Identification of New Microfoci and Genetic Characterization of Tick-Borne Encephalitis Virus Isolates from Eastern Germany and Western Poland.

(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.

Vector-Borne and Zoonotic Pathogens in Raccoon Dogs (Nyctereutes procyonoides) and Raccoons (Procyon lotor) from Schleswig-Holstein, Germany.

Raccoon dogs (Nyctereutes procyonoides) and raccoons (Procyon lotor) are invasive alien species originating from East Asia and North America, respectively. They are discussed as vectors and reservoirs for various infectious diseases, including vector-borne and zoonotic pathogens, and are therefore a potential threat to human and domestic animal health, as well as to biodiversity and conservation. In the years 2021 and 2022, 110 raccoon dogs (Nyctereutes procyonoides) and 30 raccoons (Procyon lotor) were screened via qPCR for the presence of Leptospira spp., Rickettsia spp. and Borreliella spp. in the German federal state of Schleswig-Holstein as part of a health and risk assessment study. Borreliella spp. were confirmed in one raccoon dog and one raccoon, identified as Borreliella afzelii in the raccoon. Leptospira spp. were found in 21 (19.44%) raccoon dogs and 2 (6.90%) raccoons. In five raccoon dogs, Leptospira spp. were identified as Leptospira borgpetersenii, Leptospira kirschneri and Leptospira interrogans.

Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

Spotted Fever Group Rickettsiae in Ticks and Small Mammals from Grassland and Forest Habitats in Central Germany.

Rickettsiae of the spotted fever group (SFG) are zoonotic tick-borne pathogens. Small mammals are important hosts for the immature life stages of two of the most common tick species in Europe, Ixodes ricinus and Dermacentor reticulatus. These hosts and vectors can be found in diverse habitats with different vegetation types like grasslands and forests. To investigate the influence of environmental and individual factors on Rickettsia prevalence, this study aimed to analyse the prevalence of SFG rickettsiae in ticks and small mammals in different small-scale habitats in central Germany for the first time. Small mammals of ten species and ticks of two species were collected from grasslands and forests in the Hainich-Dün region, central Germany. After species identification, DNA samples from 1098 ticks and ear snips of 1167 small mammals were screened for Rickettsia DNA by qPCR targeting the gltA gene. Positive samples were retested by conventional PCR targeting the ompB gene and sequencing. Rickettsia DNA was detected in eight out of ten small mammal species. Small mammal hosts from forests (14.0%) were significantly more often infected than those from grasslands (4.4%) (p < 0.001). The highest prevalence was found in the mostly forest-inhabiting genus Apodemus (14.8%) and the lowest in Microtus (6.6%), which inhabits grasslands. The prevalence was higher in D. reticulatus (46.3%) than in the I. ricinus complex (8.6%). Adult ticks were more often infected than nymphs (p = 0.0199). All sequenced rickettsiae in I. ricinus complex ticks were R. helvetica, and the ones in D. reticulatus were R. raoultii. Unlike adults, questing nymphs have had only one blood meal, which explains the higher prevalence in I. ricinus adults. Interestingly, habitat type did influence infection probability in small mammals, but did not in ticks. A possible explanation may be the high prevalence in Apodemus flavicollis and A. sylvaticus which were more abundant in the forest.

Preliminary Study on Artificial versus Animal-Based Feeding Systems for Amblyomma Ticks (Acari: Ixodidae).

