Towards delimitation of the Echinococcus multilocularis parasite's southernmost range in France.

Alveolar echinococcosis is a severe, potentially fatal, parasitic disease caused by ingestion of microscopic eggs of Echinococcus multilocularis. The lifecycle of the parasite is essentially sylvatic, and based on a prey-predator relationship between red foxes and small rodents. A westward expansion from the eastern historical focus has been reported in France, though the parasite has also been detected in the southern Alps. While the focus in the Auvergne region (central France) was described in the 1980s, the southern delimitation of the actual endemic area, especially in the south, was unknown in the absence of dedicated surveys. Red fox samples were collected from 2013 to 2020 in the framework of other transversal epidemiological studies in five sampling areas from southwestern and southeastern France. One hundred and seven intestines were analysed by SSCT, and 221 faecal samples from intestines were analysed by copro-qPCR. None of the 328 foxes exhibited E. multilocularis worms or DNA. Although the presence of E. multilocularis cannot be totally excluded in the departments from the study areas, the sample size tested argues for an absence of the parasite in these studied areas, which is in accordance with the currently known endemic situation in France. These new data are helpful in determining the southernmost limit of E. multilocularis distribution in France. The warm, dry Mediterranean climate in the southeastern areas is less favourable to the transmission of E. multilocularis and especially to the survival of eggs in the environment than the climate in the French Alps or Liguria (Italy) climate where the parasite is present. The intermediate area between the southwestern study areas and the historical focus of Auvergne, which is separated by around 150 km, will be investigated in the coming years. Moreover, an ongoing national surveillance programme on E. multilocularis in foxes is targeting French departements along the edge of the known endemic area both in the southeast and southwest. The data produced will supplement the results of this study, thus greatly helping to define the current distribution of E. multilocularis in France and to target prevention measures to reduce human exposure.

Experimental Infection of Captive Red Foxes (Vulpes vulpes) with Mycobacterium bovis.

In Europe, animal tuberculosis (TB) due to Mycobacterium bovis involves multi-host communities that include cattle and wildlife species, such as wild boar (Sus scrofa), badgers (Meles meles) and red deer (Cervus elaphus). Red fox (Vulpes vulpes) infections have also been recently reported in some TB endemic regions in the Iberian Peninsula and France, with some of the infected animals shedding M. bovis in urine and feces. In order to understand the pathogenesis of M. bovis infection in foxes and the associated risk of transmission, 12 captive foxes (6 females and 6 males) were inoculated orally with 2 × 107 colony-forming units of a French field isolate of M. bovis. Clinical samples (urine, feces and oropharyngeal swabs) were collected every four weeks and tested for molecular diagnosis and bacteriology. Serological responses were measured by IDEXX M. bovis Ab Test and Multi Antigen Print Immunoassay (MAPIA). At a post-mortem examination performed 12 weeks post infection (wpi), tissues were tested for the presence of M. bovis and associated gross and microscopic TB-like lesions. M. bovis was detected by PCR in bladder swabs of 3 animals at 12 wpi. It was also detected pre-mortem at different time points of the experiment in the oropharyngeal mucus of three individuals and in the feces of nine foxes, with two of them confirmed by bacteriology. All 12 foxes had at least 4 PCR positive samples (out of the 23 tested), and all but 1 fox had at least 1 culture positive sample. The culture negative fox was PCR positive in both retropharyngeal and mesenteric lymph nodes, in line with the results of the other animals. Seroconversion was observed in all foxes except one during the experiment, and in nine at the final time point. No gross visible lesions were found in any animal at the post-mortem examination. The histology showed small granulomas within the lymph nodes, tonsils, liver and lungs from eight animals, with the presence of few acid-fast bacilli. These results confirmed that all orally-infected foxes developed mild TB lesions but they were able to shed mycobacteria in about 75% of cases, 1 month post-infection (9 out 12 foxes). These results show that it is possible to induce typical TB infection experimentally in captive foxes, with measurable M. bovis excretion; such an experimental system could be useful for future evaluations of diagnostics and vaccines in this species.

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.

Discovery of a polyomavirus in European badgers (Meles meles) and the evolution of host range in the family Polyomaviridae.

Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.

Pigs and wild boar in Corsica harbor Echinococcus canadensis G6/7 at levels of concern for public health and local economy.

Cystic echinococcosis (CE) is a parasitic zoonosis widespread in the Mediterranean area. The parasite is commonly maintained in a domestic cycle involving dogs and livestock species. As no new data have been made available for the last 15 years concerning the French Mediterranean island of Corsica, a cross-sectional survey at the slaughterhouse was conducted in 2009-2010 to describe the current presence of Echinococcus granulosus sensu lato in intermediate hosts. Only pig infections with the G6/7 genotype of Echinococcus canadensis were observed. No infection was detected in other breeding species but this should be interpreted with caution because 75% of the cows inspected during the survey were calves, and all sheep and goats were younger than two months old. In parallel four wild boars harvested during the 2010-2011 hunting season were also infected by the same genotype. These data constitute the first report of E. canadensis in France and the first molecular characterization of E. granulosus sensu lato in a wild species in France. The current prevalence observed in pigs (5.9%, n=2527) highlights the fact that CE is still of economic concern on Corsica, an island where certain regional products are produced using pig's liver ("Figatelli"). This prevalence, and the similar one observed in wild boars (4.0%, n=101), is a consequence of certain breeding practices and hunting practices which enable circulation of the parasite in the environment in close contact with humans.

Nutrias and muskrats as bioindicators for the presence of Echinococcus multilocularis in new endemic areas.

Nutrias (Myocastor coypus) and muskrats (Ondatra zibethicus) are large invasive semi-aquatic or aquatic rodents, naturalized throughout Europe. They are regarded as pests, and can be infected with several pathogens and parasites transmissible to wildlife, livestock, pets and humans. As a rule, in Europe the life cycle of the cestode Echinococcus multilocularis involves red foxes (Vulpes vulpes) as main definitive hosts and common voles as intermediate hosts. The suitability of nutrias and muskrats as intermediate hosts has already been described. Here, we investigate the occurrence of E. multilocularis in the context of a wide-scale health study on nutrias and muskrats in 12 "départements" in the western part of France. During the sampling period, a total of 817 aquatic rodents were trapped in five rivers or ponds in each "départements". During post-mortem examinations, lesions were observed on the livers of 21 nutrias and 104 muskrats, and analyzed by PCR and sequencing of the mitochondrial cox1 gene for specific identification. Several non-zoonotic parasites were identified: Taenia taeniaformis, Taenia mustelae, Taenia polyacantha and Taenia martis. Four livers from 2 nutrias and 2 muskrats exhibited E. multilocularis infection. One of the muskrats was infected with fertile E. multilocularis lesions. The 4 animals came from 3 French "départements" where foxes have recently been found to be infected by E. multilocularis. These results lead us to consider nutrias and muskrats as relevant bioindicators for the presence of E. multilocularis in this environment. Our results also suggest that, when listed as pests and targeted by large trapping campaigns, nutrias and muskrats could be used to detect the presence of E. multilocularis in areas considered free of this parasite.