Antigenic and genomic relatedness among Ehrlichia risticii, Ehrlichia sennetsu, and Ehrlichia canis.

Antigenic and genomic relatedness among Ehrlichia risticii, E. sennetsu, and E. canis was analyzed by enzyme-linked immunosorbent assay, Western blotting (immunoblotting) and DNA-DNA hybridization. E. risticii and E. sennetsu were serologically related, and their Western blot antigen profiles were nearly identical. Two antigens of E. sennetsu corresponding to the 28- and 51-kDa antigens of E. risticii were apparently larger than the E. risticii antigens, and the 55-kDa antigen of E. risticii appeared to be unique to this species. The 110-, 70-, and 44-kDa antigens of these two species were identical, as determined by the use of monospecific antibodies. DNA homology between these two species was high. On the other hand, E. canis was antigenically least reactive with the antisera to E. risticii and E. sennetsu. However, a dog convalescent-stage E. canis antiserum recognized antigens in the other two species which were different from those recognized by their homologous antisera. Similarly, homology between the DNA of E. canis and the DNAs of the other two species was very minimal. These results indicate that E. risticii and E. sennetsu are closely related both at the genomic and antigenic levels and that the relationship of these two species with E. canis is minimal.

Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).

Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.

Molecular cloning and analysis of recombinant major antigens of Ehrlichia risticii.

The genome of Ehrlichia risticii, the etiologic agent of Potomac horse fever, was cloned in the lambda gt11 expression vector. The efficiency of recombinant phage production with different restriction fragments of E. risticii DNA was generally between 20 and 95%. The antigen-positive frequency, detected by immunoscreening with E. risticii antibodies, was between 8 and 40 per 10(4) recombinants. Four (70, 55, 51, and 44 kDa) major antigens of E. risticii were identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. Characterization of three (70, 55, and 44 kDa) of these recombinant antigens indicated that the 70- and 44-kDa polypeptides were beta-galactosidase fusion products that were dependent on isopropylthiogalactoside induction for expression; they contained about 50 and 73%, respectively, of the native polypeptides. The 55-kDa antigen was a nonfusion protein expressed independently of isopropylthiogalactoside induction; it was a complete protein with a molecular weight identical to that of its native counterpart. The cloned E. risticii DNAs from of the recombinants expressing 70-, 55-, and 44-kDa proteins were 3.5, 3.9, and 4.8 kb, respectively, in size, and they were unique. The insert DNAs hybridized to multiple restriction fragments of the genomic DNA, the sum of the sizes of which was much greater than that of the corresponding insert. Mice immunized with the affinity-purified 55-kDa recombinant antigen produced a high titer of antibody in serum as measured by an enzyme-linked immunosorbent assay and gave a monospecific reaction by Western immunoblotting. Challenge infection of these immunized mice showed low protection from clinical infection.

DNA restriction endonuclease cleavage pattern and protein antigen profile of Ehrlichia risticii.

Molecular characterization of Ehrlichia risticii, the etiological agent of Potomac horse fever, was performed. Restriction endonuclease cleavage of E. risticii DNA generated distinct patterns by different enzymes. The DNA cleavage patterns of E. risticii isolates obtained from different geographic regions were similar. Protein analysis identified thirty-five distinct proteins with molecular weights ranging from 160 to 16 kilodalton (kDa). Antigenic analysis by radioimmunoprecipitation using 125I surface labeled E. risticii and by Western blotting determined the presence of eighteen antigens (160, 110, 86, 84, 81, 70, 55, 51, 49, 44, 41, 36, 33, 31, 28, 24, 22 and 16 kDa) of which nine (110, 86, 70, 55, 51, 49, 44, 33, and 28 kDa) were major antigens. Fourteen of these antigens, which included the major antigens, were apparent surface components. There were no heat-modifiable proteins but lipopolysaccharide components of 245 and 14 kDa, resistant to proteinase K and of non-antigenic character, were detected in the organism.

Molecular cloning of Ehrlichia risticii and development of a gene probe for the diagnosis of Potomac horse fever.

A gene bank of Ehrlichia risticii was constructed in plasmid vector pUC13. Five clones representing discrete regions of the E. risticii genome were tested for their ability to hybridize specifically to E. risticii DNA. None of the clones cross-hybridized with Ehrlichia equi DNA, whereas four of these clones cross-hybridized with Ehrlichia canis and Ehrlichia sennetsu DNAs. However, one clone carrying a 1-kilobase HindIII fragment of E. risticii DNA failed to cross-react with the genomes of E. sennetsu, E. canis, and E. equi in dot blot hybridization assays. The sensitivity of this probe for the detection of E. risticii DNA was approximately 0.5 pg. By using this probe, the E. risticii DNA was detected in the peripheral blood mononuclear cells of 30 experimentally infected horses by 7 days postinfection (p.i.); the detection of E. risticii DNA peaked between 14 and 17 days p.i., a period immediately after the peak of the second rise in body temperature, during leukopenia and at the onset of diarrhea. E. risticii DNA was not detectable by 25 to 30 days p.i. E. risticii DNA was not detected in noninfected control horses.