Epidemiological update of cystic echinococcosis in livestock and assessment of practices related to its control in the Mediterranean area.

Cystic echinococcosis (CE), caused by the tapeworm Echinococcus granulosus, is a zoonotic parasitic disease that still represents a serious threat to human and animal health worldwide. The Mediterranean basin is recognized as one of the major hotspots of CE due to several factors, including the presence of diverse intermediate host species as well as socio-economic and cultural conditions of local communities. This study aims to take a closer look at epidemiological data on CE in the Mediterranean area and assess the knowledge attitudes and practices of shepherds towards this disease in four countries (Algeria, Greece, Italy and Tunisia), highly endemic for CE, with the final goal of identifying highly endemic risk areas and practices in use which might potentially allow the persistence of E. granulosus infection in these areas. To update the epidemiological scenario of CE in Mediterranean areas, a comprehensive review of peer-reviewed literature on CE prevalence data published during the 2017-2023 period was carried out and, through a geographical information system (GIS), a map displaying the current CE distribution in the Mediterranean area was generated. In addition, a questionnaire survey was conducted through in-depth interviews of the farmers to collect information on their management system as well as knowledge attitudes and practices towards CE. From the farmer-participatory survey some risky practices emerged including the non-regular deworming of dogs or the use of ineffective drugs or dosing, as well as the provision of uncooked animal viscera to dogs. Finally, lower levels of knowledge and awareness of the disease was observed among farmers from North Africa compared with those of European countries. In conclusion, the results obtained highlight that CE is still a very serious problem in Mediterranean areas and increased efforts are needed to promote awareness among farmers and to turn research results into policy in order to reduce the spread of this disease, according to the One Health perspective.

Baiting not-owned dogs against Echinococcus granulosus: innovative tools for integrated control.

Cystic echinococcosis (CE), caused by the larval stage of the cestode Echinococcus granulosus, is one of the most widespread zoonoses in Mediterranean countries. Baiting not-owned dogs with praziquantel (PZQ), due to their key role in the maintaining the transmission of CE, currently appears to be the most effective way to limit the transmission of CE, as well as an important aspect to introduce for the control of this parasitic disease. Therefore, this study aims to test 3 types of PZQ-based baits by evaluating different parameters (integrity over time, attractiveness and palatability for dogs, and mechanical resistance after release to different altitudes) and the bait acceptance in field by target animals, i.e. not-owned dogs, by using camera traps. The double PZQ-laced baits (with a double layer of highly palatable chews) showed the greatest resistance in the environment while also preserving the attractiveness and palatability up to 10 days, also withstood heights of 25 m, thus resulting as the most suitable also for drone delivery. The results on the field showed that most of the baits were consumed by not-owned dogs (82.2%), while the remaining were consumed by wild boars (8.9%), foxes (6.7%), badgers (1.1%) and hedgehogs (1.1%), confirming the specific and high attractiveness of the double PZQ-laced baits for the target population and highlights how an anthelmintic baiting programme may be a viable tool for the management of E. granulosus among free-ranging dog populations in endemic rural areas.

An innovative strategy for deworming dogs in Mediterranean areas highly endemic for cystic echinococcosis.

