HisAK70: progress towards a vaccine against different forms of leishmaniosis.

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.

Resistance to non-quinolone antimicrobials in commensal Escherichia coli isolates from chickens treated orally with enrofloxacin.

The aim of the present study was evaluate how oral administration of enrofloxacin affected the frequency of resistance to different antimicrobials in commensal Escherichia coli isolates from healthy chickens. A further objective of this study was to characterize the mechanisms of resistance in these isolates. A trend towards increased resistance to enrofloxacin, doxycycline and amoxicillin of E. coli isolates from chickens after enrofloxacin administration was observed. The increase in the resistance to doxycycline and amoxicillin was probably due to a co-selection of tetracycline and ß-lactam resistance genes by the administration of enrofloxacin. The detection of tetM was much higher than expected (50%), which indicates that this gene may play an important role in tetracycline resistance in E. coli from chickens.

Detection and linkage to mobile genetic elements of tetracycline resistance gene tet(M) in Escherichia coli isolates from pigs.

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.

Toxoplasma gondii and Neospora caninum seroprevalences in domestic South American camelids of the Peruvian Andes.

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.

Differences in virulence gene expression between atypical enteropathogenic Escherichia coli strains isolated from diarrheic and healthy ruminants.

Differences in the pathogenicity of atypical enteropathogenic Escherichia coli (EPEC) strains may be due, at least partially, to different expression patterns of some virulence genes. To investigate this hypothesis, the virulence gene expression patterns of 6 atypical EPEC strains isolated from healthy and diarrheic ruminants were compared using quantitative real-time reverse transcription polymerase chain reaction after growing the bacteria in culture medium alone or after binding it to HeLa epithelial cells. Some virulence genes in strains from diarrheic animals were upregulated relative to their expression in strains from healthy animals. When bacteria were cultured in the presence of HeLa cells, the ehxA and efa1/lifA genes, previously associated with the production of diarrhea, were expressed at higher levels in strains from diarrheic animals than in strains from healthy animals. Thus, the expression levels of some virulence genes may help determine which atypical EPEC strains cause diarrhea in ruminants.

Des différences dans la pathogénicité de souches atypiques d'Escherichia coli entéropathogènes (EPEC) peuvent être dues, au moins en partie, à différents patrons d'excrétion de quelques-uns des gènes de virulence. Pour étudier cette hypothèse, les patrons d'expression des gènes de virulence de six souches atypiques d'EPEC isolées de ruminants en santé et avec diarrhée ont été comparés par une épreuve quantitative en temps réel de réaction d'amplification en chaîne par la polymérase par transcription réverse après avoir fait croître la bactérie dans un milieu de culture seul ou après l'avoir liée à des cellules épithéliales HeLa. Quelques gènes de virulence dans des souches provenant d'animaux avec diarrhée étaient régulés à la hausse relativement à leur expression dans les souches provenant d'animaux en santé. Lorsque les bactéries étaient cultivées en présence de cellules HeLa, les gènes ehxA et efa1/lifA, précédemment associés avec la production de diarrhée, étaient exprimés à des niveaux plus élevés dans les souches provenant d'animaux diarrhéiques que dans les souches provenant d'animaux en santé. Ainsi les niveaux d'expression de certains gènes de virulence pourraient aider à déterminer quelles souches atypiques d'EPEC causent de la diarrhée chez les ruminants.(Traduit par Docteur Serge Messier).

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.

High prevalence of Tritrichomonas foetus infection in Asturiana de la Montaña beef cattle kept in extensive conditions in Northern Spain.

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 Montaña (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-coding genes in verotoxin-producing Escherichia coli strains from sheep and goats differ from those from cattle.

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.

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.

Kinetics and role of antibodies against intimin beta in colostrum and in serum from goat kids and longitudinal study of attaching and effacing Escherichia coli in goat kids.

The presence of antibodies to the intimin beta-binding region (Int280-beta) of attaching and effacing Escherichia coli (AEEC) in serum from 20 goat kids from 2 herds, as well as in goat colostrum, was investigated by enzyme-linked immunosorbent assay. In addition, the onset and subsequent pattern of shedding of AEEC from the same goat kids over a 6-mo period was investigated. All the colostrum and serum samples tested contained antibodies against Int280-beta. The association between the antibody titer and the isolation of AEEC suggests that antibodies to intimin beta do not prevent colonization of the intestine by AEEC in goat kids. The AEEC were generally shed only transiently. Most AEEC isolated from the kids belonged to serogroup O26. Three isolates belonged to serogroup O157. These data show that goat kids may be a reservoir of AEEC that are potentially pathogenic for humans.

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.

Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coil.

The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain's belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.

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 microg/ml) but not to ciprofloxacin (MIC = 1 microg/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.

Detection of the saa gene in verotoxin-producing Escherichia coli from ruminants.

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.

Prevalence and characteristics of attaching and effacing strains of Escherichia coli isolated from diarrheic and healthy sheep and goats.

OBJECTIVE: To determine the prevalence and characteristics of attaching and effacing Escherichia coli (AEEC) in diarrheic and healthy small ruminants. ANIMALS: 502 lambs and kids with diarrhea and 511 healthy sheep and goats. PROCEDURE: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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.

Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene.

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.

Influence of Temperature of Incubation on Staphylococcus aureus Growth and Enterotoxin Production in Homemade Mayonnaise.

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.