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Bovine trichomonosis (BT) is a sexually transmitted disease that is considered a cause of early reproductive failure in cattle under extensive management conditions. Recently, Tritrichomonas foetus was detected in 41.5% of herds from one representative beef cattle breed (Asturiana de la Montaña; AM) reared in traditional mountain systems in Spain. The objective of the present study was to evaluate the effect of BT on reproductive performance and the economic consequences in AM herds. The benefits of a control program based on testing and culling infected bulls were also studied by comparing T. foetus prevalence and reproductive data before and after the implementation of the control measures. In infected herds, T. foetus infection increased calving intervals by 79 days (P0.0001) and resulted in a higher percentage of cows-not-in calf (36% vs. 19%; P0.001). An economic analysis showed that BT could reduce income by 68.7% in AM herds. The implementation of the control program decreased calving intervals (P0.0001) and increased calving percentage (P0.05). T. foetus prevalence showed a significant decline compared with the prevalence before implementing the control program (P0.05). Nevertheless, after 2 years, the herd prevalence did not decrease (12.7-13.6%; P0.05) and the herd incidence was 22.72%. The testing and culling policy was effective in improving reproductive efficiency but the complete elimination of BT without substantial changes in management appears unlikely because putative risk factors associated with the disease are present in the management of this breed.

Las Escherichia coli enteroagregativas (ECEA) causan tanto diarrea aguda como persistente en niños y adultos en países desarrollados y en vías de desarrollo. Además, el gran brote de transmisión alimentaria que sucedió en el verano de 2011 fue causado por una cepa de ECEA O104:H4 productora de verotoxinas. La amenaza a la Salud Pública debida a las ECEA hace importante identificar los reservorios animales a partir de los cuales se podrían originar epidemias o brotes en humanos. Basados en un único estudio en ovejas y unos pocos en vacas, algunos investigadores han sugerido que los ECEA no se aíslan de rumiantes. Para ampliar los datos disponibles acerca de esta cuestión, en este estudio se usó la reacción en cadena de la polimerasa para detectar ECEA en 920 muestras fecales de vacas, ovejas y cabras. Ninguna de las muestras resultó positiva a los genes de ECEA analizados, sugiriendo que las cabras, al igual que las vacas y ovejas, no son probablemente un reservorio importante de ECEA., Enteroaggregative Escherichia coli (EAEC) causes both acute and persistent diarrhoea among children and adults in developing and developed countries. In addition, the large outbreak of food-borne illness in Europe in the summer of 2011 was caused by a verotoxin-producing EAEC O104:H4 strain. The public health threat posed by EAEC makes it important to identify animal reservoirs that might lead to epidemics in humans. Based on only one study in sheep and a few in cattle, researchers have suggested that EAEC are not found in ruminants. To expand the available data on this question, the present study applied polymerase chain reaction to faecal samples from 920 cattle, sheep and goats to detect EAEC. None of the samples tested positive for any of the EAEC genes tested, suggesting that goats, like cattle and sheep, are unlikely to be a significant reservoir of EAEC.

Bovine trichomonosis (BT) and bovine genital campylobacteriosis (BGC) are sexually transmitted diseases that can be important infectious causes of reproductive failure in extensively managed beef cattle where natural mating is a common practice. However, their prevalence in Europe was thought to be insignificant or very low. The purpose of this study was to investigate the prevalence and risk factors associated with BT and BCG in a representative beef cattle breed, Asturiana de la Montana (AM), which is usually managed extensively in the mountain areas of Northern Spain and putative risk factors associated with the two diseases are present on most farms holding AM cattle. Preputial smegma samples were collected from 103 bulls belonging to 65 herds. Pathogen detection was undertaken using culture and PCR. Two scraping methods for sample collection (AI pipette and plastic scraper), as well as different culture media and DNA extraction methods were evaluated on field samples. Campylobacter fetus veneralis infection was not detected in any animal in any herd. However, Tritrichomonas foetus infection was demonstrated in 32% (33/103) and 41.5% (27/65) of bulls and herds tested, respectively. AM bulls older than 3 years (39.7%) were more likely to be infected than young bulls (16%) (OR = 3.45, CI = 1.07–11.19). An increase in repeat breeder cows was reported in herds from which T. foetus was detected (OR = 5.2, CI = 1.5–17.18). These findings highlight the re-emergence of this disease in extensively managed beef cattle in Spain. For routine diagnosis, the use of a culture technique and PCR in combination is advisable for testing smegma samples under field conditions.

