Analysis of genetic identity of North American Anaplasma phagocytophilum strains by pulsed-field gel electrophoresis.

Biological and geographic heterogeneity of anthropozoonosis caused by Anaplasma phagocytophilum is poorly understood. Seven North American A. phagocytophilum strains were compared by PFGE. The average genome size was 1.58 Mbp, and restriction patterns were identical. New World strains of A. phagocytophilum have a large genome and a high degree of genetic uniformity.

Serial measurements of hematologic counts during the active phase of human granulocytic ehrlichiosis.

To describe the changes that occur in blood count parameters during the natural course of human granulocytic ehrlichiosis, we designed a retrospective cross-sectional case study of 144 patients with human granulocytic ehrlichiosis and matched controls who had a different acute febrile illness. Patients from New York State and the upper Midwest were evaluated from June 1990 through December 1998. Routine complete blood counts and manual differential leukocyte counts of peripheral blood were performed on blood samples that were collected during the active illness, and values were recorded until the day of treatment with an active antibiotic drug. Thrombocytopenia was observed more frequently than was leukopenia, and the risk of having ehrlichiosis varied inversely with the granulocyte count and the platelet count. Patients with ehrlichiosis displayed relative and absolute lymphopenia and had a significant increase in band neutrophil counts during the first week of illness. Knowledge of characteristic complete blood count patterns that occur during active ehrlichiosis may help clinicians to identify patients who should be evaluated specifically for ehrlichiosis and who should receive empiric antibiotic treatment with doxycycline.

Human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis is a recently recognized tick-borne infectious disease, and to date >600 patients have been identified in the United States and Europe. Most patients have presented with a non-specific febrile illness occurring within 4 weeks after tick exposure or tick bite. The risk for serious illness or death increases with advancing age and delayed onset of therapy. Routine laboratory testing may reveal reduced white blood cell and platelet concentrations and mildly elevated hepatic transaminase activity in peripheral blood. A high index of suspicion is necessary to arrive at a timely clinical diagnosis. Patients suspected of having human granulocytic ehrlichiosis (HGE) should be treated with a tetracycline-class antibiotic while awaiting the outcome of confirmatory laboratory testing.

ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats.

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.

Improved sensitivity of PCR for diagnosis of human granulocytic ehrlichiosis using epank1 genes of Ehrlichia phagocytophila-group ehrlichiae.

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi probably comprise variants of a single Ehrlichia species now called the Ehrlichia phagocytophila genogroup. These variants share a unique 153-kDa protein antigen with ankyrin repeat motifs encoded by the epank1 gene. The epank1 gene was investigated as an improved target for PCR diagnosis of HGE compared with the currently used 16S rRNA gene target. Primers for epank1 flanking a region that spans part of the 5' ankyrin repeat coding region and part of the unique 3' region were synthesized. Blood samples from 31 patients with suspected HGE who were previously tested by 16S rRNA gene (16S) PCR and indirect immunofluorescent antibody test (IFA) were retrospectively tested with the epank1 primers. Eleven patients were 16S PCR positive and had a seroconversion detected by IFA (group A), 10 patients were 16S PCR negative but had a seroconversion detected by IFA (group B), and 10 patients were 16S PCR negative and seronegative (group C). Ten of the 11 group A patients were epank1 PCR positive, all 10 of the group B patients were epank1 PCR positive, and all of the PCR-negative and seronegative patients (group C) were epank1 PCR negative. The epank1 primers are more sensitive than the previously used 16S rRNA gene primers and therefore may be more useful in diagnostic testing for HGE.

Serum cytokine responses during acute human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group. The disease ranges from subclinical to fatal. We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), gamma interferon (IFN-gamma), IL-10, and IL-4 concentrations were studied. IFN-gamma (1,035 +/- 235 pg/ml [mean +/- standard error of the mean]) and IL-10 (118 +/- 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P

Inter- and intralaboratory comparison of Ehrlichia equi and human granulocytic ehrlichiosis (HGE) agent strains for serodiagnosis of HGE by the immunofluorescent-antibody test.

Human granulocytic ehrlichiosis (HGE) is usually diagnosed by immunofluorescent antibody (IFA) serology with Ehrlichia equi-infected neutrophils or HGE agent-infected cultured HL60 cells. The HGE agent and E. equi are antigenically diverse, and interpretation of serologic results is also often variable. Thus, we investigated the sensitivity and specificity of various HGE agent and E. equi antigens used for IFA diagnosis by three different laboratories. Serum samples from 28 patients with well-characterized HGE and 9 patients with suspected HGE who were investigated by PCR, blood smear examinations, and serology were used, along with 9 serum samples from patients with other rickettsial and ehrlichial infections. Each serum sample was tested with up to 10 different antigen preparations. Overall, qualitative IFA results agreed in 70% of the samples. Titers among antigens were similar (r = 0.89 to 0. 96), but titers of individual samples varied by fourfold or more in 5 of 81 (6%) of the serum samples. Sensitivity ranged from 100% to 82%, and specificity varied from 100% to 67%, but these differences were not significant, even among those tested in the same laboratory or between two different laboratories. Antibodies were detected in 14 to 44% of acute-phase sera from confirmed HGE patients. Most false-positive reactions resulted with Ehrlichia chaffeensis; when these sera were excluded, the specificity of most antigens was 91 to 100%. These data indicate that IFA results often agree and that IFA is useful for diagnosis of HGE in convalescence. However, without further standardization, variability among serologic tests using E. equi and HGE agent isolates for diagnosis of HGE will occasionally provide discrepant results and confound diagnosis.

Seroprevalence of human granulocytic ehrlichiosis among permanent residents of northwestern Wisconsin.

Four-hundred seventy-five permanent residents of Wisconsin were tested for antibodies to the agent of human granulocytic ehrlichiosis (HGE) by indirect immunofluorescent antibody (IFA) testing with Ehrlichia equi as antigen marker. Each resident completed a standard survey questionnaire about outdoor activities, animal and tick exposure, and any febrile illness during the preceding 12 months. Seventy-one serum samples (14.9%) contained E. equi antibodies. The mean IFA titer for seropositive residents was 250 (range, 80-10,240). Seropositive residents were older than seronegative ones (62 vs. 56 years; P = .019). None of the seropositive residents had a history suggestive of ehrlichiosis. There was no association between the IFA test outcome and specific demographic variables or history of tick bites. HGE appears to be a common subclinical or mild infection among residents in northwestern Wisconsin.

Serologic evidence of a natural infection of white-tailed deer with the agent of human granulocytic ehrlichiosis in Wisconsin and Maryland.

White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.

The discovery of human granulocytotropic ehrlichiosis.

Human granulocytotropic ehrlichiosis (HGE) is a tick-borne, acute, nonspecific febrile illness that was first described in 1994. At this writing more than 300 cases have been recognized in areas where the presumed tick vector Ixodes scapularis occurs endemically. Ehrlichiosis is a nonspecific influenza-like illness, and associated laboratory alterations are variable. Characteristic morulae (clusters of bacteria in leukocyte cytoplasm) can frequently be found in the cytoplasm of circulating neutrophils after careful inspection of the peripheral blood smear. The diagnosis is confirmed by demonstrating seroconversion to the HGE agent, positive polymerase chain reaction, or growth of the HGE agent in tissue culture. Doxycycline provides rapid and effective treatment.

Major antigenic proteins of the agent of human granulocytic ehrlichiosis are encoded by members of a multigene family.

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.

Human ehrlichioses: newly recognized infections transmitted by ticks.

Human ehrlichioses are tick-borne infections caused by bacteria in the genus Ehrlichia. Human monocytic ehrlichiosis is caused by Ehrlichia chaffeensis and human granulocytic ehrlichiosis is caused by an agent similar to Ehrlichia equi. E. chaffeensis infects mononuclear phagocytes and is transmitted by Lone Star ticks (Amblyomma americanum) found in the south central and eastern United States. The agent of human granulocytic ehrlichiosis infects mostly neutrophils, it transmitted by Ixodes species ticks, and occurs mostly in the upper midwest and northeast United States. Despite the undifferentiated presentation of both ehrlichioses with fever, headache, myalgias, leukopenia, thrombocytopenia, and elevated liver enzyme activities, the diagnostic methods are distinct. Occasional severe complications include meningoencephalitis, adult respiratory distress syndrome, shock, and opportunistic infections. Immunocompromised patients are at high risk for death. An adverse outcome is associated with delayed diagnosis and therapy; thus, empirical treatment is advocated. Treatment with doxycycline usually results in prompt defervescence and cure.

Antigenic diversity of granulocytic Ehrlichia isolates from humans in Wisconsin and New York and a horse in California.

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi are very similar. HGE is of variable severity. Genetic and antigenic differences among 3 human isolates (Webster, Spooner, and NY-8) and 1 horse isolate (MRK) were evaluated. The 16S rRNA gene sequences were identical in all human isolates. By use of 5 homologous antisera from these 3 humans and 1 horse and an additional 5 antisera in heterologous reactions, the immunodominant antigens of each isolate were noted to differ in molecular size: 43 kDa in the Webster (Wisconsin) isolate, 46 kDa in the Spooner (Wisconsin) isolate, 42 and 45 kDa in the NY-8 (New York State) isolate, and a 42 kDa doublet in the E. equi MRK isolate from California. Two sera from a Wisconsin patient reacted weakly or not at all with the NY-8 isolate. Antigenic structural diversity exists among otherwise indistinguishable granulocytic ehrlichial isolates.

Serological evidence of human granulocytic ehrlichiosis in Norway.

Human granulocytic ehriichoisis was first described in 1994. This tick-transmitted illness is increasingly recognized in the USA as well as in Europe in areas where ixodes ticks and Lyme borreliosis are endemic. Blood samples from 58 Norwegian patients with physician-diagnosed Lyme borreliosis were examined for the presence of antibodies to Ehrlichia equi, a surrogate marker of the agent of human granulocytic ehrlichiosis. The results indicated that 10.2% of the patients may have been co-infected with human granulocytic ehrlichiosis and Lyme borreliosis. Human granulocytic ehrlichiosis appears to be established in southern Norway.

Human granulocytic ehrlichiosis in Wisconsin and Minnesota: a frequent infection with the potential for persistence.

Human granulocytic ehrlichiosis (HGE) is a tickborne illness caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila. The clinical presentation is nonspecific, and diagnosis is made infrequently. Sixty-six sera were obtained from 54 patients in Minnesota and Wisconsin with undifferentiated fever during the summer and fall of 1993. Serologic reactivity with E. equi was observed in 6 (11%), including 4 seroconversions, 1 stable titer, and 1 seroreversion. Of the seropositive patients, HGE agent DNA was detected by polymerase chain reaction in the first serum from 4 of 5 patients and was present in the serum of 1 of 2 untreated patients after 21 days, even when E. equi antibodies also were present. HGE is a significant and potentially frequent cause of undifferentiated fever in Wisconsin and Minnesota during seasons with tick activity. The agent may persist in untreated patients for at least 1 months or may be cleared earlier, even if not treated with doxycycline or tetracycline.