Pilar Horcajo

Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Enterohemorrhagic Escherichia Coli and Atypical Enteropathogenic E. Coli Strains from Ruminants

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, bla TEM (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.

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.

Subtilase Cytotoxin-Coding Genes in Verotoxin-Producing Escherichia coli Strains from Sheep and Goats Differ from Those from Cattle

ABSTRACT 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.

Characterization of Fluoroquinolone Resistance in Escherichia Coli Strains from Ruminants

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.

Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation

Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 106 Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes as vehicles for DNA vaccines.

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.

Mitigating an undesirable immune response of inherent susceptibility to cutaneous leishmaniosis in a mouse model: the role of the pathoantigenic HISA70 DNA vaccine

Leishmania major is the major cause of cutaneous leishmaniosis (CL) outside of the Americas. In the present study we have cloned six Leishmania genes (H2A, H2B, H3, H4, A2 and HSP70) into the eukaryotic expression vector pCMVβ-m2a, resulting in pCMV-HISA70m2A, which encodes all six pathoantigenic proteins as a single polyprotein. This expression plasmid has been evaluated as a novel vaccine candidate in the BALB/c mouse model of CL. The DNA vaccine shifted the immune response normally induced by L. major infection away from a Th2-specific pathway to one of basal susceptibility. Immunization with pCMV-HISA70m2A dramatically reduced footpad lesions and lymph node parasite burdens relative to infected control mice. Complete absence of visceral parasite burden was observed in all 12 immunized animals but not in any of the 24 control mice. Moreover, vaccinated mice produced large amounts of IFN-γ, IL-17 and NO at 7 weeks post-infection (pi), and they showed lower arginase activity at the site of infection, lower IL-4 production and a weaker humoral immune response than infected control mice. Taken together, these results demonstrate the ability of the HISA70 vaccine to shift the murine immune response to L. major infection away from an undesirable, Th2-specific pathway to a less susceptible-like pathway involving Th1 and Th17 cytokine profiles.

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

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 <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.

Vaccines for bovine neosporosis: current status and key aspects for development

Bovine neosporosis is a worldwide concern due to its global distribution and great economic impact. Reproductive failure in cattle due to abortion leads to major economic losses associated with the disease. Currently, there is no treatment or vaccine available against abortion or transmission caused by Neospora caninum infection in cattle. However, vaccination is considered the best measure of control against bovine neosporosis. Several host and parasite factors can influence the dynamics of the infection in bovines. Moreover, the availability of well‐defined infection models is a key factor for the evaluation of vaccine candidates. However, working with cattle is not easy due to difficult handling, facilities and costs, and therefore, ‘more affordable’ models could be used for screening of promising vaccines to establish proof of concept. So far, live‐attenuated vaccines have shown good efficacy against exogenous transplacental transmission; however, they have relevant disadvantages and associated risks, which render inactivated or subunit vaccines the best way forward. The identification of novel potential targets and vaccines, and the application of innovative vaccine technologies in harmonized experimental animal models, will accelerate the development of an effective vaccine against bovine neosporosis.

Association of Vt1C with Verotoxin-Producing Escherichia Coli from Goats and Sheep

A total of 232 verotoxin 1 (VT1)-positive, VT-producing Escherichia coli (VTEC) strains isolated from goats, sheep, and cattle were analyzed for the presence of the vt1c gene by polymerase chain reaction. One hundred and forty of the 144 (97.2%) caprine strains and 55 of the 63 (87.3%) ovine strains possessed the vt1c gene. In contrast, the gene was not detected in any of the 25 bovine strains. These results show that the vt1c gene is found in caprine VTEC strains and confirm that gene is associated with ovine VTEC strains.