Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool

The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.     

Characterization of the major antigenic protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and its application for serodiagnosis of ehrlichiosis

Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.     

Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and -negative human sera by using native and recombinant E. chaffeensis and E. canis antigens

Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related to E. canis. The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E. chaffeensis and E. canis, respectively. Western immunoblotting was performed to analyze the antibody responses of the 37 E. chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E. chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E. canis antigens. The 28-kDa E. chaffeensis and 30-kDa E. canis native proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed an E. ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.     

Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis

Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.     

Western Blot Analysis of Sera Reactive to Human Monocytic Ehrlichiosis and Human Granulocytic Ehrlichiosis Agents

Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeensis and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with E. chaffeensis, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.     

Potential value of major antigenic protein 2 for serological diagnosis of heartwater and related ehrlichial infections

Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae.     

Molecular cloning and characterization of the 120-kilodalton protein gene of Ehrlichia canis and application of the recombinant 120-kilodalton protein for serodiagnosis of canine ehrlichiosis

The 120-kDa outer membrane protein (p120) is a potential adhesin of Ehrlichia chaffeensis, and recombinant p120 is very useful for serodiagnosis of human monocytotropic ehrlichiosis. The analogous gene of p120 in Ehrlichia canis was cloned, sequenced, and expressed. Like the E. chaffeensis p120, the E. canis p120 contains tandem repeat units. However, neither the repeat number nor the amino acid sequences in the repeats are identical in the two Ehrlichia species. The repeat units are hydrophilic and by probability analysis are predicted to be surface exposed in both species. The repeat regions of the p120s of the two species have common amino acid sequences that are predicted to be surface exposed. The overall amino acid sequence of the E. canis p120 is 30% homologous to that of E. chaffeensis p120. Protein immunoblotting demonstrated that the recombinant E. canis p120 reacted with convalescent sera from dogs with canine ehrlichiosis. These results indicate that the recombinant p120 is a potential antigen for the serodiagnosis of canine ehrlichiosis.     

Serologic diagnosis of human monocytic ehrlichiosis by immunoblot analysis.

Human monocytic ehrlichiosis is caused by Ehrlichia chaffeensis, an intracellular bacterium probably transmitted by the tick Amblyomma americanum in the United States. Despite its lack of specificity in discriminating among infections by closely related Ehrlichia spp., immunofluorescence assay (IFA) is the most frequently used serological diagnostic method. To improve the specificity of the serological diagnosis, we compared antigenic profile of E. canis and E. chaffeensis antigen with homologous and heterologous sera, searching for the specificity of the presence of low-molecular-weight proteins. Western immunoblot analysis of IFA-positive human sera revealed 27- and 29-kDa proteins which are not found in E. canis IFA-positive sera from dogs. IFA-positive sera from dogs revealed a low-molecular-weight group of proteins (20 to 28 kDa) which were not found in human E. chaffeensis-positive sera except for a weak band at 22 kDa. The presence o antibodies directed against the 27- and 29-kDa proteins on Western blots is specific for E. chaffeensis infection, and we suggest that the Western blot might complete IFA in cases with low positive predictive value.     

Comparison of serological detection methods for diagnosis of Ehrlichia canis infections in dogs

We determined the value of four serological assays for the diagnosis of canine monocytic ehrlichiosis by comparing them to the indirect fluorescent-antibody assay "gold standard." The specificity of Dip-S-Ticks was significantly lower than that of all of the other tests evaluated. The sensitivity of Dip-S-Ticks was significantly higher than that of Snap3Dx or the Snap Canine Combo. The sensitivity of the rMAP2 enzyme-linked immunosorbent assay (ELISA) was significantly higher than that of the Snap Canine Combo. The accuracy levels of the rMAP2 ELISA, Snap3Dx, Dip-S-Ticks, and Snap Canine Combo were 97.0, 89.8, 85.1, and 82.9%, respectively.     

Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays

Two hundred seventy one serum samples from South Korean patients were tested to detect antibodies against Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) and Ehrlichia chaffeensis (the human monocytic ehrlichiosis agent) by indirect fluorescent-antibody assay (IFA) and the Western blot assay. These sera were collected from patients with symptoms of high fever. The rate of seropositivity for Orientia tsutsugamushi was 50.9% by IFA at the Public Health & Environmental Research Institute and National Institute of Health in South Korea. By IFA, 30 (11.1%) and 39 (14.4%) of the serum samples reacted with A. phagocytophilum and E. chaffeensis antigens, respectively. By the Western blot assays, 24 (8.9%) and 29 (10.7%) of the serum samples reacted with purified A. phagocytophilum and E. chaffeensis protein antigens, respectively. This report strengthens other evidence regarding the presence of A. phagocytophilum and E. chaffeensis infections in humans in South Korea.     

Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Comparison of Ehrlichia chaffeensis Recombinant Proteins for Serologic Diagnosis of Human Monocytotropic Ehrlichiosis

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.     

Serodiagnosis of Human Granulocytic Ehrlichiosis by a Recombinant HGE-44-Based Enzyme-Linked Immunosorbent Assay

Current antibody testing for human granulocytic ehrlichiosis relies predominantly on indirect fluorescent-antibody assays and immunoblot analysis. Shortcomings of these techniques include high cost and variability of test results associated with the use of different strains of antigens derived from either horses or cultured HL-60 cells. We used recombinant protein HGE-44, expressed and purified as a maltose-binding protein (MBP) fusion peptide, as an antigen in a polyvalent enzyme-linked immunosorbent assay (ELISA). Fifty-five normal serum samples from healthy humans served as a reference to establish cutoff levels. Thirty-three of 38 HGE patient serum samples (87%), previously confirmed by positive whole-cell immunoblotting, reacted positively in the recombinant ELISA. In specificity analyses, serum samples from patients with Lyme disease, syphilis, rheumatoid arthritis, and human monocytic ehrlichiosis (HME) did not react with HGE-44-MBP antigen, except for one sample (specificity, 98%). We conclude that recombinant HGE-44 antigen is a suitable antigen in an ELISA for the laboratory diagnosis and epidemiological study of HGE.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State.

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In E. chaffeensis we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of E. chaffeensis membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels. E. chaffeensis was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the E. chaffeensis inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the E. chaffeensis inclusion compartment.     

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis.

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death. Only one isolate of E. chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based. We report the isolation and characterization of a new strain of E. chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness. The new isolate grows best in culture with careful control of pH. The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E. chaffeensis-specific monoclonal antibody 1A9. Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene. Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E. chaffeensis Arkansas and 91HE17 strains. Isolate 91HE17 lacked an epitope identified in E. chaffeensis Arkansas by the monoclonal antibody 6A1. This new E. chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans. Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans.     

Immunodiagnosis of Ehrlichia canis infection with recombinant proteins

Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections.     

Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis.

BACKGROUND: Human ehrlichiosis is a recently recognized tick-borne infection. Four species infect humans: Ehrlichia chaffeensis, E. sennetsu, E. canis, and the agent of human granulocytic ehrlichiosis. METHODS: We tested peripheral-blood leukocytes from 413 patients with possible ehrlichiosis by broad-range and species-specific polymerase-chain-reaction (PCR) assays for ehrlichia. The species present were identified by species-specific PCR assays and nucleotide sequencing of the gene encoding ehrlichia 16S ribosomal RNA. Western blot analysis was used to study serologic responses. RESULTS: In four patients, ehrlichia DNA was detected in leukocytes by a broad-range PCR assay, but not by assays specific for E. chaffeensis or the agent of human granulocytic ehrlichiosis. The nucleotide sequences of these PCR products matched that of E. ewingii, an agent previously reported as a cause of granulocytic ehrlichiosis in dogs. These four patients, all from Missouri, presented between May and August 1996, 1997, or 1998 with fever, headache, and thrombocytopenia, with or without leukopenia. All had been exposed to ticks, and three were receiving immunosuppressive therapy. Serum samples obtained from three of these patients during convalescence contained antibodies that reacted with E. chaffeensis and E. canis antigens in a pattern different from that of humans with E. chaffeensis infection but similar to that of a dog experimentally infected with E. ewingii. Morulae were identified in neutrophils from two patients. All four patients were successfully treated with doxycycline. CONCLUSIONS: These findings provide evidence of E. ewingii infection in humans. The associated disease may be clinically indistinguishable from infection caused by E. chaffeensis or the agent of human granulocytic ehrlichiosis.     

Molecular cloning and characterization of the Ehrlichia chaffeensis variable-length PCR target: an antigen-expressing gene that exhibits interstrain variation

A clone expressing an immunoreactive protein with an apparent molecular mass of 44 kDa was selected from an Ehrlichia chaffeensis Arkansas genomic library by probing with anti-E. chaffeensis hyperimmune mouse ascitic fluid. Nucleotide sequencing revealed an open reading frame (ORF) capable of encoding a 198-amino-acid polypeptide. The ORF contained four imperfect, direct, tandem 90-bp repeats. The nucleotide and deduced amino acid sequences did not show close homologies to entries in the molecular databases. PCR with primers whose sequences matched the sequences flanking the ORF was performed with DNA samples extracted from cell cultures infected with nine different isolates of E. chaffeensis, blood samples from seven patients with monocytic ehrlichiosis, and Amblyomma americanum ticks collected in four different states. The resulting amplicons varied in length, containing three to six repeat units. This gene, designated the variable-length PCR target, is useful for PCR detection of E. chaffeensis and differentiation of isolates.