Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains.

BACKGROUND: Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin? METHODOLOGY/PRINCIPAL FINDINGS: To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin. CONCLUSIONS/SIGNIFICANCE: These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

Local mitochondrial DNA haplotype databases needed for domestic dog populations that have experienced founder effect.

Biological material from pets is often collected as evidence from crime scenes. Due to sample type and quality, mitochondrial DNA (mtDNA) is frequently evaluated to identify the potential contributor. MtDNA has a lower discriminatory power than nuclear DNA with multiple individuals in a population potentially carrying the same mtDNA sequence, or haplotype. The frequency distribution of mtDNA haplotypes in a population must be known in order to determine the evidentiary value of a match between crime scene evidence and the potential contributor of the biological material. This is especially important in geographic areas that include remote and/or isolated populations where founder effect may have lead to a decrease in genetic diversity and a non-random distribution of haplotypes relative to the population at large. Here we compared the haplotype diversity in dogs from the noncontiguous states of Alaska and Hawaii relative to the contiguous United States (US). We report a greater proportion of dogs carrying an A haplotype in Alaska relative to any other US population. Significant variation in the distribution of haplotype frequencies was discovered when comparing the haplotype diversity of dogs in Hawaii to that of the continental US. Each of these regions exhibits reduced genetic diversity relative to the contiguous US, likely due to founder effect. We recommend that specific databases be created to accurately represent the mitochondrial haplotype diversity in these remote areas. Furthermore, our work demonstrates the importance of local surveys for populations that may have experienced found effect.

Forensic informativity of ~3000 bp of coding sequence of domestic dog mtDNA.

The discriminatory power of the noncoding control region (CR) of domestic dog mitochondrial DNA alone is relatively low. The extent to which the discriminatory power could be increased by analyzing additional highly variable coding regions of the mitochondrial genome (mtGenome) was therefore investigated. Genetic variability across the mtGenome was evaluated by phylogenetic analysis, and the three most variable ~1 kb coding regions identified. We then sampled 100 Swedish dogs to represent breeds in accordance with their frequency in the Swedish population. A previously published dataset of 59 dog mtGenomes collected in the United States was also analyzed. Inclusion of the three coding regions increased the exclusion capacity considerably for the Swedish sample, from 0.920 for the CR alone to 0.964 for all four regions. The number of mtDNA types among all 159 dogs increased from 41 to 72, the four most frequent CR haplotypes being resolved into 22 different haplotypes.

Next-generation sequencing of the Trichinella murrelli mitochondrial genome allows comprehensive comparison of its divergence from the principal agent of human trichinellosis, Trichinella spiralis.

The mitochondrial genome's non-recombinant mode of inheritance and relatively rapid rate of evolution has promoted its use as a marker for studying the biogeographic history and evolutionary interrelationships among many metazoan species. A modest portion of the mitochondrial genome has been defined for 12 species and genotypes of parasites in the genus Trichinella, but its adequacy in representing the mitochondrial genome as a whole remains unclear, as the complete coding sequence has been characterized only for Trichinella spiralis. Here, we sought to comprehensively describe the extent and nature of divergence between the mitochondrial genomes of T. spiralis (which poses the most appreciable zoonotic risk owing to its capacity to establish persistent infections in domestic pigs) and Trichinella murrelli (which is the most prevalent species in North American wildlife hosts, but which poses relatively little risk to the safety of pork). Next generation sequencing methodologies and scaffold and de novo assembly strategies were employed. The entire protein-coding region was sequenced (13,917 bp), along with a portion of the highly repetitive non-coding region (1524 bp) of the mitochondrial genome of T. murrelli with a combined average read depth of 250 reads. The accuracy of base calling, estimated from coding region sequence was found to exceed 99.3%. Genome content and gene order was not found to be significantly different from that of T. spiralis. An overall inter-species sequence divergence of 9.5% was estimated. Significant variation was identified when the amount of variation between species at each gene is compared to the average amount of variation between species across the coding region. Next generation sequencing is a highly effective means to obtain previously unknown mitochondrial genome sequence. Particular to parasites, the extremely deep coverage achieved through this method allows for the detection of sequence heterogeneity between the multiple individuals that necessarily comprise such templates.

Deep resequencing of Trichinella spiralis reveals previously un-described single nucleotide polymorphisms and intra-isolate variation within the mitochondrial genome.

The phylogeny and historical dispersal of Trichinella spp. have been studied, in part, by sequencing portions of the mitochondrial genome. Such studies rely on two untested beliefs: that variation in a portion is representative of the entire mitochondrial genome, and that each isolate is characterized by only one mitochondrial haplotype. We have used next generation DNA sequencing technology to obtain the complete mitochondrial genome sequence from a second isolate of T. spiralis. By aligning it to the only previously sequenced genome, we sought to establish whether the exceptionally deep sequencing coverage provided by such an approach could detect regions of the genome which had been misassembled, or nucleotide positions which may vary within an isolate. The new data broadly confirm the gene order and sequence assembly for protein-coding regions. However, in the repetitive non-coding region, alignment to the previously published genome sequence proved difficult. Such discrepancies may represent true biological variation, but may rather result from methodological or algorithmic sources. Within the 13,902bp protein-coding region, 7 polymorphisms were identified. Six of these polymorphisms occurred within protein-coding genes and three alter an amino acid sequence, one occurred in a tRNA-Ile sequence, and four were found to vary within our isolate. Thus, comparing only two isolates of T. spiralis has enabled the discovery of previously unrecognized variation within the species. Characterizing diversity within and among the mitochondrial genomes of additional species of Trichinella would undoubtedly yield further insights into the diversification history of the genus. Our study affirms that next generation DNA sequencing technology can reliably characterize a complete mitochondrial genome.

Mitochondrial genome DNA analysis of the domestic dog: identifying informative SNPs outside of the control region.

While the mitochondrial control region has proven successful for human forensic evaluations by indicating ethnic origin, domestic dogs (Canis lupus familiaris) of seemingly unrelated breeds often form large groups based on identical control region sequences. In an attempt to break up these large haplotype groups, we have analyzed the remaining c. 15,484 base pairs of the canine mitochondrial genome for 79 dogs and used phylogenetic and population genetic methods to search for additional variability in the form of single nucleotide polymorphisms (SNPs). We have identified 356 SNPs and 65 haplotypes in the remainder of the mitochondrial genome excluding the control region. The exclusion capacity was found to be 0.018. The mitochondrial control region was also evaluated for the same 79 dogs. The signals from the different fragments do not conflict, but instead support one another and provide a larger fragment of DNA that can be analyzed as forensic evidence.

Identification of forensically informative SNPs in the domestic dog mitochondrial control region.

Dog hair is often found at crime scenes either due to the dog's involvement in the crime or secondary transfer. As little nuclear DNA is present in shed hair, a 1000 base pair fragment of the mitochondrial control region (mtCR) from 552 dogs was assessed for forensically useful sequence variation. Through pairwise alignment to a standard reference sequence, existing haplotypes were further described and 36 new haplotypes and 24 new single nucleotide polymorphisms were identified. The probability of exclusion was found to be 0.957. Breeds were found to have similar sequences, although not identical. No genetic basis was found for grouping dogs by either purebred or mixed or geographic location within the continental United States. Our research demonstrates that the domestic dog mtCR has not been thoroughly surveyed for sequence variation and that a single database comprised of purebred and mixed breed dogs is sufficient for the continental United States.

Forensic utility of the mitochondrial hypervariable region 1 of domestic dogs, in conjunction with breed and geographic information.

The 608-bp hypervariable region 1 (HV1) sequences from 36 local dogs were analyzed to characterize the population genetic structure of canid mitochondrial DNA (mtDNA). Sixteen haplotypes were identified. A 417-bp segment of this sequence was compared with GenBank sequences from a geographically representative sample of 201 dogs, two coyotes, and two wolves. Sixty-six haplotypes were identified including 62 found only in domestic dogs. Fourteen of these correspond to the 16 local haplotypes and were among the most frequent haplotypes. The local sample was judged to be representative of the much broader geographic sample. No correlation was observed between local haplotypes and the owner's characterization of dog breed. A 60-bp variation "hotspot" within the canid HV1 was identified as a potentially valuable molecular tool, particularly for assaying limited or degraded DNA samples.