Predicting the emergence of tick-borne infections based on climatic changes in Korea

Granulocytic anaplasmosis (GA) and monocytic ehrlichiosis (ME) are maintained in wild rodent reservoirs and tick vectors in the Republic of Korea. This study investigated the prevalence of 2 tick-borne pathogens, Anaplasma phagocytophilum and Ehrlichia chaffeensis, in wild rodents and ticks in central Korea to identify any significant associations with existing or changing climatic conditions. Specifically, the goal of this study was to develop simple models for the probability of occurrence of an epidemic of GA or ME as a function of climate in an area in a given year. Climatic data from 2 regions, Munsan and Dongducheon, Gyeonggi, in central Korea (between the Demilitarized Zone and Seoul, latitude between 37 degrees N-38 degrees N and longitude between 127 degrees E-128 degrees E), were analyzed with respect to the prevalence of GA and ME in Paju, Yoncheon, Pocheon, and Dongducheon for the period from 2001 to 2005. Rates of A. phagocytophilum and E. chaffeensis decreased as the total yearly precipitation levels and daily humidity increased, and as the daily mean sunshine hours decreased. Rates of A. phagocytophilum and E. chaffeensis from rodent ticks and rodents increased in the fall season. Linear regression analyses evaluating the numbers of positive samples by sample type found that rodent ticks were 6.64 times more likely to be actively infected with A. phagocytophilum than grass ticks or rodents, though the likelihood of any samples testing positive for this pathogen decreased by 0.17 as the annual mean level of precipitation increased by 1 mm. For E. chaffeensis, rodents were 15.67 times more likely to be infected than ticks. Logistic regression analyses evaluating each sample separately found that the odds of infection with A. phagocytophilum were nearly 5 times greater for rodents than ticks. In these analyses, precipitation was one potential factor to account for the prevalence of tickborne diseases.     

查菲埃立克体和人单核细胞埃立克体病研究进展

埃立克体病是由埃立克体属微生物引起的一类人兽共患急性传染病,其特征为高热、头疼、贫血、白细胞减少、消瘦和黄疸等。查菲埃立克体是埃立克体属中代表性的蜱媒人畜共患病病原体,对人、畜均有较强致病性,在人体引起人单核细胞埃立克体病（HME）。本文较为全面地总结了查非埃立克体和HME的病原学、流行病学、临床表现、诊断和防治等方面的研究进展。     

Anaplasma phagocytophilum groEL gene heterogeneity in Ixodes ricinus larvae feeding on roe deer in Northeastern Italy

Anaplasma phagocytophilum is an emerging tick-borne pathogen with both veterinary and human health implications. The role of wildlife hosts for this pathogen are not well defined, even thought roe deer (Capreolus capreolus) has been suggested to contribute to the occurrence of this tick-borne diseases in Europe. Therefore the aim of the present study was to investigate the potential role of this ungulate species as a reservoir of human pathogenic strains of A. phagocytophilum in a tick-borne diseases endemic area in Northeastern Italy. Ixodes ricinus feeding on roe deer were collected and analyzed for the presence for A. phagocytophilum by a molecular approach targeting 16S rRNA and groEL genes. The mean prevalence of A. phagocytophilum recorded was 5.11%, highlighting the ability of roe deer to infect the I. ricinus larval stage. The results of further genetic characterization of the strains of A. phagocytophilum herein isolated, based on phylogenetic information contained in groEL gene sequences, showed substantial heterogeneity among sequences analyzed. Nevertheless, these findings suggest that the roe deer population of the Trentino region of Italy harbors strains of A. phagocytophilum of unknown pathogenicity for humans.     

Regional differences in the association between land cover and West Nile virus disease incidence in humans in the United States

West Nile virus (WNV) is generally considered to be an urban pathogen in the United States, but studies associating land cover and disease incidence, seroprevalence, or infection rate in humans, birds, domesticated and wild mammals, and mosquitoes report varying and sometimes contradictory results at an array of spatial extents. Human infection can provide insight about basic transmission activity; therefore, we analyzed data on the incidence of WNV disease in humans to obtain a comprehensive picture of how human disease and land cover type are associated across the United States. Human WNV disease incidence in Northeastern regions was positively associated with urban land covers, whereas incidence in the Western United States was positively associated with agricultural land covers. We suggest that these regional associations are explained by the geographic distributions of prominent WNV vectors: Culex pipiens complex (including Cx. pipiens and Cx. quinquefasciatus) in the Northeast and Cx. tarsalis in the Western United States.     

