The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.rickettsiales ͉ bacterial artificial chromosome ͉ St. Maries strain

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp 2 and msp 3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Anaplasmosis is one of several tick-borne diseases severely constraining cattle production and usage in many parts ofthe world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginae carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates ofA. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the msplk gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.

Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4؉ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membranevaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.