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Recent anthropic activity related to the construction of the Bosquesur Green Park in a large urban setting in Madrid (Spain) has resulted in the largest reported community outbreak of human leishmaniosis in Europe. Previous phylogenetic and molecular-typing studies of parasite isolates have implicated the Leishmania infantum ITS-Lombardi genotype in this outbreak. In an unusual scenario, visceral leishmaniosis (VL) is affecting a significant number of individuals, suggesting that an increase in parasite virulence has occurred. In this work, using an in vivo BALB/c model of VL, we aimed to investigate the properties of emergent virulence of the L. infantum POL2FL7 and BOS1FL1 isolates obtained from Phlebotomus perniciosus collected in the outbreak area and compare them with those of the well-characterized strain BCN150 MON-1 isolated from a dog. The P. perniciosus specimens were collected during an entomological survey conducted in the transmission season of 2012. We observed a range of virulence phenotypes from moderately to highly aggressive after 5 weeks of infection. IV challenge of mice with outbreak isolates from sand flies induced higher splenic and liver parasite burdens, higher serological titres of specific anti-Leishmania antibodies and impaired capacities to control infection, as revealed by the arginine metabolism and low ratios of Th1/Th2 cytokine profiles analysed, compared with the corresponding measures evaluated in mice infected with the BCN150 strain. The BOS1FL1 isolate showed the highest degree of virulence among the isolates, superior to that of POL2FL7, as evidenced by the analysed biomarkers and the histopathological severity of liver lesions. These results provide insight into how L. infantum isolates from sand flies collected in the outbreak area have been able to affect not only immunosuppressed patients but also middle-aged people with normal immunocompetence in the largest human VL outbreak in Europe.

HisAK70 candidates have successfully been tested in cutaneous (CL) and visceral leishmaniosis (VL) mouse models. Here, we analyse different biomarkers in dog trials after a heterologous immunization strategy with a HisAK70 candidate (plasmid DNA plus adoptive transfer of peripheral blood-derived dendritic cells (DCs) pulsed with the same pathoantigen and CpG ODN as an adjuvant) to explore the antileishmanial activity in an ex vivo canine co-culture system in the presence of Leishmania infantum parasites. In the canine model, the heterologous HisAK70 vaccine could decrease the infection index in the DC-T cell co-culture system by up to 54% after 30 days and reach almost 67% after 100 days post-immunization, respectively, compared to those obtained in the control group of dogs. The observed security and potential to fight ex vivo L. infantum infection highlight a HisAK70 heterologous immunization strategy as a promising alternative to evaluate its effectiveness against canine VL.

Here, we describe a novel approach that exploits an attenuated mutant of Salmonella enterica serovar Choleraesuis as carrier to deliver a plasmid encoding protein HisAK70. Subsequently, dendritic cells (DCs) were pulsed with this vaccine vector. The aim of this study was to evaluate the effectiveness of the prepared HisAK70-S. Choleraesuis-pulsed DCs (HisAK70-SAL DCs) against visceral leishmaniosis (VL). In our ex vivo model of infection, the prepared formulations could decrease parasite growth by up to 80% by augmenting the production of IL-12p40 and by reducing arginase activity (ARG). Also, BALB/c mice when immunised with this formulation showed significant reduction in parasite burden in both spleen (20% of reduction) and liver (75% of reduction). The balance of the immune ratios IFN-γ/IL-10, TNF-α/IL-10, and IgG2a/IgG1 reflected the acquisition of an improved resistant phenotype in HisAK70-SAL DCs vaccinated mice compared to control mice. Our results suggest that HisAK70-SAL DCs could be a promising alternative approach for vaccine delivery that has the potential to fight Leishmania infantum (L. infantum) infection.

