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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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cryptosporidium is an apicomplexan protozoan parasite that primarily infects the gastrointestinal epithelium in humans and domestic and wild animals. The majority of studies have been focused on human, livestock, and pet infections. Hence, Cryptosporidium spp. in wildlife, including wild carnivores, remained neglected. There are several studies reporting the occurrence of Cryptosporidium spp. in wild foxes, but these are only a few molecular surveys; no data is available concerning the occurrence of this parasite in raccoon dogs and martens in Europe, and to the best of our knowledge to date, only one study has reported Cryptosporidium from badgers in Spain. Therefore, we used molecular analyses to identify and genotype Cryptosporidium spp. in wild-living mesocarnivores in Poland. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine, and beech martens were collected and then analyzed for the presence of Cryptosporidium spp. using the nested PCR method. The appearance of PCR products in the reaction with Cryptosporidium -specific primers against the 18S rRNA and actin genes demonstrated that Cryptosporidium spp. occurred in 23.0% of all examined species of animals. Performed sequence analyses showed the presence of the Cryptosporidium skunk genotype, Cryptosporidium vole genotype II, Cryptosporidium canis dog and fox genotypes, as well as Cryptosporidium erinacei , Cryptosporidium ditrichi , Cryptosporidium suis, and Cryptosporidium alticolis, in these hosts. Molecular data presented here indicate that examined mesocarnivores may be a significant reservoir of specific and non-specific Cryptosporidium species, including those with zoonotic potential. Most studies of carnivores have described the presence of non-specific Cryptosporidium spp. in carnivore hosts, and this is probably the result of the transfer of these parasites from prey species through the digestive tract or the transfer of the parasite from a contaminated environment.

Wild living mesocarnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. Individual tissue samples derived from 284 specimens of six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine marten and stone marten, were used for molecular investigations with the nested PCR method. The animals were sampled in the Ruszów Forest District (Poland). We aimed to examine the relative importance of the studied mesocarnivores as hosts of Borrelia spp. and investigated their role in this spirochaete's transmission cycle. We also aimed to trace the reservoir competence of these invasive and native predators and borreliosis eco-epidemiology in the context of a dilution effect. The overall prevalence of Borrelia spp. in the tested carnivores was 8.8%. Almost all of the consensus sequences of the partial flaB gene shared identity with a sequence of specific Borrelia species, i.e., B. afzelii , B. garinii and B. burgdorferi . Our results suggest that raccoons may play a role as reservoir hosts for these spirochaetal bacteria. The role of invasive species seems to be worthy of further analysis with reference to the circulation of vector-borne pathogens as well as in the context of the "dilution effect" hypothesis.

Background: The raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon's biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways., Methods: Tissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software., Results: Rickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis, Rickettsia raoultii, and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA, amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively., Conclusions: To the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required., (© 2022. The Author(s).)

The raccoon ( Procyon lotor ) and the raccoon dog ( Nyctereutes procyonoides ) were introduced to Europe and, in the past decades, their populations have increased and adapted to synanthropic environments across Europe. In view of their possible further spread in Europe, the invasive species should be examined as potential reservoirs for helminths, including tapeworms. This study aims to investigate the prevalence and diversity of tapeworms in introduced wild carnivores in Poland by identifying cestode species based on copro-DNA analysis. A total of 214 individual fecal samples from non-native invasive carnivores, i.e., raccoons and raccoon dogs, and additionally 47 samples from native carnivores, i.e., European badgers ( Meles meles ), were analyzed for the presence of cestodes. PCR analysis of fecal samples targeting a fragment of mitochondrial (mt) 12S rRNA gene revealed the presence of cestode DNA in 19 of 103 (18.4%) raccoons, in 13 of 111 (11.7%) raccoon dogs and in 23 of 47 (48.9%) badgers. Sequence analysis demonstrated the presence of Mesocestoides litteratus in raccoons and raccoon dogs, while Mesocestoides lineatus was identified only in two samples derived from raccoon dogs. Moreover, in this study, Atriotaenia incisa was for the first time molecularly characterized by using fragments of mt 12S rRNA gene, and the DNA of this cestode species was detected in the fecal samples of all the examined host species.

Information on the prevalence on Rickettsia spp. in free-ranging mustelids and their specific ectoparasites is scarce. However, stone martens (Martes foina), pine martens (Martes martes) and European badgers (Meles meles) are common predators in many regions of Poland. In the present study we used tissue fragments to determine Rickettsia prevalence in these carnivores by molecular biology techniques. In addition, we included a data on several species of invertebrates that commonly feed on badgers.