Tick-borne diseases in North Carolina: is "Rickettsia amblyommii" a possible cause of rickettsiosis reported as Rocky Mountain spotted fever?

Cases of Rocky Mountain spotted fever (RMSF) in North Carolina have escalated markedly since 2000. In 2005, we identified a county in the Piedmont region with high case numbers of RMSF. We collected ticks and examined them for bacterial pathogens using molecular methods to determine if a novel tick vector or spotted fever group rickettsiae (SFGR) might be emerging. Amblyomma americanum, the lone star tick, comprised 99.6% of 6,502 specimens collected in suburban landscapes. In contrast, Dermacentor variabilis, the American dog tick, a principal vector of Rickettsia rickettsii, comprised < 1% of the ticks collected. Eleven of 25 lone star tick pools tested were infected with "Rickettsia amblyommii," an informally named SFGR. Sera from patients from the same county who were presumptively diagnosed by local physicians with a tick-borne illness were tested by an indirect immunofluorescence antibody (IFA) assay to confirm clinical diagnoses. Three of six patients classified as probable RMSF cases demonstrated a fourfold or greater rise in IgG class antibody titers between paired acute and convalescent sera to "R. amblyommii" antigens, but not to R. rickettsii antigens. White-tailed deer, Odocoileus virginianus, are preferred hosts of lone star ticks. Blood samples collected from hunter-killed deer from the same county were tested by IFA test for antibodies to Ehrlichia chaffeensis and "R. amblyommii." Twenty-eight (87%) of 32 deer were positive for antibodies to E. chaffeensis, but only 1 (3%) of the deer exhibited antibodies to "R. amblyommii," suggesting that deer are not the source of "R. amblyommii" infection for lone star ticks. We propose that some cases of rickettsiosis reported as RMSF may have been caused by "R. amblyommii" transmitted through the bite of A. americanum.     

Identification of Ehrlichia chaffeensis,Anaplasma phagocytophilum,and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea

A total of 1,467 tick (1,463 of Haemaphysalis longicornis, three of Ixodes persulcatus and one of I. turdus) collected from nine provinces of Korea were examined by TaqMan real-time PCR for the presence of Ehrlichia and Anaplasma species. One set of primers and a probe were designed for detection of all of the Ehrlichia and Anaplasma species. Template DNAs (total 803) were prepared either from pools of larvae, nymphs, adult males and females, or from the salivary gland and midgut of adult ticks. Only DNAs positive in TaqMan PCR were examined for A. phagocytophilum with nested PCR and for E. chaffeensis with PCR. Four A. phagocytophilum 16S rRNA gene PCR products were sequenced for comparison with sequences previously reported. Amplification of a 16S rRNA gene fragment of Ehrlichia and Anaplasma species was observed in 364 tick DNAs (45.3% of the total). Of these 364 positive ticks, species-specific PCRs confirmed that 35 H. longicornis and one I. persulcatus were positive for A. phagocytophilum and one I. persulcatus was positive in E. chaffeensis. Except for one (AB-GGHL, GenBank accession number [GAN] AF470698), three of the four 16S rRNA gene fragment sequences of the A. phagocytophilum-positive samples were similar or identical to the sequences of variants of A. phagocytophilum deposited in GenBank. The 16S rRNA gene fragment sequence of AB-GGHL was similar to that of Anaplasma (Ehrlichia) bovis 16S rRNA (GAN U03775). The identities of the Anaplasmataceae genus and species DNA in the 327 ticks that could not be confirmed infected with either E. chaffeensis, A. phagocytophilum, or A. bovis are not known. This study is the first to demonstrate the presence of E. chaffeensis, A. phagocytophilum and A. bovis in Korean ticks.     

