Agnieszka Perec-Matysiak’s research while affiliated with University of Business in Wrocław and other places

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

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.

Recreational horses host many groups of parasites which in some cases can also be a threat to humans. The presented study focuses on intestinal protozoa and nematodes. The aim of this study was to determine the taxonomic variety of selected intestinal parasites found in recreational horses and their molecular identification in faecal samples. The stool samples were obtained from recreational horses from various studs in Lower Silesia. Molecular methods were used to detect intestinal parasites. After isolation of genetic material from stool samples, PCR reactions were performed using different molecular markers – 18S rRNA, gp60 and ITS rRNA. The products were sequenced. Among the 203 tested stool samples, 34 were found to contain the desired intestinal protozoa. Prevalence of Cryptosporidium spp. was 5.4%, and prevalence of Eimeriidae was 14.9%. This study allowed to identify Cryptosporidium horse genotype. The presence of Eimeria sp., Isospora sp. and Cryptosporidium spp. was also confirmed in the positive samples. Nematodes belonging to subfamily Cyathostominae were identified by DNA sequencing. Additionally among nematodes Oxyuris and Parascaris were noted. Co-infections of nematodes with microparasites were also recorded. „Research project supported/partly supported by program „Excellence initiative – research university” for years 2020- 2026 for University of Wrocław” (BPIDUB.4610.7.2021.KP.A)

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. Graphical abstract

In recent decades, populations of the raccoon ( Procyon lotor ) and the raccoon dog ( Nyctereutes procyonides ) have increased and adapted to peri-urban and urban environments in many parts of the world. Their ability to rapidly colonize new territories, high plasticity and behavioral adaptation has enabled these two species to be considered two of the most successful invasive alien species. One of the major threats arising from continually growing and expanding populations is their relevant role in maintaining and transmitting various vector-borne pathogens among wildlife, domestic animals and humans. According to the WHO, over 17% of infectious diseases are vector-borne diseases, including those transmitted by ticks. Every year tick-borne pathogens (TBPs) create new public health challenges. Some of the emerging diseases, such as Lyme borreliosis, anaplasmosis, ehrlichiosis, babesiosis and rickettsiosis, have been described in recent years as posing important threats to global health. In this review we summarize current molecular and serological data on the occurrence, diversity and prevalence of some of the TBPs, namely Babesia , Theileria , Hepatozoon , Borrelia , Rickettsia , Bartonella , Anaplasma and Ehrlichia , that have been detected in raccoons and raccoon dogs that inhabit their native habitats and introduced areas. We draw attention to the limited data currently available on these invasive carnivores as potential reservoirs of TBPs in different parts of the world. Simultaneously we indicate the need for more research in order to better understand the epidemiology of these TBPs and to assess the future risk originating from wildlife. Graphical Abstract

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.