Baylisascaris columnaris subsp. prevalence

Choi, Youna, Mason, Sara, Ahlborn, Michael, Zscheile, Brook & Wilson, Eric, 2017, Partial molecular characterization of the mitochondrial genome of Baylisascaris columnaris and prevalence of infection in a wild population of Striped skunks, International Journal for Parasitology: Parasites and Wildlife 6 (2), pp. 70-75 : 72

3.1. B. columnaris prevalence in a wild population of skunks

We first sought to determine the prevalence of B. columnaris in a wild population of skunks. The intestinal tracts of 16 skunks were collected and the prevalence of nematode infection determined by gross examination of the intestinal contents. Ten of the 16 skunks examined were found to be infected with roundworms, with numbers of worms ranging from 1 to 10 in infected animals ( Table 2). All worms were identified as B. columnaris based on gross morphology and confirmed by detailed sequence homology to the published partial sequence of B. columnaris Cox 1 (GenBank: KC543474.1).

3.2. DNA sequence heterogeneity between B. procyonis and B. columnaris

Due to the essential function of Cox1, this gene is present in a wide variety of organisms yet has sufficient variation to distinguish closely related species ( Hebert et al., 2003). Consequently, the sequence of the Cox1 gene is a frequently used marker for population genetic and phylogenetic studies ( Ai et al., 2011; Xie et al., 2011b). In this study, the complete Cox1 gene (1578 bp) of eight Baylisascaris worms isolated from eight different hosts was determined ( Table 3). Our analysis revealed 11 novel loci, which consistently distinguished B. columnaris from B. procyonis , including the three previously reported by Franssen et al. ( Fig. 1 ). The majority of these species specific SNPs are found in the 3’ portion of the gene with 8 of the 11 being found between nucleotides 1002 and 1506 of the Cox1 gene. Previously published work has shown a relatively high degree of intraspecies heterogeneity in the Cox1 gene of B. columnaris ( Franssen et al., 2013) . In our analysis of Cox1, 11 total intraspecies SNPs were identified 10 of these having not previously been reported ( Fig. 1 ).

Previous sequencing of a partial sequence of the B. columnaris Cox 2 gene has shown one specific SNP which was useful in differentiating B. procyonis from B. columnaris as well as three intragenic SNPs ( Franssen et al., 2013). These previous studies were done in a European population of skunks. In our sequencing analysis of a North American population, we found six intragenic SNPs within the Cox2 gene. Four of these SNPs were previously identified by Franssen et al. (2013). Importantly, our data showed that none of these nucleotide variations were species specific, whereas previous analysis had suggested the SNP at position 168 was useful in differentiating B. columnaris from B. procyonis ( Fig. 2 ). There have been no previous reports of sequencing of the B. columnaris ND 2 gene. In our sequencing of this gene and subsequent analysis, three SNPs were identified which consistently differentiated B. procyonis from B. columnaris . In addition, two intraspecies SNP were identified in the ND2 gene of B. columnaris ( Fig. 3 ).

Alignment of eight of the B. columnaris concatenated sequences and homologous regions of B. procyonis were used to determine the sequence of several tRNA genes from B. columnaris . This analysis revealed two SNPs and an indel within mt-tRNA genes which differentiated B. procyonis from B. columnaris . In addition, five intragenic SNPs were identified (one of them being in the same position as the indel of tRNA S ( Fig. 4 )). Based on the high degree of similarity between these organisms, we were initially skeptical of this degree of variation in tRNA genes. Therefore, we next compared the tRNA genes from all mt-DNA genomes of sequenced Baylisascaris species. This comparison demonstrated a relatively high degree of sequence variation in the tRNA genes of several closely related Baylisascaris species ( Fig. 4 ). This level of heterogeneity in tRNA sequences from numerous Baylisascaris species lends confidence in this unanticipated level of heterogeneity if tRNA genes. Additionally, concatenated sequences were used to generate maximum likelihood relationships of B. columnaris with other Ascarid species. Results of these analyses demonstrated maximum likelihood relationships in agreement with previous findings ( Franssen et al., 2013).

Figures

Fig. 1. Single nucleotide polymorphisms in the Cox1 gene of B. columnaris, compared to B. procyonis. Nucleotide position numbers are shown at the top of the figure. Italicized numbers represent the position number from a previously published partial sequence of the B.columnaris Cox1 gene (Franssen et al., 2013). Species-specific SNPs are shown in bold.

Fig. 2. Single nucleotide polymorphisms in the Cox2 gene of B. columnaris, compared to B. procyonis. Nucleotide position numbers are shown at the top of the figure. Italicized numbers represent the position number from a previously published partial sequence of the B. columnaris Cox2 gene (Franssen et al., 2013).

Fig. 3. Single nucleotide polymorphisms in the ND2 gene of B.columnaris, compared to B. procyonis. Nucleotide position numbers are shown at the top of the figure. Speciesspecific SNPs are shown in bold.

Fig. 4. Single nucleotide polymorphisms in several tRNA genes of B. columnaris, compared to B. procyonis, B. transfuga and B. schroederi. Nucleotide position numbers are shown at the top of the figure. SNPs which distinguish B. columnaris from other Baylisascaris species are shown in bold.