Subtilase cytotoxin (SubAB) from verotoxin (VT)-producing Escherichia coli (VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negative E. coli strains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes ( subA and subB ). In this study, we investigated the presence of subAB genes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessed subAB genes. The presence of subAB in a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants of subAB . We therefore sequenced the subA gene in 12 strains and showed that the subA gene in most of the subAB -positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while the subA gene in most of the subAB -positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the terms subAB1 to describe the SubAB-coding genes resembling that in the 98NK2 strain and subAB2 to describe those resembling that in the ED 591 strain.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 μg/ml) but not to ciprofloxacin (MIC = 1 μg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83→Leu and Asp-87→Gly) and a double amino acid substitution in the ParC protein (Gly-78→Cys and Ser-80→Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.

Background: Leishmania major and Leishmania infantum are among the main species that are responsible for cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL), respectively. The leishmanioses represent the second-largest parasitic killer in the world after malaria. Recently, we succeeded in generating a plasmid DNA (pCMV-HISA70m2A) and demonstrated that immunized mice were protected against L. major challenge. The efficacy of the DNA-vaccine was further enhanced by the inclusion of KMP-11 antigen into the antibiotic-free plasmid pVAX1-asd. Methods: Here, we describe the use of a HisAK70 DNA-vaccine encoding seven Leishmania genes (H2A, H2B, H3, H4, A2, KMP11 and HSP70) for vaccination of mice to assess the induction of a resistant phenotype against VL and CL. Results: HisAK70 was successful in vaccinated mice, resulting in a high amount of efficient sterile hepatic granulomas associated with a hepatic parasite burden fully resolved in the VL model; and resulting in 100 % inhibition of parasite visceralization in the CL model. Conclusions: The results suggest that immunization with the HisAK70 DNA-vaccine may provide a rapid, suitable, and efficient vaccination strategy to confer cross-protective immunity against VL and CL., This work was partially supported by grants from the Spanish Ministry of Economy and Competitiveness (AGL2010-17394 and AGL2013-44100R) and PLATESA (P2013/ABI-2906) from the Comunidad de Madrid (Spain).

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.

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.

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 β type intimin gene, but seven ovine strains possessed a variant γ type intimin gene (γV). Analysis of deduced amino acid sequences of the eae gene revealed that the sequences of β intimin of ovine and caprine strains were virtually identical to those of β intimin of rabbit EPEC, human EPEC clone 2 and swine AEEC, whereas the γV intimin present in seven ovine strains had 75–76% identity with γ 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 (espBα), espB of human EHEC clone 1 (espBγ) and espB of rabbit EPEC and human EPEC clone 2 (espBβ). There was close correlation between the intimin β type and the espBβ gene subtype in the ovine and caprine AEEC strains. The seven ovine strains possessing the γV intimin gene possessed the espBα gene subtype. None of the strains studied possessed the espBγ 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 γV intimin gene than between strains with the β intimin gene. This study showed that most of the AEEC strains isolated from diarrhoeic lambs and goat kids possessed β intimin and espB genes identical to those of rabbit EPEC, and they may be associated with enteric disease in small ruminants.

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 (p0.01) 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.

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.

Degenerate oligonucleotide primers based on internal peptide sequences obtained by HPLC from purified Staphylococcus aureus catalase were used to locate the S. aureus and S. aureus subsp. anaerobius kat regions by PCR. Southern hybridization analysis with a probe derived from a 1.1 kb PCR-amplified fragment showed that a single copy of the putative catalase gene was present in the S. aureus and S. aureus subsp. anaerobius chromosome. The nucleotide sequence of S. aureus katA revealed a 1518 bp open reading frame for a protein with 505 amino acids and a predicted molecular mass of 58347 Da, whereas S. aureus subsp. anaerobius katB is 1368 nt long and encodes a polypeptide of 455 amino acids with a predicted molecular mass of 52 584 Da. These catalases are highly homologous to typical monofunctional catalases from prokaryotes. The active-site residues, proximal and distal haem-binding ligands and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in S. aureus KatA. Escherichia coli cells carrying cloned katA had a catalase activity approximately 1000 times that of untransformed E. coli, but no detectable increase in catalase activity was observed with E. coli carrying cloned katB. Northern blotting showed the presence of a kat-specific transcript in S. aureus subsp. anaerobius, suggesting that the lack of catalase activity in this bacterium is due to a post-transcriptional alteration. Compared to the nucleotide sequence of katA, katB showed a single base-pair deletion and six mis-sense mutations, and these alterations were present in three other S. aureus subsp. anaerobius strains analysed. The deletion, located at 1338 bp from the initiation codon, originates a shift of the nucleotide reading frame and is responsible for the premature translation termination at 1368 bp, generating a KatB polypeptide 50 amino acid residues shorter than KatA. Moreover, four of the mis-sense mutations present in katB lead to non-conservative amino acid replacements, the most significant being that located at residue 317 (Pro in KatA-->Ser in KatB) because the affected amino acid is involved in determining the proximal haem-binding site. Both the main alterations found in KatB (the deletion and the substitution in residue 317) seem to contribute to the lack of catalase activity in S. aureus subsp. anaerobius, as deduced from results obtained with chimeric catalase constructs.

