Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103-HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.

Assessment by electron-microscopy of recombinant Vibrio cholerae and Salmonella vaccine strains expressing enterotoxigenic Escherichia coli-specific surface antigens

Diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) requires adhesion of microorganisms to enterocytes. Hence, a promising approach to immunoprophylaxis is to elicit antibodies against colonisation factor antigens (CFAs). Genes encoding the most prevalent ETEC-specific surface antigens were cloned into Vibrio cholerae and Salmonella vaccine strains. Expression of surface antigens was assessed by electron-microscopy. Whereas negative staining was effective in revealing CFA/I and CS3, but not CS6, immunolabelling allowed identification of all surface antigens examined. The V. cholerae vaccine strain CVD103 did not express ETEC-specific colonisation factors, whereas CVD103-HgR expressed CS3 only. However, expression of both CFA/I and CS3 was demonstrated in Salmonella Ty21a.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007

Equine influenza virus (EIV) surveillance is important in the management of equine influenza. It provides data on circulating and newly emerging strains for vaccine strain selection. To this end, antigenic characterisation by haemaggluttination inhibition (HI) assay and phylogenetic analysis was carried out on 28 EIV strains isolated in North America and Europe during 2006 and 2007. In the UK, 20 viruses were isolated from 28 nasopharyngeal swabs that tested positive by enzyme-linked immunosorbent assay. All except two of the UK viruses were characterised as members of the Florida sublineage with similarity to A/eq/Newmarket/5/03 (clade 2). One isolate, A/eq/Cheshire/1/06, was characterised as an American lineage strain similar to viruses isolated up to 10 years earlier. A second isolate, A/eq/Lincolnshire/1/07 was characterised as a member of the Florida sublineage (clade 1) with similarity to A/eq/Wisconsin/03. Furthermore, A/eq/Lincolnshire/1/06 was a member of the Florida sublineage (clade 2) by haemagglutinin (HA) gene sequence, but appeared to be a member of the Eurasian lineage by the non-structural gene (NS) sequence suggesting that reassortment had occurred. A/eq/Switzerland/P112/07 was characterised as a member of the Eurasian lineage, the first time since 2005 that isolation of a virus from this lineage has been reported. Seven viruses from North America were classified as members of the Florida sublineage (clade 1), similar to A/eq/Wisconsin/03. In conclusion, a variety of antigenically distinct EIVs continue to circulate worldwide. Florida sublineage clade 1 viruses appear to predominate in North America, clade 2 viruses in Europe.

Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences

Genetic relationships among bacterial strains belonging to the genus Aeromonas were inferred from 16S rRNA, gyrB and rpoB gene sequences. Twenty-eight type or collection strains of the recognized species or subspecies and 33 Aeromonas strains isolated from human and animal specimens as well as from environmental samples were included in the study. As reported previously, the 16S rRNA gene sequence is highly conserved within the genus Aeromonas, having only limited resolution for this very tight group of species. Analysis of a 1.1 kb gyrB sequence confirmed that this gene has high resolving power, with maximal interspecies divergence of 15.2 %. Similar results were obtained by sequencing only 517 bp of the rpoB gene, which showed maximal interspecies divergence of 13 %. The topologies of the gyrB- and rpoB-derived trees were similar. The results confirm the close relationship of species within the genus Aeromonas and show that a phylogenetic approach including several genes is suitable for improving the complicated taxonomy of the genus.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Phylogenetic analysis of Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis clinical strains reveals a clear species clustering

Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis are oral pathogens from the family Bacteroidaceae, regularly isolated from cases of gingivitis and periodontitis. In this study, the phylogenetic variability of these three bacterial species was investigated by means of 16S rRNA (rrs) gene sequence comparisons of a set of epidemiologically and geographically diverse isolates. For each of the three species, the rrs gene sequences of 11 clinical isolates as well as the corresponding type strains was determined. Comparison of all rrs sequences obtained with those of closely related species revealed a clear clustering of species, with only a little intraspecies variability but a clear difference in the rrs gene with respect to the next related taxon. The results indicate that the three species form stable, homogeneous genetic groups, which favours an rrs-based species identification of these oral pathogens. This is especially useful given the 7% sequence divergence between Prevotella intermedia and Prevotella nigrescens, since phenotypic distinction between the two Prevotella species is inconsistent or involves techniques not applicable in routine identification.

Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination-the classic presentation of CDV infection-was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Antimicrobial resistance in Escherichia coli strains isolated from Swiss weaned pigs and sows

Based on Directive (EC) No 99/2003, monitoring programs on the development of antimicrobial resistance in bacteria from livestock are implemented in many European countries. The aim of the present study was (i) to establish comprehensive baseline data on the antimicrobial resistance situation in Escherichia coli isolates obtained from healthy pigs (pooled fecal samples) originating from 60 Swiss pig-breeding farms, and (ii) to analyze differences in the resistance frequency between Escherichia coli isolates from weaned pigs and sows. Susceptibility testing (disc diffusion method) was performed on 429 isolates from weaned pigs and 431 isolates from sows. Overall, 17.7% of the isolates from weaned pigs and 22.5% of the Escherichia coli isolates from sows were susceptible to all antibiotics tested. Low resistance prevalence was found for amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, ciprofloxacin, colistin, florfenicol, and gentamicin. The most frequently found resistances were against streptomycin (60.6% of the isolates from weaners and 64.3% of the isolates from sows), sulfonamide (51.5% and 26.9%), tetracycline (35.2% and 22.0%), and trimethoprim (27.5% and 11.1%). With exception of colistin, most resistances were found for those antibiotics commonly used on the farms. Except for ciprofloxacin and streptomycin, isolates from weaned pigs showed higher resistance prevalence than those from sows. This difference was significant for cefquinome, florfenicol, sulfonamide, tetracycline, and trimethoprim (p<0.05).

Unravelling the distinct strains of Tharu ancestry

The northern region of the Indian subcontinent is a vast landscape interlaced by diverse ecologies, e.g. the Gangetic plain and the Himalayas. A great number of ethnic groups are found there, displayed as a multitude of languages and cultures. The Tharu represent one of the largest and linguistically most diversified such groups, scattered across the Tarai region of Nepal and bordering Indian states. Their origins are uncertain. Hypotheses have been advanced about an Austroasiatic affinity, Tibeto-Burman origins, as well as aboriginal roots in the Tarai. Several Tharu groups speak a variety of Indo-Aryan languages, but have traditionally been described by ethnographers as representing an East Asian phenotype. Their ancestry and intra-population diversity had previously been tested only for haploid (mtDNA and Y-chromosome) markers in a small portion of the population. This study presents the first systematic genetic survey of the Tharu from both Nepal and the Indian states of Uttaranchal and Uttar Pradesh, using genome-wide SNPs and haploid (mtDNA and Y-chromosome) markers. The results suggest that the 'ethnic' construct of Tharu is likely to have lain in the Tarai region, with a reconstructible radiation to Uttaranchal and Uttar Pradesh. Despite extensive admixture with other local communities, Tharu sub-populations preserve strong genetic signatures that indicate a common ancestry.

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.

Highly pathogenic adapted HIV-1 strains limit host immunity and dictate rapid disease progression.

OBJECTIVE: The study of HIV-1 rapid progressors has been limited to specific case reports. Nevertheless, identification and characterization of the viral and host factors involved in rapid progression are crucial when attempting to uncover the correlates of rapid disease outcome. DESIGN: We carried out comparative functional analyses in rapid progressors (n = 46) and standard progressors (n = 46) early after HIV-1 seroconversion (≤1 year). The viral traits tested were viral replicative capacity, co-receptor usage, and genomic variation. Host CD8 T-cell responses, humoral activity, and HLA immunogenetic markers were also determined. RESULTS: Our data demonstrate an unusual convergence of highly pathogenic HIV-1 strains in rapid progressors. Compared with standard progressors, rapid progressor viral strains show higher in-vitro replicative capacity (81.5 vs. 67.9%; P = 0.025) and greater X4/DM co-receptor usage (26.3 vs. 2.8%; P = 0.006) in early infection. Limited or absent functional HIV-1 CD8 T-cell responses and neutralizing activity were measured in rapid progressors. Moreover, the increase in common HLA allele-restricted CD8 T-cell escape mutations in rapid progressors acts as a signature of uncontrolled HIV-1 replication and early impairment of adaptive cellular responses. CONCLUSION: Our data support a dominant role for viral factors in rapid progressors. Robust HIV-1 replication and intrinsic viral properties limit host adaptive immune responses, thus driving rapid disease progression.

Antimicrobial susceptibility of canine Clostridium perfringens strains from Switzerland

Fifty Clostridium perfringens strains were isolated from individual dogs with acute diarrhoea that were not given antibiotics. Toxin types and minimal inhibitory concentrations of 15 antibiotics were determined for each of them. All strains harboured the alpha-toxin gene, 12 of them had both the alpha- and entero-toxin gene and 5 had both the alpha- and beta2-toxin gene. Eighteen percent of the isolates showed resistance to tetracycline and 54 % showed decreased susceptibility to metronidazole which is one of the most frequently used antibiotics in the treatment of canine diarrhoea. Apart from that, all isolates were susceptible to the remaining antibiotics tested. These findings lead to the conclusion that despite a general susceptibility to antibiotics in C. perfringens, resistance is developing in isolates from dogs. Therefore, careful identification of the pathogenic agent and antibiotic susceptibility testing should be performed prior to therapy in order to minimise further selection of antibiotic resistance.