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Anaplasma phagocytophila is an obligatory intragranulocytic bacterium that causes human granulocytic ehrlichiosis. Immunodominant 44-kDa outer membrane proteins of A. phagocytophila are encoded by a p44 multigene family. In the present study, expression profiles of p44 genes in the blood of acutely infected patients in the year 2000 were characterized. A single p44 gene was predominantly expressed in peripheral blood leukocytes from one patient, while up to 17 different p44 genes were transcribed without a single majority in the other two patients. The cDNA sequences of the central hypervariable region of several p44 genes were identical among the isolates from the three patients and a 1995 A. phagocytophila isolate. A. phagocytophila was isolated by cell culture from all of the three 2000 patients. Genomic Southern blot analysis of the three 2000 and two 1995 A. phagocytophila isolates with probes specific to the most dominant p44 transcript in each patient showed that the p44 loci in the A. phagocytophila genome were conserved. Analysis of the predicted amino acid sequences of 43 different p44 genes including 19 new sequences found in the present study, revealed that five amino acids were absolutely conserved. The hypervariable region was subdivided into five domains, including three extremely hypervariable central domains. These results suggest that variations in the sequences of p44 are not random but are restricted. Furthermore, several p44 genes are not hypermutatable in nature, based on the conservation of gene sequences and loci among isolates obtained 5 years apart.

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti- Ehrlichia equi serum recognized the rP44 protein. This suggests that rP44 is an HGE- E. equi group-specific antigen. Neither human anti- Ehrlichia chaffeensis serum nor rabbit anti- Borrelia burgdorferi serum reacted with rP44. Sera from two patients coinfected with the HGE agent and B. burgdorferi reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.

Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody-positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test.

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.

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.

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.