Distribution of ehrlichiae in tissues as determined by in-situ hybridization.

Specific identification of ehrlichiae in the tissues and determination of their distribution is difficult. In this study, an in-situ hybridization method was developed to detect ehrlichial 16S rRNA in tissue specimens from mice experimentally infected with the HF strain. This strain is closely related to Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis. HF strain-specific 16S rRNA was detected in endothelial cells and monocyte-macrophages in the liver, lungs, bone marrow, spleen, lymph nodes, and large and small intestinal tissues. The results suggest that the in-situ hybridization method with a digoxigenin-labelled RNA probe specific to ehrlichial 16S rRNA will be useful for post-mortem diagnosis and for the histopathological investigation of ehrlichial infection.

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.

Transcriptional analysis of p30 major outer membrane multigene family of Ehrlichia canis in dogs, ticks, and cell culture at different temperatures.

Ehrlichia canis, an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis. E. canis immunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs and Rhipicephalus sanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs in E. canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups of R. sanguineus ticks (adult males and females and nymphs) were separately infected with E. canis by feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower in E. canis cultivated in dog monocyte cell line DH82 at 25 degrees C than in E. canis cultivated at 37 degrees C. Analysis of differential expression of p30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E. canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

Molecular and antigenic comparison of Ehrlichia canis isolates from dogs, ticks, and a human in Venezuela.

We previously culture isolated a strain of Ehrlichia canis, the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with E. canis in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis using E. canis-specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four Rhipicephalus sanguineus ticks each were positive. An ehrlichial agent (Venezuelan dog Ehrlichia [VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human Ehrlichia isolate (VHE) and was closely related (99.9%) to that of E. canis Oklahoma. The 5' (333-bp) and 3' (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional E. canis-positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and E. canis Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human E. canis infection and that R. sanguineus, which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of E. canis directly from naturally infected ticks.

Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'.

Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.

Analysis of transcriptionally active gene clusters of major outer membrane protein multigene family in Ehrlichia canis and E. chaffeensis.

Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis and E. chaffeensis. Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensis genomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis, in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5'-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from -8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3'-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster in E. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5' and the 3' sides. Although omp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them.

Ehrlichial proliferation and acute hepatocellular necrosis in immunocompetent mice experimentally infected with the HF strain of Ehrlichia, closely related to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp.

Sensitive detection of Ehrlichia chaffeensis in cell culture, blood, and tick specimens by reverse transcription-PCR.

Ehrlichia chaffeensis is an obligatory intracellular bacterium of monocytes and macrophages and the etiologic agent of human monocytic ehrlichiosis, an emerging zoonosis. The Lone Star tick (Amblyomma americanum) has been implicated as the primary vector of E. chaffeensis. The present study examined the sensitivity of the nested reverse transcription (RT)-PCR based on the 16S rRNA gene relative to that of the nested PCR for detection of E. chaffeensis in infected DH82 cells, experimentally infected dog peripheral blood mononuclear cells, or experimentally infected A. americanum tick samples. The RT-PCR was found to be approximately 100 times more sensitive than the PCR for detection of E. chaffeensis regardless of the nature of the specimens. Thus, this RT-PCR is useful for detection of E. chaffeensis when a high sensitivity is required. Positive results by RT-PCR also imply the presence of viable pathogens. This is the first demonstration of RNA of E. chaffeensis in infected blood and acquisition-fed male, nymphal, and larval A. americanum ticks.

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Human granulocytic ehrlichiosis agent inhibits superoxide anion generation by human neutrophils.

The human granulocytic ehrlichiosis (HGE) agent, which replicates in neutrophils, was found not to induce superoxide anion (O(2)(-)) generation or extracellular release by human peripheral blood neutrophils, as measured by a luminol-dependent chemiluminescence assay or a cytochrome c reduction assay, respectively. Furthermore, the HGE agent completely prevented O(2-) release by neutrophils upon stimulation with phorbol myristate acetate (PMA), formylmethionyl-leucyl-phenylalanine, or Escherichia coli. The inhibition was HGE agent dose dependent, required ehrlichial contact with the host cells, and was reversible upon removal of the extracellular HGE agent bound to the host cells prior to PMA stimulation. Structural integrity of or new protein synthesis by the HGE agent was not required for the inhibition; carbohydrate but not surface protein of the HGE agent was required. The HGE agent did not prevent O(2-) generation in human peripheral blood monocytes derived from the same individual. This neutrophil-specific prevention of O(2-) generation by the HGE agent would be critical in survival of the HGE agent. This is the first demonstration of the rapid inhibition of preexisting NADPH oxidase in human neutrophils by the HGE agent.

Analysis of 16S rRNA and 51-kilodalton antigen gene and transmission in mice of Ehrlichia risticii in virgulate trematodes from Elimia livescens snails in Ohio.

Operculate snails (the family Pleuroceridae: Elimia livescens) were collected between June and October 1998 from a river in central Ohio where repeated cases of Potomac horse fever (PHF) have occurred. Of collected snails, consistently 50 to 80% carried a combination of cercariae and sporocysts of digenetic virgulate trematodes. The trematodes obtained from each snail were pooled and tested for Ehrlichia risticii, the agent of PHF, by nested PCR using primers specific to the 16S rRNA gene. Out of a total of 209 trematode pools, 50 pools were found to be positive by PCR. The DNA sequence of the 16S rRNA gene identified in one trematode pool was identical to that of the type strain of E. risticii, and the sequence of the gene identified in another pool differed from that of the type strain by 1 nucleotide. Comparison of the deduced amino acid sequence of the partial 51-kDa antigen gene from various sources revealed that Maryland, Ohio (except Ohio 081), and Kentucky strains are in a cluster distinct from the sequences obtained from sources in California and Oregon. Ohio 081 was shown previously by antigenic composition analysis to be distinct from other groups. However, all sequences examined were not segregated according to their sources: horse blood or infected trematodes. E. risticii was found to be transmittable from trematodes to mice and was subsequently passaged from infected mice to additional mice, as determined by PCR analysis. Our findings suggest the evolution of E. risticii in the natural reservoir in separate geographic regions and persistent infection of trematode populations with E. risticii during summer and early fall. The study also suggests that the mouse can be used to isolate E. risticii from the infected trematode.

Comparison of two recombinant major outer membrane proteins of the human granulocytic ehrlichiosis agent for use in an enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris.

Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

Expression of interleukin-1beta, tumor necrosis factor alpha, and interleukin-6 in human peripheral blood leukocytes exposed to human granulocytic ehrlichiosis agent or recombinant major surface protein P44.

Human granulocytic ehrlichiosis (HGE) is an emerging febrile systemic disease caused by the HGE agent, an obligatory intracellular bacterium of granulocytes. The pathogenicity- and immunity-related mechanisms of HGE are unknown. In this study, several cytokines generated in human peripheral blood leukocytes (PBLs) incubated with the HGE agent or a recombinant 44-kDa major surface protein (rP44) of the HGE agent were examined by reverse transcription-PCR and a capture enzyme-linked immunosorbent assay. The HGE agent induced expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNAs and proteins in PBLs in a dose-dependent manner to levels as high as those resulting from Escherichia coli lipopolysaccharide stimulation. The kinetics of induction of these three cytokines in PBLs by rP44 and by the HGE agent were similar. Proteinase K treatment of the HGE agent or rP44 eliminated the ability to induce these three cytokines. Induction of these cytokine mRNAs was not dependent on superoxide generation. These results suggest that P44 proteins have a major role in inducing the production of proinflammatory cytokines by PBLs. Expression of IL-8, IL-10, gamma interferon, transforming growth factor beta, and IL-2 mRNAs in response to the HGE agent was not remarkable. Among PBLs, neutrophils and lymphocytes expressed IL-1beta mRNA but not TNF-alpha or IL-6 mRNA in response to the HGE agent, whereas monocytes expressed all three of these cytokine mRNAs. These observations suggest that induction of proinflammatory-cytokine gene expression by the major outer membrane protein of the HGE agent in monocytes, which are not the primary host cells of the HGE agent, contributes to HGE pathogenesis and immunomodulation.

New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan.

Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup.

Intracellular infection by the human granulocytic ehrlichiosis agent inhibits human neutrophil apoptosis.

In patients with human granulocytic ehrlichiosis (HGE), the HGE agent has been seen only in the peripheral blood granulocytes, which have a life span too short for ehrlichial proliferation. To determine if the HGE agent delays the apoptosis of human peripheral blood neutrophils for its advantage, peripheral blood granulocytes consisting mostly of neutrophils were incubated with freshly freed host cell-free HGE agent in vitro. The HGE agent induced a significant delay in morphological apoptosis and the cytoplasmic appearance of histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the HGE agent freshly freed from the host cells, noninfectious purified HGE agent stored frozen and thawed also had antiapoptotic effect, which was lost with proteinase K treatment but not with periodate treatment. Treatment of neutrophils with a transglutaminase inhibitor, monodansylcadaverine, blocked the antiapoptotic effect of the HGE agent. Addition of oxytetracycline, however, did not prevent or reverse the antiapoptotic effect of the HGE agent. These results suggest that binding of a protein component(s) of the HGE agent to neutrophils and subsequent cross-linking and/or internalization of the receptor and ehrlichiae are required for antiapoptotic signaling, but ehrlichial protein synthesis and/or proliferation is not required. MG-132, a proteasome inhibitor, and cycloheximide accelerated the apoptosis of neutrophils and overrode the antiapoptotic effect of the HGE agent. Studies with specific inhibitors suggest that protein kinase A, NF-kappaB, and interleukin 1beta are not involved in the antiapoptotic mechanism of the HGE agent.