Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginale and related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2 sequences to the promoter and polycistron.Ehrlichiae are major causes of tick-borne diseases, including the recently emergent human monocytic and two granulocytic ehrlichioses and the most prevalent tick-borne infection in cattle worldwide, anaplasmosis (6). These pathogens, classified in genogroups I and II of the tribe Ehrlichieae, have a complex life cycle characterized by acute and persistent infection in the mammalian host and several replicative and developmental stages within the tick vector (10, 17). Notably, this complexity is achieved using a small genome, only 0.8 to 1.5 Mb in a single chromosome (1,25). Persistence of Anaplasma marginale in cattle, which is fundamental for continued transmission, reflects sequential expression of antigenically variant outer membrane proteins that are encoded by the msp2 multigene family (21). The outer membrane proteins of different ehrlichial organisms are significantly similar to one another in amino acid sequence, are all encoded by multigene families, and possess one to four variable regions (8, 13, 18-20, 22, 26, 29, 32). In A. marginale-infected cattle, three to six MSP2 variants are expressed in each sequential rickettsemic cycle, which recur every 4 to 8 weeks during persistent infection (8,9,15,16). Thus, over the 7-year period in which A. marginale has been shown to persist, over 500 variants may be expressed. Although the cyclic emergence and immune control of A. marginale is similar to that occurring in African trypanosomiasis, the mechanisms used by the organism to persist in mammalian hosts are unknown. We show here that variation of msp2 in erythrocyte stages of A. marginale proceeds through the formation of different sequence mosaics in a polycistronic expression site. MATERIALS AND METHODSIsolation and sequencing of A. marginale genomic DNA. Florida and South Idaho strains of A. marginale were maintained as liquid nitrogen-cryopreserved stabilates of infected bovine erythrocytes in dimethyl sulfoxide-phosphate-buffered saline that were then used to infect cattle (20). A. marginale genomic DNA ...

The isolates were similar to those that cause disease in humans.

Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.

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Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1␣ genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.