Hard ticks pose a threat to animal and human health. Active life stages need to feed on a vertebrate host in order to complete their life cycle. To study processes such as tick-pathogen interactions or drug efficacy and pharmacokinetics, it is necessary to maintain tick colonies under defined laboratory conditions, typically using laboratory animals. The aim of this study was to test a membrane-based artificial feeding system (AFS) applicable for Amblyomma ticks using Amblyomma tonelliae as a biological model. Adult ticks from a laboratory colony were fed in a membrane-based AFS. For comparison, other A. tonelliae adults were fed on calf and rabbit. The proportions of attached (AFS: 76%; calf/rabbit: 100%) and engorged females (AFS: 47.4%; calf/rabbit: 100%) in the AFS were significantly lower compared to animal-based feeding (p = 0.0265). The engorgement weight of in vitro fed ticks (x¯ = 658 mg; SD ± 259.80) did not significantly differ from that of ticks fed on animals (p = 0.3272, respectively 0.0947). The proportion of females that oviposited was 100% for all three feeding methods. However, the incubation period of eggs (x¯ = 54 days; SD ± 7) was longer in the AFS compared to conventional animal-based feeding (p = 0.0014); x¯ = 45 days; SD ± 2 in the rabbit and (p = 0.0144). x¯ = 48 days; SD ± 2 in the calf). Egg cluster hatching (x¯ = 41%; SD ± 44.82) was lower in the AFS than in the other feeding methods (rabbit: x¯ = 74%; SD ± 20; p = 0.0529; calf: x¯ = 81%; SD ± 22; p = 0.0256). Although the attachment, development, and the hatching of AFS ticks were below those from animal-based feeding, the method may be useful in future experiments. Nevertheless, further experiments with a higher number of tick specimens (including immature life stages) and different attractant stimuli are required to confirm the preliminary results of this study and to evaluate the applicability of AFS for Amblyomma ticks as an alternative to animal-based feeding methods.

White-Toothed Shrews (Genus Crocidura): Potential Reservoirs for Zoonotic Leptospira spp. and Arthropod-Borne Pathogens?

Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored (Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens (Leptospira spp., Coxiella burnetii, Brucella spp., Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis and Bartonella spp.). We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.

Horses as Sentinels for the Circulation of Flaviviruses in Eastern-Central Germany.

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

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.

Diversity of Borrelia burgdorferi sensu lato in ticks and small mammals from different habitats.

BACKGROUND: Ixodid ticks are important vectors for zoonotic pathogens, with Ixodes ricinus being the most important in Europe. Rodents are hosts of immature life stages of I. ricinus ticks and are considered main reservoirs for tick-borne pathogens, e.g. Borrelia burgdorferi. The aim of this study was to analyse the prevalence as well as genospecies and sequence type (ST) diversity of Borrelia burgdorferi sensu lato in ticks and small mammals from central Germany and to elaborate on the influence of environmental and/or individual host and vector factors on Borrelia prevalence. METHODS: After species identification, 1167 small mammal skin samples and 1094 ticks from vegetation were screened by B. burgdorferi sensu lato real-time polymerase chain reaction, and positive samples were characterized by multilocus sequence typing. Generalized linear (mixed) models were used to estimate how seasonality, small mammal species/tick life stage and habitat affect individual infection status. RESULTS: In total, 10 small mammal species and three tick species, Ixodes ricinus, Ixodes inopinatus (both considered members of the I. ricinus complex) and Dermacentor reticulatus, were investigated. Borrelia DNA was detected in eight host species, i.e. the striped field mouse (Apodemus agrarius), the yellow-necked field mouse (Apodemus flavicollis), the wood mouse (Apodemus sylvaticus), the water vole (Arvicola amphibius), the bank vole (Clethrionomys glareolus), the field vole (Microtus agrestis), the common vole (Microtus arvalis), and the common shrew (Sorex araneus). Two species were Borrelia negative, the greater white-toothed shrew (Crocidura russula) and the pygmy shrew (Sorex minutus). The average prevalence was 6.2%, with two genospecies detected, Borrelia afzelii and Borrelia garinii, and at least three STs that had not been previously reported in small mammals. Borrelia prevalence in small mammals did not differ between seasons. Six genospecies of Borrelia-Borrelia afzelii, Borrelia valaisiana, Borrelia garinii, Borrelia lusitaniae, Borrelia spielmanii, and Borrelia burgdorferi sensu stricto-and 25 STs of Borrelia, of which 12 have not been previously described at all and five have not been previously reported in Germany, were detected in 13% of I. ricinus complex ticks. Prevalence was highest in adult females (25.3%) and lowest in nymphs (11.4%). Prevalence was significantly higher in ticks from grassland (16.8%) compared to forests (11.4%). CONCLUSIONS: The high level of small mammal diversity in this region of Germany seems to be reflected in a wide variety of genospecies and STs of B. burgdorferi.

Emerging rodent-associated Bartonella: a threat for human health?

BACKGROUND: Species of the genus Bartonella are facultative intracellular alphaproteobacteria with zoonotic potential. Bartonella infections in humans range from mild with unspecific symptoms to life threatening, and can be transmitted via arthropod vectors or through direct contact with infected hosts, although the latter mode of transmission is rare. Among the small mammals that harbour Bartonella spp., rodents are the most speciose group and harbour the highest diversity of these parasites. Human-rodent interactions are not unlikely as many rodent species live in proximity to humans. However, a surprisingly low number of clinical cases of bartonellosis related to rodent-associated Bartonella spp. have thus far been recorded in humans. METHODS: The main purpose of this review is to determine explanatory factors for this unexpected finding, by taking a closer look at published clinical cases of bartonellosis connected with rodent-associated Bartonella species, some of which have been newly described in recent years. Thus, another focus of this review are these recently proposed species. CONCLUSIONS: Worldwide, only 24 cases of bartonellosis caused by rodent-associated bartonellae have been reported in humans. Possible reasons for this low number of cases in comparison to the high prevalences of Bartonella in small mammal species are (i) a lack of awareness amongst physicians of Bartonella infections in humans in general, and especially those caused by rodent-associated bartonellae; and (ii) a frequent lack of the sophisticated equipment required for the confirmation of Bartonella infections in laboratories that undertake routine diagnostic testing. As regards recently described Bartonella spp., there are presently 14 rodent-associated Candidatus taxa. In contrast to species which have been taxonomically classified, there is no official process for the review of proposed Candidatus species and their names before they are published. This had led to the use of malformed names that are not based on the International Code of Nomenclature of Prokaryotes. Researchers are thus encouraged to propose Candidatus names to the International Committee on Systematics of Prokaryotes for approval before publishing them, and only to propose new species of Bartonella when the relevant datasets allow them to be clearly differentiated from known species and subspecies.

Influence of Season, Population and Individual Characteristics on the Prevalence of Leptospira spp. in Bank Voles in North-West Germany.

Leptospirosis is a worldwide zoonotic disease with more than 1 million human cases annually. Infections are associated with direct contact to infected animals or indirect contact to contaminated water or soil. As not much is known about the prevalence and host specificity of Leptospira spp. in bank voles (Clethrionomys glareolus), our study aimed to evaluate Leptospira spp. prevalence and genomospecies distribution as well as the influence of season, host abundance and individual characteristics on the Leptospira prevalence. Bank voles, which are abundant and widely distributed in forest habitats, were collected in the years 2018 to 2020 in North-West Germany, covering parts of North Rhine-Westphalia and Lower Saxony. The DNA of 1817 kidney samples was analyzed by real-time PCR targeting the lipl32 gene. Positive samples were further analyzed by targeting the secY gene to determine Leptospira genomospecies and multilocus sequence typing (MLST) to determine the sequence type (ST). The overall prevalence was 7.5% (95% confidence interval: 6.4-8.9). Leptospira interrogans (83.3%), L. kirschneri (11.5%) and L. borgpetersenii (5.2%) were detected in bank voles. Increasing body weight as a proxy for age increased the individual infection probability. Only in years with high bank vole abundance was this probability significantly higher in males than in females. Even if case numbers of human leptospirosis in Germany are low, our study shows that pathogenic Leptospira spp. are present and thus a persisting potential source for human infection.

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

First Detection of Bartonella spp. in Small Mammals from Rice Storage and Processing Facilities in Myanmar and Sri Lanka.

(1) Background: Bartonella spp. are zoonotic bacteria with small mammals as main reservoirs. Bartonella spp. prevalence in small mammals from Myanmar and Sri Lanka are yet unknown. (2) Methods: Small mammals were snap trapped in Sri Lanka and Myanmar in urban surroundings. Spleens-derived DNA was screened for Bartonella spp. using conventional PCR based on three target genes. Positive samples were sequenced. (3) Results: 994 small mammals were collected comprising 6 species: Bandicota bengalensis, Bandicota indica, Rattus exulans, Rattus rattus, Mus booduga, and Suncus murinus. In Myanmar, the Bartonella prevalence in Bandicoot rats (68.47%) was higher than in Rattus rattus (41.67%), Rattus exulans (21.33%), and Suncus murinus (3.64%). Furthermore the prevalence in Myanmar (34%, n = 495) was twice as high as in Sri Lanka (16%, n = 499). In Sri Lanka, Bartonella spp. occurred almost exclusively in R. rattus. In Myanmar, Bartonella kosoyi was mainly detected (56%), followed by Bartonella sp. KM2529 (15%), Bartonella sp. SE-Bart D (12%) and Bartonella henselae (1%). In Sri Lanka, B. phoceensis (60%) and Bartonella sp. KM2581 (33%) were predominant. (4) Conclusions: Bartonella spp. were detected in all investigated small mammal species from Myanmar and Sri Lanka for the first time. Bartonella kosoyi and B. henselae are zoonotic. As these small mammals originated from urban settlements, human bartonellosis seems likely to occur.