BACKGROUND: Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus sensu lato, is a zoonotic parasitic disease of economic and public health importance worldwide, especially in the Mediterranean area. Canids are the main definitive hosts of the adult cestode contaminating the environment with parasite eggs released with feces. In rural and peri-urban areas, the risk of transmission to livestock as well as humans is high because of the free-roaming behavior of owned/not owned dogs. Collecting data on animal movements and behavior using GPS dataloggers could be a milestone to contain the spread of this parasitosis. Thus, this study aims to develop a comprehensive control strategy, focused on deworming a dog population in a pilot area of southern Italy (Campania region) highly endemic for CE. METHODS: Accordingly, five sheep farms, tested to be positive for CE, were selected. In each sheep farm, all shepherd dogs present were treated every 2 months with praziquantel. Furthermore, 15 GPS dataloggers were applied to sheep and dogs, and their movements were tracked for 1 month; the distances that they traveled and their respective home ranges were determined using minimum convex polygon (MCP) analysis with a convex hull geometry as output. RESULTS: The results showed that the mean daily walking distances traveled by sheep and dogs did not significantly differ. Over 90% of the point locations collected by GPS fell within 1500 mt of the farm, and the longest distances were traveled between 10:00 and 17:00. In all the sheep farms monitored, the area traversed by the animals during their daily activities showed an extension of < 250 hectares. Based on the home range of the animals, the area with the highest risk of access from canids (minimum safe convex polygon) was estimated around the centroid of each farm, and a potential scheme for the delivery of praziquantel-laced baits for the treatment of not owned dogs gravitating around the grazing area was designed. CONCLUSIONS: This study documents the usefulness of geospatial technology in supporting parasite control strategies to reduce disease transmission.

Development of an oral nanovaccine for dogs against Echinococcus granulosus.

Dogs are the main source of animal and human cystic echinococcosis caused by the Cestode parasite Echinococcus granulosus. Dog vaccination seems to be a good strategy to control this parasitic disease. Here we present the development of a polymeric nanoparticle-based oral vaccine for dogs against Echinococcus granulosus delivered in enteric-coated capsules. To achieve our target, we encapsulated two recombinant antigens into biodegradable polymeric nanoparticles in the presence of Monophosphoryl lipid A as an adjuvant to ensure efficient delivery and activation of a protective mucosal immune response. The formulated delivery system showed a nanoparticle size less than 200 nm with more than 80 % antigen encapsulation efficiency and conserved integrity and immunogenicity. The nanoparticle surface was coated with chitosan to enhance adhesion to the gut mucosa and a subsequent antigen delivery. Chitosan-coated nanoparticles showed a higher cell internalization in murine macrophages and dendritic cells as well as a higher penetration into Caco-2 cells in vitro. Antigen-loaded nanoparticles were freeze-dried and enteric-coated capsules were filled with the obtained powder. The obtained results show a promising nanoparticles delivery system for oral vaccination.

Prevalence, risk factors and emergence of extended-spectrum ß-lactamase producing-, carbapenem- and colistin-resistant Enterobacterales isolated from wild boar (Sus scrofa) in Tunisia.

Antimicrobial resistance (AMR) is recognized as an emerging and growing public health problem worldwide. In Tunisia, knowledge is still limited to domestic animals and humans, and only few data are available regarding the role of wildlife. This research determined the antibiotic susceptibility profiles of Beta-lactamase producing Gram-negative bacteria isolated from the faeces of 110 wild boars (Sus scrofa) in northern Tunisia. Fecal samples, obtained post mortem from boar carcasses, were cultured on MacConkey agar and MacConkey agar containing 2 mg/L of cefotaxime. A total of 102 Enterobacterales isolates were identified from 94(85%) fecal samples. Escherichia coli (56, 54%), Citrobacter freundii (14, 13%), Klebsiella oxytoca (11, 10%), and Klebsiella pneumoniae (7, 6%) were the most predominantly identified Enterobacterales. However, Pantoea spp. (4, 4%), Enterobacter spp. (3,3%), Enterobacter cloacae (1, 1%), Enterobacter gergoviae (2, 2%), Proteus mirabilis (2, 2%), Yersinia sp. (1, 1%), and Citrobacter diversus (1, 1%) were rarely identified. Antimicrobial susceptibility tests revealed that 55% (57/102) of the identified strains were multidrug resistant (MDR). A total of 30% (31/102) of the tested isolates were recognized as Extended Spectrum ß-Lactamase (ESBL)-producing strains and blaCTX-M-G1, blaTEM, blaSHV ß-lactamases were the main encoding genes revealed. Furthermore, identified isolates showed a high level of AMR, especially for amoxicillin-clavulanic acid (77.67%), ticarcillin-clavulanic acid (71.85%), streptomycin (76.69%), amoxicillin (75.73%), and cephalotin (74.76%). Alarming levels of resistance to colistin (2.9%) and ertapenem (9.7%) were revealed and confirmed by the detection of mcr-1, and blaIMP and blaVIM genes, respectively. Various phenotypes of AMR were obtained in this study highlighting the important role of wild boars as hosts and even carriers for several resistant Enterobacterales isolates. This may represents a focal risk factor allowing the transmission of these strains between domestic, wild animals, environment and humans.

