Identification of 19 Polymorphic Major Outer Membrane Protein Genes and Their Immunogenic Peptides inEhrlichia ewingiifor Use in a Serodiagnostic Assay

Abstract

Ehrlichia ewingii, a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii. These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii-infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.

Heartwater – Ehrlichia ruminantium infection

“…Current serological tests are based on the detection of antibodies to the immunodominant E. ruminantium outer membrane protein MAP1, and the most reliable of these tests uses a recombinant fragment of MAP1 (MAP1B) in an indirect enzyme-linked immunosorbent assay (ELISA) format (19). It must be emphasised, however, that all serological tests for E. ruminantium may exhibit false-positive reactions, owing to the presence of closely related homologs of map1 in other Anaplasmataceae species (20,21,22). Serological tests for heartwater also exhibit false-negative results, mostly in cattle, as antibody levels are often too low to be detected, even in animals that are under continuous natural challenge by infected ticks (23,24).…”

Ehrlichiosis in Brazil

“…Although this technique is still widely used, a significant number of false positives may occur due to cross-reactivity with other organisms from the genera Ehrlichia, Anaplasma and Neorickettsia (RISTIC et al, 1981;HARRUS et al, 2002;OLANO;WALKER, 2002;PADDOCK;CHILDS, 2003). Several other serological tests are now commercially available to diagnose ehrlichiosis (e.g., Enzyme Linked Immunosorbent Assay (ELISA), immunoblot, competitive Enzyme Linked Immunosorbent Assay (cELISA)) (OHASHI et al, 1998;WANER et al, 2000;ALLEMAN et al, 2001;LÓPEZ et al, 2007;ZHANG et al, 2008). Diagnostic accuracy has been greatly enhanced by the introduction of culture and molecular techniques.…”

Cloning of the Major Outer Membrane Protein Expression Locus in Anaplasma platys and Seroreactivity of a Species-Specific Antigen

“…In the present study, we could identify several A. platys-specific amino acid sequences within P44 proteins that can be used as serologic test antigens to provide differential diagnosis from other Anaplasma species infections. Additionally, Ehrlichia OMP-1/P28/P30/MAP families are immunodominant major outer membrane proteins that are useful for serodiagnosis (45,62,63,68). Our alignment results showed a distinct fragment (ϳ20 amino acids) in A. platys Omp-1X that was not observed in the closest homologs from Anaplasma and Ehrlichia spp.…”

“…p44ES sequences were amplified by nested touchdown PCR using primer pairs F2-R3 and F3-R4. Amplification was performed as previously described (68). The amplified DNA fragments were cloned using a TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced with M13 forward or M13 reverse sequencing primers.…”

“…phagocytophilum antibodies. The horseradish peroxidase substrate 2,2Ј-azido-di-(3-ethyl)-benzthiazoline-6-sulfonic acid (Sigma, St. Louis, MO) in 70 mM citrate buffer (pH 4.2) was applied, and absorbance values at 415 and 492 nm were measured in an ELISA plate reader (Molecular Devices, Sunnyvale, CA) as previously described (68). The results are presented as the optical density at 415 nm minus that at 492 nm (OD 415 Ϫ OD 492 ), and the cutoff for a positive reaction was set at greater than the mean of the value for the formula OD 415 Ϫ OD 492 ϩ 3 standard deviations (SD) for the negative-control samples (OD Ͼ 0.165).…”