Experimental infection of calves with Escherichia coli O157:H7

Three 3-month-old Japanese Black calves were experimentally infected with Escherichia coli O157:H7 to define the magnitude (CFU/g) and duration of fecal shedding of the organism. In two of the three calves, fecal shedding of E. coli O157:H7 ceased in 5 and 9 weeks. The remaining calf continued shedding E. coli O157:H7 for more than 31 weeks, and the magnitude of the shedding ranged from 10(1) to 10(4) CFU/g of feces. The possibility is suggested that a percentage of animals naturally infected with E. coli O157:H7 on farms may become long-term shedders, transmitting the organism to other animals in the herd and to the proximate environment.     

Fecal Escherichia coli O157:H7 shedding patterns of orally inoculated calves.

To assess the duration of fecal shedding upon initial infection, the duration of shedding after subsequent re-infection and the effects of dietary restriction and antibiotic treatment on shedding recrudescence, four, one-week-old calves were orally inoculated on three separate occasions with 5x10(8) cfu of Escherichia coli O157:H7 strain 86-24 Nal-R. Fecal shedding was followed by serial culture three times weekly. Following the first inoculation, the calves shed E. coli O157:H7 in their feces for a mean of 30 days, with a range of 20 to 43 days. Following the second and third inoculations, the calves shed E. coli O157:H7 in their feces for 3-8 days. In each of the three inoculations, feed was withheld from the calves for 24 h after they had become fecal culture negative. Two calves resumed shedding, one for 1 day and the other for 4 days, after food was withheld after the third inoculation, but not in the first two inoculations. In the third inoculation, one calf resumed shedding for one day after treatment with oxytetracycline. No E. coli O157:H7 strain 86-24 Nal-R was found in the calves at necropsy. These calves did not exhibit persistent low-level shedding, and did not appear to be persistently colonized with E. coli O157:H7.     

Escherichia coli O157:H7 in the gallbladders of experimentally infected calves.

Fifteen weaned calves (age 89-141 days) were treated with dexamethasone (0.25 mg/kg, IV) for 3 days before, the day of, and the day after inoculation with 10 colony-forming units of either Escherichia coli O157:H7 (strain 86-24, which produces Shiga toxin 2 and intimin; n = 13) or nonpathogenic E. coli (strain 123, which does not produce Shiga toxin or intimin; n = 2). All calves were necropsied 4 days after inoculation. Histologic lesions of attaching and effacing bacteria were observed in the large intestine (12/13) and in the gallbladder mucosa (5/13) of calves inoculated with E. coli 86-24. Cholecystitis was present in 12 of 13 calves that received E. coli 86-24. Inoculum bacteria were recovered from the distal colons or feces (13/13) and gallbladders (3/4) of calves inoculated with 86-24.     

Escherichia coli O157:H7 and other Shiga toxin-producing E. coli in white veal calves

The aims of the study were to determine the prevalence of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) and other Shiga toxin-producing E. coli (STEC) in feces of white veal calves in an operation in Ontario, to evaluate exposure of the calves to EHEC O157, and to investigate the milk replacer diet and antimicrobial resistance as factors that might influence the prevalence of EHEC O157. Feces from three cohorts of 20-21 calves were collected weekly for 20 weeks and processed for isolation of EHEC O157:H7 and detection of STEC by an ELISA. Exposure to EHEC O157 was also investigated by measuring IgG and IgM antibodies to the O157 lipopolysaccharide (O157 Ab) in sera by ELISA. The prevalences of EHEC O157 were 0.17% of 1151 fecal samples and 3.2% of 62 calves, and for STEC were 68% of 1005 fecal samples and 100% of 62 calves. Seroconversion to active IgG and IgM O157 Ab responses in some calves was not associated with isolation of EHEC O157. The milk replacer contained low levels of antibodies to EHEC antigens and without antimicrobial drugs, it did not inhibit the growth of EHEC O157 in vitro. Two E. coli O157:H7 that were isolated were totally drug sensitive whereas 60 commensal E. coli isolates that were examined were highly resistant. Antibodies in milk replacer that might be protective in vivo, and susceptibility to antimicrobial agents in the milk replacer may contribute to the low prevalence of EHEC O157 in white veal calves.     

