F.J. van der Wal

Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

ABSTRACT The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [stx1, stx 2a/stx 2c, q21/Q933, tir(A255T), and rhsA(C3468G)]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir(A255T) SNP and the stx2a/stx 2c gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx 2c (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx 2c was 36.5%, while the frequencies of stx 2a and stx 2a plus stx 2c were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir(255A) (54.8%) and tir(255T) (45.2%), while human isolates were dominated by the tir(255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx 2c and tir(255T), while LSPA6 lineage II was dominated by tir(255A) (86.4%) and stx 2c (90.9%). LSPA6 lineage I/II isolates were all genotype tir(255T) but showed more variation in stx 2 types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North America.

A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

Multiple detection of pathogens in ticks: development of a high throughput real time PCR chip used as a new epidemiologic investigative tool

Worldwide, ticks transmit more pathogens than other arthropods. Around 60 bacteria, 30 parasites and 100 viruses have been registered as tick-borne pathogens; a third of these pathogens are responsible for zoonoses. Usually, detection of tick-borne pathogens depends on the tick species collected: assays are performed for a restricted number of pathogens that are known to be transmitted by a particular tick species collected at a particular site. To better understand the epidemiology of tick-borne pathogens, it will be important to detect for each sample (one tick or one pool of ticks) most of the diseases they potentially transmit, regardless of the tick species. The aim is therefore to develop a new epidemiologic investigative tool which could detect high number of tick-borne pathogens by real time PCR. We developed a chip (BioMark™ dynamic arrays, Fluidigm Corporation) targeting pathogens of worldwide distribution transmitted by ticks. The designed epidemiologic arrays may detect 48 pathogens in 48 samples corresponding to 2304 qPCR reactions on the same time. Specific primers and probe have been designed for each pathogen and their specificity have been tested in silico with Blast. To begin, we targeted: (i) 37 pathogens whose Francisella tularensis, Coxiella burnetii, Neoehrlichia mikurensis, 5 species of Anaplasma, 3 species of Ehrlichia, 8 species of Borrelia, 2 species of Bartonella, 4 species of Rickettsia, 10 species of Babesia and 2 species of Theileria, (ii) 5 species of ticks whose 3 species of Ixodes and 2 species of Dermacentor. Sensitivity of primers and probe has been tested on a dilution range of reference DNAs of the targeted pathogens on a Lightcycler 480. Specificity then has been tested on a Biomark™dynamic array. The chip was secondly evaluated on field samples corresponding to 47 pools of 25 nymphs collected in two sites in France, the Netherlands and Denmark (corresponding to 7050 nymphs in total). We succesfully detected and determined the prevalence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia helvetica, Bartonella henselae, five different genospecies of Borrelia burgdorferi s.l., the recently identified pathogen Borrelia miyamotoi, and two parasite species Babesia divergens and Babesia venatorum. This fast and low-cost tool allows comprehensive testing of tick-borne pathogens and can be customized to fit regional demands or to accommodate new or emerging pathogens. The tool represents a major improvement for surveillance and future epidemiological studies.

Bead-based suspension array for simultaneous detection of antibodies against the Rift Valley fever virus nucleocapsid and Gn glycoprotein

A multiplex bead-based suspension array was developed that can be used for the simultaneous detection of antibodies against the surface glycoprotein Gn and the nucleocapsid protein N of Rift Valley fever virus (RVFV) in various animal species. The N protein and the purified ectodomain of the Gn protein were covalently linked to paramagnetic Luminex beads. The performance of the resulting multiplex immunoassay was evaluated by testing a comprehensive and well-characterized panel of sera from sheep, cattle and humans. The suitability of this multiplex immunoassay to differentiate infected from vaccinated animals (DIVA) was investigated by testing sera from lambs vaccinated with a paramyxovirus vaccine vector expressing the RVFV surface glycoproteins Gn and Gc. The results suggest that the bead-based suspension array can be used as a DIVA assay to accompany several recently developed experimental vaccines that are based on RVFV glycoproteins, and are devoid of the N protein.

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli

Aims:  To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer.

A bead-based suspension array for the serological detection of Trichinella in pigs

The feasibility of using bead-based suspension arrays to detect serological evidence of Trichinella in pigs was assessed. Trichinella spiralis excretory-secretory antigen was covalently coupled to paramagnetic beads and used to bind serum antibodies, which were subsequently detected using anti-swine antibody. The assay was evaluated by testing pig sera from farms where trichinellosis was endemic and comparing the results with those obtained using two commercially available ELISAs. With cut-offs established by receiver operating characteristic (ROC) analysis, digestion-negative sera from a Trichinella-free population of pigs were deemed seronegative. When anti-swine antibody was replaced with protein A/G, higher test sensitivity (94% vs. 88%) at similar test specificity (95%), was achieved. The potential use of this assay in species other than swine was also demonstrated by testing human sera.

Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

A SpoT polymorphism correlates with chill stress survival and is prevalent in clinical isolates of Campylobacter jejuni

Resistance of Campylobacter jejuni to environmental stress is regarded as a risk factor for the transmission of C. jejuni from poultry or poultry products to humans. So far, the mechanisms underlying the capacity of C. jejuni to survive environmental stress conditions are not fully understood. In this study, we searched for polymorphisms in C. jejuni genes, potentially involved in resistance to chill stress. To this end, we assessed 3 groups of C. jejuni isolates (clinical, retail chicken meat, and feces) for survival of experimentally induced chill stress. For each isolate we sequenced 3 genes encoding the C. jejuni sigma factors FliA, RpoD, and RpoN as well as the genes for the transcriptional regulator SpoT and the periplasmic protein HtrA. Data suggest a higher prevalence of a specific polymorphism in spoT in clinical isolates compared with poultry meat or farm isolates. Moreover, this genotype correlated with enhanced survival of chill stress. The observation that the prevalence of this SNP is relatively high in clinical isolates, which most likely have been exposed to multiple forms of stress, suggest that this SNP may be a biomarker for enhanced survival of stress.