Mixed infections, cryptic diversity, and vector-borne pathogens: evidence from Polygenis fleas and Bartonella species

Abstract

Coinfections within hosts present opportunities for horizontal gene transfer between strains and competitive interactions between genotypes and thus can be a critical element of the lifestyles of pathogens. Bartonella spp. are Alphaproteobacteria that parasitize mammalian erythrocytes and endothelial cells. Their vectors are thought to be various biting arthropods, such as fleas, ticks, mites, and lice, and they are commonly cited as agents of various emerging diseases. Coinfections by different Bartonella strains and species can be common in mammals, but little is known about specificity and coinfections in arthropod vectors. We surveyed the rate of mixed infections of Bartonella in flea vectors (Polygenis gwyni) parasitizing cotton rats (Sigmodon hispidus) in which previous surveys indicated high rates of infection. We found that nearly all fleas (20 of 21) harbored one or more strains of Bartonella, with rates of coinfection approaching 90%. A strain previously identified as common in cotton rats was also common in their fleas. However, another common strain in cotton rats was absent from P. gwyni, while a rare cotton rat strain was quite common in P. gwyni. Surprisingly, some samples were also coinfected with a strain phylogenetically related to Bartonella clarridgeiae, which is typically associated with felids and ruminants. Finally, a locus (pap31) that is characteristically borne on phage in Bartonella was successfully sequenced from most samples. However, sequence diversity in pap31 was novel in the P. gwyni samples, relative to other Bartonella previously typed with pap31, emphasizing the likelihood of large reservoirs of cryptic diversity in natural populations of the pathogen.

FIG. 1.

Bayesian phylogeny of the genus Bartonella, including many of the described species. The tree is rooted with B. bacilliformis and is based on partial sequences from five concatenated loci, with the exception of those shown in bold (see Table 1 for GenBank accession numbers). The bold taxa represent type isolates of the genogroups (designated A thru D) discovered in previous surveys of cotton rats in the southeastern United States (32, 33). Only gltA sequences are available for these. All nonterminal resolved nodes had clade credibility values of >98, based on the Bayesian analysis. The overall topology was supported by parsimony analysis. Arrows indicate the phylogenetic placement of the different P. gwyni-derived isolates on the constrained Bartonella phylogeny, based on neighbor-joining placement of the amplicons on the tree. With the exception of the isolates similar to B. clarridgeiae and B. rochalimae, most were >99% similar to the designated A or B genogroup. Most amplicons were confirmed by redundant sequencing of multiple cloned products.

FIG. 2.

Matrix of genetic similarity (fraction of identical sites) in a 337-bp fragment of gltA cloned from Polygenis gwyni fleas collected from cotton rats (Sigmodon hispidus). Only representative flea-Bartonella samples are shown. Many fleas contained mixed infections, and examples are shown (numbers 2, 4, and 11). The grey shading highlights the values that compare the P. gwyni strains to the corresponding strains A1 through A5 and B1 through B5 previously cultured from S. hispidus (33). The bold values represent the highest similarity values for each flea-associated Bartonella sample to the various A or B genogroups described from cotton rats. B. clarridgeiae and the newly described B. rochalimae (18) are flagellated species distantly related to Bartonella species described from cotton rats but nevertheless are genetically similar to amplicons from the fleas of cotton rats.

FIG. 3.

Consensus tree of Bartonella taxa based on partial pap31 sequences. Numbers above interior branches represent clade credibility values from the Bayesian analysis (above) and 100 maximum likelihood bootstrap replicates (below). Most amplicons were confirmed by redundant sequencing of multiple cloned products. Three distinct genotypic groups are evident from the fleas of cotton rats, as shown. The genetic distance between groups II and III, based on 152 bp of alignable transmembrane domain sequences, was approximately 1.4% (uncorrected p distance). Group I differed from both by approximately 11 to 12%. The Bartonella isolates in these groups have no clear identity based on BLAST searches of pap31 sequences. Highest BLAST scores were returned for B. henselae or B. quintana, but this probably reflects the limited taxonomic sampling of pap31 across the genus.

FIG. 4.

Amino acid fragments of pap31 homologs from various taxa and a subset amplified from representative flea samples in the present study. The fragment corresponds to an outer membrane loop sequence between conserved transmembrane domains 3 and 4, as described in reference . These loops may be composed of nearly random host chromosomal sequences (56), as evident in the lack of conservation between the related B. henselae and B. quintana. Seven of the nine P. gwyni isolates exhibit strong conservation, likely indicating a recent common ancestor. Some samples align more closely to B. henselae strains, as indicated by gray shading. In one group (clade II from Fig. 3), a 1-bp deletion (grey hatched box) causes a UAA stop codon downstream. A gap has been inserted to maintain the alignment in these samples. The black box highlights an insertion of three residues. Dots indicate matches with the topmost sequence, dashes indicate gaps, and question marks indicate uncertainties due to unresolvable ambiguities in the nucleotide sequences. The black line separates the pap31 amplicons from the present study and those from known species. Dissimilar amplicons were cloned from the same flea samples, as illustrated by P. gwyni numbers 1, 9, and 11.