Prevalence and characteristics of human and bovine verotoxigenic Escherichia coli strains isolated in Galicia (north-western Spain)

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H–, 091:H21, 0103:H2, 0105:H18, 0111:H–, 0113:H21, 0126:H–, 0128:H– and 0157:H7 or H–) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (026:H11 and 086:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes, 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT 1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1⁺ strains of serotype 026:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (026:H11, 0111:H–, 0157:H–and 0157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.     

Serotypes and colonization factors of enterotoxigenic Escherichia coli isolated in various countries

One hundred and six enterotoxigenic E. coli (ETEC) isolated from many geographical areas were serotyped and investigated for the presence of colonization factor antigens CFA/I and CFA/II, the expression of mannose-resistant haemagglutination (MRHA) and the levels of surface hydrophobicity. CFA/I was found in 6 (17%) of 36 LT⁺STa⁺ strains and in 15 (54%) of 28 STa⁺ strains; CFA/II was found in 16 (44%) of 36 LT⁺STa⁺ strains. None of 42 LT⁺ strains showed CFA/I or CFA/II. CFA/I was found in ETEC of serotypes O63:K?:H?, O78:K80, O128:K67 and O153:K?:H45, whereas CFA/II was found in serotypes O6:H?, O6:K15:H16 and 06:K?:H40. Of the 69 CFA/I^? CFA/II^? ETEC strains, 9 (13%) showed MRHA with some of the seven erythrocyte species used and 21 (30%) were hydrophobic. Among the 21 hydrophobic strains CFA-negative we have detected: (i) 6 LT⁺ strains of serogroup O25 negative for MRHA, (ii) 5 strains O159 (4 LT⁺ and 1 LT⁺ STa⁺) also negative for MRHA, and (iii) 3 STa⁺ strains of serotype O27:K?:H7 that haemagglutinated calf and sheep erythrocytes when grown on Minca-Is. The 106 ETEC strains belonged to 20 different 0 serogroups. However, 77 (73%) were of one of nine serogroups (O6, O8, O25, O27, O78, O148, O153, O159 and O167). E. coli strains belonging to O6 and O153 groups predominated among ETEC isolated in Spain, O159 strains in the Central African Republic, O25 and O148 strains in Japan, and O15 and O78 strains in India.     

Prevalence of bovine verotoxin-producing Escherichia coli in Argentina

Faecal swabs obtained from 126 calves and 118 cows in Argentina were investigated for the presence of verotoxin-producing Escherichia coli (VTEC). VTEC strains were recovered from 10 (23%) of 43 calves with diarrhoea, from 24 (29%) of 83 healthy calves, from 40 (44%) of 91 healthy cows waiting at the slaughterhouse, and from 6 (22%) of 27 healthy grazing cattle. PCR showed that 21 (9%) of animals carried VT1⁺ strains, 49 (20%) VT2⁺ strains and 10 (4%) VT1⁺ VT2⁺ strains. VT1⁺ strains predominated among calves (16% versus 0.8%; p < 0.001). The presence of eae gene was significantly more frequent among VTEC strains isolated from calves (78%; 46/59) than from cows (2%; 1/65) (p < 0.001). Furthermore, eae gene was more prevalent in VT1⁺ strains (97%; 32/33) than in VT2⁺ strains (14%; 10/70) (p < 0.001) and in VT1⁺ VT2⁺ strains (24%; 5/21) (p < 0.001). Sorbitol negative high virulent strains serogroups O157 were not detected. This study indicates that cattle are a reservoir of VTEC strains, and that eae gene is associated with VT1⁺ strains that are predominating among young animals. Fortunately, only adult animals are taken to the slaughterhouse, among which VTEC strains negative for eae gen are predominating.     

Nachweis Verotoxin-bildender E-coli (VTEC) bei gesunden Mitarbeitern eines Fleisch verarbeitenden Betriebes

A survey on the occurrence of verotoxigenic E. coli (VTEC) was carried out during 21 months in collaboration with a meat processing plant. Stool samples (n=467) from 22 healthy workers among the staff were assayed monthly. Products (shortly ripened raw sausages), samples of meat juice and surface swabs from sanitary and production facilities were sampled weekly. Two asymptomatic shedders of VTEC were detected. VTEC could be cultivated from both of the detected shedders, among which the first excreted serotype O40:H8 (VT2+, eaeA-, E-hly-) intermittently during a period of 7 weeks. An intermittent excretion of VTEC up to nearly 10 months was proven in case of the second worker and O91:H14 (VT1+, eaeA-, E-hlyl+) detected as the dominant serotype. Moreover, in some stool samples O91:H21 and O91:H- were found. The results of the product analyses (n=623) gave no indication on a possible source of infection. A total of 11.1% showed to be VT-positive on the basis of the PCR-screening. Isolation of VTEC was possible for 2.1%. All isolates were eaeA negative and showed different virulence markers. They belonged to the non-O157 group and could be assigned to 13 different O-serotypes (O5:H38, O₂2:H8, O₂3:H15, O₂6:H11, O56:H24, O62:H30, O88:H8, O91:H?, O91:H21, O103:H2, O115:H?, O116:H21 and rough:H?). From meat juice samples (n=168) only one VTEC (rough:H?) could be isolated. PCR-screening of swab samples from production facilities was positive in 3.2% cases without microbiological determination by isolation of VTEC-colonies. In one out of 256 swab samples from sanitary facilities serotype O40:H8 (VT2+, eaeA-, E-hly-) was found which indicated a contamination by one of the asymptomatic shedders. The study presented here documents for the first time the long shedding of VTEC by healthy staff of a food producing company and points to the importance of these findings in context with food safety.     

Verocytotoxin-producing Escherichia coli in foodstuffs of animal origin

While much research effort has been targeted at the verocytotoxin-producing Escherichia coli (VTEC) serotype O157:H7, it is becoming more evident that other VTEC serotypes can also be associated with human foodborne disease. An increasing number of these non-O157 serotypes have been isolated from food sources and from humans suffering from haemolytic-uraemic syndrome and diarrhoea. The aim of our work was to investigate the prevalence of VTEC O157 and non-O157 in foodstuffs of animal origin using two rapid enzymatic procedures. Various types of food samples, 352 in total, were tested: 233 with the Premier EHEC, a screening test which directly detects the presence of verocytotoxin, regardless of serotype, while 119 of these with the Vidas ECO, which is a specific screening test for E. coli O157:H7, together with the Premier EHEC. Two samples were positive for VTEC, one of serogroup O126 and the other was non-serotypable. Another sample was positive in the test specific for E. coli O157:H7, but was not confirmed by culture. This study suggests that VTEC strains are not prevalent in Italy, and that the isolation of serogroup O157 is relatively infrequent. This leads us to conclude that there is little chance of exposure to pathogen for the average consumer in Italy.     

Enterotoxigenic and necrotizing Escherichia coli in human diarrhoea in Spain

Enterotoxigenic Escherichia coli (ETEC) strains of serotype 0153: K-:H45 CFA/I⁺ STa⁺ were associated with two outbreaks of neonatal diarrhoea that occurred in two different hospitals of Madrid, in one of which several children died. Two other outbreaks were associated with ETEC strains of serotypes 0159: K-: H21 (LT⁺) and 0159: K-: H4 (LT⁺ STa⁺) without CFA/I and CFA/II colonization factors. Necrotizing E. coli (NTEC) strains of serotype 06: K13, producing the cytotoxic necrotizing factor CNFI and -haemolysin, were also associated with two outbreaks of neonatal diarrhoea that occured in a hospital in Madrid and in a hospital in Talavera de la Reina. The results of the characterization of some ETEC and NTEC strains isolated from sporadic cases of diarrhoea are also discussed.Corresponding author.     

Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria

The relationship between the presence of bacterial virulence factors and the severity of urinary tract infection (UTI) was analized in this study. The production of -hemolysin (Hly), the expression of P-fimbriae and the mannose-resistant hemagglutination (MRHA) type IVa (associated with the presence of P-fimbriae), were all detected more frequently in Escherichia coli strains from acute pyelonephritis than in strains isolated from cystitis and asymptomatic bacteriuria. In contrast, the production of cytotoxic necrotizing factor type 1 (CNF1) and the expression of MRHA types III and IVb were distributed uniformly between strains causing different clinical categories of UTI. Thus 88% of the E. coli strains from acute pyelonephritis showed some of the virulence factors investigated in this study, whereas only 60% (p < 0.01) and 56% (p < 0.01) repectively of the strains isolated from cystitis and asymptomatic bacteriuria possessed virulence factors. There were no significant differences in the prevalence of virulence properties between strains isolated from patients with or without complicating factors. Only 16% (p < 0.001) of the fecal isolates from healthy individuals showed virulence factors. The virulence factors were concentrated in strains belonging to 10 (O1, O2, O4, O6, O7, O14, O18, O22, O75 and 083) of the 12 serogroups most frequently detected in uropathogenic E.coli strains. The majority of uropathogenic O4, O6, O14, O22, O75 and O83 E.coli strains were Hly⁺CNF1⁺ and expressed P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly⁺CNFI^– and P-fimbriated. Among O1 and O7 strains we found Hly^– CNF1^–strains that expressed P-fimbriae. Among O2 strains we found Hly⁺CNF1⁺ strains that expressed P-fimbriae or MRHA type III and other Hly^–CNF1^–strains that possessed P-fimbriae. We conclude that E.coli strains isolated from pyelonephritis show virulence factors more frequently than those from cystitis and asymptomatic bacteriuria, and that strains that cause urinary tract infections in Spain belong to the same serogroups as uropathogenic E.coli isolated in other areas of the world. Our results support the special pathogenicity theory and suggest that many cases of serious urogenital disease may be caused by a limited number of P-fimbriated E.coli strains that usually produce -hemolysin.     

Nachweis Verotoxin-bildender E-coli (VTEC) bei gesunden Mitarbeitern eines Fleisch verarbeitenden Betriebes

In einem Herstellungsbetrieb für streichf?hige Rohwürste wurden über einen Zeitraum von 21 Monaten insgesamt 467 Stuhlproben von 22 Mitarbeitern aus dem Produktionsbereich in monatlichen Abst?nden auf VTEC untersucht. Parallel dazu wurden w?chentlich Produkt-, Tropfsaft- sowie Tupfersammelproben aus dem Sanit?r- und Verarbeitungsbereich in die Untersuchung einbezogen. Zwei Mitarbeiter wurden als klinisch symptomlose VTEC-Ausscheider ermittelt, wobei einer E. coli O40:H8 (VT2+, eaeA-, E-hly-) über sieben Wochen intermittierend ausschied und beim zweiten eine intermittierende VTEC-Langzeitausscheidung über nahezu zehn Monate festgestellt wurde. Als dominanter Serotyp wurde E. coli O91: H14 (VT1+, eaeA-, E-hly+) nachgewiesen, vereinzelt traten auch die Serotypen O91:H21 und O91:H- auf. Die Untersuchung der 623 zeitgleich untersuchten Produkte (streichf?hige Rohwurst) ergab keinen Hinweis auf ein erh?htes Infektionsrisiko durch das Arbeitsumfeld: 11,1% der Produkte erwiesen sich im PCR-Screening auf Verotoxine positiv, in 2,1% gelang die Isolierung von VTEC, die 13 unterschiedlichen Serotypen (O5:H38, O₂2:H8, O₂3:H15, O₂6:H11, O56:H24, O62:H30, O88:H8, O91:H−, O91:H21, O103:H2, O115:H−, O116:H21 und Rauhform:H−) angeh?rten. S?mtliche Isolate waren eaeA-negativ und unterschieden sich bezüglich der Virulenzmerkmale Verotoxin und EHEC-H?molysin. Aus den Tropfsaftproben (n=168) wurde ein VTEC-Isolat (Rauhform:H−) angezüchtet, die Tupfersammelproben aus dem Verarbeitungsbereich (n=762) waren im Screening zu 3,2% positiv, allerdings gelang in keinem Fall eine Isolierung von VTEC. E. coli O40:H8 (VT2+, eaeA-, E-hly) wurde in einer Tupferprobe aus dem Sanit?rbereich (n=256) detektiert, was auf eine Kontamination durch den Ausscheider I hindeutet. Die vorliegende Studie dokumentiert erstmals ein mehrmonatiges Ausscheidertum durch einen gesunden Mitarbeiter in einem Fleisch verarbeitenden Betrieb, womit auf die nicht zu untersch?tzende Bedeutung des Personals als m?gliche Eintragsquelle für VTEC hingewiesen wird.     

Reduced faecal shedding of Escherichia coli O157:H7 in cattle following systemic vaccination with γ-intimin C₂₈₀ and EspB proteins

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is the most prevalent EHEC serotype that has been recovered from patients with haemolytic uremic syndrome (HUS) worldwide. Vaccination of cattle, the main reservoir of EHEC O157:H7, could be a logical strategy to fight infection in humans. This study evaluated a vaccine based on the carboxyl-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C₂₈₀) and EspB, two key colonization factors of E. coli O157:H7. Intramuscular immunization elicited significantly high levels of serum IgG antibodies against both proteins. Antigen-specific IgA and IgG were also induced in saliva, but only the IgA response was significant. Following experimental challenge with E. coli O157:H7, a significant reduction in bacterial shedding was observed in vaccinated calves, compared to control group. These promising results suggest that systemic immunization of cattle with intimin and EspB could be a feasible strategy to reduce EHEC O157:H7 faecal shedding in cattle.     

An epidemiological study on Verotoxin-producing Escherichia coli (VTEC) infection among population of northern region of Iran (Mazandaran and Golestan provinces)

Faecal samples from 3268 randomly selected inhabitants of two provinces in the northern region of Iran were screened to detect Verotoxin-producing Escherichia coli (VTEC) using colony sweep polymyxin-B extraction method. Non-sorbitol fermentation phenotype and slide agglutination with O157 and H7 antisera used to detect this serotype. We found that 0.7% of the population were infected with VTEC, however none of the isolates belonged to O157:H7 serotype. We also found that children < 6 years of age were at highest risk of infection with VTEC (p < 0.05). Moreover, a significant association was found between the VTEC and diarrhoeal cases at the same age group (p < 0.001). Overall distribution of the VTEC isolates in the general population was found to be random, though a kind of clustered distribution could be noticed.     

Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers.

The distribution of the Escherichia coli attaching and effacing (eae) gene in strains of verotoxin-producing E. coli (VTEC) isolated from cattle and humans was studied. The majority of strains isolated from humans with bloody diarrhoea or HUS and cattle with severe diarrhoea were eae positive (82 and 83% respectively). In contrast, 59% of VTEC isolated from asymptomatic cattle were eae negative and of the remaining 41% that were eae positive, the majority were serotype O157. H7. The nucleotide sequence of the 3'' end of the eae gene of enteropathogenic E. coli (EPEC) of serotype O55. H7 was found to be almost identical to that of serotype O157. H7. Specific primers are described which detect the eae sequences of VTEC serotypes O157. H7, O157. H-, and EPEC serotypes O55. H7 and O55. H-. The nucleotide sequence of the 3'' end of the eae gene of serotype O111. H8 differed significantly from that of O157. H7. Primers were developed to specifically identify the eae sequences of VTEC serotypes O111. H- and O111. H8. We conclude that whereas the majority of VTEC associated with disease in cattle and humans possess the eae gene, the gene itself may not be necessary to produce haemorrhagic colitis and HUS. Sequence heterogeneity in the 3'' end of eae alleles of VTEC permits specific identification of subsets of these organisms.     

Serotypes of CNF1-producingEscherichia coli strains that cause extraintestinal infections in humans

The O:K:H serotypes of 137 necrotoxigenicEscherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.     

Evaluation of hha and hha sepB mutant strains of Escherichia coli O157:H7 as bacterins for reducing E. coli O157:H7 shedding in cattle

Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 10⁹ heat-killed cells of the hha⁺ wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha⁺ wild-type bacterins. Following oral inoculations with 10¹⁰ CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha⁺ wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce.     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario.

This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA gene showed that 151 (35.2%) strains were positive for the eaeA gene. All isolates (9-21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7-34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA-negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle.     

Review of epidemiological surveys on the prevalence of contamination of healthy cattle with Escherichia coli serogroup O157 : H7

This paper gathers and critically analyses the results of 26 published epidemiological surveys on the prevalence of contamination of cattle with verocytotoxin-producing E. coli (VTEC) serogroup O157 : H7. These surveys have been conducted since 1986 on farms in North America (10 studies), on farms in Europe (6 studies) and at slaughterhouses prior to or just after slaughter (7 studies) or after skinning and evisceration (3 studies). The purpose of this review is to understand the first stages of the epidemiology of the infection in animals and humans (the infection process being obscure in many points) and to prepare herd-based control measures to reduce the risk of O157 : H7 human infection.The different statistical methods employed in these surveys, as well as the various laboratory screening methods used for detecting positive animals are presented. The observed frequencies of infected animals (animal prevalence) and herds (herd prevalence) are given as a function of localisation, year, type of industry (beef or dairy) and age. From these measured prevalence values, the risk of contamination of ground beef by E. coli O157 : H7 in the first stages of the farm-to-fork continuum is assessed. First, we follow the evolution of contamination frequencies from the living animal on-farm to carcasses before transformation. Then, within each set of measurements (i. e., on farm or at slaughterhouse), we identify the effects of the following factors: target population, sampling strategies and laboratory procedures. We argue that the prevalence values inferred from these measurements are very likely underestimated, due to insufficient sampling and not enough sensitive laboratory procedures (one exception being the immunomagnetic bead separation technique). No firm conclusion can be drawn as to the effects of geographical localisation and season. In those surveys, the effect of hygiene level at slaughterhouse on prevalence values is not quantitatively assessed. In addition, there is growing evidence of other sources of E.coli O157 : H7 than live cattle in the farm environment, such as feed, water and water-troughs.     

High prevalence of clade 8 Escherichia coli O157:H7 isolated from retail meat and butcher shop environment

Escherichia coli O157:H7 is an enteric pathogen associated with food safety threats and with significant morbidity and mortality worldwide. In Argentina, post-enteric hemolytic uremic syndrome (HUS) is endemic, with > 70% of cases associated with E. coli O157 infection. To date the biological basis behind the severity among E. coli O157 infections is unknown. However, single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades, of which clade 8 strains are associated with severe disease cases. The aim of this study was to characterize a collection of 20 STEC O157:H7 strains isolated between 2011 and 2013 from ground beef and different environmental samples from butcher shops of Argentina. All strains harbored the eae, ehxA, fliC_H7, efa, iha, and toxB genes, with stx_2a/stx_2c as the predominant genotype (75%). The XbaI-PFGE analysis showed that the E. coli O157 strains had high genetic diversity. Nine strains were grouped in four XbaI-PFGE clusters, whereas 11 strains showed unique XbaI-PFGE patterns. In contrast, the SNP analysis allowed us to separate the strains in two distinct lineages representing clade 8 (70%) and clade 6 (30%). Our results show the molecular characterization of E. coli O157 strains isolated from ground beef and environmental samples from Argentinean butcher shops.     

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.     

A longitudinal study of Vero cytotoxin producing Escherichia coli in cattle calves in Sri Lanka.

Two cohorts of 10 and 16 calves were followed at weekly or fortnightly intervals from 4-28 and 1-9 weeks respectively to determine whether natural infection by Vero cytotoxin (VT) producing Escherichia coli (VTEC) occurred. Ninety-one of 171 (53%) faecal specimens were VTEC positive and 20-80% of animals at any given time excreted VTEC. Of 104 VTEC strains studied further, 6 different serogroups (O 22.H16; O 25.H5; O 49.H-; O 86.H26; O 88.H25; O 153.H12) and an untypable strain (O? .H21) were identified. All strains belonging to the same serotype had identical profiles of reactivity with DNA probes to toxins VT1 or 2, LTI or II and a probe (CVD419) derived from a plasmid carried by enterohaemorrhagic Escherichia coli O 157.H7. Four of these serotypes were found in the faecal flora of the calves, taken as a group, throughout the 4-month study period. Sixty percent of the strains hybridized with the probe for VT1, 4% with the probe for VT2, and 36% with both probes. Faecal VTEC were significantly associated with overt diarrhoeal illness in animals < 10 weeks of age, but no characteristic profile of markers (serotype or hybridization pattern) in E. coli isolates was associated with diarrhoea. A serological response to VT1 was detected in some animals, but faecal VT1 VTEC excretion persisted in spite of seroconversion. VT1 seroconversion was not associated with diarrhoea. A serological response to VT2 was not detected even in those animals excreting VT2 VTEC in the faeces.