A systemic vaccine based on Escherichia coli O157:H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157:H7 in calves

Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine.     

Effect of combined rotavirus and Escherichia coli in neonatal gnotobiotic calves

Gnotobiotic calves (24 hours old) were monoinfected with calf rotavirus (CRV) strain NCDV, an enterotoxigenic Escherichia coli (ETEC) strain B44 (K99+), or a nonenterotoxigenic E coli (NETEC) strain 123 (K99-). Calves also were dually infected with CRV and either ETEC or NETEC. Eighteen calves equally allotted between 6 treatment groups were used in these studies: Noninfected controls--group A; CRV--group B; ETEC--group C; NETEC--group D; CRV + ETEC--group E; and CRV + NETEC--group F. Severe diarrhea and villous atrophy were observed in calves of treatment groups B, C, E, and F. Mortality was present only in treatment groups C and E as result of ETEC infection. There were no significant differences in the clinical responses or enteric lesions between treatments B and F, although a significant increase in the concentrations of NETEC was demonstrated in calves dually infected with CRV + NETEC (group F) as compared with calves monoinfected with NETEC (group D). Calves inoculated with ETEC (group C) had severe villous atrophy, neutrophilic infiltration of intestinal lumen, and moderate enterocyte necrosis. Calves dually inoculated with CRV + ETEC (group E) had the most extensive and severe lesions, similar to those in group C, plus a pronounced necrotic fibrino-hemorrhagic enteritis. Infection of enterocytes by CRV did not affect in any way the adherence of ETEC to the intestinal mucosa. Dual viral and bacterial infections of the same enterocytes were evident.     

Isolation and characterisation of Shiga toxin-producing Escherichia coli O157:H7 from calves in Argentina

Four hundred and twenty-two calves were examined for intestinal carriage of Shiga toxin-producing Escherichia coli O157:H7 using conventional plating. Two (0.5%) E. coli O157 were recovered. They were compared with 96 Argentine strains of different origin by pulsed-field gel electrophoresis, phage typing and PCR-RFLP of stx2 genes. One strain isolated from a calf, was closely related with 18 strains of clinical origin.     

Experimental infection in calves with a specific subtype of verocytotoxin-producing Escherichia coli O157:H7 of bovine origin

Background

In Sweden, a particular subtype of verocytotoxin-producing Escherichia coli (VTEC) O157:H7, originally defined as being of phage type 4, and carrying two vtx₂ genes, has been found to cause the majority of reported human infections during the past 15 years, including both sporadic cases and outbreaks. One plausible explanation for this could be that this particular subtype is better adapted to colonise cattle, and thereby may be excreted in greater concentrations and for longer periods than other VTEC O157:H7 subtypes.

Methods

In an experimental study, 4 calves were inoculated with 10⁹ colony forming units (cfu) of strain CCUG 53931, representative of the subtype VTEC O157:H7 (PT4;vtx₂;vtx_2c). Two un-inoculated calves were co-housed with the inoculated calves. Initially, the VTEC O157:H7 strain had been isolated from a dairy herd with naturally occurring infection and the farm had previously also been linked to human infection with the same strain. Faecal samples were collected over up to a 2-month period and analysed for VTEC O157 by immuno-magnetic separation (IMS), and IMS positive samples were further analysed by direct plating to elucidate the shedding pattern. Samples were also collected from the pharynx.

Results

All inoculated calves proved culture-positive in faeces within 24 hours after inoculation and the un-inoculated calves similarly on days 1 and 3 post-inoculation. One calf was persistently culture-positive for 43 days; in the remainder, the VTEC O157:H7 count in faeces decreased over the first 2 weeks. All pharyngeal samples were culture-negative for VTEC O157:H7.

Conclusion

This study contributes with information concerning the dynamics of a specific subtype of VTEC O157:H7 colonisation in dairy calves. This subtype, VTEC O157:H7 (PT4;vtx_2;vtx_2c), is frequently isolated from Swedish cattle and has also been found to cause the majority of reported human infections in Sweden during the past 15 years. In most calves, inoculated with a representative strain of this specific subtype, the numbers of shed bacteria declined over the first two weeks. One calf could possibly be classified as a high-shedder, excreting high levels of the bacterium for a prolonged period.     

