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Foodborne diseases continue to represent an important threat to public health in many parts of the world and are particularly widespread in developing countries. They are essentially acquired through an oro-fecal route via the consumption of uncooked fruits and vegetables. This study evaluated the parasitological contamination of vegetables for sale to humans in Tunisian retail markets. A total of 240 samples of fresh vegetables were examined for helminth eggs and protozoan cysts and oocysts (collectively, (oo)cysts) contamination. The parasitic elements (helminth eggs and protozoan (oo)cysts) were concentrated by sucrose flotation and identified by microscopic examination. The molecular identification of Echinococcus granulosus eggs was carried out using PCR. Helminth eggs and protozoan (oo)cysts eggs were found in 12.5% of the unwashed vegetables, and the most common parasites observed in vegetables were coccidian oocysts (4.1%), Toxocara spp. (2.5%), hookworm (2.1%), and Taenia spp. (1.25%) eggs, followed by Pseudolimax butschlii (1.6%) and Entamoeba coli (1.6%) protozoan cysts. Furthermore, parasite contamination differed significantly from one city to another. Taeniid eggs were identified by PCR as E. granulosus sensu stricto (s.s.) (genotype G1). To our knowledge, this study highlights for the first time in Tunisia that fresh vegetables for sale in markets are contaminated with helminths and protozoan cysts, which are potentially pathogenic for humans. The control of these pathogens is in part a question of sanitary education, especially for retail vendors, and in part of improvement in hygiene measures throughout the food production chain, from the field to the consumer. HIGHLIGHTS

Background Cystic echinococcosis (CE) has a worldwide distribution and is especially prevalent in North African countries. With a mean annual surgical incidence (ASI) of CE of 12.7 per 100,000 inhabitants, Tunisia is one of the most CE endemic countries in the Mediterranean area. Tataouine governorate is considered to be the most CE hypoendemic region in Tunisia (ASI = 0.92) despite favourable socioeconomic conditions that enable maintenance of the Echinococcus granulosussensu lato (s.l.) life-cycle and a significant environmental contamination with E. granulosuss.l. eggs. The aim of this study was to assess human CE seroprevalence, prevalence of CE in food animals and environmental contamination by E. granulosus s.l. eggs in different districts of Tataouine governorate. Methods This study was conducted from January to December 2018. A total of 374 human sera samples were tested for the presence of immunoglobulin G antibodies against E. granulosus using a commercial ELISA kit. Specimens were also collected from animals slaughtered at the Tataouine abattoir (n = 8609) and examined for the presence of hydatid cysts; 111 hydatid cysts were genotyped. Eggs of E. granulosuss.l. were identified by PCR and DNA sequencing from dog faecal samples (n = 288). Results Serological tests showed that 8.5% of the sera samples tested were positive for E. granulosus-specific antibodies. The average prevalence of hydatidosis in livestock was 1.6%, and CE infection was more prevalent in cattle than in sheep, goats and dromedaries. The contamination rate of dog faeces by E. granulosus sensu stricto eggs varied significantly from 0 to 23.5% depending on the collection area. Molecular analyses only revealed the presence of the G1 genotype for cysts and eggs. Conclusions Based on our findings, CE is likely to be more endemic in the Tataouine governorate than previously described. Thus, to implement an effective control programme against CE, a national survey should be carried out to determine human CE prevalence in the different Tunisian governorates. Graphical Abstract

International audience; Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

Hydatidosis has become a real concern for health care institutions and animal rearers in Tunisia. The Tunisian endemicity is aggravated by the growing number of dogs and the difficulty of getting rid of contaminated viscera because of the lack of equipment in most slaughterhouses. Therefore, microscopic and molecular tools were applied to evaluate the role of slaughterhouses in canine infection and Echinococcus granulosus sensu lato (s. l.) egg dissemination. Exposure risk to E. granulosus s. l. eggs in urban and rural areas was explored in order to implant preventive and adapted control strategies. Microscopic examinations detected taeniid eggs in 152 amongst 553 fecal samples. The copro-PCR demonstrated that 138 of 152 taeniid samples analyzed were positive for E. granulosus s. l. DNA. PCR-RFLP demonstrated that all isolated samples belonged to E. granulosus sensu stricto (s. s.). An important environmental contamination index (25.0%) by E. granulosus s. l. eggs was demonstrated. The average contamination index from the regions around slaughterhouses (23.3%; 95% CI: 17.7-28.9%) was in the same range as detected in areas located far from slaughterhouses (26.0%, 95% CI: 21.3-30.8%). Echinococcosis endemic areas were extended in both rural (29.9%, 95% CI: 24.8-34.9%) and urban locations (18.1%, 95% CI: 13.0-22.9%). The pathogen dissemination is related neither to the presence/absence of slaughterhouses nor to the location in urban or rural areas, but is probably influenced by human activities (home slaughtering) and behavior towards the infected viscera.

It has been demonstrated that human hydatidosis was generally due to G1 genotype of Echinococcus granulosus throughout the world. Nevertheless, some other genotypes, such as G3, were recently identified for human cyst. The present work confirms the predominance of the sheep strain G1 in humans, bovine and ovine and demonstrates for the first time the occurrence of the buffalo strain G3 in human and bovine in Tunisia.

G1 genotype of Echinococcus granulosus sensu stricto is the major cause of hydatidosis in Northern Africa, Tunisia included. The genetic relationship between lung and liver localization were studied in ovine, bovine and human hydatid cysts in Tunisia. Allozyme variation and single strand conformation polymorphism were used for genetic differentiation. The first cause of genetic differentiation was the host species and the second was the localization (lung or liver). The reticulated genetic relationship between the liver or the lung human isolates and isolates from bovine lung, is indicative of recombination (sexual reproduction) or lateral genetic transfer. The idea of two specialized populations (one for the lung one for the liver) that are more or less successful according to host susceptibility is thus proposed.

International audience

Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP) was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst = 0.46). No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing) was found in a limited number of camel isolates., Les Echinococcus granulosus de Tunisie ont été identifiés comme appartenant aux lignées G1 et G6 en se fondant sur le polymorphisme de la cytochrome oxydase CO1. Le polymorphisme de conformation simple brin (SSCP) a été utilisé afin d’évaluer la variabilité génétique intra et inter-isolats des lignées G1 et G6, et pour estimer le taux d’autofécondation. Les isolats issus de dromadaires sont génétiquement distincts des isolats ovins (valeur de Fst : 0,46). Il n’y a pas de déficit significatif en hétérozygotes chez les isolats ovins. Un déficit en hétérozygotes est présent dans les isolats du dromadaire, ce qui suggère l’autofécondation comme l’un des modes de reproduction.