Wild Boar Sera Research Articles

Epidemiological study on Japanese encephalitis virus distribution in Ishikawa prefecture, Japan, by serological investigation using wild boar sera.

Thirty-seven specimens of wild boar sera were collected from August 2016 to March 2018 in Ishikawa prefecture, Japan. Thirty-two specimens (86.5%) were positive for neutralizing antibodies against Japanese encephalitis virus (JEV). Eight specimens (21.6%) were positive for IgM antibodies against JEV. One sample was obtained from a wild boar captured in February during the winter season. Four other serum specimens obtained during the winter season were positive using a JEV gene-specific PCR assay. Based on IgM and PCR assays, wild boars were infected with JEV during the winter season, suggesting that the prevalence of JEV antibodies in wild boars in Ishikawa is high and JEV activity is possible during winter in this region. In addition, wild boars may play an important role in the infection cycle of JEV.

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs.

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV-2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV-3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV-3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV-3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV-3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV-3 infection. The high prevalence and theabsence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations.

Frequent infection of wild boar with atypical porcine pestivirus (APPV).

The recently identified atypical porcine pestivirus (APPV) was demonstrated to be the causative agent of the neurological disorder "congenital tremor" in newborn piglets. Despite its relevance and wide distribution in domestic pigs, so far nothing is known about the situation in wild boar, representing an important wild animal reservoir for the related classical swine fever virus. In this study, 456 wild boar serum samples obtained from northern Germany were investigated for the presence of APPV genomes and virus-specific antibodies. Results of real-time RT-PCR analyses revealed a genome detection rate of 19%. Subsequent genetic characterization of APPV (n=12) from different hunting areas demonstrated close genetic relationship and, with exception of APPV from one location, displayed less than 3.3% differences in the analysed partial NS3 encoding region. Furthermore, indirect Erns ELISA revealed an antibody detection rate of approx. 52%, being in line with the high number of viremic wild boar. Analysis of fifteen wild boar samples from the Republic of Serbia by Erns antibody ELISA provided evidence that APPV is also abundant in wild boar populations outside Germany. High number of genome and seropositive animals suggest that wild boar may serve as an important virus reservoir for APPV.

Antibody Prevalence to Influenza Type A in Wild Boars of Northern Ukraine

ObjectiveA preliminary serological survey was carried out to assess thelikelihood of Influenza A (IA) infection in wild boars and begin tocharacterize the role of wild boars in the epidemiology of the IA virus.IntroductionDomestic swine have been viewed as important for the adaptationand spillover of IA from birds into human populations as they aresensitive to both avian and mammalian (including human) influenzaviruses [1]. However, in much of Eurasia and North America wildswine are geographically widespread, abundant and often come inclose contact with humans in rural and agricultural settings. Untilrecently, little attention has been paid to this as an alternate routefor IA transmission to human and domestic populations and itssignificance is not clear.Therefore, the monitoring of the exposure of wild mammals toIA was viewed as essential as potential vectors impacting domesticanimals and public health.MethodsFrom September to December 2014, wild boar sera were collectedby professional hunters in 4 Oblasts of Ukraine: Volyn, Rivne,Zhytomyr, and Chernihiv. Blood was collected from jugular veins.Sera were collected in Eppendorf type tubes, separated from wholeblood without centrifugation and stored at -20C until serologicallytested. To detect antibodies to IA, a blocking ELISA was used.Serum samples were tested using commercial test kits “InfluenzaA Ab Test” (IDEXX, USA). Specific antibodies in wild boarserum samples were detected based on manufacturer’s instructions.Briefly, sera were diluted 1:10, and incubated in test wells for60 minutes at room temperature, followed by three washes. Anti-IAHorseradish Peroxidase HRP conjugate was then added and incubatedfor 30 minutes at room temperature. Following three washes,3',5,5’-tetramethylbenzidine (TMB), as a substrate, was addedand incubated for 15 minutes. Absorbencies were measured at 650A using a iMark Microplate Absorbance Reader and data wereanalyzed using Microsoft Excel. Based on the manufacturer’sinstructions, a serum sample was considered positive if the sample/negative control ratio (S/N) did not exceed a threshold of 0.60.Statistical analyses were performed with the program “StatisticsCalculator”.ResultsSera from 120 wild boars that were shot in 2014 were tested. Thirtyboars from each of 4 Oblasts were collected in the north central andnorthwestern regions of Ukraine. Antibodies against IAV weredetected using ELISA in 27 samples (22.5 %), (Table 1). Antibodiesto IA virus were detected in at least some of the wild boars from all ofthe 4 Oblasts. The highest percentages of seropositive samples weredetected in wild boar from Volyn and Zhytomyr Oblasts (Fig. 1).The prevalence differences were statistically significant only betweensamples from Volyn and Chernihiv Oblasts (P<0.05). The averageS/N value of all positive serum samples was 0.36±0.03.ConclusionsThis preliminary survey of IA antibodies in wild boar populationsof northern Ukraine indicates a substantial presence of exposure toIAV throughout the region.Infection of wild boar populations provides an alternative oradditional route for spillover from wild populations to domesticanimals and humans. This potential has received relatively littleattention until recently, likely in part because feral swine populationshave not been viewed as a serious challenge in most regions of theworld where the natural history of IA has received serious study.Table 1Seroprevalence of IA virus in wild boars in UkraineFigure 1Serological surveillance of wild boars for IA virus innorthern Ukraine

Prevalence of Anti-Hepatitis E Virus Antibodies and First Detection of Hepatitis E Virus in Wild Boar in Slovenia

Hepatitis E is an emerging zoonotic disease caused by hepatitis E virus (HEV). In this study, we investigated HEV presence in a wild boar (Sus scrofa) population of Slovenia. A total of 288 wild boar serum samples were collected throughout the country, and HEV infection was investigated by serology, using enzyme-linked immunosorbent assay (ELISA) and by HEV RNA detection using a real-time PCR assay. Antibodies against HEV were detected in 30.2% (87/288) of animals tested, whereas HEV RNA was detected in only one sample. This is the first evidence of HEV presence in the wild boar population in Slovenia, and these results suggest that these animals are part of the HEV epidemiological cycle in the country.

A picture of trends in Aujeszky's disease virus exposure in wild boar in the Swiss and European contexts.

BackgroundIn parallel to the increase of wild boar abundance in the past decades, an increase of exposure to the Aujeszky’s disease virus (ADV) has been reported in wild boar in several parts of Europe. Since high animal densities have been proposed to be one of the major factors influencing ADV seroprevalence in wild boar populations and wild boar abundance has increased in Switzerland, too, a re-evaluation of the ADV status was required in wild boar in Switzerland. We tested wild boar sera collected from 2008–2013 with a commercial ELISA for antibodies against ADV. To set our data in the European context, we reviewed scientific publications on ADV serosurveys in Europe for two time periods (1995–2007 and 2008–2014).ResultsSeven out of 1,228 wild boar sera were positive for antibodies against ADV, resulting in an estimated seroprevalence of 0.57 % (95 % confidence interval CI: 0.32–0.96 %). This is significantly lower than the prevalence of a previous survey in 2004–2005. The literature review revealed that high to very high ADV seroprevalences are reported from Mediterranean and Central-eastern countries. By contrast, an “island” of low to medium seroprevalences is observed in the centre of Europe with few isolated foci of high seroprevalences. We were unable to identify a general temporal trend of ADV seroprevalence at European scale.ConclusionsThe seroprevalence of ADV in wild boar in Switzerland belongs among the lowest documented in Europe. Considering the disparity of seroprevalences in wild boar in Europe, the fact that seroprevalences in Switzerland and other countries have decreased despite increasing wild boar densities and the knowledge that stress leads to the reactivation of latent ADV with subsequent excretion and transmission, we hypothesize that not only animal density but a range of factors leading to stress - such as management - might play a crucial role in the dynamics of ADV infections.

Results of Serological and Molecular Analysis of African and Classical Swine Fever in the Population of Wild Boars in Ukraine

Abstract The results of the study of lymphoid organs and sera of wild boars for the presence of DNA of African swine fever (ASF) virus and RNA of classical swine fever (CSF) virus are presented, as well as the results of a serological examination for the presence of ASF and CSF virus antibodies. The study was conducted in Ukraine between 2008 and 2013. Biological samples were obtained from wild boars shot during the hunting season, and were examined by real-time PCR and ELISA. In total, 5759 sera were tested for CSF virus antibodies and 4856 for ASF virus antibodies by ELISA. Samples of lymphoid organs totalling 1129 were examined by PCR for the detection of CSF virus RNA and 8102 such samples were examined for the detection of ASF virus DNA. CSF virus antibodies were detected in 6.56% of wild boar sera. RNA of CSF virus was also identified in 1 out of 1129 samples tested. ASF virus antibodies or DNA in lymphoid organ samples were not detected.