Isolation of a rare Escherichia coli O157:H7 strain from farm animals in Greece

A strain of Escherichia coli O157:H7 was isolated from goat faeces during a surveillance study on the prevalence of this serotype of E. coli in farm animals in Greece. Three hundred and fifty one faecal samples were collected from goat, sheep and cattle breeding farms in the area of Epirus, Northwestern Greece. The E. coli O157:H7 isolate was nonsorbitol-fermenter, produced only VT2 and showed a beta-glucuronidase positive activity, a rather unusual biochemical feature for the E. coli O157:H7 serotype. No other strain of E. coli O157:H7 was isolated from the faecal samples of the rest farm animals examined, thus the overall prevalence of animal carriage was found to be 0.2%. The findings also indicate that goats can be a reservoir of E. coli O157:H7 and goat milk, dairy products and meat may serve as a vehicle for the pathogen transmission to humans.     

Effect of antibiotics in milk replacer on fecal shedding of Escherichia coli O157:H7 in calves

The objective of this study was to compare the concentration and duration of fecal shedding of Escherichia coli O157:H7 between calves fed milk replacer with or without antibiotic (oxytetracycline and neomycin) supplementation. Eighteen 1-wk-old Holstein calves were orally inoculated with a strain of E. coli O157:H7 (3.6 x 10(8) cfu/calf) made resistant to nalidixic acid (NA). Rectal samples were obtained three times weekly for 8 wk following oral inoculation. Fecal shedding of NA-resistant E. coli O157:H7 was quantified by direct plating or detected by selective enrichment procedure. Eight weeks after inoculation, calves were killed, necropsied, and tissues (tonsils, retropharyngeal and mesenteric lymph nodes, and Peyer's patches) and gut contents (rumen, omasum, abomasum, ileum, cecum, colon, and rectum) were sampled to quantify or detect NA-resistant E. coli O157:H7. The percentage of calves shedding NA-resistant E. coli O157:H7 in the feces in the antibiotic-fed group was higher (P < 0.001) early in the study period (d 6 and 10) compared with the control group fed no antibiotics. There was no difference between treatment and control groups in the concentration of E. coli O157 in feces that were positive at quantifiable concentrations. A comparison of the duration of fecal shedding between treated and untreated calves showed no significant difference between groups. At necropsy, E. coli O157:H7 was recovered from the rumen and omasum of one calf in the control group and from retropharyngeal lymph node and Peyer's patch of two calves in the antibiotic group. Supplementation of milk replacer with antibiotics may increase the probability of E. coli O157:H7 shedding in dairy calves, but the effect seems to be of low magnitude and short duration.     

用PCR鉴定大肠杆菌O157:H7

根据大肠杆菌Ｏ１５７：Ｈ７的编码ｅａｅ蛋白的ｅａｅ Ａ基因和大肠杆菌编码Ｈ７抗原的ｆｌｉＣ基因的核苷酸序列,合成了２对窦核苷酸引物,建立了一个检测大肠杆菌Ｏ１５７：Ｈ７的ＰＣＲ方法,对１１株已知大肠杆菌Ｏ１５７：Ｈ７（ＮＭ：无运动性）株和其他不同属的４２株已知肠道致病菌的检测结果表明,该方法只从大肠杆菌Ｏ１５７：Ｈ７（ＮＭ）株的ＤＮＡ中产生预期的扩增产物,而从其他菌株的ＤＮＡ中未扩增出任何ＤＮＡ不     

Occurrence and characteristics of enterohemorrhagic Escherichia coli O157 in calves associated with diarrhoea