Zoonotic vector-borne bacteria in wild rodents and associated ectoparasites from Tunisia.

Wild rodents are considered as potential carriers of several zoonotic vector-borne bacteria but their epidemiology is poorly understood in Tunisia. A total of 305 biological samples (100 spleens, 100 livers, 100 kidneys, and 5 pooled ectoparasites (Xenopsylla cheopis, Laelaps echidninus, Ornithonyssus sp., Hoplopleura sp. and eggs of the rat fleas)) were collected from 100 wild rodents from three Tunisian governorates. Molecular screening was performed to reveal infections with main vector-borne bacteria. Captured rodents belonged to three rodent genera and species including Rattus rattus (n = 51, 51%), Meriones shawi (n = 24, 24%) and Mus musculus (n = 25, 25%). Examined rodents were found to be heavily infested by the rat flea X. cheopis (n = 32, 47%) and the rat mite L. echidninus (n = 22, 32.3%). However, the rat mite Ornithonyssus sp. (n = 13, 19.1%) and the rat lice Hoplopleura sp. (n = 1, 1.5%) were rarely identified. Based on 16S rRNA and msp4 genes, infection with Anaplasmataceae bacteria was detected in six specimens of R. rattus and one M. shawi. Pathogenic A. phagocytophilum (n = 1), A. phagocytophilum-like 1 (Anaplasma sp. Japan) (n = 1), and A. ovis (n = 5) were identified. On the basis of ompB, ompA and gltA genes, infection with Rickettsia spp. was identified in three specimens of R. rattus and one of M. shawi. Five Rickettsia species of the spotted fever group, corresponding to R. monacensis, R. helvetica, R. massiliae, R. africae, and R. aeschlimannii, were detected in mixed infections. Bartonella henselae DNA was also found in two R. rattus, based on rpoB partial sequences. All revealed Anaplasma, Rickettsia and Bartonella bacteria were detected in spleen samples. Ehrlichia, Coxiella and Borrelia spp. were not identified in any of the tested samples. In Tunisia, this is the first report indicating infections with Anaplasma, Rickettsia and Bartonella spp. in wild rodents, particularly present alongside domestic livestock and human. This represents a serious risk of potential bacterial transmission. Thus, controlling rodent population in animal herds, residential areas and sensitizing local people to this risk seem absolutely necessary.

First survey of ticks, tick-borne pathogens (Theileria, Babesia, Anaplasma and Ehrlichia) and Trypanosoma evansi in protected areas for threatened wild ruminants in Tunisia.

