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European wildcats (Felis silvestris silvestris) have not been investigated in large numbers for blood-associated pathogens in Germany, because wildcats, being a protected species, may not be hunted, and the collection of samples is therefore difficult. Thus, spleen tissue and whole blood from 96 wildcats from Germany found as roadkill or dead from other causes in the years 1998–2020 were examined for the prevalence of blood associated pathogens using molecular genetic tools. PCR was used to screen for haemotrophic Mycoplasma spp., Hepatozoon spp., Cytauxzoon spp., Bartonella spp., Filarioidea, Anaplasmataceae, and Rickettsiales, and positive samples were subsequently sequenced. Phylogenetic analyses were performed for Mycoplasma spp. and Hepatozoon spp. by calculating phylogenetic trees and DNA haplotype networks. The following pathogens were found: Candidatus Mycoplasma haematominutum (7/96), Mycoplasma ovis (1/96), Hepatozoon silvestris (34/96), Hepatozoon felis (6/96), Cytauxzoon europaeus (45/96), and Bartonella spp. (3/96). This study elucidates the prevalence of blood-associated pathogens in wildcats from Germany.

Abstract Background The range of the ornate dog tick Dermacentor reticulatus is rapidly expanding in Europe. This tick species is the vector of canine babesiosis, caused by Babesia canis, and also plays a role in the transmission of Theileria equi and Babesia caballi in equids. Methods The geographic range of D. reticulatus in the Czech Republic was re-assessed, and an up-to-date distribution map is presented based on material and data obtained during a nationwide citizen science campaign. Received and flagged individuals of D. reticulatus were also analysed for the presence of B. canis DNA. Results In striking contrast to historical records, D. reticulatus was found in all regions of the Czech Republic, with most reports coming from the southeast and northwest of the country. Between February 2018 and June 2021, the project team received 558 photo reports of ticks and 250 packages containing ticks. Of the former, 71.1% were identified as Dermacentor sp. with the remainder identified as Ixodes sp., Haemaphysalis sp., Argas sp. or Hyalomma sp. The majority of specimens in the subset of ticks that were received (N = 610) were D. reticulatus (N = 568, 93.7%), followed by Ixodes ricinus and Hyalomma spp. A total of 783 adult D. reticulatus, either received (568) or collected by flagging (215), were tested for the presence of B. canis DNA using species-specific nested PCR targeting part of the 18S rRNA gene; B. canis DNA was demonstrated in 22 samples (2.81%). Conclusions The continuous spread of D. reticulatus in the Czech Republic was documented in this study. In addition, DNA of B. canis was also detected in a number of ticks, suggesting the establishment of B. canis in the Czech Republic. These results suggest that veterinarians need to consider the possibility of canine babesiosis even in dogs without a history of travel. Graphical Abstract

Haemoparasites of the genus Babesia infect a wide range of domestic and wild animals. Feline babesiosis is considered endemic in South Africa, while data on Babesia spp. infection in felids in Europe is scarce. Using samples from 51 wild felids, 44 Felis silvestris and 7 Lynx lynx, the study aimed to determine the presence and genetic diversity of Babesia spp. in wild felids in Romania by analyzing the 18S rDNA and two mitochondrial markers, cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) genes. By 18S rDNA analyses, Babesia spp. DNA was detected in 20 European wild felids. All sequences showed 100% similarity to B. canis by BLAST analysis. Conversely, Cytb and COI analyses revealed the presence of two Babesia spp., B. pisicii n. sp., which we herein describe, and B. canis. The pairwise comparison of both mitochondrial genes of B. pisicii n. sp. showed a genetic distance of at least 10.3% from the most closely related species, B. rossi. Phylogenetic analyses of Cytb and COI genes revealed that B. pisicii n. sp. is related to the so-called “large” canid-associated Babesia species forming a separate subclade in a sister position to B. rossi.

Abstract Background Ixodes inopinatus was described from Spain on the basis of morphology and partial sequencing of 16S ribosomal DNA. However, several studies suggested that morphological differences between I. inopinatus and Ixodes ricinus are minimal and that 16S rDNA lacks the power to distinguish the two species. Furthermore, nuclear and mitochondrial markers indicated evidence of hybridization between I. inopinatus and I. ricinus. In this study, we tested our hypothesis on tick dispersal from North Africa to Southern Europe and determined the prevalence of selected tick-borne pathogens (TBPs) in I. inopinatus, I. ricinus, and their hybrids. Methods Ticks were collected in Italy and Algeria by flagging, identified by sequencing of partial TROSPA and COI genes, and screened for Borrelia burgdorferi s.l., B. miyamotoi, Rickettsia spp., and Anaplasma phagocytophilum by polymerase chain reaction and sequencing of specific markers. Results Out of the 380 ticks, in Italy, 92 were I. ricinus, 3 were I. inopinatus, and 136 were hybrids of the two species. All 149 ticks from Algeria were I. inopinatus. Overall, 60% of ticks were positive for at least one TBP. Borrelia burgdorferi s.l. was detected in 19.5% of ticks, and it was significantly more prevalent in Ixodes ticks from Algeria than in ticks from Italy. Prevalence of Rickettsia spotted fever group (SFG) was 51.1%, with significantly greater prevalence in ticks from Algeria than in ticks from Italy. Borrelia miyamotoi and A. phagocytophilum were detected in low prevalence (0.9% and 5.2%, respectively) and only in ticks from Italy. Conclusions This study indicates that I. inopinatus is a dominant species in Algeria, while I. ricinus and hybrids were common in Italy. The higher prevalence of B. burgdorferi s.l. and Rickettsia SFG in I. inopinatus compared with that in I. ricinus might be due to geographical and ecological differences between these two tick species. The role of I. inopinatus in the epidemiology of TBPs needs further investigation in the Mediterranean Basin. Graphical Abstract

Abstract Background Ixodes ricinus is an important vector of several pathogens, primarily in Europe. Recently, Ixodes inopinatus was described from Spain, Portugal, and North Africa and then reported from several European countries. In this study, a multiplex polymerase chain reaction (PCR) was developed to distinguish I. ricinus from I. inopinatus and used in the surveillance of I. inopinatus in Algeria (ALG) and three regions in the Czech Republic (CZ). Methods A multiplex PCR on TROSPA and sequencing of several mitochondrial (16S rDNA, COI) and nuclear markers (TROSPA, ITS2, calreticulin) were used to differentiate these two species and for a subsequent phylogenetic analysis. Results Sequencing of TROSPA, COI, and ITS2 separated these two species into two subclades, while 16S rDNA and calreticulin could not distinguish I. ricinus from I. inopinatus. Interestingly, 23 nucleotide positions in the TROSPA gene had consistently double peaks in a subset of ticks from CZ. Cloning of these PCR products led to a clear separation of I. ricinus and I. inopinatus indicating hybridization and introgression between these two tick taxa. Based on a multiplex PCR of TROSPA and analysis of sequences of TROSPA, COI, and ITS2, the majority of ticks in CZ were I. ricinus, no I. inopinatus ticks were found, and 10 specimens showed signs of hybridization. In contrast, most ticks in ALG were I. inopinatus, four ticks were I. ricinus, and no signs of hybridization and introgression were detected. Conclusions We developed a multiplex PCR method based on the TROSPA gene to differentiate I. ricinus and I. inopinatus. We demonstrate the lack of evidence for the presence of I. inopinatus in Central Europe and propose that previous studies be re-examined. Mitochondrial markers are not suitable for distinguishing I. inopinatus from I. ricinus. Furthermore, our data indicate that I. inopinatus and I. ricinus can hybridize, and the hybrids can survive in Europe. Graphical abstract

Abstract Background Vector-borne pathogens (VBPs) are a major threat to humans, livestock and companion animals worldwide. The combined effect of climatic, socioeconomic and host composition changes favours the spread of the vectors, together with the expansion of invasive carnivores contributing to the spread of the pathogens. In Europe, the most widespread invasive species of carnivores are raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides). This study focused on the detection of four major groups of VBPs namely Babesia, Hepatozoon, Anaplasma phagocytophilum and Bartonella in invasive and native carnivores in the Czech Republic, with the emphasis on the role of invasive carnivores in the eco-epidemiology of said VBPs. Methods Spleen samples of 84 carnivores of eight species (Canis aureus, Canis lupus, Lynx lynx, P. lotor, Martes foina, Lutra lutra, Mustela erminea and N. procyonoides) were screened by combined nested PCR and sequencing for the above-mentioned VBPs targeting 18S rRNA and cytB in hemoprotozoa, groEL in A. phagocytophilum, and using multilocus genotyping in Bartonella spp. The species determination is supported by phylogenetic analysis inferred by the maximum likelihood method. Results Out of 84 samples, 44% tested positive for at least one pathogen. Five different species of VBPs were detected in P. lotor, namely Bartonella canis, Hepatozoon canis, Hepatozoon martis, A. phagocytophilum and Bartonella sp. related to Bartonella washoensis. All C. lupus tested positive for H. canis and one for B. canis. Three VBPs (Hepatozoon silvestris, A. phagocytophilum and Bartonella taylorii) were detected in L. lynx for the first time. Babesia vulpes and yet undescribed species of Babesia, not previously detected in Europe, were found in N. procyonoides. Conclusions Wild carnivores in the Czech Republic are hosts of several VBPs with potential veterinary and public health risks. Among the studied carnivore species, the invasive raccoon is the most competent host. Raccoons are the only species in our study where all the major groups of studied pathogens were detected. None of the detected pathogen species were previously detected in these carnivores in North America, suggesting that raccoons adapted to local VBPs rather than introduced new ones. Babesia vulpes and one new, probably imported species of Babesia, were found in raccoon dogs. Graphical abstract

Abstract Background Although the tick-borne pathogen Anaplasma phagocytophilum is currently described as a single species, studies using genetic markers can distinguish groups of variants associated with different hosts, pathogenicity, zoonotic potential and biotic and geographic niches. The objective of our study was to investigate the genetic diversity of A. phagocytophilum and Ixodes ricinus ticks attached to people. Methods In collaboration with a commercial diagnostic company, a total of 52 DNA samples were obtained from ticks that tested positive for A. phagocytophilum by quantitative PCR. The genetic profile of each sample was determined using the groEL and ankA genes. Identification of the tick species was confirmed by partial sequencing of the COI subunit and a portion of the TROSPA gene. Results All 52 ticks were identified as I. ricinus. Two protocols of nested PCR amplifying 1293- and 407-bp fragments of groEL of A. phagocytophilum yielded amplicons of the expected size for all 52 samples. Among all sequences, we identified 10 unique genetic variants of groEL belonging to ecotype I and ecotype II. The analysis targeting ankA was successful in 46 of 52 ticks. Among all sequences, we identified 21 unique genetic variants phylogenetically belonging to three clusters. Conclusions Our results indicate that ticks attached to people harbor distant genetic variants of A. phagocytophilum, some of which are not recognized as zoonotic. Further studies are needed to determine the risk of human infection by genetic variants other than those designated as zoonotic. Graphical Abstract

Abstract Background Anaplasma phagocytophilum is characterized by a worldwide distribution and distinguished from other Anaplasmataceae by the broadest range of mammalian hosts and high genetic diversity. The role carnivores play in the life cycle of A. phagocytophilum in Europe is uncertain. Currently, only the red fox is considered a suitable reservoir host. In this study, we focused on native and invasive medium-sized carnivore species that live in sympatry and represent the most abundant species of wild carnivores in Poland. Methods A total of 275 individual spleen samples from six carnivore species (Vulpes vulpes, Meles meles, Procyon lotor, Nyctereutes procyonoides and Martes spp.) were screened combining nested PCR and sequencing for A. phagocytophilum targeting a partial groEL gene with subsequent phylogenetic analysis inferred by the maximum likelihood method. Results The DNA of A. phagocytophilum was detected in 16 of 275 individuals (5.8%). Eight unique genetic variants of A. phagocytophilum were obtained. All detected haplotypes clustered in the clade representing European ecotype I. Three variants belonged to the subclade with European human cases together with strains from dogs, foxes, cats, and wild boars. Conclusions While carnivores might have a restricted role in the dissemination of A. phagocytophilum due to their relatively low to moderate infection rates, they hold significance as hosts for ticks. Consequently, they could contribute to the transmission of tick-borne infections to humans indirectly, primarily through tick infection. This underscores the potential risk of urbanization for the A. phagocytophilum life cycle, further emphasizing the need for comprehensive understanding of its ecological dynamics . Graphical Abstract

Abstract Background Endemic lizards of the genus Gallotia are of high ecological value to the terrestrial ecosystem of the archipelago of the Canary Islands, being potent seed spreaders as well as an important component of the diet of other vertebrates. The endemic lizard Gallotia galloti in Tenerife has recently been reported to be a paratenic host of Angiostrongylus cantonensis, an invasive metastrongylid with zoonotic potential that is associated with rats as definitive hosts. However, microscopic examination of G. galloti tissue samples also revealed the presence of other metastrongylid larvae inside granulomas on the liver of this reptile. The aim of this study was to investigate the presence of helminths other than A. cantonensis in tissues of G. galloti from Tenerife. Methods A multiplex-nested PCR targeting the internal transcribed spacer 1 was designed that enabled the species-specific detection of A. cantonensis, Angiostrongylus vasorum, Aelurostrongylus abstrusus, Crenosoma striatum and Crenosoma vulpis. Liver samples from 39 G. galloti were analysed. Results Five metastrongylids were detected: A. cantonensis (15.4% of samples analysed), A. vasorum (5.1%), Ae. abstrusus (30.8%), C. striatum (30.8%) and undetermined metastrongylid sequences (12.8%). Co-infection was highly prevalent among the lizards which tested positive. Conclusions The study provides a new specific tool for the simultaneous detection of a range of metastrongylids of veterinary importance as well as new data on the circulation of metastrongylids in an ecosystem dominated by lizards. Graphical Abstract

Abstract Background A high prevalence of parasites may result from life-long persistence of infection or from high reinfection rates. We have studied blood parasites in a breeding population of the accipitrid raptor, Eurasian sparrowhawk (Accipiter nisus), to determine parasite diversity and turnover. Methods During this 7-year study, 210 adult Eurasian sparrowhawks breeding in the city of Prague were checked for parasites using several diagnostic methods. Results In both female and male raptors, parasites of the genus Leucocytozoon were the most prevalent (92% and 85%, respectively) followed in decreasing order of prevalence by those of genus Trypanosoma (74% and 68%, respectively) and genus Haemoproteus (46% and 16%, respectively). The prevalence of all parasites increased with age in both sexes, with the females at each respective age having the higher prevalence. There was a positive association between Haemoproteus and Leucocytozoon infections. Persistence at the individual level was higher than incidence for Trypanosoma and Haemoproteus. In the case of Leucocytozoon and Trypanosoma, most individuals probably become infected in their first year of life or even before dispersal from the nest. The detected parasites belonged to Trypanosoma avium sensu stricto, Leucocytozoon sp. (haplotypes ACNI1 and ACNI3) and Leucocytozoon mathisi (haplotype ACNI4) and two new lineages of the Haemoproteus elani complex (ACCNIS6 and ACCNIS7). Detailed analysis of parasite lineages in individuals that were repeatedly sampled revealed lineage turnover that would otherwise remain hidden. Phylogenetic analysis revealed that the detected Haemoproteus belongs to a phylogenetically distant group whose taxonomic position requires further analysis. Conclusions All three genera of blood parasites persist in infected individuals, thus enabling sustainability of vector transmission cycles. Prevalence increases with age; however, there is a high turnover of Leucocytozoon lineages. No clear evidence of parasite-induced mortality was found, and most of the individuals were infected early in life, particularly in the case of Leucocytozoon. Graphical abstract

The red fox (Vulpes vulpes) is the most widespread free-living carnivore in the world. Over the years, foxes have been recognized as hosts for a number of tick-borne pathogens. However, their role as reservoirs for zoonotic tick-borne diseases is poorly understood. The aim of our study was to investigate tick-borne pathogens in the red fox population in the Czech Republic. Out of 117 red foxes, 110 (94.02%) individuals tested positive for the presence of at least one pathogen by the combined PCR and sequencing approach. Hepatozoon canis was the most frequently detected pathogen (n = 95; 81.2%), followed by Babesia vulpes (n = 75; 64.1%). Babesia canis was not detected in our study. Four (3.42%) red foxes were positive for Candidatus Neoehrlichia sp., 3 (2.56%) for Anaplasma phagocytophilum, and one red fox (0.85%) tested positive for the presence of Ehrlichia sp. DNA. Overall, DNA of spirochetes from the Borrelia burgdorferi s.l. complex was detected in 8.6% of the foxes and B. miyamotoi in 5.12% of the samples. As a carnivore found in all ecosystems of Central Europe, foxes obviously contribute to transmission of tick-borne pathogens such as A. phagocytophilum, B. burgdorferi s.l., and B. myiamotoi. In addition, foxes apparently harbour a community of pathogens, associated with this host in local ecological context, dominated by H. canis and B. vulpes (possibly also Candidatus Neoehrlichia sp.). These species have the potential to spread to the domestic dog population and should be included in the differential diagnosis of febrile diseases with hematologic abnormalities in dogs.

Abstract Background Rats (Rattus spp.) invaded most of the world as stowaways including some that carried the rat lungworm, Angiostrongylus cantonensis, the cause of eosinophilic meningoencephalitis in humans and other warm-blooded animals. A high genetic diversity of A. cantonensis based on short mitochondrial DNA regions is reported from Southeast Asia. However, the identity of invasive A. cantonensis is known for only a minority of countries. The affordability of next-generation sequencing for characterisation of A. cantonensis genomes should enable new insights into rat lung worm invasion and parasite identification in experimental studies. Methods Genomic DNA from morphologically verified A. cantonensis (two laboratory-maintained strains and two field isolates) was sequenced using low coverage whole genome sequencing. The complete mitochondrial genome was assembled and compared to published A. cantonensis and Angiostrongylus malaysiensis sequences. To determine if the commonly sequenced partial cox1 can unequivocally identify A. cantonensis genetic lineages, the diversity of cox1 was re-evaluated in the context of the publicly available cox1 sequences and the entire mitochondrial genomes. Published experimental studies available in Web of Science were systematically reviewed to reveal published identities of A. cantonensis used in experimental studies. Results New A. cantonensis mitochondrial genomes from Sydney (Australia), Hawaii (USA), Canary Islands (Spain) and Fatu Hiva (French Polynesia), were assembled from next-generation sequencing data. Comparison of A. cantonensis mitochondrial genomes from outside of Southeast Asia showed low genetic diversity (0.02–1.03%) within a single lineage of A. cantonensis. Both cox1 and cox2 were considered the preferred markers for A. cantonensis haplotype identification. Systematic review revealed that unequivocal A. cantonensis identification of strains used in experimental studies is hindered by absence of their genetic and geographical identity. Conclusions Low coverage whole genome sequencing provides data enabling standardised identification of A. cantonensis laboratory strains and field isolates. The phenotype of invasive A. cantonensis, such as the capacity to establish in new territories, has a strong genetic component, as the A. cantonensis found outside of the original endemic area are genetically uniform. It is imperative that the genotype of A. cantonensis strains maintained in laboratories and used in experimental studies is unequivocally characterised.

Abstract Background Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania. Methods A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing. Results The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades. Conclusion Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries.

Abstract Background Jackals are medium-sized canids from the wolf-like clade, exhibiting a unique combination of ancestral morphotypes, broad trophic niches, and close phylogenetic relationships with the wolf and dog. Thus, they represent a potential host of several pathogens with diverse transmission routes. Recently, populations of the Eurasian golden jackal Canis aureus have expanded into the Western Palaearctic, including most of Europe. The aim of our study was to examine Eurasian golden jackals from Romania, Czech Republic and Austria for a wide spectrum of vector-borne protists and to evaluate the role of this species as a reservoir of disease for domestic dogs and/or humans. Results Diagnostic polymerase chain reaction (PCR) DNA amplifications revealed 70% of jackals to be positive for Hepatozoon, 12.5% positive for piroplasms, and one individual positive for Leishmania infantum. Phylogenetic analyses of partial 18S rDNA sequences invariably placed sequenced isolates of Hepatozoon into the H. canis clade. For piroplasms, both the 18S and cox1 sequences obtained confirmed the presence of Babesia canis and “Theileria annae” in 5 and 2 individuals, respectively, providing the first records of these two piroplasmids in Eurasian golden jackals. A single animal from Dolj County (Romania) was PCR-positive for L. infantum, as confirmed also by sequencing of ITS1-5.8S. Conclusions Apparently, expanding populations of jackals can play a significant role in spreading and maintaining new Babesia canis foci in Central Europe. The role of jackals in the epidemiology of “Theileria annae” and H. canis is probably similar to that of red foxes and should be taken into account in further research on these parasites. Also the presence of L. infantum deserves attention. Our study confirms that once established, the populations of Eurasian golden jackals constitute natural reservoirs for many canine vector-borne diseases, analogous to the role of the coyotes in North America.