Katarzyna Buńkowska-Gawlik

Intermediate and paratenic hosts in the life cycle of Aelurostrongylus abstrusus in natural environment

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.

Small rodents as reservoirs of Cryptosporidium spp. and Giardia spp. in south-western Poland

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.

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.

Spotted fever rickettsiae in wild-living rodents from south-western Poland

BackgroundRickettsiae 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.ResultsIn 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.ConclusionsThis 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.

The occurrence of Anaplasmataceae in European populations of invasive carnivores

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.

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.

The molecular identification of Calodium hepaticum in the wild brown rat (Rattus norvegicus) in Poland

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.

Co-occurrence of Babesia microti, Bartonella spp., Borrelia burgdorferi s.l. and Anaplasma phagocytophilum in rodents from Lower Silesia, Poland

This study aims to establish the relative contribution of rodent populations from diverse habitats to the occurrence of rodent-borne pathogens of public health significance. Rodents (n = 492) represented by Apodemus agrarius, A. flavicollis and Myodes glareolus, were captured in live traps in four localities of south-western Poland (2009-2012). For the analysis of co-occurrence of pathogens, Babesia microti, Borrelia burgdorferi s.l., Bartonella spp. and Anaplasma phagocytophilum, both blood and spleen samples were obtained from selected rodent specimens. The choice of genetic markers and primers was based on the literature data and our preliminary results. Conventional PCR was used for the detection of DNA of examined pathogens. Selected PCR positive products were purified and sequenced. BLAST searches were conducted in order to elucidate any homologies with previously deposited sequences in GenBank. The DNA of pathogens was detected in 66.7% of the rodents tested. We observed that among infected rodents, 40.5% were infected with at least two pathogens, while only 4.7% with all four pathogens. All three of the rodent species were infected with each of the examined pathogens. In examined rodent populations the prevalence of B. microti was 40.0%, Bartonella spp. 37.7%, B. burgdorferi s.l. 28.2% and A. phagocytophilum 17.7%. In each of the tissue samples (blood or spleen), the prevalences of Babesia microti and Bartonella spp. were recorded as comparable, on rather high levels. Interestingly, the occurrence of these pathogens in both blood and spleen was only detected in 13% of the rodents. While examining the blood and spleen-derived DNA, it was found that as many as 92.7% of A. agrarius harbored at least one pathogen. Co-occurrence of 3-4 pathogens was most common in this rodent species (32.1% infected). By examining the spleen and blood samples of rodents at the same time, we estimate that the prevalence of pathogens in these rodents is higher than the literature indicates. This would implicate the examined rodent species as a significant reservoir of pathogens with zoonotic potential. Additionally, the role of A. agrarius, now widespread in some regions of Europe, as a reservoir host needs to be emphasized. A. agrarius can act as a bridge between woodland habitats and periurban environments frequented by humans.