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RNA polymerase beta-subunit-based phylogeny of Ehrlichia spp., Anaplasma spp., Neorickettsia spp. and Wolbachia pipientis.

Sequence analysis of rpoB, the gene encoding the beta-subunit of RNA polymerase, was used in a phylogenetic investigation of nine species from the genera Ehrlichia, Neorickettsia, Wolbachia and Anaplasma. The complete nucleotide sequences obtained for Anaplasma phagocytophilum (HGE agent), Ehrlichia chaffeensis, Neorickettsia sennetsu, Neorickettsia risticii, Anaplasma marginale and Wolbachia pipientis were amongst the longest rpoB sequences in GenBank and ranged from 4074 bp for N. sennetsu to 4311 bp for W. pipientis. Additional partial rpoB sequences were obtained for Ehrlichia canis, Ehrlichia ruminantium and Ehrlichia muris. Identical phylogenetic trees were inferred from multiple sequence alignments of the nucleotide sequences and the derived amino acid sequences using either distance, maximum-likelihood or parsimony methods. This study confirms the phylogeny previously inferred from sequence analyses of the 16S rRNA gene, groESL and gltA and allows the confirmation of four monophyletic clades. The rpoB nucleotide sequences were more variable than the 16S rRNA gene and groESL sequences at the species level.

Anaplasma↗

Analysis of p51, groESL, and the major antigen P51 in various species of Neorickettsia, an obligatory intracellular bacterium that infects trematodes and mammals.

The p51 gene that encodes the major antigenic 51-kDa protein in Neorickettsia risticii was identified in strains of Neorickettsia sennetsu and the Stellantchasmus falcatus agent but not in Neorickettsia helminthoeca, suggesting that p51-based diagnosis would be useful to distinguish among them. groESL sequencing results delineated the phylogenic relationships among Neorickettsia spp.

Anaplasmataceae Infections↗

Budget genome.

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Anaplasma phagocytophilum↗

Mechanisms to create a safe haven by members of the family Anaplasmataceae.

Members of the family Anaplasmataceae are obligatory intracellular bacteria with unique host cell specificities. Depending on each bacterial species, granulocytes, platelets, endothelial cells, monocytes, macrophages, red blood cells, and cells of invertebrates are specifically infected. This unique host cell specificity has been the major hurdle to overcome in order to cultivate this group of bacteria. Because these bacteria cannot survive outside host cells, once released from a host cell, they need to rapidly induce signals for their own internalization into another host cell unique to each species. How these bacteria enter and continue to survive and replicate within the host milieu, then exit the host cell is largely unknown. Recently, however, unique strategies employed by some of these bacteria for successful parasitism of mammalian leukocytes have begun to be uncovered. When these bacteria interact with host cells, signals are transduced both inside the host cells and inside the bacteria. These signals disable the alarm system, as well as microbicidal mechanisms, of the leukocytes and condition the host cells to accept these intruders to share space and nutrient resources. Signals transduced inside the bacteria allow them to finely tune their metabolism and physiology in the new host cell environment and to disguise themselves as "insiders" so that their sojourn does not upset the host cell physiology until they have sufficiently multiplied. This paper discusses our recent findings on these topics.

Adaptation, Physiological↗

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.

Anaplasma↗

Molecular detection of an Ehrlichia-like agent in rainbow trout (Oncorhynchus mykiss) from Northern California.

Ehrlichia DNA was identified by nested PCR in rainbow trout (Oncorhynchus mykiss) collected from a creek in northern California where Potomac horse fever is endemic. Ehrlichia DNA was found in tissues from several organs including the gills, heart, spleen, liver, kidneys and intestine of trout and from three different adult digenetic trematodes (Deropegus sp., Crepidostomum sp., Creptotrema sp.) parasitizing the gallbladder and/or the intestine of the trout. Sequencing of PCR-amplified DNA from the 16S rRNA gene indicated that the source organism was most closely related to the sequences of E. risticii (level of sequence similarity 96.0%), the SF agent (95.9%), E. sennetsu (95.8%), and Neorickettsia helminthoeca (95.3%). The data suggest that trout and parasitic trematodes may be involved in the epidemiology of an Ehrlichia-like agent belonging to the E. sennetsu genogroup. Whether the fish agent infects horses, dogs, or human beings, and whether it causes disease, remain to be determined.

Animals↗

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia.

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

Animals↗

Real-time PCR duplex assay for Rickettsia prowazekii and Borrelia recurrentis.

Rickettsia prowazekii, the etiologic agent for epidemic typhus, and Borrelia recurrentis, the etiologic agent of relapsing fever, both utilize the same vector, the human body louse (Pediculus humanus), to transmit human disease. We have developed an assay to detect both bacterial pathogens in a single tube utilizing real-time PCR. Assays for both agents are specific. The R. prowazekii and B. recurrentis assays do not detect nucleic acid from R. typhi, R. canada, or any of eight spotted fever rickettsiae. In addition they did not react with Neorickettsia risticii, N. sennetsu, Franciscella persica, Bartonella quintana, Legionella pneumophila, Proteus mirabilis, Salmonella enterica, Escherichia coli, and Staphylococcus aureus. Moreover, the B. recurrentis assay did not detect B. duttonii, B. coriaceae, B. afzelii, B. garinii, B. hermsii, or B. burgdorferi nucleic acid. Both assays detected repeatedly only R. prowazekii or B. recurrentis either when tested alone or together in one test tube.

Animals↗

Comparative genomics of emerging human ehrlichiosis agents.

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens.

Animals↗

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines.

