Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Continuous presence of genetically diverse rustrela virus lineages in yellow-necked field mouse reservoir populations in northeastern Germany.

Rustrela virus (RusV; species Rubivirus strelense, family Matonaviridae) was discovered in different zoo animal species affected by fatal encephalitis. Simultaneous RusV RNA detection in multiple yellow-necked field mice (Apodemus flavicollis) suggested this rodent as a reservoir of RusV. Here, we investigated 1,264 yellow-necked field mice and sympatric other small mammals from different regions in Germany for RusV RNA using an optimized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6 per cent) yellow-necked field mice but absence in other sympatric species. RT-qPCR-determined tissue distribution of RusV RNA revealed the highest viral loads in the central nervous system, with other tissues being only very rarely affected. The histopathological evaluation did not reveal any hints of encephalitis in the brains of infected animals despite the detection of viral RNA in neurons by in situ hybridization (ISH). The positive association between the body mass of yellow-necked field mice and RusV RNA detection suggests a persistent infection. Phylogenetic analysis of partial E1 and full-genome sequences showed a high diversification with at least four RusV lineages (1A-1D) in northeastern Germany. Moreover, phylogenetic and isolation-by-distance analyses indicated evolutionary processes of RusV mostly in local reservoir populations. A comparison of complete genome sequences from all detected RusV lineages demonstrated a high level of amino acid and nucleotide sequence variability within a part of the p150 peptide of the non-structural polyprotein and its coding sequence, respectively. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of the rubella virus. The broad range of detected RusV spillover hosts in combination with its geographical distribution in northeastern Germany requires the assessment of its zoonotic potential and further analysis of encephalitis cases in mammals. Future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.

Seroprevalence of Tick-Borne Encephalitis (TBE) Virus Antibodies in Wild Rodents from Two Natural TBE Foci in Bavaria, Germany.

Tick-borne encephalitis (TBE) is Eurasia's most important tick-borne viral disease. Rodents play an important role as natural hosts. Longitudinal studies on the dynamics of the seroprevalence rates in wild rodents in natural foci over the year are rare, and the dynamics of the transmission cycle still need to be understood. To better understand the infection dynamics, rodents were captured in a capture-mark-release-recapture-study in two natural foci in Bavaria, Germany, monthly from March 2019 to October 2022. Overall, 651 blood and thoracic lavage samples from 478 different wild rodents (Clethrionomys glareolus and Apodemus flavicollis) were analyzed for antibodies against tick-borne encephalitis virus (TBEV) by indirect immunofluorescence assay (IIFA) and confirmed using a serum neutralization test (SNT). Furthermore, a generalized linear mixed model (GLMM) analysis was performed to investigate ecological and individual factors for the probability of infection in rodents. Clethrionomys glareolus (19.4%) had a higher seroprevalence than A. flavicollis (10.5%). Within Cl. glareolus, more males (40.4%) than females (15.6%) were affected, and more adults (25.4%) than juveniles (9.8%). The probability of infection of rodents rather depends on factors such as species, sex, and age than on the study site of a natural focus, year, and season. The high incidence rates of rodents, particularly male adult bank voles, highlight their critical role in the transmission cycle of TBEV in a natural focus and demonstrate that serologically positive rodents can be reliably detected in a natural focus regardless of season or year. In addition, these data contribute to a better understanding of the TBEV cycle and thus could improve preventive strategies for human infections.

Investigation and response to a large outbreak of leptospirosis in field workers in Lower Saxony, Germany.

Between June and August 2014, 45 cases of leptospirosis were notified among workers on two strawberry farms in North-West Germany. We describe the characteristics of the outbreak and the actions taken to prevent further cases. The activities of the local, federal and national public health and veterinary authorities included collection of case data, laboratory testing of human specimens and of small mammals trapped on the fields, investigation of weather data, as well as information provided to farmers, field workers, physicians and to the authorities in Poland and Romania. Of the 45 identified cases (median age 22, 60% male), 47% were hospitalized. Characteristic symptoms were fever ≥38.5°C, generalized muscle pain and an increase in renal or liver enzymes. Thirteen cases were laboratory confirmed by serological and/or molecular methods. ELISA tests for Leptospira IgG and IgM-antibodies were positive in those samples taken >5 days after hospitalization. The probable causative agent was identified as Leptospira kirschneri serovar Grippotyphosa. Leptospira-specific DNA was found in kidneys of 67% of 64 trapped small mammals and was further identified as Leptospira kirschneri multi locus sequence type 110. During the estimated time period of human infections, the affected region faced warm weather with heavy rainfalls. The results of this investigation are in accordance with the theory of a chain of infection from mice to field workers during warm and humid weather. In 2015, a campaign was initiated to inform physicians, farmers and workers to enhance prevention measures, such as the use of personal protective equipment and early consultation of physicians in case of illness. Since then, no further outbreak occurred.