West Nile virus serosurveillance in pigs, wild boars, and roe deer in Serbia

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, but it also infects many other vertebrates, including humans and horses, in which it can induce severe neurological diseases; however, data about virus circulation in other mammals is scarce. WNV has a history of recent outbreaks in Europe, including Serbia, where it was identified for the first time in 2010 in mosquitoes and in 2012 in birds and humans, being responsible for over 300 confirmed human cases and 35 deaths there along 2013. To assess WNV circulation among mammals in the country, 688 samples obtained from 279 farm pigs, 318 wild boars, and 91 roe deer were investigated for the presence of antibodies to WNV by enzyme-linked immunosorbent assay (ELISA) and viral neutralization test (VNT), and the specificity of their reactivity was assayed against Usutu virus (USUV). ELISA-reactive sera were identified in 43 (15.4%) pigs, 56 (17.6%) wild boars, and 17 (18.7%) roe deer. Of these, 6 (14%), 33 (59%), and 4 (23.5%) respectively, neutralized WNV. One out of the 45 ELISA negative sera tested, from a roe deer, neutralized WNV. Cross-reactivity neutralization test indicated that all deer and pigs neutralizing sera were WNV specific, while in 5 (15.2%) of the wild boar samples the specificity could not be established. Four wild boar sera showed USUV specificity. All these data confirm the circulation of both flaviviruses in Serbia, and highlight the need for the implementation of global coordinated surveillance programs in the region.

Phylogenetic analysis of hepatitis E virus in domestic swine and wild boar in Germany

Hepatitis E virus (HEV) is an emerging non-enveloped positive strand RNA virus with worldwide distribution that can cause acute liver disease in humans. The virus has also been detected in both domestic and wild animals. In this study we investigated the presence of HEV in free-living wild boar as well as in domestic swine. A total of 105 domestic swine fecal samples and 124 wild boar sera were tested for the presence of HEV RNA by RT-PCR. A 241 nucleotide (nt) fragment from the capsid gene of HEV from one domestic swine and from 18 wild boars were amplified and sequenced. In addition, the complete capsid of three HEV sequences found in wild boar and the complete genomic sequence of the domestic swine HEV were obtained. Phylogenetic analyses based on both the 241 nt fragments as well as four complete capsid gene sequences demonstrated that all sequences belong to genotype HEV-3.

Prevalence of antibodies against tick-borne encephalitis virus in wild game from Saxony, Germany

Tick-borne encephalitis (TBE) is the most important tick-transmitted viral disease in Europe and is caused by the flavivirus TBE-virus (TBEV). In Germany TBE is unevenly distributed with the vast majority of cases occurring in the south in so-called risk areas defined as regions with an incidence of at least 1 case in 100,000 inhabitants. However, in low endemic areas with lesser TBE cases the respective risk assessment is hard to achieve. We therefore intend to use the prevalence of antibodies against TBEV in wildlife to trace TBEV endemic areas as a surrogate marker for the notification of human cases. This study was conducted in Saxony, Germany, where 34 autochthonous cases were reported since 2001, thereby not allowing a geographic allocation within the state. A total of 1,851 sera from wild boar and 35 sera from roe deer from all Saxon districts shot between April 2011 and March 2013 were screened for the presence of antibodies against TBEV. The overall seropositivity for Saxony was 10.5%. Among the wild boar sera, most positive samples could be found in the districts Meißen (23%) and Vogtlandkreis (20%) followed by Dresden (18%), Erzgebirgskreis and Görlitz (both 10%). We conclude that seroprevalence studies in game animals represent a promising surrogate marker and should be considered for future determination of risk areas. Although we are currently unable to explain the discrepancy of the few human cases and the high seroprevalence in some districts, vaccination against TBE should be considered for people planning outdoor activities in Dresden, Meißen or Vogtlandkreis.

