Within-host competition between Borrelia afzelii ospC strains in wild hosts as revealed by massively parallel amplicon sequencing

Abstract

Infections frequently consist of more than one strain of a given pathogen. Experiments have shown that co-infecting strains often compete, so that the infection intensity of each strain in mixed infections is lower than in single strain infections. Such within-host competition can have important epidemiological and evolutionary consequences. However, the extent of competition has rarely been investigated in wild, naturally infected hosts, where there is noise in the form of varying inoculation doses, asynchronous infections and host heterogeneity, which can potentially alleviate or eliminate competition. Here, we investigated the extent of competition between Borrelia afzelii strains (as determined by ospC genotype) in three host species sampled in the wild. For this purpose, we developed a protocol for 454 amplicon sequencing of ospC, which allows both detection and quantification of each individual strain in an infection. Each host individual was infected with one to six ospC strains. The infection intensity of each strain was lower in mixed infections than in single ones, showing that there was competition. Rank-abundance plots revealed that there was typically one dominant strain, but that the evenness of the relative infection intensity of the different strains in an infection increased with the multiplicity of infection. We conclude that within-host competition can play an important role under natural conditions despite many potential sources of noise, and that quantification by next-generation amplicon sequencing offers new possibilities to dissect within-host interactions in naturally infected hosts.

Keywords: Borrelia afzelii; mixed infections; ospC; virulence; within-host competition.

Figure 1.

Correlation between strain-specific infection intensities measured from two linked B. afzelii loci (ospC and rrs-rrlA IGS) in naturally infected small mammals (bank voles (N = 50), yellow-necked mice (N = 50) and common shrews (N = 24) sampled at Kalvs mosse, Sweden). Five linkage groups of the two loci [13] are indicated with different colours. The infection intensity of each strain was estimated by 454 amplicon sequencing of the two loci in combination with qPCR of B. afzelii flaB.

Figure 2.

Frequency distribution of infections with different number of B. afzelii ospC strains in three species of naturally infected small mammals (bank voles (N = 45), yellow-necked mice (N = 27) and common shrews (N = 22)) sampled at Kalvs mosse, Sweden. The number of ospC strains per individual was determined from 454 amplicon sequencing data.

Figure 3.

Box plot of infection intensity per B. afzelii ospC strain in infections with different number of strains in three small mammal species (bank voles (N = 45), yellow-necked mice (N = 27) and common shrews (N = 22)) sampled at Kalvs mosse, Sweden. The box plots indicate the median, first and third quartiles, and range of the data. Relative infection intensity of each ospC strain in an infection was determined from 454 amplicon sequencing of ospC. Total infection intensity was estimated with qPCR of B. afzelii flaB.

Figure 4.

Rank-abundance plot of natural B. afzelii infections with different number of strains in small mammals (N = 94). The box plots indicate the (log₁₀) median, quartiles and range of the proportions of the most abundant strain in each infection (abundance rank = 1), the second most abundant (abundance rank = 2), and so on, in infections with two to six ospC strains. The slope of the relationship between relative abundance and rank indicates the evenness of relative abundance in infections with a certain number of strains; a more shallow slope means higher evenness. Relative abundance of the strains was determined by 454 amplicon sequencing of B. afzelii ospC.