Circulation of Anaplasma phagocytophilum among invasive and native carnivore species living in sympatry in Poland.

BACKGROUND: Anaplasma phagocytophilum is characterized by a worldwide distribution and distinguished from other Anaplasmataceae by the broadest range of mammalian hosts and high genetic diversity. The role carnivores play in the life cycle of A. phagocytophilum in Europe is uncertain. Currently, only the red fox is considered a suitable reservoir host. In this study, we focused on native and invasive medium-sized carnivore species that live in sympatry and represent the most abundant species of wild carnivores in Poland. METHODS: A total of 275 individual spleen samples from six carnivore species (Vulpes vulpes, Meles meles, Procyon lotor, Nyctereutes procyonoides and Martes spp.) were screened combining nested PCR and sequencing for A. phagocytophilum targeting a partial groEL gene with subsequent phylogenetic analysis inferred by the maximum likelihood method. RESULTS: The DNA of A. phagocytophilum was detected in 16 of 275 individuals (5.8%). Eight unique genetic variants of A. phagocytophilum were obtained. All detected haplotypes clustered in the clade representing European ecotype I. Three variants belonged to the subclade with European human cases together with strains from dogs, foxes, cats, and wild boars. CONCLUSIONS: While carnivores might have a restricted role in the dissemination of A. phagocytophilum due to their relatively low to moderate infection rates, they hold significance as hosts for ticks. Consequently, they could contribute to the transmission of tick-borne infections to humans indirectly, primarily through tick infection. This underscores the potential risk of urbanization for the A. phagocytophilum life cycle, further emphasizing the need for comprehensive understanding of its ecological dynamics.

The genetic structure of populations of Isthmiophora melis (Schrank, 1788) (Digenea: Echinostomatidae). Does the host's diet matter?

BACKGROUND: Here we provide a comparative analysis of the genetic structure of populations (based on nad1 mtDNA) of Isthmiophora melis isolated from the American mink (Neogale vison), an introduced invasive species, commonly occurring in the territory of Poland, and from the striped field mouse (Apodemus agrarius). METHODS: A total of 133 specimens of I. melis were obtained from naturally infected N. vison collected from six localities in Poland (108 samples) and 25 individuals of I. melis from A. agrarius. All sequences of the nad1 gene obtained during the present study were assembled and aligned. The standard statistics for haplotype composition, i.e., the number of haplotypes, haplotype diversity, nucleotide diversity, and average number of nucleotide differences, were calculated. Haplotype analysis and visualization of haplotype frequency among populations were performed using a median-joining network. RESULTS: Based on the samples collected from different localities in Poland, our study revealed that the overall genetic diversity of I. melis isolated from the American mink and of the striped field mouse do not differ significantly. The median-joining network showed that the three main haplotypes are in the centre of a star-like structure, with the remaining haplotypes as the satellites, reflecting the recent expansion of the populations. CONCLUSIONS: The overall genetic diversity of I. melis isolated from the American mink and striped field mouse reveals a high level of homogeneity. Moreover, regional differences in the food composition of the definitive hosts play an important role in shaping the genetic structure of the trematode populations.

Occurrence of Dirofilaria repens in wild carnivores in Poland.

Dirofilaria repens is an expanding vector-borne zoonotic parasite of canines and other carnivores. Sub-clinically infected dogs constitute the most important reservoir of the parasite and the source of infection for its mosquito vectors. However, occurrence of D. repens infection in wild animals may contribute to the transmission of the parasite to humans and may explain the endemicity of filariae in newly invaded regions. The aim of the current study was to determine the occurrence of D. repens in 511 blood and spleen samples from seven species of wild carnivores (wolves, red foxes, Eurasian badgers, raccoons, raccoon dogs, stone martens, and pine martens) from different regions of Poland by means of a PCR protocol targeting the 12S rDNA gene. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodeships in four of the seven regions of Poland: Masovia, Lesser Poland, Pomerania and Warmia-Masuria. The highest prevalence was found in Masovia region (8%), coinciding with the highest previously recorded prevalence in dogs in Central Poland. The DNA of Dirofilaria was detected in 16 samples of three species (total prevalence 3.13%). A low and similar percentage of positive samples (1.9%, 4.2% and 4.8%) was recorded among badgers, red foxes, and wolves, respectively. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodships. Based on detection in different voivodeships, D. repens-positive animals were recorded in four out of the seven regions of Poland: in Masovia, Lesser Poland, Pomerania, and Warmia-Masuria. The highest prevalence of filariae was found in Masovia region (8%), reflecting the highest previously recorded prevalence in dogs (12-50%) in Central Poland. In summary, we conducted the first comprehensive study on the epidemiology of D. repens in seven species of wild hosts in all seven regions of Poland and identified the first case of D. repens infection in Eurasian badgers in Poland and the second in Europe.