Ehrlichiae are strict intracellular bacterial pathogens that parasitize leukocytes or other blood cells. Only six agents of the tribe Ehrlichieae, namely, Cowdria ruminantium, Neorickettsia helminthoeca, Ehrlichia risticii, Ehrlichia sennetsu, Ehrlichia canis, and Ehrlichia chaffeensis, have been adapted to growth in continuous cell lines. E. chaffeensis, the agent of human ehrlichiosis, has been cultured only in a cell line of canine origin. We adapted purified cell-free E. chaffeensis for growth in human embryonic lung (HEL) fibroblasts (HEL 299), green monkey kidney cells (Vero), and a human cervical epithelioid carcinoma (HeLa) cell line. We observed a cytopathic effect with both Vero cells and HEL cells and plaque formation with cellular lysis when infected Vero cells were cultured in agar. Human fibroblasts are already commonly used for the isolation of viruses, coexiellae, and rickettsiae. Furthermore, the capability of these cells to support the growth of ehrlichiae suggests that they may be useful for primary isolation of ehrlichiae as well. The cytopathic effect produced in Vero or HEL cells offers a very helpful indicator of the infection. Plaque formation in Vero cells is a new phenomenon not yet reported for ehrlichiae and will allow the titration of inocula and clonal purification of this bacterium.

Adaptation, Physiological↗

Neorickettsia-like organism isolated from metacercaria of a fluke, Stellantchasmus falcatus.

"Hyuga" fever, reportedly caused by Rickettsia sennetsu, occurring in the west of Japan was suspected to be an endoparasite-borne rickettsiosis. No R. sennetsu, however, was detected in metacercariae found in the muscles of grey mullets (Mugil cephalus) ingested raw by most "Hyuga" fever patients. Instead, another neorickettsia-like organism (SF strain) was isolated occasionally from metacercariae of Stellantchasmus falcatus. A dog was inoculated with SF strain. It developed mild fever after an incubation period of 10 days. No other clinical sign nor any macroscopic change upon autopsy was noted. The organisms were detected in its blood, lymph nodes and spleen, but not further transmissible. From these findings, the pathogenicity of SF strain to the dog seemed to be much lower than that of Neorickettsia helminthoeca or the Elokomin fluke fever agent of neorickettsia. These experiments support our presumption that SF strain may be a new member of neorickettsia, although no comparison of the antigenicity between SF strain and other neorickettsiae has been made.

Animals↗

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.

Anaplasmataceae↗

Cross-reacting antigens between Neorickettsia helminthoeca and Ehrlichia species, shown by immunofluorescence and Western immunoblotting.

Dogs orally infected with Neorickettsia helminthoeca developed immunoglobulin G titers against Erlichia risticii, Erlichia sennetsu, and Erlichia canis similar to those against N. helminthoeca antigen, as determined by immunofluorescence. Western immunoblotting showed that the major common antigens shared among the microorganisms were 80- or 78-kDa and 64-kDa polypeptides. In contrast, horse anti-E. risticii and anti-E. sennetsu and dog anti-E. canis sera reacted more weakly to N. helminthoeca antigen than to homologous antigens in both immunofluorescence and Western immunoblotting. Antisera raised in other species of animals, i.e., mouse anti-E. canis and rabbit anti-E. risticii and anti-E. sennetsu sera, however, all reacted with the 64-kDa antigen of N. helminthoeca. This strong antigenic cross-reactivity and similarity in Western immunoblotting reaction profiles indicate that N. helminthoeca is antigenically closely related to E. risticii and E. sennetsu and less so to E. canis. In both immunofluorescence and Western immunoblotting, E. canis shared fewer common antigens with E. risticii and E. sennetsu than N. helminthoeca did. It is reasonable to conclude that these results may have both diagnostic and taxonomic significance.

Animals↗

Characterization of ehrlichial organisms isolated from a wild mouse.

An infectious agent was isolated from the enlarged spleen of a wild mouse, Eothenomys kageus, by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed splenomegaly, and the agent was maintained by serial passage of spleen homogenates in laboratory mice. The agent in the spleen homogenate was inactivated after incubation at 37 or 50 degrees C. Tetracyclines were effective in preventing infection of mice with this agent, but penicillin and sulfonamides were ineffective. Cytoplasmic inclusion bodies were observed in the peritoneal macrophages of infected mice. Electron microscopy revealed numerous small pleomorphic cocci within membrane-lined vacuoles in the cytoplasm of splenic macrophages. Morphologically similar to the ehrlichial organisms, each organism was surrounded by a distinct plasma membrane and rippled outer cell membrane without a distinct peptidoglycan layer. The agent did not grow in chicken embryos, and the Weil-Felix test result was negative. In the indirect fluorescent-antibody test, the agent reciprocally cross-reacted with Ehrlichia canis and cross-reacted somewhat with Ehrlichia sennetsu but did not cross-react with Ehrlichia risticii, Neorickettsia helminthoeca, Rickettsia tsutsugamushi, or Chlamydia spp. The mouse antiserum against this agent reacted with 64-, 47-, 46-, 44-, and 40-kDa proteins of E. canis by Western blotting (immunoblotting). Since E. canis and closely related Ehrlichia chaffeensis and Ehrlichia ewingii are not known to proliferate or cause splenomegaly in mice, these results suggest that the agent is a new species within the tribe Ehrlichieae of the family Rickettsiaceae. The finding suggests that wild rodents may serve as reservoirs for pathogenic ehrlichiae.

Animals↗

Characterization of the SF agent, an Ehrlichia sp. isolated from the fluke Stellantchasmus falcatus, by 16S rRNA base sequence, serological, and morphological analyses.

The organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.

Animals↗