E. coli F18.     

Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli

The goal of this study was to isolate and characterize phages that might be used in prevention and treatment of porcine post-weaning diarrhea due to O149 enterotoxigenic E. coli (ETEC). Serotype O149:H10:F4 was especially targeted because this is the dominant ETEC serotype. Mixtures of 10 strains of O149:H10:F4 ETEC and of 10 O149:H43:F4 ETEC were used as hosts for isolation of phages in sewage from 38 Ontario pig farms. Six phages (GJ1-GJ6) that lysed O149:H10:F4 ETEC and three (GJ7-GJ9) that lysed O149:H43:F4 ETEC were isolated. All phages produced large, clear plaques. All nine phages had necks and contractile tails and therefore belonged to the Myoviridae. Their estimated genome sizes were 48.3-50.7kb and their restriction enzyme fragments suggested that they were closely related. Phages GJ1-GJ6 lysed 99-100% of 85 O149:H10:F4 ETEC, 0-12% of 42 O149:H43:F4 ETEC, 3-35% of 37 non-O149 porcine ETEC, and 6-68% of the 72 strains of the ECOR collection. Phages GJ7-GJ9 lysed 86-98% of the O149:H43:F4 ETEC, 2-53% of the O149:H10:F4 ETEC, and 24-41% of the non-O149 porcine ETEC. Titres of the nine phages were unaffected by exposure for 16h to pH 5-9. Among phages GJ1-GJ6, resistance of O149:H10:F4 ETEC to one phage was generally not accompanied by resistance to other phages. It is concluded that the nine phages are suitable candidates for prophylaxis and therapy of porcine post-weaning diarrhea due to O149 ETEC.     

Dietary supplementation of Bacillus subtilis influenced intestinal health of weaned pigs experimentally infected with a pathogenic E. coli

Background: There is growing evidence to support the beneficial effects of supplementing direct-fed microbials(DFM) on performance, health status, and immune responses of weaned pigs. Therefore, the objective of this study was to investigate dietary supplementation of Bacillus subtilis(DSM 25841) on growth performance, diarrhea, gut permeability and immunity of weaned pigs experimentally infected with a pathogenic F-18 Escherichia coli(E. coli).Results: The F18 E. coli infection reduced(P 0.05) growth performance and intestinal villi height, whereas increased(P 0.05) diarrhea and transcellular and paracellular permeability in the jejunum compared with non-challenged control. Supplementation of Bacillus subtilis linearly enhanced average daily gain of E. coli infected pigs from d 0 to 5 post-inoculation(PI)(P 0.05) and d 0 to 11 PI(P = 0.058). Supplementation of high dose of Bacillus subtilis reduced(P 0.05) both transcellular and paracellular permeability on d 5 and d11 PI compared with the E. coli infected pigs fed with control diet. E. coli infection up-regulated(P 0.05)the m RNA expression of SLC5 A10(soluble carrier family 5 member 10) and MUC2(mucin 2) on d 5 PI, but down-regulated(P 0.05) expression of SLC5 A10, MUC2, and CLDN1 on d 11 PI in jejunal mucosa when pigs were fed with the control diet. Supplementation of Bacillus subtilis linearly up-regulated(P 0.05) the m RNA expression of CFTR and ZO1 on d 5 PI and SLC5 A10 and MUC2 on d 11 PI in jejunal mucosa of E. coli infected pigs. In addition, E. coli infection increased(P 0.05) the m RNA expression of several immune genes(IL1 A, IL1 B, and IL7 on d 5 PI, and IL1 B, IL6, IL7, and TNF on d 11 PI) in the ileal mucosa of weaned pigs. Inclusion of Bacillus subtilis to control diet linearly down-regulated gene expression of IL1 A on d 5 PI(P = 0.07) and IL6 on d 11 PI(P 0.05) in ileal mucosa of E. coli infected pigs.Conclusions: Supplementation of Bacillus subtilis(DSM 25841) enhanced growth rate and improved gut barrier function of weaned pigs experimentally infected with a pathogenic E. coli.     

Replicon typing of F18 fimbriae encoding plasmids of enterotoxigenic and verotoxigenic Escherichia coli strains from porcine postweaning diarrhoea and oedema disease

The presence of fimbrial adhesin F18 is frequently found in enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) strains responsible for diarrhoea and oedema disease of weaned pigs. The F18 adhesin occurs in two antigenic variants: F18ab is characteristic of VTEC while F18ac is more typical for ETEC. F18 encoding plasmids of 17 phenotypically characterized porcine E. coli isolates (10 ETEC, 6 VTEC and 1 ETEC/VTEC) were tested with a DNA probe for F18 fimbrial adhesin and with replicon probes for the RepFIa, RepFIb and for the RepFIc family of basic replicons. In all the cases, the F18 probe hybridized to only one plasmid band of size higher than 42MDa. All F18 plasmids were determined to be unireplicon plasmids belonging to the RepFIc replicon family of the F incompatibility complex. There was no difference between F18ac plasmids of ETEC and F18ab plasmids of VTEC strains in terms of replicon type or subtype. However, the size of F18ab plasmids of the VTEC strains varied between 42 and 98MDa, in contrast to F18ac plasmids of ETEC strains (constantly approximately 98MDa).     

Prevalence of enteropathic Escherichia coli in dogs with acute and chronic diarrhoea

Samples of faeces from 57 dogs with acute diarrhoea, 82 dogs with chronic diarrhoea, 34 clinically healthy household dogs and 88 kennelled control dogs were analysed by hybridisation, using DNA probes to detect enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E coli (ETEC), verocytotoxin-producing E coli (VTEC), enterohaemorrhagic E coli (EHEC), enteroinvasive E coli (EIEC) and enteroaggregative E coli (EAggEC). Samples of duodenal juice from 60 of the 82 dogs with chronic diarrhoea were also examined. Significantly more of the dogs with diarrhoea were excreting EPEC (acute 35.1 per cent, chronic 31.7 per cent) and VTEC (acute 24.6 per cent, chronic 28 per cent) than the kennelled dogs (EPEC 17.1 per cent, VTEC 0 per cent) or the household control dogs (EPEC 6 per cent, VTEC 5.9 per cent). Enteropathic E coli was also detected in the duodenal juice of 23 of 60 (38.3 per cent) of the dogs with chronic diarrhoea. The EPEC attaching and effacing A (eaeA) gene and the verocytotoxin 1 (VR1) gene coding for VTEC were often found together. There was good agreement between in vitro studies and hybridisation for the detection of eaeA and VT1. Isolates from the dogs with diarrhoea adhered significantly more to Hep-2 cells, and VT1-positive strains from the dogs with diarrhoea consistently killed more than 50 per cent of Vero cells.