A validated method to identify Echinococcus granulosus sensu lato at species level.

The zoonotic tapeworm Echinococcus granulosus sensu lato (s.l.) represents a species complex encompassing multiple causative agents of cystic echinococcosis, a neglected tropical disease affecting more than one million people in the world. At least eight genotypes, grouped in five species, are currently recognized within this species complex, and they differ in terms of relative public health impact. Here we present a molecular method that first identifies the common E. granulosus sensu stricto (s.s.) (genotypes G1 and G3) based on a PCR-RFLP assay, and can further identify the remaining species based on a multiplex PCR assay. We demonstrate the applicability of the method to DNA extracted from parasitic cyst material of human and animal origin, preserved in ethanol or frozen. The method has been developed and validated at the European Union Reference Laboratory for Parasites (EURLP), according to the ISO/IE 17025.

Efficacy of novel albendazole salt formulations against secondary cystic echinococcosis in experimentally infected mice.

In this study, we evaluated the efficacy, expressed as a mean weight decrease of the whole echinococcal cyst mass, of novel benzimidazole salt formulations in a murine Echinococcus granulosus infection model. BALB/c mice were intraperitoneally infected with protoscoleces of E. granulosus (genotype G1). At 9 months post-infection, treatment with albendazole (ABZ), ricobendazole (RBZ) salt formulations, and RBZ enantiomer salts (R)-(+)-RBZ-Na and (S)-(-)-RBZ-Na formulations were initiated. Drugs were orally applied by gavage at 10 mg kg-1 body weight per day during 30 days. Experimental treatments with benzimidazole sodium salts resulted in a significant reduction of the weight of cysts compared to conventional ABZ treatment, except for the (S)-(-)-RBZ-Na enantiomer formulation. Scanning electron microscopy and histological inspection revealed that treatments impacted not only the structural integrity of the parasite tissue in the germinal layer, but also induced alterations in the laminated layer. Overall, these results demonstrate the improved efficacy of benzimidazole salt formulations compared to conventional ABZ treatment in experimental murine cystic echinococcosis.

Laboratory Diagnosis of Echinococcus spp. in Human Patients and Infected Animals.

Among the species composing the genus Echinococcus, four species are of human clinical interest. The most prevalent species are Echinococcus granulosus and Echinococcus multilocularis, followed by Echinococcus vogeli and Echinococcus oligarthrus. The first two species cause cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively. Both diseases have a complex clinical management, in which laboratory diagnosis could be an adjunctive to the imaging techniques. To date, several approaches have been described for the laboratory diagnosis and followup of CE and AE, including antibody, antigen and cytokine detection. All of these approaches are far from being optimal as adjunctive diagnosis particularly for CE, since they do not reach enough sensitivity and/or specificity. A combination of several methods (e.g., antibody and antigen detection) or of several (recombinant) antigens could improve the performance of the adjunctive laboratory methods, although the complexity of echinococcosis and heterogeneity of clinical cases make necessary a deep understanding of the host-parasite relationships and the parasite phenotype at different developmental stages to reach the best diagnostic tool and to make it accepted in clinical practice. Standardization approaches and a deep understanding of the performance of each of the available antigens in the diagnosis of echinococcosis for the different clinical pictures are also needed. The detection of the parasite in definitive hosts is also reviewed in this chapter. Finally, the different methods for the detection of parasite DNA in different analytes and matrices are also reviewed.

Correlation of serum sHLA-G levels with cyst stage in patients with cystic echinococcosis: is it an immune evasion strategy?

Patients with cystic echinococcosis (CE) can harbour cysts for years or even decades, apparently without effect of the immune system on the metacestode. Although several immune evasion mechanisms by echinococcal cysts have been described, it is unclear whether the human leucocyte antigen (HLA) system plays a role in the susceptibility or resistance to CE in humans. HLA-G molecules are known to exert a suppressive action on dendritic cells maturation and on natural killer (NK) cells functions, therefore hampering T-cell responses and NK cytolysis. HLA-G plays an important role in immune tolerance, is involved in foetus and in allotransplant tolerance, and may be involved in tumoral and viral immune evasion. In this study, we assessed the presence and levels of soluble HLA-G (sHLA-G) in patients with CE using a commercial ELISA kit to determine whether host's HLA-G may have a role in the course of human CE.

The Italian registry of cystic echinococcosis (RIEC): the first prospective registry with a European future.

Cystic echinococcosis (CE), a worldwide zoonosis, is highly endemic in southern and eastern Europe. Its actual prevalence is unknown due to the lack of efficient reporting systems designed to take into account the particular features of the disease. Neglect of CE makes diagnosis and clinical management difficult outside referral centres, with inconsistencies in clinical practice and often unnecessary procedures carried out that have associated risks and costs. The Italian registry of CE (RIEC) is a prospective multicentre registry of CE patients seen from January 2012 in Italian health centres; data are voluntarily submitted to the registry. Its aims are to show the prevalence of CE in Italy, bring the importance of this infection to the attention of health authorities, encourage public health policies towards its control, and stimulate biological, epidemiological and clinical research on CE. From January 2012 to February 2014, a total 346 patients were enrolled in 11 centres, outnumbering national reports of many CE-endemic European countries. We discuss preliminary data and challenges of the RIEC, template for the European registry of CE, which has been implemented within the Seventh Framework Programme project HERACLES (Human cystic Echinococcosis ReseArch in CentraL and Eastern Societies) since September 2014.

Spatial distribution and genetic diversity of Echinococcus multilocularis in Hungary.

Human alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is the most pathogenic helminthic zoonosis in the temperate and arctic region of Europe. Between November 2008 and February 2009, 840 red fox (Vulpes vulpes) carcasses, were randomly collected from the whole Hungarian territory. The intestinal mucosa from all the foxes was tested by sedimentation and counting technique. E. multilocularis adult worms were detected in foxes of 16 out of the 19 Hungarian counties and in the suburban areas of the capital, Budapest. The prevalence and abundance of infection was significantly (P<0.001) higher in the north-western half (16.2%, CI=14.5-17.9; m ± SE=165.5 ± 112.4) than in the south-eastern half of the country (4.2%, CI=3.2-5.2; m ± SE=3.6 ± 2.1). The highest prevalence (26.6%, CI=22.5-30.8%) and abundance (m ± SE=614.2 ± 469.3) was observed in the Northern Mountain Region bordering Slovakia. The multi-locus microsatellite analysis of 81 worms showed the presence of four out of the five main European profiles. The H profile was the most common profile (55.5%) with nine genotypes, followed by the G (18.5%) with two genotypes, E (13.6%) with one genotype and D (12.4%) with two genotypes. The genetic distance was not statistically correlated with the geographical distance of the samples, supporting the hypothesis that the geographical distance is only a minor factor among those involved in the genetic distribution of this parasite in Europe. These data indicate that Hungary should be considered as a peripheral area of a single European focus, where the dispersal movement of foxes resulted in the spreading of the parasite from one county to another within a time period short enough to avoid a substantial genetic drift.

Multi-locus microsatellite analysis supports the hypothesis of an autochthonous focus of Echinococcus multilocularis in northern Italy.

Echinococcus multilocularis is characterised by a wide geographical distribution, encompassing three continents (North America, Asia and Europe) yet very low genetic variability is documented. Recently, this parasite has been detected in red foxes (Vulpes vulpes) circulating in an Alpine region of Italy, close to Austria. This finding raised the question as to whether an autochthonous cycle exists in Italy or whether the infected foxes originated from the neighbouring regions of Austria. Studies have shown that multi-locus microsatellite analysis can identify genomic regions carrying mutations that result in a local adaptation. We used a tandem repeated multi-locus microsatellite (EmsB) to evaluate the genetic differences amongst adult worms of E. multilocularis collected in Italy, worms from neighbouring Austria and from other European and extra-European countries. Fluorescent PCR was performed on a panel of E. multilocularis samples to assess intra-specific polymorphism. The analysis revealed four closed genotypes for Italian samples of E. multilocularis which were unique compared with the other 25 genotypes from Europe and the five genotypes from Alaska. An analysis in the Alpine watershed, comparing Italian adult worms with those from neighbouring areas in Austria, showed a unique cluster for Italian samples. This result supports the hypothesis of the presence of an autochthonous cycle of E. multilocularis in Italy. EmsB can be useful for 'tracking' the source of infection of this zoonotic parasite and developing appropriate measures for preventing or reducing the risk of human alveolar echinococcosis.

Echinococcus multilocularis in north Italy.

Alveolar echinococcosis is a zoonotic infection caused by the metacestode of the tapeworm Echinococcus multilocularis. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes were detected in Bolzano province (Trentino Alto Adige region) near Austrian border. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples belonging to red fox populations from five Italian regions. A total of 522 faecal samples were analysed from foxes shot in Valle d'Aosta (N = 65), Liguria (N = 44), Lombardy (N = 105), Veneto (N = 67), and Trentino Alto Adige (N = 241) regions. Among these, 24 samples, all from the Trentino Alto Adige region, were found positive. Moreoever, 1406 faecal samples of red foxes were analyzed by CA-ELISAs commercial test kit. This paper provides an update of the epidemiological knowledge of this parasite in north Italy.

Echinococcus multilocularis in red foxes (Vulpes vulpes) of the Italian Alpine region: is there a focus of autochthonous transmission?

Alveolar echinococcosis, caused by the metacestode of Echinococcus multilocularis, is a zoonosis with a wider distribution area than described in the past. Fox populations living in the Alpine regions of Italy had been considered free from this parasite until 2002, when two infected foxes (Vulpes vulpes) were detected in the Bolzano province (Trentino Alto Adige region) near the Austrian border. The aim of this work was to evaluate the prevalence of infection in red fox populations from five Italian regions. A modified nested PCR analysis was used to detect E. multilocularis DNA in faecal samples. Amplicons were confirmed by sequencing. Of 500 faecal samples from foxes shot in Valle d'Aosta (n=57), Liguria (n=44), Lombardy (n=102), Veneto (n=56), and Trentino Alto Adige (n=241) regions, 24 animals, all from the Trentino Alto Adige region, were found positive. Twenty-two positive animals originated from the Bolzano province and two positive animals from the Trento province. Several localities of the Bolzano province, in which positive foxes were detected, are the same as those where alveolar echinococcosis had been described in humans in the second half of the 19th century, suggesting an old endemicity for the investigated area, which is adjacent to endemic areas of Austria. Therefore, the question arises if we are observing an increase and expansion of foci, or if the new records are due to the more sensitive and specific methods used to detect the worm DNA.

Trichinella papuae and Trichinella zimbabwensis induce infection in experimentally infected varans, caimans, pythons and turtles.

The discovery of Trichinella zimbabwensis in farm crocodiles of Zimbabwe has opened up a new frontier in the epidemiology of the Trichinella genus. The objective of the present study was to investigate the infectivity of encapsulated species (T. spiralis, T. nativa, T. britovi, T. murrelli and T. nelsoni) and non-encapsulated species (T. pseudospiralis, T. papuae and T. zimbabwensis) in caimans (Caiman crocodilus), varans (Varanus exanthematicus), pythons (Python molurus bivittatus) and turtles (Pelomedusa subrufa) raised at their natural temperature range (26-32 degrees C). Mice and chickens were used as controls. At 6 days post-infection (p.i.), adult worms were detected in the small intestine of reptiles infected with T. papuae and T. zimbabwensis, of chickens infected with T. pseudospiralis and of mice infected with all encapsulated and non-encapsulated species. At 60 days p.i., T. papuae and T. zimbabwensis adult worms were collected from the intestine of varans and caimans and larvae from muscles of the four reptile species, T. pseudospiralis larvae from muscles of chickens, and larvae of all Trichinella species from mouse muscles. The highest reproductive capacity index of both T. papuae and T. zimbabwensis was observed in varans. The results show that T. papuae and T. zimbabwensis are able to complete their entire life-cycle in both poikilothermic and homoiothermic animals.

Molecular identification of natural hybrids between Trichinella nativa and Trichinella T6 provides evidence of gene flow and ongoing genetic divergence.

To date, there are no data available on the population genetics of Trichinella due to the lack of genetic markers and the difficulty of working with such small parasites. In the Arctic region of North America and along the Rocky Mountains, there exist two genotypes of Trichinella, Trichinella nativa and Trichinella T6, respectively, which are well differentiated by biochemical and molecular characters. However, both are resistant to freezing, show other common biological characters (e.g. low or no infectivity to rodents and swine) and produce fertile F1 offspring upon interbreeding. To data, these two genotypes have been considered allopatric. In this study, we detected both genotypes in wolves of the same wolf packs in Alaska, suggesting sympatry. A single GTT trinucleotide present in the ITS-2 sequence of T. nativa but not in Trichinella T6 was used as a genetic marker to study gene flow for this character in both a murine infection model and in larvae from naturally-infected Alaskan wolves. Only F1 larvae originating from a cross between T. nativa male and Trichinella T6 female were able to produce F2 offspring. Larvae (F1) originating from a cross between Trichinella T6 male and T. nativa female were not reproductively viable. As expected, all F1 larvae showed a heterozygote pattern for the GTT character upon heteroduplex analysis; however, within the F2 population, the number of observed heterozygotes (n=52) was substantially higher than expected (n=39.08), as supported by the F(is) index, and was not in the Hardy-Weinberg equilibrium. Larvae from two of the 16 Trichinella positive Alaskan wolves, showed the Trichinella T6 pattern or the T. nativa/Trichinella T6 hybrid pattern. Our data demonstrate that T. nativa and Trichinella T6 live in sympatry at least in Alaskan wolves, where T. nativa occurs more frequently (69%) than Trichinella T6 (31%). One explanation for this phenomenon is that glacial periods may have caused a geographical relocation, colonisation and independent evolution of T. nativa within the Rocky Mountains, resulting in a bifurcation of the freeze-resistant genotype. Additional studies will be required to test this hypothesis.

Combined use of maternal, paternal and bi-parental genetic markers for the identification of wolf-dog hybrids.

The identification of hybrids is often a subject of primary concern for the development of conservation and management strategies, but can be difficult when the hybridizing species are closely related and do not possess diagnostic genetic markers. However, the combined use of mitochondrial DNA (mtDNA), autosomal and Y chromosome genetic markers may allow the identification of hybrids and of the direction of hybridization. We used these three types of markers to genetically characterize one possible wolf-dog hybrid in the endangered Scandinavian wolf population. We first characterized the variability of mtDNA and Y chromosome markers in Scandinavian wolves as well as in neighboring wolf populations and in dogs. While the mtDNA data suggested that the target sample could correspond to a wolf, its Y chromosome type had not been observed before in Scandinavian wolves. We compared the genotype of the target sample at 18 autosomal microsatellite markers with those expected in pure specimens and in hybrids using assignment tests. The combined results led to the conclusion that the animal was a hybrid between a Scandinavian female wolf and a male dog. This finding confirms that inter-specific hybridization between wolves and dogs can occur in natural wolf populations. A possible correlation between hybridization and wolf population density and disturbance deserves further research.

Trichinella infection in wildlife of the southwestern United States.

Several potential mammalian reservoirs of sylvatic species of Trichinella were examined from Texas, New Mexico, and Arizona. During 1998-99, tongues were collected from a black bear (Ursus americanus) in Arizona; from 9 black bears, a coyote (Canis latrans), and a mountain lion (Felis concolor) in New Mexico; and from 154 coyotes, 32 raccoons (Procyon lotor), 13 opossums (Didelphis marsupialis), 4 ocelots (Leopardus pardalis), 3 bobcats (Lynx rufus), and 5 feral hogs (Sus scrofa) in southern Texas. Larvae of Trichinella murrelli were identified by a multiple-polymerase chain reaction analysis in 1 black bear (11.1%) from New Mexico and in 7 coyotes (4.5%) of Texas, whereas Trichinella spiralis larvae were detected in the black bear of Arizona. This is the first report of Trichinella infection in wildlife of New Mexico and Texas and extends the distribution of T. murrelli into the southwestern United States near the border of Mexico.

Sylvatic trichinellosis in Texas.

There are no published reports of domestic or sylvatic trichinellosis in Texas. The aim of the present survey was to determine the presence of Trichinella species in selected representative species of potential wildlife reservoirs in southern Texas. In 1998-99, tongues of 211 wild mammals were collected in southern Texas: 154 coyotes (Canis latrans), three bobcats (Lynx rufus), 32 racoons (Procyon lotor), 13 opossum (Didelphis marsupialis), four ocelots (Leopardus pardalis) and five wild boars (Sus scrofa). Presence of Trichinella sp. larvae was investigated by artificial digestion and larvae of positive samples were identified at the species level by a multiple-polymerase chain reaction analysis. Nine (5.8%) coyotes had trichinellosis; in the muscles of seven of these coyotes, the larvae were identified as Trichinella murrelli. This is the first report of sylvatic trichinellosis in Texas.

High prevalence of Trichinella nativa infection in wolf (Canis lupus) populations of Tvier and Smoliensk regions of European Russia.

Domestic and sylvatic trichinellosis have frequently been documented in European regions of Russia, with the highest prevalence reported in wolves (Canis lupus). From 1998 to 2000, 75 carcasses of wolves shot by hunters were tested for Trichinella larvae, and 73 (97.3%) of them were found to be positive. This very high prevalence of infection, the highest ever detected in a natural population of carnivores, could be explained by the human impact on the natural ecosystem. In fact, the diet of wolves living in the region under study mainly consists of carcasses of dogs and wolves, which are left in the forest or used as bait by hunters.

Organochlorine pesticide, polychlorinated biphenyl and heavy metal concentrations in wolves (Canis lupus L. 1758) from north-west Russia.

The wolf Canis lupus is a major terrestrial predator in eastern Europe and, as a top carnivore, may be exposed to high concentrations of contaminants that are readily transferred through the food chain. Despite this, there are few published data on pollutant and pesticide levels in wolves. This study utilised tissues from animals legally killed by hunters for other reasons (animals were not killed for the purposes of this study) to carry out the only detailed investigation of contaminants in wolves in Europe and the first in animals from Eastern Europe. The livers of 58 wolves from the Tver and Smoliensk regions of northwest Russia (54 degrees N 31 degrees E to 57 degrees N 35 degrees E) were analysed for seven organochlorine pesticides, 24 PCB congeners, Aroclor 1254-matched summed PCBs (sigmaPCBs), total mercury, cadmium and lead. Cadmium, most of the organochlorine pesticides and many PCB congeners were not detectable in any of the wolves. Hexachlorobenzene, alpha-HCH, pp'DDE, PCB congeners 118, 138, 149 and 156 and lead were detected in up to 6% of livers. Dieldrin, PCB congeners 153, 170 and 180, sigmaPCBs and mercury were detected more frequently. Contaminant levels were generally low; maximum wet weight concentrations of any of the organochlorine pesticides, sigmaPCBs and mercury were less than 0.1, 1 and 0.25 microg g(-1), respectively. PCB congeners 153, 170 and 180 accounted for 41% of the sigmaPCBs. Dieldrin, sigmaPCBs and mercury concentrations did not vary significantly between males and females nor between adult and juvenile (< 12 months old) wolves apart from the sigmaPCB concentration, which was on average five times higher in adults than juveniles. Liver residues were generally below the level normally associated with adverse effects except for lead levels which exceeded the critical 5 microg g(-1) dry wt. concentration in three of the 58 animals examined.

Hunting practices increase the prevalence of Trichinella infection in wolves from European Russia.

From 1998 to 2000, 184 animals (82 wolves, 29 red foxes, 55 mustelids, 5 raccoon dogs, and 13 domestic dogs), mainly shot by hunters in the Tvier and Smoliensk regions of northwest European Russia, were tested for Trichinella larvae; 98 animals (53.3%) were found to be positive. The highest prevalence was detected in wolf (97.5%). Trichinella nativa was the most common species detected (98%). The diet of wolves was investigated by examining the stomach contents of 62 animals (75.6% of the total number of wolves examined for Trichinella). It consisted mainly of dog (36.4% of the total number of occurrences of all food items, PFO) and moose (31.2 PFO); however, during the hunting seasons of 1998-1999 and 1999-2000, skinned wolf carcasses were left in the forest as bait (567 carcasses, about 18,000 kg). This very high prevalence of Trichinella infection, the highest ever detected in a natural population of carnivores, could be explained by carnivore-carnivore transmission, influenced by the hunting practices adopted in the study area.