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.

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.

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.

The objective of this study was to investigate the presence of Toxoplasma gondii- and Neospora caninum-specific antibodies in domestic South American camelids (SAC) (llamas and alpacas) from the Peruvian Andes through a cross-sectional study. A wide panel of serum samples collected from 1,845 llamas and 2,874 alpacas from the two main SAC production areas of Peru was selected. Immunofluorescence antibody technique was employed to detect and titrate specific anti-T. gondii and anti-N. caninum immunoglobulins G in serum samples. The association between T. gondii and N. caninum seroprevalence and the geographical origin (Central and South Peruvian Andes) was evaluated. Anti-T. gondii antibodies were found in 460 (24.9 %) llamas and 706 (24.6 %) alpacas, whereas anti-N. caninum antibodies were detected in 153 (8.3 %) llamas and 425 (14.8 %) alpacas. Toxoplasma gondii infection was strongly associated with the South Peruvian Andes where moderate climate conditions, larger human population, compared to the Central region, and the presence of wildlife definitive hosts could favor horizontal transmission to SAC. In contrast, N. caninum infection was not associated with the geographical region. These results indicate that T. gondii and N. caninum infections are highly and moderately widespread, respectively, in both species of domestic SAC studied in the sampled areas and appropriate control measures should be undertaken to reduce the prevalence of both parasitic infections.

This note reports an outbreak of abscess disease caused by Staphylococcus aureus subsp. anaerobius occurring in a 250 Assaf sheep flock located in Salamanca (north-central Spain). Thirty-nine of the 55 animals (71%) of the young female group (6–7 months old) selected for breeding were affected by the disease. No abscesses were detected in the adult ewes. Clinical cases were first detected 3 weeks after shearing. In consideration of known data on experimental infections of sheep with S. aureus subsp. anaerobius , it is highly probable that the animals in the present outbreak became infected about shearing time. Therefore, shearing was the risk factor most probably associated with the outbreak.

Background: In Escherichia coli the genes involved in the acquisition of tetracycline resistance are mainly tet(A) and tet(B). In addition, tet(M) is the most common tetracycline resistance determinant in enterococci and it is associated with conjugative transposons and plasmids. Although tet(M) has been identified in E. coli, to our knowledge, there are no previous reports studying the linkage of the tet(M) gene in E. coli to different mobile genetic elements. The aim of this study was to determine the occurrence of tet(A), tet(B), and tet(M) genes in doxycycline-resistant E. coli isolates from pigs, as well as the detection of mobile genetic elements linked to tet(M) in E. coli and its possible transfer from enterococci. Results: tet(A) was the most frequently detected gene (87.9%) in doxycycline-resistant isolates. tet(M) was found in 13.1% E. coli isolates. The tet(M) gene was detected in relation with conjugative transposons in 10 out of 36 enterococci isolates analyzed but not in any of E. coli isolates positive for tet(M). Southern blot showed that in E. coli and in most of the enterococci isolates the tet(M) gene was carried on a plasmid. According to the phylogenetic analysis, E. coli contained a new tet(M) allele grouping separately. Mating experiments revealed that tet(M) was carried on a mobile element successfully transferred between enterococci and between enterococci and E. coli. Conclusions: The detection of tet(M) in E. coli isolates from pigs was higher than expected. In our study, tet(M) detected in E. coli seems not to have been transferred from enterococci, although it can not be ruled out that the horizontal transfer of this gene occurred from other intestinal tract bacteria.

