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Abstract Background European wildcats (Felis silvestris) are widely distributed in Europe and a strictly protected species in Germany. Lately, anthropogenic protective efforts lead to increasing numbers of wildcats in southwestern Germany. Moreover, in recent years the numbers of domestic cats are increasing. Thus, the contact between domestic and wildcats may lead to the spread of zoonotic pathogens in both animal species. As data on vector-borne pathogens (VBPs) in wildcats from Germany are limited to date, the objective of this study was to investigate the presence and current distribution of VBPs in wildcats from southwestern Germany. Methods Skin and spleen samples from 117 European wildcats, originating from a regional carcass-monitoring program in southwestern Germany, were examined by real-time and conventional polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Rickettsia spp., Bartonella spp., and Piroplasmida. Results In total, 6.8% (n = 8) of the wildcats were Rickettsia-positive, specified as R. helvetica. Three wildcats were positive for A. phagocytophilum (2.6%), one for Bartonella spp., namely B. taylorii (0.8%), and 84 for Cytauxzoon spp. (71.8%). Out of these 84 samples, 23 were further sequenced revealing very high identity levels (99.84–100%) to C. europaeus, which is considered to be pathogenic for domestic cats. All wildcats were negative for the presence of N. mikurensis DNA. Conclusions European wildcats in southwestern Germany are hosting several VBPs. With the exception of Cytauxzoon spp., low prevalence rates of most examined pathogens suggest that wildcats are primarily incidental hosts for sylvatic pathogens associated with rodents, in contrast to domestic cats. However, the high prevalence of the cat-associated pathogen C. europaeus suggests that wildcats in southwestern Germany may serve as reservoirs for this pathogen. Graphical Abstract

Ixodid ticks are obligate blood-feeding arthropods and important vectors of pathogens. In Mallorca, almost no data on the tick fauna are available. Herein, we investigated ticks and tick-borne pathogens in ticks collected from dogs, a cat and humans in Mallorca as result of a citizen science project. A total of 91 ticks were received from German tourists and residents in Mallorca. Ticks were collected from March to October 2023 from dogs, cat and humans, morphologically and genetically identified and tested for pathogens by PCRs. Six tick species could be identified: Ixodes ricinus (n = 2), Ixodes ventalloi (n = 1), Hyalomma lusitanicum (n = 7), Hyalomma marginatum (n = 1), Rhipicephalus sanguineus s.l. (n = 71) and Rhipicephalus pusillus (n = 9). Rhipicephalus sanguineus s.l. adults were collected from dogs and four females from a cat and the 16S rDNA sequences identified it as Rh. sanguineus s.s. Hyalomma lusitanicum was collected from 1 human, 1 dog and 5 specimens were collected from the ground in the community of Santanyi, together with one H. marginatum male. This is the first report of Hyalomma marginatum in Mallorca. Both I. ricinus were collected from humans and I. ventalloi female was collected from a dog. All ticks tested negative for Anaplasma phagocytophilum, Coxiella spp., Francisella spp., and piroplasms. In 32/71 (45%) specimens of Rh. sanguineus s.s., Rickettsia spp. could be detected and in 18/32 (56.2%) sequenced tick DNAs R. massiliae was identified. Ixodes ventalloi female and both I. ricinus tested positive in the screening PCR, but the sequencing for the identification of the Rickettsia sp. failed.

Abstract 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. Graphical abstract

(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;

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.

Abstract 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. Graphical abstract

Abstract 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. Graphical Abstract

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.

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.

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.

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.

Abstract 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.

Abstract 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.

