Baylisascaris (Sprent, 1968)

Camp, Lauren E., Radke, Marc R., Shihabi, Danny M., Pagan, Christopher, Yang, Guangyou & Nadler, Steven A., 2018, Molecular phylogenetics and species-level systematics of Baylisascaris, International Journal for Parasitology: Parasites and Wildlife 7 (3), pp. 450-462 : 458-459

4.2. Discrimination of raccoon and skunk Baylisascaris

Baylisascaris procyonis and B. columnaris are believed to be closely related, but distinct, species. However, discriminating between B. procyonis and B. columnaris is difficult. Morphologically, they are almost indistinguishable, and identifying the species of larval or adult B.

procyonis or B. columnaris based on morphology alone is not always possible even for specialists ( Kazacos, 2001; Graeff-Teixeira et al., 2016). Previous authors have also attempted to discriminate between B. columnaris and B. procyonis based on pathogenicity in paratenic hosts, protein electrophoresis ( Berry, 1985), larval excretory-secretory antigens ( Dangoudoubiyam et al., 2010), and DNA sequence data ( Dangoudoubiyam et al., 2009; Gatcombe et al., 2010; Franssen et al., 2013; Choi et al., 2017). The most pronounced difference between skunk- and raccoon-derived worms is in pathogenicity for paratenic hosts. Clinical baylisascariasis can occur when paratenic hosts ingest infective eggs of either B. procyonis or B. columnaris , but fewer B. procyonis eggs are needed to cause disease ( Kazacos, 2001).

Morphological characters proposed to discriminate between B. columnaris and B. procyonis include shape of the tail tip in males, rough areas near the cloaca in males, and lip denticle shape. Subsets of these characters have recently been used in research focused on distinguishing B. columnaris from B. procyonis (e.g. Franssen et al., 2013) or describing new species ( B. potosis, Tokiwa et al., 2014 ). However, when Berry (1985) analyzed these characters for multiple individuals of B. columnaris , B. procyonis , and B. laevis , he determined that they were too variable to be useful for discrimination. Therefore, most contemporary studies have turned to molecular data in attempts to discriminate between B. procyonis and B. columnaris .

Phylogenetic analyses based on molecular data have consistently resolved skunk- and raccoon-derived Baylisascaris specimens as members of the same clade (e.g., Tranbenkova and Spiridonov, 2017) but they have been unclear with regard to further delimiting B. procyonis and B. columnaris . The inclusion of variable nuclear genes (msp, ard1, and hars1) in the current phylogenetic analysis resolved reciprocal monophyly of specimens derived from raccoon and skunk hosts in a single tree: the BI tree for combined genes including hars1 ( Fig. S1 ). However, both clades lacked strong support (BPP 0.64 for B. columnaris specimens and 0.63 for B. procyonis specimens). In some analyses, the B. columnaris specimens were monophyletic, but the B. procyonis specimens were not (cox1, cox2, mitochondrial genes, and certain combined data, Tables S7 and S8, Figs. 1 and 3 ). In other cases, there was either no resolution or the resolved clades included individuals of both species (Tables S7 and S8, Fig. 2 and Fig. S2 ). The alternative topology depicting reciprocal monophyly for B. columnaris and B. procyonis was significantly worse for two datasets (nuclear genes with hars1, and combined data without hars1) but was not significantly worse than the original topologies for the other three datasets (Table S9).

In order to more fully utilize the data collected from multiple loci, we employed a multispecies coalescent approach using the program BP &P ( Yang, 2015) to test the hypothesis that B. procyonis and B. columnaris are separate species. Given a prior of three species (skunk-derived worms, raccoon-derived worms, and B. transfuga ALB as the test taxa), support for B. columnaris and B. procyonis as separate species was resolved by two combinations of Θ and τ 0 priors that we tested. These priors correspond to a shallow divergence time with either a large or moderate effective population size ( Table 4). With large population size and shallow divergence time, support for B. procyonis and B. columnaris as separate species was PP 0.72, and support for B. transfuga ALB as a species was higher (PP 0.93; Table 4). With priors for a moderate population size and shallow divergence, support for separate species was stronger (PP 0.91), and support for B. transfuga ALB was absolute ( Table 4). Effective population sizes for Baylisascaris species are probably similar to Ascaris in that they are large, but smaller than trichostrongylids in domesticated ruminants ( Anderson and Jaenike, 1997), which tend to have thousands of worms per host. Raccoons have been documented to have hundreds of worms in their small intestines ( Kazacos, 2001, 2016; Weinstein, 2016), although average infrapopulations are smaller. For the divergence time prior, it is unlikely that the split between B. procyonis and B. columnaris was deep given their low sequence divergence across genes included in this and other studies. Prior combinations corresponding to a shallow divergence were the most reasonable based on the posterior distributions obtained using the BP&P program (module A00). Future analyses using BP&P would benefit from including more nuclear loci and additional geographic isolates and individuals of skunk- and raccoon-derived worms.

Recent attempts at using molecular data to distinguish B. columnaris and B. procyonis were conducted by Franssen et al. (2013) and Choi et al. (2017). Franssen et al. (2013) analyzed specimens obtained from skunk hosts in the Netherlands, and from raccoon hosts in Indiana, USA and Norway, and reported a GA repeat region in ITS-2 with unique patterns for individuals of B. procyonis (nine repeats) and B. columnaris (six or seven repeats). In addition, the authors reported 14 single nucleotide polymorphisms (SNPs) among several genes. Five of these SNPs were reported to be identical in isolates from skunk hosts, but different in isolates from raccoons: three SNPs in cox1 and one SNP each in cox2 and ITS-1. These comparisons were based on 12 B. procyonis and 15 B. columnaris for ITS-1; 19 B. procyonis and 43 B. columnaris for cox1; and 10 B. procyonis and 38 B. columnaris for cox2.

Choi et al. (2017) obtained mitochondrial sequence data for 10 skunk-derived Baylisascaris in Salt Lake County, Utah, USA. They compared complete sequences of 11 mitochondrial genes – cox1, cox2, nd2, and 8 tRNA genes – with mitochondrial genome sequences of one B. procyonis specimen ( Xie et al., 2011b) and with cox1 and cox2 sequences obtained by Franssen et al. (2013). In total, Choi et al. (2017) reported 11 SNPs that were presumed diagnostic for skunk- and raccoon-derived Baylisascaris : six SNPs in cox1; three SNPs in nd2; one SNP in tRNA-Leu; and one SNP in tRNA-Ser. In contrast to Franssen et al. (2013) no diagnostic SNPs were identified in cox2. The SNPs identified by Choi et al. (2017) were based on one B. procyonis and 34 B. columnaris for cox1; one B. procyonis and 9 B. columnaris for cox2; and one B. procyonis and 10 B. columnaris for nd2 and the tRNA genes.

In order to assess the reliability of putatively diagnostic SNPs reported by Franssen et al. (2013) and Choi et al. (2017), we compared their sequences with cox1, cox2, ITS-1, and ITS-2 sequences from additional isolates of raccoon- and skunk-derived worms generated in our lab. For ITS, cox1, and cox2, we compared additional sequences of B. columnaris (two, six, and two, individuals, respectively) and B. procyonis (two, 44, and two, respectively). We did not have additional sequences for nd2 or tRNA and could not further assess SNPs in those genes ( Choi et al., 2017).

When more individuals from the United States were compared (based on RFLP screening of individuals followed by confirmation by sequencing) with the individuals sequenced by Franssen et al. (2013) and Choi et al. (2017), intraindividual polymorphism at the SNP from ITS-1 (position 201 from Franssen et al., Table 4 – T in B. columnaris and C in B. procyonis ) was revealed. The pattern of GA repeats in ITS-2 was the same in our specimens as in those of Franssen and colleagues (six for B. columnaris isolates and nine for B. procyonis isolates, Table 5). For cox1 SNPs, none of those identified as diagnostic by Franssen et al. (2013) were unique to skunk- or raccoon-derived worms. Choi and colleagues identified five additional putatively diagnostic SNPs in full-length cox1 ( Fig. 1 , Choi et al. (2017)) at the following positions: 231; 1266; 1315; 1491; and 1506. Partial cox1 sequences from skunk- and raccoon-derived worms generated in our lab were used to assess cox1 SNPs at positions 1266 and 1315 of Choi et al. (2017), but SNPs at other positions could not be assessed due to lack of sequence overlap. The SNPs at positions 1266 and 1315 of cox1 were not specific for skunk- and raccoon-derived worms. For the cox2 SNP, B. procyonis CA had the same sequence as the skunk isolates sequenced by Franssen et al. (position 66 from Table 3 in Franssen et al.). Considering all three studies, these comparisons are based on 14 B. procyonis and 17 B. columnaris for full length ITS, 13 B. procyonis and 49 B. columnaris for cox2, and 64 B. procyonis and 83 B. columnaris for cox1. Further testing of the ITS-2 GA repeat diagnostic region is needed, however, this is made more difficult by the need to clone PCR products to obtain high quality sequence from this region. These results emphasize the importance of having sufficient sample sizes and appropriate sampling from all hosts and geographic regions for testing SNPs as species diagnostic markers. For example, in comparison to Baylisascaris species, Ascaris from humans and pigs have been broadly geographically sampled for comparative genetics ( Betson et al., 2013), and geographic variation in the cox1 gene of Ascaris (e.g., Betson et al., 2011) has revealed more than 50 haplotypes.

One potential problem with using only molecular methods to distinguish B. columnaris and B. procyonis is the possibility that these species lack strict host specificity for skunks and raccoons. Nadler (2010, unpublished) used comparisons of ITS sequences for skunk- and raccoon-derived worms to develop a restriction fragment length polymorphism test based on a SNP difference in their ITS-1 sequences; this is the same SNP reported by Franssen et al. (2013). The restriction enzyme Apo I recognizes the sequence 5′-RAATTY; at the SNP site, sequences derived from raccoon Baylisascaris have the sequence AAACTT, whereas sequences from skunks have AAATTT. Raccoon Baylisascaris ITS lacks the Apo I recognition site relative to skunk Baylisascaris specimens. Using this RFLP test, 148 individuals from raccoons were assignable to B. procyonis , 34 individuals from skunks were assignable to B. columnaris , but 12 individuals from raccoon hosts had the polymorphic pattern, and a single individual from a raccoon was assignable to B. columnaris . The polymorphic ITS pattern found in some raccoon Baylisascaris could reflect an ancestral polymorphism maintained in B. procyonis rDNA despite concerted evolution of multicopy rDNA, or perhaps even past hybridization event(s) with subsequent backcrosses to B. procyonis individuals ( Camp et al., 2011). These polymorphic individuals call into question the species-specific diagnostic value of this ITS-1 SNP.

Figures

Fig. 1. Bayesian consensus tree based on combined FULL data (8 genes; not including hars1). Branch lengths are scaled to the expected number of substitutions per site. Numbers above nodes are Bayesian posterior probabilities, shown when 0.90 and greater.

Fig. 3. Bayesian consensus tree based on FULL mitochondrial gene sequences (3 genes). Branch lengths are scaled to the expected number of substitutions per site. Numbers above nodes represent Bayesian posterior probabilities, shown when 0.90 and greater.

Fig. 2. Bayesian consensus tree based on FULL data from nuclear genes (5 genes; not including hars1). Branch lengths are scaled to the expected number of substitutions per site. Numbers above nodes represent Bayesian posterior probabilities, shown when 0.90 and greater.