The Occurrence of Cryptosporidium spp. in Wild-Living Carnivores in Poland-A Question Concerning Its Host Specificity.

Cryptosporidium is an apicomplexan protozoan parasite that primarily infects the gastrointestinal epithelium in humans and domestic and wild animals. The majority of studies have been focused on human, livestock, and pet infections. Hence, Cryptosporidium spp. in wildlife, including wild carnivores, remained neglected. There are several studies reporting the occurrence of Cryptosporidium spp. in wild foxes, but these are only a few molecular surveys; no data is available concerning the occurrence of this parasite in raccoon dogs and martens in Europe, and to the best of our knowledge to date, only one study has reported Cryptosporidium from badgers in Spain. Therefore, we used molecular analyses to identify and genotype Cryptosporidium spp. in wild-living mesocarnivores in Poland. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine, and beech martens were collected and then analyzed for the presence of Cryptosporidium spp. using the nested PCR method. The appearance of PCR products in the reaction with Cryptosporidium-specific primers against the 18S rRNA and actin genes demonstrated that Cryptosporidium spp. occurred in 23.0% of all examined species of animals. Performed sequence analyses showed the presence of the Cryptosporidium skunk genotype, Cryptosporidium vole genotype II, Cryptosporidium canis dog and fox genotypes, as well as Cryptosporidium erinacei, Cryptosporidium ditrichi, Cryptosporidium suis, and Cryptosporidium alticolis, in these hosts. Molecular data presented here indicate that examined mesocarnivores may be a significant reservoir of specific and non-specific Cryptosporidium species, including those with zoonotic potential. Most studies of carnivores have described the presence of non-specific Cryptosporidium spp. in carnivore hosts, and this is probably the result of the transfer of these parasites from prey species through the digestive tract or the transfer of the parasite from a contaminated environment.

Occurrence of Borrelia sp. among Wild Living Invasive and Native Mesocarnivores in Poland.

Wild living mesocarnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. Individual tissue samples derived from 284 specimens of six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine marten and stone marten, were used for molecular investigations with the nested PCR method. The animals were sampled in the Ruszów Forest District (Poland). We aimed to examine the relative importance of the studied mesocarnivores as hosts of Borrelia spp. and investigated their role in this spirochaete's transmission cycle. We also aimed to trace the reservoir competence of these invasive and native predators and borreliosis eco-epidemiology in the context of a dilution effect. The overall prevalence of Borrelia spp. in the tested carnivores was 8.8%. Almost all of the consensus sequences of the partial flaB gene shared identity with a sequence of specific Borrelia species, i.e., B. afzelii, B. garinii and B. burgdorferi. Our results suggest that raccoons may play a role as reservoir hosts for these spirochaetal bacteria. The role of invasive species seems to be worthy of further analysis with reference to the circulation of vector-borne pathogens as well as in the context of the "dilution effect" hypothesis.

Life history strategies of Cotylurus spp. Szidat, 1928 (Trematoda, Strigeidae) in the molecular era - Evolutionary consequences and implications for taxonomy.

Species of Cotylurus Szidat, 1928 (Diplostomoidea: Strigeidae) are highly specialized digeneans that parasitize the gastrointestinal tract and bursa of Fabricius of water and wading birds. They have a three-host life cycle; the role of first intermediate host is played by pulmonate snails, while a wide range of water snails (both pulmonate and prosobranch) and leeches are reported as second intermediate hosts. Unfortunately, species richness, molecular diversity and phylogeny of metacercariae of Cotylurus spp. (tetracotyle) occurring in snails remain poorly understood. Thus, we have performed the parasitological and taxonomical examination of tetracotyles form freshwater snails from Poland, supplemented with adult Strigeidae specimens sampled from water birds. In this study we report our use of recently obtained sequences of two molecular markers (28S nuclear large ribosomal subunit gene (28S rDNA) and the cytochrome c oxidase subunit 1 (CO1) fragment), supplemented by results of a method of species delimitation (GMYC) and haplotype analysis to analyse some aspects of the ecology, taxonomy, and phylogeny of members of the genus Cotylurus. The provided phylogenetic reconstructions discovered unexpectedly high molecular diversity within Cotylurus occurring in snails, with clearly expressed evidence of cryptic diversity and the existence of several novel-species lineages. The obtained results revealed the polyphyletic character of C. syrius Dubois, 1934 (with three separate molecular species-level lineages) and C. cornutus (Rudolphi, 1809) Szidat, 1928 (with four separate molecular species-level lineages). Moreover, we demonstrated the existence of two divergent phylogenetical and ecological lineages within Cotylurus (one using leeches and other snails as second intermediate hosts), differing significantly in their life history strategies.

A molecular survey of spotted fever group rickettsiae in introduced raccoons (Procyon lotor).

BACKGROUND: The raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon's biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways. METHODS: Tissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software. RESULTS: Rickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis, Rickettsia raoultii, and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA, amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively. CONCLUSIONS: To the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required.

Invasive raccoon (Procyon lotor) and raccoon dog (Nyctereutes procyonoides) as potential reservoirs of tick-borne pathogens: data review from native and introduced areas.

In recent decades, populations of the raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonides) have increased and adapted to peri-urban and urban environments in many parts of the world. Their ability to rapidly colonize new territories, high plasticity and behavioral adaptation has enabled these two species to be considered two of the most successful invasive alien species. One of the major threats arising from continually growing and expanding populations is their relevant role in maintaining and transmitting various vector-borne pathogens among wildlife, domestic animals and humans. According to the WHO, over 17% of infectious diseases are vector-borne diseases, including those transmitted by ticks. Every year tick-borne pathogens (TBPs) create new public health challenges. Some of the emerging diseases, such as Lyme borreliosis, anaplasmosis, ehrlichiosis, babesiosis and rickettsiosis, have been described in recent years as posing important threats to global health. In this review we summarize current molecular and serological data on the occurrence, diversity and prevalence of some of the TBPs, namely Babesia, Theileria, Hepatozoon, Borrelia, Rickettsia, Bartonella, Anaplasma and Ehrlichia, that have been detected in raccoons and raccoon dogs that inhabit their native habitats and introduced areas. We draw attention to the limited data currently available on these invasive carnivores as potential reservoirs of TBPs in different parts of the world. Simultaneously we indicate the need for more research in order to better understand the epidemiology of these TBPs and to assess the future risk originating from wildlife.

Copro-Molecular Identification of Tapeworms in Introduced Invasive Carnivores in Poland.

The raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonoides) were introduced to Europe and, in the past decades, their populations have increased and adapted to synanthropic environments across Europe. In view of their possible further spread in Europe, the invasive species should be examined as potential reservoirs for helminths, including tapeworms. This study aims to investigate the prevalence and diversity of tapeworms in introduced wild carnivores in Poland by identifying cestode species based on copro-DNA analysis. A total of 214 individual fecal samples from non-native invasive carnivores, i.e., raccoons and raccoon dogs, and additionally 47 samples from native carnivores, i.e., European badgers (Meles meles), were analyzed for the presence of cestodes. PCR analysis of fecal samples targeting a fragment of mitochondrial (mt) 12S rRNA gene revealed the presence of cestode DNA in 19 of 103 (18.4%) raccoons, in 13 of 111 (11.7%) raccoon dogs and in 23 of 47 (48.9%) badgers. Sequence analysis demonstrated the presence of Mesocestoides litteratus in raccoons and raccoon dogs, while Mesocestoides lineatus was identified only in two samples derived from raccoon dogs. Moreover, in this study, Atriotaenia incisa was for the first time molecularly characterized by using fragments of mt 12S rRNA gene, and the DNA of this cestode species was detected in the fecal samples of all the examined host species.

Rickettsiae among mustelids - new data from south-west Poland.

Information on the prevalence on Rickettsia spp. in free-ranging mustelids and their specific ectoparasites is scarce. However, stone martens (Martes foina), pine martens (Martes martes) and European badgers (Meles meles) are common predators in many regions of Poland. In the present study we used tissue fragments to determine Rickettsia prevalence in these carnivores by molecular biology techniques. In addition, we included a data on several species of invertebrates that commonly feed on badgers.

Zoonotic Genotypes of Enterocytozoon bieneusi in Wild Living Invasive and Native Carnivores in Poland.

Wild carnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. The role of wild living reservoir hosts cannot be ignored because of their known carriage of E. bieneusi zoonotic genotypes. In the past decades, populations of wild living carnivores, i.e., native, such as red foxes, and invasive, such as raccoon dogs and raccoons, have increased and adapted to synanthropic environments across Europe, including Poland. The knowledge concerning E. bieneusi genotype identification and distribution in wild carnivores is limited worldwide. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine and beech martens, were collected and then analysed for the presence of E. bieneusi using the nested PCR method. Overall prevalence of the pathogen was estimated to be as high as 27.3%. The infection rates for E. bieneusi varied between the carnivore species, from 13.7% in beech martens to 40.4% in raccoon dogs. Based on sequence analysis of the ITS region of the rRNA gene marker, we detected five known genotypes of E. bieneusi in examined animals. In the invasive species, E. bieneusi NCF2 and D genotypes have been identified, whereas in the native ones, E. bieneusi NCF2, D, C, EbCar2 and Type IV genotypes were identified. All E. bieneusi genotypes recorded in this survey clustered in Group 1, showing their zoonotic potential. Our results provide the first description of the occurrence and genotypes of the microsporidian E. bieneusi in wild living population of raccoon dogs in Europe. Our findings are important for the study of pathogen epidemiology and emphasize the fact that the invasive and the native wild living carnivores, both widely distributed, should be considered more seriously as significant sources of zoonotic pathogens hazardous to domestic and farmed animals and humans.

Leeches as the intermediate host for strigeid trematodes: genetic diversity and taxonomy of the genera Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928.

BACKGROUND: Leeches (Hirudinida) play a significant role as intermediate hosts in the circulation of trematodes in the aquatic environment. However, species richness and the molecular diversity and phylogeny of larval stages of strigeid trematodes (tetracotyle) occurring in this group of aquatic invertebrates remain poorly understood. Here, we report our use of recently obtained sequences of several molecular markers to analyse some aspects of the ecology, taxonomy and phylogeny of the genera Australapatemon and Cotylurus, which utilise leeches as intermediate hosts. METHODS: From April 2017 to September 2018, 153 leeches were collected from several sampling stations in small rivers with slow-flowing waters and related drainage canals located in three regions of Poland. The distinctive forms of tetracotyle metacercariae collected from leeches supplemented with adult Strigeidae specimens sampled from a wide range of water birds were analysed using the 28S rDNA partial gene, the second internal transcribed spacer region (ITS2) region and the cytochrome c oxidase (COI) fragment. RESULTS: Among investigated leeches, metacercariae of the tetracotyle type were detected in the parenchyma and musculature of 62 specimens (prevalence 40.5%) with a mean intensity reaching 19.9 individuals. The taxonomic generic affiliation of metacercariae derived from the leeches revealed the occurrence of two strigeid genera: Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928. Phylogenetic reconstructions based on the partial 28S rRNA gene, ITS2 region and partial COI gene confirmed the separation of the Australapatemon and Cotylurus clades. Taking currently available molecular data and our results into consideration, recently sequenced tetracotyle of Australapatemon represents most probably Au. minor; however, unclear phylogenetic relationships between Au. burti and Au. minor reduce the reliability of this conclusion. On the other hand, on the basis of the obtained sequences, supplemented with previously published data, the metacercariae of Cotylurus detected in leeches were identified as two species: C. strigeoides Dubois, 1958 and C. syrius Dubois, 1934. This is the first record of C. syrius from the intermediate host. CONCLUSIONS: The results of this study suggest the separation of ecological niches and life cycles between C. cornutus (Rudolphi, 1808) and C. strigeoides/C. syrius, with potential serious evolutionary consequences for a wide range of host-parasite relationships. Moreover, phylogenetic analyses corroborated the polyphyletic character of C. syrius, the unclear status of C. cornutus and the separate position of Cotylurus raabei Bezubik, 1958 within Cotylurus. The data demonstrate the inconsistent taxonomic status of the sequenced tetracotyle of Australapatemon, resulting, in our opinion, from the limited availability of fully reliable, comparative sequences of related taxa in GenBank.

Sucking of human blood by Placobdella costata (O. F. Müller, 1846) (Hirudinida: Glossiphoniidae): Case study with notes on body form.

Four events of Placobdella costata sucking human blood are described.Human blood was sucked by both adult and juvenile specimens of P. costata.The feeding strategies of juveniles under parental care are presented.New data on juvenile specimens' body form are presented.Information on the potential role of mammals in dispersion and habitat preferences of leeches P. costata is considered.

Molecular Epidemiology and Genetic Diversity of Orthohantaviruses in Small Mammals in Western Poland.

Orthohantaviruses are negative-sense, single-stranded RNA viruses harbored by multiple small mammals. Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) cause hemorrhagic fever with renal syndrome (HFRS) in Europe. In Poland, serological surveys have demonstrated antibodies against DOBV and PUUV in patients with HFRS. Molecular evidence of DOBV and PUUV has been found in Apodemus flavicollis and Myodes glareolus, respectively, in southeastern Poland, and Seewis virus (SWSV) has been reported in Sorex araneus in central Poland. However, data on the geographic distribution and phylogeny of orthohantaviruses are unavailable for other regions in Poland. To ascertain the prevalence and genetic diversity of orthohantaviruses in western and northern Poland, lung tissues from 106 small mammals were analyzed for the presence of orthohantavirus RNA. DOBV and SWSV were detected in two of 42 (4.8%) Apodemus agrarius and in three of 10 (30%) S. araneus, respectively. Phylogenetic analyses of partial L- and S-segment sequences of DOBV indicated a shared genetic lineage with the Kurkino genotype from Slovakia, Russia, and Hungary, whereas the partial M segment of DOBV clustered with the Kurkino genotype from Germany. Phylogenetic relationships of the SWSV L and S segments showed a geographic lineage with SWSV strains from central Poland, Czech Republic, and Germany. In conclusion, the study provides insights into the molecular prevalence, phylogenetic diversity, and evolutionary relationship of DOBV in A. agrarius and SWSV in S. araneus. This report increases awareness among physicians for HFRS outbreaks in western Poland.

Molecular Identification and Phylogenetic Analysis of Heterakis dispar Isolated from Geese.

PURPOSE: Heterakidosis is a common parasitic infection caused in domestic birds by Heterakis species: Heterakis gallinarum, H. isolonche, and H. dispar. Among them, the best described species is H. gallinarum, noted mainly in gallinaceous birds. In waterfowl, H. dispar is the predominant species. The variations in morphology and host specificity qualify H. dispar as a different species, but the phylogenetic relationships between heterakids were unclear for a long time, because of a lack of H. dispar sequences. METHODS: The authors provided the molecular data for H. dispar and analyzed the obtained sequences of the partial 18S rRNA gene and region ITS1-5.8SrRNA-ITS2 with the homological sequences. RESULTS: The 18S rRNA PCR product of H. dispar was about 800 bp, and the ITS-5.8S-ITS2 PCR product was about 920 bp, noticeably smaller size compared to H. gallinarum product. The BLAST analysis of H. dispar 18S sequence showed a 99% similarity with the sequences of Heterakis gallinarum and Ascaridia galli, 98% with A. nymphii, but only 94% with the sequence of Heterakis sp. Our ITS sequence of H. dispar was almost identical to the H. isolonche isolate, there is only one nucleotide of difference among the 943 sites analyzed. It also showed a lower similarity to the ITS sequences of H. gallinarum (88%), H. spumosa (87%), and H. dahomensis (87%). CONCLUSIONS: In our phylogenetic analysis, it is the first attempt at the reconstruction of relationships within this superfamily Heterakoidea based on 18S rDNA and ITS region.

Correction to: Description and Phylogenetic Relationships of Pojmanskatrema balcanica n. gen., n. sp. (Digenea: Dicrocoeliidae) from the Eurasian Water Shrew Neomys fodiens (Mammalia: Soricidae) in Bulgaria.

Results section, 3rd paragraph: in the 4th enumeration the explanation is missing.

Molecular phylogeny provides new insights on the taxonomy and composition of Lyperosomum Looss, 1899 (Digenea, Dicrocoeliidae) and related genera.

Lyperosomum Looss, 1899 is one of the largest genera of the Dicrocoeliidae and is one of the best examples of the systematic complexity and taxonomic instability within this family. We present the molecular analyses based on novel sequences of nuclear and mitochondrial genes obtained from 56 isolates of adult flukes and larval stages of dicrocoeliids belonging to Lyperosomum, Skrjabinus, Zonorchis as well as previously available sequence data. According to obtained results we propose to return Zonorchis clathratus and Z. petiolatus into Lyperosomum, and to recognize L. alagesi as a synonym of L. petiolatum. Our study shows that L. petiolatum commonly occurs in Europe in corvids as well as in several species of migratory songbirds, e.g. Sylvia atricapilla. At the same time, the Turdidae appear to host a distinct species of Lyperosomum. The phylogenetic analysis has clearly demonstrated the paraphylepic nature of Lyperosomum and indicated the need of its thorough revision preferably using specimens from type hosts and type territories of nominal species. In addition, inclusion of numerous not yet sequenced dicrocoeliid genera into future phylogenetic studies is necessary to clarify the interrelationships of taxa within the family and stabilize its system.

Description and Phylogenetic Relationships of Pojmanskatrema balcanica n. gen., n. sp. (Digenea: Dicrocoeliidae) from the Eurasian Water Shrew Neomys fodiens (Mammalia: Soricidae) in Bulgaria.

BACKGROUND: We describe a new dicrocoeliid collected from the Eurasian water shrew Neomys fodiens in Bulgaria based on the combination of morphological characteristics and molecular data. RESULTS: Pojmanskatrema balcanica n. sp., n. gen. is distinguished from all other species of family Dicrocoeliidae Looss, 1900 in having a postovarian vitellarium situated in the posterior half of the body, postacetabular, nearly tandem, lobed testes, and relatively short body. CONCLUSIONS: Molecular phylogenetic analysis based on 28S rDNA sequences of the new species and previously sequenced dicrocoeliid taxa has demonstrated close relationship of Pojmanskatrema n. gen. with the genus Eurytrema. Considering the non-monophyletic nature of previously recognized dicrocoeliid sub-families we support the recently proposed dissolution of the sub-families within this family.

The opportunistic pathogen Encephalitozoon cuniculi in wild living Murinae and Arvicolinae in Central Europe.

Encephalitozoon spp. is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. This study was conducted to determine the prevalence of Encephalitozoon spp. in wild living rodents from Poland, the Czech Republic and Slovakia. Faecal and spleen samples were collected from individuals of Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, and Myodes glareolus (n = 465) and used for DNA extraction. PCR, targeting the ITS region of the rRNA gene was performed. The overall prevalence of microsporidia was 15.1%. The occurrence of Encephalitozoon cuniculi in the abovementioned host species of rodents has been presented for the first time, with the highest infection rate recorded for A. flavicollis. Sequence analysis showed that the most frequent species was E. cuniculi genotype II (92.5%). E. cuniculi genotypes I (1.5%) and III (6.0%) were also identified.

Paralogs vs. genotypes? Variability of Babesia canis assessed by 18S rDNA and two mitochondrial markers.

Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory.