Antigenic and molecular characterisation of Border disease virus associated with high mortality in lambs in Spain.

INTRODUCTION: Border disease virus (BDV) causes congenital disorders in sheep and results in severe, but underestimated, economic losses worldwide. However, information about BDV strains affecting several ruminants worldwide is scarce. Therefore, antigenic and genetic classification of isolates from different geographical regions is important to enhance the knowledge of the epidemiology of BDV. MATERIALS AND METHODS: Five pestiviruses isolated from lambs in an epidemic outbreak with an unusually high mortality in Spain in 1997 were characterised antigenically with a panel of monoclonal antibodies and genetically by sequencing within the 50 untranslated (50UTR) region of the genome. RESULTS: All the isolates were classified as BDV and showed a high homology with the Aveyron strain (Av), which was associated with an epidemic reported in sheep from the Aveyron region of France in 1984. CONCLUSIONS: Classification of the isolates from this study provides valuable information on the molecular epidemiology of BDV.

A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds.

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.

Occurrence and preliminary study of antimicrobial resistance of enterococci isolated from dairy goats in Spain.

The present study performed in Spain was designed to investigate the occurrence and antimicrobial resistance of enterococci in faecal and bulk tank milk samples from 222 healthy Murciano-Granadina dairy goats reared on 12 farms. Enterococci were isolated in 54.5% and 63.6% of the faecal and bulk tank milk samples, respectively. Enterococci were detected more frequently from goat kids (70%) than from replacement animals (44.4%) and adults (56.4%). Seven species were found in the faecal samples but the most common species detected were Enterococcus faecium (32.3%), Enterococcus faecalis (27.6%) and Enterococcus hiriae (22%). In contrast, only E. faecium and E. faecalis were found in the bulk tank milk samples. According to NCCLS (2002), of the 134 strains studied, 17 (12.7%) were resistant to at least 7 antimicrobials and 14 of these strains were resistant to vancomycin.

Detection of the astA (EAST1) gene in attaching and effacing Escherichia coli from ruminants.

A total of 206 attaching and effacing Escherichia coli strains from ruminants were analysed for the presence of the astA (EAST1) and bfpA genes. None of these strains was bfpA-positive. The percentage of enteropathogenic E. coli (EPEC) strains astA-positive found in healthy cattle (15.6%) suggests that this animal species may be a significant reservoir of atypical EPEC potentially pathogenic for humans.

Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coli isolated from healthy dairy goats in Spain.

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.

Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants.

The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR. Six types of the eae gene: beta (beta), gamma1 (gamma-1), gamma2 (gamma-2), epsilon (epsilon), zeta (zeta) and iota (iota), and three types of the espB gene: alpha, beta and gamma were identified in the strains studied. Moreover, three strains were negative to all the types of the eae gene tested. The types beta and gamma2 in healthy cattle, beta, gamma2 and epsilon in healthy sheep and goats, and beta in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied. Although the eaebeta gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7-100%) were higher than those found in healthy animals (33.3-40.6%). Thus, these data suggest that AEEC strains with the eaebeta gene are associated with neonatal diarrhoea in ruminants. The eaegamma1, eaezeta and eaeiota genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively). All the types of the eae gene, except the type iota, showed a close correlation with the types of the espB gene: the eaebeta and eae epsilon genes with the espBbeta gene, the eaegamma2 and eaezeta genes with the espBalpha gene and the eaegamma1 gene with the espBgamma gene.

Prevalence and characterization of Vero cytotoxin-producing Escherichia coli isolated from diarrhoeic and healthy sheep and goats.

Faecal samples from 146 diarrhoeic lambs and goat kids, and from 511 healthy sheep and goats were screened for the presence of Vero cytotoxin-producing Escherichia coli (VTEC). In healthy sheep and goats, VTEC were isolated in 24.4 and 16.2% of the animals, respectively. Moreover, VTEC were detected in 3.1 and 5.9% of the diarrhoeic lambs and goat kids, respectively. These data suggest that VTEC seems not to be associated with diarrhoea in lambs and goat kids. Only four VTEC strains were eae-positive. The absence of the eae gene in most of these VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, almost half (42.9%) and 12.2% of VTEC strains isolated from healthy sheep and goats, respectively, belonged to serotypes associated with severe diseases in humans.

Verotoxin-producing Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) isolated from healthy cattle in Spain.

