The Use of Specific Antibodies to Demonstrate the Glycocalyx and Spatial Relationships of a K99-, F41- Enterotoxigenic Strain of Escherichia coli Colonizing the Ileum of Colostrum-deprived Calves

Electron microscopy was used to study the interaction between the glycocalyx of enterotoxigenic Escherichia coli strain 210 (09:K30+;K99-;F41-:H-) and the glycocalyx of epithelial cells in then ileum of experimentally infected newborn colostrum-deprived calves. Fixation of tissues in anti-K30 antibody and ruthenium red was used to stabilize the bacterial glycocalyx so that the spatial relationship between the bacteria and the intestinal epithelial cells could be characterized.

When strain 210 was grown in vitro and reacted with anti-K30 antibody prior to staining with ruthenium red, the extensive glycocalyx could be clearly visualized surrounding the bacterial cells. By negative staining, an unidentified pilus was also seen. Sections of ileum from infected calves, which were not fixed in antibody nor stained with ruthenium red, revealed attached bacteria which were surrounded by an electron-translucent zone and no visible bacterial glycocalyx. When ruthenium red staining was used, the bacterial glycocalyx partially collapsed during the dehydration steps of fixation, but could be seen as either a fibrous capsule or an electron-dense accretion on the bacterial cell surface. When ileal tissue was reacted for one hour in anti-K30 antibody before staining with ruthenium red, the bacterial glycocalyx was seen as a discrete electron-dense structure up to 1.0µm thick which was in intimate contact with the glycocalyx of the epithelial cells. The importance of the bacterial exopolysaccharide to microcolony formation on the villi could be clearly visualized.

     

Evaluation of a monoclonal antibody to the K99 fimbrial adhesin produced by Escherichia coli enterotoxigenic for calves, lambs and piglets

All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).     

Pathogenicity of porcine enterotoxigenic Escherichia coli that do not express K88, K99, F41, or 987P adhesins.

Three-week-old weaned and colostrum-deprived neonatal (less than 1 day old) pigs were inoculated to determine the pathogenicity of 2 enterotoxigenic Escherichia coli isolates that do not express K88, K99, F41, or 987P adhesins (strains 2134 and 2171). Strains 2134 and 2171 were isolated from pigs that had diarrhea after weaning attributable to enterotoxigenic E coli infection. We found that both strains of E coli adhered in the ileum and caused diarrhea in pigs of both age groups. In control experiments, adherent bacteria were not seen in the ileum of pigs less than 1 day old or 3 weeks old that were noninoculated or inoculated with a nonpathogenic strain of E coli. These control pigs did not develop diarrhea. Antisera raised against strains 2134 and 2171 and absorbed with the autologous strain, grown at 18 C, were used for bacterial-agglutination and colony-immunoblot assays. Both absorbed antisera reacted with strains 2134 and 2171, but not with strains that express K99, F41, or 987P adhesins. A cross-reaction was observed with 2 wild-type K88 strains, but not with a K12 strain that expresses K88 pili. Indirect immunofluorescence with these absorbed antisera revealed adherent bacteria in frozen sections of ileum from pigs infected with either strain. We concluded that these strains are pathogenic and express a common surface antigen that may be a novel adhesin in E coli strains that cause diarrhea in weaned pigs.     

The role of K antigens of enteropathogenic Escherichia coli in colonization of the small intestine of calves.

The colonizing and proliferating abilities of enterotoxigenic acapsular or K99- mutants of bovine enteropathogenic Escherichia coli strains were compared with those of their capsulated and K99+ parent strains in the small intestine of infected colostrum-fed calves. Calves infected with the enteropathogenic E. coli parent strains developed profuse diarrhea and severe dehydration. None of the calves which received the acapsular mutant developed diarrhea and one of three calves inoculated with the K99- enterotoxigenic mutant developed moderate diarrhea. The parent enteropathogenic E. coli strains colonized the middle and lower small intestine; in these areas a layer of specific immunofluorescence against the enteropathogenic E. coli covered most villi and 80% of the organisms were associated with the intestinal wall. The acapsular mutant strain failed to colonize the small intestine and fluorescent bacteria were not observed in any area of the small intestine. The K99- mutant proliferated to a lesser extent than did the K99+ parent strain in all areas of the small intestine but moderately colonized the lower small intestine where fluorescent bacteria were observed to cover parts of the intestinal villi.     

Importance of Escherichia coli in young beef calves from northwestern Quebec.