From trees to fleas: masting indirectly affects flea abundance on a rodent host.

Mast seeding causes strong fluctuations in populations of forest animals. Thus, this phenomenon can be used as a natural experiment to examine how variation in host abundance affects parasite loads. We investigated fleas infesting yellow-necked mice in beech forest after 2 mast and 2 non-mast years. We tested 2 mutually exclusive scenarios: (1) as predicted by classical models of density-dependent transmission, an increase in host density will cause an increase in ectoparasite abundance (defined as the number of parasites per host), versus (2) an increase in host density will cause a decline in flea abundance ("dilution," which is thought to occur when parasite population growth is slower than that of the host). In addition, we assessed whether masting alters the relationship between host traits (sex and body mass) and flea abundance. We found a hump-shaped relationship between host and flea abundance. Thus, the most basic predictions are too simple to describe ectoparasite dynamics in this system. In addition, masting modified seasonal dynamics of flea abundance, but did not affect the relationship between host traits and flea abundance (individuals with the highest body mass hosted the most fleas; after controlling for body mass, parasite abundance did not vary between sexes). Our results demonstrate that pulses of tree reproduction can indirectly, through changes in host densities, drive patterns of ectoparasite infestation.

Leptospira interrogans Sensu Lato in Wild Small Mammals in Three Moravian Localities of the Czech Republic.

Leptospirosis is a widespread zoonosis, affecting humans, domestic animals and wildlife, with small mammals as a reservoir of this infection. In recent years, this disease has been re-emerging and affects approximately 1 million people all over the world each year. Due to this disease having a significant health impact, it is important to identify the source and method of infection. The risk of Leptospira sp. infection is higher mainly in the cities of developed and industrialised countries. The aim of the study was the detection of antibodies against Leptospira sp. in some wild small mammals captured in the Czech Republic. In total, samples of 855 animals captured in three locations of Moravia during a six-year study (2010-2015) were examined by a microscopic agglutination test, using eight serovars of Leptospira interrogans sensu lato, representing serogroups Grippotyphosa, Icterohaemorrhagiae, Australis, Canicola, Sejroe, Javanica, Pomona and Pyrogenes, as antigens. Antibodies to Leptospira sp. were detected in 6.1% (52/855) of animals, with a prevalence of 6.4% (51/801) and 1.9% (1/54) in rodents and insectivores, respectively. The only statistically significant difference (p ≤ 0.05) was in prevalence between individual species (0-33%), while there were no differences in sex (6.7% in females and 5.1% in males), locality (1.8-8%) and year of trapping (0-8.4%). Only two serovars, L. interrogans serovar Pomona and L. interrogans serovar Grippotyphosa, were detected in 5.5% and 0.5% of animals, respectively. The prevailing serovar of pathogenic L. interrogans s.l. can be identified in a number of infected people in the Czech Republic. The composition of vaccines should be based on the current occurrence of Leptospira serovars in the actual territory. For this reason, the occurrence of Leptospira and its serovars should therefore be regularly monitored.

Identification of Dobrava-Belgrade Virus in Apodemus flavicollis from North-Eastern Italy during Enhanced Mortality.

Hantaviruses include several zoonotic pathogens that cause different syndromes in humans, with mortality rates ranging from 12 to 40%. Most commonly, humans get infected through the inhalation of aerosols or dust particles contaminated with virus-containing rodent excreta. Hantaviruses are specifically associated with the host species, and human cases depend on the presence and the dynamics of reservoir hosts. In this letter, we report the identification of Dobrava-Belgrade virus (DOBV) in the yellow-necked mouse (Apodemus flavicollis) from Italy. The virus was detected in the mountainous area of the province of Udine, bordering Austria and Slovenia, during an event of enhanced mortality in wild mice and voles. Despite serological evidence in rodents and humans that suggested the circulation of hantaviruses in Italy since 2000, this is the first virological confirmation of the infection. Phylogenetic analyses across the whole genome of the two detected viruses confirmed the host-specificity of DOBV sub-species and showed the highest identity with viruses identified in Slovenia and Croatia from both A. flavicollis and humans, with no signs of reassortment. These findings highlight the need for ecologists, veterinarians and medical doctors to come together in a coordinated approach in full compliance with the One Health concept.

