Molecular investigation of tick-borne pathogens in ticks from grazing cattle in Korea

This study was carried out to identify the tick species that infest grazing cattle and to determine the presence of tick-borne pathogens transmitted by these ticks in Korea. A total of 903 ticks (categorized into 566 tick pools) were collected from five provinces during 2010–2011. The most prevalent tick species was Haemaphysalis longicornis, followed by three Ixodes spp. ticks. The collected ticks were infected with both rickettsial and protozoan pathogens. In all, 469 (82.9%) tick pools tested positive for the Anaplasma/Ehrlichia 16S rRNA gene, whereas 67 (11.8%) were positive for the Babesia/Theileria 18S rRNA gene. Among the rickettsial pathogens, E. canis was detected with the highest rate (22.3%), followed by A. platys (20%), E. chaffeensis (19.4%), E. ewingii (19.3%), Rickettsia sp. (12.4%), A. phagocytophilum (5.5%) and E. muris (0.5%). Among the protozoan pathogens, T. equi was detected with the highest rate (7.2%), followed by T. sergenti/T. buffeli (3.7%) and B. caballi (0.35%). Simultaneous infections with up to seven pathogens were also identified. In particular, ticks infected with rickettsial pathogens were also infected with protozoan pathogens (22 samples). All five provinces investigated infected with tick-borne pathogens.     

Tick-borne rickettsial pathogens in ticks and small mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

Comparative Experimental Infection Study in Dogs with Ehrlichia canis,E. chaffeensis,Anaplasma platys and A. phagocytophilum

Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.     

Detection of <Emphasis Type="Italic">Anaplasmataceae</Emphasis> agents and co-infection with other tick-borne protozoa in dogs and <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> sensu lato ticks

Anaplasmosis and ehrlichiosis are of serious health concern worldwide for animals and humans. In the present study, we report the occurrence of Anaplasma platys and Ehrlichia canis in dogs and Rhipicephalus sanguineus sensu lato (s.l.) ticks from Peninsular Malaysia using a nested polymerase chain reaction assay based on amplification of the 16S rRNA gene. Anaplasma platys was detected from dogs and ticks with prevalence rates of 3.3% (8/240) and 2.9% (4/140), respectively. On the other hand, 12.9% (31/240) of the dogs and 0.7% (1/140) of the ticks were tested positive for E. canis. Additionally, co-infections of A. platys and E. canis with Babesia or Hepatozoon protozoa were also noted in this study. Double infection (E. canis?+?B. gibsoni) was observed in tick, whereas triple infections (E. canis?+?A. platys?+?B. vogeli and E. canis?+?A. platys?+?H. canis) were found in dogs. This study represents the first evidence of A. platys DNA in R. sanguineus s.l. in Malaysia.     

Predominance of <Emphasis Type="Italic">Ehrlichia chaffeensis</Emphasis> in <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks from kennel-confined dogs in Limbe,Cameroon

Rhipicephalus sanguineus ticks (n = 63) collected from five dogs (two adults and three puppies) housed in a kennel were screened for Ehrlichial agents (Ehrlichia canis, E. chaffeensis, and E. ewingii) using a species-specific multicolor real-time TaqMan PCR amplification of the disulphide bond formation protein (dsb) gene. Ehrlichia chaffeensis DNA was detected in 33 (56%) ticks, E. canis DNA was detected in four (6%) ticks, and one tick was coinfected. The E. chaffeensis and E. canis nucleotide sequences of the amplified dsb gene (374 bp) obtained from the Cameroonian R. sanguineus ticks were identical to the North American genotypes.     

Species,developmental stage and infection with microbial pathogens of engorged ticks removed from dogs and questing ticks

Research into tick‐borne diseases implies vector sampling and the detection and identification of microbial pathogens. Ticks were collected simultaneously from dogs that had been exposed to tick bites and by flagging the ground in the area in which the dogs had been exposed. In total, 200 ticks were sampled, of which 104 came from dogs and 96 were collected by flagging. These ticks were subsequently examined for DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Rickettsia spp. and Babesia canis. A mixed sample of adult ticks and nymphs of Ixodes ricinus (Ixodida: Ixodidae) and Haemaphysalis concinna (Ixodida: Ixodidae) was obtained by flagging. Female I. ricinus and adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks dominated the engorged ticks removed from dogs. Rickettsia spp. were detected in 17.0% of the examined ticks, A. phagocytophilum in 3.5%, B. canis in 1.5%, and B. burgdorferi s.l. in 16.0%. Ticks with multiple infections were found only among the flagging sample. The ticks removed from the dogs included 22 infected ticks, whereas the flagging sample included 44 infected ticks. The results showed that the method for collecting ticks influences the species composition of the sample and enables the detection of a different pattern of pathogens. Sampling strategies should be taken into consideration when interpreting studies on tick‐borne pathogens.     

Molecular analysis of tick-borne protozoan and rickettsial pathogens in small ruminants from two South African provinces

Tick-borne protozoan and rickettsial diseases are a major threat to livestock in tropical and sub-tropical regions of Africa. In this study we investigated the presence and distribution of Theileria spp., Babesia ovis, Anaplasma ovis, Anaplasma phagocytophilum, Ehrlichia ruminantium and SFG Rickettsia in sheep and goats from Free State and KwaZulu-Natal provinces. A total of 91 blood samples were screened in this study, 61 from goats and 30 from sheep. PCR assay was conducted using primers based on Theileria spp. 18S rRNA, Babesia ovis (BoSSU rRNA), Anaplasma ovis (AoMSP4), Anaplasma phagocytophilum epank1, Ehrlichia ruminantium pCS20 and SFG Rickettsia OmpA. Overall infection rates of Theileria spp., Anaplasma ovis and Ehrlichia ruminantium were 18 (19.8%), 33 (36.3%) and 13 (14.3%), respectively. The co-infection of two pathogens were detected in 17/91 (18.7%) of all samples, goats having higher rates of co-infection compared to sheep. Phylogenetic tree analysis sequence of pCS20 gene of E. ruminantium of this study was found to be in the same clade with Kumm2 and Riverside strains both from South Africa. The phylogram of SSU rRNA of Theileria ovis had longer branch length compared to all other sequences most of which were from Asia and Middle East. This study provides important data for understanding the tick-borne diseases occurrence in the study area and it is expected to improve the approach for the diagnosis and control of these diseases.     

Longitudinal Analysis of Tick Densities and Borrelia, Anaplasma, and Ehrlichia Infections of Ixodes ricinus Ticks in Different Habitat Areas in The Netherlands

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m²). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m². The highest tick density was found in the dune area (139 to 551/100 m²). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.     

A survey of tick‐borne pathogens in dogs and their ticks in the Pantanal biome,Brazil

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick‐borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick‐borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick‐borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human‐biting ticks in Brazil.     

Ticks Parasitizing Wild Birds in Portugal: Detection of Rickettsia aeschlimannii, R. helvetica and R. massiliae

From January 2002 to December 2004, 152 ticks were collected from 40 wild birds recovered in Santo André Natural Reserve and Monsanto Forestal Park, Portugal mainland. Five ticks species were identified from 22 species of birds, and new host record were provided for some species. In addition, 32 (21%) ticks were screened by PCR to detect infections with agents belonging to order Rickettsiales: Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Rickettsia spp. PCR amplicons were obtained in 5 (15.6%) tick samples. Rickettsia DNA exhibiting gltA sequences similar to those of Rickettsia aeschilimannii, R. helvetica and R. massiliae were identified in Hyalomma marginatum, Ixodes ventalloi and in Rhipicephalus turanicus, respectively. This is the first report of rickettsiae infections in ticks collected from wild birds in Portugal. Giving the results presented above wild birds play an important role in the maintenance and dissemination of several tick species and associated rickettsiae.     

First molecular detection of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in the hard tick <Emphasis Type="Italic">Rhipicephalus haemaphysaloides</Emphasis> in Taiwan

Anaplasma phagocytophilum is transmitted mainly by hard ticks and can cause potentially fatal granulocytic anaplasmosis in humans, but its occurrence in ticks in Taiwan has never been investigated although this pathogen has been detected in Taiwanese rodents before. Ticks collected from small mammals in Hualien, eastern Taiwan, were assayed for Anaplasma infections; infections of Rickettsia and Apicomplexa protozoans were also studied. Of the 270 individually assayed Rhipicephalus haemaphysaloides ticks, A. phagocytophilum was identified in a nymphal tick. Parasites most similar to Anaplasma bovis, Rickettsia rickettsii, Rickettsia sp. TwKM01, and at least seven apicomplexan species (including genera Cryptosporidium, Hepatozoon, and Theileria) were also identified. This study shows that A. phagocytophilum does occur in the hard tick in Taiwan, although whether R. haemaphysaloides can vector this pathogen remains to be determined. This work also reveals a high diversity of tick-borne bacteria and protozoans circulating in a small region and calls for further research on their potential risks for human health.     

Molecular survey of canine vector-borne diseases in stray dogs in Thailand

Despite the large population of stray dogs in Thailand, there is limited information on the prevalence of canine vector-borne diseases (CVBDs). In this study, a molecular survey was conducted to determine the prevalence of Babesia spp., Ehrlichia canis, Hepatozoon spp., Anaplasma platys and Mycoplasma spp. in dogs in Thailand. Of the 181 dog blood samples tested by PCR, 78/181 (43.1%) were found to be infected with one or more pathogens. The overall prevalence rates of Mycoplasma spp., Hepatozoon spp., Babesia spp., A. platys and E. canis infections were 19.9%, 18.8%, 9.4%, 4.4% and 3.9%, respectively. To the authors' knowledge, this is the first report of Mycoplasma infection in Thailand in dogs. The current findings are important for future surveillance of CVBDs and designing appropriate approaches for diagnosis and control for the diseases in Thailand.     

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province,Korea

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.     

Tick-borne pathogens in Dermacentor reticulatus collected from dogs in eastern Poland

In recent years, the distribution of Dermacentor reticulatus ticks has expanded into new territories in many European countries, including Poland, with increased population densities in areas of their regular occurrence. The spread of D. reticulatus enhances the risk of exposure of domestic animals and their owners to tick-borne diseases. The objective of this study was to assess the prevalence of infection of D. reticulatus ticks feeding on dogs with the pathogens Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. The study material comprised 152 D. reticulatus ticks collected from dogs in the northeastern part of Lublin Province (eastern Poland). A ready-made AmpliSens® TBEV, B.burgdorferi sl, A.phagocytophilum, E.chaffeensis/E.muris-FRT PCR kit was used for qualitative detection and differentiation of tick-borne infections. The assessment of the degree of infection of the analyzed ticks with the two pathogens revealed that 9.2% (14/152) of the examined ticks were infected with one of the pathogens. No co-infections with the pathogens were detected in any of the ticks. The highest specific percentage of infections (8.6%, 13/152) was associated with A. phagocytophilum. The presence of B. burgdorferi s.l. was detected in only one of the examined ticks (0.7%). The spread of D. reticulatus to new territories and the increase in population density in areas of their regular occurrence implies the need for further studies of the prevalence of pathogens with medical and veterinary importance in order to assess the risk of tick-borne diseases.

     

Rickettsial agents in Egyptian ticks collected from domestic animals

To assess the presence of rickettsial pathogens in ticks from Egypt, we collected ticks from domestic and peridomestic animals between June 2002 and July 2003. DNA extracts from 1019 ticks were tested, using PCR and sequencing, for Anaplasma spp., Bartonella spp., Coxiella burnetii, Ehrlichia spp., and Rickettsia spp. Ticks included: 29 Argas persicus, 10 Hyalomma anatolicum anatolicum, 55 Hyalomma anatolicum excavatum, 174 Hyalomma dromedarii, 2 Hyalomma impeltatum, 3 Hyalomma marginatum rufipes, 55 unidentified nymphal Hyalomma, 625 Rhipicephalus (Boophilus) annulatus, 49 Rhipicephalus sanguineus, and 17 Rhipicephalus turanicus. Ticks were collected predominantly (>80%) from buffalo, cattle, and camels, with smaller numbers from chicken and rabbit sheds, sheep, foxes, a domestic dog, a hedgehog, and a black rat. We detected Anaplasma marginale, Coxiella burnetii, Rickettsia aeschlimannii, and four novel genotypes similar to: “Anaplasma platys,” Ehrlichia canis, Ehrlichia spp. reported from Asian ticks, and a Rickettsiales endosymbiont of Ixodes ricinus.     

Morphological,molecular and MALDI-TOF MS identification of ticks and tick-associated pathogens in Vietnam

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising and reliable tool for arthropod identification, including the identification of alcohol-preserved ticks based on extracted leg protein spectra. In this study, the legs of 361 ticks collected in Vietnam, including 251 Rhiphicephalus sanguineus s.l, 99 Rhipicephalus (Boophilus) microplus, two Amblyomma varanensis, seven Dermacentor auratus, one Dermacentor compactus and one Amblyomma sp. were submitted for MALDI-TOF MS analyses. Spectral analysis showed intra-species reproducibility and inter-species specificity and the spectra of 329 (91%) specimens were of excellent quality. The blind test of 310 spectra remaining after updating the database with 19 spectra revealed that all were correctly identified with log score values (LSV) ranging from 1.7 to 2.396 with a mean of 1.982 ± 0.142 and a median of 1.971. The DNA of several microorganisms including Anaplasma platys, Anaplasma phagocytophilum, Anaplasma marginale, Ehrlichia rustica, Babesia vogeli, Theileria sinensis, and Theileria orientalis were detected in 25 ticks. Co-infection by A. phagocytophilum and T. sinensis was found in one Rh. (B) microplus.     

Prevalence of Tick-Borne Pathogens in Ixodes ricinus and Dermacentor reticulatus Ticks from Different Geographical Locations in Belarus

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.     

Analysis of pathogen co-occurrence in host-seeking adult hard ticks from Serbia

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis. As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified (Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. concinna and I. ricinus) and analyzed for the presence of seven pathogens. Anaplasma ovis, A. phagocytophilum, Babesia canis, B. burgdorferi s.l., Coxiella burnetii, Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella-like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species (D. reticulatus, H. concinna and I. ricinus). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.     

Detection of Borrelia burgdorferi s.l., Anaplasma phagocytophilum and Babesia spp. in Dermacentor reticulatus ticks found within the city of Białystok,Poland—first data

Pathogens carried by ticks pose a threat to both human and animal health across the world. Typically associated with rural landscapes, ticks appear to adapt well to life in urban recreational areas. Although Dermacentor reticulatus is commonly found across Europe, data on the prevalence of pathogens in this tick species, in an urban environment, are very limited. PCR was used to examine 368 D. reticulatus individuals collected in the Zwierzyniecki Forest Nature Reserve in Bia?ystok, Poland. In total, 10.3% of ticks were infected, with Babesia spp. (9.2%), Anaplasma phagocytophilum (0.8%) and Borrelia burgdorferi sensu lato (0.3%). Rickettsia spp., Bartonella spp., and Coxiella burnetii were not detected. Sequence analysis for Babesia-positive samples identified 79.4% of them as Babesia canis, 8.8% as Babesia microti, 5.9% as Babesia spp., 2.9% as Babesia venatorum, and 2.9% as Babesia vogeli. Results obtained in this study indicate that D. reticulatus ticks found within the urban premises of the study area are infected with at least three pathogens and therefore are an important factor in public health risk for tick-borne diseases.