The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosis of small ruminant lentiviruses (SRLVs). Detection of the proviral genome by PCR, though sensitive, is difficult due to the heterogeneity of the SRLV genomes. One of the PCR techniques amplifies a fragment in the pol gene (pol-PCR) and the other PCR targets the LTR region of the proviral genome (LTR-PCR). Milk from 194 sheep and 163 goats from farms in the Central Spain was analyzed by both techniques and compared to results obtained by ELISA. When compared to the serologic assay, the agreement of both PCR techniques was very low (0.024 and 0.020 in sheep, and 0.124 and 0.114 in goats). In view of these results, it may be concluded that the efficacy of PCR for the diagnosis of SRLVs is low and a combination of PCR and ELISA should be used for diagnosis.

Bovine trichomonosis (BT) is a sexually transmitted disease of cattle caused by infection with Tritrichomonas foetus. In a recent study, T. foetus infection was detected in 41.5% of herds of an endangered beef breed, the Asturiana de la Montana (AM), which is farmed under extensive, mountain pastoral systems in northern Spain. The objective of this study was to determine the prevalence of this pathogen in the more production-centred Asturiana de los Valles (AV) beef breed farmed in the same region, and to identify potential associated management risk factors. Infection was detected in a significantly smaller number (5.2%) of AV herds, despite the fact that both populations share the same ecological niche. Communal grazing was not identified as significant risk factor and study results suggest the prevalence of BT is likely to vary considerably depending on how the cattle are managed.

Five hundred and eighty four strains of Escherichia coli isolated from 146 diarrhoeic lambs and goat kids in Spain were analysed for the presence of F5 and F41 fimbriae by slide agglutination. Only two strains positive for both F5 and F41 fimbriae from one lamb were isolated. These two strains F5 and F41 positive for slide agglutination were also heat-stable enterotoxin a, F5 and F41 positive by PCR. Thus, our results show that F5 and/or F41 enterotoxigenic E. coli are a very infrequent cause of small ruminant diarrhoea in Spain.

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50–70% of strains; and other genes such as tccP, espP, katP and toxB were detected in

The susceptibility of the ovine mammary gland to experimental infection with Staphylococcus aureus subsp anaerobius was studied in five lactating Churra ewes. The left mammary gland of each ewe was inoculated with bacteria while the right halves remained untreated and served as a control. A rapid cellular response was elicited in all inoculated glands. The reaction of inoculated mammary glands varied from that of clinical mastitis (ewes C and D) to subclinical mastitis (ewes B and E) or merely to a transient increase of somatic cell counts (ewe A). S aureus subsp anaerobius is less pathogenic for ewes by the intramammary route than S aureus since doses of about 108 bacteria caused clinical mastitis in only two of the inoculated animals.

A group of 17 strains of coagulase negative staphylococci isolated from slaughtered lambs, and which could not be identified with the conventional methods, exhibited high levels of DNA homology (92%) with the S. caseolyticus reference strain. The isolates described in this study provide a more extensive comprehension of S. caseolyticus. The original description of this species was based on only two strains isolated from milk. To our knowledge, S. caseolyticus had never been previously associated with animal microflora.

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.

A total of 163 verotoxin-producing Escherichia coli (VTEC) strains isolated from diarrheic and healthy cattle, sheep, and goats were analyzed for the presence of the saa gene by polymerase chain reaction. Seventeen (45.9%) and 5 (29.4%) of the VTEC isolated from healthy cattle and diarrheic calves, respectively, had the saa gene. None of the saa-positive strains carried the eae gene, but 20 of the 22 saa positive were ehxA positive. In contrast with cattle VTEC, none of the VTEC isolated from small ruminants were saa positive. These results show that the saa gene is commonly associated with bovine eae-negative VTEC strains but not with ovine or caprine VTEC strains.

The age of the sheep donating neutrophils had a marked influence in vitro on their phagocytic ability with respect to Staphylococcus aureus and S aureus subspecies anaerobius. Neutrophils from lambs five to eight weeks old phagocytosed these organisms significantly less efficiently (P

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.