One unidentified, Gram-stain-positive, catalase-negative coccus-shaped organism was recovered from a subcutaneous abscess of the udder of a sheep and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolate was tentatively assigned to the genus Streptococcus , although the organism did not appear to match any recognized species. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus and showed that the nearest phylogenetic relatives of the unknown coccus corresponded to Streptococcus moroccensis and Streptococcus cameli (95.9 % 16S rRNA gene sequence similarity). The sodA sequence analysis showed less than 89.3 % sequence similarity with the currently recognized species of the genus Streptococcus . The novel bacterial isolate was distinguished from close relatives of the genus Streptococcus by using biochemical tests. A mass spectrometry profile was also obtained for the novel isolate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a representative of a novel species of the genus Streptococcus , Streptococcus ovuberis sp. nov. The type strain of Streptococcus ovuberissp. nov. is VB15-00779T (=CECT 9179T=CCUG 69612T).

Escherichia coli is a natural colonizer of the urogenital mucosa of healthy females; however it is one of the pathogens associated to reproductive failures in cows and sows. A better knowledge about the characteristics of native E. coli will allow us to differentiate them from pathogenic strains. Ninety autochthonous isolates from the reproductive tract of sows and cows were characterized to determine the phylogenetic profile, antibiotic resistance and virulence factors; also, comparisons between different breeding systems were performed. Vaginal colonization of E. coli was statistically higher in cows (57.5%) than sows (23.8%), and most isolates belonged to the phylogenetic group A: 79.69 and 80.77%, respectively; moreover phylo-groups B1 (12.5 and 11.54%) and D (7.81 and 7.69%) were significantly lower; however, none was classified as B2. Positive associations between virulence factors and group D were found. Isolates with antimicrobial susceptibility were associated with group A and the MDR (Multiple Drug Resistance) was related to the porcine source. These results contribute to the knowledge of extra-intestinal E. coli populations; which could affect the reproductive performance of females. Fil: Torres Luque, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Gonzalez Moreno, Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina Fil: Pasteris, Sergio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Orden, José A.. Universidad Complutense de Madrid; España Fil: Leon de la Fuente, Ricardo Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Complutense de Madrid; España Fil: Otero, María Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Staphylococcus aureus subsp. anaerobius, a microaerophilic, catalase-negative bacteria, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, characterized by the formation of necrotic lesions that are typically located in superficial lymph nodes. In this study, molecular analysis including pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and accessory gene regulator (agr) typing was carried out on 94 S. aureus subsp. anaerobius strains isolated in different countries (79 were isolated from 35 outbreaks of the disease in Spain from 1981 to 2009, 9 were isolated in Italy, 3 in Denmark and 3 in Sudan). All of the 94 S. aureus subsp. anaerobius isolates examined belonged to one PFGE type, within which four minority subtypes were identified. Representative isolates of all PFGE subtypes as well of all countries belonged to the same sequence type (ST), ST1464, which was a singleton, and to the agr type II. Our results support the view that abscess disease is caused by a single bacterial clone worldwide. This bacterium has existed for at least a century and, thus, has undergone long-term small ruminant host restriction.

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.

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially ΔphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and ΔphoP also in pigs. In addition, ΔrpoS and ΔphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the ΔrpoS and ΔphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs. © 2008 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

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 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

We investigated the use of two previously described attenuated strains of Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis), DeltaphoP and DeltarpoS, compared with the commercial attenuated SC-54 strain, as bactofection vehicles, to deliver an epitope model (3xFLAG) to the intestinal immune system. The gene encoding the epitope 3xFLAG was subcloned into the pCMVbetam2A mammalian expression vector (creating pCMV3xFLAGm2A) and introduced into S. Choleraesuis strains. The 3xFLAG epitope was expressed efficiently in murine macrophage J774A.1 cell cultures infected with Salmonella DeltaphoP and DeltarpoS vehicles but not with SC-54, as shown by gene-specific quantitative real-time reverse-transcriptase PCR. The stability of pCMV3xFLAGm2A in each strain was determined in vitro in the absence of antibiotic selection, and in vivo following oral immunisation of BALB/c mice. Administration of the DNA vaccine to mice led to the production of 3xFLAG-specific serum IgG and intestinal IgA antibody responses in DeltarpoS and SC-54, and spleen cell secretion of IFN-gamma following specific 3xFLAG stimulation in DeltaphoP. All together, these results indicate that DeltaphoP, DeltarpoS and SC-54 that expressed 3xFLAG from pCMV3xFLAGm2A elicited a different biased immune response, in which the T-helper-1-like cellular immune response was predominant in DeltaphoP, whilst IgA-related mucosal immunity predominated in DeltarpoS and SC-54. We conclude that DeltaphoP and DeltarpoS of S. Choleraesuis are new promising candidates as vaccine bactofection vectors.

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.

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.

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.

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.

Las fluoroquinolonas son una de las clases de agentes antimicrobianos más útiles empleadas hoy en día en medicina humana y animal debido a su espectro y a sus propiedades fisicoquímicas. El uso de quinolonas en animales es un asunto de especial preocupación porque podría contribuir a la adquisición de resistencia en bacterias transmitidas por alimentos (tales como Salomonella spp., Campylobacter spp. y Escherichia coli) y esto, a su vez, podría conducir a una reducción en la eficacia de tales componentes en el tratamiento de infecciones en los seres humanos. Sin embargo, la relación causal entre el uso de fluoroquinolonas en medicina veterinaria y el aislamiento de bacterias resistentes a fluoroquinolonas en humanos no ha sido generalmente probada y, además, el uso de fluoroquinolonas en animales es sólo uno de los muchos factores implicados en la resistencia a estos antimicrobianos. A pesar de ello, la vigilancia de la resistencia a fluoroquinolonas en bacterias aisladas de animales y alimentos y el uso adecuado de estos antimicrobianos en animales deben tener la máxima prioridad.

From 160 staphylococci isolated from ovine mastitis, 125 were identified as coagulase-positive staphylococci (CPS) and 35 as coagulase-negative staphylococci (CNS). Of these, 108 (87.8%) S. aureus produced at least one of the staphylococcal enterotoxins (SE) described. However, no CNS was found to be enterotoxigenic. Enterotoxin C (SEC) was the type most frequently produced. TSST-1 was shown to be produced by 91 (74.0%) of S. aureus, almost invariably in combination with SEC. Three CNS strains were also found to produce TSST-1 (two strains of S. xylosus and one strain of S. epidermidis).

Summary Three monoclonal antibodies (MAbs) which react specifically with staphylococcal enterotoxin B (SEB) were studied for their suitability for use in ELISA. One of them (MAb B14) worked well as a coating antibody; MAb B12 was shown to be a good probing antibody (conjugated with peroxidase) when ELISA plates were coated with MAb B14. This effective pair of MAbs (B14–B12PO) is able to detect 0.625 ng SEB ml, and to distinguish between SEB and other proteins present in food extracts.

Resume La capacite de produire des enterotoxines et du TSST-1 est etudiee chez 53 souches sauvages de Staphylococcus aureus subsp. anaerobius et chez 48 mutants aerotolerants obtenus par incubation des souches sauvages en aerobiose. La proteine A est produite par 4 souches sauvages et 3 souches aerotolerantes. Le TSST-1 est produit par 2 souches sauvages aux concentrations de 7,0 et de 6,8 ng/ml, concentrations definies par ELISA. Aucune enterotoxine (A-E) n'est detectee, quel que soit l'extrait.

The ratio of the different types of S. aureus surviving cells immediately after sub-lethal heat treatment (52 degrees C for 15 min) was studied. A high ratio of cells which are unable to form colonies even in a common medium such as TSA was observed. This fact has not been reported previously for common media through recovery after heat shock has been widely studied. After the treatment, staphylococcal protein A, but no enterotoxin, was seen to be released into the extracellular environment.

Summary The production of staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 by 40 coagulase negative staphylococci isolated from sheep, goat and cow mastitis was studied. Both ELISA double sandwich and Western blot were used to detect the production of these toxins. Only two strains of S.xylosus were enterotoxigenic, producing SEC. TSST-1 was seen to be produced by 5 strains of S. xylosus, 1 S. sciuri and 2 S. epidermidis. Results obtained by ELISA and by Western blot agreed in all cases except in one strain of S. epidermidis which was only positive using ELISA.