Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.     

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.     

Cryptosporidium parvum and Giardia intestinalis in calf diarrhoea in Sweden

The objective of this study conducted in 75 herds was to investigate the presence and significance of Criptosporidium parvum and Giardia intestinalis in Swedish dairy calves in comparison with rotavirus, coronavirus and Escherichia coli K99+. The farmers were asked to collect faecal samples from each heifer calf that had diarrhoea between birth and 90 days of age, and also from a healthy calf of the same age. In total, 270 samples were collected and analysed. C. parvum, either alone or together with G. intestinalis and/or rotavirus, was detected in 16 (11%) and 6 (5%) of the samples from diarrhoeic and healthy calves, respectively. Even though a higher proportion of diarrhoeic calves shed C. parvum, the difference between the groups was not statistically significant (p = 0.067), possibly due to the low number of positive samples. G. intestinalis was found in 42 (29%) of the diarrhoea samples and in 29 (23%) of the samples from healthy calves. Rotavirus and coronavirus were demonstrated in 24% and 3% of the diarrhoea samples, respectively, whereas E. coli K99+ was only found in samples from 2 healthy calves. C. parvum and G. intestinalis were found in samples from calves 7 to 84 days of age and during all seasons. The results confirm that C. parvum is present in Swedish dairy herds and might have clinical significance. G. intestinalis was the most common agent found but the importance of this parasite remains unclear. Both parasites have suggested zoonotic potential and thus warrant further attention. In addition, rotavirus is a major pathogen in neonatal enteritis in Sweden, whereas coronavirus and E. coli K99+ seem to be of less importance.     

Prevalence of four enteropathogens in the faeces of young diarrhoeic dairy calves in Switzerland

The prevalences of Cryptosporidium parvum, rotavirus, bovine coronavirus (bcv), and enterotoxigenic Escherichia coli (E coli k99) were determined in diarrhoeic dairy calves aged one to 21 days on 71 dairy farms in western Switzerland during the winter of 2005 to 2006. Faecal samples from 147 untreated calves suffering from acute diarrhoea were analysed by standardised diagnostic methods, and the immunoglobulin status of each calf was evaluated. The prevalences of C parvum, rotavirus, bcv and E coli k99 were 55.0 per cent, 58.7 per cent, 7.8 per cent and 5.5 per cent, respectively. The proportions of herds positive for the respective pathogens among the herds with diarrhoeic calves were 41.7 per cent, 52.1 per cent, 2.1 per cent and 2.1 per cent. The immunoglobulin concentration in the serum of 90.5 per cent of the diarrhoeic calves was below 8 g/l.     

Role of Cryptosporidium parvum as a pathogen in neonatal diarrhoea complex in suckling and dairy calves in France.

This study was carried out to find the importance of Cryptosporidium parvum in diarrhoea of neonatal calves in two types of breeding - suckling and dairy calves - in France. Different agents causing neonatal diarrhoea, E. coli, rotavirus, coronavirus, Salmonella and Cryptosporidium were systematically researched in faeces. 1. Suckling calves: In 40 livestock farms selected for diarrhoea, 311 calves 4 to 10 days old which had diarrhoea for less than 24h or no diarrhoea, were included in the study. A prophylaxis of neonatal diarrhoea had been carried out in 21 of the 40 livestock farms. On D0 (inclusion day), the mean age was 6 days, 82% presented a good initial general condition and 76.2% had a good appetite; 48.6% were diarrhoeic but 91.3% presented no sign of dehydration. Only 6.1% were infected by E. coli K99, 14.3% by rotavirus, 6.8% by coronavirus, 0.3% by Salmonella but 50% excreted C. parvum oocysts. This later percentage increases up to 84% and 86% by D3 and D7, respectively . We note that 16% of the 4-day-old calves on D0 are excreting oocysts and this percentage increases as a function of the age of the calf on D0 to reach 90% to 95% by the age of 8 days. 10 out of 12 dead calves excreted C. parvum oocysts. From D0 to D14 the other pathogen agents show a relative or a decreasing stability. 2. Dairy calves: 382 calves which had diarrhoea for less than 24 h or no diarrhoea, aged 8 to 15 days coming from six industrial livestock farms were included in the study. On D0, 99% of the calves presented a good initial general condition, 99.7% had a good appetite and no calf was dehydrated. At this date (D0), 16.8% of the calves excreted cryptosporidia. This percentage increases up to 23% and 51.8% on D3 and D8, respectively, then decreases to 31.9% on D14. The pressure of the other pathogenicagents remains relatively stable, excepted for rotavirus on D7 (from 9.9% on D0 to 27.2% on D7, then 12.6% on D14) which does not explain the concomitantpeak in diarrhoea because the infection by rotavirus on D7 is more frequent in non-diarrhoeic calves than in diarrhoeic calves. Our results show that Cryptosporidium prevalence is higher in suckling than in dairy calves and C. parvum constitutes actually in both cases the major aetiological agent of neonatal diarrhoea.     