Little is known of the association of enterohemorrhagic Escherichia coli O157:H7/NM (EHEC O157) with disease in naturally infected calves, although cattle have been known as a major source for EHEC O157 outbreaks in humans. In this study, we investigated the occurrence of EHEC O157 in calves associated with/without diarrhoea to examine if EHEC O157 is involved in calf diarrhoea and to characterize the isolates. Four hundred and ninety eight diarrhoeic and non-diarrhoeic young calves from 115 different farms were examined. Of 244 diarrhoeic calves, 24 (9.8%) were positive for EHEC O157, and of 254 non-diarrhoeic calves, 7 (2.8%) were positive. EHEC O157 was recovered from 12/76 (15.79%) of diarrhoeic calves less than 2-week-old, and no EHEC O157 was detected in this age group of non-diarrhoeic calves. This implicates EHEC O157 as a possible cause of the disease in naturally infected neonatal calves. The occurrence of EHEC O157 was relatively lower in the older calves (aged older than 8 weeks) and no significant difference was found in the occurrence rates between these diarrhoeic and non-diarrhoeic calves. PCR analysis of virulence markers revealed that the isolates carried various virulence genes such as Ehly, eae, stx1 and stx2, which underlines the potential importance of these attributes for the infection, colonization and possible pathogenesis of calf diarrhoea.     

Escherichia coli O157:H7 in healthy dairy cows

Faecal samples were taken from 371 cows originating from 55 dairy farms and slaughtered at one slaughterhouse; tonsils were taken from 215 of these animals. Escherichia coli 0157:H7 was found in the faeces of only two animals and was not found in any tonsils. The farm supplying the first positive cow detected at the slaughterhouse was visited 3 months later and 160 animals (80 cows and 80 heifers) were tested by rectal swabs; E. coli 0157:H7 was not isolated.     

Prevalence of Escherichia coli O157:H7 in white-tailed deer sharing rangeland with cattle.

OBJECTIVE: To determine the prevalence of fecal shedding of Escherichia coli O157:H7 in white-tailed deer (Odocoileus virginianus) with access to cattle pastures. DESIGN: Survey study. SAMPLE POPULATION: 212 fecal samples from free ranging white-tailed deer. PROCEDURE: Fresh feces were collected on multiple pastures from 2 farms in north central Kansas between September 1997 and April 1998. Escherichia coli O157:H7 was identified by bacterial culture and DNA-based methods. RESULTS: Escherichia coli O157:H7 was identified in 2.4% (5/212) of white-tailed deer fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: There is considerable interest in the beef industry in on-farm control of E coli O157:H7 to reduce the risk of this pathogen entering the human food chain. Results of our study suggest that the design of programs for E coli O157:H7 control in domestic livestock on pasture will need to account for fecal shedding in free-ranging deer. In addition, the results have implications for hunters, people consuming venison, and deer-farming enterprises.     

Non-O157 Shiga toxin-producing Escherichia coli isolated from diarrhoeic calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

用PCR鉴定大肠杆菌O157∶H7

根据大肠杆菌Ｏ１５７∶Ｈ７的编码ｅａｅ蛋白的ｅａｅＡ基因和大肠杆菌编码Ｈ７抗原的ｆｌｉＣ基因的核甘酸序列,合成了２对寡核苷酸引物,建立了一个检测大肠杆菌Ｏ１５７∶Ｈ７的ＰＣＲ方法。对１１株已知大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ;无运动性）株和其他不同属的４２株已知肠道致病菌的检测结果表明,该方法只从大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ）株的ＤＮＡ中产生预期的扩增产物,而从其他菌株的ＤＮＡ中未扩增出任何ＤＮＡ产物。该方法从基因水平直接确定大肠杆菌的血清型,特异性强,克服了以往血清学方法有非特异性反应的缺陷,为检测和鉴定大肠杆菌Ｏ１５７∶Ｈ７（ＮＭ）提供了一个新方法。     

肠出血性大肠杆菌O157:H7感染小鼠模型的研究

目前制约肠出血性大肠杆菌(EHEC)疫苗发展的瓶颈之一,是国内外尚没有理想的动物感染模型评价疫苗保护效果,像其他致病菌一样EHEC对不同动物易感性是不同的。有报道100~200个EHECO157活菌就可以导致人发病,但多数实验动物对EHEC O157则不易感。为了更好地评价疫苗的安全性和有效