The aim of the study was to identify ticks present in the environment and wild Tunisian ruminants and to detect tick-borne pathogens and Trypanosoma evansi DNA in these specimens. Sampling was done throughout each season from the environment in three protected areas around Tunisia: El Feidja, Haddaj and Oued Dekouk. Ticks were collected also, from one fawn of Barbary red deer and eight naturally deceased wild ruminants (one Barbary red deer, five Scimitar-horned oryx, one Addax antelope and one Dorcas gazelle), all of which lived in various protected areas. PCR and nested PCRs were performed to detect the presence of Theileria spp., Babesia spp., Trypanosoma evansi, Ehrlichia spp., Anaplasma spp., Anaplasma bovis and Anaplasma phagocytophilum DNA in these tick specimens. A total of 352 ticks were collected, belonging to six different species: Hyalomma excavatum (80.6%), Hyalomma dromedarii (10.2%), Hyalomma marginatum (0.5%), Rhipicephalus bursa (0.5%), Rhipicephalus sanguineus sensu lato (5.1%) and Ixodes ricinus (2.8%). Pathogens have been detected in 25% of H. dromedarii, 9.1% of H. excavatum and 5% of R. sanguineus sensu lato. The percentage of detection of T. evansi was 0.2%. Ehrlichia spp.-Anaplasma spp. were detected in 10.1% of ticks. Anaplasma spp. and A. bovis were detected in 7.6%, and 0.8% of examined ticks, respectively. None of the Theileria spp., Babesia spp., or A. phagocytophilum DNA was detected in the tested ticks. To our knowledge, the present study represents the first identification of these six tick species and the first detection of rickettsial pathogens and T. evansi in North African wild ruminants' species. These results extend the knowledge about the diversity of ticks and tick-borne pathogens in wildlife and justify further investigations of the possible role of R. sanguineus sensu lato in the transmission of T. evansi.

Cystic echinococcosis in the Eastern Mediterranean region: Neglected and prevailing!

Cystic echinococcosis (CE) is distributed worldwide, extending from China to the Middle East and from Mediterranean countries to the sub-Saharan Africa and South America. According to WHO, one million people around the world are suffering from CE with an estimated burden of 183,573 DALYs. The annual monetary burden of the disease due to treatment costs and CE-related livestock losses has been estimated at US$ 3 billion. CE is endemic in all countries within the WHO Eastern Mediterranean Regional Office (EMRO). The region, which includes most of the Middle East and North Africa, is one of the most ancient foci of the domestic cycle of CE and is recognized as one of the major hotspots of CE. There are 22 countries in the EMRO, where about 688 million people are living at risk of CE. In many EMRO countries, little is known about CE epidemiology and transmission. WHO included echinococcosis in a list of 17 neglected tropical diseases (NTDs) and 12 neglected zoonotic diseases (NZDs). Accordingly, different regional offices of WHO organized several initiatives for CE control and prevention. WHO's Western Pacific regional office considered echinococcosis as one of the region's major health topics, and several preventive measures have been implemented in the American region with the support of Pan American Health Organization (PAHO) in Argentina, Peru, Uruguay, and Chile. Although CE is endemic in all 22 EMRO countries, surprisingly, CE is absent from the health topics list of diseases and conditions in this region. Therefore, CE clearly requires further attention in the WHO EMRO agenda, and the need for elaboration of specific measures for CE control is becoming apparent in EMRO countries, where substantial collaborations among the member states and WHO EMRO is of paramount importance. Major topics of collaborative activities include training programs and health communication on different aspects of CE control, analysis of CE burden, national and international surveillance and disease registry systems, technical support to promote epidemiological studies for collecting baseline data, cost-benefit analysis of control interventions, and intersectoral cooperation among the agriculture, veterinary, medical, and health sectors.

Cystic echinococcosis and other helminth infections of wild boar in northeastern and northwestern regions of Tunisia.

This study identified helminth species of wild boar (Sus scrofa) originating from northeastern and northwestern regions of Tunisia using 297 lungs, 297 livers, 264 intestinal tracts, 120 samples of muscle tissue (tongue, masseter, diaphragm, inter-costal) and 232 faecal samples derived from a total of 591 animals. Host gender was registered for the lung and liver wild boar group, which included 163 males and 134 females. All animals, excluding those used to retrieve muscular samples, were classified into three age classes, <2 (n = 212), 2-3 (n = 208) and ⩾4 years old (n = 141). Helminth fauna of the examined wild boar included 14 parasite species: one trematode (adult, Brachylaemus suis), three cestodes (metacestodes of Echinococcus granulosus, Taenia hydatigena cysticercus, adult, Hymenolepis diminuta), nine nematodes (adults of Metastrongylus apri, Metastrongylus pudendotectus, Ascarops strongylina, Globocephalus urosubulatus, Physocephalus sexalatus, Gnathostoma hispidum, Gongylonema pulchrum and eggs of Strongyloides ransoni and Capillaria spp.) and one acanthocephalan (adult, Macracanthorhynchus hirudinaceus). Trichinella larvae were not recovered from any of the 30 wild boar examined. Results showed a 73.5% global prevalence of infection with visceral helminths, 67.3% of which were lung and hepatic infections and 80.3% of helminths were recovered from the gastrointestinal tract. The most prevalent parasite was M. hirudinaceus (61.7%) while the highest intensity of infection was observed for Metastrongylus spp. The most prevalent cestode was E. granulosus (18.9%). This is the first detailed study on helminth infections of wild boar from a North African country.