Detection of Anaplasma phagocytophilum from Wild Boars and Deer in Japan

This study was conducted to determine the prevalence and geographic distribution of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, in wild deer and boars in Japan. We analyzed the blood samples of the study animals using PCR-targeting the 16S rDNA followed by DNA sequencing. Wild deer infected with Anaplasma spp., including Anaplasma bovis and Anaplasma centrale were detected in the region from Hokkaido to Kyushu. The infection rates of A. phagocytophilum, A. bovis, and A. centrale in deer were 15.6, 21.9, and 37.5%, respectively, and the corresponding infection rates in wild boar were 3.6, 17.9, and 3.6%, respectively. However, p44/msp2 genes specific to A. phagocytophilum were not detected among the 16S rDNA-positive samples on PCR analysis. In addition, the 16S rDNA sequences of A. phagocytophilum were 100% similar to those detected previously in the deer in Japan and 98.6% similar to those of A. phagocytophilum detected in the United States and Europe, and from the tick, Ixodes ovatus and Ixodes persulcatus in Japan. These findings suggested that the A. phagocytophilum-related sequences detected from deer and wild boars in Japan were different from those of typical A. phagocytophilum strains.     

Spatial analysis of the distribution of Ehrlichia chaffeensis, causative agent of human monocytotropic ehrlichiosis, across a multi-state region

Ehrlichia chaffeensis, which causes human monocytotrophic ehrlichiosis (HME), is an important emerging tick-borne pathogen in the southeastern and southcentral United States. The endemnicity probability of E. chaffeensis and, by implication, locations with risk for HME, was predicted by using two modeling methods. This is first large-scale study to use geospatial analyses to estimate the distribution of E. chaffeensis, and it was conducted using data from a prototypic surveillance system that used white-tailed deer as natural sentinels. Analyses included the E. chaffeensis serostatus for 563 counties from 18 states. Both kriging and logistic regression models provided very reliable portrayals of E. chaffeensis occurrence and predicted that E. chaffeensis distribution had good concordance with human case data. The integration of a deer surveillance system with geospatial analyses was useful in developing HME risk maps that will be useful for identifying high-risk areas for public health interventions such as prevention and control efforts.     

Experimental and field studies on the suitability of raccoons (Procyon lotor) as hosts for tick-borne pathogens

We investigated the experimental susceptibility and natural exposure of raccoons (Procyon lotor) to five tick-borne pathogens of human and veterinary importance, Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum (ApVariant 1 and Ap-ha HGE-1 strains), and Borrelia lonestari. Infections were assessed by polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) testing, and/or culture isolation methods for at least 30 days postinoculation (DPI). Two E. chaffeensis-inoculated raccoons seroconverted and were transiently PCR positive. One raccoon was culture positive. Laboratory raised Amblyomma americanum nymphs fed on a third infected raccoon failed to become infected. Two A. phagocytophilum (HGE-1)-inoculated raccoons became PCR positive and seroconverted. Both remained positive for at least 74 DPI. In contrast, raccoons inoculated with A. phagocytophilum (Ap-Variant 1) were only transiently PCR positive and only seroconverted with low titers. No evidence of infection was observed for E. ewingii- and B. lonestari-inoculated raccoons. Only one E. canis-inoculated raccoon was PCR positive 3 DPI. Serologic testing of wild raccoons from five populations (3 infested with ticks) in Georgia and Florida showed antibodies reactive with E. chaffeensis in the 3 tick-infested populations (range of 30%-46%), E. canis in the same three populations (8%-23%), A. phagocytophilum in a single raccoon from Florida (12%), and Borrelia spp. in all 5 populations (8%-53%). All raccoons were PCR negative for tick-borne pathogens. These data suggest that raccoons are likely not important reservoirs of E. canis, E. ewingii, or B. lonestari. However, raccoons are experimentally susceptible and naturally exposed to E. chaffeensis, and these data support the previous finding that raccoons may be involved in the natural history of A. phagocytophilum.     

