the baylisascaris genus of nematodes ( family : ascarididae ) is comprised of nine recognized species , each parasitizing distinct definitive hosts and a vast array of intermediate hosts ( bauer , 2013 , kazacos , 2001 , sorvillo et  al . , 2002 ) .
the most widely studied of these is b.  procyonis , which primarily utilizes the common raccoon ( procyon lotor ) as its principal definitive host ( kazacos , 2001 ) .
embryonated eggs of baylisascaris species are infectious to over 90 species of mammals and birds including humans .
larval infections with baylisascaris species can lead to irreversible neural , optical and visceral damage , in both wild animals as well as humans ( gavin et  al .
the severity of pathology of baylisascaris infection in the intermediate host is known to vary depending on the species of the infecting parasite ( tiner , 1953 ) .
understanding which species of baylisascaris a host is infected with may provide valuable information concerning prognosis and treatment , as well as the source of the infection . as with other nematodes ,
baylisascaris species have been traditionally identified through morphometric data . however , distinguishing larval nematodes to the species level is problematic ( graeff - teixeira et  al . , 2016 ) .
with the current widespread availability of molecular tools , the use of genetic analysis to rapidly and accurately identify organisms to the species level is increasingly standard .
however , differentiation of closely related species such as b.  procyonis and b.  columnaris has proven difficult ( dangoudoubiyam et  al . , 2009 , gatcombe et  al . , 2010 ) .
extensive previous research has documented the life - cycle , distribution and prevalence of b.  procyonis ( graeff - teixeira et  al . , 2016 ,
additionally , the complete mt genome of b.  procyonis , has been sequenced ( xie et  al .
in contrast to the relative abundance of studies on b.  procyonis , little is known of its closest relative , b.  columnaris .
previous research using copromicroscopic detection has shown b.  columnaris infection prevalence of 25% in a population of eastern spotted skunks ( spilogale putorius ) ( lesmeister et  al . , 2008 ) and 25% prevalence in captive striped skunks in europe ( d'ovidio et  al . , 2017 ) .
to our knowledge , the prevalence of b.  columnaris in wild populations of striped skunks has not previously been reported .
( 2013 ) reported the cloning and sequencing of partial sequences of b.  columnaris for the cox1 ( 413 bp ) and cox2 ( 483 bp ) genes .
additionally , in this work franssen et  al . identified three single nucleotide polymorphisms ( snps ) in the partial cox1 sequence and one snp found in the partial cox2 sequence which are useful in differentiating b.  procyonis from b.  columnaris .
the partial sequence of these mitochondrial genes has provided a reference which has facilitated the differentiation of b.  procyonis from b.  columnaris by dna sequencing .
in addition to the four snps which allow for the molecular differentiation of b.  procyonis and b.  columnaris , franssen et  al . also reported a number of intragenic differences in b.  columnaris mitochondrial sequences suggesting there is a high degree of genetic diversity in this species . in this study we sought to determine the prevalence of b.  columnaris in a wild population of striped skunks ( mephitis mephitis ) in salt lake county , utah , usa .
in addition to determining the prevalence of these parasites in a native skunk population we report the complete sequence of 11 mitochondrial genes ( cox1 , cox2 , nd2 and 8 trna genes ) comprising 3638 bp of the b.  columnaris mitochondrial genome .
results revealed several novel snps in these genes , which further facilitates and improves the molecular distinction between b.  procyonis and b.  columnaris .
additionally , several intragenic snps were identified in b.  columnaris worms . in summary ,
these results demonstrate the prevalence of b.  columnaris in a wild population of skunks and extend the number of known genetic differences between b.  procyonis and b.  columnaris .
skunks were acquired through nuisance animal calls from the public in salt lake county , utah , usa during the fall and winter of 2013 . institutional animal care and use committee ( iacuc ) or
ethics committee approval was not necessary , as animals were not sacrificed for research purposes .
skunks were captured in live traps ( tru - catch , belle fourche , south dakota , usa ) .
skunks were euthanized by chemical immobilization with 5/1 ketamine / xylazine followed by intracardiac injection of potassium chloride .
the presence of nematode parasites was determined by emptying the small intestinal contents through manual extrusion using finger / grip pressure and the visual examination of intestinal contents .
species determination of collected worms was performed by extracting dna from one or more worms from each skunk and performing dna sequencing of the cox1 gene .
sequences were aligned with the previously published cox1 to determine worm genus and species ( genbank : kc543474.1 ) ( https://blast.ncbi.nlm.nih.gov ) .
a total of 34 worms were collected and the cox1 gene of 22 of these worms was sequenced .
a single worm from several animals was then used for more extensive dna sequencing of a number of genes as described below .
pcr primers were designed based on the mitochondrial genome of b.  procyonis ( ac . no .
primers were designed to flank the gene of interest by 100300 bp from the 5 and 3 ends .
this primer design resulted in the amplification of segments of dna which contained the complete gene of interest ( cox1 , cox2 , nd2 ) as well as several small trna genes contained at the 5 and 3 ends of each amplicon ( table  1 ) .
three separate amplicons were generated with amplicon 1 containing the sequences for the trna genes q , r , i , s , and l , as well as the complete nd2 gene .
amplicon 2 contained the complete cox1 gene and amplicon 3 contained the trna genes d , g , and h as well as the complete cox2 gene .
primer sequences for each amplicon are contained in table  1 . upon collection of parasites ( described above ) , individual specimens were preserved by immediately freezing each worm at  80  c. for genetic analysis , specimens were thawed and a 2.0  mm portion of each worm macerated with a razor blade .
the macerated tissue samples were then placed in a 1.5  ml microcentrifuge tube and dna extracted using the qiagen dneasy blood & tissue kit ( qiagen inc . ,
pcr reactions were carried out in a final reaction volume of 50  l containing 2  l of dna , 25  l of onetaq dna polymerase ( new england biolabs , ipswich , ma usa ) , 0.5  l of each primer with 22  l of molecular grade water .
polymerase chain reaction conditions used to amplify the cox1 and cox2 containing amplicons ( amplicons 2 and 3 ) , involved a 30  s initial denaturation at 94  c followed by 30 cycles of denaturation at 94  c for 30  s , annealing at 55  c for 30  s , and elongation at 68  c for 80  s , followed by a 10-min final extension at 68  c .
the pcr amplification of the nd2 target region ( amplicon 1 ) was identical to the aforementioned profile , with the exception of the annealing temperature being 63  c .
the pcr amplified products of interest for each amplicon were then extracted and gel purified using the wizard sv gel and pcr clean - up system ( promega madison , wi ) .
the sequencing of each amplicon was performed using primers designed to bind approximately every 500 bp ( supplemental fig .  1 ) .
cycle sequencing was performed by the brigham young university dna sequencing center using an abi 3730xl automated sequencer .
primers were designed using the forward dna strand and sequencing was performed on a single strand .
contigs for each sample were assembled by mapping sample reads to the mitochondrial genome of b.  procyonis ( ac .
sequence reads were determined using geneious software ( biomatters limited , san francisco , ca , usa ) .
snp analysis , multiple alignments , and prediction of transmembrane and cytoplasmic regions were performed using geneious software .
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 cox1 ( genbank : kc543474.1 ) . 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
our analysis revealed 11 novel loci , which consistently distinguished b.  columnaris from b.  procyonis , including the three previously reported by franssen et  al .
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 cox2 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 .
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 nd2 gene . in our sequencing of this gene and subsequent analysis ,
in addition , two intraspecies snp were identified in the nd2 gene of b.  columnaris ( fig . 
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 . 
