The Use of the Antigenically Variable Major Surface Protein 2 in the Establishment of Superinfection during Natural Tick Transmission of Anaplasma marginale in Southern Ghana.

Many vector-borne pathogens, including Anaplasma spp., Borrelia spp., Trypanosoma spp., and Plasmodium spp., establish persistent infection in the mammalian host by using antigenic variation. These pathogens are also able to establish strain superinfection, defined as infection of an infected host with additional strains of the same pathogen despite an adaptive immune response. The ability to establish superinfection results in a population of susceptible hosts even with high pathogen prevalence. It is likely that antigenic variation, responsible for persistent infection, also plays a role in the establishment of superinfection. Anaplasma marginale, an antigenically variable, obligate intracellular, tickborne bacterial pathogen of cattle, is well suited for the study of the role of antigenically variant surface proteins in the establishment of superinfection. Anaplasma marginale establishes persistent infection by variation in major surface protein 2 (msp2), which is encoded by approximately six donor alleles that recombine into a single expression site to produce immune escape variants. Nearly all cattle in regions of high prevalence are superinfected. By tracking the acquisition of strains in calves through time, the complement of donor alleles, and how those donor alleles are expressed, we determined that simple variants derived from a single donor allele, rather than multiple donor alleles, were predominant. Additionally, superinfection is associated with the introduction of new donor alleles, but these new donor alleles are not predominantly used to establish superinfection. These findings highlight the potential for competition among multiple strains of a pathogen for resources within the host and the balance between pathogen fitness and antigenic variation.

Both Coinfection and Superinfection Drive Complex Anaplasma marginale Strain Structure in a Natural Transmission Setting.

Vector-borne pathogens commonly establish multistrain infections, also called complex infections. How complex infections are established, either before or after the development of an adaptive immune response, termed coinfection or superinfection, respectively, has broad implications for the maintenance of genetic diversity, pathogen phenotype, epidemiology, and disease control strategies. Anaplasma marginale, a genetically diverse, obligate, intracellular, tick-borne bacterial pathogen of cattle, commonly establishes complex infections, particularly in regions with high transmission rates. Both coinfection and superinfection can be established experimentally; however, it is unknown how complex infections develop in a natural transmission setting. To address this question, we introduced naive animals into a herd in southern Ghana with a high infection prevalence and high transmission pressure and tracked the strain acquisition of A. marginale through time using multilocus sequence typing. As expected, the genetic diversity among strains was high, and 97% of animals in the herd harbored multiple strains. All the introduced naive animals became infected, and three to four strains were typically detected in an individual animal prior to seroconversion, while one to two new strains were detected in an individual animal following seroconversion. On average, the number of strains acquired via superinfection was 16% lower than the number acquired via coinfection. Thus, while complex infections develop via both coinfection and superinfection, coinfection predominates in this setting. These findings have broad implications for the development of control strategies in high-transmission settings.

Sequence and immunologic conservation of Anaplasma marginale OmpA within strains from Ghana as compared to the predominant OmpA variant.

A primary challenge in developing effective vaccines against obligate, intracellular, bacterial tick-borne pathogens that establish persistent infection is the identification of antigens that cross protect against multiple strains. In the case of Anaplasma marginale, the most prevalent tick-borne pathogen of cattle found worldwide, OmpA is an adhesin and thus a promising vaccine candidate. We sequenced ompA from cattle throughout Ghana naturally infected with A. marginale in order to determine the degree of variation in this gene in an area of suspected high genetic diversity. We compared the Ghanaian sequences with those available from N. America, Mexico, Australia and Puerto Rico. When considering only amino acid changes, three unique Ghanaian OmpA variants were identified. In comparison, strains from all other geographic regions, except one, shared a single OmpA variant, Variant 1, which differed from the Ghanaian variants. Next, using recombinant OmpA based on Variant 1, we determined that amino acid differences in OmpA in Ghanaian cattle as compared to OmpA Variant 1 did not alter the binding capacity of antibody directed against OmpA Variant 1, supporting the value of OmpA as a highly conserved vaccine candidate.

Identification of a T-Cell Epitope That Is Globally Conserved among Outer Membrane Proteins (OMPs) OMP7, OMP8, and OMP9 of Anaplasma marginale Strains and with OMP7 from the A. marginale subsp. centrale Vaccine Strain.

Within the protective outer membrane (OM) fraction of Anaplasma marginale, several vaccine candidates have emerged, including a family of OM proteins (OMPs) 7 to 9, which share sequence identity with each other and with the single protein OMP7 in the vaccine strain A. marginale subsp. centrale. A. marginale OMPs 7 to 9 are logical vaccine candidates because they are surface exposed, present in the OM immunogen and protective cross-linked OM proteins, recognized by immune serum IgG2 and T cells in cattle immunized with OM, and recognized by immune serum IgG2 from cattle immunized with the A. centrale vaccine strain. We report the identification of a globally conserved 9-amino-acid T-cell epitope FLLVDDAI/VV shared between A. centrale vaccine strain OMP7 and the related A. marginale OMPs 7 to 9, where position 8 of the peptide can be isoleucine or valine. The epitope is conserved in American A. marginale strains, in the Australia Gypsy Plains strain, and in multiple field isolates from Ghana. This epitope, together with additional T-cell epitopes that are present within these proteins, should be considered for inclusion in a multivalent vaccine for A. marginale that can provide protection against disease caused by globally distributed bacterial strains.

