MURDOCH RESEARCH REPOSITORY This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination. The definitive version is available at http://dx.doi.org/10.1016/j.exppara.2015.11.001 Yang, R., Brice, B. and Ryan, U. (2016) Morphological and molecular characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa: Eimeriidae, 1989, Paperna and Landsberg) in a western bearded dragon (Pogona minor minor). Experimental Parasitology, 160 . pp. 11-16. http://researchrepository.murdoch.edu.au/29213/ Copyright: © 2015 Published by Elsevier Inc. Accepted Manuscript Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western bearded dragon (Pogona minor minor) Rongchang Yang, Belinda Brice, Una Ryan PII: S0014-4894(15)30059-X DOI: 10.1016/j.exppara.2015.11.001 Reference: YEXPR 7150 To appear in: Experimental Parasitology Received Date: 18 June 2015 Revised Date: 22 October 2015 Accepted Date: 9 November 2015 Please cite this article as: Yang, R., Brice, B., Ryan, U., Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western bearded dragon (Pogona minor minor), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 60, 58, 65 JF419354 E. sp. WGK2346 JF419344 E. sp. WGK2534 KF225639 E. setonicis FJ829322 E. trichosuri clone G JF419351 E. sp. WGK2298 65, 67, 72 JF419336 E. sp. Euro38K JF419337 E. sp. RK38N 66,72, 87 JF419359 E. sp. WGK2336 66,72, 87AB243082 E. gruis AB205169 E. gruis 99, 100, 99 Eimeria spp. and Cyclospora spp. 100, 100, 100 AB544327 E. reichenowi 76 AB544326 E. reichenowi AF324215 E. pilarensis JX464223 E. cf. tenggilingi 75, 86, 60 AF307877 E. rioarribaensis JX839286 E. sp. 2 RCY-2012 AF111185 Cyclospora cercopitheci 77, 84, 82 AB769592 E. bukidnonensis JQ993661 E. sp. ex Apodemus sylvaticus JQ993664 E. sp. ex Gerbillus dasyurus JQ993663.1 100, 100, 100 JX839287 E. tiliquae n. sp. CloneMANUSCRIPT 1 55, 60, 65 JX839288 E. tiliquae n. sp. Clone 2 AF324217 E. tropidura 78, 80, 82 KR360730 C. scincorum AY043207 C. sp. 100, 100, 100 KR779871 C. pogonae n. sp Choleoeimeria. spp . 66, 63, 58 KR360728 C. gallotiae 78, 79, 76 KR360733 C. wiegmanniana GU479650 G. balatonica GU479643 G. sp. BMR-2011d GU479671 G. balatonica 100, 99, 98 GU479655 E. subepithelialis GU479638 G. balatonica Goussia spp . and Eimeria. spp. GU479637 G. vargai GU479664 G. desseriACCEPTED 90, 86, 90 GU479666 G. desseri 88, 96, 96 GU479665 G. desseri EF472967 Toxoplasma gondii 0.01 1 Morphological and Molecular Characterization of Choleoeimeria pogonae n. sp. 2 coccidian parasite (Apicomplexa:Eimeriidae, 1989, Paperna and Landsberg) in a Western 3 bearded dragon (Pogona minor minor ) 4 Rongchang Yang a* , Belinda Brice b and Una Ryan a 5 6 aSchool of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, 6150. 7 bKanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076. 8 9 __________________________________ ACCEPTED MANUSCRIPT 10 *Corresponding author. Mailing address: School of Veterinary and Life Sciences, Murdoch 11 University, Murdoch, Western Australia, Australia, 6150. Phone: 61 89360 2495. Fax: 61 89310 12 4144. 13 E-mail: [email protected] 14 15 MANUSCRIPT ACCEPTED 16 ABSTRACT 17 A new species, Choleoeimeria pogonae n. sp. is described from a Western bearded dragon 18 (Pogona minor minor ) in Western Australia . Sporulated oocysts (n = 48) were cylindroidal in 19 shape. Oocyst length, 27.0 (26.0 - 28.3) µm, oocyst width, 15.2 (14.0 -16.5) µm, oocyst 20 length/width ratio (L/W) 1.8 (1.6-1.9), each with 4 sporocysts ( Eimeria -like) and a polar granule, 21 but lacking a micropyle and oocyst residuum. Sporocysts are ovoidal in shape, sporocyst length, 22 10.0 (9.0-11.0) µm, sporocyst width 8.5 (7.0-9.5) µm, sporocyst L/W ratio, 1.2 (1.1-1.3). Stieda, 23 substieda and parasubstieda bodies were all absent. Molecular analysisACCEPTED was MANUSCRIPT conducted at the 18S 24 rRNA and cytochrome c oxidase I (COI) loci. Phylogenetic analysis of 18S sequences revealed that 25 C. pogonae n. sp. grouped together with another four Choleoeimeria spp. and exhibited 99.1% to 26 99.4% genetic similarity. At the COI locus, C. pogonae n. sp. was in an independent clade and had 27 the highest similarity (80.4%) to Eimeria cf. mivati from a chicken ( Gallus gallus domesticus ). 28 According to the morphological and molecular data, this isolate is a new species of coccidian 29 parasite. This study further supports the taxonomy of Choleoeimeria spp . as a new genus based on 30 molecular phylogenetic analysis. 