To determine the prevalence and characteristics of attaching and effacing Escherichia coli (AEEC) in diarrheic and healthy small ruminants.502 lambs and kids with diarrhea and 511 healthy sheep and goats.Fecal samples from diarrheic and healthy sheep and goats were screened for the eae gene. In addition, E coli isolates with positive results for the eae gene (E coli eae+) were analyzed for the espB gene, production of verotoxins (VT), and serogroup.A significantly higher prevalence of healthy lambs and kids were infected with AEEC, compared with diarrheic lambs and kids and healthy adult sheep and goats. Some differences in the characteristics of E coli eae strains isolated from diarrheic and healthy animals were detected. Thus, the espB gene was detected more frequently among E coli eae+ strains isolated from healthy animals than in those isolated from diarrheic animals, and VT production was only detected in E coli eae+ strains isolated from healthy lambs and kids. The E coli eae+ isolates belonged to several O serogroups. However, 17 of 40 (42.5%) isolates from diarrheic lambs and only 4 of 168 (2.4%) isolates from healthy sheep belonged to serogroup 026.Our results suggest that E coli eae+ 026 strains may play a role in diarrheal disease in lambs, whereas E coli eae+ strains that also had VT production and eae+ strains that had positive results for the espB gene did not appear to be associated with diarrhea in small ruminants.

The in vitro activities of several cephalosporins and quinolones against 195 strains of Escherichia coli isolated from dairy 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 results 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.

The in vitro activities of 14 anti-microbial agents were determined against 92 strains of E. coli isolated from lambs (60 strains) and kids (32 strains) affected by neonatal diarrhoea. The overall percentage of resistant strains to streptomycin, sulphadimethoxine and tetracycline was very high (above 70%). A high level of resistance (from 30% to 50%) to ampicillin, kanamycin, neomycin and chloramphenicol was also detected. The E. coli strains were highly susceptible to cephalosporins, polymyxin and quinolones. Most of the strains showed multiresistance: 77.2% of isolates were resistant to at least two antibiotics, 55.4% were resistant to at least four antibiotics and 33.7% were resistant to at least six antibiotics. A total of 34 antibiotypes could be distinguished.

In this study the susceptibility of 91 methicillin-resistant and -susceptible Staphylococcus intermedius strains (MRSI and MSSI, respectively) against 15 antimicrobial agents was determined. The activity of the antimicrobial agents was studied at pH 7.2 and pH 8.5. Methicillin was more active at pH7.2 (28 strains methicillin-resistant) than at pH 8.5 (55 strains methicillin-resistant). Gentamicin showed excellent activity, with only 3 strains resistant at pH 8.5. However, gentamicin would have to be administered parenterally. Oxytetracy-cline cannot be recommended for treatment of canine staphylococcal dermatitis, due to the high percentage (over 25%) of strains that were found to be resistant. Clindamycin showed little activity in inhibiting growth of the strains studied, the percent resistance at pH 7.2 was 93.4%. Rifampin behaved differently at the two pH values. However, a close relationship was noted between methicillin-resistant and rifampin-resistant strains, particularly at the lower pH. Of the fluoroquinolones, ciprofloxacin or enrofloxacin would be a good useful alternative for the treatment of methicillin-resistant strains of S. intermedius. Lastly, very high resistance to sulphamethoxypyridazine was found, as was the case with trimethoprim and a combination of trimethoprim/ sulphamethoxypyridazine, against not only MRSI but also MSSI strains.

A study was made of the susceptibility of 91 strains of Staphylococcus intermedius, isolated from staphylococcal dermatitis of dogs, to 11 beta-lactam antimicrobial agents. The activity of these antimicrobial agents against all strains was tested at pH 7.2 and 8.5. Finally, a comparative study was made of the susceptibility of these micro-organisms, dividing them into six groups: beta-lactamase positive and negative, methicillin resistant and susceptible and oxacillin resistant and susceptible. Only three of the 11 antimicrobial agents studied exhibited satisfactory behaviour at the two pH levels studied: mezlocillin, cefazolin and cefalotine. There was a good correlation at pH 8.5 between methicillin-resistant strains and those resistant to penicillin G, ampicillin, amoxycillin, cefamandole and cefuroxime. This correlation was not observed, however, between oxacillin-resistant strains and strains resistant to the other antimicrobial agents studied.

The production of staphylococcal enterotoxins A (SEA) and B (SEB) was studied by inoculating six well-defined staphylococcal collection strains into cow's, goat's, or sheep's milk (individually or as a 50% mixture of cow's + goat's or cow's + sheep's), into brain heart infusion, and into a medium generally used to enhance the synthesis of enterotoxins (3+3 medium). Four of the strains used are considered to be SEB producers, another is considered an SEA producer, and the remaining strain is nonenterotoxigenic but produces large quantities of staphylococcal protein A. Staphylococcal protein A masked the results in most cases. Only one strain secreted exclusively SEB, while the other three SEB producers synthesized SEA in different amounts. We conclude that enterotoxin production depends on the natural substrate and may differ from the results obtained when the strain is grown on cellophane over agar to determine its toxigenicity.

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.

Two hundred and twenty-six attaching and effacing Escherichia coli (AEEC) strains (20 enterohemorrhagic E. coli and 206 atypical enteropathogenic E. coli) isolated from calves, lambs, and goat kids with diarrhea and from healthy cattle, sheep, and goats were tested for their resistance to 10 antimicrobial agents by the disc diffusion method. Resistant and intermediate strains were analyzed by polymerase chain reaction for the presence of the major resistance genes. The overall percentage of resistant strains to tetracycline, streptomycin, erythromycin, and sulfamethoxazole was very high (>65%). Moreover, a high level of resistance (approximately 30%) to ampicillin, chloramphenicol, trimethoprim, and trimethoprim-sulfamethoxazole was also detected. The AEEC strains were very susceptible (>90%) to gentamicin and colistin. Because AEEC from ruminants can cause diseases in human beings, the high frequency of antimicrobial resistance detected in the current study is a source of concern. For each antimicrobial agent, the predominant resistance genes in the resistant strains were ampicillin, blaTEM (97.1%); tetracycline, tetA (76.7%); gentamicin, aac(3)II (80%); streptomycin, strA/strB (76.7%) and aadA (71.7%); chloramphenicol, catI (85.1%); trimethoprim, dhfrI (76.3%); and sulfamethoxazole, sul1 (60%) and sul2 (63.3%). In the majority of cases, resistance to a given antimicrobial, except for streptomycin, was caused by a single gene. A negative association between tetA and tetB, between aac(3)II and aac(3)IV, and between dhfrI and dhfrV was observed. The present study gives baseline data on frequency and molecular basis of antimicrobial resistance in AEEC strains from ruminants.

The in vitro activities of four fluoroquinolone compounds were tested against 92 Escherichia coli strains of ovine and caprine origin under aerobic and anaerobic incubation conditions. The four fluoroquinolones proved to be highly effective against the E. coli isolates tested. When bacteria were cultured anaerobically, at least fourfold increases in the MICs of enoxacin for the strains occurred and no detectable changes in enrofloxacin, sparfloxacin, and ciprofloxacin MICs occurred.

Forty-five ovine and caprine nonenterotoxigenic Escherichia coli strains producing F17-related fimbriae were characterized with respect to the fimbrial structural subunit and adhesin subtypes produced. In addition, several characteristics related to the virulence of strains producing F17 fimbriae were studied. Most of the strains (73%) possessed the f17cA structural subunit gene, whereas the f17aA and f17dA genes were detected only on three (6%) and two (4%) strains, respectively. The f17bA gene was not detected. All but one of these strains possessed the f17G genes of the adhesin subfamily II. The only strain having the f17G gene of subfamily I possessed the structural subunit gene f17dA . Sequencing of the f17A and f17G genes of four selected strains confirmed the association of f17cA and f17dA structural subunit genes with the f17G genes of the adhesin subfamily II. These results indicated that adhesins of the subfamily II are prominent among ovine and caprine isolates and that they are indistinctly associated with the F17 structural subunit subtypes on these field strains. CS31A- and CNF2-related genes were not detected. Most of the strains adhered in vitro to ovine intestinal brush borders (36 of 45) and agglutinated the erythrocytes of different species in the presence of d -mannose (39 of 45). F17-positive strains produced colicin V (57%) and were resistant to the bactericidal effect of serum (91%) in significantly higher percentages than F17-negative strains (34% produced colicin V, and 66% were serum resistant). Thus, most of the studied ovine and caprine strains showed phenotypic characteristics of septicemic strains.

Homemade mayonnaise, in which pH had been adjusted to a range between 5.0 and 5.8 by the addition of vinegar, was inoculated with eight Staphylococcus aureus strains known to be enterotoxigenic. They were incubated for a maximum of 7 days at 22, 28, 37, and 44°C. Periodically, staphylococcal growth and pH were determined. Mayonnaise samples were examined on d 7 for the presence of enterotoxins A, B, C, and D. Staphylococcal growth was higher at 22°C (average log10 7.21 cfu/g), than at the other temperatures tested (log10 7.15, 6.77, and 5.93 cfu/g, respectively for 28, 37, and 44°C), suggesting a better growth in mayonnaise at low room temperature. Enterotoxin synthesis took place mainly at 28°C, as 33.3% of the total enterotoxins produced were detected at this temperature. However, some strains synthesized high amounts of enterotoxin even at 22°C.