BACKGROUND
oxyspirura petrowi is a spirurian nematode  that infects the eyes of quail and other birds  <cit> .
in texas, a 47–56% prevalence has been reported in northern bobwhites  and scaled quail   <cit> .
similar infections caused by this genus of parasites have also been reported in other animals including poultry and zoo animals, where some of them were described as ocular oxyspiruriasis or oxyspirurosis  <cit> .
given that bobwhites are experiencing long-term declines throughout their range in north america, there is a recognition that populations are declining even where suitable habitat conditions exist , thereby raising concerns that parasites such as o. petrowi may be a contributing factor .
it is likely that infection may cause host eye damage and physically impair vision, making birds less competitive in feeding and more susceptible to predators .
oxyspirura petrowi
adult worms in the eye of a northern bobwhite collected in texas in february,  <dig> demonstrating their potential to cause visual obstruction in addition to a pathological response resulting from infection.
although the eye worm has been considered as a possible contributing factor for the decline of wild quail populations in the rolling plains, little is known of the parasite’s biology, particularly at the molecular and genomic levels .
previous knowledge on the relationship of this parasite with other nematodes was solely acquired by morphology, which also needs to be validated at the molecular level.
in fact, only a single nucleotide sequence is present in the database for the whole genus oxyspirura .
the lack of molecular data severely hampers our efforts in studying molecular epidemiology and transmission routes of o. petrowi, which may be useful for developing effective strategies to treat and control ocular oxyspiruriasis in wild quail.
to fill the knowledge gap, we have performed a small-scale genome sequence survey  that provides the first batch of genomic sequence data for this nematode.
additionally, we have cloned the 18s rrna, internal transcribed spacer  <dig> ,  <dig> s rrna, its <dig> and partial 28s rrna genes.
the small random gss effort rapidly generated ~240 kb of sequence information that provided not only a snapshot of the quail eye worm genome, but also a large amount of microsatellite sequences for future genotyping and population genetic analysis.
based on the newly obtained rrna sequences, we determined the evolutionary affinity of oxyspirura with other nematodes among spirurians with available 18s rrna sequences.
we also designed specific primers based on the its <dig> sequences, and performed real-time quantitative pcr -based molecular detection of o. petrowi from dna extracted from fecal samples collected from northern bobwhite and scaled quail in texas.
understanding the biology of this parasitic nematode at molecular levels will enable us to effectively determine the prevalence by detecting parasite-specific dna in feces, as well as to identify infected intermediate hosts that is otherwise difficult  based on morphology of larval stages.
molecular tools would enable further study of potential drug targets and target-based drug discovery to treat this important nematode.
methods
isolation of genomic dna and genome sequence survey
adult o. petrowi worms were isolated from the eyes of northern bobwhites collected in texas as part of a 3-year integrated research project called operation idiopathic decline, which was initiated to further our understanding of potentially pathogenic parasites occurring within the rolling plains ecoregion of texas and western oklahoma.
all animal experiments were performed in accordance with procedures approved by the institutional animal care and use committee of texas a&m university .
after microscopic examination for species validation, four worms were rinsed with pbs, placed in lysis buffer of the dneasy blood & tissue kit , and grinded with a plastic microtube grinder.
genomic dna  was isolated from the ground worms according to manufacturer’s protocol for animal tissues.
for the construction of a genomic library, gdna was first subjected to whole genome amplification using a genomeplex complete whole genome amplification  kit according the manufacturer’s standard protocol .
amplified gdna products were fractionated in agarose gels and fractions containing fragments between  <dig>  – 2 kb were collected and purified using a gel extraction kit .
after an incubation at 72°c for 20 min in a regular pcr reaction buffer to add a single adenine overhang to the 3’-end, the products were ligated into pcr <dig> -topo vector using a topo-ta cloning kit .
after transformation, escherichia coli oneshot top10f' chemically competent cells  were plated onto lb plates spread with 40 μl of 40 mg/ml x-gal and 5 μl of 200 mm/ml iptg, and incubated at 37°c overnight.
bacteria from a single white colony were collected into 10 μl milli-q water in a microtube, from which 2 μl of suspension was used directly as template in pcr reactions to determine the presence of inserts using a pair of m <dig> forward and m <dig> reverse primers.
colonies containing inserts with desired sizes were further incubated in lb broth containing 50 μg/ml kanamycin.
plasmids were prepared using a plasmid dna mini kit , and sequenced by the sanger dye-terminator sequencing method at the gene technology laboratory at texas a&m university.
vector and adaptor sequences were removed using a cross-match algorithm, and long inserts were assembled using the phrap method implemented in the macvector program  .
all sequences were used as queries to search the non-redundant protein and nucleotide databases at the national center for biotechnology information  by the blastn, blastx and tblastx algorithms using the koriblast program  .
additional annotations were performed using the blast2go program , which included interproscan for identifying protein domains and gene ontology  analysis.
go_slim was performed at the categorizer server   <cit> .
contigs were also mapped onto the metabolic pathways at the kyoto encyclopedia of genes and genomes  using the kegg automatic annotation server    <cit> .
candidate trna sequences were examined at the trnascan-se server   <cit> .
microsatellite sequences  were identified using the phobos  program , in which only perfect matches with a minimal length of  <dig> nt and a minimal score at  <dig> were reported.
molecular cloning of parasite ribosomal rna  genes
the 18s rrna gene and downstream its <dig>   <dig> s rrna and its <dig> regions from o. petrowi were cloned by pcr using two pairs of primers: 1) nema18s_f <dig>  and nema18s_r <dig>  for the nearly whole 18s region; and 2) nema18s_f <dig>  and nema28s_r <dig>  for the region between the 18s and 28s rrna genes.
pcr was performed using a jumpstart redtaq readymix pcr reaction kit containing hot-start high-fidelity dna polymerase .
after treating with regular taq dna polymerase at 72°c for 10 min, pcr amplicons were similarly cloned into the pcr <dig> -topo vector as described above.
at least  <dig> independent clones from each reaction were sequenced, and all reads were assembled by phrap as described above.
regions representing 18s, its <dig>   <dig> s, its <dig> and partial 28s sequences were determined by rfam   <cit> .
phylogenetic reconstructions
the assembled o. petrowi 18s rrna sequence was used as a query to search and identity nematode orthologs from the ncbi nucleotide databases.
up to  <dig>  gene sequences were initially retrieved, subjected to multiple sequence alignments using the muscle program    <cit> , followed by simple phylogenetic reconstructions by the neighbor-joining  method using the tamura-nei nucleotide substitution model.
based on the alignment and nj trees, short or identical sequences were individually removed, and the same procedure was repeated until a balanced dataset containing  <dig> sequences representing all major nematode taxonomic groups were identified.
the dataset was subjected to phylogenetic reconstructions by bayesian inference  using mrbayes   and the maximum likelihood  method using treefinder    <cit> .
this approach determined the phylogenetic relationships among major taxonomic groups, in which o. petrowi was placed within the spirurians, but the relationship among spirurians was not well resolved.
therefore, we resampled the sequences to include only taxa within spirurida and ascaridida as these two groups displayed a sister relationship by this study and previous analyses  <cit> .
this also allowed us to include more taxa within these two groups.
the second dataset contained  <dig> taxa with  <dig>  nucleotide positions and was subjected to phylogenetic reconstructions using bi and ml methods.
to further resolve the o. petrowi position, we also compiled a third dataset containing only taxa with close relationship with o. petrowi.
this small dataset included only  <dig> taxa with  <dig>  nucleotide positions, and was also subjected to bi and ml analyses.
in all datasets, gaps were removed and only positions that could be unambiguously aligned were used in subsequent phylogenetic analyses.
in the bi analysis,  <dig>  million generations of searches for the first and second datasets  were performed with  <dig> independent chains running.
searches reached convergence as determined by the average standard deviation  of split frequencies reaching <  <dig> , and potential scale reduction factor  values for various approaching  <dig>   <cit> .
bootstrapping ml analyses were derived from  <dig> replicated sequences.
in both bi and ml methods, the general time reversal  nucleotide substitution model was used with the consideration of fraction of invariance and 4-rate of discrete gamma .
majority rule consensus trees were visualized using figtree , followed by tree annotations using adobe illustrator cs <dig>
molecular detection of o. petrowi
sequence comparison of the rrna regions between o. petrowi and other nematodes indicated that 18s rrna sequences were less suitable for designing species-specific primers, as they were highly conserved among nematodes.
we hence designed primers based on the its <dig> region sequences for specific molecular detection for o. petrowi: qew_2417f  and qew_2578r  with a predicted product size of 162 bp.
to ensure desired specificity of detection, we used dna samples isolated from aulonocephalus pennula, a cecal worm collected from bobwhite quail in northern texas as negative control.
we applied real-time quantitative pcr  to detect eye worm dna from fecal samples from northern bobwhite and scaled quail in texas.
feces from individual or pooled birds were collected at rolling plains quail research ranch  in fisher county, texas in the spring,  <dig> via the seasonal trap-and-release program in a separate conservation research project.
feces were mixed, weighed and placed in lysis buffer included in the qiaamp dna stool mini kit .
after one freeze/thaw cycle in liquid nitrogen, samples were homogenized with glass beads in a mini-beadbeater- <dig>  at full-speed for 4 min, followed by dna isolation according to the manufacturer’s protocol for the stool dna isolation kit.
a sybr-green-based qpcr detection was performed in 20 μl reactions containing iq sybr green supermix reagent , the qew_2417f/qew_2578r primer pair  and 2 μl stool dna in a bio-rad icycle iq real-time pcr detection system.
the reactions started with sample denaturation at 95°c for 5 min, followed by  <dig> thermal cycles at 95°c for 30 sec, 56°c for 30 sec, and 72°c for 30 sec.
specificity of detection was confirmed by melting curve analysis and agarose gel electrophoresis of selected pcr products.
selected individual or pooled pcr products were also sequenced to confirm their identities.
nucleotide sequence accession numbers
nucleotide sequences generated in this study were deposited into the genbank database with accession numbers  and  for rrna sequences containing type  <dig> and type  <dig> its <dig>  and  to  for gss sequences.
RESULTS
characterization of the o. petrowi genome
in this small genome sequence survey, we have obtained valid sequences for  <dig> clones.
among them, six sequences were determined to be bacterial contaminants, representing  <dig> % of the sequenced clones.
there were no contaminants from birds and other organisms, suggesting that the prepared o. petrowi genomic libraries were of high quality.
the limited bacterial sequences  were likely derived from commensal bacteria in o. petrowi.
the remaining  <dig> valid o. petrowi sequences resulted in  <dig>  bp of genomic sequences, which ranged from  <dig> to  <dig>  bp  for individual contigs.
the scale of the survey was relatively small, but it was sufficient to provide a first snapshot of the genome features for this nematode.
the o. petrowi genome was generally at-rich with gc contents ranging from 18% to 64% for individual contigs .
various blast searches yielded no hits for  <dig> contigs , suggesting that these sequences might be unique to oxyspirura or closely related species.
among the  <dig> contigs with hits from the databases,  <dig>  had top hits on nematode sequences, particularly on those from loa, brugia and wuchereria, but only very few on ascaris or caenorhabditis, which was congruent with the evolutionary relationship of oxyspirura with filarioidea, ascaridida and rhabditida .
by combining blast with interproscan searches, more than half of the contigs with hits were able to be assigned into major functional categories  .
the functionally undefined  <dig> contigs were mainly hypothetical proteins with some containing low complexity sequences.
among the  <dig> annotatable contigs, the largest group was enzymes  that were separated into general enzymes involved in various metabolic pathways  and those involved in protein metabolism such as protein kinases  and proteases .
examples of enzymes included glycogen synthase , glycosyl-transferase , histone acetyltranferase , and succinate dehydrogenase ; while those for protein metabolism included a ulp <dig> protease family member , as well as protein kinases in the agc/ndr , cam/camkl/nuak  and ck1/worm <dig>  families.
fourteen contigs encoded proteins involved in dna/rna metabolism, e.g., splicing factor  <dig> , dna replication licensing factor mcm- <dig> , and protein containing double-stranded rna binding motif .
there were  <dig> contigs containing genes encoding extracellular membrane  proteins, in which  <dig> contigs were associated with the nematode-specific cuticle formation, such as cuticle collagen precursors , cuticulin- <dig> , and nematode cuticle collage domain containing proteins .
other groups include those involved in ribosomal biogenesis , molecular interactions , ion transporters , cytoskeletal proteins , membrane proteins associated with cell adhesion , and those involved in gene expression .
interestingly, there were three contigs encoding nematode-specific major sperm protein , which was grouped together with a fic protein under the “cell development” category in figure  <dig>  there were also three contigs derived from the mitochondrial genomes, including gene fragments encoding cytochrome b, cytochrome c oxidase subunit iv  and nahd dehydrogenase subunit  <dig>  finally,  <dig> contigs were found to contain retroelements, such as tigger transposable element-derived protein 1-like proteins  and retrotransposon protein .
microsatellite sequences identified by the genome survey
one major purpose of the study was to identify microsatellite sequences  for future genotyping and population genetics analyses on this quail eye worm.
in fact, o. petrowi appears to be rich in microsatellites, in which a total of  <dig> units of perfect ssrs were identified with a minimal length of  <dig> nt .
these included mononucleotides , dinucleotides , trinucleotides , tetranucleotides  and  <dig> repeats with 5– <dig> nucleotides.
at least  <dig> contigs contained two or more ssrs, and  <dig> contigs contained 3– <dig> ssrs .
examples included qew_ <dig> with  <dig> for mono-, tri- or tetra-nucleotide ssrs; qew_ <dig> with  <dig> mono-, tetra- or octa-nucleotide ssrs, and qew_ <dig> with  <dig> di-, tri- or penta-nucleotide ssrs .
we also looked at the distribution of microsatellites with repeat units of ≥ <dig> nt, which revealed ~ <dig> or ~ <dig>  times more microsatellite sequences are present in contigs with no hits in blast/interproscan searches  or with hits but unknown function  than in the annotatable contigs  .
in summary, the eye worm genome contains a rich number of microsatellite sequences with the potential to be further validated as potential genetic markers.
oxyspirura petrowi
identified by the genome sequence survey
* see additional file 2: table s <dig> for a complete list of microsatellite sequences.
phylogenetic position of o. petrowi based on 18s rrna genes
our first phylogenetic analyses based on a large 18s rrna dataset with bi and ml methods produced trees that agreed with those produced by others.
while o. petrowi was clustered within the spirurida clade, it was close to a branch consisting of tetrameres fissipina and an unknown onchoceridae species.
this was likely a result caused by a long branch attraction  artifact based on the unusual long branch formed by t. fissipina and the onchoceridae species, as well as by the obvious high numbers of nucleotide substitutions in these two sequences .
we also observed potential sequencing mistakes for the long 18s rrna sequence of thelazia lacrymalis .
therefore, we removed these three sequences from subsequent analyses.
because spirurida was consistently placed as a sister group with ascaridida, as observed in this study and by others  <cit> , our second dataset contained only sequences from these two groups, which allowed more reliable alignment with more nucleotide positions to better resolve the position of o. petrowi within spirurida using ascaridida as outgroup.
gnathastoma sequences were also excluded from the second dataset, as they have been shown to be seperate from the rest of the spirurids  <cit> .
both bi and ml trees inferred from the second dataset distinctly separated ascaridida from spirurida .
within the spirurida clade, dracunculoidea and camallanoidea formed two major sister branches, whereas the third branch comprised of the remaining families including spiruroidea, acuarioidea, physalopteroidea, filarioidea, habronematoidea and thelazioidea.
further phylogenetic analysis based only on sequences from the third branch produced similar tree topology, but with slightly better resolution and statistical support .
acuarioidea, physalopteroidea, filarioidea and habronematoidea were monophyletic, whereas spiruroidea was paraphyletic, intermixed with other families.
among them, o. petrowi was clustered with streptopharagus and spirocerca, which in turn formed a sister branch to the filarioidea, albeit with low posterior probability and bootstrap proportion support .
at the moment, more sophisticated phylogenetic analyses were unachievable due to the lack of more sequences from closely related species, and the lack of sufficient sequence data such as the mitochondrial genomes and proteins within spirurida, particularly among thelazioidea.
nonetheless, our study revealed that thelazioidea, including quail eye worm, was closely related to filarial nematodes, which implies that therapeutic strategies for filariasis such as those for l. loa might be referential in developing treatments for the thelazoidea eye worms.
feature of internal transcribed regions and molecular detection of o. petrowi
in addition to the nearly complete 18s rrna gene, we have also determined the complete sequences of the its <dig>   <dig> s rrna and its <dig> regions.
two types of its <dig> sequences have been observed , in which the type  <dig> sequence contained a 17-nucleotide insertion at positions  <dig> –  <dig> .
additionally, there were two nucleotide differences between the two types of its <dig> sequences at positions  <dig> and  <dig> .
the insertion/deletion was further confirmed by pcr using primers flanking the region and subsequent sequencing of pcr amplicons.
while the  <dig> s rrna gene shared significant sequence homology to those of other nematodes available the ncbi databases , both the its <dig> and its <dig> regions displayed high sequence diversity .
because of the insertion/deletion at the its <dig> region, we initially designed two pairs of primers based on the its <dig> sequence for the potential of using nested pcr-based molecular detection of o. petrowi: an external primer pair qew_2373f  and qew_2681r , and an internal primer pair qew_2417f and qew_2578r  that would give 309 bp and 162 bp products, respectively.