Species delimitation based on mtDNA genes suggests the occurrence of new species of Mesocestoides in the Mediterranean region.

BACKGROUND: This study is the first contribution to the molecular taxonomy of Mesocestoides spp. from domestic and wild carnivores in the Mediterranean area. A total of 13 adult worms and 13 larval stages of Mesocestoides spp. were collected from domestic and wild carnivore hosts in Italy and Tunisia. Samples collected in the Slovak Republic were used as comparative samples from outside the Mediterranean. The genes cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) of the mitochondrial genome were used as molecular markers to investigate the presence of cryptic Mesocestoides species in the area analysed. RESULTS: Results were consistent in showing three well-supported clusters of Mesocestoides spp. in southern Italy and Tunisia, which were strongly divergent from Mesocestoides litteratus, M. corti and M. lineatus. High levels of genetic variation and no evidence of geographical structuring was found between the clusters. CONCLUSIONS: Considering the low dispersal capability of the intermediate hosts of Mesocestoides spp., the lack of geographical structuring among the Mediterranean regions could be due to a high potential for dispersion of the definitive hosts. This study provides a foundation for future formal descriptions of new species of the genus Mesocestoides in the Mediterranean area.

Identification of first-stage dorsal-spined lungworm larvae of Tunisian barbary red deer: First report of Varestrongylus sagittatus and Elaphostrongylus cervi in Africa.

Barbary red deer (Cervus elaphus barbarus) is a protected rare subspecies of red deer. The study of its Protostrongylidae fauna based only on sporadic necropsy of naturally dead animals is difficult. Therefore diagnosis of lungworms rely mainly on the identification of the first stage larvae (L1). The L1 of the different species are not readily diagnosed on morphological basis since much variation is recorded within and among dorsal-spined larvae belonging to various species. The aim of this study was to identify the dorsal-spined lungworm larvae of the Barbary red deer. A discriminant function was established, using the measurements of L1 lungworms recorded from red deer in the literature, then applied to identify 220 dorsal-spined larvae extracted from 25 Tunisian Barbary red deer fresh fecal samples. Also the ITS2 region of rDNA of four pools of larvae (n = 25-60) were amplified, sequenced and analyzed. Using discriminant analysis of morphological traits, Elaphostrongylus cervi and Varestongylus sagittatus were identified. Molecular identification confirmed the presence of E. cervi which is the most prevalent species. This study represents the first identification of V. sagittatus and E. cervi in North Africa.

Molecular identification of parasitic nematodes (Nematoda: Strongylida) in feces of wild ruminants from Tunisia.

In Tunisia and other North African countries, there is a lack of knowledge about parasite biodiversity within threatened wild ruminants and there are not any studies on their gastrointestinal nematodes. Thus the aim of this study was to identify gastrointestinal fauna in the faecal samples of Tunisian wild ruminants. A total of 262 faecal samples were collected from domestic sheep and goat, and wild ruminants (Addax, Barbary sheep, Barbary red deer, Dorcas gazelle, Slender-horned gazelle and Scimitar-horned Oryx) living in protected areas. Samples were examined with floatation (saturated sodium chloride solution), polymerase chain reaction and sequencing of the second internal transcribed spacer region of the rDNA. Microscopic analysis allowed the identification of only Nematodirus genus or molecular tools allowed a first identification of five gastrointestinal nematode species in North African wild ruminants: Chabertia ovina (1.6%), Camelostrongylus mentulatus (1.6%), Marshallagia marshalli (4.7%), Nematodirus helvetianus (62.5%) and Nematodirus spathiger (29.7%). This study reported the first records of C. mentulatus and M. marshalli in Addax and of M. marshalli in Dorcas gazelle and it was the first reported record of N. helvetianus and M. marshalli in Tunisia.

Reprint of "Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia".

Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A. nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

A preliminary investigation into the genetic variation and population structure of Taenia hydatigena from Sardinia, Italy.

Cysticercosis caused by the metacestode stage of Taenia hydatigena is endemic in Sardinia. Information on the genetic variation of this parasite is important for epidemiological studies and implementation of control programs. Using two mitochondrial genes, the cytochrome c oxidase subunit 1 (cox1) and the NADH dehydrogenase subunit 1 (ND1) we investigated the genetic variation and population structure of Cysticercus tenuicollis from Sardinian intermediate hosts and compared it to that from other hosts from various geographical regions. The parsimony cox1 network analysis indicated the existence of a common lineage for T. hydatigena and the overall diversity and neutrality indices indicated demographic expansion. Using the cox1 sequences, low pairwise fixation index (Fst) values were recorded for Sardinian, Iranian and Palestinian sheep C. tenuicollis which suggested the absence of genetic differentiation. Using the ND1 sequences, C. tenuicollis from Sardinian sheep appeared to be differentiated from those of goat and pig origin. In addition, goat C. tenuicollis were genetically different from adult T. hydatigena as indicated by the statistically significant Fst value. Our results are consistent with biochemical and morphological studies that suggest the existence of variants of T. hydatigena.

Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia.

Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A.nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

Echinococcus equinus and Echinococcus granulosus sensu stricto from the United Kingdom: genetic diversity and haplotypic variation.

Cystic echinococcosis is endemic in Europe including the United Kingdom. However, information on the molecular epidemiology of Echinococcus spp. from the United Kingdom is limited. Echinococcus isolates from intermediate and definitive animal hosts as well as from human cystic echinococcosis cases were analysed to determine species and genotypes within these hosts. Echinococcus equinus was identified from horse hydatid isolates, cysts retrieved from captive UK mammals and copro-DNA of foxhounds and farm dogs. Echinococcus granulosus sensu stricto (s.s.) was identified from hydatid cysts of sheep and cattle as well as in DNA extracted from farm dog and foxhound faecal samples, and from four human cystic echinococcosis isolates, including the first known molecular confirmation of E. granulosus s.s. infection in a Welsh sheep farmer. Low genetic variability for E. equinus from various hosts and from different geographical locations was detected using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), indicating the presence of a dominant haplotype (EQUK01). In contrast, greater haplotypic variation was observed for E. granulosus s.s. cox1 sequences. The haplotype network showed a star-shaped network with a centrally placed main haplotype (EgUK01) that had been reported from other world regions.

Genetic variability and haplotypes of Echinococcus isolates from Tunisia.

BACKGROUND: The species/genotypes of Echinococcus infecting a range of intermediate, canid and human hosts were examined as well as the intraspecific variation and population structure of Echinococcus granulosus sensu lato (s.l.) within these hosts. METHODS: A total of 174 Echinococcus isolates from humans and ungulate intermediate hosts and adult tapeworms from dogs and jackals were used. Genomic DNA was used to amplify a fragment within a mitochondrial gene and a nuclear gene, coding for cytochrome c oxidase subunit 1 (cox1; 828 bp) and elongation factor 1-alpha (ef1a; 656 bp), respectively. RESULTS: E. granulosus sensu stricto was identified from all host species examined, E. canadensis (G6) in a camel and, for the first time, fertile cysts of E. granulosus (s.s.) and E. equinus in equids (donkeys) and E. granulosus (s.s.) from wild boars and goats. Considerable genetic variation was seen only for the cox1 sequences of E. granulosus (s.s.). The pairwise fixation index (Fst) for cox1 E. granulosus (s.s.) sequences from donkeys was high and was statistically significant compared with that of E. granulosus populations from other intermediate hosts. A single haplotype (EqTu01) was identified for the cox1 nucleotide sequences of E. equinus. CONCLUSIONS: The role of donkeys in the epidemiology of echinococcosis in Tunisia requires further investigation.