AIMS: To determine the prevalence and characteristics of verotoxigenic Escherichia coli (VTEC), enteropathogenic E. coli (EPEC) and necrotoxigenic E. coli (NTEC) in healthy cattle. METHODS AND RESULTS: Faecal samples from 412 healthy cattle were screened for the presence of VTEC, EPEC and NTEC. Four isolates from each sample were studied. VTEC, EPEC and NTEC were isolated in 8.7%, 8.2% and 9.9% of the animals, respectively. VTEC and NTEC were isolated more frequently from calves and heifers than from adults. Seventy (4.2%), 69 (4.2%) and 74 (4.5%) of the 1648 E. coli isolates were VTEC, EPEC and NTEC, respectively. Seventeen (24.3%) of the VTEC strains were eae-positive. Thirty-six (51.4%) of VTEC strains belonged to E. coli serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. The serogroups most prevalent among the EPEC strains were O10, O26, O71, O145 and O156. CONCLUSIONS: Healthy cattle are a reservoir of VTEC, EPEC and NTEC. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most of the VTEC strains were eae-negative, a high percentage of VTEC strains belonged to serogroups associated with severe disease in humans.

Quinolone resistance in potentially pathogenic and non-pathogenic Escherichia coli strains isolated from healthy ruminants.

Quinolone resistance was studied in potentially pathogenic and non-pathogenic Escherichia coli strains from healthy ruminants. In cattle, 5.9% of the strains were resistant to nalidixic acid and 4.9% were resistant to enrofloxacin and ciprofloxacin, whereas in sheep and goats only 0.5% and 1.4%, respectively, of the strains were resistant to nalidixic acid and none to fluoroquinolones. Most of the strains resistant to quinolones were non-pathogenic strains isolated from cattle. However, the results of this study do not show that the potentially pathogenic E. coli strains isolated from healthy ruminants are more susceptible to quinolones than the non-pathogenic E. coli strains.

Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids.

Attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic lambs and goat kids were characterized for intimin (eae) and EspB (espB) gene subtypes by PCR and sequencing, and for genetic relatedness by PFGE. Fifty (23 ovine and 27 caprine) AEEC strains of 398 (246 ovine and 152 caprine) analysed were detected by colony blot hybridization. These strains were epidemiologically unrelated since they were isolated from different outbreaks of neonatal diarrhoea over a long period. Ovine AEEC strains belonged to serogroups O2, O4, O26, O80, O91 or were untypable, and caprine strains belonged to serogroups O3, O153 and O163. Two intimin subtypes were detected among the ovine and caprine strains studied. Most of the strains (43/50) had the beta type intimin gene, but seven ovine strains possessed a variant gamma type intimin gene (gamma(V)). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of beta intimin of ovine and caprine strains were virtually identical to those of beta intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the gamma(V) intimin present in seven ovine strains had 75-76% identity with gamma intimin of human EHEC clone 1 strains, and 96% of identity with intimin of the human EHEC strain 95NR1 of serotype O111:H-. A PCR test was developed to identify the three different espB gene subtypes, espB of human EPEC clone 1 (espBalpha), espB of human EHEC clone 1 (espBgamma) and espB of rabbit EPEC and human EPEC clone 2 (espBbeta). There was close correlation between the intimin beta type and the espBbeta gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the gamma(V) intimin gene possessed the espBalpha gene subtype. None of the strains studied possessed the espBgamma gene found in human O157:H7 EHEC strains. PFGE analysis of genomic DNA of selected strains showed a great diversity among strains. Cluster analysis of PFGE patterns showed greater divergence between strains with the gamma(V) intimin gene than between strains with the beta intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed beta intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

In vitro susceptibility of Escherichia coli strains isolated from diarrhoeic dairy calves to 15 antimicrobial agents.

The in vitro activities of 15 antimicrobial agents against 195 strains of Escherichia coli isolated from dairy calves affected by neonatal diarrhoea were determined. Of these strains 137 produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin and the eae gene), but the remaining 58 strains did not produce any of these factors. The overall percentage of resistant strains to streptomycin, tylosin and tetracycline was very high (above 65%). A high level of resistance (from 23 to 50%) to ampicillin, neomycin, kanamycin, spectinomycin, chloramphenicol, sulphadimethoxine and trimethoprim was also detected. The E. coli strains were very susceptible (89-95%) to apramycin and gentamicin and highly susceptible (99-100%) to polymyxin B, florfenicol and nitrofurazone. Some significant differences (P < 0.05) in the frequencies of resistance to some of the antimicrobials tested and in the rates of multi-drug resistance among the strains producing potential virulence factors and non-fimbriated, non-toxigenic, eae-negative strains were found. Most of the strains showed multi-resistance: 76.9% of the isolates were resistant to at least two antibiotics, 67.7% were resistant to at least four antibiotics and 50.3% were resistant to at least six antibiotics.

Rotavirus and concurrent infections with other enteropathogens in neonatal diarrheic dairy calves in Spain.