Disaggregating Tropical Disease Prevalence by Climatic and Vegetative Zones within Tropical West Africa.

Tropical infectious disease prevalence is dependent on many socio-cultural determinants. However, rainfall and temperature frequently underlie overall prevalence, particularly for vector-borne diseases. As a result these diseases have increased prevalence in tropical as compared to temperate regions. Specific to tropical Africa, the tendency to incorrectly infer that tropical diseases are uniformly prevalent has been partially overcome with solid epidemiologic data. This finer resolution data is important in multiple contexts, including understanding risk, predictive value in disease diagnosis, and population immunity. We hypothesized that within the context of a tropical climate, vector-borne pathogen prevalence would significantly differ according to zonal differences in rainfall, temperature, relative humidity and vegetation condition. We then determined if these environmental data were predictive of pathogen prevalence. First we determined the prevalence of three major pathogens of cattle, Anaplasma marginale, Babesia bigemina and Theileria spp, in the three vegetation zones where cattle are predominantly raised in Ghana: Guinea savannah, semi-deciduous forest, and coastal savannah. The prevalence of A. marginale was 63%, 26% for Theileria spp and 2% for B. bigemina. A. marginale and Theileria spp. were significantly more prevalent in the coastal savannah as compared to either the Guinea savanna or the semi-deciduous forest, supporting acceptance of the first hypothesis. To test the predictive power of environmental variables, the data over a three year period were considered in best subsets multiple linear regression models predicting prevalence of each pathogen. Corrected Akaike Information Criteria (AICc) were assigned to the alternative models to compare their utility. Competitive models for each response were averaged using AICc weights. Rainfall was most predictive of pathogen prevalence, and EVI also contributed to A. marginale and B. bigemina prevalence. These findings support the utility of environmental data for understanding vector-borne disease epidemiology on a regional level within a tropical environment.

Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector.

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity.

Many vector-borne pathogens evade clearance via rapid variation in their immunogenic surface expressed proteins. This is exemplified by Anaplasma marginale, a tick-borne bacterial pathogen that generates major surface protein 2 (Msp2) variants to provide for immune escape and allow long-term pathogen persistence. In contrast to persistence following infection, immunization with a surface protein complex, which includes Msp2, induces a response that prevents infection upon challenge. We hypothesized that the immune response induced by immunization altered the anti-Msp2 antibody repertoire as compared to that induced during infection, shifting the immune response toward conserved and thus broadly protective epitopes. The antibody response to the conserved (CR) and hypervariable (HVR) regions encoded by the full set of msp2 variant alleles was determined for immunized animals prior to challenge and non-immunized, infected animals. While both groups of animals had a similar antibody repertoire in terms of breath and magnitude, the titers to the Msp2 CR were strongly correlated (p<0.005) with control of bacteremia only in the infected animals. Among the immunized animals, there was no correlation between the breadth or magnitude of the anti-Msp2 antibody response and either complete protection from infection or control of bacteremia. This is consistent with separate immunologic mechanisms being responsible for control of bacteremia in infected animals as compared to immunized animals and suggests that conserved outer membrane proteins other than Msp2 are responsible for the complete clearance observed following challenge of vaccinees.

Generation of antigenic variants via gene conversion: Evidence for recombination fitness selection at the locus level in Anaplasma marginale.

Multiple bacterial and protozoal pathogens utilize gene conversion to generate antigenically variant surface proteins to evade immune clearance and establish persistent infection. Both the donor alleles that encode the variants following recombination into an expression site and the donor loci themselves are under evolutionary selection: the alleles that encode variants that are sufficiently antigenically unique yet retain growth fitness and the loci that allow efficient recombination. We examined allelic usage in generating Anaplasma marginale variants during in vivo infection in the mammalian reservoir host and identified preferential usage of specific alleles in the absence of immune selective pressure, consistent with certain individual alleles having a fitness advantage for in vivo growth. In contrast, the loci themselves appear to have been essentially equally selected for donor function in gene conversion with no significant effect of locus position relative to the expression site or origin of replication. This pattern of preferential allelic usage but lack of locus effect was observed independently for Msp2 and Msp3 variants, both generated by gene conversion. Furthermore, there was no locus effect observed when a single locus contained both msp2 and msp3 alleles in a tail-to-tail orientation flanked by a repeat. These experimental results support the hypothesis that predominance of specific variants reflects in vivo fitness as determined by the encoding allele, independent of locus structure and chromosomal position. Identification of highly fit variants provides targets for vaccines that will prevent the high-level bacteremia associated with acute disease.

Superinfection as a driver of genomic diversification in antigenically variant pathogens.

A new pathogen strain can penetrate an immune host population only if it can escape immunity generated against the original strain. This model is best understood with influenza viruses, in which genetic drift creates antigenically distinct strains that can spread through host populations despite the presence of immunity against previous strains. Whether this selection model for new strains applies to complex pathogens responsible for endemic persistent infections, such as anaplasmosis, relapsing fever, and sleeping sickness, remains untested. These complex pathogens undergo rapid within-host antigenic variation by using sets of chromosomally encoded variants. Consequently, immunity is developed against a large repertoire of variants, dramatically changing the scope of genetic change needed for a new strain to evade existing immunity and establish coexisting infection, termed strain superinfection. Here, we show that the diversity in the alleles encoding antigenic variants between strains of a highly antigenically variant pathogen was equal to the diversity within strains, reflecting equivalent selection for variants to overcome immunity at the host population level as within an individual host. This diversity among strains resulted in expression of nonoverlapping variants that allowed a new strain to evade immunity and establish superinfection. Furthermore, we demonstrated that a single distinct allele allows strain superinfection. These results indicate that there is strong selective pressure to increase the diversity of the variant repertoire beyond what is needed for persistence within an individual host and provide an explanation, competition at the host population level, for the large genomic commitment to variant gene families in persistent pathogens.

Maintenance of antibody to pathogen epitopes generated by segmental gene conversion is highly dynamic during long-term persistent infection.

Multiple bacterial and protozoal pathogens utilize gene conversion to generate rapid intrahost antigenic variation. Both large- and small-genome pathogens expand the size of the variant pool via a combinatorial process in which oligonucleotide segments from distinct donor loci are recombined in various combinations into expression sites. Although the potential combinatorial diversity generated by this segmental gene conversion mechanism is quite large, the functional variant pool depends on whether immune responses against the recombined segments are generated and maintained, regardless of their specific combinatorial context. This question was addressed by tracking the Anaplasma marginale variant population and corresponding segment-specific immunoglobulin G (IgG) antibody responses during long-term infection. Antibody was induced early in A. marginale infection, predominately against the surface-exposed hypervariable region (HVR) rather than against the invariant conserved flanking domains, and these HVR oligopeptides were most immunogenic at the time of acute bacteremia, when the variant population is derived via recombination from a single donor locus. However antibody to HVR oligopeptides was not consistently maintained during persistent infection, despite reexpression of the same segment, although in a different combinatorial context. This dynamic antibody recognition over time was not attributable to the major histocompatibility complex haplotype of individual animals or use of specific msp2 donor alleles. In contrast, the position and context of an individual oligopeptide segment within the HVR were significant determinants of antibody recognition. The results unify the genetic potential of segmental gene conversion with escape from antibody recognition and identify immunological effects of variant mosaic structure.

Selection for simple major surface protein 2 variants during Anaplasma marginale transmission to immunologically naïve animals.

Anaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginale expresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.

Insights into mechanisms of bacterial antigenic variation derived from the complete genome sequence of Anaplasma marginale.

Persistence of Anaplasma spp. in the animal reservoir host is required for efficient tick-borne transmission of these pathogens to animals and humans. Using A. marginale infection of its natural reservoir host as a model, persistent infection has been shown to reflect sequential cycles in which antigenic variants emerge, replicate, and are controlled by the immune system. Variation in the immunodominant outer-membrane protein MSP2 is generated by a process of gene conversion, in which unique hypervariable region sequences (HVRs) located in pseudogenes are recombined into a single operon-linked msp2 expression site. Although organisms expressing whole HVRs derived from pseudogenes emerge early in infection, long-term persistent infection is dependent on the generation of complex mosaics in which segments from different HVRs recombine into the expression site. The resulting combinatorial diversity generates the number of variants both predicted and shown to emerge during persistence.

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants.

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.

Transmission of Anaplasma marginale by Boophilus microplus: retention of vector competence in the absence of vector-pathogen interaction.

Whether arthropod vectors retain competence for transmission of infectious agents in the long-term absence of vector-pathogen interaction is unknown. We addressed this question by quantifying the vector competence of two tick vectors, with mutually exclusive tropical- versus temperate-region distributions, for genetically distinct tropical- and temperate-region strains of the cattle pathogen Anaplasma marginale. The tropical cattle tick Boophilus microplus, which has been eradicated from the continental United States for over 60 years, was able to acquire and transmit the temperate St. Maries (Idaho) strain of A. marginale. Similarly, the temperate-region tick Dermacentor andersoni efficiently acquired and transmitted the Puerto Rico strain of A. marginale. There were no significant quantitative differences in infection rate or number of organisms per tick following feeding on cattle with persistent infections of either A. marginale strain. In contrast, the significantly enhanced replication of the Puerto Rico strain in the salivary gland of B. microplus at the time of transmission feeding is consistent with adaptation of a pathogen strain to its available vector. However, the transmission of both strains by B. microplus demonstrates that adaptation or continual interaction between the pathogen and vector is not required for retention of vector competence. Importantly, the results clearly show that reestablishment of acaricide-resistant B. microplus in the United States would be associated with A. marginale transmission.