The objectives of this study were: (i) to investigate the prevalence of Escherichia coli producing F5 (K99), F41, or F165 fimbriae and STa enterotoxin; (ii) to determine serum antibody levels against these fimbriae; (iii) and to examine the association between bacteriological and serological results and the presence of diarrhea, in beef calves from northwestern Quebec. A total of 373 live three to four week old calves and 27 dead calves were sampled between January and March 1991. No isolates positive for F5 were detected in live calves, and only one E. coli producing STa and F41 was isolated. Escherichia coli producing F41-like surface antigens or F165 fimbriae were isolated from 17.43% and 5.63% of live calves, respectively. Antibodies against F5, F41 and F165 were low. Escherichia coli isolates positive for F41-like surface antigen were most often observed in calves born between January and March. No association was found between bacteriological and serological findings, nor between these findings and diarrhea. Calves born from dams vaccinated against E. coli had higher median antibody levels than those born from unvaccinated dams. No E. coli positive for F5 or F41 fimbriae were isolated from dead calves. Escherichia coli with F41-like surface antigen or F165 were found in 55.56% and 11.11% of ileal samples; 4% and 16% of cecal samples, and 0% and 7.4% of colon samples, respectively. Escherichia coli positive for F41-like surface antigen were detected significantly more frequently in the ileum (chi (2)2df = 31.01, p < 0.001).     

Relationship between virulence and adherence of various enterotoxigenic Escherichia coli strains to isolated intestinal epithelial cells from Chinese Meishan and European large white pigs

In vitro adherence to intestinal epithelial cells by enterotoxigenic Escherichia coli strains bearing K88, K99, F41, or 987P adhesins and of their variants not bearing adhesins (K88-, K99-, or F41-) was investigated in European Large White and Chinese Meishan pigs. Possible relationship between adherence and virulence was also examined. The K88-positive (K88+) strain strongly adhered to intestinal epithelial cells from 26 of 28 Large White pigs. This strain had previously been found to be highly virulent for Large White pigs. The only surviving pig was of nonadherent phenotype, and cells from 4 dehydrated moribund pigs had strong adherence. By contrast, the same K88+ strain found previously to have little pathogenicity for Meishan pigs adhered with variable intensity to cells from 17 of 23 Meishan pigs; correlation was not evident between adherence and virulence. The K99+ F41+ strain of porcine origin and the F41+ strain generally adhered strongly to cells from 24 and 23 Meishan pigs, respectively, and to cells from 25 of 26 Large White pigs. Correlation was not found between adherence and virulence for the 2 strains. A K99+ F41+ strain of bovine origin adhered to cells from 20 of 22 Meishan and 22 of 23 Large White pigs, and a K99- F41+ variant adhered to cells from 19 of 23 Meishan and 23 of 24 Large White pigs. The adhesin-negative variants never adhered to intestinal epithelial cells. Strain 987 known not to readily produce 987P adhesin after in vitro growth never adhered to cells during the test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in galicia (north-western Spain)

A total of 289 Escherichia coli colonies isolated from 78 diarrhoeic calves were studied for production of heat-labile (LT) and heat-stable (STa) enterotoxins, verotoxin (VT), cytotoxic necrotizing factor (CNF) and K99 antigen, and they were serotyped. Production of STa was detected in a single strain possessing both K99 and F41 antigens; the serotype was 09:K (A) 35. LT-producing strains were not detected. From 16 (20.5%) calves, 51 VT-producing colonies of E. coli were isolated. Production of the necrotic factor was detected in 33 E. coli colonies isolated from 14 (17.9%) calves. Serotype was a useful marker for production of VT and CNF. Among the 51 VT-producing colonies, 24 were untypable and the remainder belonged to serotypes O2:K?, O103:K--, O104:K?, O128:K?, O153:K-- and O157:K--:H7. Four of the 33 CNF-producing colonies were untypable and the majority of the remaining colonies belonged to serotypes O15:K14, O78:(K80), O123:K-- and O139:K--. Both VT and CNF were lethal for mice, but only CNF showed necrotizing reaction in rabbit skin. Our results indicate that VT-producing and CNF-producing E. coli strains are frequently isolated from diarrhoeic calves and that according to the serotypes exhibited, some of them might be considered potential pathogens for humans. The role of VT-producing and CNF-producing strains in calf diarhoea remains to be established.     

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.     

