Xiang et al. BMC Microbiology 2013, 13:233 http://www.biomedcentral.com/1471-2180/13/233 RESEARCH ARTICLE Open Access Gene discovery, evolutionary affinity and molecular detection of Oxyspirura petrowi, an eye worm parasite of game birds Lixin Xiang1,2*, Fengguang Guo1, Haili Zhang1, Lloyd LaCoste3, Dale Rollins3,4, Andrea Bruno5, Alan M Fedynich5 and Guan Zhu1* Abstract Background: 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. Results: We have generated ~240 kb of genome sequence data derived from 348 clones by a random genome survey of an O. petrowi genomic library. The eye worm genome is AT-rich (i.e., 62.2% AT-content), 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 ITS2 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 2013 are O. petrowi positive. Conclusions: 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 infectionisofpotentialconcerntoquailmanagersinTexas. Background some of them were described as ocular oxyspiruriasis or Oxyspirura petrowi is a spirurian nematode (Order oxyspirurosis [5-10]. Given that bobwhites are experien- Spirurida) that infects the eyes of quail and other birds [1]. cing long-term declines throughout their range in North In Texas, a 47–56% prevalence has been reported in America, there is a recognition that populations are Northern Bobwhites (Colinus virginianus) and Scaled declining even where suitable habitat conditions exist (e.g., Quail (Callipepla squamata) [2-4]. Similar infections Rolling Plains ecoregion of Texas), thereby raising con- caused by this genus of parasites have also been reported cerns that parasites such as O. petrowi maybeacontribut- in other animals including poultry and zoo animals, where ing factor (e.g., see a more detailed description at http:// www.quailresearch.org). It is likely that infection may cause host eye damage and physically impair vision, mak- * Correspondence: [email protected]; [email protected] ing birds less competitive in feeding and more susceptible 1Department of Veterinary Pathobiology, College of Veterinary Medicine & to predators (Figure 1). Biomedical Sciences, Texas A&M University, College Station, Texas, USA Although the eye worm has been considered as a pos- 2College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China Full list of author information is available at the end of the article sible contributing factor for the decline of wild quail © 2013 Xiang et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Xiang et al. BMC Microbiology 2013, 13:233 Page 2 of 9 http://www.biomedcentral.com/1471-2180/13/233 the prevalence by detecting parasite-specific DNA in feces, as well as to identify infected intermediate hosts that is otherwise difficult (if not impossible) 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 Idio- pathic Decline, which was initiated to further our under- standing of potentially pathogenic parasites occurring Figure 1 Oxyspirura petrowi adult worms in the eye of a within the Rolling Plains Ecoregion of Texas and Northern Bobwhite collected in Texas in February, 2013 western Oklahoma. All animal experiments were demonstrating their potential to cause visual obstruction in performed in accordance with procedures approved by addition to a pathological response resulting from infection. the Institutional Animal Care and Use Committee of Texas A&M University (protocol # 2011–193). populations in the Rolling Plains, little is known of the After microscopic examination for species validation, parasite’s biology, particularly at the molecular and gen- four worms were rinsed with PBS, placed in lysis buffer of omic levels (i.e., no molecular data were available in the the DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, GenBank databases prior to this study). Previous know- CA), and grinded with a plastic microtube grinder. ledge on the relationship of this parasite with other Genomic DNA (gDNA) was isolated from the ground nematodes was solely acquired by morphology, which worms according to manufacturer’s protocol for animal also needs to be validated at the molecular level. In fact, tissues. For the construction of a genomic library, gDNA only a single nucleotide sequence is present in the da- was first subjected to whole genome amplification using a tabase for the whole genus Oxyspirura (i.e., a 689-bp GenomePlex Complete Whole Genome Amplification partial rRNA gene from O. conjuctivalis [GenBank: (WGA) kit according the manufacturer’s standard proto- EF417873]). The lack of molecular data severely ham- col (Sigma-Aldrich Co., St. Louis, MO). Amplified gDNA pers our efforts in studying molecular epidemiology and products were fractionated in agarose gels and fractions transmission routes of O. petrowi, which may be useful containing fragments between 0.5 – 2 kb were collected for developing effective strategies to treat and control and purified using a Gel Extraction Kit (Omega Bio-Tek, ocular oxyspiruriasis in wild quail. Norcross, GA). After an incubation at 72°C for 20 min in To fill the knowledge gap, we have performed a small- a regular PCR reaction buffer to add a single adenine over- scale genome sequence survey (GSS) that provides the hang to the 3’-end, the products were ligated into pCR2.1- first batch of genomic sequence data for this nematode. TOPO vector using a TOPO-TA cloning kit (Invitrogen, Additionally, we have cloned the 18S rRNA, internal tran- Life Technologies, Grand Island, NY). scribed spacer 1 (ITS1), 5.8S rRNA, ITS2 and partial 28S After transformation, Escherichia coli OneShot TOP10F' rRNA genes. The small random GSS effort rapidly gener- chemically competent cells (Invitrogen) were plated onto LB ated ~240 kb of sequence information that provided not plates spread with 40 μLof40mg/mLX-galand5μLof only a snapshot of the quail eye worm genome, but also a 200 mM/mL IPTG, and incubated at 37°C overnight. Bac- large amount of microsatellite sequences for future geno- teria from a single white colony were collected into 10 μL typing and population genetic analysis. Based on the newly Milli-Q water in a microtube, from which 2 μLofsuspen- obtained rRNA sequences, we determined the evolution- sion was used directly as template in PCR reactions to deter- ary affinity of Oxyspirura with other nematodes among minethepresenceofinsertsusingapairofM13forward spirurians with available 18S rRNA sequences. We also and M13 reverse primers. Colonies containing inserts with designed specific primers based on the ITS2 sequences, desired sizes were further incubated in LB broth containing and performed real-time quantitative PCR (qPCR)-based 50 μg/mL kanamycin. Plasmids were prepared using a Plas- molecular detection of O. petrowi from DNA extracted mid DNA Mini kit (Omega Bio-Tek), and sequenced by the from fecal samples collected from Northern Bobwhite and Sanger dye-terminator sequencing method at the Gene Scaled Quail in Texas. Technology Laboratory at Texas A&M University. Understanding the biology of this parasitic nematode Vector and adaptor sequences were removed using a at molecular levels will enable us to effectively determine cross-match algorithm, and long inserts were assembled Xiang et al. BMC Microbiology 2013, 13:233 Page 3 of 9 http://www.biomedcentral.com/1471-2180/13/233 using the Phrap method implemented in the MacVector by simple phylogenetic reconstructions by the neighbor- program (version 12.7.4) (http://www.macvector.com). joining (NJ) method using the Tamura-Nei nucleotide All sequences were used as queries to