Evaluation of a prototype Ehrlichia chaffeensis surveillance system using white-tailed deer (Odocoileus virginianus) as natural sentinels

The natural history of Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, includes the lone star tick (LST, Amblyomma americanum) as a vector and white-tailed deer (WTD; Odocoileus virginianus) as both a natural reservoir of E. chaffeensis and a major host of LST. The goal of the current study was to implement and evaluate a prototype surveillance system to delineate the geographic distribution of E. chaffeensis using WTD as natural sentinels. To accomplish this goal, serologic testing using the indirect immunofluorescent antibody (IFA) test was performed on WTD serum samples, and to confirm serologic results, polymerase chain reaction (PCR) assays and culture isolation were conducted. Considerations relevant to the applicability of a surveillance system utilizing WTD were analyzed (e.g., age and gender relationships to serologic status, adequacy of sample sizes needed to distinguish between uninfected and infected populations, presence of LST, and ability to detect stability and spread of E. chaffeensis in WTD populations). Of 3275 WTD serologically tested, 549 (47%) from 17 of 18 states had antibodies reactive to E. chaffeensis (IFA titer > or = 1:128). No difference between age groups or gender was noted with serologic testing, thus these variables would not be a concern for a surveillance system using WTD. Significantly more deer in younger age groups (< or = 1.5 yr) were PCR and culture positive, and 46% of 122 seropositive WTD populations were confirmed positive by PCR or culture isolation. A significant association between LST infestation and E. chaffeensis seroreactivity was noted. Furthermore, the surveillance system was able to detect stability of E. chaffeensis within WTD populations and also spread to new populations, both of which were associated with LST status. These data clearly demonstrate that WTD are useful as natural sentinels for this emerging human pathogen, and establish a prototypical framework for a WTD surveillance system.     

Serosurveillance for Anaplasma phagocytophilum antibodies in white-tailed deer (Odocoileus virginianus) in Iowa, USA

Cases of human granulocytic anaplasmosis have increased in number and are being identified in new geographic areas since its discovery in 1994. White-tailed deer (WTD) become infected with the causative agent, Anaplasma phagocytophilum, and serve as natural sentinels for this organism. In order to determine if A. phagocytophilum is present in the state of Iowa, sera collected from 628 WTD in 2004 from 13 sites and from 282 WTD in 1999 from a single, common site were tested by enzyme-linked immunosorbent assay and Western immunoblotting. A seroprevalence of 9.1% was found among the 2004 samples, and there was no change in seropositivity rates from 1999 to 2004 at the single, common site. As A. phagocytophilum is another tick-borne human pathogen to be identified in the state of Iowa, this study has important implications for health care providers.     

Evidence of tick-borne organisms in mule deer (Odocoileus hemionus) from the western United States

Free-ranging mule deer (MD; Odocoileus hemionus) from Arizona and California were tested for evidence of infection with several tick-borne pathogens, including species of Ehrlichia, Anaplasma, Babesia, and Borrelia. Of 125 mule deer tested from Arizona, 29 (23%) and 11 (9%) had antibodies reactive to E. chaffeensis and A. phagocytophilum by indirect immunofluorescent antibody testing, respectively; none of the six MD tested from California were seropositive. Using a commercial competitive ELISA kit, antibodies reactive to Anaplasma spp. were detected in 19 (15%) MD from Arizona and four of six (67%) MD from California. Polymerase chain reaction (PCR) testing for tick-borne pathogens was conducted on blood samples from 29 MD from Arizona and 11 MD from California. Twenty-two of 29 (75.9%) MD from Arizona had PCR evidence of infection with at least one tick-borne pathogen. We detected an Anaplasma sp. in 19 of 29 (65.5%) MD and a Babesia sp. in 10 of 29 (34%) MD. Sequencing of these amplicons indicated that the Anaplasma sp. was the same that had previously been detected in MD from California and the Babesia sp. was similar to one previously detected in a reindeer (Rangifer tarandus tarandus) from California. All of the California MD had evidence of infection with a tick-borne pathogen. Two different species of Anaplasma spp. were detected in MD from California, eight of of 11 MD were infected with an Anaplasma sp., and three of 11 MD were infected with A. ovis. This is the first report of a mule deer naturally infected with A. ovis. Ten of 11 MD from California were infected with a Babesia-like organism previously associated with human disease, and a single MD was PCR positive for Borrelia coriaceae, which has been associated with epizootic bovine abortion. Together, these data suggest that MD in northern Arizona and eastern California are exposed to several pathogens of human and veterinary importance.     