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 ) .
both skunks as well as raccoons commonly live in urban areas facilitating human contact and potential ingestion of embryonated eggs which can cause visceral , ocular and neural larval migrans ( roussere et  al . , 2003 ,
the molecular identification of b.  columnaris was not possible due to the lack of any dna sequences in public databases . in 2009 , a partial ( 529 bp ) sequence of the b.  columnaris cox2 gene was generated ( dangoudoubiyam et  al . , 2009 ) .
in 2013 work by franssen et  al . resulted in partial sequences for b.  columnaris cox1 ( 413 bp ) and cox2 ( 483 bp ) genes ( franssen et  al . ,
the availability of these sequences has facilitated the molecular identification of these parasites for us as well as other researchers ( d'ovidio et  al . , 2017 ) . in this study , we utilized gene sequencing to identify roundworms as b.  columnaris . in this population of wild skunks
we then extended earlier findings by sequencing a total of 3638 bp of the mitochondrial genome of b.  columnaris .
in so doing several novel snps were identified , facilitating the molecular discrimination of b.  columnaris from b.  procyonis .
these snps provide additional species specific targets for the molecular differentiation of b.  columnaris from b.  procyonis .
importantly this data demonstrate that three previously reported species specific snps do not accurately differentiate b.  procyonis from b.  columnaris ( 168 of cox2 and 804 and 834 of cox1 ) .
all of these snps involved transitions between the purines g and a. this unique finding , compared to previously published work , is likely due to the previous study being done in the netherlands , while skunks ( and by extension parasites of skunks ) are native to the americas .
parasites infecting european skunks are likely from a relatively small founder population of nematodes infecting skunks transported outside of north america .
it is logical to assume that the nematodes ( as well as the skunks ) would have less genetic diversity than a native , free roaming population of animals .
a recent study on the prevalence of b.  columnaris in captive european skunks found that 25% of skunks tested were infected with b.  columnaris ( d'ovidio et  al .
data indicates that in our study area there is a much higher infection rate of striped skunks than reported in studies of captive striped skunks in europe .
this is likely due to wild animals encountering other wild skunks as well as infected intermediate hosts more commonly than captive skunks .
as expected , more intragenic variation of parasites was observed from this population of native wild skunks compared to the imported non - native population studied previously ( franssen et  al . , 2013 )
. the higher prevalence of parasites in our study compared to a previous study of spotted skunks ( lesmeister et  al . , 2008 )
is likely due to the difficulty in accurately identifying infected animals through fecal analysis compared to our method of visual inspection of the small intestine . additionally , several of the animals in our study had very few worms infecting them .
animals infected with a single male worm would not be detected by microscopic analysis of feces but would easily be identified through visual inspection on the intestinal contents .
differences in the susceptibility to baylisascaris infection by these two types of skunks , as well as other environmental factors may also play a role in variations in infection prevalence . in summary , in this study several novel intragenic snps were identified .
additionally , nine novel polymorphisms are identified which aid in the molecular differentiation of b.  procyonis from b.  columnaris .
three polymorphisms , which were previously thought to differentiate these two species were shown to in fact be intragenic rather than species specific snps .
 members of the genus baylisascaris utilize omnivores or carnivores as their definitive hosts . 
 the best known member of this genus is baylisascaris procyonis , which is an intestinal parasite of raccoons . 
 the closest relative of b.  procyonis is b.  columnaris , which utilizes the common skunk as its definitive host . 
 although b.  procyonis has been extensively studied , relatively little is known of b.  columnaris . for example , the mitochondrial genome of b.  procyonis has been sequenced in its entirety . 
 conversely , the mitochondrial genome of b.  columnaris remains largely unexplored . 
 likewise , the prevalence of this parasite in its wild host has not been documented . in this study 
 , we collected parasites from a wild population of skunks in the state of utah , united states . 
 the cytochrome c oxidase subunit 1 and 2 genes , nadh dehydrogenase 2 and several trna genes were sequenced from the mitochondrial genome of these parasites . 
 we also determined the prevalence of b.  columnaris in a wild population of skunks . in this work 
 we identify several novel polymorphic genetic loci between b.  procyonis and b.  columnaris . 
 these findings provide additional molecular targets for the differentiation of baylisascaris species through clarification of genetic differences between b.  columnaris and b.  procyonis . 