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Anaplasma phagocytophilum is an emerging tick-borne pathogen. Great genetic diversity of A. phagocytophilum has been described in animals and ticks. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. During 1996–2008, there were 66 serologically confirmed patients with human granulocytic anaplasmosis. Of these, 46 were tested with a screening PCR for a small part of the 16S rRNA gene of A. phagocytophilum and 28 (60.9%) were positive. Positive samples were additionally tested with a PCR targeting the groESL operon and a larger fragment of the 16S rRNA gene. All amplicons were further sequenced and analyzed. The homology search and the alignment of the groESL sequences showed only one genetic variant. Sequence analysis of the 16S rRNA gene revealed 100% identity among amplicons. Slovenia is a small country with diverse climate, vegetation, and animal representatives. In previous studies in deer, dogs, and ticks, great diversity of the groESL operon was found. In contrast, in wild boar and in human patients from this study, only one genetic variant was detected. The results suggest that only one genetic variant might be pathogenic for humans or is competent enough to replicate in humans. To support this theory, other genetic markers and further studies need to be performed.

Anaplasma phagocytophilum is a gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.

We report a patient with febrile illness and epidemiologic and clinical findings consistent with human granulocytic ehrlichiosis and tickborne encephalitis, in whom infection with Anaplasma phagocytophilum was demonstrated by polymerase chain reaction and seroconversion. Tickborne encephalitis virus infection was established by serum immunoglobulin (Ig) M and IgG antibodies.

In order to assess the validity of definitions for human granulocytic ehrlichiosis (HGE), epidemiological, clinical and laboratory findings for 13 patients with confirmed HGE and 21 patients fulfilling criteria for probable HGE were compared. The patients were identified during a prospective study on the aetiology of febrile illness occurring after a tick bite, performed in Slovenia from 1995 to 2002. Significant differences between the 2 groups were found for age of patients, duration of fever before the first examination, and several clinical and laboratory parameters including the occurrence of chills, myalgia, arthralgia, leukopenia, thrombocytopenia, abnormal liver function tests results, and elevated concentration of C-reactive protein. All the abnormalities were established more often in patients with confirmed HGE than in those with probable HGE. Patients with confirmed HGE exhibited higher geometric mean titre values and higher peak antibody titres to Anaplasma phagocytophilum that developed later in the course of their illness. The differences indicate that in some patients fulfilling criteria for probable HGE the signs and symptoms most probably are not the result of a recent infection with A. phagocytophilum.

Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at -70 degrees C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 x 10(6) neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.

Several reports indicate the presence of infection with Anaplasma phagocytophila in Europe. The first case with confirmed human granulocytic ehrlichiosis was reported from Slovenia in 1997. By 2002, 13 patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis was established by molecular identification of ehrlichial organisms and/or by seroconversion to A. phagocytophila. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States. The severity of the illness was mild to moderately severe. All patients recovered rapidly and without sequelae. HGE should be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Slovenia.

Objective: To establish epidemiological, clinical and laboratory differences between adult patients with proven acute human granulocytic ehrlichiosis (HGE) and patients with the initial phase of tick-borne encephalitis (TBE)., Methods: Epidemiological, clinical and laboratory findings were compared in nine patients with acute HGE (established by the presence of specific DNA sequences of Ehrlichia phagocytophila in whole blood by polymerase chain reaction and/or by seroconversion or at least a four-fold change of serum antibody titres to E. phagocytophila by indirect immunofluorescence assay) and 29 patients with the initial phase of TBE (demonstrated in ELISA tests by the presence of serum IgM antibodies to TBE virus)., Results: Findings were similar for the majority of the general parameters examined, which included sex, age, incubation period, duration of fever, and frequency of additional tick bites. However, a statistically significant difference was found for the duration of fever before the first visit (median 7 days in patients with acute HGE and 4 days in patients with the initial phase of TBE; p = 0.0169), and for the frequency of hospitalisation, which was higher for patients with the initial phase of TBE (29/29) than for those with acute HGE (5/9; p = 0.0017). Comparison of clinical signs and symptoms and laboratory findings in the two groups revealed significant differences: in patients with acute HGE there was a higher frequency of chills (4/9 versus 3/29, respectively; p = 0.0407), myalgia (8/9 versus 13/29, respectively; p = 0.0263), and arthralgia (7/9 versus 3/29, respectively; p = 0.0003), and elevated values of lactate dehydrogenase (7/9 versus 3/29, respectively; p = 0.0003) and concentration of C-reactive protein (9/9 versus 6/29, respectively; p = 0.0000)., Conclusions: In a patient with febrile illness occurring after a tick bite, a clinical report of chills, myalgia and arthralgia, and laboratory findings of elevated values for lactate dehydrogenase and concentration of C-reactive protein direct towards a diagnosis of acute HGE rather than the initial phase of TBE.

Objective: Human granulocytic ehrlichiosis (HGE) was recently recognized as an emerging tick-borne infection in Europe. The disease is caused by Anaplasma (previously Ehrlichia) phagocytophila. The first confirmed acute human disease caused by A. phagocytophila was reported from Slovenia in 1998. The tick Ixodes ricinus was identified as the likely vector for this pathogen of humans and animals in Europe. In order to assess the possibility that roe and red deer in Slovenia serve as potential reservoir hosts for A. phagocytophila, materials from both species were examined., Methods: Samples were obtained from 32 red deer (Cervus elaphus) and 56 roe deer (Capreolus capreolus). Polyvalent antibodies to the USG3 isolate of Anaplasma phagocytophila were detected by indirect immunofluorescence assay (IFA). DNA was extracted from spleen tissue. The 16S rRNA gene and a portion of the groESL heat shock operon were used for PCR detection and subsequent direct sequencing of amplified products., Results: Serological and PCR results indicated that high proportions of roe and red deer were infected with A. phagocytophila. Infection was confirmed in 74% of the animals by IFA and in 86% of animals by PCR. While similar prevalences by PCR were seen in the two species (approximately 86%), the prevalence of antibodies was much higher in roe deer (94% vs. 35% in red deer). Sequence analysis of a 1256-bp fragment of the groESL operon revealed genetic diversity among collected samples. Identity of sequences ranging from 98% to 100%. None of the A. phagocytophila groESL and 16S rRNA gene sequences from red or roe deer were identical to the sequences previously obtained from human patients with ehrlichiosis from Slovenia or elsewhere in the world. All red deer sequences clustered with those obtained from humans, whereas all but two sequences from roe deer clustered separately., Conclusions: The results of our study indicate that a high percentage of red deer and roe deer in Slovenia are infected with A. phagocytophila. Analysis of groESL and 16S rRNA gene sequences revealed two distinct genetic lineages. Among deer, one variant was primarily associated with roe deer. Although none of the sequences from red deer was identical to those found in humans, they were more closely related.

Herein we report on the first confirmed pediatric case of acute human granulocytic ehrlichiosis in Europe. Presentation in this 11-year-old girl was comparable to clinical findings seen in adult European patients with human granulocytic ehrlichiosis; i.e., she had self-limited febrile illness with leukopenia, thrombocytopenia, and elevated serum C-reactive protein concentration. It is of interest that the patient not only had a fourfold change in antibody titer to Ehrlichia phagocytophila but also developed antibodies to Ehrlichia chaffeensis and that her PCR test result was positive on the third as well as on the 22nd day after the onset of illness, that is, 16 days after spontaneous defervescence.

Background: Little is known about the seroprevalence of ehrlichiosis in adults and much less about the same in children., Methods: One hundred and forty-three healthy children and young adults (6-24 years of age, male to female ratio, 1:1) were assessed for the presence of antibodies to the agents of human granulocytic ehrlichiosis (HGE), human monocytic ehrlichiosis (HME), Borrelia burgdorferi sensu stricto (BB), and tick-borne encephalitis (TBE) virus in Slovenia, where tick-related infections are endemic. Antibodies to HGE and HME agents were assayed by indirect immunofluorescence, and antibodies to BB and TBE by enzyme-linked immunosorbent assay. A questionnaire about tick exposure was answered by all subjects. In the event of a positive result, a detailed interview was conducted., Results: Of 143 study subjects, 22 (15.4%) had detectable antibodies to HGE agent, 22 (15.4%) were positive to BB, 18 (12.6%) were positive to TBE virus (12 of these were vaccinated) and 4 (2.8%) were positive to the HME agent. The history of persons seropositive to an HGE agent had been uneventful., Conclusions: Our study documents a high seroprevalence of HGE in children and young adults in Slovenia, similar to the seroprevalence of LB and higher than that of TBE and HME. Although the majority of these infections are probably asymptomatic or mild, active surveillance for acute HGE infections in children in areas endemic for tick-related infections is necessary.

The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.

Objectives: The aim of our study was to establish clinical and laboratory differences between patients with acute human granulocytic ehrlichiosis (HGE) and patients with the initial phase of tick-borne encephalitis (TBE)., Methods: Clinical features and laboratory results of four patients with acute HGE (established by the presence of the specific DNA sequences of the HGE agent in whole blood by polymerase chain reaction and/or by seroconversion to the HGE agent by indirect immunofluorescence assay) and 12 patients with the initial phase of TBE (demonstrated by the presence of serum IgM antibodies to TBE virus) were compared. All these patients were uncovered at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia, during 1995-1996, in a prospective study on the aetiology of febrile illnesses occurring within 6 weeks after a tick bite., Results: Findings were similar for the majority of the examined parameters including severity of illness, level and duration of fever, presence of headache, leukopenia, thrombocytopenia, and liver - function test abnormalities. Statistically significant differences were found only for arthralgia (P=0.026) and elevated concentration of C-reactive protein (P=0.003); both variables were found more often in patients with acute HGE., Conclusions: In a patient residing in the central part of Slovenia, who reports a tick bite followed by a febrile illness with leukopenia and/or thrombocytopenia, the presence of arthralgias and/or an elevated C-reactive protein value directs toward the diagnosis of acute HGE and against the initial phase of TBE.

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

Ehrlichioses are tick-transmitted diseases associated with illnesses of animals for decades, but recently recognised to be emerging human diseases. In the last ten years increasing number of cases of human infections caused by Ehrlichia chaffeensis and granulo-cytic ehrlichia were described in the United States. Several reports also indicate the presence of infection with the human granulocytic ehrlichiosis (HGE) agent in Europe. The first confirmed acute human disease caused by HGE agent was reported from Slovenia. Until 1997, five patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis of acute HGE was established by seroconversion to the HGE agent and/or molecular identification of ehrlichial organisms. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only three patients received antibiotic therapy (of whom only two were treated with doxycycline). Many ehrlichiosis cases could go undetected due to a lack of physician awareness, lack of public knowledge, or limited investigation. HGE should now be also included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Febrile illnesses following a tick bite in patients from Slovenia were evaluated for an ehrlichial etiology. A case of acute human granulocytic ehrlichiosis (HGE) was confirmed by seroconversion to the HGE agent or molecular identification of ehrlichial organisms. Acute infection with the HGE agent was confirmed in four patients. None of the patients had detectable antibodies to the HGE agent at their first visit, but polymerase chain reaction analysis was positive for three patients. All four patients subsequently seroconverted to the HGE agent as shown by high titers of antibody. Clinical features and laboratory findings were similar to those in reports from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only two received antibiotic therapy (of whom only one was treated with doxycycline). HGE is an emerging tick-borne disease in the United States and should now be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Human granulocytic ehrlichiosis (HGE) was recently described in North America. It is caused by an Ehrlichia species closely related to Ehrlichia phagocytophila and Ehrlichia equi, recognized to infect mostly ruminants and horses, respectively. The vector in North America is the tick Ixodes scapularis, which is also the vector of the Lyme disease agent, Borrelia burgdorferi. Previous serologic studies in patients with a diagnosis of Lyme borreliosis indicate that HGE may exist in Europe. We report the first documented case of HGE in Europe. The diagnosis was established by seroconversion to E. equi and the HGE agent and by PCR with sequence analysis of the gene encoding the HGE agent 16S rRNA. Interestingly, the patient presented with a self-limited but moderately severe illness. Thus, European physicians need to be aware that HGE exists in Europe and that the diagnosis should be considered in febrile patients with tick bites in areas where Lyme disease is endemic.