31 Keywords: 18S rRNA ; Cytochrome c oxidase I; Choleoeimeria pogonae n. sp; morphology; 32 molecular characterization. MANUSCRIPT 33 34 ACCEPTED 2 35 1. Introduction 36 The bearded dragon ( Pogona minor) is a species of lizard (Family: Agamidae) found on the 37 southwest coast and interior of Western Australia and includes the sub-species, the western bearded 38 dragon, (Pogona minor minor) and the Abrolhos bearded dragon (Pogona minor minima). The 39 latter is confined to the Abrolhos Islands, a chain of 122 islands, and associated coral reefs, in the 40 Indian Ocean off the west coast of Australia (Wilson and Swan, 2004). The Western bearded 41 dragon lives in a variety of habitats including woodlands, heath, arid desert and even coastal dunes. 42 It is regarded as semi- arboreal, and is often observed basking onACCEPTED rocks or fallen MANUSCRIPT trees (Ziring, 43 1999). 44 Choleoeimeria is a coccidian genus, proposed by Paperna and Landsberg (1989) that includes 45 tetrasporocystic, dizoic and Eimeria –like coccidians infecting reptilian gall bladder epithelium (El– 46 Toukhy et al., 2014). Choleoeimeria species possess bivalved sporocysts and lack a Stieda body and 47 have cylindroidal or ellipsoidal oocysts. So far, over 40 Choleoeimeria species have been described 48 morphologically and four species have been reported from Australia; C. egerniae from White's 49 skink (Egernia whitii) (Modrý et al., 2006), C. allogehyrae from the house gecko (Gehyra 50 australis), C. boulii from the Tree Dtella (Gehyra variegata) and C. heteronotis from Bynoe's 51 gecko ( Heteronotia binoei) (Paperna, 2007). To date no Choleoeimeria species have been reported 52 from the Western bearded dragon or from any other animal in Western Australia.MANUSCRIPT Molecular 53 characterization of Choleoeimeria species is in its infancy and therefore only four partial 18S rRNA 54 sequences from Choleoeimeria species were available in GenBank, one from the gall bladder of a 55 diadem snake ( Spalerosophis diadema ) from the Czech Republic (accession number: AY043207). 56 The other three species reported from Spain were Choleoeimeria gallotiae from a Tenerife lizard 57 (Gallotia galloti ) (accession number: KR360728), Choleoeimeria scincorum from a skink, Mabuya 58 (s. l.) sp. Fitzinger 1826 (accession number: KR360730)ACCEPTED and Choleoeimeria wiegmanniana from a 59 checkerboard worm lizard ( Trogonophis wiegmanni ) (accession number: KR360733). In the present 60 study, we characterized a novel Choleoeimeria sp . in a western bearded dragon from Western 3 61 Australia , both at morphological and molecular levels and propose the name Choleoeimeria 62 pogonae n. sp . This is the first report of Choleoeimeria from lizard hosts of the family Agamidae. 63 64 65 2. Materials and methods 66 67 2.1. Sample collection ACCEPTED MANUSCRIPT 68 The Western bearded dragon ( Pogona minor minor ) came into care at the Kanyana Wildlife 69 Rehabilitation Centre (KWRC), Perth. On admission it was noted that it had a depressed demeanor, 70 was extremely dehydrated and was reluctant to open it’s eyes. A diagnosis of conjunctivitis was 71 made and the appropriate treatment implemented. A faecal sample was obtained a few days after 72 admission and microscopy (wet mount) revealed large numbers of amoeba, coccidian oocysts and 73 occasional nematodes. The lizard was subsequently treated with Metronidazole and Toltrazuril. 74 This lizard was euthanased three months later as it remained inactive and would not self-feed. It had 75 lost 19% of its body weight over this period, despite being force fed daily. A faecal sample obtained 76 a few days prior to euthanasia showed no evidence of coccidian oocysts, either by wet mount or 77 faecal flotation. MANUSCRIPT 78 Faecal flotation was done using a standard protocol as described by Yang et al. (2014). 79 Sporulated oocysts were observed using an Olympus DP71 digital micro-imaging camera and 80 images were taken using Nomarski contrast imaging system with a 100x oil immersion objective. 81 82 2.2. DNA isolation 83 Total DNA was extracted from 200 mg of faecalACCEPTED sample using a Power Soil DNA Kit 84 (MolBio, Carlsbad, California) with some modifications as described by Yang et al. (2012). Briefly, 85 the faeces for DNA extraction were subjected to four cycles of freeze/thaw (liquid nitrogen 4 86 followed by boiling water) to ensure efficient lysis of oocysts before being processed using the 87 manufacturer’s protocol. A negative control (no faecal sample) was used in each extraction group. 88 89 2.3. PCR amplification and sequencing 90 91 A partial Choleoeimeria 18S rRNA sequence (1,285 bp) was amplified using a nested PCR 92 with the following primers EiF1 (Power et al., 2010) and EiR3 (Yang et al., 2012) for the external 93 reaction and EiF4: 5’ -CTAT GGC TAA TAC ATG CGC AAT -3’ (This study) and EiR4 – ACT ACCEPTED MANUSCRIPT 94 CAA AAG ATT ACC TAG AC-3 (This study) for the internal reaction.