Modelling the transmission dynamics of cystic echinococcosis in donkeys of different ages from Tunisia.

During the period from March 2006 to July 2009 a total of 2040 slaughtered donkeys were examined for cystic echinococcosis (CE). CE prevalence in donkeys was 8.48% and the infection pressure (0.0088 infections per year) and infection rate (0.0448 cysts per year) appeared to be lower than those previously reported for cattle, sheep, dromedaries and goats in Tunisia. However, the number of cysts per infection was relatively high (5.07 cysts per infection). Among the 901 collected hydatid cysts the majority were located in the liver (89.9%), 10.09% in the lungs and 4.77% were fertile (43/901). The amplification of a fragment within the mitochondrial gene coding for cytochrome c oxidase subunit 1 (cox1) revealed that donkeys were infected with both Echinococcus equinus (horse strain, G4 genotype) and Echinococcus granulosus (sheep strain, G1 genotype). E. granulosus G1 developed into fertile cysts (15,112 protoscoleces/ml) with a protoscoleces viability of 65.78%. This investigation is the first detailed epidemiological report on cystic echinococcosis infection in donkeys for any endemic region.

Intestinal helminths of golden jackals and red foxes from Tunisia.

Forty wild canids including 31 golden jackals (Canis aureus Linné, 1758) and 9 red foxes (Vulpes vulpes Linné, 1758) collected between 2008 and 2011 in the northeast, northwest and center of Tunisia were necropsied and examined for intestinal helminth parasites. All jackals and foxes were found infected with a prevalence rate of 95% for cestodes, 82.5% for nematodes and 7.5% for acanthocephalans. A total of twelve helminth species were recorded in red foxes: cestodes, Dipylidium caninum (55.6%), Diplopylidium noelleri (55.6%), Mesocestoïdes lineatus (55.6%), Mesocestoïdes litteratus (33%), Mesocestoïdes corti (22%); nematodes, Ancylostoma caninum (11%), Uncinaria stenocephala (44%), Spirura rytipleurites (11%), Trichuris vulpis (33%), Pterygodermatites affinis (67%), Oxynema linstowi (33%) and the acanthocephalan Macracanthorhynchus hirudinaceus (22%). The fifteen recovered helminth species in jackals were Echinococcus granulosus (9.7%), D. caninum (16%), D. noelleri (16%), M. lineatus (74%), M. litteratus (23%), M. corti (12.9%), Taenia pisiformis (3.2%), Taenia spp. (19%), Toxocara canis (16%), Toxascaris leonina (6.5%), A. caninum (9.7%), U. stenocephala (68%), P. affinis (6.5%), O. linstowi (3.2%) and Macracanthorhynchus hirudinaceus (3.2%). This is the first report on the presence of P. affinis, D. noelleri and O. linstowi in Tunisia. E. granulosus was found in young jackals, aged less than 4 years old, with a higher abundance in females (8.9 worms). M. lineatus presented the highest mean intensity of 231.86 and 108.8 tapeworms respectively in jackals and foxes. Canids from the northwest region had the highest prevalence (77.5%) and highest intensity (243.7) of helminth species compared to those from the northeast and central areas. U. stenocephala and O. linstowi had the highest mean intensity for nematodes in both jackals and foxes at 14.3 and 88 worms respectively.

Development of three PCR assays for the differentiation between Echinococcus shiquicus, E. granulosus (G1 genotype), and E. multilocularis DNA in the co-endemic region of Qinghai-Tibet plateau, China.

To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples.