V. Šnábel

Cystic echinococcosis in Turkey: genetic variability and first record of the pig strain (G7) in the country

A sample of 22 Echinococcus granulosus isolates collected from 12 sheep and ten humans from a focus of cystic echinococcosis in western Turkey was examined by DNA sequencing of four mitochondrial genes (cox1, atp6, nad1, rrnS). Results demonstrated the presence of two species of E. granulosus complex, E. granulosus sensu stricto and E. canadensis. Of E. granulosus sensu stricto, the G1 genotype (including three microvariants) was found in 17 isolates from humans and sheep, the G3 genotype and an intermediate form G1/G3 in one isolate each (both from sheep). Of E. canadensis, the pig strain G7 was found in three isolates from sheep and human. This is the first report of this strain in Turkey. Its presence has implications for local control programs due to its shorter maturation rate in dogs compared with E. granulosus sensu stricto. Goat and/or wild boar are likely reservoirs for G7 in the region. We provided further data on the pattern and frequency of nucleotide substitutions within the G1/G3 cluster. Based on our results and GenBank records, G2 (Tasmanian sheep strain) is not considered as a discrete genotypic unit, as its sequences at polymorphic sites conform to microvariants of both G1 and (more often) G3.

Morphological and genetic characterization of Echinococcus granulosus in the Slovak Republic

The present study was undertaken to characterize more thoroughly aetiological agent of cystic echinococcosis that still represents a health problem in the Slovak Republic. Sequencing of the mitochondrial ND1 gene, Random Amplified Polymorphic DNA (RAPD) analysis and isoenzyme analyses were used to characterize genetically protoscolices of 37 isolates from pigs and cattle and two isolates from humans collected in different regions of the Slovak Republic. Solely the G7 genotype (pig strain) was identified by ND1 sequences in all 14 pigs isolate examined by this method. This genotype was also found in two human patients and in two cattle. The exclusive finding of the G7 variant has implications for the implementation of control strategies given its unique developmental and biological properties. Despite striking morphological variability, a low degree of the intra-strain variability was found in the Slovak Echinococcus granulosus isolates with the genetic methods used. This was particularly associated with the shape and size of hooks, number of testes and shape of ovaries and uterus. GPI and MDH enzymes were found suitable for the discrimination of G7 and G1 (=cosmopolitan sheep strain) variant.

Echinococcus multilocularis is a frequent parasite of red foxes (Vulpes vulpes) in Latvia

Summary45 red foxes (Vulpes vulpes) from various regions of Latvia were examined by necropsy between 2003 and 2008 for the presence and infection intensity of Echinococcus multilocularis. The overall prevalence was 35.6 %, with the intensity of infection ranging from 1 to 1438. Although a limited number of foxes were examined, the prevalence was high and infected animals were found throughout the country. Considering the increasing number of humans infected with echinococcosis in Latvia since 2002, the situation is highly suggestive of a recent emergence of E. multilocularis. DNA sequences of four fox isolates were examined by multilocus analysis of mitochondrial and nuclear genes (cox1, nad1, rrnS, atp6, actII). All isolates were allocated to the predominant genetic form in Europe, with partial affinity of one isolate to a genotype that had previously been reported from southern Germany.

Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

Background The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the worlds most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples. Methodology 151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions. Principal Findings No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum. Conclusions/Significance These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.

Molecular and morphological circumscription of Mesocestoides tapeworms from red foxes (Vulpes vulpes) in central Europe.

Here we examine 3157 foxes from 6 districts of the Slovak Republic in order to determine for the first time the distribution, prevalence and identity of Mesocestodes spp. endemic to this part of central Europe. During the period 2001-2006, an average of 41.9% of foxes were found to harbour Mesocestoides infections. Among the samples we confirmed the widespread and common occurrence of M. litteratus (Batsch, 1786), and report the presence, for the first time, of M. lineatus (Goeze, 1782) in the Slovak Republic, where it has a more restricted geographical range and low prevalence (7%). Using a combination of 12S rDNA, CO1 and ND1 mitochondrial gene sequences together with analysis of 13 morphometric characters, we show that the two species are genetically distinct and can be differentiated by discrete breaks in the ranges of the male and female reproductive characters, but not by the more commonly examined characters of the scolex and strobila. Estimates of interspecific divergence within Mesocestoides ranged from 9 to 18%, whereas intraspecific variation was less than 2%, and phylogenetic analyses of the data showed that despite overlapping geographical ranges, the two commonly reported European species are not closely related, with M. litteratus more closely allied to North American isolates of Mesocestoides than to M. lineatus. We confirm that morphological analysis of reproductive organs can be used to reliably discriminate between these often sympatric species obtained from red foxes.

Trichinella spiralis-outbreak in the Slovak Republic.

Background:Trichinellosis is a zoonosis caused by nematode worms of the genus Trichinella and acquired through the consumption of raw or undercooked meat or meat products that harbour parasite larvae. The present report was designed to evaluate the course and circumstances surrounding the trichinellosis outbreak that occurred due to consumption of pork from a backyard pig in the southwest area of Slovakia in 2001.Patients and Methods:Patients suspected of acquiring infection were serologically examined by ELISA, sera of patients with symptoms of clinical trichinellosis were additionally tested by Western Blot. In symptomatic patients haematological and biochemical parameters were assessed on day 45 p.i.Results:An epidemiological investigation showed that the disease was linked to the consumption of infected pork and/or smoked pork products and affected the household members of four families. Out of 23 persons who had consumed the infected meat, 11 showed anti-Trichinella IgA, IgM and IgG antibodies in their sera by an ELISA against somatic and excretory-secretory antigens. Six of them developed clinical symptoms. Sera from symptomatic patients recognised bands of 64, 47, 45 and 43 kDa of crude T. spiralis antigens by Western blot. Using multiplex PCR analysis, parasite larvae isolated from pork were identified as T. spiralis, rarely occurring in Slovakia.Conclusion:The outbreak of human trichinellosis in an area where feral animals have been previously considered free of Trichinella was unexpected. Following the suspicion of trichinellosis being addressed, larvae were detected in meat and meat products from pig and the course of disease in patients was successfully controlled and managed.

Host specificity and geographical distribution of Eubothrium in European salmonid fish

The host specificity and distribution of Eubothrium crassum (Bloch, 1779) and Eubothrium salvelini (Schrank, 1790), morphologically fairly similar pseudophyllidean tapeworms parasitizing salmonid fish, were critically assessed on the basis of morphological and genetic evaluation of extensive material collected from different definitive hosts and geographical regions in Europe. Eubothrium crassum occurs in fish of the genera Salmo, i.e. salmon (S. salar - both freshwater and marine), sea trout (S. trutta trutta), brown trout (S. trutta fario), and lake trout (S. trutta lacustris), and also in Danubian salmon (Hucho hucho) and vendace (Coregonus albula). Eubothrium salvelini parasitizes Arctic char (Salvelinus alpinus) and brook trout (Salvelinus fontinalis) in Europe, and also whitefish (Coregonus wartmanni). Rainbow trout (Oncorhynchus mykiss), which is not a native European fish species, was found to be a suitable definitive host for both Eubothrium species, which may occur simultaneously in the same fish. Previous records of E. crassum in Arctic char and brook trout, and those of E. salvelini in fish of the genus Salmo were most probably misidentifications. Most studies of Eubothrium have involved salmonids from the northern part of Europe, with few records from southern and south-eastern Europe. This study also confirmed the reliability of the morphology of the apical disc for the discrimination of E. crassum and E. salvelini.

Genetic polymorphism in Proteocephalus exiguus shown by enzyme electrophoresis

Enzyme electrophoresis has been used to examine genetic diversity in a population of Proteocephalus exiguus La Rue, 1911 (Cestoda: Proteocephalidae), parasitizing salmonid and coregonid fishes. Among 16 loci tested, three polymorphic loci ( Ada, Got, Pgm-2 ) were found. Six different genotypes at the Got locus distributed in Hardy-Weinberg equilibrium suggest remarkable genetic flexibility of P. exiguus . Balancing selection is proposed as the mechanism maintaining genetic variation within the species. Data of genetic variability parameters (H o = 0.064; H e = 0.07; P = 0.19) and outcrossing rate (t = 0.842) of P. exiguus population have been provided. Possible factors affecting these data are discussed.

Morphological and genetic comparison of two Proteocephalus species (Cestoda: Proteocephalidae)

TwoProteocephalus species,P. neglectus La Rue, 1911 andP. exiguus La Rue, 1911, parasites of salmonid and coregonid fishes, were compared morphologically, biometrically, and electrophoretically. The study revealed that the two taxa cannot be differentiated by the morphological criteria used. No fixed allelic difference was found. In all, 12 of 13 enzymes detected by isoelectric focusing exhibited virtually identical isoenzyme profiles in both species. Only in the glutamate oxaloacatate transaminase enzyme locus was a difference observed. The negligible differences in zymograms and morphology, including similar biometric variability of the taxa studied, suggest that the species could be conspecific.

Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions

The study of the genetic polymorphism of pathogens is important for phylogenetic and biogeographic studies and, in the case of foodborne pathogens, to trace the origin of food infection. Since its discovery in 1972, the nonencapsulated species Trichinella pseudospiralis has been detected in mammals and birds, and human infection has occurred, in some cases resulting in death. We studied DNA polymorphism among ten T. pseudospiralis isolates from the Palearctic, Nearctic, and Australian regions, screening the sequences of nine genes [18sRNA, a random amplified polymorphism DNA derived sequence, mitochondrial cytochrome oxidase subunit I (COI), cytochrome P450, cynate lyase, epithelial fusion failure-1, and three unknown genes of Tp3, Tp8, and Tp26]. A high identity of sequence for the nine gene loci was obtained among the seven isolates from the Palearctic region and between the two isolates from the Nearctic region. Genetic identity analysis indicated the distinct polymorphism among the three geographical origins. To easily identify T. pseudospiralis genotypes, a polymerase chain reaction-restriction fragment length polymorphism analysis of COI gene was performed, and the results confirmed the DNA polymorphism within T. pseudospiralis, corresponding to the three regions of origin. We have named the three genotypes as “T. pseudospiralis Palearctic genotype” (code T4P), “Nearctic genotype” (code T4N), and “Australian genotype” (code T4A). To further investigate polymorphism among the nonencapsulated Trichinella species, the sequences of four gene loci (COI, P450, cynate lyase, and SB147D) of T. pseudospiralis, T. papuae, and T. zimbabwensis were analyzed, and the results showed high polymorphism among the three species, strongly supporting their classification as separate species.