Increasing incidence of Ehrlichia chaffeensis and Anaplasma phagocytophilum in the United States, 2000-2007

Ehrlichia chaffeensis causes human monocytic ehrlichiosis, and Anaplasma phagocytophilum causes human granulocytic anaplasmosis. These related tick-borne rickettsial organisms can cause severe and fatal illness. During 2000-2007, the reported incidence rate of E. chaffeensis increased from 0.80 to 3.0 cases/million persons/year. The case-fatality rate was 1.9%, and the hospitalization rate was 49%. During 2000-2007, the reported incidence of A. phagocytophilum increased from 1.4 to 3.0 cases/million persons/year. The case-fatality rate was 0.6%, and the hospitalization rate was 36%. Rates among female patients were lower than among male patients for ehrlichiosis (rate ratio = 0.68) and anaplasmosis (rate ratio = 0.70). Most (80%) ehrlichiosis and anaplasmosis cases met only a probable case definition, although, use of a polymerase chain reaction to confirm infections increased during 2000-2007. Heightened reporting of these diseases will likely continue with improving recognition, changing surveillance practices, and appropriate application of diagnostic assays.     

The root-vole Microtus oeconomus (Pallas, 1776): a new potential reservoir of Anaplasma phagocytophilum

Anaplasma phagocytophilum infection is a recently emerged tick-borne zoonosis. The bacterium's reservoirs likely comprise cervids, some ruminants, rodents, and perhaps other small and intermediate-size mammals; the main vector in Europe is the Ixodes ricinus tick. The Bia?owieza Primeval Forest is an ecosystem with a known prevalence of tick-borne pathogens. We studied the root-vole Microtus oeconomus to evaluate the natural infection of A. phagocytophilum. Intragranulocytic bacterial clusters (morulae) were not seen, but the A. phagocytophilum-specific nested polymerase chain reaction (PCR) product, targeting the rrs gene, was detected in two out of 30 rodent samples (GenBank accession nos. DQ361024 and DQ361025). Twenty-six root vole (86.6%) hosted ticks, mainly Dermacentor reticulatus larvae and nymphs. Only two rodents were parasitazed by I. ricinus single larvae. These data show the presence of natural infection of A. phagocytophilum among the root-vole M. oeconomus in the Bia?owieza Primeval Forest ecosystem.     

Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA

Rabbits have been shown to harbor a suite of zoonotic organisms, including a Babesia species, Borrelia burgdorferi, and Anaplasma phagocytophilum. In this study, we conducted a molecular survey for various tick-borne pathogens in three species of rabbits from Texas and Georgia. Of 18 black-tailed jackrabbits (Lepus californicus) tested from Texas, six (28%) were polymerase chain reaction (PCR) positive for Babesia, and nucleotide sequencing revealed two distinct species or strains. Two jackrabbits were infected with a Babesia species or strain (Babesia sp. A) that was nearly identical (99.9%) to a piroplasm previously detected in humans from Washington state, and the remaining four jackrabbits were infected with a Babesia species (Babesia sp. B) that was most similar (99.7%) to a Babesia species detected in cottontail rabbits from Massachusetts and humans from Kentucky and Missouri. Eleven (61%) black-tailed jackrabbits were positive for A. bovis, and one was positive for A. phagocytophilum. Two of four desert cottontails (Sylvilagus audubonii) from Texas were positive for the Babesia sp. B, and one desert cottontail each was positive for A. bovis and A. phagocytophilum. One of these desert cottontails was coinfected with the Babesia sp. B and A. phagocytophilum, and five jackrabbits were coinfected with Babesia species and A. bovis. Of 19 eastern cottontails (S. floridanus) from Georgia, only one (5.3%) was positive for A. phagocytophilum, and three (15.8%) were positive for A. bovis. No rabbits from Texas or Georgia were positive for Borrelia species. The only tick species detected on the Texas and Georgia rabbits was the rabbit tick, Haemaphysalis leporispalustris. These data extend the geographic and host range of these pathogens, and because both the Babesia species and A. phagocytophilum are potential zoonotic pathogens, it is important to be aware that these organisms are enzootic in parts of the southern United States.     

Ehrlichiosis and human anaplasmosis

Human ehrlichiosis and anaplasmosis are acute febrile tick-borne diseases caused by various species of the genera Ehrlichia and Anaplasma (Anaplasmataceae). To date, only cases of human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum (formerly human granulocytic Ehrlichia, Ehrlichia phagocytophila, and E. equi) have been diagnosed in Europe. HGA and Lyme borreliosis are closely related diseases that share vector and reservoirs. In addition to HGA, human monocytic ehrlichiosis caused by E. chaffeensis has been reported in North America, as well as cases of infection due to E. ewingii in immunocompromised hosts. Ehrlichia spp. and A. phagocytophilum have tropism for blood cells, especially leukocytes and platelets, causing a considerable decrease of both components in these patients. HGA should be suspected in tick-bitten patients or those who have visited an endemic area and show symptoms of flu-like fever, leukopenia and thrombocytopenia.     

The common shrew (Sorex araneus): a neglected host of tick-borne infections?

Although the importance of rodents as reservoirs for a number of tick-borne infections is well established, comparatively little is known about the potential role of shrews, despite them occupying similar habitats. To address this, blood and tick samples were collected from common shrews (Sorex araneus) and field voles (Microtus agrestis), a known reservoir of various tick-borne infections, from sites located within a plantation forest in northern England over a 2-year period. Of 647 blood samples collected from shrews, 121 (18.7%) showed evidence of infection with Anaplasma phagocytophilum and 196 (30.3%) with Babesia microti. By comparison, of 1505 blood samples from field voles, 96 (6.4%) were positive for A. phagocytophilum and 458 (30.4%) for Ba. microti. Both species were infested with the ticks Ixodes ricinus and Ixodes trianguliceps, although they had different burdens: on average, shrews carried almost six times as many I. trianguliceps larvae, more than twice as many I. ricinus larvae, and over twice as many nymphs (both tick species combined). The finding that the nymphs collected from shrews were almost exclusively I. trianguliceps highlights that this species is the key vector of these infections in this small mammal community. These findings suggest that common shrews are a reservoir of tick-borne infections and that the role of shrews in the ecology and epidemiology of tick-borne infections elsewhere needs to be comprehensively investigated.     

Ehrlichia species in Rhipicephalus sanguineus ticks in Cameroon

Ehrlichia and Anaplasma species are tick-transmitted obligately intracellular bacteria that commonly cause disease in dogs worldwide. In addition to causing disease in canines, Ehrlichia chaffeensis, Ehrlichia ewingii, and Anaplasma phagocytophilum are responsible for emerging and life-threatening human zoonoses in the United States. We previously reported a high prevalence of E. canis infection in Cameroonian dogs based on serologic and molecular evidence. This study was undertaken to determine the Ehrlichia species (E. canis, E. chaffeensis, E. ewingii) present in Rhipicephalus sanguineus ticks (n = 92) collected from those dogs (n = 51). Ehrlichial DNA was detected by real-time polymerase chain reaction (PCR) in 28 (30%) unengorged R. sanguineus ticks attached to dogs. E. canis, the causative agent of canine monocytic ehrlichiosis, was detected in 19 (21%) ticks from 15 dogs, E. ewingii was detected in six (6%) ticks from 6 dogs, and E. chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis, was detected in 4 (4%) ticks. Notably, 2 ticks were coinfected with E. chaffeensis and E. canis, one tick with E. canis and E. ewingii, and one tick with E. chaffeensis and E. ewingii. These findings further support our previous conclusion that multiple Ehrlichia species are present in Cameroon and identify R. sanguineus ticks primarily infected with E. canis, but suggest that they may be infected with and transmit other ehrlichial agents in Cameroon, potentially to humans.     

Molecular survey of tick-borne pathogens in Ixodid ticks collected from hunted wild animals in Tuscany,Italy

Objective: To determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. Methods: PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. Results: A total of 432 ticks were collected: 30(6.94%) Haemaphysalis punctata, 72(16.7%) Dermacentor marginatus and 330(76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58(75.32%) resulted infected and among them 14(18.18%) showed co-infections. In particular, 29(37.66%) pools were positive for Bartonella spp., 23(29.87%) for Anaplasma phagocytophilum, 16(20.78%) for Rickettsia spp., and 5(6.49%) for Borrelia burgdorferi s.l. All samples were negative for Coxiella burnetii. Conclusions: The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks.