Diversity and geographic distribution of haplotypes of Dirofilaria immitis across European endemic countries.

BACKGROUND: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. METHODS: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). RESULTS: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1-4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). CONCLUSION: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene.

Remotely sensed localised primary production anomalies predict the burden and community structure of infection in long-term rodent datasets.

The increasing frequency and cost of zoonotic disease emergence due to global change have led to calls for the primary surveillance of wildlife. This should be facilitated by the ready availability of remotely sensed environmental data, given the importance of the environment in determining infectious disease dynamics. However, there has been little evaluation of the temporal predictiveness of remotely sensed environmental data for infection reservoirs in vertebrate hosts due to a deficit of corresponding high-quality long-term infection datasets. Here we employ two unique decade-spanning datasets for assemblages of infectious agents, including zoonotic agents, in rodents in stable habitats. Such stable habitats are important, as they provide the baseline sets of pathogens for the interactions within degrading habitats that have been identified as hotspots for zoonotic emergence. We focus on the enhanced vegetation index (EVI), a measure of vegetation greening that equates to primary productivity, reasoning that this would modulate infectious agent populations via trophic cascades determining host population density or immunocompetence. We found that EVI, in analyses with data standardised by site, inversely predicted more than one-third of the variation in an index of infectious agent total abundance. Moreover, in bipartite host occupancy networks, weighted network statistics (connectance and modularity) were linked to total abundance and were also predicted by EVI. Infectious agent abundance and, perhaps, community structure are likely to influence infection risk and, in turn, the probability of transboundary emergence. Thus, the present results, which were consistent in disparate forest and desert systems, provide proof-of-principle that within-site fluctuations in satellite-derived greenness indices can furnish useful forecasting that could focus primary surveillance. In relation to the well-documented global greening trend of recent decades, the present results predict declining infection burden in wild vertebrates in stable habitats; but if greening trends were to be reversed, this might magnify the already upwards trend in zoonotic emergence.

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.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite.

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

Candidatus Neoehrlichia mikurensis and Hepatozoon sp. in voles (Microtus spp.): occurrence and evidence for vertical transmission.

Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. are important vector-borne parasites of humans and animals. CNM is a relatively recently discovered pathogen of humans. Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide. The present study aimed to determine the prevalence of CNM and Hepatozoon spp. in three species of Microtus and to assess the occurrence of vertical transmission in naturally-infected voles. Molecular techniques were used to detect pathogen DNA in blood and tissue samples of captured voles and their offspring. The prevalence of CNM in the vole community ranged 24-47% depending on Microtus species. The DNA of CNM was detected in 21% of pups from three litters of six infected Microtus dams (two Microtus arvalis and one M. oeconomus) and in 3/45 embryos (6.6%) from two litters of eight CNM-infected pregnant females. We detected Hepatozoon infection in 14% of M. arvalis and 9% of M. oeconomus voles. Hepatozoon sp. DNA was detected in 48.7% of pups from seven litters (6 M. arvalis and 1 M. oeconomus) and in two embryos (14.3%) obtained from one M. arvalis litter. The high prevalence of CNM infections in the Microtus spp. community may be a result of a relatively high rate of vertical transmission among naturally infected voles. Vertical transmission was also demonstrated for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.

Long term patterns of association between MHC and helminth burdens in the bank vole support Red Queen dynamics.

Major histocompatibility complex (MHC) genes encode proteins crucial for adaptive immunity of vertebrates. Negative frequency-dependent selection (NFDS), resulting from adaptation of parasites to common MHC types, has been hypothesized to maintain high, functionally relevant polymorphism of MHC, but demonstration of this relationship has remained elusive. In particular, differentiation of NFDS from fluctuating selection, resulting from changes in parasite communities in time and space (FS), has proved difficult in short-term studies. Here, we used temporal data, accumulated through long-term monitoring of helminths infecting bank voles (Myodes glareolus), to test specific predictions of NFDS on MHC class II. Data were collected in three, moderately genetically differentiated subpopulations in Poland, which were characterized by some stable spatiotemporal helminth communities but also events indicating introduction of new species and loss of others. We found a complex association between individual MHC diversity and species richness, where intermediate numbers of DRB supertypes correlated with lowest species richness, but the opposite was true for DQB supertypes-arguing against universal selection for immunogenetic optimality. We also showed that particular MHC supertypes explain a portion of the variance in prevalence and abundance of helminths, but this effect was subpopulation-specific, which is consistent with both NFDS and FS. Finally, in line with NFDS, we found that certain helminths that have recently colonized or spread in a given subpopulation, more frequently or intensely infected voles with MHC supertypes that have been common in the recent past. Overall, our results highlight complex spatial and temporal patterns of MHC-parasite associations, the latter being consistent with Red Queen coevolutionary dynamics.

Pathogens detected in the tick Haemaphysalis concinna in Western Poland: known and unknown threats.

In recent years, a new focus of the relict tick Haemaphysalis concinna was discovered in Western Poland, near Wolsztyn, Greater Poland voivodeship. This species may play an important role in the circulation of pathogens of medical and veterinary importance. In the present study we tested 880 juvenile ticks collected from rodents, including 427 H. concinna, 443 Ixodes ricinus and 10 Dermacentor reticulatus for three of the most common pathogens vectored by ticks in Poland: Rickettsia and Babesia spp. and Borrelia burgdorferi s.l. Additionally, molecular techniques were applied for accurate identification of tick host species (the voles Microtus and Alexandromys). Our study found differences in the range and prevalence of vectored pathogens between the three tick species. DNA of all three pathogens was found in I. ricinus. In juvenile H. concinna, DNA of Babesia microti, Borrelia afzelii and Rickettsia sp. was identified. Moreover, DNA of a new unnamed Babesia species related to B. crassa, was found in two H. concinna nymphs. This genotype of Babesia was previously identified in H. concinna in the Far East and then in Central Europe. DNA of Rickettsia raoulti and B. afzelii was detected in D. reticulatus nymphs. Among rodent hosts, Alexandromys oeconomus seems to be host of the highest significance for juvenile tick stages and was the only host species with B. afzelii detected in blood samples. Using phylogenetic methods, we confirmed a clear division between rodents from the genera Microtus and Alexandromys. Moreover, we found that A. oeconomus trapped in Western Poland clustered with a Central European A. oeconomus allopatric phylogroup.

Seroprevalence of Toxoplasma gondii among Sylvatic Rodents in Poland.

Toxoplasma gondii is an intracellular Apicomplexan parasite with a broad range of intermediate hosts, including humans and rodents. Rodents are considered to be reservoirs of infection for their predators, including cats, felids, pigs, and wild boars. We conducted a multi-site, long-term study on T. gondii in northeastern Poland. The study aimed to monitor the seroprevalence of T. gondii in the four abundant vole species found in the region (Myodes glareolus, Microtus arvalis, Microtus agrestis, and Alexandromys oeconomus) and to assess the influence of both extrinsic (year of study and study site) and intrinsic (host sex and host age) factors on seroprevalence. A bespoke enzyme-linked immunosorbent assay was used to detect antibodies against T. gondii. We examined 577 rodent individuals and detected T. gondii antibodies in the sera of all four rodent species with an overall seroprevalence of 5.5% [4.2-7.3] (3.6% [2.6-4.9] for M. glareolus and 20% [12-30.9] for M. arvalis, M. agrestis, and A. oeconomus). Seroprevalence in bank voles varied significantly between host age and sex. Seroprevalence increased with host age and was higher in females than males. These results contribute to our understanding of the distribution and abundance of T. gondii in voles in Poland and confirm that T. gondii also circulates in M. glareolus and M. arvalis, M. agrestis and A. oeconomus. Therefore, they may potentially play a role as reservoirs of this parasite in the sylvatic environment.

Emerging risk of Dirofilaria spp. infection in Northeastern Europe: high prevalence of Dirofilaria repens in sled dog kennels from the Baltic countries.

Dirofilariasis is a fast-spreading disease of dogs and humans in Europe. We investigated whether Dirofilaria spp. have spread northwards in Europe, invading the Baltic countries. Altogether, 424 blood samples were collected from eight countries in the period 2017-2019, including 227 samples from sled dogs and 197 samples from other dogs. PCR amplification and sequencing were conducted employing three genetic markers (mitochondrial [mt] 12S rDNA, mt cytochrome c oxidase subunit I [COI] gene and mt dehydrogenase subunit I [NAD1] gene). The SNAP test (IDEXX) for detection of D. immitis infections was also implemented. The DNA of D. repens was detected in 59 of 424 dogs (prevalence 13.9%). D. repens was found in sled dogs from Lithuania, Latvia, Poland and Belarus. Only one dog from Estonia was infected, apparently an imported case. The highest prevalence was recorded in Lithuania (38%). Among pet dogs from the Ukraine, six dogs tested positive (3.8%). Our study has revealed a high prevalence of D. repens infections in Lithuania and Latvia, but no evidence for spread of the heartworm D. immitis. We conclude that sled dog kennels constitute hot spots for D. repens transmission.

Zoonotic Viruses in Three Species of Voles from Poland.

Rodents are known to be reservoir hosts for a plethora of zoonotic viruses and therefore play a significant role in the dissemination of these pathogens. We trapped three vole species (Microtus arvalis, Alexandromys oeconomus and Microtus agrestis) in northeastern Poland, all of which are widely distributed species in Europe. Using immunofluorescence assays, we assessed serum samples for the presence of antibodies to hantaviruses, arenaviruses and cowpox viruses (CPXV). We detected antibodies against CPXV and Puumala hantavirus (PUUV), the overall seroprevalence of combined viral infections being 18.2% [10.5-29.3] and mostly attributed to CPXV. We detected only one PUUV/TULV cross-reaction in Microtus arvalis (1.3% [0.1-7.9]), but found similar levels of antibodies against CPXV in all three vole species. There were no significant differences in seroprevalence of CPXV among host species and age categories, nor between the sexes. These results contribute to our understanding of the distribution and abundance of CPXV in voles in Europe, and confirm that CPXV circulates also in Microtus and Alexandromys voles in northeastern Poland.

Ectoparasites of red foxes (Vulpes vulpes) with a particular focus on ticks in subcutaneous tissues.

In the present study, we identified the ectoparasite communities of red foxes in three regions of Poland that encompassed two endemic regions for the occurrence of Dermacentor reticulatus, as well as a region that is free of this tick species ('gap' area). Our study sites were selected to enable the role of foxes as hosts for juvenile (nest dwelling) and adult (exophilic) D. reticulatus ticks to be determined, and to assess their contribution to the spread of this important vector of Babesia canis. We compared also ectoparasite communities between adult foxes with those of fox cubs. Finally, we carried out a systematic search for subcutaneous ticks determining their prevalence and abundance. In 2016-2018, 366 adult foxes and 25 live-trapped cubs were examined for ectoparasites. Ectoparasites were identified based on morphological features, PCR amplification and sequencing. The total prevalence of ectoparasites was higher in cubs (68%) than in adults (62.8%). In adults, 15 parasite species were recorded, including four tick species, seven flea species, scabies, and one Anopluran species each in the genera Felicola and Lipoptena. In cubs, six ectoparasite species were found, including Ixodes kaiseri, a species not found in adults. Although Ixodes ricinus and D. reticulatus were the dominant tick species on adult foxes, no D. reticulatus ticks were found on cubs. Subcutaneous ticks were common (38%) and abundant in all areas. Molecular analysis of subcutaneous nodules allowed the identification of 17 I. ricinus and five D. reticulatus. In conclusion, red foxes play a minor role as hosts of D. reticulatus.

Parallel evolution of arborescent carrots (Daucus) in Macaronesia.

PREMISE: Despite intensive research, the pathways and driving forces behind the evolution of derived woodiness on oceanic islands remain obscure. The genus Daucus comprises mostly herbs (therophytes, hemicryptophytes) with few rosette treelets (chamaephytes) endemic to various Macaronesian archipelagos, suggesting their independent evolution. To elucidate the evolutionary pathways to derived woodiness, we examined phylogenetic relationships and the habit and secondary xylem evolution in Daucus and related taxa. METHODS: Sixty taxa were surveyed for molecular markers, life history, and habit traits. Twenty-one species were considered for wood anatomical characters. A dated phylogeny was estimated using Bayesian methods. The evolution of selected traits was reconstructed using parsimony and maximum likelihood. RESULTS: Daucus dispersed independently to the Canary Islands (and subsequently to Madeira), Cape Verde, and the Azores in the late Miocene and Pleistocene. Life span, reproductive strategy, and life form were highly homoplastic; the ancestor of Daucus was probably a monocarpic, biennial hemicryptophyte. Rosette treelets evolved independently in the Canarian-Madeiran lineage and in Cape Verde, the latter within the last 0.13 Myr. Treelets and hemicryptophytes did not differ in wood anatomy. Pervasive axial parenchyma in wood occurred more often in polycarpic rather than monocarpic species. CONCLUSIONS: Life span and life form in Daucus are evolutionarily labile and may change independently of wood anatomy, which is related to plant reproductive strategy rather than to life form. Insular woodiness may evolve rapidly (as demonstrated in D. bischoffii), and in Daucus, it does not seem to be an adaptation to lower the risk of xylem embolism.

Rodents as intermediate hosts of cestode parasites of mammalian carnivores and birds of prey in Poland, with the first data on the life-cycle of Mesocestoides melesi.

BACKGROUND: Rodents constitute an important part of the diet of many carnivore species. This predator-prey food chain is exploited by helminth parasites, such as cestodes, whose larval stages develop in rodents and then mature to the adult stage in predators. The main aim of our study was to use molecular techniques for identification of cestode species recovered from both intermediate and definitive hosts, with a particular focus on the genus Mesocestoides. METHODS: Larval cestodes were obtained during our long-term studies on rodent helminth communities in the Mazury Lake District in the north-east Poland in 2000-2018. Cestode larvae/cysts were collected from body cavities or internal organs (e.g. liver) during autopsies. Adult tapeworms were derived from nine red foxes, three Eurasian badgers and one Eurasian lynx. PCR amplification, sequencing and phylogenetic analyses were conducted employing three genetic markers: 18S rDNA, mitochondrial (mt) 12S rDNA and the mt cytochrome c oxydase subunit 1 (cox1) gene fragment. RESULTS: Altogether 19 Mesocestoides samples were analyzed, including 13 adult tapeworms from definitive hosts and six larval samples from 4 bank voles and 2 yellow-necked mice. Phylogenetic analyses revealed three well-supported trees of similar topology. In each case the Mesocestoides samples formed two separate clades. All isolates from foxes, the lynx isolate and two isolates from rodents grouped with Mesocestoides litteratus. Four isolates from rodents and all three isolates from Eurasian badgers were resolved in a separate clade, most similar to North American M. vogae (syn. M. corti). Examination of fixed, stained adult specimens from Eurasian badgers revealed consistency with the morphology of Mesocestoides melesi. Therefore, this clade is likely to represent M. melesi, a species first described in 1985 from the Eurasian badger Meles meles. Molecular analysis allowed also the identification of Taenia crassiceps, Hydatigera kamiyai and Cladotaenia globifera among larvae derived from rodents. CONCLUSIONS: Molecular and phylogenetic analyses support the recognition of M. melesi as a valid species. Our data represent the first record of the larvae of this species in rodents. This is the first report on the occurrence of H. kamiyai in rodents from Poland.

Seroprevalence of Tick-Borne Encephalitis Virus in Three Species of Voles (Microtus spp.) in Poland.

Rodents play a role as reservoir hosts for tick-borne encephalitis virus (TBEV). We trapped 77 voles of the genus Microtus and tested their blood using an enzyme-linked immunosorbent assay for the presence of antibodies against TBEV. We detected TBEV antibodies in three adult individuals (4%). Voles might play a role in the circulation of TBEV.

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples - the way to eco-epidemiological studies.

INTRODUCTION AND OBJECTIVE: It is easier and non-invasive to obtain faecal samples compared with blood samples. Molecular techniques may enable detection of parasites even in tiny amounts of blood-containing faeces. We aimed to compare the sensitivity of detection of three Babesia species and Hepatozoon canis in blood and faecal samples, including samples derived from naturally infected hosts. MATERIAL AND METHODS: Three groups were involved: 1) Nine BALB/c mice infected with Babesia microti sampled during acute (n=3), post-acute (n=3) and chronic phases of infection (n=3); 2) Eight dogs with symptoms of babesiosis; 3) Six red foxes infected with B. vulpes, one fox infected with B. canis, four foxes infected with H. canis. Genomic DNA was extracted from blood and faeces by use of commercial kits and amplified with genus-specific primers in one-step or nested PCR reactions. Selected PCR products were sequenced. RESULTS: No positive results for faecal samples were obtained from H. canis-positive foxes in contrast to Babesia spp. infections. Positive results from PCRs were obtained for all BALB/c mice (100%), five dogs (62.5%) and four of seven foxes (57.1%). Successful sequencing was obtained for six selected murine samples (B. microti), four canine samples (B. canis) and for one fox sample (B. vulpes). The success of B. microti detection in murine faecal samples from acute, post-acute and chronic phases was identical (100%). CONCLUSIONS: Detectability of Babesia spp. infections was lower in naturally infected dogs and foxes, compared to experimentally infected mice. Detection of DNA in faecal samples can be useful in the detection of Babesia infection in populations from which blood samples are hard to obtain, but due regard must be given to the possibility that prevalence of infection may be severely underestimated.

Zoonotic Virus Seroprevalence among Bank Voles, Poland, 2002-2010.

Bank voles in Poland are reservoirs of zoonotic viruses. To determine seroprevalence of hantavirus, arenavirus, and cowpox virus and factors affecting seroprevalence, we screened for antibodies against these viruses over 9 years. Cowpox virus was most prevalent and affected by extrinsic and intrinsic factors. Long-term and multisite surveillance is crucial.

Seroprevalence of Trichinella spp. infection in bank voles (Myodes glareolus) - A long term study.

Rodents play an important role as reservoir hosts of zoonotic diseases. As a component of our long-term programme of monitoring parasitic infections in bank vole populations in three ecologically similar sites in NE Poland, we screened blood samples for signs of a serological response to the presence of Trichinella spp. The overall seroprevalence of Trichinella spp. was 1.52%, but prevalence was largely concentrated in one of our three study sites and confined to the oldest individuals in the study. Seroprevalence of Trichinella spp. did not differ between the sexes. Although a local prevalence of 1.52% may seem low, when this is extrapolated to the national population of bank voles in peak years, perhaps numbering hundreds of millions of animals, the number of infected bank voles on a country wide scale is likely to be huge. Our results suggest that bank voles may be reservoirs of Trichinella spp. However, on the basis of our results we consider their importance as epidemiologically significant hosts for Trichinella spp. to be moderate and their role in this context to require further investigation.

The role of juvenile Dermacentor reticulatus ticks as vectors of microorganisms and the problem of 'meal contamination'.

Juvenile Dermacentor reticulatus ticks inhabit nests and burrows of their rodent hosts and cannot be collected from vegetation. To detect vertical transmission of Babesia canis in D. reticulatus, we studied larvae and nymphs collected from rodents. However, the molecular techniques used for detection of pathogen DNA are sensitive enough to detect not only pathogens vectored by ticks but also those taken up with current or previous blood meals ('meal contamination') or just present in the environment and on the tick or host surface ('environmental contaminations'). Thus, an additional aim of our study was to evaluate the extent of such contamination while studying feeding ticks collected from rodents. Juvenile D. reticulatus were collected from 140 rodents: 91 bank voles trapped in two forest sites in the Mazury Lake District and 49 rodents (Apodemus and Microtus spp.) from an open habitat near the town of Bialobrzegi in Central Poland. Altogether 504 D. reticulatus ticks, comprising 266 individually evaluated nymphs and 238 larvae assigned to 50 larval pools, were studied for the presence of Babesia, Bartonella and Rickettsia spp. DNA. Statistical analyses were conducted to (1) evaluate the effect of rodent host factors (species, sex and age) on prevalence of infection in ticks, and (2) to compare the frequency of positive samples between groups of pathogen-positive and pathogen-negative rodent hosts. To complete the last aim, blood samples obtained from 49 rodents from Bialobrzegi were studied for the presence of Babesia and Bartonella DNA. Infestation of rodent hosts with juvenile ticks ranged between 46 and 78%, with a mean abundance of 3.6 ticks/rodent for D. reticulatus and 4.8 ticks/rodent for Ixodes ricinus. The highest prevalence of PCR-positive D. reticulatus samples was obtained for Rickettsia spp. (28%) and R. raoultii was identified in 22 sequenced PCR products. Babesia DNA was detected in 20 (7.5%), including B. microti in 18 (6.8%) and B. canis in two (0.8%) of 266 D. reticulatus nymphs that were analyzed. Babesia microti DNA was also detected in four pools of D. reticulatus larvae (4/50 pools = 8%). The detection success of B. microti in D. reticulatus was associated with the species of the rodent hosts of the ticks (much higher for typical B. microti-host-species such as Microtus spp. than for Apodemus spp.) and host age (3 × higher in ticks collected from adult hosts in comparison to juvenile ones). Moreover, the DNA of B. microti was detected in 68% of D. reticulatus nymphs collected from B. microti-positive rodents in comparison to only 1.6% of nymphs collected from B. microti-negative rodents. Bartonella DNA was detected in 18% of D. reticulatus tick samples (38% of larval pools, 14% of nymphs). Again, host factors played important roles for 'tick positivity'-the highest prevalence of positive ticks was on Apodemus spp., which are regarded as Bartonella reservoirs. Bartonella DNA was detected in 42% of nymphs and 57% of larval pools collected from Bartonella-positive rodents in comparison to 28% of nymphs and 11% of larvae collected from Bartonella-negative rodents. Vertical transmission of B. canis in D. reticulatus ticks was confirmed in the field. Additionally, we demonstrated that 'meal contamination' generates a confounding signal in molecular detection of pathogen DNA extracted from ticks collected from infected hosts and must be taken into account in evaluating the competence of tick species as vectors.

A new focus of the tick Haemaphysalis concinna in Western Poland.

The relict tick Haemaphysalis concinna has a fragmented and focal distribution in Central Europe and Asia. Although in the majority of neighboring countries the occurrence of this tick species is well-documented (i.e., in Germany, Czech Republic, Slovakia, Ukraine), to-date its occurrence in Poland has been registered only once, in 1953 in Troszyn in North-Western Pomerania, close to the German-Polish border. In the present study we report the first documented finding of H. concinna in Western Poland, confirmed both by collection of juvenile ticks from rodent hosts and questing ticks from vegetation. Trapping of rodents took place in the summer of 2018 in three locations in Western Poland (Slonin, Nowy Mlyn 1, Nowy Mlyn 2). Rodents were inspected for ectoparasites, which were detached and fixed in 70% ethanol. All the collected ticks were assigned to species and developmental stages using appropriate morphological keys, and representative individuals were genotyped by molecular methods. A total of 1482 feeding ticks were collected from 106 rodents from three sites. The common tick Ixodes ricinus was found in abundance on small rodents at all three sites; Dermacentor reticulatus ticks were identified at two sites in small numbers and, finally, numerous juvenile H. concinna (n = 427) were found at one of our study sites (Nowy Mlyn 2). The highest prevalence and abundance of H. concinna were recorded on voles, Microtus agrestis and M. oeconomus, from this site in August. Additionally, questing nymphs and adult H. concinna were collected locally from vegetation (n = 20). Genotyping and phylogenetic analysis confirmed the species as H. concinna. A new focus of H. concinna has been described in Western Poland. Our long-term field work monitoring the expansion of the distribution of D. reticulatus in Poland, during which all collected ticks are identified, suggests that H. concinna is still very rare in the country.

Bartonella infections in three species of Microtus: prevalence and genetic diversity, vertical transmission and the effect of concurrent Babesia microti infection on its success.

BACKGROUND: Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival. RESULTS: We sampled 124 Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos (47.1%; 40/85) and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%). CONCLUSIONS: High prevalence of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.