Diversity of Ehrlichia ruminantium Major Antigenic Protein 1-2 in Field Isolates and Infected Sheep

Abstract

Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.

Progress and obstacles in vaccine development for the ehrlichioses

“…Multilocus sequence typing has revealed that eight strains in one small African locality had different phylogenies for each gene target examined, providing an example of the heterogeneity that occurs in the natural population [46]. Similar findings for the MAP genes suggest a high level of diversity that reveals divergent evolution and no correlation among MAP genotypes, geographic distribution and timing of strain introduction [44,47,48]. The inconsistency of any single strain to protect against experimental needle challenge is at least partially associated with the antigenic differences in the challenge strain.…”

“…Until recent genome sequences were obtained from three different strains, E. ruminantium was considered to be relatively homogenous [40,41]. It is evident that E. ruminantium exhibits substantial genetic hetero geneity, and this may have contributed to the inconsistent efficacy of various vaccines for heartwater [42-44]. In fact, eight different 16S rRNA ( rrs ) genotypes are well documented, and there are probably many more [11,45].…”

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium , from the Panola Mountain Ehrlichia

“…MAP‐1B peptides for ER (Gardel, DWDGVKTPSSDSGNNSIIFTEKDYSFKYENNPFLGFAGAIGYSMNGPRI) (Barbet et al., ), PME (DWDGVKSTASTSLFTEKDYSFKYENNPFLGFAGAIGYSMNGPRI) and Ehrlichia chaffeensis (DWDGSAIPHTQSSTPFTVSNYSFKYENNPFLGFAGAIGYSMDGPRI) were synthesized by EZBiolabs (Carmel, IN, USA). All reactions were performed on equivalently coated plates with 3 μg/ml of each MAP‐IB peptide in coating buffer of 35 m m sodium bicarbonate and 15 m m sodium carbonate as previously described (Barbet et al., ). To determine the extent of cross‐reactivity, the MAP‐1B peptides described above were reacted with known positive sera (experimentally infected livestock or seroreactive clinically ill human patients) in homologous and heterologous reactions.…”

“…To determine the extent of cross‐reactivity, the MAP‐1B peptides described above were reacted with known positive sera (experimentally infected livestock or seroreactive clinically ill human patients) in homologous and heterologous reactions. Panola Mountain Ehrlichia‐positive sera from experimentally inoculated goats were collected in a previous study (Loftis et al., ), as were ER‐positive sera from experimentally inoculated sheep and a bovine (Barbet et al., ). Ehrlichia chaffeensis ‐positive human sera from clinically ill, naturally infected human patients were de‐identified and graciously provided by Dr. Susan Wong (Wadsworth Center, New York State Department of Health, Albany, NY, USA).…”

Tick-borne diseases in cattle: Applications of proteomics to develop new generation vaccines

“…One of the most promising alternatives is the inactivated vaccine; not only because it has proven to protect against homologous and heterologous challenge under controlled conditions but also a complete industrial process is now readily available for the production, purification and formulation of large amounts of E. ruminantium at low cost [19,20]. Nevertheless, the problems caused by antigenic strain differences and high diversity shown in restricted areas still remain, hampering the development of a fully effective vaccine [21,22].…”