Zoonotic Genotypes of Enterocytozoon bieneusi in Wild Living Invasive and Native Carnivores in Poland.

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.

The opportunistic pathogen Encephalitozoon cuniculi in wild living Murinae and Arvicolinae in Central Europe.

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.

Survey for Zoonotic Microsporidian Pathogens in Wild Living Urban Rooks (Corvus frugilegus).

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.

Wildlife as an environmental reservoir of Enterocytozoon bieneusi (Microsporidia) ­ analyses of data based on molecular methods

Enterocytozoon bieneusi is the most commonly identified Microsporidia in humans and has also been detected worldwide in a large group of wild living and domestic animals. The identification of E. bieneusi in wildlife has raised the question of the importance of animal reservoirs in the epidemiology of microsporidiosis and the implications of the infection with this pathogen in hosts. This review summarizes the available molecular data on the variety of E. bieneusi genotypes, both potentially zoonotic and host-specific isolated from wild living mammals and birds. In contrast to microsporidial infections of humans and domestic animals, wildlife deserves attention as a source of significant environmental reservoir of E. bieneusi.

Cryptosporidium spp. and Enterocytozoon bieneusi in introduced raccoons (Procyon lotor)-first evidence from Poland and Germany.

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 among small rodents in southwestern Poland.

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.