Rodent control programmes can integrate Echinococcus multilocularis surveillance by facilitating parasite genotyping: the case of Arvicola terrestris voles screening in France.

The tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, the most serious parasitic disease for humans in Europe. In Europe, the E. multilocularis lifecycle is based on a prey-predator relationship between the red fox and small rodents. Over the last three decades, the surveillance of E. multilocularis infection in red foxes has led to the description of a wider distribution pattern across Europe. France constitutes the current European western border, but only the north-eastern half of the country is considered endemic. The red fox is the host mainly targeted in E. multilocularis surveillance programmes, but surveys targeting small rodents may be useful for obtaining molecular data, especially when the time-consuming trapping is already carried out in dedicated pest-control programmes. Here, we screened for parasitic lesions in the livers of 1238 Arvicola terrestris voles originating from the historical, but neglected focal area located in central France (Auvergne region) and from Hautes-Alpes, a recently identified endemic department in south-eastern France. This screening identified six voles infected with E. multilocularis in Hautes-Alpes and none in Puy-de-Dôme (Auvergne region) after molecular confirmation. The absence of infected rodents from Puy-de-Dôme can be mainly explained by the generally low prevalence reported in intermediate hosts. The infected Hautes-Alpes samples come all from the same trapping site situated at around 5 km from one of the three fox faecal samples with E. multilocularis DNA collected 15 years prior, thereby confirming the existence and persistence of the E. multilocularis lifecycle in the area. All the rodent E. multilocularis samples from Hautes-Alpes showed the same EmsB microsatellite marker profile. This profile has previously been described in Europe only in the Jura department (central eastern France), located at least 180 km further north. Successive migrations of infected foxes from the historical focal area, including from Jura, to Hautes-Alpes may explain the detection of the parasite in A. terrestris in Hautes-Alpes. Existing trapping efforts in areas where farmers trap A. terrestris for surveillance and pest control can be an effective complement to sampling foxes or fox faeces to obtain E. multilocularis molecular profiles.

National survey and molecular diagnosis of Echinococcus granulosus sensu lato in livestock in France, 2012.

The parasitic species of the Echinococcus granulosus sensu lato (sl) complex are the causative agents of cystic echinococcosis in humans. The lifecycle of E. granulosus sl is essentially domestic, and is based on the consumption by dogs of hydatid cysts in viscera of livestock species. The aim of this study was to survey E. granulosus sensu lato in livestock in France. A 1-year national survey of E. granulosus sl in livestock at the slaughterhouse was organized in 2012 in France, with systematic molecular confirmation. The prevalence of E. granulosus ss nationally was 0.002% in sheep, mainly focused in the Alpine area, and 0.001% in cattle, with the distribution of cases throughout the country. Echinococcus canadensis G6/7 was observed only in Corsica in pigs, with a prevalence of nearly 1% in the island. A national prevalence of 0.0002% was estimated for E. ortleppi in cattle, due to seven cases distributed in two foci. The results of this survey are of particular interest because of the zoonotic risk associated with the presence of these parasite species, for which systematic control at the slaughterhouse should enable their elimination.

Identification and molecular characterization of Echinococcus canadensis G6/7 in dogs from Corsica, France.

Recent surveys at slaughterhouses confirmed the presence of three different species of Echinococcus granulosus sensu lato in France: E. granulosus sensu stricto, E. ortleppi, and E. canadensis G6/7. The latter species was only identified on the French Mediterranean island of Corsica, with a high prevalence in pigs and wild boar. In order to investigate the life cycle of E. canadensis in this region, dog feces were collected in 31 municipalities, mainly from individual kennels. The analysis of fecal samples from 259 dogs by multiplex real-time PCR shows no infection by E. granulosus sensu stricto, but three dogs were infected by E. canadensis G6/7. Genetic analyses of mitochondrial genes (cox1, nad1, nad3, atp6) revealed in two dogs a haplotype previously identified in pigs. The third dog was infected by a new haplotype differing only from the two others from dogs by two mutations in the nad3 gene. This latter haplotype is genetically closer to those identified in pigs rather than those from wild boars. Analysis of questionnaires completed by the owners revealed that the sampled dog population was almost exclusively composed of hunting dogs that had been infrequently dewormed. Most of the owners (78%) leave carcasses of hunter-harvested wild boar in close proximity to their dogs. Nevertheless, genetic results seem to indicate that the three dogs were infected due to their consumption of a pig's infected viscera following home slaughtering. This study confirms the role of dogs as definitive hosts of E. canadensis G6/7 in Corsica. Further molecular studies, notably in human cases, are needed to assess the zoonotic impact of E. canadensis G6/7 in this region.