Prevalence of four enterotoxin (STaP, STaH, STb, and LT) and four adhesin subunit (K99, K88, 987P, and F41) genes among Escherichia coli isolates from cattle

Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+, STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+, STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.     

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.     

Enterotoxigenic colibacillosis in colostrum-fed calves: pathologic changes.

Enterotoxigenic colibacillosis was experimentally produced in 8 of 9 colostrum-fed calves orally given 10(11) Escherichia coli. The eight calves developed profuse diarrhea accompanied by dehydration and depression. At 12 hours after exposure, all calves were euthanatized for necropsy and for collection of tissues for microscopic examination. Histopathologic changes included stunted villi in the jejunum and ileum, focal degeneration and exfoliation of absorptive epithelial cells at the tips of jejunal and ileal villi, and focal emigration of neutrophils which was especially prominent above the dome area of aggregated lymphatic follicles (Peyer's patches). A layer of E coli adhered to the epithelial surface of the jejunum and ileum. In the duodenum, lesions were minimal or absent and bacteria were not adhering to the mucosa. Histopathologic changes were not observed in other tissues. In two calves examined 24 hours after they were inoculated and in two calves euthanatized 24 to 36 hours after spontaneously developing enteric colibacillosis, lesions were similar to those observed in the calves at 12 hours after exposure.     

Characterization and purification of the F17 adhesin on the surface of bovine enteropathogenic and septicemic Escherichia coli

The F17 antigen from bovine enterotoxigenic Escherichia coli strain (E coli 25KHO9), which adhered to calf intestinal villi, was isolated. An enterotoxin-negative derivative (25KHO9st) was used for further studies. Using an immunogold-labeling technique, the F17 antigen was characterized as a fimbrial protein. Pure fimbriae with a subunit molecular weight of 20,000 were obtained by homogenization and use of a sucrose gradient. The adhesion of E coli 25KHO9st was mediated by the F17 fimbriae, as both F17 antibodies and F17 protein blocked the adhesion of the strain 25KHO9st. The F17 fimbriae were serologically distinct from K88, K99, F41, and 987P fimbriae and did not agglutinate bovine, ovine, guinea pig, human, or chicken erythrocytes. Peptide fingerprint analysis revealed F17 and F(Y) adhesins to be homologous, if not identical.     

Protection of lambs against enteric colibacillosis by vaccination of ewes

Pregnant ewes were vaccinated twice, seven weeks and three weeks before lambing, with a multivalent formalin-killed Escherichia coli vaccine containing an added K99, F41 antigen preparation. Lambs born to vaccinated and unvaccinated ewes were exposed to oral infection with E coli B44 (09:K30, K99, F41). All 10 lambs from vaccinated ewes were protected whereas all 10 control lambs developed severe diarrhoea and five died or were killed in extremis. In the following year, previously immunised ewes were given a single dose of the vaccine two weeks before lambing. Eleven of their 12 lambs were protected against a similar challenge, which caused the death of six of eight control lambs and severe diarrhoea in the two survivors. Higher levels of antibody to the K99, F41 preparation were detected by enzyme-linked immunosorbent assay in the serum and colostrum from vaccinated ewes and in the serum of their lambs when compared with similar samples from control ewes and lambs.     

Phenotypic expression of K88 adhesin alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41

F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens. Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium. K99 antigen could be detected in each strain bearing K99 plasmid. Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen. Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all. Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen. None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20°C as found for K99 and F41 ETEC natural strains, respectively. These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains. In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.     

Susceptibility of Chinese Meishan and European large white pigs to enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, K99, or F41

Conventionally raised Chinese Meishan and European Large White pigs were intragastrically challenge exposed with 2.1 x 10(10) enterotoxigenic Escherichia coli strains bearing colonization factor K88, 987P, F41, or F41 plus K99. In response to challenge exposure with the K88-positive (K88+) organisms, 96% of Large White pigs died within 48 hours, whereas none of the Meishan pigs died. Both breeds of pigs had similar susceptibility to strains bearing 987P or F41. Lastly, Meishan pigs were found to be more susceptible than Large White pigs to a strain expressing K99 and F41. In pigs with diarrhea, challenge-exposure strains intensively colonized the jejunum (10(8) to 10(10) bacteria/g of tissue) and, to less extent, the duodenum (except K88+ strain, which comprised 10(8)/g). In most cases, jejunal concentrations of the challenge-exposure strains were substantially lower in pigs that did not have diarrhea. Half the resistant Meishan pigs eliminated the K88+ strain from the intestines. Colostral antibody titer that agglutinated challenge-exposure strains did not differ between Meishan and Large White gilts. Results indicate that resistance of pigs to the K88+ strain did not extend to enterotoxigenic strains bearing other well-known factors. They indicate, in addition, that genetic resistance to K88+ strains described in pigs in Europe may exist in pigs in China.     

Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs.

A novel common colonization factor was detected in enterotoxigenic strains of Escherichia coli isolated from intestinal contents of piglets affected with postweaning diarrhoea. This factor was antigenically distinct from the previously described K88, K99, F41, 987P, CFAI, CFAII and Att25 fimbrial antigens. E. coli strains possessing this factor adhered to the pig intestinal brush borders and one strain, used in experimental infection in weanlings, colonized the intestinal epithelium and induced diarrhoea. Examination of 212 toxigenic strains of E. coli isolated from weanlings revealed the presence of the novel common colonization factor in 83 strains, belonging to serogroups O25, O108, O138, O141, O147 and O157. The antigen K88 was detected in 47 strains belonging to serogroups O8, O141, O147 and O149.     

The diagnostic value of immunoperoxidase in detecting K99+ Escherichia coli on histological sections

The small intestine of 51 calves was examined for the presence of K99+ Escherichia coli by means of both an immunoperoxidase procedure performed on paraffin sections and by the slide agglutination test after isolation. Twelve cases resulted immunoperoxidase positive (23.5%) and 8 of them were also agglutination positive. Results of the 2 diagnostic tests agreed in 46 cases (90.2%). In immunoperoxidase positive sections a thick layer of immunoreactive bacteria was seen on the luminal surface of the enterocytes. Post-mortem autolysis or prolonged fixation did not seem to affect the immunoperoxidase reactivity of the sample.     

Ultrastructural changes in the small intestine of neonatal calves with enteric colibacillosis

The small intestines of calves inoculated orally with the enteropathogenic strain of Escherichia coli 0101:K'B41',K99 were examined by electron microscopy at 3, 6, 12, 16, 21, 36, 69, 70 and 72 hours after inoculation. The challenge organism adhered to the mucosa of the distal small intestine from six hours post-inoculation. Bacteria were separated from the microvillous brush border by a gap of 200 to 300 nm in which bacterial fimbriae and the microvillous glycocalyx were seen. Bacteria never were found in epithelial cells but were present in macrophages in the lamina propria from 12 hours. At three and six hours, cytopathic changes were not seen in the small intestine, but from 12 hours epithelial cells on affected villi had blunt and thick microvilli and contained cytoplasmic inclusions. Epithelial cells were seen frequently in the process of extrusion from the villi, either singly, in small groups, or as ribbons of cells. Intervillous bridges, characteristic of villous fusion, were seen frequently from 69 hours.     

Protection of neonatal calves against fatal enteric colibacillosis by administration of egg yolk powder from hens immunized with K99-piliated enterotoxigenic Escherichia coli.

The protective effects of egg yolk powder prepared from hens vaccinated with heat-extracted antigens from K99-piliated enterotoxigenic Escherichia coli (ETEC) strain 431 were evaluated in a colostrum-fed calf model of ETEC-induced diarrhea caused by a heterologous strain (B44). The antibody powder was obtained by spray-drying the water-soluble protein fraction of egg yolks after removing the lipid and fatty components by precipitation with hydroxypropylmethylcellulose phthalate. A total of 16 colostrum-fed calves were studied to determine whether the orally administered antibody powder would prevent fatal bovine colibacillosis caused by a virulent ETEC strain. Clinical response of individual calves was monitored and evaluated in the context of these variables: fecal consistency score, intestinal colonization, weight loss, and mortality. Control calves that were treated with vehicle (milk with egg yolk powder from nonimmunized hens) had severe diarrhea and dehydration and died within 72 hours after infection was manifested. In contrast, calves fed milk containing egg yolk powder with antipili agglutinin titers of 1:800 and 1:1,600 had transient diarrhea, 100% survival, and good body weight gain during the course of the study. Results indicate that the orally administered egg yolk powder protected against ETEC-induced diarrhea in neonatal calves and that the protective components may have been the antibodies raised by vaccination of chickens against ETEC.     

The studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of Escherichia coli strains by multiplex PCR

The purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty-two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme-linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat-stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves.