Ehrlichial DNA Research Articles

Features of human monocytic ehrlichiosis laboratory diagnostics

Objective. Using the data obtained in Perm Region as an example, to identify the effectiveness of polymerase chain reaction (PCR) for the diagnosis of Human Monocytic Ehrlichiosis (HME) at different periods from the onset of the disease, and to determine the role of HME in the structure of infections transmitted by ixodic ticks using PCR and enzyme-linked immunosorbent assay (ELISA)
 Materials and methods. A thorough clinical and epidemiological examination of 583 patients with acute febrile diseases developed after the suction of ticks was carried out. To detect E. muris DNA, 1586 whole blood samples were examined by PCR at different periods from the onset of the disease. For the purpose of serological verification of HME, all patients were examined with ELISA for the presence of immunoglobulins M and G against E. chaffeensis.
 Results. In total, using the PCR method, ehrlichial DNA was detected in 76 (4.8 %) blood samples from 53 patients. Based on two research methods (ELISA and PCR) HME was diagnosed in 58 (9.9 %) persons, while in 50 (86.2 %) of them, the diagnosis was confirmed only by PCR. The timing of E. muris genomic material detection in the blood of patients varied from 1 to 58 days from the moment of the disease. The greatest effectiveness of PCR (up to 69.4 % of positive samples) was noted by us from the 1st to the 7th day of illness. HME was found in the form of monoinfection in 9 (15.5 %), mixed infection in 49 (84.5 %) persons. The following was revealed: HME+Ixodid tick-borne borreliosis (ITBB) in 35 (60.3 %), HME+ITBB+Human granulocytic anaplasmosis (HGA) in 6 (10.3 %), HME+ITBB+HGA+Tick-borne encephalitis (TBE) in 4 (6.9 %), HME+TBE in 2 (3.5 %), HME+TBE+ITBB in 2 (3.5 %).
 Conclusions. In the diagnosis of HME, PCR significantly increased the number (up to 86.2 %) of confirmed cases, and most often in the acute period of the disease (up to 69.4 15.3 % of positive samples in the first week of the disease). For laboratory verification of HME, it is advisable to combine ELISA with the PCR method, especially in case of negative results of serological studies.

Genetic characterization of a novel Ehrlichia chaffeensis genotype from an Amblyomma tenellum tick from South Texas, USA

Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis (HME), a disease that ranges in severity from mild to fatal infection. Ehrlichia chaffeensis is maintained in a zoonotic cycle involving white-tailed deer (Odocoileus virginianus) as the main vertebrate reservoir and lone star ticks (Amblyomma americanum) as its principal vector. Through complete genomic analysis from human ehrlichial isolates and DNA sequences obtained from deer and tick specimens, nine strains of E. chaffeensis have been characterized. Few studies have examined the genetic diversity of E. chaffeensis in ticks, and some of these investigations have identified that the genetic sequences coincide with the circulating strains reported so far. Here, we report the first evidence of E. chaffeensis DNA from an unfed Amblyomma tenellum (formerly Amblyomma imitator) collected in South Texas. We characterized the genetic variation of this E. chaffeensis genotype using conserved gene markers such as rRNA, dsb, and groEL. We also used gene targets useful to distinguish genotypes, such as the variable length PCR target gene (VLPT) and 120-kDa gene, encoding the tandem-repeat proteins TRP32 and TRP120, respectively. Our results suggest a novel E. chaffeensis genotype that exhibited greater variability than other genotypes of E. chaffeensis and highlights the role for A. tenellum as a potential vector of E. chaffeensis.