Prevalence of carriage of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among slaughtered adult cattle in France.

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these "top five" STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples.O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P<0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover,simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.

Intimin gene (eae) subtype-based real-time PCR strategy for specific detection of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in cattle feces.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, ß1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

Phylogenetic grouping and virulence potential of extended-spectrum-ß-lactamase-producing Escherichia coli strains in cattle.

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.

Asian Admixture in European Echinococcus multilocularis Populations: New Data From Poland Comparing EmsB Microsatellite Analyses and Mitochondrial Sequencing.

The cestode Echinococcus multilocularis is the causative agent of a severe zoonotic disease: alveolar echinococcosis (AE). The parasite is distributed over a vast area in northern Eurasia and North America, but the impact of AE on human health is highly uneven between different regions. One hypothetical reason for this difference in virulence may be the genetic structure of E. multilocularis which-based on mitochondrial sequences and EmsB microsatellite profiles-forms four distinct clades. These clades correspond approximately to their continents of origin: Asia, Europe, and North America, with a fourth clade apparently restricted to Mongolia and neighboring regions, even though this clade has not yet been described by EmsB genotyping. However, there are various records of genetic variants from the "wrong" region, e.g., "European" haplotypes in Western Canada, which may be the result of introduction or natural migration of host animals. One such example, prompting this study, is the recent record of an "Asian" mitochondrial haplotype in worms from foxes in Poland. At the time, this could not be confirmed by EmsB microsatellite analysis, a method that has proven to possess greater discriminatory power with the E. multilocularis nuclear genome than sequencing of mitochondrial markers. Therefore, worms collected from foxes in Poland were examined both by EmsB analysis and sequencing of the full mitochondrial cox1 gene in order to allocate the samples to the European or Asian cluster. Based on EmsB analyses of 349 worms from 97 Polish red foxes, 92% of the worms clearly showed "European-type" EmsB profiles, but 27 worms (8%) from seven foxes showed profiles that clustered with samples of Asian origin. According to cox1 sequences, a total of 18 worms from 8 foxes belonged to the Asian cluster of haplotypes. The two methods did not fully agree: only 13 worms from three foxes belonged to Asian clusters by both EmsB and cox1, whereas 18 worms from nine foxes belonged to different clusters, according to each marker. Cross-fertilization between worms of Asian origin and those from the European Polish population may explain these conflicting results. The presence of clearly Asian elements in the Polish E. multilocularis population could be the result of introduction of E. multilocularis with host animals (e.g., domestic dogs), or the migration of foxes. In the absence of genetic data from eastern European countries, especially those bordering Poland, it cannot be concluded whether this Asian admixture is typical for a larger area toward central/eastern Europe, or the Polish parasite population is the western extreme of a gradient where both European and Asian elements mingle. Further studies are needed on this subject, preferably using both mitochondrial sequencing and EmsB microsatellite analysis.

Evaluation of six TaqMan RT-rtPCR kits on two thermocyclers for the reliable detection of rabies virus RNA.

Rabies is diagnosed postmortem in animals, based on tests prescribed by the World Organization for Animal Health (OIE), such as the fluorescent antibody test, the direct rapid immunohistochemistry test, or pan-lyssavirus PCR assays. Several reverse-transcription real-time PCR (RT-rtPCR) methods have been developed and validated for rapid and accurate detection of lyssaviruses. We evaluated the performance of 6 TaqMan RT-rtPCR kits using different commercial master mixes and 2 real-time thermocyclers. Changing the master mix overall did not influence the TaqMan RT-rtPCR performance, regardless of the thermocycler used. The limits of detection at the 95% confidence level were 18.1-25.8 copies/µL for the Rotor-Gene Q MDx thermocycler and 16.7-21.5 for the Mx3005P thermocycler. Excellent repeatability was demonstrated for rabies virus (RABV) RNA samples of 100, 50, and 25 copies/µL regardless of the thermocycler used. RABV field samples ( n = 35) isolated worldwide gave positive results using the most efficient of the 6 kits tested, with a copy number of 6.03 × 102 to 6.78 × 107 RNA copies per reaction. The TaqMan RT-rtPCR assay provides sensitive and rapid amplification of RABV RNA.

Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.