Aetiology of diarrhoea in young calves

Faeces samples were collected from 302 untreated calves on the day of onset of diarrhoea and from 49 healthy calves at 32 farms experiencing outbreaks of diarrhoea. At least four diarrhoeic calves were sampled on each farm, and samples were examined for rotavirus, coronavirus, cryptosporidium, enterotoxigenic Escherichia coli and Salmonella species. Although all these enteropathogens were excreted more frequently by the diarrhoeic than by the healthy calves, the difference was significant overall only for rotavirus. Rotavirus was excreted by 18 per cent of healthy calves, coronavirus by 4 per cent, cryptosporidium by 14 per cent, and no enterotoxigenic E coli or Salmonella species were detected. The most common enteropathogen in diarrhoeic calves was rotavirus, which was excreted by more than half the diarrhoeic calves on 18 farms. Coronavirus was excreted at a similar high prevalence on one farm, cryptosporidium on five farms and enterotoxigenic E coli on three farms. Concurrent infection with two or more microorganisms occurred in 15 per cent of diarrhoeic calves. There was no difference in the isolation rate of campylobacters between diarrhoeic and healthy calves.     

Virulence gene profiles and intimin subtypes of Shiga toxin-producing Escherichia coli isolated from healthy and diarrhoeic calves

The virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrhoeic and non-diarrhoeic calves were compared. The strains were also tested for O157:H7, O111 and O26 serotypes, using PCR and conventional serotyping methods. E coli strains isolated from 297 faecal samples, from 200 diarrhoeic and 97 non-diarrhoeic calves, were screened by multiplex PCR assay for the stx1, stx2, eae and Ehly virulence genes. STECs were recovered from 8 per cent of diarrhoeic calves and 10.3 per cent of non-diarrhoeic calves. The predominant virulence gene profile was stx1/eae/Ehly (47.3 per cent) among isolates from diarrhoeic calves and eae/Ehly (36.8 per cent) among isolates from non-diarrhoeic calves. Among three tested serogroups, the predominant serogroup was O26 (18.4 per cent), and O157:H7 was not detected. Intimin subtyping by restriction fragment length polymorphism analysis revealed only three intimin subtypes (β, γ and ). A significant difference was observed in the distribution of Int- between two groups. Int- was present in 50 per cent of the isolates from diarrhoeic calves and in 11.1 per cent of the isolates from non-diarrhoeic calves; this difference was statistically significant (P=0.01).     

Microbiology of calf diarrhoea in southern Britain

Faeces samples from calves with diarrhoea in 45 outbreaks were examined for six enteropathogens. Rotavirus and coronavirus were detected by ELISA in 208 (42 per cent) and 69 (14 per cent) of 490 calves respectively; calici-like viruses were detected by electron microscopy in 14 of 132 calves (11 per cent). Cryptosporidium were detected in 106 of 465 (23 per cent), Salmonella species in 58 of 490 (12 per cent) and enterotoxigenic Escherichia coli bearing the K99 adhesin (K99+ E coli) in nine of 310 calves (3 per cent). In the faeces of 20 per cent of calves with diarrhoea more than one enteropathogen was detected; in 31 per cent no enteropathogen was found. Faces samples from 385 healthy calves in the same outbreaks were also examined. There was a significant statistical association of disease with the presence of rotavirus, coronavirus, Cryptosporidium and Salmonella species (P less than 0.001). Healthy calves were not examined for calici-like viruses and the association of K99+ E coli with disease was not analysed because there were too few positive samples. Rotavirus infections were more common in dairy herds and single suckler beef herds whereas Salmonella infections were more often found in calf rearing units. Cryptosporidium were more common in single and multiple suckler beef herds. K99+ E coli were found in one dairy herd and one multiple suckler beef herd both with unhygienic calving accommodation. Variations in coronavirus detection among different farm types were not statistically significant. In this survey rotavirus was the most commonly detected agent in calf diarrhoea and Cryptosporidium were found in approximately one quarter of affected calves. Infection with Salmonella species was widespread, but K99+ E coli infections were less common in the United Kingdom than in other countries.     