Molecular Identification of Ehrlichia canis in Rhipicephalus Sanguineus Ticks from Siirt Province

Abstract This study was performed on Ehrlichia canis positive ticks collected from dogs to perform sequencing of their 16S rRNA genetic section using the PCR method. The collection of ticks was performed from a total of 60 dogs in the Siirt province, Turkey. A total of 250 ticks were collected and morphologically investigated. All ticks were identified as Rhipicephalus sanguineus sensu lato (s.l). Ehrlichial DNA was detected by the PCR method performed on 38 (15.2 %) of the ticks. The E. canis strains obtained as a result of the sequence analysis were found to be 100% identical to the American Texas (MH620194), Indian (KX766395), and Egyptian (MG564254) strains. This study thereby has identified a zoonotic agent from the R. sanguineus ticks collected from the dogs in the Siirt province.

First detection of Ehrlichia minasensis in Hyalomma marginatum ticks collected from cattle in Corsica, France.

Ehrlichiosis are severe, feverish tick‐borne illnesses caused by specific species within the genus Ehrlichia (Anaplasmataceae family). Recent data suggest that ruminants in Corsica area reservoir for several Anaplasmataceae species. The purpose of our study was to determine whether Ehrlichia species could be detected in ticks collected in Corsican ruminants by using molecular methods. Ticks were collected in northern Corsica: (i) in May 2016 from sheep bred in one farm located in a 5000‐inhabitants village and (ii) from cattle in June and July 2016 in a slaughterhouse. There sheep and cattle whole skin was inspected and ticks were collected manually. A total of 647 ticks was collected in northern Corsica during this study: 556 (86%) belonged to the Rhipicephalus bursa species and 91 (14%) to Hyalomma marginatum. The 91 H. marginatum ticks were organized in 27 pools, of which one (3.7%) was found positive for the presence of E. minasensis; this pool consisted of six ticks collected from a cow bred and raised northwestern Corsica. Ehrlichial DNA was not detected in R. bursa ticks. The 16S rRNA and groEL gene sequences of Ehrlichia detected in the H. marginatum pool showed 100% (303/303 bp) and 99.8% (555/556) of nucleotide identity with E. minasensis, respectively. Phylogenetic analyses demonstrated the highest closeness with E. minasensis UFMG‐EV genotype than to any other E. canis strains. To our knowledge, this is the first report of E. minasensis outside of Brazil, Ethiopia and Canada. This identification of E. minasensis in H. marginatum merits to be further investigated and pleads for translational studies addressing the potential impact of vector‐borne diseases of human and veterinary impact through large‐scale research and surveillance programmes in Corsica.

Isolation and molecular detection of Ehrlichia species from ticks in western, central, and eastern Japan

Ehrlichiosis is a tick-borne bacterial disease caused by pathogens of the Ehrlichia genus. Although human ehrlichiosis has not been reported in Japan, Ehrlichia spp., which are closely related to Ehrlichia chaffeensis, were detected in several species of ixodid ticks. In this study, the presence of Ehrlichia spp. in ticks in Japan was studied by using isolation and molecular detection methods. In total, 1237 ticks were collected from vegetation in western, central, and eastern parts of Japan. The ticks were tested for detection of ehrlichial DNA with a nested polymerase chain reaction and/or isolation by inoculation of mice with the homogenate. Ehrlichial DNA was detected in 29 of these ticks. The ehrlichial DNAs, groEL and 16S rRNA genes, detected in Ixodes turdus showed a high similarity to those of E. chaffeensis with 94.7% and 99.2% identity, respectively. Ehrlichia sp. HF and Candidatus Neoehrlichia mikurensis were also detected in I. ovatus. Furthermore, Ehrlichia sp. HF was isolated from laboratory mice that were intraperitoneal inoculated with I. ovatus tick homogenate. Some ehrlichial agents detected in Ixodes ticks might be a previously unknown Ehrlichia species. In this study, Candidatus N. mikurensis was detected in I. ovatus ticks. Because I. ovatus is distributed widely and cases of its tick bite in humans are ubiquitously reported in Japan, there is a potential for ehrlichiosis to be endemic to Japan, necessitating intensive surveillance of this infectious disease.

A novel Ehrlichia genotype strain distinguished by the TRP36 gene naturally infects cattle in Brazil and causes clinical manifestations associated with ehrlichiosis

A novel Ehrlichia genotype most closely related to E. canis was reported in North American cattle in 2010, and a similar agent was subsequently identified in the hemolymph of Brazilian Rhipicephalus (Boophilus) microplus ticks and isolated in 2012. The purpose of this study was to determine whether this or other novel ehrlichial agents naturally infect Brazilian cattle. Using PCR targeting the genus-conserved dsb gene, DNA from this novel ehrlichial agent in Brazilian cattle was detected. Attempts to isolate the organism in vitro were performed using DH82 cells, but morulae and ehrlichial DNA could only be detected for approximately one month. In order to further molecularly characterize the organism, PCR was performed using primers specific for multiple E. canis genes (dsb, rrs, and trp36). Sequence obtained from the conserved rrs and dsb genes demonstrated that the organism was 99–100% identical to the novel Ehrlichia genotypes previously reported in North American cattle (rrs gene) and Brazilian ticks (rrs and dsb genes). However, analysis of the trp36 gene revealed substantial strain diversity between these Ehrlichia genotypes strains, including divergent tandem repeat sequences. In order to obtain preliminary information on the potential pathogenicity of this ehrlichial agent and clinical course of infection, a calf was experimentally infected. The calf showed clinical signs of ehrlichiosis, including fever, depression, lethargy, thrombocytopenia, and morulae were observed in peripheral blood monocytes. This study reports a previously unrecognized disease-causing Ehrlichia sp. in Brazilian cattle that is consistent with the genotype previously described in North America cattle and ticks from Brazil. Hence, it is likely that this is the organism previously identified as Ehrlichia bovis in Brazil in 1982. Furthermore, we have concluded that strains of these Ehrlichia genotypes can be molecularly distinguished by the trp36 gene, which has been widely utilized to define E. canis strain diversity.

Levamisole enhances global and differential leukocyte numbers in peripheral blood of dogs with ehrlichiosis

Ehrlichiosis is a disease caused by Ehrlichia, a genus of obligatory intracellular bacteria that parasitize leukocytes and provoke several hematological alterations. In this work, we investigated the effect of levamisole associated with doxycycline on peripheral leukocytes in dogs PCR-positive for ehrlichial DNA. Twenty-one Ehrlichia-infected dogs were randomly distributed in 2 groups: a control group (n = 10) and a levamisole-treated group (n = 11). Blood samples were collected from each animal for hematological and DNA analysis both before initiating the experiment and 5 days later. All dogs were treated with doxycycline (10 mg/kg body weight), cimetidine (5 mg/kg of body weight), and fluid therapy for 5 consecutive days. In addition to receiving antibiotic therapy, the levamisole-treated dogs received a single subcutaneous injection (1 mg/kg body weight) of levamisole on the first day of the experiment. The results show that levamisole associated with doxycycline significantly enhanced global leukocyte, lymphocyte, and monocyte numbers in the peripheral blood of dogs with ehrlichiosis. Those data suggest that the combination of doxycycline with levamisole may improve canine ehrlichiosis treatment through impairment of leukopenia, which is not controlled by antibiotic therapy alone.

Prevention of transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs treated with a combination of fipronil, amitraz and (S)-methoprene (CERTIFECT®)

The ability of CERTIFECT® (a combination of fipronil, amitraz and (S)-methoprene) to prevent transmission of Ehrlichia canis was studied in two groups of eight dogs. One group was treated with CERTIFECT while the other group remained untreated. All dogs were exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and again on day 28 post-treatment by releasing ticks into the kennels of the dogs to simulate the natural way of infestation. Animals were examined in situ for ticks on Days 9, 16 and 23 and any ticks present were counted and removed on Day 30. The efficacy of CERTIFECT against R. sanguineus was 100%, since no ticks were found on the treated dogs at any time. Clinical examinations (including monitoring body temperature and blood collections for PCR analysis and serology) were performed at intervals throughout the study until Day 56. Five out of 8 untreated control dogs (62.5%) became infected with E. canis, as demonstrated by detection of specific E. canis antibodies and the presence of E. canis DNA in blood samples by PCR. These dogs displayed fever and thrombocytopenia and were rescue-treated with doxycline. None of the 8 dogs treated with CERTIFECT became infected with E. canis, in comparison to the 5/8 control dogs, as confirmed by the lack of specific antibodies and absence of any ehrlichial DNA in blood samples by PCR. CERTIFECT prevented transmission of E. canis and effectively provided protection against monocytic ehrlichiose for at least 4 weeks post treatment.

Longitudinal quantification of Ehrlichia canis in experimental infection with comparison to natural infection

Ehrlichia canis is a major tick-borne bacterial pathogen of dogs. Quantitative real-time PCR was evaluated for the detection of E. canis in naturally (NI) and experimentally infected (EI) dogs. DNA was extracted from blood, spleen and conjunctival swabs of experimentally infected dogs pre- and post-infection (PI), and during doxycycline therapy, and from blood and conjunctivas of naturally infected dogs. The primers and probe were designed to amplify a 93 bp fragment of the single copy E. canis 16S rRNA gene with the TaqMan system. All EI dogs were positive for E. canis DNA by 7 d PI and developed clinical ehrlichiosis by 9–12 d PI. A rapid increase in ehrlichial DNA in EI dogs correlated with the appearance of severe clinical signs of disease. The mean spleen and blood DNA copies significantly increased by more than 10-folds from 7 d PI to 10 and 12 d PI ( p < 0.05). E. canis DNA was undetectable in the blood by day 9 post-treatment. Although the spleen was more frequently positive than blood (15/15 specimens vs. 13/15), no significant differences were found between the mean ehrlichial DNA copies in the spleen and blood on each day of examination. In 12 naturally infected dogs, the mean blood DNA copies was similar to the number found in EI 7 d PI, but significantly lower than the means of 10 and 12 d PI ( p < 0.0001). Although the conjunctivas of all EI dogs were positive by 12 d PI, only 3/5 (60%) NI dogs were positive also by conjunctival PCR. In conclusion, the kinetics of E. canis during acute experimental infection with complete pathogen clearance following doxycyline treatment was demonstrated for the first time by real-time PCR. The value of real-time PCR was shown in NI dogs as well as in EI dogs with spleen and blood sampling more sensitive than non-invasive conjunctival PCR.

EhrlichiaSpecies inRhipicephalus sanguineusTicks 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.

Study on seasonal activity in dogs and ehrlichial infection in Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) from southern Uruguay

ABSTRACT The seasonal activity on dogs of Rhipicephalus sanguineus in southern Uruguay has been studiedfrom samples collected in veterinary clinics of the country. From a total of 744 specimens, adults(92%) were active on dogs between August and March (peaking in October) being absent betweenApril and July. Immatures represent more than 80% of captures on dogs in summer. This pattern is wellcorrelated with the climate records in the zone. The search for ehrlichial DNA from tick-derivedextracts (36 pools from 180 ticks) was negative in all the cases. While R. sanguineus has beenincriminated as vector of these pathogens, additional studies are required to understand the role ofthis tick in the transmission of ehrlichial organisms. Key words : Rhipicephalus sanguineus , seasonal activity, dogs, absence of ehrlichial DNA,Uruguay. INTRODUCTIONAbout 74 species are currently recognized inthe tick genus Rhipicephalus Koch, 1844 1 . Rhipicephalus sanguineus is probably the mostwidely distributed tick in the World. It is foundcircumglobally approximately between thelatitudes 50oN and 42oS. Its preferences for dogsas main host have facilitated its worldwidedistribution

Serological survey of Ehrlichia and Anaplasma infection of feral raccoons ( Procyon lotor) in Kanagawa Prefecture, Japan

Numbers of feral raccoon; the possible reservoir animal of Ehrlichia and Anaplasma, are increasing in Japan. Thus serological methods were utilized to examine Ehrlichia and Anaplasma infection in raccoons from Kanagawa Prefecture, Japan. By using an indirect immunofluorescence assay, among 187 feral raccoons examined, 1 (0.5%) serologically reacted with Ehrlichia canis, 3 (1.6%) with Ehrlichia chaffeensis and 1 (0.5%) with Anaplasma phagocytophilum with the titers of 1:40 or more. Although screening PCR for Ehrlichia and Anaplasma species failed to detect the presence of ehrlichial DNA in serum samples, results of the serological tests suggested that the feral raccoons might be infected with some species of Ehrlichia and Anaplasma.

Evaluation of an Improved PCR Diagnostic Assay for Human Granulocytic Ehrlichiosis.

Background: Human granulocytic ehrlichiosis (HGE) is a tick-borne disease caused by an agent similar to Ehrlichia equi. The etiologic agent has only recently been cultivated, and clinical and laboratory findings are nonspecific. Severity and fatal outcome are associated with delays in diagnosis and therapy. Methods and Results: The sensitivity of the polymerase chain reaction (PCR) technique for HGE was enhanced at least 250-fold for identification of the 16S ribosomal RNA gene of the HGE agent in blood by shortening cycle segments, using 35 rounds of amplification, employing eLONGase polymerase, and by using SYBR Green I for detection of amplified products. From 17 patients with suspected HGE, acute-phase blood was tested by PCR and for E. equi antibodies in acute and convalescent sera. The HGE PCR detected ehrlichial DNA in 1 ng of genomic leukocyte DNA and in as little as 0.6 infected leukocyte per microliter of blood. Of the 17 patients with suspected HGE, 7 (41%) were serologically confirmed, and six of these seven had positive PCRs (86% sensitivity). Ten seronegative patients had negative PCRs, and no patient had a positive PCR and negative serology (100% specificity). Conclusions: The polymerase chain reaction for the HGE agent 16S rRNA gene is a sensitive and specific method for the early confirmation of HGE at a time when therapeutic decisions are required.

Detection of Ehrlichial DNA in Small Rodents Captured in a Woodland Area of Hokkaido, the Northernmost Island of Japan, Where Lyme Disease Is Endemic

Detection of Ehrlichial DNA in Small Rodents Captured in a Woodland Area of Hokkaido, the Northernmost Island of Japan, Where Lyme Disease Is Endemic

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.

Detection of ehrlichial DNA in Haemaphysalis ticks recovered from dogs in Japan that is closely related to a novel Ehrlichia sp. found in cattle ticks from Tibet, Thailand, and Africa.

Tick DNA samples from dogs in Japan were examined for Ehrlichia infection by 16S rRNA gene-based PCR and sequencing. Three positive samples were detected from Haemaphysalis ticks, and higher levels of similarity (98.46 to 99.06%) were found to recently detected Ehrlichia spp. from cattle ticks in Tibet, Thailand, and Africa.

Ehrlichiosis in anemic, thrombocytopenic, or tick-infested dogs from a hospital population in South Brazil

Ehrlichia canis has a worldwide distribution, but clinical manifestations may vary geographically. We selected 129 dogs to determine prevalence of ehrlichiosis in dogs with anemia, thrombocytopenia, or ticks presented to a Veterinary Teaching Hospital in South Brazil. Of the 129 dogs, 68 carried the brown dog tick ( Rhipicephalus sanguineus), 61 had thrombocytopenia (platelet count <150,000/μl), and 19 had anemia (PCV < 22%). Twenty dogs fulfilled more than one inclusion criteria. Ehrlichiosis was diagnosed by positive amplification of ehrlichial DNA by PCR using primers ECC and ECB that amplify a sequence of the 16S rRNA gene. Presence of E. canis was confirmed by cleavage of the amplified DNA using endonucleases HaeIII and AvaI. Fourteen of 68 (21%) dogs with ticks had ehrlichiosis, whereas 12 of 61 (20%) dogs presented with thrombocytopenia and 4 of 19 (21%) anemic dogs had ehrlichiosis. Similar results were obtained in dogs with thrombocytopenia and anemia (one of eight positive) and in dogs with thrombocytopenia and ticks (two of seven positive). All four dogs with anemia and ticks, and the dog that fulfilled all inclusion criteria yield no amplification of ehrlichial DNA by PCR. Based on our results, one in each five dogs infested by the brown dog tick, with anemia or thrombocytopenia had ehrlichosis. Contrary to widespread believe, ehrlichiosis was not the main cause for thrombocytopenia in our region.

Infection rates of Amblyomma americanum and Dermacentor variabilis by Ehrlichia chaffeensis and Ehrlichia ewingii in southwest Missouri.

Both Ehrlichia chaffeensis and Ehrlichia ewingii are causative agents of human ehrlichiosis. Both pathogens are transmitted to humans through the bite of an infected lone star tick (Amblyomma americanum). Since Missouri has a high incidence of human monocytic ehrlichiosis, we investigated the prevalence of E. chaffeensis- and E. ewingii-infected A. americanum and Dermacentor variabilis (American dog tick) ticks to help assess the relative risk for humans exposed to these vectors. We used a nested polymerase chain reaction assay for the detection of ehrlichial DNA in the collected ticks. Infection rates for both ehrlichial species were calculated from the assay results for each of the tick species. E. chaffeensis was found to be present in 9.8% of adult A. americanum ticks (57 of 579) and 6.7% of D. variabilis ticks (eight of 120). E. ewingii DNA was present at an infection rate of 5.4% in adult A. americanum (31 of 579) and 3.3% of D. variabilis ticks (four of 120). A minimum infection rate for nymph pools of A. americanum was 1.7% for E. chaffeensis and 0.6% for E. ewingii.

Serologic evidence of infection with granulocytic ehrlichiae in black bears in Pennsylvania.

Serum samples from 381 black bears (Ursus americanus) killed in Pennsylvania (USA) on 24 November 1997 were analyzed for antibodies reactive to the agent of human granulocytic ehrlichiosis (HGE; Ehrlichia sp.) by indirect immunofluorescence assay. Antibody reactivity to HGE antigen was detected in 21% (81/381) of the samples collected. Reactive samples were reported from 56% (14/25) of the counties where bear samples were collected. Endpoint antibody titer ranged from 1:8 to 1:16, 192, with a geometric mean titer of 1:582. There was no significant difference in antibody prevalence between male and female bears (P < 0.01). However, adult bears were significantly more likely to have reactive antibodies than juvenile bears (P < 0.01). Attempts to amplify and detect granulocytic ehrlichial DNA from corresponding bear blood clots (n = 181) through nested polymerase chain reaction assays were unsuccessful. Further studies are needed for identification of the pathogen-responsible for induction of HGE-reactive. This is the first description of antibodies reactive to the HGE agent in black bears and suggests these mammals are infected with the agent of HGE or an antigenically related ehrlichial species.

Granulocytic Ehrlichiae in Ixodes persulcatus ticks from an area in China where Lyme disease is endemic.

A total of 372 adult Ixodes persulcatus ticks were collected from vegetation in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be endemic. The ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with primers derived from the 16S rRNA gene. Of 310 ticks obtained from the Dahe forestry farm, two pools (each containing 5 ticks) were found positive, with a minimum infection rate of 0.6%. Ehrlichial DNA was also detected in one female (1.6%) of 62 ticks collected from the Yulin forestry farm. The overall minimum infection rate of the 372 I. persulcatus adults was 0.8%. The nucleotide sequences of 919-bp PCR products from the three positive tick specimens were identical to each other and very closely related to the members of the Ehrlichia phagocytophila genogroup. This is the first identification of granulocytic ehrlichiae in ticks in Asia and the first report of infection in I. persulcatus anywhere.