Wild carnivores, both introduced and native species, are able to adapt well to peri-urban environments, facilitating cross-species pathogen transmission with domestic animals, and potentially humans. The role of wild living reservoir hosts cannot be ignored because of their known carriage of E. bieneusi zoonotic genotypes. In the past decades, populations of wild living carnivores, i.e., native, such as red foxes, and invasive, such as raccoon dogs and raccoons, have increased and adapted to synanthropic environments across Europe, including Poland. The knowledge concerning E. bieneusi genotype identification and distribution in wild carnivores is limited worldwide. A total of 322 individual fecal samples from six carnivore species, i.e., raccoon, raccoon dog, red fox, European badger, pine and beech martens, were collected and then analysed for the presence of E. bieneusi using the nested PCR method. Overall prevalence of the pathogen was estimated to be as high as 27.3%. The infection rates for E. bieneusi varied between the carnivore species, from 13.7% in beech martens to 40.4% in raccoon dogs. Based on sequence analysis of the ITS region of the rRNA gene marker, we detected five known genotypes of E. bieneusi in examined animals. In the invasive species, E. bieneusi NCF2 and D genotypes have been identified, whereas in the native ones, E. bieneusi NCF2, D, C, EbCar2 and Type IV genotypes were identified. All E. bieneusi genotypes recorded in this survey clustered in Group 1, showing their zoonotic potential. Our results provide the first description of the occurrence and genotypes of the microsporidian E. bieneusi in wild living population of raccoon dogs in Europe. Our findings are important for the study of pathogen epidemiology and emphasize the fact that the invasive and the native wild living carnivores, both widely distributed, should be considered more seriously as significant sources of zoonotic pathogens hazardous to domestic and farmed animals and humans.

Encephalitozoon spp. is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. This study was conducted to determine the prevalence of Encephalitozoon spp. in wild living rodents from Poland, the Czech Republic and Slovakia. Faecal and spleen samples were collected from individuals of Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, and Myodes glareolus (n = 465) and used for DNA extraction. PCR, targeting the ITS region of the rRNA gene was performed. The overall prevalence of microsporidia was 15.1%. The occurrence of Encephalitozoon cuniculi in the abovementioned host species of rodents has been presented for the first time, with the highest infection rate recorded for A. flavicollis. Sequence analysis showed that the most frequent species was E. cuniculi genotype II (92.5%). E. cuniculi genotypes I (1.5%) and III (6.0%) were also identified., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonoides) belong to a group of the invasive species. The introduced species as potential reservoirs for vector-borne pathogens have been the subject of recent research, though there are still no data with reference to the European population of the raccoon, and few studies concern only the raccoon dog. This study shows the occurrence of Anaplasmataceae representatives in raccoons and a sympatric population of the raccoon dogs obtained from the area of Poland and Germany. During the study, the occurrence of Anaplasma phagocytophilum ecotype I in the introduced raccoon in northwestern Poland was revealed. Additionally, Candidatus Neoehrlichia sp. (FU98) was identified for the first time in the raccoon dog in Central Europe and thereby the raccoon dog is a new host for this pathogen., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Calodium hepaticum is a parasitic nematode found primarily in rodents but known to infect numerous other mammal species, and causing in humans the hepatic calodiasis. Herein, we present the first finding of C. hepaticum in Rattus norvegicus in Poland. In this study, we have used the combined pepsin-HCl digestion and molecular techniques to detect C. hepaticum infection in the liver. The results confirm the usefulness of molecular approaches for investigating the biology and epidemiology of C. hepaticum. Additionally in this study, the parts of the small subunit rRNA (18S rRNA) gene of Aonchotheca annulosa from bank vole, Aonchotheca erinacei and Eucoleus sp. from hedgehog were amplified, yielding the first 18S rRNA gene sequences of these Capillariinae nematodes.

Background: Rickettsiae are obligate intracellular alpha-proteobacteria. They are transmitted via arthropod vectors, which transmit the bacteria between animals and occasionally to humans. So far, much research has been conducted to indicate reservoir hosts for these microorganisms, but our knowledge is still non-exhaustive. Therefore, this survey was undertaken to investigate the presence of Rickettsia spp. in wild-living small rodents from south-western Poland., Results: In total, 337 samples (193 from spleen and 144 from blood) obtained from 193 wild-living rodents: Apodemus agrarius, Apodemus flavicollis, and Myodes glareolus were tested by qPCR for Rickettsia spp. based on a fragment of gltA gene. The prevalence of infection was 17.6% (34/193). Subsequently, the positive samples were analysed by conventional PCR targeting the gltA gene fragment. The genus Rickettsia was confirmed by sequence analysis in four samples from the blood. In two blood samples from A. agrarius, the identified pathogen was Rickettsia helvetica. The Rickettsia obtained from A. flavicollis was assigned to Rickettsia felis-like organisms group. One isolate from A. agrarius could be determined only to the genus level., Conclusions: This study shows the presence of Rickettsia DNA in tissues of wild-living rodents, suggesting some potential role of these animals in temporarily maintaining and spreading the bacteria in enzootic cycles.

Microsporidia are opportunistic pathogens in nature infecting all animal phyla. There is a potential risk of microsporidian spores transmission from urban rooks inhabiting some metropolitan cities to people through casual interactions. The aim of this study was to identify microsporidia species in the droppings of rooks in Wroclaw, Poland. A total of 15 collective sets of droppings were examined using nested-PCR method. Amplification of ITS rRNA gene revealed the presence of Enterocytozoon bieneusi D, Peru 6, and Encephalitozoon hellem 1A genotypes. This study indicates that excreta of urban rooks can be an important source of human infection with these pathogens., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

The raccoon (Procyon lotor) carnivore native to North America is a fast spreading, invasive species in the Europe now. At the moment, the highest population occupies areas near the German-Polish border. The data on the occurrence of Cryptosporidium spp. and microsporidia in raccoons is limited to North America's territory and is totally lacking in the case of their introduction to Europe. Therefore, the objective of this study was to investigate the occurrence of microparasites, i.e., Cryptosporidium spp. and microsporidia in the introduced raccoons obtained from localities in Poland and Germany. A PCR-based approach that permitted genetic characterization via sequence analysis was applied to raccoon fecal samples (n = 49), collected during 2012-2014. All fecal samples were simultaneously tested with the use of genetic markers, and DNA of microsporidia and Cryptosporidium spp. was detected among the examined raccoons. The results of our research confirmed the presence of Cryptosporidium skunk genotype and Enterocytozoon bieneusi NCF2 genotype. The results suggest a possible role of raccoons in the contamination of the environment, including urban areas, with pathogens of zoonotic significance as well as their role in the transmission and introduction of new genotypes of microparasites in the areas where P. lotor has not been observed yet. To our knowledge, there has been no literature data on the above genotypes detected previously in humans or animals from the examined study sites so far.

Diversity of Enterocytozoon bieneusi genotypes in wild small rodent populations still remains incomplete and only few molecular studies have been conducted among these hosts. Therefore, the aim of this study was to determine whether small rodents, i.e., Apodemus agrarius, Apodemus flavicollis, Mus musculus and Myodes glareolus act as hosts of E. bieneusi and can play an important role in spore spreading in the environment of south-western Poland. Molecular analyses were conducted to determine pathogen genotypes. A total of 191 fecal and 251 spleen samples collected from 311 rodent individuals were examined for the occurrence of E. bieneusi by PCR amplifying ITS gene. The overall prevalence of E. bieneusi in rodent samples was 38.9%. The nucleotide sequences of ITS region of E. bieneusi revealed the presence a total of 12 genotypes with two being already known, i.e., D and gorilla 1 genotypes. The remaining ten are novel genotypes (WR1-WR10) which segregated into three groups in a neighbor joining phylogeny. This study reports for the first time E. bieneusi occurrence in wild living rodents in Poland and shows extensive genetic diversity within E. bieneusi isolates of rodent origin., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cryptosporidium spp. and Giardia spp. have been detected in a range of host species, including rodents. The aim of this study was to determine the distribution of these pathogens and recognition of the reservoir role of rodents in the maintenance of these pathogens in south-western Poland. Additionally, preliminary molecular studies were conducted to elucidate the species and genotypes of Cryptosporidium and Giardia identified in this study. Stool samples (n=266) from A. agrarius, A. flavicollis and M. glareolus, were subjected for analyses. Values of prevalence were 61.7, 68.3 and 68.1%, respectively, for Cryptosporidium spp. and 41.7, 24.4 and 38.4%, respectively, for Giardia spp. There was a statistically significant correlation between host species and Giardia infection where A. agrarius was the species of the highest prevalence. Statistically significant differences were not found for comparisons made for study sites and occurrence of Giardia spp. and Cryptosporidium spp. Due to preliminary nested PCR results, specific amplifications of Cryptosporidium COWP and SSU rRNA genes were obtained for several isolates taken from rodent host species. One isolate recovered from A. agrarius (from a semi-aquatic, urban area) was identified as C. parvum and revealed 100% similarity with sequences obtained from humans. To the best of the knowledge of the authors, this is the first record of the C. parvum zoonotic species from the striped field mouse. Also recorded were the first findings of C. ubiquitum from three small rodent species.

The cat lungworm Aelurostrongylus abstrusus affects the domestic cats and other felids all over the world. Feline aelurostrongylosis is of importance in clinical feline medicine. Snails and slugs are the intermediate hosts, but the cat is probably infected by eating paratenic hosts, e.g., rodents, birds, amphibians and reptiles. Herein we present the first finding of A. abstrusus in a naturally infected invasive synantropic slugs Arion lusitanicus (intermediate host) and wild living rodents Apodemus agrarius (paratenic host). The results confirm the usefulness of molecular approaches for investigating the biology, ecology and epidemiology of A. abstrusus, the agent of feline aelurostrongylosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)