Evaluating Transmission Paths for Three Different Bartonella spp. in Ixodes ricinus Ticks Using Artificial Feeding.

Bartonellae are facultative intracellular alpha-proteobacteria often transmitted by arthropods. Ixodes ricinus is the most important vector for arthropod-borne pathogens in Europe. However, its vector competence for Bartonella spp. is still unclear. This study aimed to experimentally compare its vector competence for three Bartonella species: B. henselae, B. grahamii, and B. schoenbuchensis. A total of 1333 ticks (1021 nymphs and 312 adults) were separated into four groups, one for each pathogen and a negative control group. Ticks were fed artificially with bovine blood spiked with the respective Bartonella species. DNA was extracted from selected ticks to verify Bartonella-infection by PCR. DNA of Bartonella spp. was detected in 34% of nymphs and females after feeding. The best engorgement results were obtained by ticks fed with B. henselae-spiked blood (65.3%) and B. schoenbuchensis (61.6%). Significantly more nymphs fed on infected blood (37.3%) molted into adults compared to the control group (11.4%). Bartonella DNA was found in 22% of eggs laid by previously infected females and in 8.6% of adults molted from infected nymphs. The transovarial and transstadial transmission of bartonellae suggest that I. ricinus could be a potential vector for three bacteria.

Host-parasite interactions of rodent hosts and ectoparasite communities from different habitats in Germany.

BACKGROUND: Small mammals are important maintenance hosts of ectoparasites as well as reservoir hosts for many arthropod-borne pathogens. In Germany, only a few studies have investigated ectoparasite communities on small mammals in their natural habitats. The aim of this study was to assess the species diversity and parameters influencing the mean intensity and prevalence of macroscopically visible ectoparasites, such as fleas, predatory mites and ticks. METHODS: A total of 779 small mammals and 3383 ticks were available from earlier investigations for the data analysis of the current study from three differently structured study sites. In addition, fleas and predatory mites were collected from the captured rodents and taxonomically identified. Regression analyses were conducted on the group (ticks/mites/fleas) and species levels using hurdle models for the abundance of ectoparasite groups and a negative binomial model for the abundance of species. RESULTS: Nearly 90% of the small mammals analyzed were infested with ectoparasites, with an average of 7.3 specimens per host. Hosts were infested with up to six species of ectoparasites simultaneously. In total, 12 flea, 11 mite and three tick species were detected. Ticks were more prevalent than fleas or mites, with > 80% of the hosts in urban and forest areas hosting ticks and around 60% of hosts presenting fleas, and only 20-40% of hosts presenting mites. Polyparasitism had a statistically significant influence on the prevalence of the investigated tick, mite and flea species, with odds ratios of > 1.0. Trapping location, season and host characteristics had significant influences on some-but not all-of the investigated species. CONCLUSIONS: The diversity of flea species was unexpectedly high and higher than that reported in comparable studies, which can be explained by the differently structured habitats and regions examined in this study. Polyparasitism was a key influencing factor and had a positive effect on the prevalence and/or abundance of the predominant tick, flea and mite species occurring on small mammals. Season, trapping location, host species and sex of the host species also had an influence on the prevalence and mean intensity of certain, but not all, ectoparasite species.

The Red Fox (Vulpes vulpes) as Sentinel for Tick-Borne Encephalitis Virus in Endemic and Non-Endemic Areas.

