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In the present study, we identified the ectoparasite communities of red foxes in three regions of Poland that encompassed two endemic regions for the occurrence of Dermacentor reticulatus, as well as a region that is free of this tick species (‘gap’ area). Our study sites were selected to enable the role of foxes as hosts for juvenile (nest dwelling) and adult (exophilic) D. reticulatus ticks to be determined, and to assess their contribution to the spread of this important vector of Babesia canis. We compared also ectoparasite communities between adult foxes with those of fox cubs. Finally, we carried out a systematic search for subcutaneous ticks determining their prevalence and abundance. In 2016–2018, 366 adult foxes and 25 live-trapped cubs were examined for ectoparasites. Ectoparasites were identified based on morphological features, PCR amplification and sequencing. The total prevalence of ectoparasites was higher in cubs (68%) than in adults (62.8%). In adults, 15 parasite species were recorded, including four tick species, seven flea species, scabies, and one Anopluran species each in the genera Felicola and Lipoptena. In cubs, six ectoparasite species were found, including Ixodes kaiseri, a species not found in adults. Although Ixodes ricinus and D. reticulatus were the dominant tick species on adult foxes, no D. reticulatus ticks were found on cubs. Subcutaneous ticks were common (38%) and abundant in all areas. Molecular analysis of subcutaneous nodules allowed the identification of 17 I. ricinus and five D. reticulatus. In conclusion, red foxes play a minor role as hosts of D. reticulatus.

Background Babesia spp. are widespread tick-borne intraerythrocytic protozoa, infecting a broad range of vertebrate hosts. Red foxes are reservoirs of Babesia vulpes, belonging to the Babesia microti -like group (clade I), and play an important role in the epidemiology of canine and wildlife babesiosis. Besides B. vulpes , another species of this genus was molecularly reported in red foxes from Israel and Iraq and provisionally named “ Babesia sp. MML-2014”; however, no morphological description of this small Babesia species was provided, preventing a proper species naming. Methods Infection with piroplasmid species was detected and described by microscopy of stained blood smears in one red fox from Southern Italy. Molecular characterization of the Babesia sp. and differentiation from B. vulpes was performed through PCR amplification of nuclear ( 18S rRNA , ITS1-5.8S-ITS2) and mitochondrial (cytochrome c oxidase subunit 1, cox1 ) gene markers, followed by DNA sequencing and phylogenetic analysis. In addition, Ixodes kaiseri ticks collected from the infected fox were screened for piroplasmids by PCR. Results Sequence comparison of piroplasmids showed 98–99% identity with the undescribed Babesia sp. MML-2014 and phylogenetic analyses confirmed that this taxon belongs to the Western group (clade III) and is differentiated by B. vulpes . Morphological and morphometric analyses further demonstrated that Babesia sp. nov. is a distinct small piroplasm and is characterized by unique Maltese cross forms. Based on the above, we named Babesia banethi sp. nov. as a new taxon. In addition, Babesia sp. nov. DNA was detected in the intestine of one engorged I. kaiseri specimen. Conclusions This study provides genetic and morphological findings of B. banethi sp. nov. A morphological description with measurements of the parasite forms in red fox erythrocytes, differential diagnosis supplemented by genetic characterization, and the deposition of the holotype in suitable collections have been made in compliance with the ICZN guidelines. Graphical Abstract

A study of ticks on wildlife was carried out in the area of Levice, Bratislava, Stupava, and Vrbovce (south-western Slovakia) during 2021 and 2022. Overall, 512 ticks were collected from 51 individuals of six wild mammalian species. Eight tick species were identified, namely Dermacentor reticulatus , D. marginatus , Haemaphysalis inermis , H. concinna , Ixodes ricinus , I. hexagonus , and two Ixodes spp. Ixodes hexagonus were collected from northern white-breasted hedgehogs ( Erinaceus roumanicus ), females belonging to Ixodes spp. were collected from red fox ( Vulpes vulpes ) and nymphs from European badger ( Meles meles ). Ixodes hexagonus and the Ixodes spp. were identified morphologically and molecularly based on sequences of fragments of two mitochondrial genes, COI and 16S rRNA. Molecular analysis of Ixodes spp. confirmed the identity of Ixodes kaiseri Arthur, 1957 and I. canisuga (Johnston, 1849). Sequence analyses show that the I. kaiseri isolate from Slovakia is identical to I. kaiseri isolates from Romania, Poland, Germany, Turkey, and Croatia. We demonstrate for the first time the presence of I. kaiseri in Slovakia using both morphological and molecular methods.

The role of red fox, Vulpes vulpes, and its associated ticks in maintaining Borrelia burgdorferi sensu lato (s.l.) was studied. A total of 1583 ticks were removed from ears of 120 infested animals and were identified as species using a nested PCR targeting the ITS2 and coxI fragments of Ixodes DNA. Ixodes kaiseri prevailed (76%), followed by I. canisuga, I. ricinus, and I. hexagonus. In total, 32.4% of 943 ticks revealed Borrelia DNA and 10 species of B. burgdorferi s.l. complex were identified. Borrelia garinii and B. afzelii comprised 70% of all infections. The other eight species included B. americana, B. bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. lanei, B. spielmanii, and B. valaisiana. Analysis of tissues from 243 foxes showed that 23.5% were infected with B. burgdorferi s.l. Borrelia garinii was detected in 91% of the infected animals, including 31% of mixed infections with B. afzelii, the second most prevalent species, followed by B. spielmanii. The predominance of B. garinii in PCR-positive animals and infected larval ticks (38.1%), suggests that this spirochete and B. afzelii are preferentially associated with foxes. Although red foxes are exposed to a high diversity of B. burgdorferi s.l. species found in engorged Ixodes ticks, their reservoir competence for most of them appears to be low.

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.