however, after our pilot experiments indicated that nested pcr was unnecessary, we used regular qpcr reactions with the qew_2417f and qew_2578r primers in subsequent detection of o. petrowi dna from wild bobwhite fecal specimens collected in texas in february – march,  <dig>  the specificity of the qew_2417f and qew_2578r primer pair for o. petrowi was also confirmed by its inability to produce products from dna isolated from the cecal worm a. pennula that is commonly present in wild quail .
on the other hand, due to the lack of molecular sequences at the its regions from very closely related species, we could not firmly conclude that the relatively short primers were absolutely specific to o. petrowi.
in fact, only six its <dig> sequences were present in the genbank database for the superfamily thelazioidea, including five from thelazia species and one from o. conjuctivalis .
however, these its <dig> sequences were highly divergent from each other and from those of o. petrowi , which indicated that their its <dig> regions were likely divergent enough for speciation.
to further ensure the quality of detection, selected individual or pooled pcr products were also sequenced to validate their identities.
when the qpcr detection system was used, melting curve analysis was performed to confirm that pcr amplicons showed the same curves as those of positive controls.
among the  <dig> fecal dna specimens, we have detected  <dig>  positive samples.
however, because the specimens were collected from both individual and pooled quail samples derived from  <dig> birds, direct calculation of positive rate  was inappropriate as pooled positive specimens might contain both positive and negative samples.
therefore, we employed two additional approaches to estimate the prevalence.
the first approach was to only calculate positive rate from the  <dig> samples collected from individual birds , in which  <dig> samples were positive, giving a  <dig> % positive rate.
the second approach employed software written by dr. brad biggerstaff as an excel add-in  , which was originally developed to determine positive rates of viral infections in pooled mosquito samples using a maximum likelihood estimation  algorithm  <cit> .
by applying a bias-corrected mle estimation, we obtained an infection rate of  <dig> % with lower and upper limits at  <dig> % and  <dig> %, respectively .
collectively, we conclude that ~30%  of the sampled wild quail were infected by the eye worm.
the actual rate might be even higher, as the fecal samples were only collected once, rather than continuously for several days, and the sensitivity of pcr detection might not be maximal due to the inhibitory substances commonly present in fecal samples as discussed below.
the detection of o. petrowi dna in feces allows rapid and sensitive detection of the presence of eye worms without the need to examine individual birds.
however, one needs to be aware of the presence of inhibitory substances in fecal samples and the difficulties in releasing dna from eggs or encysted larvae.
the presence of inhibitory substances could be minimized  using the tablets included in the dna isolation kits specifically designed for stool samples such as the qiaamp dna stool mini kit .
freeze/thaw cycles combined with homogenization with glass beads were necessary to break the eggs or encysted larvae to ensure the release of dna.
furthermore, nested pcr might also be used by the addition of a primary amplification using the external primers qew_2373f and qew_2681r to not only improve the sensitivity of pcr detection, but to also further eliminate the presence of inhibitory substances in a second amplification procedure.
the life cycle of o. petrowi is not well understood, its exact intermediate host as well as its migration details in quail.
the presence of o. petrowi dna in quail feces indicates that eggs laid by female adult worms in the eyes could be passed into the digestive system and released with the excrement via the naso-lacrimal ducts to the gut route, as previously observed in other eye worms such as o. mansoni <cit> .
this pcr detection protocol can also be easily adapted to identify intermediate host and perform surveillance, which is important in developing effective strategies to control the transmission of eye worms in the field.
our phylogenetic analysis based on 18s rrna sequences indicated that o. petrowi clustered closely with streptopharagus and spirocerca .
it is known that birds are a paratenic host for spirocerca lupi infection in their life cycle between dogs and dung beetles  <cit> .
dung beetles are also the intermediate host for streptopharagus <cit> .
these observations suggest that dung beetles might be worth examining as one of the potential intermediate hosts.
indeed, the detection of o. petrowi dna in various insects including dung beetles is currently ongoing as part of a separate project in determining the intermediate host and transmission route, and the data will be reported upon the completion of the survey.
CONCLUSIONS
we have performed a small-scale genome sequence survey , which not only rapidly generated a large number of molecular sequence data for the first time for o. petrowi, but also provided a snapshot of the genome for the eye worm in quail.
the survey also identified a large number of microsatellite sequences that may be employed in further genotyping and population genetics studies.
our phylogenetic reconstructions based on 18s rrna sequences indicated that spiruroidea was paraphyletic, while o. petrowi, streptopharagus and spirocerca formed a sister clade to the filarial nematodes.
the obtained its sequence data also permitted us to design specific primers for molecular detection of o. petrowi in fecal samples, which may also be adapted to detect this nematode in insect intermediate hosts for surveillance and developing strategies to control the transmission of eye worms from intermediate hosts to quail.
we also determined that ~28% - 33% of the birds were o. petrowi positive, suggesting that eye worm was a significant parasite in at least some quail ranches in texas.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
lx participated in experimental design and performed the majority of experiments on the genome survey including constructing genomic library, cloning and sequencing, the cloning and sequencing of rrna gene and downstream region sequences, and the isolation stool dna and pcr/qpcr detection; fg and hz participated in sample preparation; ll participated in collection of fecal samples from wild quail; ab participated in collection of adult eye worms; dr participated in fecal sample collection, writing the manuscript, and securing funding for the study; amf participated in collection and speciation of eye worm and writing manuscript; gz conceived the study, participated in its design, molecular and phylogenetic analysis, and writing the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
list of contigs with annotations and information on top blast hits.
click here for file
additional file 2: table s2
oxyspirura petrowi microsatellite sequences identified by the gss  using phobos.
click here for file
acknowledgements
major funding for this research provided by rolling plains quail research foundation  to gz, amf and dr. we thank dr. jason m. fritzler at the weber state university for his critical reading of the manuscript.