Abstract Background Ixodes spp. are vectors of zoonotic pathogens. All three active life stages (larvae, nymphs, adults) need to feed on a host in order to develop. Usually ticks parasitize attached to the external surface of their hosts’ skin. Interestingly, in some cases ticks can also be found in the subcutaneous tissue in a variety of hosts, such as red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs. Methods The visceral side of 126 red fox-furs from Germany was examined visually searching for ticks. The localization of ticks was recorded and assigned to ten specific body parts. Morphological identification of ticks was performed according to standardized taxonomic protocols. Ticks which could not be further identified were examined genetically via conventional PCR targeting the 16S rRNA and cox1 gene. Hematoxylin and eosin (H&E) staining was used for histopathological examination. Results In 111 out of 126 (88.1%) examined coats, at least one tick was found in the subcutaneous tissue. A total of 1203 ticks were removed from the subcutaneous tissue. Well-preserved ticks could be identified based on morphological criteria, but most ticks were in a progressed state of decomposition. Here, morphological species identification was not successful. Also, PCR methods did not lead to a successful species identification. The following species and development stages were found by morphological identification: Ixodes ricinus (female, n = 289; male, n = 8; nymph, n = 1), I. hexagonus (female, n = 2), I. canisuga (female, n = 1). Male I. ricinus were found individually or copulating in pairs with females. Subcutaneous ticks were localized at three predominant affected body parts: ears, axillar and inguinal region. Histological examination of subcutaneous ticks revealed a granulomatous panniculitis. Conclusions To the authors’ knowledge, this is the first finding of highly prevalent subcutaneous ticks in red foxes from Germany. Subcutaneous location of ticks seems to be very common in red foxes and the rule rather than the exception. Deep embedment of longirostra and long feeding times of females seem to put the subcutaneous location in favor. Most foxes were infested in the inguinal area, where the skin is thin and less hairy.

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.

Abstract Background Rodents are important reservoirs for zoonotic vector-borne agents. Thus, the distribution of rodents and their vicinity to humans and companion animals may have an important impact on human and animal health. However, the reservoir potential of some rodent genera, e.g. Microtus, has not yet been precisely examined concerning tick-borne pathogens in Central Europe. Therefore, we examined small mammals from Germany and the Czech Republic for the following vector-borne pathogens: Babesia spp., Bartonella spp., Anaplasma phagocytophilum, “Candidatus Neoehrlichia mikurensis” (CNM) and Coxiella burnetii. Spleen DNA from 321 small mammals belonging to four genera, Myodes (n = 78), Apodemus (n = 56), Microtus (n = 149), Sorex (n = 38), collected during 2014 in Germany and the Czech Republic were available for this study. DNA samples were examined for the presence of Babesia and Bartonella DNA by conventional PCR targeting the 18S rRNA gene and the 16S–23S rRNA intergenic spacer region, respectively. For the detection of CNM, A. phagocytophilum and C. burnetii real-time PCR assays were performed. Results Bartonella spp. DNA was detected in 216 specimens (67.3%) with 102/174 (58.6%) positive in Germany and 114/147 (77.6%) in the Czech Republic. The prevalence in each genus was 44.9% for Myodes, 63.2% for Sorex, 77.2% for Microtus and 75% for Apodemus. Four Bartonella species, i.e. Bartonella sp. N40, B. grahamii, B. taylorii and B. doshiae, as well as uncultured bartonellae, were detected. The Bartonella species diversity was higher in rodents than in shrews. In total, 27/321 (8.4%) small mammals were positive for CNM and 3/321 (0.9%) for A. phagocytophilum (S. coronatus and M. glareolus). All samples were negative for Babesia spp. and Coxiella spp. Conclusions While the detected high prevalence for Bartonella in Apodemus and Myodes spp. is confirmatory with previous findings, the prevalence in Microtus spp. was unexpectedly high. This indicates that individuals belonging to this genus may be regarded as potential reservoirs. Interestingly, only Sorex spp. and M. glareolus were positive for A. phagocytophilum in the present study, suggesting a possible importance of the latter for the maintenance of certain A. phagocytophilum strains in nature.

Abstract 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.

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.

Captive reindeer in German zoos and wildlife parks live outside their natural geographic range and are exposed to a variety of viral, bacterial and protozoan pathogens, some host-specific and some which they are not exposed to in their native habitat. Reindeer blood samples and ticks collected in 2013 from 123 reindeer at 16 different zoological facilities were available from a previous study. The aims of this study were to assess the serological status of these animals with regards to various microorganisms as well as to test ticks (Ixodes ricinus) and blood samples for the presence of Anaplasma spp. DNA in order to evaluate the exposure of captive reindeer in Germany to a variety of pathogens. A total of 119 or 118 serum samples were screened (ELISA) and antibodies were detected (seropositive/tested, prevalence, confidence interval) against alphaherpesvirus (24/119, 20.3%, CI: 13.9–28.3), bluetongue virus (BTV; 4/119, 3.4%, CI: 1.0–8.7), malignant catarrhal fever related gammaherpesvirus (MCFV-related gammaherpesvirus; 7/119, 5.9%, CI: 2.7–11.9), pestivirus (5/118, 4.2%, CI: 1.6–9.8), Schmallenberg virus (SBV; 70/118, 59.3%, CI: 50.3–67.8), smooth Brucella spp. (1/118; 0.9%, CI: 0–5.1), Neospora caninum (5/118, 4.2%, CI: 1.6–9.8), and Toxoplasma gondii (62/119, 52.1%, CI: 43.2–60.9). These results suggested the exposure of reindeer to all tested pathogens. Moreover, real-time PCR for Anaplasma phagocytophilum targeting the partial msp2 gene was performed on DNA extracted from whole blood samples from reindeer (n = 123) and from ticks (n = 49) collected from 22 reindeer in seven different facilities. In addition to the real-time PCR, a semi-nested PCR for the partial groEL gene, and a nested PCR targeting the partial 16S rRNA gene were performed. DNA of A. phagocytophilum was detected in 17 reindeer (13.8%) and 15 ticks (30.6%). Three of the five reindeer with ticks having A. phagocytophilum DNA also had such DNA in blood. These results indicate that captive reindeer can be exposed to several ruminant pathogens that they hitherto had no known exposure to through their natural geographical distribution and habitats as shown for Culicoides-borne BTV and SBV. Further, captive reindeer may serve as reservoir hosts for pathogens circulating in local domestic, captive, and wild ruminant species and populations and arthropod vectors.

Abstract Background Ticks are important carriers of many different zoonotic pathogens. To date, there are many studies about ticks and tick-borne pathogens (TBP), but only a few were carried out in Bulgaria. The present study intends to detect the prevalence of tick-borne bacteria and parasites occurring at the Black Sea in Bulgaria to evaluate the zoonotic potential of the tick-borne pathogens transmitted by ticks in this area. Methods In total, cDNA from 1541 ticks (Dermacentor spp., Haemaphysalis spp., Hyalomma spp., Ixodes spp. and Rhipicephalus spp.) collected in Bulgaria by flagging method or from hosts was tested in pools of ten individuals each for Anaplasma phagocytophilum, Babesia spp., Borrelia burgdorferi (s.l.), Rickettsia spp. and “Candidatus Neoehrlichia mikurensis” via conventional and quantitative real-time PCR. Subsequently, samples from positive pools were tested individually and a randomized selection of positive PCR samples was purified, sequenced, and analyzed. Results Altogether, 23.2% of ticks were infected with at least one of the tested pathogens. The highest infection levels were noted in nymphs (32.3%) and females (27.5%). Very high prevalence was detected for Rickettsia spp. (48.3%), followed by A. phagocytophilum (6.2%), Borrelia burgdorferi (s.l.) (1.7%), Babesia spp. (0.4%) and “Ca. Neoehrlichia mikurensis” (0.1%). Co-infections were found in 2.5% of the tested ticks (mainly Ixodes spp.). Sequencing revealed the presence of Rickettsia monacensis, R. helvetica, and R. aeschlimannii, Babesia microti and B. caballi, and Theileria buffeli and Borrelia afzelli. Conclusion This study shows very high prevalence of zoonotic Rickettsia spp. in ticks from Bulgaria and moderate to low prevalence for all other pathogens tested. One should take into account that tick bites from this area could lead to Rickettsia infection in humans and mammals.

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.

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.

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.

(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.

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.

Abstract Background Small mammals are crucial for the life history of ixodid ticks, but their role and importance in the transmission cycle of tick-borne pathogens is mostly unknown. Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum are both tick-borne pathogens, and rodents are discussed to serve as main reservoir hosts for CNM but not for the latter especially in Germany. Analysing the prevalence of both pathogens in small mammals and their ticks in endemic regions may help to elucidate possible transmission paths in small mammal populations and between small mammals and ticks. Methods In 2012 and 2013, small mammals were trapped at three different sites in Germany. DNA was extracted from different small mammal tissues, from rodent neonates, foetuses and from questing and attached ticks. DNA samples were tested for CNM and A. phagocytophilum by real-time PCR. Samples positive for A. phagocytophilum were further characterized at the 16S rRNA gene locus. Results CNM was detected in 28.6% of small mammals and in 2.2% of questing and 3.8% of attached ticks. Altogether 33 positive ticks were attached to 17 different hosts, while positive ticks per host ranged between one and seven. The prevalences for this pathogen differed significantly within small mammal populations comparing sites (χ2: 13.3987; p: 0.0004) and between sexes. Male rodents had an approximately two times higher chance of infection than females (OR: 1.9652; 95% CI: 1.32-2.92). The prevalence for CNM was 31.8% (95% CI: 22-44) in rodent foetuses and neonates (23 of 67) from positive dams, and 60% (95% CI: 35.7-80.25) of positive gravid or recently parturient rodents (9 out of 15) had at least one positive foetus or neonate. Anaplasma phagocytophilum was detected at a low percentage in rodents (0-5.6%) and host-attached ticks (0.5-2.9%) with no significant differences between rodent species. However, attached nymphs were significantly more often infected than attached larvae (χ2: 25.091; p:

Small mammals serve as most important reservoirs for Leptospira spp., the causative agents of Leptospirosis, which is one of the most neglected and widespread zoonotic diseases worldwide. The knowledge about Leptospira spp. occurring in small mammals from Germany is scarce. Thus, this study's objectives were to investigate the occurrence of Leptospira spp. and the inherent sequence types in small mammals from three different study sites: a forest in southern Germany (site B1); a National Park in south-eastern Germany (site B2) and a renaturalised area, in eastern Germany (site S) where small mammals were captured. DNA was extracted from kidneys of small mammals and tested for Leptospira spp. by real-time PCR. Positive samples were further analysed by duplex and conventional PCRs. For 14 positive samples, multi locus sequence typing (MLST) was performed. Altogether, 1213 small mammals were captured: 216 at site B1, 456 at site B2 and 541 at site S belonging to following species: Sorex (S.) araneus, S. coronatus, Apodemus (A.) flavicollis, Myodes glareolus, Microtus (Mi.) arvalis, Crocidura russula, Arvicola terrestris, A. agrarius, Mustela nivalis, Talpa europaea, and Mi. agrestis. DNA of Leptospira spp. was detected in 6% of all small mammals. At site B1, 25 small mammals (11.6%), at site B2, 15 small mammals (3.3%) and at site S, 33 small mammals (6.1%) were positive for Leptospira spp. Overall, 54 of the positive samples were further determined as L. kirschneri, nine as L. interrogans and four as L. borgpetersenii while five real-time PCR-positive samples could not be further determined by conventional PCR. MLST results revealed focal occurrence of L. interrogans and L. kirschneri sequence type (ST) 117 while L. kirschneri ST 110 was present in small mammals at all three sites. Further, this study provides evidence for a particular host association of L. borgpetersenii to mice of the genus Apodemus.

The aim of this study was to examine the presence of tick-borne rickettsial bacteria in Rhipicephalus sanguineus sensu stricto ticks collected from dogs in the Patagonian region of Argentina. Fourteen stray dogs from Valcheta, Río Negro province, Argentina were examined for the presence of R. sanguineus s.s. ticks. Ninety ticks were collected and identified to species level. DNA was extracted and analysed by conventional PCR assays for the presence of tick-borne bacteria belonging to the genera Anaplasma, Ehrlichia and Rickettsia. Thirty-three tick pools were tested by different PCR assays of which 3 were positive for Anaplasmataceae bacteria. From the 3 Anaplasmataceae positive samples, 2 partial 16S rDNA sequences were generated and belonging to Anaplasma platys, the causative agent of canine cyclic thrombocytopenia. Two tick samples were positive in Rickettsia specific PCR assays and were identified by phylogenetic analysis as Rickettsia massiliae, a member of the spotted fever group rickettsiae. The results of this study demonstrate the molecular detection of 2 rickettsial bacteria in R. sanguineus s.s. in a region of Argentina where no data were available so far.