Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains

Abstract

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D strain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two-to fourfold differences were observed between homologous and heterologous antigens. The differences in antigen profiles were demonstrated by Western blot (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns. Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and 85-kDa recombinant clones of the 90-12 strain showed a similar pattern with probes of the 51-and 55-kDa clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferontreated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.

Comparison of PCR and culture to the indirect fluorescent-antibody test for diagnosis of Potomac horse fever

“…The efficacies of vaccines in the field are perceived to be marginal (13). In agreement with this perception, E. risticii has been isolated from sick horses which had been vaccinated (4,26). Although, rising IFA test titers with accompanying clinical signs suggest active infection, an IFA test at a single time point is useless in diagnosing PHF in vaccinated horses.…”

Detection ofEhrlichia risticii, the Agent of Potomac Horse Fever, in Freshwater Stream Snails (Pleuroceridae:Jugaspp.) from Northern California

“…Segments of two additional ehrlichia genes were amplified by PCR. Sets of nested primers were designed to detect portions of the E. risticii homolog of the Escherichia coli groESL heat shock operon (39) and the E. risticii 51-kDa major antigen gene (12,13,41). The following primer sequences were used to amplify the heat shock operon: 5Ј-ACCAGGCTACCTCACAGGC-3Ј and 5Ј-TTGACC CTCGCATCAATG-3Ј (outer primers); and 5Ј-CACAAGTTGGTTCAATTTC TGC-3Ј and 5Ј-CCGAGATCTTCAACAGTAAGGC-3Ј (inner primers).…”

Cloning and Expression of 51‐kDa Antigenic Protein of Neorickettsia risticii NR‐JA1

“…It appears that p51 antigenic protein may be a good target for the development of effective diagnostic or immunoprophylactic agent, not only because of its immunogenic potential but also because the p51 gene is present only in N. risticii and has been found consistently in all N. risticii strains identified to date. 3,4 Moreover, p51 predicted to be an outer membrane protein by PSORT analysis (http://psort.nibb.ac.jp/). Therefore it appears that p51 antigen could be a potential target for the development of improved serological or molecular diagnostic methods and possibly a recombinant DNA vaccine against PHF.…”

“…Infected horses and mice develop a strong immunoglobulin G antibody response and protection against N. risticii infection. 3,4 Passive transfer of horse antisera to N. risticii or mouse antibodies to N. risticii (antiserum or purified immunoglobulin G) protected mice against N. risticii challenge infection, strongly indicating that antibody mediates the immunity. 3,5 Molecular analysis of N. risticii strain (strain 25-D) indicated the presence of nine major component antigens (indicated by molecular mass: 110, 70, 68, 55, 51, 50, 49, 33, and 28 kDa), all of which are apparent surface antigens, as determined by 125 I surface labeling.…”