Faeces samples from 218, one to 30 days old, diarrheic dairy calves in 65 dairy herds were screened for the presence of rotavirus and concurrent infections with coronavirus, Cryptosporidium, F5+ Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Rotavirus infection was detected in 46.9%, 45.6%, 33.8% and 48.3% of the calves in the respective age-groups. No significant differences in the detection rate of rotavirus were found among calves on the different age-groups. Rotavirus was the only enteropathogen detected in 39 of the 93 (41.9%) diarrheic calves positive to this agent. Concurrent infections with other enteropathogen(s) were detected in 31.3%, 33.3%, 20.6% and 3.4% of the rotavirus infected calves in the age-groups 1-7, 8-14, 15-21 and 22-30 d, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.01) was found. The detection rates of the other enteropathogens considered in calves with rotavirus infection were 20.4% for coronavirus, 85.2% for Cryptosporidium, 16.7% for F5+ E. coli and 1.8% for Salmonella.

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies.

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves.

Fecal samples from 246, 1-90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1-7 days, 8-14 days, 15-21 days, 22-30 days and 31-90 days) and analyses of prevalence were done by the Mantel-Haenzsel chi2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 (p<0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).

Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain.

Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5 Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22-30 days and all other age groups, and between the age group 1-7 days and the age groups 8-14 days and 15-21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1-7, 8-14, 15-21 and 22-30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.

In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves.

The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from diary calves affected by neonatal diarrhea were determined. One hundred thirty-seven of these strains produced one or more potential virulence factors (F5, F41, F17, cytotoxic necrotizing factor, verotoxin, and the eae gene), but the remaining 58 strains did not produce any of these factors. From 11 to 18% of the E. coli strains were resistant to cephalothin, nalidixic acid, enoxacin, and enrofloxacin. However, cefuroxime, cefotaxime, and cefquinome were highly effective against the E. coli isolates tested. Some significant differences (P < 0.05) in resistance to quinolones between the strains producing potential virulence factors and nonfimbriated, nontoxigenic, eae-negative strains were found. Thus, eae-positive, necrotoxigenic, and verotoxigenic (except for nalidixic acid) E. coli strains were significantly more sensitive to nalidixic acid, enoxacin, and enrofloxacin than nonfimbriated, nontoxigenic, eae-negative strains. Moreover, eae-positive strains were significantly more sensitive to enoxacin and enrofloxacin than F5-positive strains. Thus, the result of this study suggest that the bovine E. coli strains that produce some potential virulence factors are more sensitive to quinolones than those that do not express these factors.

Verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC in 1-30-days-old diarrhoeic dairy calves.

Faecal samples from 221, 1-30-days-old, diarrhoeic dairy calves were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and eae-positive non-VTEC. Calves were grouped according to their age (1-7, 8-14, 15-21 and 22-30 days) and analyses of prevalences were done by Mantel-Haenzsel chi 2-test for trend. VTEC and eae-positive non-VTEC were detected in 20 (9.0%) and 18 (8.1%) of the diarrhoeic calves, respectively. A significant age-associated increase in the prevalence of VTEC (p = 0.0001), but not in the prevalence of eae-positive non-VTEC (p = 0.381), was found. Significant differences in VTEC prevalence were found between the age-group 22-30 days and in all other age-groups. 43 (5.0%) of the 861 E. coli isolates from the 221 diarrhoeic calves were VTEC, and 30 (69.8%) of these strains produced VT1 only. More than one-half of the VTEC strains (55.8%) were positive for the eae gene and all these eae-positive VTEC strains produced VT1 only. A high percentage (76.7%) of VTEC strains belonged to E. coli serogroups (O4, O26, O39, O91, O113, O128 and O145) associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. 51 (5.9%) of the E. coli strains studied were eae-positive non-VTEC and the serogroups most prevalent among these strains were O4, O14, O26 and O123. Only four of the eae-positive strains were also espB-positive by hybridization with a probe from a human EPEC isolate and none of these strains produced VT.

Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain.

Faecal samples from 218 diarrheic dairy calves in 65 dairy herds, selected by convenience, were screened for the presence of rotavirus, coronavirus, Cryptosporidium spp., F5+ Escherichia coli and Salmonella spp. Animals surveyed were from 1 to 30 days old. Cryptosporidium and rotavirus were the most commonly detected agents (52.3% and 42.7% of the samples positive, respectively). F5+ E. coli was detected in the faeces of 11.9% of the calves and bovine coronavirus was detected in the faeces of 7.3% of the calves. Salmonella spp. was only found in the faeces of two calves (0.9%). Mixed infections with two or more agents occurred in 28% of the calves. Concurrent infection of rotavirus and Cryptosporidium was found in 21.6% of the calves. Two tests were used for the detection of rotavirus (a commercial ELISA and PAGE), F5+ E. coli (ELISA and bacterial culture) and Cryptosporidium (ELISA and microscopy). The validity of the commercial ELISA for the detection of rotavirus, F5+ E. coli and Cryptosporidium in faeces from diarrheic calves was evaluated using PAGE, bacterial culture and microscopy as gold standard, respectively. The ELISA showed a very low sensitivity (28.6%) for the detection of F5+ E. coli compared to bacterial culture.