Anaplasma marginale Superinfection Attributable to Pathogen Strains with Distinct Genomic Backgrounds

Abstract

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.

Superinfection Exclusion of the Ruminant Pathogen Anaplasma marginale in Its Tick Vector Is Dependent on the Time between Exposures to the Strains

“…The occurrence of superinfection exclusion and the failure to transmit the second strain following sequential exposure are consistent with a previous study using genetically distinct Virginia and Oklahoma strains in adult male Dermacentor variabilis ticks (17). This confirmation of superinfection exclusion within a short time window is in agreement with populationbased studies of A. marginale strain structure (2,6). Vallejo Esquerra et al reported that infected cattle, a natural host for A. marginale, in tropical regions of Mexico carried multiple strains (mean of 3.3 Ϯ 1.2 strains per animal), with up to six strains detected in an individual (2).…”

“…Individual hosts in tropical regions, characterized by long periods of warm temperatures and high relative humidity, carry multiple genetically distinct A. marginale strains, and there is broad hetero geneity at the population level (2,6,24). In contrast, hosts in temperate regions are most commonly infected with only a single A. marginale strain, and the overall population strain structure is homogeneous (25,26).…”

“…ector-borne, antigenically variant bacterial and protozoal pathogens that establish persistent infection, including those in the genera Anaplasma, Borrelia, Plasmodium, and Trypanosoma, have remarkable amounts of genetic diversity (1)(2)(3)(4), which enables establishment of persistent infection and ongoing transmission within a host population. As a consequence of this genetic diversity within the population, individual mammalian hosts may be infected with multiple genotypically distinct strains (5)(6)(7)(8)(9).…”

Primary Structural Variation in Anaplasma marginale Msp2 Efficiently Generates Immune Escape Variants

“…Even a single unique msp2 allele is sufficient to allow strain superinfection, with the requirement that the unique allele be expressed at the time of superinfection (10). Within regions of endemicity with high population immunity against a dominant strain, there is strong selective pressure for emergence of new strains, and consequently, strain superinfection is common (12,21,24,27). While there is genomic evidence that a new strain may emerge by insertions or deletions within a previously duplicated msp2 allele, the magnitude of change in the Msp2 primary structure required to generate a truly antigenically unique strain capable of superinfection is unknown.…”

“…Segmental gene conversion uses combinatorial recombination to generate hundreds to thousands of novel sequence variants not represented elsewhere in the genome other than transiently in the expression site (9). For this expansion to be functional in pathogen persistence requires that they evade the extant antibody response (12,21,22). The fraction of structural variants that represent antigenic variants has been unknown.…”