High Prevalence of Hepatitis E Virus in Wild Boar (Sus scrofa) in Yamaguchi Prefecture, Japan

Hepatitis E virus (HEV) causes a food- and water-borne disease in humans, and Japanese wild boar (Sus scrofa leucomystax) meat is one of the most important sources of infection in Japan. We tested 113 serum samples from wild boar captured in Shimonoseki City, Yamaguchi Prefecture, Japan from 2010 to 2012. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) using virus-like particles as antigen and nested reverse-transcription PCR (RT-PCR). Anti-HEV IgG antibodies were detected in 47 of the 113 wild boar serum samples (42%), and HEV RNA was detected in five samples (4%). Sequence analysis showed that the five HEV isolates belonged to genotype 4, forming a cluster with a previous isolate from a human hepatitis E case in this region in 2011. These results indicate that wild boar in this region are infected with potentially pathogenic HEV at a high prevalence.

Seroepidemiological Survey of Aujeszky's Disease Virus in Wild Boar (Sus scrofa) and Raccoon Dogs (Nyctereutes procyonoides koreensis) in Korea

Aujeszkys disease caused by Aujeszkys disease virus (ADV) is one of the most important diseases in the pig industry. In this study, we conducted a seroepidemiological survey of ADV in wild boars and raccoon dogs in South Korea. In total, 217 wild boar sera collected between March and August 2013, and 96 raccoon dogs between 2011 and 2012 were screened for the presence of antibodies against ADV. The sero-positive rates in wild boars and raccoon dogs tested for ADV were found to be 3.55% (8/225) and 0% (0/96), respectively. The presence of virus neutralization antibody titer against ADV means that small number of wild boars was infected with ADV and AD may be circulated continuously in Korean wild boar populations, and that wild boars may act as a potential reservoir of ADV. Therefore, to achieve the declaration of AD free, effective preventive measures to block transmission of AD should be taken to the wild boars.

Detection of <i>Rickettsia tamurae</i> DNA in Ticks and Wild Boar (<i>Sus scrofa leucomystax</i>) Skins in Shimane Prefecture, Japan

We used 24 wild boars trapped from December 2009 to January 2010 and a further 65 from July 2010 to August 2010 in Misato Town, Shimane Prefecture, Japan. We collected blood, spleens, skins and ticks from the wild boars, which were examined for rickettsial infections using polymerase chain reaction (PCR) primers for the genes rickettsial 17-kDa antigen and citrate synthase (gltA). We amplified Rickettsia tamurae AT-1 DNA from the tick Amblyomma testudinarium and from wild boar skins where ticks attached. Antibodies against spotted fever group Rickettsia were detected in wild boar sera using immunofluorescence, whereas blood and spleen samples contained no rickettsial DNA. This study suggests that wild boars have a role as an amplifier and a transporter of A. testudinarium, which harbor R. tamurae. One case of R. tamurae infection in humans was reported in Shimane Prefecture. Therefore, R. tamurae infections in humans might increase, if wild boar populations and their habitats expand.

Pathogenesis and Treatment of Hepatitis E Virus Infection

Hepatitis E has been considered to be a travel-associated, acute, self-limiting liver disease that causes fulminant hepatic failure in specific high-risk groups only. However, hepatitis E virus (HEV) infection can also be acquired in industrialized countries-HEV genotype 3 infection is a zoonosis, with pigs and rodents serving as animal reservoirs. In recent years, cases of chronic HEV infection that were associated with progressive liver disease have been described in several cohorts of immunocompromised individuals, including recipients of organ transplants. The topic of hepatitis E is therefore re-emerging and has raised the following important questions: what is the risk for HEV infection in Western countries (eg, from eating uncooked meat)? How frequently does chronic hepatitis E develop among human immunodeficiency virus-infected patients and recipients of organ transplants? What are the treatment options? What is the current status of vaccine development? What do we know about the pathogenesis of HEV infection, and why does it have a more severe course in pregnant women? This review summarizes the current knowledge on the pathogenesis and treatment of HEV infection.

Farmed wild boars exposed to Toxoplasma gondii and Trichinella spp.

The meat of wild boar (Sus scrofa L.) can be a source of human infections with zoonotic parasites Toxoplasma gondii and Trichinella spp. We screened 197 wild boar sera collected at slaughter from 25 Finnish farms in 2007–2008 for serological evidence of infections with these parasites. Using a commercial direct agglutination test at a serum dilution of 1:40, T. gondii-specific IgG antibodies were detected in 65 (33.0%) samples, on 14 (56.0%) farms. Females, animals older than 24 months, animals of small herds, and animals originating from south-western parts of Finland were more often T. gondii-seropositive than were males, younger animals, animals of larger herds, and animals originating from the north and east, respectively. Four (2.0%) of the sera, originating from three (12.0%) farms, tested Trichinella-seropositive with an in-house ELISA and a conservative cut-off for seropositivity. One farm had both T. gondii- and Trichinella-seropositive animals. Taken together, an infection source had been present on 16 (64.0%) farms, and 69 (35.0%) of the 197 farmed wild boars intended for human consumption had specific serological evidence of exposure to a zoonotic parasite.

Effect of haemolysis and repeated freeze-thawing cycles on wild boar serum antibody testing by ELISA

BackgroundMonitoring wildlife diseases is needed to identify changes in disease occurrence. Wildlife blood samples are valuable for this purpose but are often gathered haemolysed. To maximise information, sera often go through repeated analysis and freeze-thaw cycles. Herein, we used samples of clean and haemolysed Eurasian wild boar (Sus scrofa) serum stored at -20°C and thawed up to five times to study the effects of both treatments on the outcome of a commercial ELISA test for the detection of antibodies against Suid Herpesvirus 1 (ADV).ResultsThe estimated prevalence of antibodies against ADV was 50-53% for clean and haemolysed sera. Hence, haemolysis did not reduce the mean observed serum antibody prevalence. However, 10 samples changed their classification after repeated freeze-thawing. This included 3 (15%) of the clean sera and 7 (41%) of the haemolysed sera.ConclusionsWe recommend (1) establishing more restrictive cut-off values when testing wildlife sera, (2) recording serum quality prior to sample banking, (3) recording the number of freezing-thawing cycles and (4) store sera in various aliquots to reduce repeated usage. For instance, sera with more than 3 freeze-thaw cycles and a haemolysis of over 3 on a scale of 4 should better be discarded for serum antibody monitoring. Even clean (almost not haemolysed) sera should not go through more than 5 freeze-thaw cycles.

A long-term serological survey on Aujeszky's disease virus infections in wild boar in East Germany

Between 1985 and 2008, a total of 102,387 wild boar sera originating from Eastern Germany covering an area of 108 589 km2 were tested for the presence of Aujeszky's disease virus (ADV)-specific antibodies. From 1985 until 1991 and from 1992 until 2008, wild boar sera were exclusively investigated using either conventional seroneutralization assays (n=39 621) or commercial gB and full antigen ELISAs (n=62,766), respectively. Spatial-temporal analysis revealed an increasing ADV seroprevalence from 0·4% to 15·9%, on average, during the 24-year observation period that went along with a continuous spread of the infection in a western direction. During 2006 and 2008, 18% of the 66 affected districts had ADV seroprevalences >30%. There was a significant correlation between ADV seroprevalence and the hunting index of population density (HIPD) of wild boar in the entire study area, although this did not hold true for some regions. Seroprevalences did not differ between sexes but were age-dependent. East Germany has been officially free of Aujeszky's disease (pseudorabies) in domestic pigs since 1985. Although a risk for domestic pigs cannot be completely ruled out, experience has shown that ADV in domestic pigs could be eliminated although the virus was present in the wild boar population. Despite increasing ADV seroprevalence in the East German wild boar population no spillover infections from wild boar to domestic pigs have been reported. To further trace ADV infections in the wild boar population in Germany, a nationwide serological monitoring programme should be implemented.

Serologic Tests for Detecting Antibodies against Mycobacterium Bovis and Mycobacterium Avium Subspecies Paratuberculosis in Eurasian Wild Boar (Sus Scrofa Scrofa)

New tools to detect exposure of free-range Eurasian wild boar (Sus scrofa scrofa) to pathogenic mycobacteria would be valuable for improved disease surveillance and wildlife management. Two hundred sera from wild boar of known Mycobacterium bovis infection status were used to evaluate test suitability for the detection of antibodies against M. bovis and Mycobacterium avium subsp. paratuberculosis (or cross-reacting members of the M. avium complex). Two traditional enzyme-linked immunosorbent assays were evaluated using M. bovis purified protein derivative (bPPD) and paratuberculosis protoplasmatic antigen 3 (PPA3) as antigens, respectively, and a new point-of-care test format for bovine tuberculosis (bTB) that uses the innovative dual-path platform (DPP TB) test. The effect of individual factors (sex, age, lesions) on the diagnostic performance of the serologic tests was also determined. Although the DPP had a sensitivity of 89.6% and a specificity of 90.4%, for bPPD, the sensitivity was 79.2% and the specificity 100%. Both tests had a kappa agreement of 0.80. Sixty-five of 68 (95.6%) wild boar sera with antibodies against the PPA3 antigen corresponded to known M. bovis-infected wild boar. Significant differences were not observed in the bPPD and DPP readings among lesion categories or between age classes. A slight sex-related difference in sensitivity toward males in the DPP was found, but it was not detected in the bPPD enzyme-linked immunosorbent assay. The results support the use of antibody-based diagnostic tests for both large-scale and individual bTB testing of Eurasian wild boar and suggest that wild boar cannot be used as sentinels for infections caused by M. avium complex members.

Development and validation of an enzyme-linked immunosorbent assay for antibodies against Mycobacterium bovisin european wild boar

BackgroundBovine tuberculosis (bTB) remains a significant problem in some parts of Spain largely because of contacts between cattle and wildlife reservoirs in extensive grazing systems. European Wild boar (Sus scrofa) is one of the species involved in the transmission of the disease to other species. Fast and simple detection methods would be critical for assessing infection prevalence, study the mechanisms of pathogen transmission and monitoring the effects of TB control measures.ResultsAn enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Mycobacterium bovis in wild boar serum was developed and validated on 185 sera from TB positive and negative wild boar. Based on antigen inoculation of captive animals as well as tuberculosis compatible lesions, culture results and molecular analysis of hunted individuals, animals were allocated into two groups: tuberculosis positive group and tuberculosis negative group. After optimization of the positive to negative ratio using different combinations of serum dilutions and conjugate concentrations, the test yielded a sensitivity of 72.60% and a specificity of 96.43% for the best cut-off.ConclusionAlthough some negative group animals showed an ELISA positive reaction (< 3%), this assay showed a high potential for accurate diagnosis of TB in wild boar, as its large dynamic range supported a good discriminatory power and a satisfactory balance between sensitivity and specificity.

Segregation of genetic variants of Anaplasma phagocytophilum circulating among wild ruminants within a Bohemian forest (Czech Republic)

Seroprevalence, infection rate, and genetic variants of Anaplasma phagocytophilum were studied in a synusia of large mammals (deer, moufflon, and wild boar) which occupied a natural, largely broad-leaved forest in Central Bohemia. Almost 250 animals were harvested during 1999–2002. Their serum and/or full blood as well as spleen tissues were sampled. The sera were tested for the presence of anti- A. phagocytophilum antibodies with IFA, and the blood and spleen DNA extracts were examined with nested PCR. Selected positive samples were sequenced in both directions using BigDye chemistry. Whenever a mixture was encountered, the PCR product was cloned, and several randomly selected clones were individually sequenced, to make the mixture's constituents clear. The established sequences were compared with each other, among host species, and also with the GenBank database. All 154 ruminant sera (moufflon, red deer, roe deer, fallow deer, and sika deer) showed reactive antibodies to A. phagocytophilum, with titres ranging from 1:320 to 1:10,240. By contrast, only 8 out of 34 (24%) wild boar sera showed elevated titres, from 1:80 to 1:1280. A. phagocytophilum DNA was detected in the blood samples ( n=66) of all ruminant species. The highest prevalence was shown in red deer (18/21) and the lowest in moufflon (1/28). The difference is statistically significant ( p=0.01). Two samples of wild boar blood tested negative, but one spleen sample was positive. The paired blood/spleen samples ( n=17) showed 71% correspondence in the PCR tests. Eleven extracts of DNA of A. phagocytophilum (6 red deer, 4 roe deer, 1 moufflon) were analysed in detail, and 4 genetic variants were distinguished based on partial 16S rRNA sequence. All red deer showed dual infections with a variant already reported from this host (DV), accompanied by a variant which was undistinguishable from that associated with horse, dog, and human infections in Europe (HV). All the roe deer hosted a variant peculiar to that species (RV); this variant was either found singly or mixed with HV. The unique moufflon isolate corresponded to a variant reported from sheep in Europe (SV). Any genetic variant, with the exception of HV, was only detected within a single ruminant species. Fisher's exact test proved that the association of RV and DV with the respective roe and red deer is statistically significant ( p=0.005). This study indicates that while circulating in nature, the variants of A. phagocytophilum in wild ruminants tend to be segregated from one another by species barriers despite the close relationships of their hosts, their sympatric ranges as well as a shared population of vector ticks.