Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.     

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.     

黑龙江地区断奶仔猪腹泻大肠杆菌种系类群、血清型和毒力基因分析

揭示黑龙江地区集约化猪场仔猪断奶腹泻(PWD)直肠棉拭子分离到的123株大肠埃希菌所属种系分类群、O血清型及其与毒力基因的特征.采用常规凝集试验进行测定,种系分类群标记的ch uA、yjaA基因和TSPE4.C2DNA片段及肠毒素基因STa、STb、LT、SL T-2e采用PCR方法检测.在123株分离菌株中,除21株未定型、10株自凝外,测定出92株分离的血清型,覆盖18个血清型,其中以O8、O 9、O 149 3种血清型为主,共57株,占定型菌株的61.96％.环境共生的种系进化A群113株占91.87％,肠外强致病D种系进化群2株占1.63％.肠毒素检出菌株74,占分离株的60.16％,其中以STa、STb为主,共占肠毒素检出株的94.59％.结果显示黑龙江省PWD病原性大肠埃希菌优势血清型为O 8、O9、O149,大肠埃希菌肠毒素主要毒力因子为STa、STb,种系进化群为A.     

Serological, enterotoxin-producing and biochemical properties of Escherichia coli isolated from piglets with neonatal diarrhea in Norway

Altogether 235 strains of Escherichia coli isolated from jejunal content of piglets with neonatal diarrhea were examined for serological, enterotoxin-producing and certain biochemical properties. Of 198 strains examined, 84 (42 %) belonged to O-group 149, while 14 (7 %) strains belonged to each of the O-group s 8 and 64. Seventy strains (30%) could not be grouped with the sera used. The remaining strains were distributed among the following O-groups with only a few strains in each group: 2,6,9,32,45,98 and 141. Eighty out of 84 E. coli strains of O-group 149 possessed the K88 antigen and produced heat labile enterotoxin (LT). Besides, LT production was demonstrated in 3 out of 14 strains of E. coli 08. K88 antigen was demonstrated in only 1 strain not belonging to O-group 149. Among strains of E. coli 064 12 out of 14 were K99 positive. This antigen was not demonstrated in E. coli strains of other O-groups. A close relationship was demonstrated between strains of E. coli 0149 possessing the K88 antigen and the ability to ferment both raffinose and adonitol. This ability was only detected in 2 other strains of E. coli not belonging to O-group 149.     

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied.Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum.These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality.Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.     

Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.     

Enterotoxigenic Escherichia coli in the jejunum of piglets with neonatal diarrhea

Thirty-two Escherichia coli colonies were taken from the primary step of cultivation of the jejunal contents of each of 10 dead piglets which had suffered from diarrhea. The organisms of each colony were examined for the presence of adhesion fimbria (F4 (K88) and F5 (K99)), production of heat-stable and heat-labile enterotoxin and of colicins.The presence of heat-labile enterotoxin in the intestinal content of the necropsied pigs was also tested, and results correlated with enterotoxin production of the isolated E. coli strains. In all but 3 pigs, 50–80 % of the E. coli strains were found to produce one or both of the enterotoxins and to possess the F4 of the F5 antigen. All bacteria producing both heat-labile and heat-stable enterotoxin proved to belong toi O group 149 and to possess the F4 antigen. Strains from 1 pig belonged to O group 64 and possessed the F5 antigen; these bacteria produced heat-stable enterotoxin only. Most of the enterotoxin-producing E. coli also produced colicins.After each subcultivation, the strains produced less heat-labile enterotoxin, some becoming negative when assayed.     

特异性IgY抗仔猪断奶腹泻机理的研究概况

针对仔猪断奶腹泻（Post—weaning Escherichia coli diarrhea，PWECD）这一仔猪断奶后的常见病和多发病的病原特异性，阐述了利用K88和F18等特异性菌毛作为抗原免疫产蛋母鸡制备的抗PWECD特异性卵黄抗体对大肠埃希氏菌引起的断奶仔猪腹泻的作用机理。认为，特异性IgY用于防治PWECD，具有安全、高效、成本低、产率高、稳定性好，无药物残留，不会产生耐药性，符合现代动物管理理念等诸多优点，是一种能有效防治PWECD的绿色生物制剂，应用前景广阔。     

Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil

Ninety-one Escherichia coli isolates obtained from diarrheic and normal feces of newborn piglets (0-11 days of age) from three states of Brazil were assessed for phenotypic and genotypic characteristics associated with pathogenic processes. These isolates expressed fimbriae F18ac and type 1, but not fimbriae K88, K99, 987P or F41. Genes for toxins (LT-I, STa, SLT-I, SLT-II, SLT-IIv) either individually or combined were found to be present in most of the diarrheic strains (65.7%) and in 42.8% of the non-diarrheic ones. The eaeA gene was present in 25.7% of the diarrheic isolates and in 9.5% of the non-diarrheic ones. Colicin, hemolysin and aerobactin were also found to be produced by some strains from both sources. Because of the great variety of biological characteristics associated with different illness processes, we suggest that, in Brazil, pigs may act as a reservoir for transmission of Escherichia coli strains to other animals.     

Virulence factors of Escherichia coli isolated from cows with acute mastitis

A total of 184 Escherichia coli isolates recovered from cows with acute mastitis were examined for recognized pathogenic mechanisms and virulence factors commonly found in pathogenic groups of E coli. A modification of the Eng procedure (for detecting complement deficiencies in serum) was used to test for resistance to different animal sera. The Sereny test (for invasiveness), infant mouse test (for heat-stable enterotoxin), and Y-1 adrenal tumor cell assay (for heat-labile enterotoxin) were used. Hemagglutination tests, using rabbit, sheep, and guinea pig RBC, were done with and without added mannose. All of the 184 isolates were serum resistant in all tested sera. None of the isolates was invasive. Only 1 isolate was positive for heat-stable enterotoxin and 2 cultures were positive for heat-labile enterotoxin. Multiple patterns of hemagglutination were observed. The majority of the isolates exhibited both mannose-sensitive and mannose-resistant hemagglutinins with guinea pig and rabbit RBC. A few strains were positive only in mannose-sensitive or mannose-resistant hemagglutination tests. A few strains were negative in all hemagglutination tests. Based on our results, E. coli from cows with acute mastitis lack the virulence factors commonly observed in other E coli groups associated with disease. Serum resistance was the only characteristic that could be related to virulence.     

一规模化猪场断奶仔猪腹泻大肠杆菌毒力因子的检测

从疑似断奶仔猪腹泻的仔猪中分离、鉴定出15株病原性大肠杆菌。经O血清型鉴定,11株为0131、4株未定型,所有O131血清型菌株呈β溶血。多重PCR检测毒素基因(STa、STb、LT、SLT-2e)和大肠杆菌粘附素单抗(F4、F5、F6、F41、F18)检测菌毛,O131菌株的毒素为STa、STb、SLT-2e,表达F18粘附素,未定型菌株的毒素为STa,共表达F6和F18粘附素。对15株大肠杆菌用16种抗生素进行药敏试验,结果表明:分离株对阿米卡星、痢特灵、新霉素敏感,而对多种抗生素产生了不同程度的耐药性。运用本场大肠杆菌分离株灭活苗免疫猪群,取得良好效果。     

Virulence plasmids of enterotoxigenic Escherichia coli isolates from piglets

Virulence plasmids of 68 ETEC isolates from piglets belonging to different pathotypes and six ETEC isolates from calves with pathotypes typical of porcine ETEC were identified with seven virulence probes for the heat-stable (STa and STb) and heat-labile (LT) enterotoxins, for the F4, F5, F6, and F41 fimbrial adhesin subunit, and also with five Rep probes for the RepFIA and RepFIB basic replicons, and the RepFIC family of basic replicons. With the exception of the F41 probe, the other virulence probes hybridized with at least one plasmid band of a size range from 65 to more than 100 Mda. Common associations of virulence factor-encoding genes on plasmid bands were: STb/LT, STa/F5, STa/F6, STa/STb. Other associations, STa/F4, STa/F4/F6, and STa/STb/LT/F6, were rarer. On the other hand the F4 adhesin-encoding genes were isolated on one plasmid band in all but three F4+ isolates. All but one of the 92 virulence plasmids which were studied have Rep probe hybridization profiles and replicon types typical of the uni- or multireplicon plasmids belonging to the various incompatibility groups of the F incompatibility complex.     

Virulence factors of Escherichia coli isolated from bovine clinical mastitis.

Escherichia coli isolates from bovine mastitis were examined for a selection of virulence factors. The strains originated from Finland and Israel, which have differences in the proportion of mastitis caused by E. coli, clinical pictures of coliform mastitis, environmental conditions and herd management. The genes of nine virulence factors were detected by polymerase chain reaction. Presence of K1 and K5 capsules was assessed by use of specific bacteriophages. Serum resistance was tested by a turbidimetric assay. Out of 160 Finnish isolates, 37% had traT, 14% cnf2, 8% cnf1, 11% aer, 9% f17, 8% sfa, 7% pap, 1% afa8D and 1% afa8E. Out of 113 Israeli isolates, 41% had traT, 4% aer, 3% cnf2, 1% cnf1, 1% sfa and 1% f17. Some of the genes were distributed among two major pathotype groups, with either f17 family or sfa, pap and cnf1 as major determinants. Genes for F17a, CS31A, Afa7D and Afa7E were not detected. Altogether 49% of Finnish and 42% of Israeli isolates had at least one virulence gene, but genes other than traT were present in only 24% of Finnish and 5% of Israeli isolates. Serum resistance was more common among Finnish (94/160) than Israeli isolates (19/113). K1 and K5 capsules were not detected.     

Invasive ability of Escherichia coli O18 isolated from swine neonatal diarrhea

Neonatal diarrhea occurred at two swine breeding farms in Hokkaido. Ten piglets aged 2 to 4 days were examined. Grossly, significant changes were confined to the small intestine. The mucous membrane was muddy and thickened. The intraluminal contents from the jejunum to the colon were liquid and yellow. In the small intestine, numerous Gram-negative bacilli preferentially adhered to the apex of villi. The mucosa was erosive with villous atrophy. There were bacilli also in the lamina propria and in the cytoplasm of degenerated enterocytes. Nonhemolytic Escherichia coli strains, belonging to serogroup E. coli O18 and possessing K88 fimbriae, were isolated from the small intestine. They could not be classified into any of the diarrheagenic E. coli groups because of the absence of genes of LT, STh, STp, VT1, VT2, eae, invE, and ipaH. After inoculation of the isolates on HEp-2 cells, some bacilli were engulfed by cytoplasmic projections resembling membrane ruffles and subsequently were localized in cytoplasmic vacuoles or free in the cytoplasm. These findings support the view that the present E. coli O18 is a new invasive strain enteropathogenic to piglets.     

Virulence factors and antimicrobial susceptibility of Escherichia coli isolated from calves in Turkey

Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin.     

Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea

In vitro resistance to 8 antimicrobials among enterotoxigenic Escherichia coli from piglets and calves over a 13-year period was evaluated. Least resistance occurred against ceftiofur for all, followed by apramycin and gentamicin for porcine and florfenicol for bovine isolates. No significant differences were found between the first 8 and last 5 years.     

Escherichia coli post-weaning diarrhoea occurrence in piglets with monitored exposure to creep feed

The objective of the study was to investigate the effect of offering supplementary creep feed to piglets during the suckling period on the faecal shedding of haemolytic Escherichia coli bacteria and occurrence of spontaneous post-weaning diarrhoea 0-5 days after weaning. Supplementary creep feed was offered to half of the piglets in 12 litters, from 2 weeks of age until weaning at 4 weeks, and the individual feed contact was recorded by direct observations. It was found that diarrhoea occurrence was associated with faecal shedding of haemolytic E. coli bacteria (p=0.003), specifically E. coli O149 (p=0.004). Occurrence of diarrhoea and faecal shedding of haemolytic E. coli was not associated with creep feeding per se. However, the faecal E. coli O149 shedding occurred significantly less often in piglets that were offered creep feed in the suckling period but only showed limited interest in the feed (i.e. contacted the feed less than or equal to the median level of contact) compared to piglets that had frequent creep feed contact or piglets that had not had access to creep feed at all (p=0.015). Correspondingly, the diarrhoea occurrence tended to be lower in these piglets (p=0.081). Piglets with low creep feed contact during the suckling period ate the same total amount of feed during the 5 days after weaning, however, they ate less feed on days 0-2 after weaning compared to the piglets with frequent creep feed contact and non-creep fed piglets. It is suggested that intestinal function associated with a voluntary low creep feed contact during the suckling period leads to decreased feed intake just after weaning, and thus reduces the intestinal proliferation of E. coli O149 in these piglets.     

Virulence factors and markers in Escherichia coli from calves with bacteremia

Relative pathogenicity of 151 Escherichia coli isolates from 36 calves with bacteremia after necropsy was studied by measurement of the LD50 after mice were inoculated IP with E coli isolates. Study of virulence factors and markers revealed that the pathogenicity of E coli was associated with the production of hydroxamate siderophores and with resistance to serum bactericidal effects. Production of colicins, including colicin V, and of surface antigen 31A was correlated with virulence. The close association between phenotypic expression of virulence factors and markers was consistent with a hypothesis of a localization of genes coding for virulence factors and markers on the same plasmid.     

Development of a novel live vaccine delivering enterotoxigenic Escherichia coli fimbrial antigens to prevent post-weaning diarrhea in piglets

The efficacy of a novel, live delivery vaccine was examined for protection against post-weaning diarrhea in pigs. An expression/secretion plasmid harboring genes encoding enterotoxigenic Escherichia coli K88ab, K88ac, FedA and FedF fimbriae was constructed and harbored in an attenuated Salmonella, which was used as the vaccine candidate. Groups A (n=3) and B (n=3) sows were orally immunized with the candidate vaccine and PBS as a control, respectively, at 8 and 11 weeks of pregnancy. All group piglets were challenged with two challenge strains at 5-week-old. All immunized sows had significantly increased IgG and IgA levels in both serum and colostrum to individual adhesins compared to the control (p ≤ 0.05). Immune response in Group A piglets were significantly increased (p ≤ 0.05). Furthermore, no clinical signs were observed in Group A piglets after the challenge and no challenge strains were detected in rectal swabs, while diarrhea was observed in 47.8% control piglets and challenge strains were isolated from all the diarrheic piglets. These results show that immune response of sucking piglets can maintain at higher levels through the milk of the immunized sows and vaccination of sows with the candidate may protect colibacillosis in weaned piglets.