Rotavirus and enterotoxigenic Escherichia coli infections of calves on a closed Finnish dairy farm

Rotavirus and enterotoxigenic Escherichia coli infections of calves were surveyed during 2 successive years on a closed Finnish dairy farm consisting of 90–105 milking cows. From a clinical standpoint, diarrhoea was of moderate to high severity during the first year, compared to the milder disease in the second year of the study. Diarrhoea or abnormal faeces were found only in calves less than 8 weeks old, with the peak occurring during the first 2 weeks of life.In the first year, rotavirus was detected throughout the calving season in diarrhoeic or abnormal faeces of calves aged 1 day to 7 weeks. In the second calving season, rotavirus was detected only during the 4 autumn months and in calves aged 11 days to 8 weeks. Rotavirus was detected in only 1 sample of normal faeces in both years. Electron microscopy revealed no enteropathogenic viruses other than rotaviruses. Enterotoxigenic K99 E. coli was found in about half of diarrhoeic or abnormal faeces in both years and throughout the calving seasons. K99 E. coli was also found in 5–10 % of normal faeces.In the second year of the study, 45 of 101 pregnant dams were vaccinated with 2 doses of E. coli antigen. The vaccination trial did not prevent or reduce altered faeces in calves whose dams had been vaccinated compared with calves whose dams had not been vaccinated in the same year. Comparing the 2 years, the earlier uptake of colostrum together with better cleaning and disinfection of the calf house, contributed to the later and rarer occurrence of rotavirus infection in the second year of the study. The earlier uptake of colostrum together with better cleaning and disinfection of the salf house, in the second year, could not prevent enterotoxigenic E. coli infections in calves but partly prevented and modified the disease.     

Characterization of Escherichia coli isolated from adult horses with and without enteritis

In the present study E. coli strains isolated from the faeces of ten horses with diarrhoea and 14 horses without diarrhoea were characterized. All horses were culture negative for Salmonella species. Nine colonies of E. coli from each faecal sample were picked at random and a DNA fingerprint was made by means of a polymerase chain reaction (PCR) using Enterobacterial Repetitive Intergenic Consensus (ERIC) primers. The number of E. coli genotypes did not differ significantly between horses with and without diarrhoea. In addition, all E. coli strains with different DNA fingerprints were tested by PCR for genes encoding the virulence factors K88, F41, F17, CS31a, Sta1, LT1, VT2, CNF, BFP, and intimin. Genes coding for K88, F41, BFP, STa1, VT2, and CS31A were not detected. Genes for CNF were found in strains from one horse with diarrhoea and one horse with normal faeces. Genes for LT1 (n=1) and intimin (n=1) were found only in strains from horses with normal faeces. Genes for F17 fimbriae were found in strains from three horses with diarrhoea (30%) and in none of the strains from healthy horses. In two of these horses, E. coli strains with different DNA polymorphism patterns were F17 positive; however, none of these strains possessed LT1, Sta1, or CNF genes. Haemolytic E. coli strains were only isolated from two horses with diarrhoea and from none of the healthy horses. Nineteen percent of all E. coli strains did not ferment lactose. Eight per cent of these lactose-negative strains were from horses with diarrhoea, whereas 32% were from horses without diarrhoea. In conclusion, virulence factors were present in E. coli isolates from horses with and without diarrhoea, except for F17, which was only found in E. coli isolated from horses with diarrhoea. F17-positive E. coli might have importance as cause of diarrhoea in horses, but further studies are needed.     

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.     

Prevalence of enterotoxigenic Escherichia coli in calves in Scotland and northern England

Eighty-eight of 1529 (5.7 per cent) Escherichia coli isolates from diarrhoeic and clinically normal calves in Scotland and northern England were found to possess the K99 pilus antigen (K99+). There was complete correlation between possession of K99 antigen, heat stable enterotoxin production and ability to dilate intestinal loops. The diagnosis of calf enterotoxigenic E coli infections may therefore be based on the detection of K99 antigen alone. Enterotoxigenic E coli was isolated from 23 of 306 (7.5 per cent) diarrhoeic calves from eight of 70 (11.4 per cent) farms and was not isolated from clinically normal calves. Infected calves were between one and three days old. A survey by an enzyme-linked immunosorbent assay found 3.0 per cent and 3.9 per cent of sera from calves and cows respectively to contain antibodies to K99 antigen. The prevalence of other enteropathogenic organisms in calf faeces is also discussed.     

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.     

Enteric infections in veal calves: a longitudinal study on four veal calf units

Forty-five calves on four veal calf units were monitored during the first four weeks after their arrival. Faecal samples were collected on alternate days and screened for the presence of rotaviruses, bovine coronavirus, Cryptosporidium oocysts, K99 positive strains of E. coli and Salmonella spp. Rotaviruses and Cryptosporidium were the most commonly detected agents (78% and 60% respectively of the calves). Bovine coronavirus was detected in the faeces of 18% of the calves, whilst K99 positive E. coli was only found in 2 samples from one calf. Salmonella spp. were not isolated from any of the 646 faecal samples examined. Shedding of rotaviruses occurred in a bimodal pattern beginning in the first week of the survey. Cryptosporidium oocysts were detected most frequently in the interval between the two peaks of rotavirus shedding. The presence of rotaviruses or Cryptosporidium oocysts in faeces was not strongly associated with scour, nor were combined infections with these agents or the cases of bovine coronavirus infection. The condition of the calves throughout the survey was generally satisfactory.     

Pathology of calves with diarrhoea in southern Britain

Twenty-one moribund calves with diarrhoea were purchased from 11 farms, their faeces examined for enteropathogens and samples of intestinal tissue removed under anaesthesia. Lesions and presence of enteropathogens on the mucosal surface were scored by histological examination of immunostained paraffin sections. Two or more enteropathogens were detected in 19 calves. Cryptosporidium appeared to be the principal cause of diarrhoea in six calves, rotavirus in four, Salmonella typhimurium in two, bacteria adherent to the surface of the large intestine in two, coronavirus in one and K99+ Escherichia coli in one calf. Diarrhoea in four calves was the consequence of mixed infections in which no one enteropathogen appeared to predominate. In one calf no enteropathogen was detected. Diarrhoea was associated with infections and lesions throughout the small and large intestines. The enteropathogens most frequently associated with lesions in the small intestines were rotavirus, coronavirus and cryptosporidium; in the large intestines they were coronavirus and bacteria apparently adherent to the mucosal surface.     

Agents associated with neonatal diarrhoea in Ethiopian dairy calves

Faeces samples collected from diarrhoeic dairy calves in the first 8 weeks of life were examined for the presence of 5 enteropathogens. The majority of the 108 diarrhoea cases occurred in the first 5 weeks of life and a commercial ELISA kit detected bovine enteric coronavirus (BEC) in 38.9%, serogroup A rotavirus (RV) in 16.7% and K99 (F5) fimbrial adhesin-positive Escherichia coli (K99 ETEC) in 11.1 per cent. Concurrent infections of these enteropathogens were detected in 14.8% of samples (30.8% of samples positive for these agents). No evidence of cryptosporidial infection was found using a differential staining method on faecal smears nor was salmonella excretion detected. On 2 of the 8 farms only BEC was present; the other 6 farms were positive for all 3 agents. It is concluded that BEC is the major infectious cause of neonatal calf diarrhoea in the Ethiopian dairy herds studied with RV and K99 ETEC also contributing to morbidity, either alone or as mixed infections.     

Studies of Enteric Pathogens and γ-Globulin Levels of Neonatal Calves in Sweden

Viring, S., S.-O. Olsson, S. Alenius, U. Emanuelsson, S.-O. Jacobsson, B. Larsson, N. Linde and A. Uggla: Studies of enteric pathogens and γ-globulin levels of neonatal calves in Sweden. Acta vet. scand. 1993, 34, 271-279.– Faecal and blood samples were taken from 10-30% of calves, 36 hours to 14 days old, in 47 dairy herds in different regions of Sweden from September 1987 to October 1988 (Olsson et al 1993). Faecal samples from 279 calves were analysed for the presence of Escherichia coli (K99⁺), rotavirus and Cryptosporidium sp. Twenty (7.2%) of these samples were from diarrhoeic calves. An ELISA was developed and used for the rotavirus analysis. E. coli K99⁺ was detected in 11.5%, Cryptosporidium sp. in 6.1% and rotavirus in 5.4% of the faecal samples. The presence of rotavirus alone and the combination rotavirus and E. coli (K99⁺) was found to be associated with diarrhoea (p<0.001 and p<0.01 respectively).Blood samples from 327 calves were analysed for the level of total protein and γ-globulin. In 43 of these samples (13%) γ-globulin did not separate from the ß₂-region by electrophoresis. The mean total protein concentration was 53.6 g/1 in calves free from diarrhoea. The mean γ-globulin concentration, adjusted to 7 days age was 5.9 g/1. The 20 diarrhoeic calves had lower levels of both total protein and γ-globulin, compared with calves without diarrhoea, but the difference was not significant. One litre more of colostrum at the first feed increased the level of total protein of the calves’ sera by 1.4 g/1 (p = 0.05). Calves born between May and September had a 2.0 g/1 higher (p<0.001) serum concentration of γ-globulin than calves born between October and April.     

Prevalence of enteric pathogens in the feces of healthy beef calves

Fecal specimens from 136 healthy beef calves (1 day to 12 weeks of age) were examined for the presence of infectious agents known to cause enteric disease in calves. The calves were selected from 22 herds in which all calves were free of clinically apparent enteric disease. Salmonella sp, enterotoxigenic Escherichia coli, Cryptosporidium, and coronavirus were not detected in any of the calves. Three calves were infected with rotavirus and 1 calf was infected with Yersinia enterocolitica. Campylobacter-like bacteria were isolated from 50 of 130 calves, with 36 of the calves positive for C jejuni. Seemingly, clinically normal calves may be infected more often with enterotoxigenic E coli, Cryptosporidium, coronavirus, or rotavirus in herds in which some calves have enteric disease than in herds free of major enteric disease. Campylobacter jejuni was well adapted to the bovine host and was of similar prevalence in diarrheal and nondiarrheal calves. The K99 positive, nonenterotoxigenic E coli was isolated from the feces of 16 healthy calves. Information in addition to the presence of K99 antigen is useful when diagnosing enterotoxic colibacillosis in calves.     

牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立

通过多重PCR扩增产肠毒素大肠杆菌（enterotoxigentic E.coli，ETEC）的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg^2＋、引物浓度以及退火温度等因素进行优化，在优化条件的基础上，确定多重PCR的特异性和灵敏性，以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测，结果2株为F41、K99和STa阳性，4株为F41、STa阳性，1株为K99STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明，该方法特异性强、敏感性高、简便、快速，适用于临床鉴定和检测牛ETEC菌株。     

Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea

Duplex real-time PCR assays were used as modules to cover partially automated detection of 12 genes encoding adhesins, enterotoxins and Shiga toxins in faecal E. coli isolates. For this a total of 194 E. coli isolates from pigs suffering from post-weaning diarrhoea (PWD), including 65 isolates with haemolytic activity, and 83 isolates from calves with diarrhoea were examined. Data obtained by PCR were compared with O-typing and with haemolytic activity as indirect virulence markers. E. coli O-types O139:K82, O141:K85, and O149:K91 accounted for 43.8% (n = 85) of all porcine strains and for 55.4% (n = 36) of the porcine strains, which exhibited haemolytic activity. These strains carried virulence genes by 65.9% (n = 56) and 80.6% (haemolytic E. coli, n = 29), respectively. The E. coli O-types O139:K82 and O141:K85 were significantly associated with the adhesin gene F18, and O149:K81 with the F4 gene. In this context, detection of the gene encoding F18 was coupled predominantly with the genes responsible for the production of the toxins ST-I, ST-II and Stx2, and the F4 gene with those of the enterotoxins ST-I, ST-II and LT. Both virulence patterns were detected more pronounced in E. coli strains with haemolytic activity. Fifty-six of a total of 83 E. coli isolates originating from calves were O-typed as O101 (O101:K28, O101:K30, O101:K32; n = 29), O78:K80 (n = 23), and O9:K35 (n = 4). Most of the E. coli O78:K80 strains carried the F17 gene (69.6%, n = 16). Virulence genes encoding for F4, F5 or ST-I were detected only in single cases. Intimin and Shiga toxin genes that are present in enterohaemorrhagic E. coli (EHEC) were not detected.