Temporal Partitioning between Forest-Dwelling Small Rodents in a Mediterranean Deciduous Woodland.

Temporal partitioning is reported as one of the main strategies adopted by coexisting mammal species to limit interspecific competition and behavioural interference. In the last decades, camera-trapping surveys have provided valuable insights in assessing temporal niche and activity rhythms of medium and large-sized mammalian species. Conversely, this method has been poorly applied to small rodents. In this work we aimed at assessing temporal niche partitioning between two species of forest-dwelling small rodents-Apodemus flavicollis and Clethrionomys glareolus-by means of intensive camera-trapping. Camera traps were placed in areas where previous genetic analyses have confirmed the only presence of A. flavicollis amongst wood mice species, to prevent misinterpretation of records. We collected 124 independent records of A. flavicollis and 67 records of C. glareolus over three years. The former was mostly nocturnal, with activity peaking after midnight, whereas the latter was mostly active at dawn and dusk. Therefore, a limited temporal overlap was observed, confirming the potential for interspecific competition. Intraguild interference competition between A. flavicollis and C. glareolus may play a pivotal role forcing C. glareolus to be more active in daylight hours where, the more strictly nocturnal A. flavicollis is present. Nocturnal activity of C. glareolus was limited and not influenced by moon phases, whereas A. flavicollis was mostly active in the darkest nights, avoiding bright moonlight nights.

Lead content in soil, plants, rodents, and amphibians in the vicinity of a heating plant's ash waste.

This study supplements previous research focused on environmental condition in the vicinity of waste ash material. The main aim of our study was the comparative analysis of lead levels in soil, plant, and animal organisms in the area of the tailings pond and surroundings, using x-ray. Findings confirm that the level of Pb in the top layer of soil is in the range of 20-135 ppm. Lead content in Calamagrostis plant tissues was confirmed only at the tailings pond area, with the highest lead concentrations measured in above-ground components; stems with blooms followed by roots and ground floor sheats. The livers, kidneys, and hearts of Apodemus flavicollis were examined, with findings of higher values in the tailings pond area than in the reference site, and average values of 14.5 ppm for livers, 16.0 ppm for kidneys, and 16.6 ppm for hearts. No significant differences were discovered based on sex and body length/body weight of A. flavicollis individuals. Values for Bombina variegata liver tissue reached an average of 12.3 ppm for individuals caught in a water reservoir without ash sediments, versus 15.7 ppm in those trapped by the edge of then tailings pond area. Females had lower concentrations of lead than males, but with no statistically significant differences found. Despite lower lead levels in soil and ash than expected, concentrations in mammalian and amphibian organs suggest a possible transition of this element into the food chain, and therefore further research in this area is recommended.

Divergence in Coding Sequence and Expression of Different Functional Categories of Immune Genes between Two Wild Rodent Species.

Differences in immune function between species could be a result of interspecific divergence in coding sequence and/or expression of immune genes. Here, we investigate how the degree of divergence in coding sequence and expression differs between functional categories of immune genes, and if differences between categories occur independently of other factors (expression level, pleiotropy). To this end, we compared spleen transcriptomes of wild-caught yellow-necked mice and bank voles. Immune genes expressed in the spleen were divided into four categories depending on the function of the encoded protein: pattern recognition receptors (PRR); signal transduction proteins; transcription factors; and cyto- and chemokines and their receptors. Genes encoding PRR and cyto-/chemokines had higher sequence divergence than genes encoding signal transduction proteins and transcription factors, even when controlling for potentially confounding factors. Genes encoding PRR also had higher expression divergence than genes encoding signal transduction proteins and transcription factors. There was a positive correlation between expression divergence and coding sequence divergence, in particular for PRR genes. We propose that this is a result of that divergence in PRR coding sequence leads to divergence in PRR expression through positive feedback of PRR ligand binding on PRR expression. When controlling for sequence divergence, expression divergence of PRR genes did not differ from other categories. Taken together, the results indicate that coding sequence divergence of PRR genes is a major cause of differences in immune function between species.

B Chromosomes' Sequences in Yellow-Necked Mice Apodemus flavicollis-Exploring the Transcription.

B chromosomes (Bs) are highly polymorphic additional chromosomes in the genomes of many species. Due to the dispensability of Bs and the lack of noticeable phenotypic effects in their carriers, they were considered genetically inert for a long time. Recent studies on Bs in Apodemus flavicollis revealed their genetic composition, potential origin, and spatial organization in the interphase nucleus. Surprisingly, the genetic content of Bs in this species is preserved in all studied samples, even in geographically distinct populations, indicating its biological importance. Using RT-PCR we studied the transcription activity of three genes (Rraga, Haus6, and Cenpe) previously identified on Bs in A. flavicollis. We analysed mRNA isolated from spleen tissues of 34 animals harboring different numbers of Bs (0-3).The products of transcriptional activity of the analysed sequences differ in individuals with and without Bs. We recorded B-genes and/or genes from the standard genome in the presence of Bs, showing sex-dependent higher levels of transcriptional activity. Furthermore, the transcriptional activity of Cenpe varied with the age of the animals differently in the group with and without Bs. With aging, the amount of product was only found to significantly decrease in B carriers. The potential biological significance of all these differences is discussed in the paper.

Climate change has cascading effects on tree masting and the breeding performance of a forest songbird in a primeval forest.

Forest habitats change significantly under the influence of global warming. It is important to predict the effects of these changes, especially in primeval forests which currently represent a small percentage of temperate forests. Such changes often manifest themselves in an acceleration of the frequency of mass seeding of trees, which causes cascading effects in various organisms. We evaluated changes in: tree masts (oak Quercus robur and hornbeam Carpinus betulus), rodent abundance (yellow-necked mouse Apodemus flavicollis), folivorous caterpillar abundance (winter moth Operophtera brumata), and the breeding success of a cavity-nesting songbird (collared flycatcher Ficedula albicollis) in over a 30-year period in the Bialowieza Forest (E Poland). We also analysed temperature, precipitation and snow cover to determine the effects of weather on each trophic level. Previous studies have exposed the indirect effect of tree masting on songbirds breeding in open nests. Our study uniquely highlights the relationships between trees, rodents, caterpillars, and a cavity-nesting bird. Precipitation was positively correlated with the fructification of trees, abundance of caterpillars, and the breeding losses of flycatchers (in July, August, October in the previous year, in May in the current year, respectively). We found that along with the changing climate, the frequency of mast years of oak increased, which caused an increasing frequency of rodent outbreaks. The abundance of mice was positively correlated with the predation on flycatcher broods (current year) and negatively - with the abundance of caterpillars (following year). We predict that current global trends in climate change will have a negative impact on the flycatcher due to the cascading effects from the above species. Bearing in mind that F. albicollis is one of the most numerous bird species, it can be assumed that more frequent masting will result in substantial changes in the entire bird assemblage, and presumably also other groups of animals.

"Parasite turnover zone" at secondary contact: A new pattern in host-parasite population genetics.

We describe here a new pattern of population genetic structure in a host-parasite system that can arise after secondary contact of previously isolated populations. Due to different generation times, and therefore different tempos of molecular evolution, the host and parasite populations reach different degrees of genetic differentiation during their separation (e.g., in refugia). Consequently, upon secondary contact, the host populations are able to re-establish a single panmictic population across the area of contact, while the parasite populations stop their dispersal at the secondary contact zone and create a narrow hybrid zone. From the host's perspective, the parasite's hybrid zone functions on a microevolutionary scale as a "parasite turnover zone": while the hosts are passing from area A to area B, their parasites turn genetically from the area A genotypes to the area B genotypes. We demonstrate this novel pattern with a model composed of Apodemus mice and Polyplax lice by comparing maternally inherited markers (complete mitochondrial genomes, and complete genomes of the vertically transmitted symbiont Legionella polyplacis) with single nucleotide polymorphisms derived from louse genomic data. We discuss the circumstances that may lead to this pattern and possible reasons why it has been overlooked in studies of host-parasite population genetics.

Population structure of Apodemus flavicollis and comparison to Apodemus sylvaticus in northern Poland based on RAD-seq.

BACKGROUND: Mice of the genus Apodemus are one the most common mammals in the Palaearctic region. Despite their broad range and long history of ecological observations, there are no whole-genome data available for Apodemus, hindering our ability to further exploit the genus in evolutionary and ecological genomics context. RESULTS: Here we present results from the double-digest restriction site-associated DNA sequencing (ddRAD-seq) on 72 individuals of A. flavicollis and 10 A. sylvaticus from four populations, sampled across 500 km distance in northern Poland. Our data present clear genetic divergence of the two species, with average p-distance, based on 21377 common loci, of 1.51% and a mutation rate of 0.0011 - 0.0019 substitutions per site per million years. We provide a catalogue of 117 highly divergent loci that enable genetic differentiation of the two species in Poland and to a large degree of 20 unrelated samples from several European countries and Tunisia. We also show evidence of admixture between the three A. flavicollis populations but demonstrate that they have negligible average population structure, with largest pairwise FST<0.086. CONCLUSION: Our study demonstrates the feasibility of ddRAD-seq in Apodemus and provides the first insights into the population genomics of the species.

Chronic exposure to lead and cadmium pollution results in genomic instability in a model biomonitor species (Apodemus flavicollis Melchior, 1834).

Polymetal dust is a common industrial pollutant. While the use of remediation filters and equipment in lead smelters has reduced pollutant emission, surrounding areas remain contaminated due to the long-term transfer of heavy metals along the food chain. Here we assess the mutagenic potential of the lead-zinc smelter near Plovdiv (Bulgaria) situated in an area that has been contaminated with heavy metals for 60 years. We aimed to evaluate the genomic response of the yellow-necked mouse (A. flavicollis), a biomonitor species, in three sampling sites along the pollution gradient. Mice from Strandzha Natural Park were used as a negative control. The bioaccumulation rate of two non-essential heavy metals, lead (Pb) and cadmium (Cd), in liver tissues was determined by atomic absorption spectroscopy. Genetic alterations attributable to chronic exposure to trace levels of heavy metals were assessed in different blood cell populations using two independent methods: a micronucleus test was applied to evaluate the clastogenic and aneugenic alterations in erythrocytes, while a comet assay was used to assess DNA instability, as evidenced by single- and double-stranded breaks and alkali-labile sites, in leucocytes. We observed elevated levels of Pb and Cd in livers derived from mice from the impacted area: the mean Pb concentration (21.38 ± 8.77 µg/g) was two-fold higher than the lowest-observed-adverse-effect levels (LOAELs), while the mean Cd concentration (13.95 ± 9.79 µg/g) was extremely close to these levels. The mean levels of Pb and Cd in livers derived from mice from the impacted area were 31-fold and 63-fold higher, respectively, than the levels measured in mice from the control area. The mean frequency of micronuclei was significantly higher (four-fold) than that observed in the control animals. Furthermore, parameters measured by the comet assay, % tail DNA, tail length and tail moment, were significantly higher in the impact area, indicating the degree of genetic instability caused by exposure to heavy metals. In conclusion, this study shows that despite the reported reduction in lead and cadmium emissions in Bulgaria in recent years, A. flavicollis individuals inhabiting areas subject to long-term contamination exhibit significant signs of DNA damage.

Bartonella spp. in Small Mammals and Their Fleas in Differently Structured Habitats From Germany.

Most Bartonella spp. are transmitted by fleas and harbored by small mammals which serve as reservoirs. However, little is known about the composition of fleas and their Bartonella spp. from small mammals in Central Europe. Therefore, the aims of this study were to investigate flea communities on small mammals from three differently structured sites (urban, sylvatic, renatured) in Germany as well as the prevalence of Bartonella spp. in small mammals and their parasitizing fleas. In total, 623 small mammals belonging to 10 different species (the majority were Myodes glareolus and Apodemus flavicollis) were available. Fleas were removed from the small mammals' fur, morphologically identified and DNA was extracted. To detect Bartonella spp., two conventional PCRs targeting the gltA gene and the 16S-23S rRNA intergenic spacer were carried out followed by sequencing. Obtained sequences were compared to those in GenBank. In total, 1,156 fleas were collected from 456 small mammals. Altogether, 12 different flea species (the majority were Ctenophthalmus agyrtes, Nosopsyllus fasciatus, and Megabothris turbidus) were detected. At the urban site mostly Leptopsylla segnis and N. fasciatus were collected which may be vectors of zoonotic pathogens to companion animals. The overall prevalence for Bartonella in small mammals was 43.3% and in fleas 49.1%. Five different Bartonella spp. were detected in small mammals namely B. grahamii, B. taylorii, B. doshiae, Bartonella sp. N40 and uncultured Bartonella sp. whereas in fleas four Bartonella spp. were found which were with the exception of B. doshiae identical to the Bartonella species detected in their small mammal hosts. While B. grahamii was the only zoonotic Bartonella sp. most Bartonella strains found in fleas and small mammals belonged to uncultured Bartonella spp. with unknown zoonotic potential. This study showed a high diversity of flea species on small mammals from Germany. Further, high prevalence rates of Bartonella species were detected both in fleas and in their mammalian hosts. Several different Bartonella species with a high genetic variability were discovered. Especially at the urban study sites, this may pose a risk for Bartonella transmission to companion animals and humans.

Parasites in brains of wild rodents (Arvicolinae and Murinae) in the city of Leipzig, Germany.

Small rodents serve as intermediate or paratenic hosts for a variety of parasites and may participate in the transmission of these parasites into synanthropic cycles. Parasites with neuroinvasive stages, such as Toxoplasma gondii or Toxocara canis, can cause detrimental damage in the brain of intermediate or paratenic hosts. Therefore, the occurrence of neuroinvasive parasite stages was evaluated in brains of wild rodents captured in the city of Leipzig, Germany. In addition, a few specimens from the cities of Hanover, Germany, and Vienna, Austria were included, resulting in a total of 716 rodents collected between 2011 and 2016. Brains were investigated for parasitic stages by microscopic examination of native tissue, artificially digested tissue as well as Giemsa-stained digestion solution to verify positive results. Infective stages of zoonotic ascarids or other helminths were not detected in any sample, while coccidian cysts were found in 10.1% (95% CI: 7.9-12.5%; 72/716) of examined brains. The most abundant rodent species in the study was the bank vole (Myodes glareolus; Arvicolinae), showing an infection rate with cerebral cysts of 13.9% (95% CI: 11.0-17.8%; 62/445), while 2.7% (95% CI: 1.0-5.8%; 6/222) of yellow-necked mice (Apodemus flavicollis; Murinae) were infected. Generalized linear modelling revealed a statistically significant difference in prevalence between M. glareolus and A. flavicollis, significant local differences as well as an effect of increasing body mass on cyst prevalence. Coccidian cysts were differentiated by amplification of the 18S rRNA gene and subsequent sequencing. The majority of identifiable cysts (97.9%) were determined as Frenkelia glareoli, a coccidian species mainly circulating between M. glareolus as intermediate and buzzards (Buteo spp.) as definitive hosts. The zoonotic pathogen Toxoplasma gondii was confirmed in one M. glareolus originating from the city of Leipzig. Overall, it can be concluded that neuroinvasion of zoonotic parasites seems to be rare in M. glareolus and A. flavicollis.

Changes in host densities and co-feeding pattern efficiently predict tick-borne encephalitis hazard in an endemic focus in northern Italy.

Tick-borne encephalitis is an important zoonosis in many parts of north-western, central and eastern Europe, Russia and the Far East, with considerable altitudinal and latitudinal shifts described during recent decades. The reported routes of transmission for TBE virus include the saliva-activated non-viraemic transmission between co-feeding ticks taking place on rodent hosts. During the period 2001-2014, a population of the yellow-necked mouse (Apodemus flavicollis), which is considered among the most efficient TBE competent host, especially in central and western Europe, was intensively live-trapped in a known TBE focus in the Province of Trento, Italy. Individual live-trapped mice were checked for the number and position of feeding ticks and serologically screened for TBEv antibodies. A combined effect of climatic conditions and density of both roe deer and mice on the number of co-feeding tick groups was observed. Specifically, the occurrence of co-feeding ticks on mice during the questing season was affected by autumnal cooling in the previous season. On the other hand, co-feeding occurrence was also positively associated with roe deer abundance, while mouse density showed a hump-shaped pattern. Individual features of A. flavicollis such as weight and sex also affected co-feeding occurrence with the heaviest (breeding adult) males carrying more co-feeding ticks. We also found that the overall number of co-feeding ticks on mice positively affected TBEv antibody detection in this species the following year. In conclusion, a specific combination of climatic conditions in conjunction with certain rodent and roe deer densities are the principal determinants of the number of co-feeding ticks on A. flavicollis and, consequently, TBEv circulation. These variables can be used to provide an early warning signal for a TBE hazard, thus representing a useful tool for Public Health authorities to prepare action for prevention and control within TBEv circulation areas.

Long-term trends of tick-borne pathogens in regard to small mammal and tick populations from Saxony, Germany.

BACKGROUND: Rodents are important in the life-cycle of ticks as hosts for immature developmental stages. Both rodents and ticks are of public health interest as they are reservoirs and vectors for different tick-borne pathogens (TBP). The aim of this study was to reassess the prevalence of TBP in previously studied areas of the city of Leipzig (Saxony, Germany). METHODS: In the years 2015-2017 rodents and ticks were collected in parks and forest areas in Saxony. DNA was extracted from the rodents, attached and questing ticks. Samples were screened for the presence of Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi (s.l.), "Candidatus Neoehrlichia mikurensis" (CNM), Bartonella spp., Hepatozoon spp. and Rickettsia spp. using PCR methods. Rodent, attached nymph and questing tick (nymph and adult) samples were tested individually, while attached larvae were further processed in pools. RESULTS: A total of 165 rodents (Apodemus agrarius, n = 1; A. flavicollis, n = 59; Arvicola terrestris, n = 1; Myodes glareolus, n = 104), 1256 attached ticks (Ixodes ricinus, n = 1164; Dermacentor reticulatus, n = 92) and 577 questing ticks (I. ricinus, n = 547; D. reticulatus, n = 30) were collected. The prevalence levels in rodents were 78.2% for Bartonella spp., 58.2% for CNM, 49.1% for B. burgdorferi (s.l.) 29.1% for Rickettsia spp. and 24.2% for Hepatozoon spp. The minimal infection rates (MIR) in attached larvae ticks were 39.8% for Rickettsia spp., 32.7% for Bartonella spp., 7.1% for CNM and 8.8% for B. burgdorferi (s.l.) and the prevalence rates in attached nymphs were 33.7% for Bartonella spp., 52.9% for Rickettsia spp., 13.5% for CNM and 11.3% for B. burgdorferi (s.l.) Both rodents and attached ticks were negative for Babesia spp. The prevalence in questing ticks was 18.2% for Rickettsia spp., 7.3% for CNM, 6.4% for B. burgdorferi (s.l.) and 1.4% for Babesia spp. All tested samples were Anaplasma-negative. Sequencing revealed the occurrence of 14 identified species. CONCLUSIONS: This research is the first evaluation of the prevalence for Hepatozoon spp. in rodents from Germany. In comparison to earlier studies, detected pathogens species remained the same; however, the prevalence for particular pathogens differed.

Regional and local patterns of genetic variation and structure in yellow-necked mice - the roles of geographic distance, population abundance, and winter severity.

The goal of this study, conducted in seven large woodlands and three areas with small woodlots in northeastern Poland in 2004-2008, was to infer genetic structure in yellow-necked mouse Apodemus flavicollis population and to evaluate the roles of environmental and population ecology variables in shaping the spatial pattern of genetic variation using 768 samples genotyped at 13 microsatellite loci. Genetic variation was very high in all studied regions. The primal genetic subdivision was observed between the northern and the southern parts of the study area, which harbored two major clusters and the intermediate area of highly admixed individuals. The probability of assignment of individual mice to the northern cluster increased significantly with lower temperatures of January and July and declined in regions with higher proportion of deciduous and mixed forests. Despite the detected structure, genetic differentiation among regions was very low. Fine-scale structure was shaped by the population density, whereas higher level structure was mainly shaped by geographic distance. Genetic similarity indices were highly influenced by mouse abundance (which positively correlated with the share of deciduous forests in the studied regions) and exhibited the greatest change between 0 and 1 km in the forests, 0 and 5 km in small woodlots. Isolation by distance pattern, calculated among regions, was highly significant but such relationship between genetic and geographic distance was much weaker, and held the linearity at very fine scale (~1.5 km), when analyses were conducted at individual level.