Diversity of Babesia in Ixodes ricinus ticks in Poland.

PURPOSE: The aims of this study were: (1) to estimate Babesia prevalence in the most common species of tick in Poland, Ixodes ricinus, in two recreational areas (Urwitalt in the Mazury Lake District and Bielanski Forest in Warsaw), and (2) to evaluate the molecular diversity of Babesia isolates in questing I. ricinus in Poland. MATERIAL AND METHODS: Questing ticks were collected from vegetation in forest areas in Urwitalt near Mikolajki and in Bielanski Forest (Warsaw). Purified genomic DNA was used with specific primers to amplify a fragment of the Babesia spp. 18S rRNA gene. RESULTS: Tick-drag indices for I. ricinus were high in both study areas, reaching somewhat higher values in Urwitalt than in Bielanski Forest. The overall prevalence of Babesia spp. in examined ticks was 1.6%. In Urwitalt, two strains of B. microti were identified using rRNA sequences: the enzootic Munich strain and an isolate close to the zoonotic Jena strain. The proportion of infections due to these two strains in questing ticks reversed over a six-year period. During 3 years of study in Bielanski Forest, all Babesia isolates obtained from I. ricinus were identical to Babesia sp. EU1 (B. venatorum), previously recognized as an agent of human babesiosis. CONCLUSIONS: This study has confirmed the presence of enzoonotic and zoonotic Babesia species/strains in the abundant human-biting tick I. ricinus in recreational areas in Poland. It has also shown that the distribution of different genotypes has changed over time, however the reasons for these fluctuations still remain to be investigated.

Differences in the ecology of Bartonella infections of Apodemus flavicollis and Myodes glareolus in a boreal forest.

The epidemiology of Bartonella species infecting Apodemus flavicollis and Myodes glareolus in a forest in Eastern Poland was followed for 2 years using mark-recapture. Infections could be acquired in any month, but prevalence, and probability of infection, peaked in the summer. There were significant differences in the pattern of infections between the two species. Both hosts were primarily infected as juveniles, but the probability of infection was highest for A. flavicollis, which, evidence suggests, experienced longer-lasting infections with a wider range of Bartonella genotypes. There was no evidence of increased host mortality associated with Bartonella, although the infection did affect the probability of recapture. Animals could become re-infected, generally by different Bartonella genotypes. Several longer lasting, poorly resolved infections of A. flavicollis involved more than 1 genotype, and may have resulted from sequential infections. Of 22 Bartonella gltA genotypes collected, only 2 (both B. grahamii) were shared between mice and voles; all others were specific either to A. flavicollis or to M. glareolus, and had their nearest relatives infecting Microtus species in neighbouring fields. This heterogeneity in the patterns of Bartonella infections in wild rodents emphasizes the need to consider variation between both, host species and Bartonella genotypes in ecological and epidemiological studies.

Effectiveness of water treatment for the removal of Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia are intestinal parasites of humans and of many other species of animals. Water constitutes an important route of transmission for human infections in both developed and developing countries. In Poland, contamination of water sources with oocysts/cysts is not routinely monitored and scientific research in this field is scarce. Our aim was to compare the contamination of surface and treated water and thus the success of water treatment processes. Water samples (n=94) of between 30 l (surface water) to over 1000 l for tap water, were taken in the period of 2008-2009 using specially constructed equipment with cartridge filtration (Filta-Max; IDEXX, USA). Immunofluorescent assay, and nested polymerase chain reaction were used for the detection of parasites. Cryptosporidium oocysts were found in 85% of surface water and in 59% of raw (intake) water samples. Oocysts were also detected in treated water (16%) but were absent in samples of swimming pool water. The highest mean number of Cryptosporidium oocysts [geometric mean (GM)=61/10 l] was found in samples of rinsing water. Giardia cysts were observed in 61% of surface water samples, in 6% of raw water and in 19% of treated water, with the highest number of cysts noted in rinsing water samples (GM=70 cysts/10 l). Our study highlights the frequent occurrence of parasites in surface waters in Poland and the effectiveness of water treatment for the removal of parasites from drinking water.

Temporal and between-site variation in helminth communities of bank voles ( Myodes glareolus) from N.E. Poland. 1. Regional fauna and component community levels.

Helminth infections were studied in bank voles (Myodes glareolus) from 3 woodland sites in N.E. Poland in the late summers of 1999 and 2002, to assess the temporal stability of derived statistics describing the regional helminth fauna and component community structure, and spatial influence on the latter. Regional helminth fauna changed dramatically between the two years, primarily due to a fall in the abundance of Syphacia petrusewiczi but was partially compensated for by an increase in Mesocestoides lineatus and Cladotaenia globifera. It was dominated by nematodes overall, but more so in 1999 than in 2002 when larval cestodes were more frequent. Most derived parameters for component community structure varied considerably between sites and the two surveys, the hierarchical order for sites not being maintained between surveys. They were susceptible to the disproportionate influence of three relatively rare, unpredictable species with the greatest overall aggregated distribution among hosts. Jaccard's similarity index was less influenced by the rare species, showing greater stability between sites and across years. In conclusion, temporal variation confounded any site-specific characteristics of the summary measures quantified in this study and their usefulness is therefore restricted to the years in which the surveys were conducted.

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri.

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L(3) of H. bakeri and then challenged with 10(4) oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-gamma and IL-12 but co-infection with H. bakeri inhibited IFN-gamma secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.

Temporal and between-site variation in helminth communities of bank voles ( Myodes glareolus) from N.E. Poland. 2. The infracommunity level.

The relative importance of temporal and spatial effects was assessed in helminth communities of bank voles (Myodes glareolus) in 3 woodland sites in N.E. Poland in the late summers of 1999 and 2002. Among common species the rank order of sites in relation to prevalence and abundance of infection was maintained between surveys. Site effects accounted for most of the deviance (in statistical models), and time was less important, so the exact location from which voles were sampled was of critical importance. The only exception was Syphacia petrusewiczi. In contrast, for derived measures such as species richness and diversity, most deviance was accounted for by host age, and the interaction between site and year was significant, implying that rank order of sites changed between years. Temporal effects on derived measures were generated primarily by a combination of relatively small changes in prevalence and abundance of the common, rather than the rare, species between the years of the study. In the medium-term, therefore, helminth communities of bank voles in N.E. Poland had a stable core, suggesting a substantial strong element of predictability.

Medium-term temporal stability of the helminth component community structure in bank voles (Clethrionomys glareolus) from the Mazury Lake District region of Poland.

The structure of helminth communities in wild rodents is subject to seasonal variation, and is dependent on host age within years. Although between-year variation has been monitored, seldom has it been assessed rigorously by appropriate multifactorial analysis with potentially confounding factors taken into account. In this study we tested the null hypothesis that despite seasonal, host age and sex effects, helminth communities should show relative stability between years. Over a period of 3 years (1998-2000) we sampled bank vole (Clethrionomys glareolus) populations (total n = 250) at 2 points in the year: in spring, at the start of the breeding season, and in autumn, after the cessation of breeding. In spite of seasonal differences and strong age effects, the between-year effects were surprisingly small. Measures of component community structure (Berger-Parker dominance index, the dominant species, S. petrusewiczi) did not vary, or varied only slightly from year to year. The majority of measures of infracommunity structure [Brillouin's index of diversity, prevalence of all helminths combined, prevalence and abundance of H. mixtum (the most prevalent helminth), mean species richness] did not differ significantly between years when other factors such as age, sex and seasonal variation had been taken into account. Some between-year variations were found (at the component community level, Simpson's index of diversity; at the infracommunity level, prevalence and abundance of S. petrusewiczi and abundance of all helminths combined), but even these were modest in comparison to seasonal and age differences, and were primarily attributable to S. petrusewiczi. We conclude that despite dynamic within-year fluctuations, helminth communities in bank voles in this region of Poland show relative stability across years. The sporadic occurrence of individual platyhelminths at low prevalence, makes little difference to the overall structure, which is largely maintained by the key roles played by the dominant intestinal nematodes of bank voles and the rarer species collectively.

Factors affecting the component community structure of haemoparasites in common voles ( Microtus arvalis) from the Mazury Lake District region of Poland.

The prevalence and abundance of infections with haemoparasites were studied over a 4-year period in Microtus arvalis (common vole, n =321) sampled from fallow grassland sites in north-eastern Poland. Total species richness was five (prevalence= Haemobartonella sp. 63.9%, Bartonella spp. 27.7%, Babesia microti 9.0%, Trypanosoma sp. 8.4% and and Hepatozoon lavieri 3.1%) with 76.9% of the voles carrying at least one species and a mean infracommunity species richness of 1.1. Variation in species richness was determined primarily by season and year, the interaction of these factors, and that of year with host age. The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species. Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal, their interaction, and interactions with intrinsic factors (age and sex), the latter playing only a minor role in their own right. However, the relative importance of these combinations varied and was distinct for each of the species in the study. Prevalence data revealed eight sets of two- and three-way associations between species, mostly dependent to some extent on one of the intrinsic and extrinsic factors in the model. Analysis of quantitative associations suggested two sets of positive two-way interactions, none of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species. These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors that shape component community structures of haemoparasites in wild rodents.

Preliminary molecular characterization of Cryptosporidium parvum isolates of wildlife rodents from Poland.

Isolates of Cryptosporidium were collected from 3 species of woodland and field rodents (Clethrionomys glareolus, Microtus arvalis, and Apodemus flavicollis) and were characterized by polymerase chain reaction amplification and sequencing of fragments of the oocyst wall protein (COWP) gene and of the 18S ribosomal RNA gene. Sequence analysis of these markers revealed that the animals were infected with C. parvum, and that the genotype involved was almost identical to the mouse genotype previously described from Mus musculus. Thus, small rodents should be considered as an important reservoir of C. parvum genotypes closely related to the zoonotic genotype 2 and potentially hazardous to humans.

Local variation in helminth burdens of bank voles (Clethrionomys glareolus) from ecologically similar sites: temporal stability and relationships with hormone concentrations and social behaviour.

Populations of bank voles (Clethrionomys glareolus) in a fragmented forest habitat in north-east Poland showed local differences in helminth infection intensity, morphometric measures and organ weights that were consistent with differences at the same locations two years previously. Although overall intensities of infection were lower than previously, and there were some differences in the relative intensities of individual helminth species, site differences remained significant and were consistent across replicated subsites. In keeping with site differences in helminth infection and adrenal gland weight and asymmetry, voles at site 1 (high intensity infection) had higher circulating concentrations of corticosterone than those at site 2 (low intensity infection). Since males were sampled outside the breeding season, and thus non-scrotal, testosterone levels were low and did not differ between sites. As previously, voles at site 1 also showed greater hind foot asymmetry. Dyadic interactions between males from the same and different sites in the laboratory showed that males from site 1 were significantly less aggressive, especially when confronted with intruder males from site 2. There was no relationship between aggressiveness and intensity of infection overall or at site 1, but a significant negative relationship emerged at site 2. Aggression thus appeared to be downregulated at the higher intensity site independently of individual levels of infection. Terminal corticosterone concentrations were greater at site 1 and lower among residents that initiated more aggression. While corticosterone concentrations rose over the period of testing, they did not correlate with the amount of aggression initiated or received.

Prevalence and abundance of Cryptosporidium parvum and giardia spp. in wild rural rodents from the Mazury Lake District region of Poland.

Prevalence and abundance of Cryptosporodium parvum and Giardia spp. were studied in 3 species of rodents from forests and abandoned agricultural fields in N.E. Poland (Clethrionomys glareolus n = 459; Microtus arvalis n = 274; Apodemus flavicollis n = 209). Overall prevalence was consistently higher in the voles compared with A. flavicollis (70.6, 73.0 and 27.8% respectively for C. parvum and 93.9, 96.3 and 48.3% respectively for Giardia spp.). Prevalence and abundance of infection also varied markedly across 3 years with 1998 being a year of higher prevalence and abundance with both species. Fewer older animals (especially C. glareolus and M. arvalis) carried infection with C. parvum and infections in these animals were relatively milder. Although seasonal differences were significant, no consistent pattern of changes was apparent. Host sex did not influence prevalence or abundance of infection with C. parvum, but made a small contribution to a 4-way interaction (in 5-way ANOVA) with other factors in the case of Giardia spp. The 2 species co-occurred significantly and in animals carrying both parasites there was a highly significant positive correlation between abundance of infection with each, even with between-year, seasonal, host age, sex and species differences taken into account. Quantitative associations were confined to the 2 vole species in the study. These results are discussed in relation to the importance of wild rodents as reservoir hosts and sources of infection for local human communities.

Local variation in endoparasite intensities of bank voles (Clethrionomys glareolus) from ecologically similar sites: morphometric and endocrine correlates.

Much interest has centred recently on the role of adaptive trade-offs between the immune system and other components of life history in determining resistance and parasite intensities among hosts. Steroid hormones, particularly glucocorticoids and sex steroids, provide a plausible mechanism for mediating such trade-offs. A basic assumption behind the hypothesis, however, is that steroid activity will generally correlate with reduced resistance and thus greater parasite intensities. Here, we present some findings from a field study of bank voles (Clethrionomys glareolus) in which we have looked at associations between parasite intensities, anatomical and morphometric measures relating to endocrine function and life history variation in three local populations inhabiting similar but mutually isolated woodland habitats. In general, sites with greater parasite intensities were those in which male C. glareolus had significantly larger adrenal glands, testes and seminal vesicles for their age and body size. Females also showed a site difference in adrenal gland weight. Some aspects of site-related parasite intensity were associated with asymmetry in adrenal gland weight and hind foot length, which may have reflected developmental effects on glucocorticoid activity.

Variation in the helminth community structure in bank voles (Clethrionomys glareolus) from three comparable localities in the Mazury Lake District region of Poland.

We tested the null hypothesis that populations of hosts trapped in isolated neighbouring locations showing comparable habitat quality, should support similar helminth parasite communities. The study was undertaken in a 2-week period in late summer in NE Poland in a single year, thereby eliminating seasonal and between-year variation in parasite burdens. A total of 139 Clethrionomys glareolus (bank vole) were sampled from 3 forest sites of similar habitat quality. Total species richness was 11 (6 nematodes and 5 cestodes) with 85.6% of the voles carrying at least 1 species and an overall mean species richness of 1.4. At the component community level, the fewest species of helminths were recorded from site 2 (n = 6, compared with 9 at each of the other sites), but site 3 had the lowest Berger-Parker Dominance Index and the highest Simpson's Index of Diversity. At the infracommunity level, site 3 had the highest mean no. of helminth species/vole, the highest mean Brillouin's Index of Diversity but the lowest mean no. of helminths/vole. Voles from sites 1 and 3 differed in the nematodes that were most common (site 1, Heligmosum mixtum-95%; site 3, Heligmosomoides glareoli -79.3%). At site 2 no species exceeded 50% but prevalence of Syphacia petrusewiczi was higher than at the other sites. The prevalence of cestodes was too low to test reliably (12.%), but the highest prevalence of adult cestodes was recorded at site 1 (22.5% compared with 4.9 and 1.7% for sites 2 and 3 respectively). Host sex did not influence infection, but mean species richness increased with age. The different sites were responsible for most of the variation in our data, and the intrinsic factors (sex and age) were less important in shaping the component community structure of helminths. We conclude that even locations in relative close proximity to one another (13-25 km), selected on the basis of similar habitat quality, have rodent populations that differ in their helminth parasite communities, although for reasons other than the factors quantified in the present study.

Interactions involving intestinal nematodes of rodents: experimental and field studies.

Multiple species infections with parasitic helminths, including nematodes, are common in wild rodent populations. In this paper we first define different types of associations and review experimental evidence for different categories of interactions. We conclude that whilst laboratory experiments have demonstrated unequivocally that both synergistic and antagonistic interactions involving nematodes exist, field work utilizing wild rodents has generally led to the conclusion that interactions between nematode species play no, or at most a minor, role in shaping helminth component communities. Nevertheless, we emphasize that analysis of interactions between parasites in laboratory systems has been fruitful, has made a fundamental contribution to our understanding of the mechanisms underlying host-protective intestinal immune responses, and has provided a rationale for studies on polyparasitism in human beings and domestic animals. Finally, we consider the practical implications for transmission of zoonotic diseases to human communities and to their domestic animals, and we identify the questions that merit research priority.

Factors affecting the component community structure of haemoparasites in bank voles (Clethrionomys glareolus) from the Mazury Lake District region of Poland.

The prevalence and abundance of infections with haemoparasites were studied over a 3 year period in Clethrionomys glareolus (bank vole, n = 420) sampled from forests in the NE of Poland. Total species richness was 5 (Prevalence = Haemobartonella sp. 63.1%, Bartonella grahamii 27.4%, Hepatozoon erhardovae 31.4%, Trypanosoma evotomys 15% and Babesia microti 1.0%) with 81.9% of the voles carrying at least 1 species and a mean infracommunity species richness of 1.4. Variation in species richness was determined primarily by season and year, and the interaction of these factors. The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species. Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal and their interaction, intrinsic factors such as age and sex playing only a minor role. However, the relative importance of specific extrinsic, and rarely intrinsic, factors varied and was distinct for each of the species in the study. Prevalence data revealed 4 sets of 2-way associations between species, mostly varyingly dependent on combinations of intrinsic and extrinsic factors. Analysis of quantitative associations suggested 4 sets of positive 2-way interactions, 3 of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species, but only one could be unequivocally accepted (Haemobartonella sp. +B. grahamii) after correction for multiple comparisons. These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors which shape component community structures of haemoparasites in wild rodents.

[Co-infection of Borrelia garinii and B. afzelii in a population of wild rodents from woodland]. / Wspólwystepowanie Borrelia garinii i B. afzelii wsród populacji gryzoni lesnych.

The maintenance of Borrelia burgdorferi s.l. in the environment is dependent on the zoonotic cycle involving tick vectors and certain reservoir hosts. It is well known, that the same species of wild rodents, as well as the vector Ixodes ricinus, are often co-infected with at least two genomospecies of B. burgdorferi s.l.: B. afzelii and B. garinii. The ticks collected from two rodent species: Clethrionomys glareolus and Apodemus flavicollis were examined for the presence of B. burgdorferi s.l., as well as for B. garinii and B. afzelii. In this study, an immunofluorescent antibody assay (IFA) and polymerase chain reaction (PCR) protocols were used. The high level of infestation in rodents (90% for C. glareolus and nearly 100% for A. flavicollis) shows that wild rodents are important hosts of the immature stages of I. ricinus. A high percent of Borrelia positive ticks collected from bank voles and yellow necked mice; above 7% determined by IFA and 2% determined by PCR, clearly revealed that these species of animals are competent zoonotic reservoirs of B. burgdorferi s.l.

[The ecology of Cryptosporidium parvum infection in small rodent populations]. / Srodowiskowe uwarunkowania zarazen Cryptosporidium parvum w populacjach drobnych gryzoni.

The prevalence and abundance of Cryptosporidium parvum were studied over a three year period (1997-1999) in three species of rodents sampled from forest and abandoned fields in the Mazury Lake District, Poland. The overall prevalence was consistently higher in voles compared with Apodemus flavicollis (70.6% in Clethrionomys glareolus, 73.0% in Microtus arvalis and 27.8% in A. flavicollis). The prevalence and abundance of infection also varied across the 3 years of the study with 1998 being the year of higher prevalence and abundance of the parasite. Fewer older animals carried the infection, and their infections were relatively mild. We found no consistent pattern of seasonal changes despite the significance of seasonal differences. Host sex did not influence either the prevalence or abundance of infection with C. parvum. A great proportion of recaptured voles developed chronic infections between consecutive trapping sessions and only a small number of animals recovered. However, yellow-necked mice seem to be much more resistant to infection that became self-limiting. Our results firmly establish that the common woodland and grassland wild rodents in the Mazury Lake District constitute a significant and hazardous reservoir of C. parvum for animals and humans.

[Usefulness of the molecular techniques for detecting and/or identifing of parasites and fungi in humans and animals or pathogens transmitted by ticks (Part I)]. / Zastosowanie technik molekularnych do wykrywania i/lub identyfikacji pasozytów i grzybów u ludzi i zwierzat oraz pa- togenów przenoszonych przez kleszcze. Czesc.

After a long period of using basic microscopic, immunological and biochemical methods for diagnosis, rapid development of nucleic acids investigation enabled introduction of specific and sensitive methods of detection of pathogenic agents on the molecular level. Among others, polymerase chain reaction (PCR), discovered in mid of 80'ies and then automatized, offered an attractive alternative to conventional testing systems. In this paper we describe reliable diagnostic tests widely used in the world, including Poland, and capable of detecting different disease agents as parasites and fungi in clinical specimens and pathogens of emerging zoonotic diseases in ticks. The possibilities of using molecular methods for determination of Plasmodium falciparum drug resistance is also discussed. Moreover, the report offers information concerning kinds of molecular tests and institutions in which there are executed.