 oxyspirura petrowi is a spirurian nematode that infects the eyes of quail and other birds. in texas, a 47–56% prevalence has been reported in northern bobwhites and scaled quail. similar infections caused by this genus of parasites have also been reported in other animals including poultry and zoo animals, where some of them were described as ocular oxyspiruriasis or oxyspirurosis. given that bobwhites are experiencing long-term declines throughout their range in north america, there is a recognition that populations are declining even where suitable habitat conditions exist, thereby raising concerns that parasites such as o. petrowi may be a contributing factor. it is likely that infection may cause host eye damage and physically impair vision, making birds less competitive in feeding and more susceptible to predators. oxyspirura petrowi adult worms in the eye of a northern bobwhite collected in texas in february, demonstrating their potential to cause visual obstruction in addition to a pathological response resulting from infection. although the eye worm has been considered as a possible contributing factor for the decline of wild quail populations in the rolling plains, little is known of the parasite’s biology, particularly at the molecular and genomic levels. previous knowledge on the relationship of this parasite with other nematodes was solely acquired by morphology, which also needs to be validated at the molecular level. in fact, only a single nucleotide sequence is present in the database for the whole genus oxyspirura. the lack of molecular data severely hampers our efforts in studying molecular epidemiology and transmission routes of o. petrowi, which may be useful for developing effective strategies to treat and control ocular oxyspiruriasis in wild quail. to fill the knowledge gap, we have performed a small-scale genome sequence survey that provides the first batch of genomic sequence data for this nematode. additionally, we have cloned the 18s rrna, internal transcribed spacer, s rrna, its and partial 28s rrna genes. the small random gss effort rapidly generated ~240 kb of sequence information that provided not only a snapshot of the quail eye worm genome, but also a large amount of microsatellite sequences for future genotyping and population genetic analysis. based on the newly obtained rrna sequences, we determined the evolutionary affinity of oxyspirura with other nematodes among spirurians with available 18s rrna sequences. we also designed specific primers based on the its sequences, and performed real-time quantitative pcr -based molecular detection of o. petrowi from dna extracted from fecal samples collected from northern bobwhite and scaled quail in texas. understanding the biology of this parasitic nematode at molecular levels will enable us to effectively determine the prevalence by detecting parasite-specific dna in feces, as well as to identify infected intermediate hosts that is otherwise difficult based on morphology of larval stages. molecular tools would enable further study of potential drug targets and target-based drug discovery to treat this important nematode. methods isolation of genomic dna and genome sequence survey adult o. petrowi worms were isolated from the eyes of northern bobwhites collected in texas as part of a 3-year integrated research project called operation idiopathic decline, which was initiated to further our understanding of potentially pathogenic parasites occurring within the rolling plains ecoregion of texas and western oklahoma. all animal experiments were performed in accordance with procedures approved by the institutional animal care and use committee of texas a&m university. after microscopic examination for species validation, four worms were rinsed with pbs, placed in lysis buffer of the dneasy blood & tissue kit, and grinded with a plastic microtube grinder. genomic dna was isolated from the ground worms according to manufacturer’s protocol for animal tissues. for the construction of a genomic library, gdna was first subjected to whole genome amplification using a genomeplex complete whole genome amplification kit according the manufacturer’s standard protocol. amplified gdna products were fractionated in agarose gels and fractions containing fragments between – 2 kb were collected and purified using a gel extraction kit. after an incubation at 72°c for 20 min in a regular pcr reaction buffer to add a single adenine overhang to the 3’-end, the products were ligated into pcr -topo vector using a topo-ta cloning kit. after transformation, escherichia coli oneshot top10f' chemically competent cells were plated onto lb plates spread with 40 μl of 40 mg/ml x-gal and 5 μl of 200 mm/ml iptg, and incubated at 37°c overnight. bacteria from a single white colony were collected into 10 μl milli-q water in a microtube, from which 2 μl of suspension was used directly as template in pcr reactions to determine the presence of inserts using a pair of m forward and m reverse primers. colonies containing inserts with desired sizes were further incubated in lb broth containing 50 μg/ml kanamycin. plasmids were prepared using a plasmid dna mini kit, and sequenced by the sanger dye-terminator sequencing method at the gene technology laboratory at texas a&m university. vector and adaptor sequences were removed using a cross-match algorithm, and long inserts were assembled using the phrap method implemented in the macvector program. all sequences were used as queries to search the non-redundant protein and nucleotide databases at the national center for biotechnology information by the blastn, blastx and tblastx algorithms using the koriblast program. additional annotations were performed using the blast2go program, which included interproscan for identifying protein domains and gene ontology analysis. go_slim was performed at the categorizer server. contigs were also mapped onto the metabolic pathways at the kyoto encyclopedia of genes and genomes using the kegg automatic annotation server. candidate trna sequences were examined at the trnascan-se server. microsatellite sequences were identified using the phobos program, in which only perfect matches with a minimal length of nt and a minimal score at were reported. molecular cloning of parasite ribosomal rna genes the 18s rrna gene and downstream its s rrna and its regions from o. petrowi were cloned by pcr using two pairs of primers: 1) nema18s_f and nema18s_r for the nearly whole 18s region; and 2) nema18s_f and nema28s_r for the region between the 18s and 28s rrna genes. pcr was performed using a jumpstart redtaq readymix pcr reaction kit containing hot-start high-fidelity dna polymerase. after treating with regular taq dna polymerase at 72°c for 10 min, pcr amplicons were similarly cloned into the pcr -topo vector as described above. at least independent clones from each reaction were sequenced, and all reads were assembled by phrap as described above. regions representing 18s, its s, its and partial 28s sequences were determined by rfam. phylogenetic reconstructions the assembled o. petrowi 18s rrna sequence was used as a query to search and identity nematode orthologs from the ncbi nucleotide databases. up to gene sequences were initially retrieved, subjected to multiple sequence alignments using the muscle program, followed by simple phylogenetic reconstructions by the neighbor-joining method using the tamura-nei nucleotide substitution model. based on the alignment and nj trees, short or identical sequences were individually removed, and the same procedure was repeated until a balanced dataset containing sequences representing all major nematode taxonomic groups were identified. the dataset was subjected to phylogenetic reconstructions by bayesian inference using mrbayes and the maximum likelihood method using treefinder. this approach determined the phylogenetic relationships among major taxonomic groups, in which o. petrowi was placed within the spirurians, but the relationship among spirurians was not well resolved. therefore, we resampled the sequences to include only taxa within spirurida and ascaridida as these two groups displayed a sister relationship by this study and previous analyses. this also allowed us to include more taxa within these two groups. the second dataset contained taxa with nucleotide positions and was subjected to phylogenetic reconstructions using bi and ml methods. to further resolve the o. petrowi position, we also compiled a third dataset containing only taxa with close relationship with o. petrowi. this small dataset included only taxa with nucleotide positions, and was also subjected to bi and ml analyses. in all datasets, gaps were removed and only positions that could be unambiguously aligned were used in subsequent phylogenetic analyses. in the bi analysis, million generations of searches for the first and second datasets were performed with independent chains running. searches reached convergence as determined by the average standard deviation of split frequencies reaching <, and potential scale reduction factor values for various approaching. bootstrapping ml analyses were derived from replicated sequences. in both bi and ml methods, the general time reversal nucleotide substitution model was used with the consideration of fraction of invariance and 4-rate of discrete gamma. majority rule consensus trees were visualized using figtree, followed by tree annotations using adobe illustrator cs molecular detection of o. petrowi sequence comparison of the rrna regions between o. petrowi and other nematodes indicated that 18s rrna sequences were less suitable for designing species-specific primers, as they were highly conserved among nematodes. we hence designed primers based on the its region sequences for specific molecular detection for o. petrowi: qew_2417f and qew_2578r with a predicted product size of 162 bp. to ensure desired specificity of detection, we used dna samples isolated from aulonocephalus pennula, a cecal worm collected from bobwhite quail in northern texas as negative control. we applied real-time quantitative pcr to detect eye worm dna from fecal samples from northern bobwhite and scaled quail in texas. feces from individual or pooled birds were collected at rolling plains quail research ranch in fisher county, texas in the spring, via the seasonal trap-and-release program in a separate conservation research project. feces were mixed, weighed and placed in lysis buffer included in the qiaamp dna stool mini kit. after one freeze/thaw cycle in liquid nitrogen, samples were homogenized with glass beads in a mini-beadbeater- at full-speed for 4 min, followed by dna isolation according to the manufacturer’s protocol for the stool dna isolation kit. a sybr-green-based qpcr detection was performed in 20 μl reactions containing iq sybr green supermix reagent, the qew_2417f/qew_2578r primer pair and 2 μl stool dna in a bio-rad icycle iq real-time pcr detection system. the reactions started with sample denaturation at 95°c for 5 min, followed by thermal cycles at 95°c for 30 sec, 56°c for 30 sec, and 72°c for 30 sec. specificity of detection was confirmed by melting curve analysis and agarose gel electrophoresis of selected pcr products. selected individual or pooled pcr products were also sequenced to confirm their identities. nucleotide sequence accession numbers nucleotide sequences generated in this study were deposited into the genbank database with accession numbers and for rrna sequences containing type and type its and to for gss sequences.  characterization of the o. petrowi genome in this small genome sequence survey, we have obtained valid sequences for clones. among them, six sequences were determined to be bacterial contaminants, representing % of the sequenced clones. there were no contaminants from birds and other organisms, suggesting that the prepared o. petrowi genomic libraries were of high quality. the limited bacterial sequences were likely derived from commensal bacteria in o. petrowi. the remaining valid o. petrowi sequences resulted in  bp of genomic sequences, which ranged from to  bp for individual contigs. the scale of the survey was relatively small, but it was sufficient to provide a first snapshot of the genome features for this nematode. the o. petrowi genome was generally at-rich with gc contents ranging from 18% to 64% for individual contigs. various blast searches yielded no hits for contigs, suggesting that these sequences might be unique to oxyspirura or closely related species. among the contigs with hits from the databases, had top hits on nematode sequences, particularly on those from loa, brugia and wuchereria, but only very few on ascaris or caenorhabditis, which was congruent with the evolutionary relationship of oxyspirura with filarioidea, ascaridida and rhabditida. by combining blast with interproscan searches, more than half of the contigs with hits were able to be assigned into major functional categories. the functionally undefined contigs were mainly hypothetical proteins with some containing low complexity sequences. among the annotatable contigs, the largest group was enzymes that were separated into general enzymes involved in various metabolic pathways and those involved in protein metabolism such as protein kinases and proteases. examples of enzymes included glycogen synthase, glycosyl-transferase, histone acetyltranferase, and succinate dehydrogenase; while those for protein metabolism included a ulp protease family member, as well as protein kinases in the agc/ndr, cam/camkl/nuak and ck1/worm families. fourteen contigs encoded proteins involved in dna/rna metabolism, e.g., splicing factor, dna replication licensing factor mcm-, and protein containing double-stranded rna binding motif. there were contigs containing genes encoding extracellular membrane proteins, in which contigs were associated with the nematode-specific cuticle formation, such as cuticle collagen precursors, cuticulin-, and nematode cuticle collage domain containing proteins. other groups include those involved in ribosomal biogenesis, molecular interactions, ion transporters, cytoskeletal proteins, membrane proteins associated with cell adhesion, and those involved in gene expression. interestingly, there were three contigs encoding nematode-specific major sperm protein, which was grouped together with a fic protein under the “cell development” category in figure  there were also three contigs derived from the mitochondrial genomes, including gene fragments encoding cytochrome b, cytochrome c oxidase subunit iv and nahd dehydrogenase subunit finally, contigs were found to contain retroelements, such as tigger transposable element-derived protein 1-like proteins and retrotransposon protein. microsatellite sequences identified by the genome survey one major purpose of the study was to identify microsatellite sequences for future genotyping and population genetics analyses on this quail eye worm. in fact, o. petrowi appears to be rich in microsatellites, in which a total of units of perfect ssrs were identified with a minimal length of nt. these included mononucleotides, dinucleotides, trinucleotides, tetranucleotides and repeats with 5– nucleotides. at least contigs contained two or more ssrs, and contigs contained 3– ssrs. examples included qew_ with for mono-, tri- or tetra-nucleotide ssrs; qew_ with mono-, tetra- or octa-nucleotide ssrs, and qew_ with di-, tri- or penta-nucleotide ssrs. we also looked at the distribution of microsatellites with repeat units of ≥ nt, which revealed ~ or ~ times more microsatellite sequences are present in contigs with no hits in blast/interproscan searches or with hits but unknown function than in the annotatable contigs. in summary, the eye worm genome contains a rich number of microsatellite sequences with the potential to be further validated as potential genetic markers. oxyspirura petrowi identified by the genome sequence survey * see additional file 2: table s for a complete list of microsatellite sequences. phylogenetic position of o. petrowi based on 18s rrna genes our first phylogenetic analyses based on a large 18s rrna dataset with bi and ml methods produced trees that agreed with those produced by others. while o. petrowi was clustered within the spirurida clade, it was close to a branch consisting of tetrameres fissipina and an unknown onchoceridae species. this was likely a result caused by a long branch attraction artifact based on the unusual long branch formed by t. fissipina and the onchoceridae species, as well as by the obvious high numbers of nucleotide substitutions in these two sequences. we also observed potential sequencing mistakes for the long 18s rrna sequence of thelazia lacrymalis. therefore, we removed these three sequences from subsequent analyses. because spirurida was consistently placed as a sister group with ascaridida, as observed in this study and by others, our second dataset contained only sequences from these two groups, which allowed more reliable alignment with more nucleotide positions to better resolve the position of o. petrowi within spirurida using ascaridida as outgroup. gnathastoma sequences were also excluded from the second dataset, as they have been shown to be seperate from the rest of the spirurids. both bi and ml trees inferred from the second dataset distinctly separated ascaridida from spirurida. within the spirurida clade, dracunculoidea and camallanoidea formed two major sister branches, whereas the third branch comprised of the remaining families including spiruroidea, acuarioidea, physalopteroidea, filarioidea, habronematoidea and thelazioidea. further phylogenetic analysis based only on sequences from the third branch produced similar tree topology, but with slightly better resolution and statistical support. acuarioidea, physalopteroidea, filarioidea and habronematoidea were monophyletic, whereas spiruroidea was paraphyletic, intermixed with other families. among them, o. petrowi was clustered with streptopharagus and spirocerca, which in turn formed a sister branch to the filarioidea, albeit with low posterior probability and bootstrap proportion support. at the moment, more sophisticated phylogenetic analyses were unachievable due to the lack of more sequences from closely related species, and the lack of sufficient sequence data such as the mitochondrial genomes and proteins within spirurida, particularly among thelazioidea. nonetheless, our study revealed that thelazioidea, including quail eye worm, was closely related to filarial nematodes, which implies that therapeutic strategies for filariasis such as those for l. loa might be referential in developing treatments for the thelazoidea eye worms. feature of internal transcribed regions and molecular detection of o. petrowi in addition to the nearly complete 18s rrna gene, we have also determined the complete sequences of the its s rrna and its regions. two types of its sequences have been observed, in which the type sequence contained a 17-nucleotide insertion at positions –. additionally, there were two nucleotide differences between the two types of its sequences at positions and. the insertion/deletion was further confirmed by pcr using primers flanking the region and subsequent sequencing of pcr amplicons. while the s rrna gene shared significant sequence homology to those of other nematodes available the ncbi databases, both the its and its regions displayed high sequence diversity. because of the insertion/deletion at the its region, we initially designed two pairs of primers based on the its sequence for the potential of using nested pcr-based molecular detection of o. petrowi: an external primer pair qew_2373f and qew_2681r, and an internal primer pair qew_2417f and qew_2578r that would give 309 bp and 162 bp products, respectively. however, after our pilot experiments indicated that nested pcr was unnecessary, we used regular qpcr reactions with the qew_2417f and qew_2578r primers in subsequent detection of o. petrowi dna from wild bobwhite fecal specimens collected in texas in february – march, the specificity of the qew_2417f and qew_2578r primer pair for o. petrowi was also confirmed by its inability to produce products from dna isolated from the cecal worm a. pennula that is commonly present in wild quail. on the other hand, due to the lack of molecular sequences at the its regions from very closely related species, we could not firmly conclude that the relatively short primers were absolutely specific to o. petrowi. in fact, only six its sequences were present in the genbank database for the superfamily thelazioidea, including five from thelazia species and one from o. conjuctivalis. however, these its sequences were highly divergent from each other and from those of o. petrowi, which indicated that their its regions were likely divergent enough for speciation. to further ensure the quality of detection, selected individual or pooled pcr products were also sequenced to validate their identities. when the qpcr detection system was used, melting curve analysis was performed to confirm that pcr amplicons showed the same curves as those of positive controls. among the fecal dna specimens, we have detected positive samples. however, because the specimens were collected from both individual and pooled quail samples derived from birds, direct calculation of positive rate was inappropriate as pooled positive specimens might contain both positive and negative samples. therefore, we employed two additional approaches to estimate the prevalence. the first approach was to only calculate positive rate from the samples collected from individual birds, in which samples were positive, giving a % positive rate. the second approach employed software written by dr. brad biggerstaff as an excel add-in, which was originally developed to determine positive rates of viral infections in pooled mosquito samples using a maximum likelihood estimation algorithm. by applying a bias-corrected mle estimation, we obtained an infection rate of % with lower and upper limits at % and %, respectively. collectively, we conclude that ~30% of the sampled wild quail were infected by the eye worm. the actual rate might be even higher, as the fecal samples were only collected once, rather than continuously for several days, and the sensitivity of pcr detection might not be maximal due to the inhibitory substances commonly present in fecal samples as discussed below. the detection of o. petrowi dna in feces allows rapid and sensitive detection of the presence of eye worms without the need to examine individual birds. however, one needs to be aware of the presence of inhibitory substances in fecal samples and the difficulties in releasing dna from eggs or encysted larvae. the presence of inhibitory substances could be minimized using the tablets included in the dna isolation kits specifically designed for stool samples such as the qiaamp dna stool mini kit. freeze/thaw cycles combined with homogenization with glass beads were necessary to break the eggs or encysted larvae to ensure the release of dna. furthermore, nested pcr might also be used by the addition of a primary amplification using the external primers qew_2373f and qew_2681r to not only improve the sensitivity of pcr detection, but to also further eliminate the presence of inhibitory substances in a second amplification procedure. the life cycle of o. petrowi is not well understood, its exact intermediate host as well as its migration details in quail. the presence of o. petrowi dna in quail feces indicates that eggs laid by female adult worms in the eyes could be passed into the digestive system and released with the excrement via the naso-lacrimal ducts to the gut route, as previously observed in other eye worms such as o. mansoni. this pcr detection protocol can also be easily adapted to identify intermediate host and perform surveillance, which is important in developing effective strategies to control the transmission of eye worms in the field. our phylogenetic analysis based on 18s rrna sequences indicated that o. petrowi clustered closely with streptopharagus and spirocerca. it is known that birds are a paratenic host for spirocerca lupi infection in their life cycle between dogs and dung beetles. dung beetles are also the intermediate host for streptopharagus. these observations suggest that dung beetles might be worth examining as one of the potential intermediate hosts. indeed, the detection of o. petrowi dna in various insects including dung beetles is currently ongoing as part of a separate project in determining the intermediate host and transmission route, and the data will be reported upon the completion of the survey.  we have performed a small-scale genome sequence survey, which not only rapidly generated a large number of molecular sequence data for the first time for o. petrowi, but also provided a snapshot of the genome for the eye worm in quail. the survey also identified a large number of microsatellite sequences that may be employed in further genotyping and population genetics studies. our phylogenetic reconstructions based on 18s rrna sequences indicated that spiruroidea was paraphyletic, while o. petrowi, streptopharagus and spirocerca formed a sister clade to the filarial nematodes. the obtained its sequence data also permitted us to design specific primers for molecular detection of o. petrowi in fecal samples, which may also be adapted to detect this nematode in insect intermediate hosts for surveillance and developing strategies to control the transmission of eye worms from intermediate hosts to quail. we also determined that ~28% - 33% of the birds were o. petrowi positive, suggesting that eye worm was a significant parasite in at least some quail ranches in texas. competing interests the authors declare that they have no competing interests. authors’ contributions lx participated in experimental design and performed the majority of experiments on the genome survey including constructing genomic library, cloning and sequencing, the cloning and sequencing of rrna gene and downstream region sequences, and the isolation stool dna and pcr/qpcr detection; fg and hz participated in sample preparation; ll participated in collection of fecal samples from wild quail; ab participated in collection of adult eye worms; dr participated in fecal sample collection, writing the manuscript, and securing funding for the study; amf participated in collection and speciation of eye worm and writing manuscript; gz conceived the study, participated in its design, molecular and phylogenetic analysis, and writing the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 list of contigs with annotations and information on top blast hits. click here for file additional file 2: table s2 oxyspirura petrowi microsatellite sequences identified by the gss using phobos. click here for file acknowledgements major funding for this research provided by rolling plains quail research foundation to gz, amf and dr. we thank dr. jason m. fritzler at the weber state university for his critical reading of the manuscript.
to fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18s rrna and the intergenic region between the 18s and 28s rrna genes, studied its phylogenetic affinity, and developed a pcr protocol for the detection of this eye worm. phylogenetic analysis based on 18s rrna sequences indicate that the spiruroidea is paraphyletic, in which oxyspirura and its closely related species are sisters to the filarial nematodes. despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of o. petrowi. the eye worm genome is at-rich, and contains a high number of microsatellite sequences. we have also developed a pcr protocol based on the its sequence that allows sensitive and specific detection of eye worm dna in feces. this eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as loa loa would be referential in developing treatments for the thelazoidea parasites. using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from northern bobwhites and scaled quail in texas in the spring of are o. petrowi positive. we have generated ~240 kb of genome sequence data derived from clones by a random genome survey of an o. petrowi genomic library. the o. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. our qpcr-based survey has confirmed that o. petrowi infection is of potential concern to quail managers in texas. the discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact northern bobwhite quail individuals and populations within texas and other regions of the united states.
 oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact northern bobwhite quail individuals and populations within texas and other regions of the united states. despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of o. petrowi. to fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18s rrna and the intergenic region between the 18s and 28s rrna genes, studied its phylogenetic affinity, and developed a pcr protocol for the detection of this eye worm.  we have generated ~240 kb of genome sequence data derived from clones by a random genome survey of an o. petrowi genomic library. the eye worm genome is at-rich, and contains a high number of microsatellite sequences. the discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. phylogenetic analysis based on 18s rrna sequences indicate that the spiruroidea is paraphyletic, in which oxyspirura and its closely related species are sisters to the filarial nematodes. we have also developed a pcr protocol based on the its sequence that allows sensitive and specific detection of eye worm dna in feces. using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from northern bobwhites and scaled quail in texas in the spring of are o. petrowi positive.  the o. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. this eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as loa loa would be referential in developing treatments for the thelazoidea parasites. our qpcr-based survey has confirmed that o. petrowi infection is of potential concern to quail managers in texas. 