Henry-Michel Cauchie

Surveillance and field investigations of Campylobacter infections require molecular tools with genetic markers appropriate for tracing purposes, i.e. based on the principle that some Campylobacter lineages acquire a host signature under adaptive selection pressure. We developed a sequence-based method targeting the quinolone resistance determining region within the subunit A of DNA gyrase (gyrA). Host specificity was evaluated by characterizing two collections of Campylobacter jejuni (N=430) and Campylobacter coli (N=302) originating from surface waters, domestic mammals and poultry. Based on nucleotide identity, a total of 80 gyrA alleles were observed. Thirty nine alleles assigned to C. coli encoding two peptides fell into three clades: two associated with surface waters and one associated with domestic mammals and poultry. The variability in GC content generated by synonymous mutations suggested that surface waters isolates originated from two distinct ecological niches. A total of 42 alleles were recorded from C. jejuni strains and encoded 8 peptides including one lying in a distinct lineage associated with wildlife. Seven of the 23 alleles encoding peptide #1 displayed the synonymous mutation G408A not identified in poultry isolates. By contrast, the substitution Ser22Gly observed in 4 different peptide groups was significantly associated with domestic birds (P=0.001). The change in amino acid sequences Thr86Ile conferring resistance to quinolones was significantly associated with poultry (PC. jejuni and C. coli with 38.7% and 67.9% of quinolone-resistant strains, respectively. The gyrA typing method presented here is an informative tool as sequences appear to be predictive of particular ecological niches. Combined with multi-locus sequence typing, it could increase the resolution of source attribution, and combined with porA/flaA typing it could be suitable for detecting temporal clusters of human cases. All gyrA alleles identified were deposited in the freely accessible online database http://pubmlst.org/campylobacter.

Aims Development of TaqMan MGB real‐time PCR assays for quantitative typing of major cattle and human‐pathogenic Cryptosporidium species. Methods and Results Three specific TaqMan MGB real‐time PCRs, based on the SSU rRNA gene, were directed towards livestock‐restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human‐pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non‐target DNA in environmental samples. Conclusions All TaqMan MGB real‐time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. Significance and Impact of the Study No molecular‐based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

Noroviruses (NoV) in 78 wastewater samples from Luxembourg were quantified, cloned, and sequenced in 2008-2009. The concentrations of NoV genogroup II and the relative occurrences of certain genotypes changed significantly during the winter season. NoV genogroup I was frequently detected by real-time reverse transcription-PCR (RT-PCR), albeit at 30-fold lower concentrations than for genogroup II, hampering attempts to assess overall genetic diversity by the cloning/sequencing approach.

Starting in 2006, a monitoring of Giardia lamblia and Cryptosporidium parvum occurrence was conducted for 2 years in the largest drinking water reservoir of Luxembourg (Esch-sur-Sûre reservoir) using microscopy and qPCR techniques. Parasite analyses were performed on water samples collected from three sites: site A located at the inlet of the reservoir, site B located 18 km downstream site A, at the inlet of the drinking water treatment plant near the dam of the reservoir and site C where the finished drinking water is injected in the distribution network. Results show that both parasites are present in the reservoir throughout the year with a higher occurrence of G. lamblia cysts compared to C. parvum oocysts. According to our results, only 25% of the samples positive by microscopy were confirmed by qPCR. (Oo)cyst concentrations were 10 to 100 times higher at site A compared to site B and they were positively correlated to the water turbidity and negatively correlated to the temperature. Highest (oo)cyst concentrations were observed in winter. In contrast, no relationship between the concentrations of (oo)cysts in the reservoir and rain events could be established. Though a correlation has been observed between both parasites and faecal indicators in the reservoir, some discrepancies highlight that the latter do not represent a reliable tool to predict the presence/absence of these pathogenic protozoa. In summer 2007, the maximal risk of parasite infection per exposure event for swimmers in the reservoir was estimated to be 0.0015% for C. parvum and 0.56% for G. lamblia. Finally, no (oo)cysts could be detected in large volumes of finished drinking water.