Tick-borne encephalitis (TBE) is one of the most important viral zoonosis caused by a neurotropic arbovirus (TBEV). In Germany, TBE is classified as a notifiable disease with an average of 350 autochthonous human cases annually. The incidence-based risk assessment in Germany came under criticism because every year, a number of autochthonous human TBE cases have been detected outside of the official risk areas. Therefore, it is necessary to find additional parameters to strengthen TBEV surveillance. The aim of this study was to examine red foxes as sentinels for TBE. Thus far, there are no published data about the sensitivity and specificity for serological methods testing fox samples. Hence, we aimed to define a system for the screening of TBEV-specific antibodies in red foxes. A total of 1233 fox sera were collected and examined by ELISA and IIFA and confirmed by micro-NT. The overall seroprevalence of antibodies against TBEV in red foxes from Germany confirmed by micro-NT was 21.1%. The seroprevalence differed significantly between risk (30.5%) and non-risk areas (13.1%), with good correlations to local TBE incidence in humans. In conclusion, serological monitoring of red foxes represents a promising surrogate marker system and may even determine unexpected TBEV foci in regions currently regarded as non-risk areas.

Cryptosporidium sp. skunk genotype in wild raccoons (Procyon lotor) naturally infected with Baylisascaris procyonis from Central Germany.

Cryptosporidium spp. are apicomplexan parasites of public health concern. They are one of the main causes of intestinal diseases in humans and animals. Contaminated water is among the main sources of infection for humans and mammals. Raccoons are an introduced species in Germany. They are anthropogenic adapters with a natural affinity for water bodies. We collected samples from wild raccoons in the Federal States of Saxony and Thuringia, Central Germany. Through molecular genotyping, we found Cryptosporidium sp. skunk genotype in one raccoon from Saxony (1/24) and in one animal from Thuringia (1/27). Both raccoons were also infected with the zoonotic nematode Baylisascaris procyonis. This is the first report of co-infection with these two parasites in raccoons from Germany. Our study highlights the potential of these animals as carriers of zoonotic pathogens. Since raccoons can thrive in human settlements, this study provides data that can be used as a baseline for preventive programs.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Rats as potential reservoirs for neglected zoonotic Bartonella species in Flanders, Belgium.

BACKGROUND: Bartonella spp. are vector-borne pathogens transmitted to humans via blood-sucking arthropods. Rodents such as the black rat (Rattus rattus) and Norway rat (R. norvegicus) are thought to be the main reservoirs. An infection with rodent-associated Bartonella spp. may cause severe symptoms in humans such as endocarditis and neuroretinitis. The current knowledge of Bartonella prevalence in rats from western Europe is scarce. METHODS: Rats and a few other rodent by-catches were trapped in the context of a rodenticide resistance study at different sites in Flanders, Belgium. During dissection, biometric data were collected, and spleen tissues were taken. DNA was extracted from spleen samples and tested for Bartonella spp. by conventional generic polymerase chain reaction (PCR). To determine the Bartonella species, a selected number of amplicons were sequenced and compared with GenBank entries. RESULTS: In total, 1123 rodents were trapped. The predominate species was R. norvegicus (99.64%). Other rodents trapped included: two water voles (Arvicola amphibius, 0.18%); one colour rat (R. norvegicus forma domestica, 0.09%); and one muskrat (Ondatra zibethicus, 0.09%). PCR analysis of 1097 rodents resulted in 410 (37.37%, 95% CI: 34.50-40.31%) Bartonella spp. DNA-positive samples. Bartonella tribocorum (94.68%, 95% CI: 88.02-98.25%) was the most frequently detected Bartonella species, followed by B. grahamii (3.19%, 95% CI: 0.66-9.04%) and B. doshiae (1.06%, 95% CI: 0.03-5.79%). An uncultured Bartonella species occurred in one water vole (1.06%, 95% CI: 0.03-5.79%). There was a significantly higher Bartonella prevalence in older rats compared to juveniles and a significant difference in Bartonella prevalence concerning the localisation of trapping sites. In contrast, there was no statistically significant difference in Bartonella prevalence regarding sex, degree of urbanisation and season. CONCLUSIONS: Based on the high prevalence found, we conclude that the Norway rat seems to be a key reservoir host for zoonotic B. tribocorum in Belgium.