Italian peninsula as a hybridization zone of Ixodes inopinatus and I. ricinus and the prevalence of tick-borne pathogens in I. inopinatus, I. ricinus, and their hybrids.

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.

Escherichia coli from Human Wounds: Analysis of Resistance to ß-Lactams and Expression of RND Efflux Pumps.

Purpose: Resistance of pathogenic strains of Escherichia coli to ß-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to ß-lactams. Materials and Methods: Twelve E. coli isolates obtained from patients' wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected ß-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of ß-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin. Results: Phenotypic resistance to ß-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin. Conclusion: Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.

Circulation of Anaplasma phagocytophilum among invasive and native carnivore species living in sympatry in Poland.

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.

Genetic analysis challenges the presence of Ixodes inopinatus in Central Europe: development of a multiplex PCR to distinguish I. inopinatus from I. ricinus.

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.

Host specificity of passerine Lankesterella (Apicomplexa: Coccidia).

Lankesterella parasites are blood coccidians that have recently gained attention as their records in common passerine species emerge. To date, their occurrence has been molecularly confirmed in several passerine genera, mainly among members of the families Paridae and Acrocephalidae. Despite their relatively high prevalence in some host populations, their life cycles remain unclear, mosquitoes or mites being the proposed vectors. The aim of this study was to reveal Lankesterella host specificity, focusing mainly on parasites of tit and warbler species (families Paridae and Acrocephalidae). We have determined the 18S rRNA gene sequences of Lankesterella from 35 individuals belonging to eight different host species. Phylogenetic analysis revealed that passerine Lankesterella are host-specific, with specificity at the host genus or species level. Besides Lankesterella, Isospora sequences were obtained from avian blood as well, pointing out the need for barcoding.

Role of invasive carnivores (Procyon lotor and Nyctereutes procyonoides) in epidemiology of vector-borne pathogens: molecular survey from the Czech Republic.

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.

Range of metastrongylids (superfamily Metastrongyloidea) of public health and veterinary concern present in livers of the endemic lizard Gallotia galloti of Tenerife, Canary Islands, Spain.

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.

Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people.

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.

Blood parasites (Trypanosoma, Leucocytozoon, Haemoproteus) in the Eurasian sparrowhawk (Accipiter nisus): diversity, incidence and persistence of infection at the individual level.

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.

Red fox (Vulpes vulpes) play an important role in the propagation of tick-borne pathogens.

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.

Molecular analysis of blood-associated pathogens in European wildcats (Felis silvestris silvestris) from Germany.

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.

The distribution of Dermacentor reticulatus in the Czech Republic re-assessed: citizen science approach to understanding the current distribution of the Babesia canis vector.

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.

A new report of adult Hyalomma marginatum and Hyalomma rufipes in the Czech Republic.

Hyalomma marginatum and Hyalomma rufipes are important vectors of Crimean-Congo Hemorrhagic Fever Virus (CCHFV) in North Africa and Southern Europe. They are occasionally also reported from Central and Western Europe where they are likely introduced from their natural range by migratory birds. In this study, we report findings and molecular identification of adults and one nymph of H. marginatum and H. rufipes, primarily from horses from different regions of the Czech Republic. While the number of the reported ticks is small, this is likely to be an underrepresentation of the actual number. Due to their vector competence for CCHFV and potential expansion into new areas with a changing climate, surveillance programs in Europe are warranted.

Species-specific PCR assay for the detection of Babesia odocoilei.

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

Detection of Anaplasma phagocytophilum in European brown hares (Lepus europaeus) using three different methods.

European brown hare (Lepus europaeus Pallas 1778) is a broadly distributed lagomorph species in Europe, recognized as a host for Ixodes ricinus and reservoir of a wide range of pathogens with zoonotic potential. Even though Lepus europaeus represents an important game animal in Central Europe, the data available on Anaplasma phagocytophilum in this lagomorph are scarce. In this study, three populations of brown hare from distinct localities in the Czech Republic were analysed for the presence of Anaplasma phagocytophilum DNA. We used standard qPCR, targeting the msp2 gene and adapted the same assay also for digital droplet PCR. Out of 91 samples, these two methods identified 9 and 12 as positive, respectively. For taxonomic analysis, we amplified the groEL gene from five of six samples that were found positive by both methods. In phylogenetic analyses, this haplotype belongs to ecotype 1, and to the subclade with isolates from cervids and I. ricinus. Our findings underline the importance of correct result interpretation and positivity cut-off set-up for different detection methods of A. phagocytophilum. This bacterium is characterized by a high intraspecific variability and highly sensitive detection itself, is not enough. Detailed molecular typing is necessary to define the zoonotic potential of different strains and their natural reservoirs.

Babesia pisicii n. sp. and Babesia canis Infect European Wild Cats, Felis silvestris, in Romania.

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.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

Theileria equi and Babesia caballi in horses in the Czech Republic.

Theileria equi and Babesia caballi are protozoan agents causing equine piroplasmosis, endemic in countries all over the world. The aim of this study was to detect antibodies to T. equi and B. caballi in horses in the Czech Republic and to investigate the origin of the infection. Blood sera from 711 horses were examined with competitive enzyme-linked immunosorbent assay; positive samples were verified with indirect fluorescence immunoassay. Antibodies to T. equi and B. caballi were detected in eight (1.1%) and three (0.4%) horses, respectively. Infection with T. equi was confirmed by PCR and sequencing in the blood of five serologically positive horses. An autochthonous origin of T. equi infection could not be excluded in two (0.3%) horses. Intensive movement of horses across European countries and the expanding occurrence of competent tick vector Dermacentor reticulatus in the Czech Republic create an increasing risk of establishing active foci of equine piroplasmosis in the country.

The Role of Peridomestic Animals in the Eco-Epidemiology of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.

Molecular survey on tick-borne pathogens and Leishmania infantum in red foxes (Vulpes vulpes) from southern Italy.

Red foxes (Vulpes vulpes) have been recognised to harbour and transmit a wide range of tick-borne pathogens (TBPs) including those of zoonotic concern. To investigate the prevalence and the distribution of TBPs and of Leishmania infantum in foxes (n = 244), spleen samples were collected within the frame of a multi-regional wildlife health surveillance program in Italy. A combined PCR/sequencing approach was performed for the detection of Anaplasma spp., Babesia spp., Borrelia spp., Ehrlichia spp., Hepatozoon spp. and L. infantum DNA. Overall, 146 foxes (59.8 %, 95 % CI: 53.6-65.8) tested positive for at least one pathogen with Hepatozoon canis being the most prevalent (i.e., n = 124; 50.8 %, 95 % CI: 44.6-57.0), followed by Babesia vulpes (n = 20; 8.2 %, 95 % CI: 5.4-12.3), different spirochete species from Borrelia burgdorferi sensu lato complex (n = 9; 3.7 %, 95 % CI: 1.9-6.9), Ehrlichia canis and L. infantum (n = 7; 2.9 % each, 95 % CI: 1.4-5.8), Anaplasma platys (n = 4; 1.6 %, 95 % CI: 0.6-4.1), Anaplasma phagocytophilum ecotype I and Candidatus Neoehrlichia sp. (n = 3; 1.2 % each, 95 % CI: 0.4-3.5). All samples scored negative for Babesia canis and Borrelia miyamotoi. This study revealed the presence of spirochetes from B. burgdorferi s.l. complex, Ca. Neoehrlichia sp., A. platys and A. phagocytophilum ecotype I in red fox population from Italy, underling the necessity to monitoring these carnivores, mainly because they live in contact with dogs and humans. Data on the tick fauna circulating on wildlife species will complement information herein obtained, instrumentally to establish preventive strategies for minimizing the risk of infection for animals and humans.