Occurrence of Dirofilaria repens in wild carnivores in Poland.

Dirofilaria repens is an expanding vector-borne zoonotic parasite of canines and other carnivores. Sub-clinically infected dogs constitute the most important reservoir of the parasite and the source of infection for its mosquito vectors. However, occurrence of D. repens infection in wild animals may contribute to the transmission of the parasite to humans and may explain the endemicity of filariae in newly invaded regions. The aim of the current study was to determine the occurrence of D. repens in 511 blood and spleen samples from seven species of wild carnivores (wolves, red foxes, Eurasian badgers, raccoons, raccoon dogs, stone martens, and pine martens) from different regions of Poland by means of a PCR protocol targeting the 12S rDNA gene. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodeships in four of the seven regions of Poland: Masovia, Lesser Poland, Pomerania and Warmia-Masuria. The highest prevalence was found in Masovia region (8%), coinciding with the highest previously recorded prevalence in dogs in Central Poland. The DNA of Dirofilaria was detected in 16 samples of three species (total prevalence 3.13%). A low and similar percentage of positive samples (1.9%, 4.2% and 4.8%) was recorded among badgers, red foxes, and wolves, respectively. Dirofilaria repens-positive hosts were identified in seven of fourteen voivodships. Based on detection in different voivodeships, D. repens-positive animals were recorded in four out of the seven regions of Poland: in Masovia, Lesser Poland, Pomerania, and Warmia-Masuria. The highest prevalence of filariae was found in Masovia region (8%), reflecting the highest previously recorded prevalence in dogs (12-50%) in Central Poland. In summary, we conducted the first comprehensive study on the epidemiology of D. repens in seven species of wild hosts in all seven regions of Poland and identified the first case of D. repens infection in Eurasian badgers in Poland and the second in Europe.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite.

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

Tick-Borne Pathogens, Babesia spp. and Borrelia burgdorferi s.l., in Sled and Companion Dogs from Central and North-Eastern Europe.

Ticks are important vectors of numerous pathogens of medical and veterinary significance. The aim of the current study was to determine the prevalence of Babesia spp. and Borrelia burgdorferi s.l. in sled and pet dogs from Central and North-Eastern Europe. Neither Babesia spp. nor Borrelia burgdorferi s.l. infections were detected in sled dogs from seven countries (Poland, Lithuania, Latvia, Estonia, Belarus, Russia and Finland). The DNA of Babesia spp. was detected in 100% of symptomatic and 5.4% of asymptomatic pet dogs from Poland. Similarly, the DNA of Babesia spp. was identified in 82% of symptomatic and 3.8% of asymptomatic pet dogs from Ukraine. The DNA of Borrelia burgdorferi s.l. was detected in 4.4% of pet dogs. Molecular typing confirmed the presence of Babesia canis and Borrelia burgdorferi sensu stricto (s.s.) in selected samples. Four dogs were co-infected by B. canis and Borrelia burgdorferi s.l. Tick-borne pathogens constitute a serious health threat to pet dogs in Central and South-Eastern Europe, but were not observed among sled dogs from the same region of Europe nor in the Baltic countries.

Update on prevalence of Babesia canis and Rickettsia spp. in adult and juvenile Dermacentor reticulatus ticks in the area of Poland (2016-2018).

Ornate dog tick, Dermacentor reticulatus is an important vector of Babesia canis, and Rickettsia spp. and other pathogens of veterinary and public health interest. The current study is the first to investigate the long-term changes in prevalence of these pathogens in expanding tick populations in Central Europe. Molecular techniques (PCR, sequencing) were applied for the detection of pathogen DNA in adult (n = 2497) and juvenile ticks (1096 larvae and 410 nymphs). DNA of Rickettsia spp. was identified in 35% of adults and 12.6% of juvenile ticks. DNA of B. canis was detected in 3% of adult ticks and only in ticks from the Eastern region (regional prevalence 6%). As previously, no B. canis-positive ticks were found in Western Poland, including ticks from Wroclaw area (n = 298). DNA of B. canis was identified in 0.33% of juvenile ticks (in 3 pools of larvae and 2 nymphs) from the Eastern region. In the current study we confirmed high occurrence of R. raoultii in adults ticks from all four zones and relatively high prevalence of B. canis in the Eastern population of D. reticulatus, corresponding well with high incidence of canine babesiosis in this area of Poland. Finally, we confirmed R. raoultii and B. canis infection in all life stages of D. reticulatus ticks.

Occurrence of juvenile Dermacentor reticulatus ticks in three regions in Poland: the final evidence of the conquest.

BACKGROUND: Two populations of Dermacentor reticulatus ticks (Western and Eastern) in Poland are among the most dynamic tick populations in Central Europe. Expansion and settlement of ticks in new localizations depend on the presence of suitable hosts, for both adult and juvenile ticks. METHODS: The current study was planned to complement our previous studies on questing adult ticks and was focused on a collection of juvenile D. reticulatus ticks from rodents from three regions in Poland, defined by the presence/absence of adult ticks (regions of the Western and Eastern tick population and the gap area between them) to confirm the existence of stable populations. Rodent trapping was conducted in open habitats (fallow lands, wasteland and submerged meadows) in 2016-2018 in June, July and/or August to encompass seasonal peaks of larvae and nymph activity. RESULTS: Altogether, three tick species were collected, 2866 D. reticulatus, 2141 Ixodes ricinus and 427 Haemaphysalis concinna. Dermacentor reticulatus was the most common (72.3%) and abundant (mean 17.94 ± 2.62 ticks/rodent) tick species on rodents from the Eastern region; in the Western region infestation of rodents was only 6.8%. Ixodes ricinus was found in all three regions and was the only tick species collected from rodents from the gap area. Haemaphysalis concinna was noted only in the Western region. The highest infestation of juvenile D. reticulatus was recorded on voles (Myodes and Microtus spp.), infestation of I. ricinus was the highest on Apodemus mice, and the majority of H. concinna ticks were collected from root voles Alexandromys oeconomus. CONCLUSIONS: Our study confirmed a stable population of D. reticulatus in Eastern and Central Poland and a lower prevalence and mean abundance of this tick species among rodents from the Western region. A lack of juvenile D. reticulatus on rodents in Niewiadów confirmed the existence of the gap area, free of D. reticulatus ticks.

Pathogens detected in the tick Haemaphysalis concinna in Western Poland: known and unknown threats.

In recent years, a new focus of the relict tick Haemaphysalis concinna was discovered in Western Poland, near Wolsztyn, Greater Poland voivodeship. This species may play an important role in the circulation of pathogens of medical and veterinary importance. In the present study we tested 880 juvenile ticks collected from rodents, including 427 H. concinna, 443 Ixodes ricinus and 10 Dermacentor reticulatus for three of the most common pathogens vectored by ticks in Poland: Rickettsia and Babesia spp. and Borrelia burgdorferi s.l. Additionally, molecular techniques were applied for accurate identification of tick host species (the voles Microtus and Alexandromys). Our study found differences in the range and prevalence of vectored pathogens between the three tick species. DNA of all three pathogens was found in I. ricinus. In juvenile H. concinna, DNA of Babesia microti, Borrelia afzelii and Rickettsia sp. was identified. Moreover, DNA of a new unnamed Babesia species related to B. crassa, was found in two H. concinna nymphs. This genotype of Babesia was previously identified in H. concinna in the Far East and then in Central Europe. DNA of Rickettsia raoulti and B. afzelii was detected in D. reticulatus nymphs. Among rodent hosts, Alexandromys oeconomus seems to be host of the highest significance for juvenile tick stages and was the only host species with B. afzelii detected in blood samples. Using phylogenetic methods, we confirmed a clear division between rodents from the genera Microtus and Alexandromys. Moreover, we found that A. oeconomus trapped in Western Poland clustered with a Central European A. oeconomus allopatric phylogroup.

Ectoparasites of red foxes (Vulpes vulpes) with a particular focus on ticks in subcutaneous tissues.

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.

Comparison of the detection efficiency of haemoparasite DNA in blood and faecal samples - the way to eco-epidemiological studies.

INTRODUCTION AND OBJECTIVE: It is easier and non-invasive to obtain faecal samples compared with blood samples. Molecular techniques may enable detection of parasites even in tiny amounts of blood-containing faeces. We aimed to compare the sensitivity of detection of three Babesia species and Hepatozoon canis in blood and faecal samples, including samples derived from naturally infected hosts. MATERIAL AND METHODS: Three groups were involved: 1) Nine BALB/c mice infected with Babesia microti sampled during acute (n=3), post-acute (n=3) and chronic phases of infection (n=3); 2) Eight dogs with symptoms of babesiosis; 3) Six red foxes infected with B. vulpes, one fox infected with B. canis, four foxes infected with H. canis. Genomic DNA was extracted from blood and faeces by use of commercial kits and amplified with genus-specific primers in one-step or nested PCR reactions. Selected PCR products were sequenced. RESULTS: No positive results for faecal samples were obtained from H. canis-positive foxes in contrast to Babesia spp. infections. Positive results from PCRs were obtained for all BALB/c mice (100%), five dogs (62.5%) and four of seven foxes (57.1%). Successful sequencing was obtained for six selected murine samples (B. microti), four canine samples (B. canis) and for one fox sample (B. vulpes). The success of B. microti detection in murine faecal samples from acute, post-acute and chronic phases was identical (100%). CONCLUSIONS: Detectability of Babesia spp. infections was lower in naturally infected dogs and foxes, compared to experimentally infected mice. Detection of DNA in faecal samples can be useful in the detection of Babesia infection in populations from which blood samples are hard to obtain, but due regard must be given to the possibility that prevalence of infection may be severely underestimated.

Molecular study of transovarial transmission of Babesia canis in the Dermacentor reticulatus tick.

The Dermacentor reticulatus tick is a main vector of Babesia canis in Europe. The risk of canine babesiosis is unpredictable, due to significant differences in the prevalence of B. canis between ticks originating from closely situated regions. This phenomenon may be explained by vertical transmission of the pathogen in a vector population. Thus, molecular techniques were applied to investigate the occurrence of transovarial transmission in D. reticulatus ticks. DNA of B. canis was detected in 20.7% (6/29) of engorged female ticks collected from dogs, in every pool of eggs laid by positive females (100%, 6/6) and in larvae hatched from these eggs. In the pools of eggs collected from two positive females (2/6; 33.3%), no larvae hatched and no embryos were observed inside the eggs. Conclusions. Transovarial transmission of B. canis can be an important mechanism supporting maintenance of the pathogen in the environment without the presence of a reservoir vertebrate host. However, the efficiency of transovarial transmission in the maintenance of B. canis in natural conditions requires further field research.