MHC class II genotype-by-pathogen genotype interaction for infection prevalence in a natural rodent-Borrelia system.

MHC genes are extraordinarily polymorphic in most taxa. Host-pathogen coevolution driven by negative frequency-dependent selection (NFDS) is one of the main hypotheses for the maintenance of such immunogenetic variation. Here, we test a critical but rarely tested assumption of this hypothesis-that MHC alleles affect resistance/susceptibility to a pathogen in a strain-specific way, that is, there is a host genotype-by-pathogen genotype interaction. In a field study of bank voles naturally infected with the tick-transmitted bacterium Borrelia afzelii, we tested for MHC class II (DQB) genotype-by-B. afzelii strain interactions for infection prevalence between 10 DQB alleles and seven strains. One allele (DQB*37) showed an interaction, such that voles carrying DQB*37 had higher prevalence of two strains and lower prevalence of one strain than individuals without the allele. These findings were corroborated by analyses of strain composition of infections, which revealed an effect of DQB*37 in the form of lower ß diversity among infections in voles carrying the allele. Taken together, these results provide rare support at the molecular genetic level for a key assumption of models of antagonistic coevolution through NFDS.

Borrelia Infection in Bank Voles Myodes glareolus Is Associated With Specific DQB Haplotypes Which Affect Allelic Divergence Within Individuals.

The high polymorphism of Major Histocompatibility Complex (MHC) genes is generally considered to be a result of pathogen-mediated balancing selection. Such selection may operate in the form of heterozygote advantage, and/or through specific MHC allele-pathogen interactions. Specific MHC allele-pathogen interactions may promote polymorphism via negative frequency-dependent selection (NFDS), or selection that varies in time and/or space because of variability in the composition of the pathogen community (fluctuating selection; FS). In addition, divergent allele advantage (DAA) may act on top of these forms of balancing selection, explaining the high sequence divergence between MHC alleles. DAA has primarily been thought of as an extension of heterozygote advantage. However, DAA could also work in concert with NFDS though this is yet to be tested explicitly. To evaluate the importance of DAA in pathogen-mediated balancing selection, we surveyed allelic polymorphism of MHC class II DQB genes in wild bank voles (Myodes glareolus) and tested for associations between DQB haplotypes and infection by Borrelia afzelii, a tick-transmitted bacterium causing Lyme disease in humans. We found two significant associations between DQB haplotypes and infection status: one haplotype was associated with lower risk of infection (resistance), while another was associated with higher risk of infection (susceptibility). Interestingly, allelic divergence within individuals was higher for voles with the resistance haplotype compared to other voles. In contrast, allelic divergence was lower for voles with the susceptibility haplotype than other voles. The pattern of higher allelic divergence in individuals with the resistance haplotype is consistent with NFDS favouring divergent alleles in a natural population, hence selection where DAA works in concert with NFDS.

Positive selection on MHC class II DRB and DQB genes in the bank vole (Myodes glareolus).

The major histocompatibility complex (MHC) class IIB genes show considerable sequence similarity between loci. The MHC class II DQB and DRB genes are known to exhibit a high level of polymorphism, most likely maintained by parasite-mediated selection. Studies of the MHC in wild rodents have focused on DRB, whilst DQB has been given much less attention. Here, we characterised DQB genes in Swedish bank voles Myodes glareolus, using full-length transcripts. We then designed primers that specifically amplify exon 2 from DRB (202 bp) and DQB (205 bp) and investigated molecular signatures of natural selection on DRB and DQB alleles. The presence of two separate gene clusters was confirmed using BLASTN and phylogenetic analysis, where our seven transcripts clustered according to either DQB or DRB homologues. These gene clusters were again confirmed on exon 2 data from 454-amplicon sequencing. Our DRB primers amplify a similar number of alleles per individual as previously published DRB primers, though our reads are longer. Traditional d N/d S analyses of DRB sequences in the bank vole have not found a conclusive signal of positive selection. Using a more advanced substitution model (the Kumar method) we found positive selection in the peptide binding region (PBR) of both DRB and DQB genes. Maximum likelihood models of codon substitutions detected positively selected sites located in the PBR of both DQB and DRB. Interestingly, these analyses detected at least twice as many positively selected sites in DQB than DRB, suggesting that DQB has been under stronger positive selection than DRB over evolutionary time.

Infection dynamics of the tick-borne pathogen "Candidatus Neoehrlichia mikurensis" and coinfections with Borrelia afzelii in bank voles in Southern Sweden.

The tick-borne bacterium "Candidatus Neoehrlichia mikurensis" has recently been recognized as a human pathogen. Together with Borrelia afzelii, it is one of the most common pathogens found in the tick Ixodes ricinus. Here, we compared the epidemiologies of "Ca. Neoehrlichia mikurensis" and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole (Myodes glareolus), using real-time PCR for detection and quantification. The prevalences of "Ca. Neoehrlichia mikurensis" and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of "Ca. Neoehrlichia mikurensis" increased significantly during the sampling season. The clearance rate of "Ca. Neoehrlichia mikurensis" was significantly higher than that of B. afzelii. We found a high frequency of double infections; 46% of all samples infected with "Ca. Neoehrlichia mikurensis" also had a coinfection with B. afzelii. The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.

Polymorphisms at the innate immune receptor TLR2 are associated with Borrelia infection in a wild rodent population.

The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture-mark-recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.

Multiple-strain infections of Borrelia afzelii: a role for within-host interactions in the maintenance of antigenic diversity?

Genetically diverse infections are common but little is known about what effects coinfecting strains have on each other in natural host-parasite systems. To explore the nature and consequences of interactions in the wild, we studied the tick-transmitted bacterium Borrelia afzelii in one of its main reservoir hosts, the bank vole Myodes glareolus. We measured overall infection intensity with quantitative polymerase chain reaction (PCR) and resolved the composition of multiple infections using strain-specific PCR assays targeting the ospC gene (which encodes an immunodominant surface protein). We found seven different strains, as defined by ospC genotype. There was little evidence for interactions affecting infection intensities, but strains were highly aggregated (i.e., there were more multiple infections than expected from random co-occurrence). Moreover, there was a positive correlation between the difference at the amino acid level between two OspC types and their degree of association. Overall, the observed patterns suggest that facilitation is more important than competition in this system and that more diverse infections have an advantage in establishing and/or maintaining infection. We propose that this advantage is one of the factors that favors antigenic diversity.

Contrasting patterns of diversity and population differentiation at the innate immunity gene toll-like receptor 2 (TLR2) in two sympatric rodent species.

Comparing patterns of diversity and divergence between populations at immune genes and neutral markers can give insights into the nature and geographic scale of parasite-mediated selection. To date, studies investigating such patterns of selection in vertebrates have primarily focused on the acquired branch of the immune system, whereas it remains largely unknown how parasite-mediated selection shapes innate immune genes both within and across vertebrate populations. Here, we present a study on the diversity and population differentiation at the innate immune gene Toll-like receptor 2 (TLR2) across nine populations of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) in southern Sweden. In yellow-necked mice, TLR2 diversity was very low, as was TLR2 population differentiation compared to neutral loci. In contrast, several TLR2 haplotypes co-occurred at intermediate frequencies within and across bank vole populations, and pronounced isolation by distance between populations was observed. The diversity and differentiation at neutral loci was similar in the two species. These results indicate that parasite-mediated selection has been acting in dramatically different ways on a given immune gene in ecologically similar and sympatric species. Furthermore, the finding of TLR2 population differentiation at a small geographical scale in bank voles highlights that vertebrate innate immune defense may be evolutionarily more dynamic than has previously been appreciated.