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.     

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     

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.     

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 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.     

All blood, No stool: enterohemorrhagic Escherichia coli O157:H7 infection

Enterohemorrhagic Escherichia coli serotype O157:H7 is a pathotype of diarrheagenic E. coli that produces one or more Shiga toxins, forms a characteristic histopathology described as attaching and effacing lesions, and possesses the large virulence plasmid pO157. The bacterium is recognized worldwide, especially in developed countries, as an emerging food-borne bacterial pathogen, which causes disease in humans and in some animals. Healthy cattle are the principal and natural reservoir of E. coli O157:H7, and most disease outbreaks are, therefore, due to consumption of fecally contaminated bovine foods or dairy products. In this review, we provide a general overview of E. coli O157:H7 infection, especially focusing on the bacterial characteristics rather than on the host responses during infection.     

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.     

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     

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.     

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.     

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。     

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.     

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.     

应用多重PCR方法检测出血性大肠杆菌O157:H7

选择O157：H7的产志贺样毒素基因stx1、stx2和β-葡糖醛酸糖苷酶基因(uidA)分别设计引物,在同一扩增体系中进行PCR反应。在优化好的多重PCR反应条件下,对菌株及其它肠道菌进行检测。试验结果为：O157：H7菌株在250,207,179bp处均出现特异条带。试验结果表明,选择3对引物的多重PCR方法可特异、快速而且灵敏地对大肠杆菌O157：H7进行检测。     

牛源大肠杆菌O157:H7的分离鉴定

为调查新疆部分地区E.coli O157：H7的感染情况和菌株致病性,从新疆阿克苏、伊犁、塔城3个地区的牛场采集新鲜粪样564份,对E.coli O157：H7进行分离与鉴定。利用E.coli营养肉汤（EC肉汤）对样品进行增菌后,用山梨醇麦康凯培养基（SMAC）平板选择性培养,再经过4-甲基伞形酮-β-D葡萄糖醛酸苷培养基（MUG）的筛选,对疑似菌株进行生化和PCR鉴定,并将分离鉴定到的菌株进行小鼠攻毒试验。结果显示,从伊犁地区采集的样品中共分离出2株E.coli O157：H7（Y166和Y226）,其检出率为0.88%;小鼠攻毒试验中,Y166和Y226试验组小鼠在48 h内全部死亡,具有一定致病性;从阿克苏、塔城所采样品中未分离到E.coli O157：H7。     

H7-HCP-Tir-Intimin高效降低小鼠粪便中大肠杆菌O157:H7排菌

构建BL21(PcoldⅠ-fliC-hcpA-tir-eae),并评价对小鼠的免疫原性,以期研制更加有效的候选免疫原,最大限度降低E.coli O157:H7对食物、环境和人类的危害。将946、423、309、811bp的fliC,hcpA,tir和eae基因被依次克隆,中间添加8个氨基酸的间隔序列,构建重组菌BL21(PcoldⅠ-fliC-hcpA-tir-eae)。进口白油和ISA50V作为佐剂与重组蛋白乳化,制备2种油乳剂疫苗,分别接种4周龄BALB/c雄鼠,二免21d,口服攻击E.coli O157:H7109 CFU/只,每天采集粪便,检测排菌。结果显示,2种重组疫苗均能够诱导高滴度血清IgG。ISA50V佐剂组抗体产生早于进口白油佐剂组,但进口白油佐剂组抗体滴度一致性更好。所有免疫组小鼠排菌极度降低,第1天排菌低于104 CFU/g,第5天检测不到排菌,整个检测期无反弹,而对照组在整个检测期排菌持续很高(>104 CFU/g)。结果表明,四价重组蛋白H7-HCP-Tir-Intimin能够高效降低口服大肠杆菌O157:H7后的排菌,极显著降低细菌的定植,很大程度减少病原传播,对环境和人类的威胁。     

Verocytotoxin-producing Escherichia coli O157:H7 in the Swedish pig population

Verocytotoxin-producing Escherichia coli O157:H7 (VTEC O157:H7) was detected in two of 2446 individual faecal samples collected from pigs slaughtered at five Swedish slaughterhouses, indicating a prevalence of 0.08 per cent, with a 95 per cent confidence interval from 0 to 0.16 per cent Four Swedish VTEC O157:H7-positive farms which kept ruminants and pigs were studied by repeated faecal sampling; VTEC O157:H7 was isolated from the ruminants and pigs on all the farms and the same strains were present in the pigs and the ruminants. On one of the farms, the organism persisted in the pig population for 11 months. On all four farms, management practices which might have influenced the isolation rate in pigs were identified. A group of young VTEC O157:H7-positive pigs was moved from one of the VTEC O157:H7-positive farms to a fattening herd where there were no ruminants. The number of VTEC O157:H7-positive faecal samples decreased gradually and after nine weeks the pigs were all negative; at slaughter none of the pigs was VTEC O157:H7-positive.