Morphological and Molecular Characteriz

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Yang, R., Brice, B., Elliot, A., Lee, E. and Ryan, U. (2014) Morphological and molecular characterization of Eimeria paludosa coccidian parasite (Apicomplexa:Eimeriidae) in a dusky moorhen (Gallinula tenebrosa, Gould, 1846) in Australia. Experimental Parasitology, 147 . pp. 16-22.

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1 Full length article 2 3 Morphological and molecular characterization of Eimeria paludosa 4 coccidian parasite (Apicomplexa:Eimeriidae) in a dusky moorhen 5 (Gallinula tenebrosa, Gould, 1846) in Australia 6 7 Q1 Rongchang Yang a,*, Belinda Brice b, Aileen Elloit a, Elvina Lee a, Una Ryan a

8 a School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia 9 b Kanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076, Australia 10 11 12 HIGHLIGHTS GRAPHICAL ABSTRACT 13 14 Re-description of E. paludosa in a 15 • 0.02 16 dusky moorhen in Australia. 17 Morphology study: Identical to • 57 AF336339 Eimeria crandallis 18 E. paludosa. 72 AB769588 Eimeria bovis 19 • Genetic study: 99.7% similar to 100 AF345998 20 Eimeria gruis (AB544336) at 18S Eimeria faurei 21 locus. 100 AB769654 Eimeria wyomingensis 56 AB769624 Eimeria cylindrica 42 100 AF080612 Isospora robini AF080613 0.1 45 Isospora gryphoni AF060975 Caryospora bigenetica 99 KJ767187 Eimeria paludosa 97 HE653904 Eimeria crecis n. sp. 100 AY613853 100 Eimeria arnyi HE653905 Eimeria crecis n. sp. 99 HE653906 n. sp. KJ000078 Eimeria hermani Eimeria nenei AB544336 Eimeria gruis 22 92 97 KJ767187 Eimeria paludosa 96 AB544330 Eimeria gruis 100 100 AB544342 Eimeria reichenowi AB544336 Eimeria gruis KJ000078 Eimeria hermani 100 AY613853 AB544330 Eimeria arnyi 100 Eimeria gruis EF472967 Toxoplasma gondii AB544342 Eimeria reichenowi EF472967 Toxoplasma gondii

23 ARTICLE INFO ABSTRACT 24 2625 Article history: An Eimeria species is described from a dusky moorhen (Gallinula tenebrosa). Sporulated oocysts (n = 40) 2827 Received 16 July 2014 are ovoid, with a pitted single-layered oocyst wall in young oocysts and a relatively smooth wall in the 3029 Received in revised form 14 October 2014 mature oocysts. Oocyst wall was 1.0 μm thick, oocysts measured 17.3 × 13.3 (16.3–17.9 × 12.7–13.9) μm, 31 Accepted 16 October 2014 32 oocyst length/width (L/W) ratio, 1.3. Oocyst residuum was absent. A large polar granule was always ob- 33 Available online 34 served in the centre of the micropyle and many small polar granules were observed when the focus was 35 36 on the wall. Sporocysts are elongate-ovoid, 8.4 × 5.1 (8.0–8.9 × 4.9–5.5) μm, sporocyst L/W ratio, 1.6 (1.5– 37 Keywords: 38 1.8), sporocyst residuum was present, composed of numerous granules in a spherical or ovoid mass. Each 39 18S rRNA × × 4140 E. paludosa sporocyst contained 2 elongate sporozoites, 7.7 2.6 (7–10 2.2–3) μm. A spherical-ellipsoid posterior 4342 Morphology refractile body was found in the sporozoites. A nucleus is located immediately anterior to the posterior 4544 Genetic characterization refractile body. When the oocyst measurements and features were compared with valid Eimeria species 4746 Mitochondrial cytochrome oxidase gene from hosts in the Rallidae family, this Eimeria species was identiﬁed as E. paludosa. This is the ﬁrst report 4948 (COI) of E. paludosa in Australia and the dusky moorhen (Gallinula tenebrosa) in a new host for this species. 5150 Phylogeny Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the

53 54 * Corresponding author. Fax: +61 89310 4144. 55 E-mail address: [email protected] (R. Yang).

Please cite this article in press as: Rongchang Yang, Belinda Brice, Aileen Elloit, Elvina Lee, Una Ryan, Morphological and molecular characterization of Eimeria paludosa coccidian parasite (Apicomplexa:Eimeriidae) in a dusky moorhen (Gallinula tenebrosa, Gould, 1846) in Australia, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.10.010 ARTICLE IN PRESS

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mitochondrial cytochrome oxidase gene (COI). At the18S locus, E. paludosa shared 97.3% genetic similarity with Eimeria gruis (GenBank accession number: AB544336). It also shared 99.2% genetic similarity with Eimeria crecis (GenBank accession numbers: HE653904 and HE653905) and 98.5% similarity with Eimeria nenei (GenBank accession numbers: HE653906), both of which were identiﬁed from a corncrake (Crex crex) in the United Kingdom. At the 28S locus, E. paludosa shared 91.4% similarity with E. papillata from achicken(Gallus gallus) in the USA. At COI locus, E. paludosa was in a clade by itself and shared 87.2% similarity with E. irresidua, from a European rabbit (Oryctolagus cuniculus) from the Czech Republic. This is the ﬁrst molecular characterization of E. paludosa. © 2014 Published by Elsevier Inc.

1. Introduction days. A faecal sample was taken and microscopy revealed Eimeria and nematode larvae. Treatment was implemented and the bird was Eimeria spp. are coccidian parasites that infect a wide range of successfully released nine days after admission. vertebrate hosts (McDonald and Shirley, 2009). With more than 1700 Faecal ﬂotation was conducted using a saturated sodium chlo- described species (Duszynski et al., 2000), the genus is one of the ride and 50% sucrose (w/v) solution. A portion of faeces was placed

most speciose eukaryotic taxa. Pathogenic eimerian species that in 2% (w/v) potassium dichromate solution (K2Cr2 O7), mixed well cause severe clinical disease and economic loss in poultry and pro- and poured into petri dishes to a depth of less than 1 cm and kept duction animals have been well characterized (Aarthi et al., 2010; at room temperature in the dark to facilitate sporulation. Sporu- Fitzgerald, 1980; Taubert et al., 2010). Traditionally, identification lated oocysts were observed using an Olympus DP71 digital micro- of Eimeria species has been based largely not only on sporulated imaging camera and images were taken using Nomarski contrast oocyst morphology but also on host species, pathology and geo- imaging system with a 100× oil immersion objective. graphic distribution (Duszynski and Wilber, 1997; Tenter et al., 2002). However, some species of Eimeria are morphologically identical and 2.2. Isolation of single Eimeria oocysts using a micromanipulator occur in several hosts and it is now recognized that molecular data are essential to accurately delimit species and infer phylogenetic A 3 axis hydraulic micromanipulator (MO-102, Nirashige, Japan) relationships among Eimeria species (Tenter et al., 2002). was used to isolate four separate single oocysts for DNA extrac- A total of 13 Eimeria species have been identified from the order tion and PCR. Gruiformes including 6 from the family Rallidae and 7 from the family Gruidae (Atkinson et al., 2008). The taxonomy of the Eimeria species in the family Rallidae is somewhat confused. Since the first 2.3. DNA isolation Eimeria species from gruiform birds (family Rallidae), Eimeria paludosa, was described by Leger and Hesse (1922) from the Eur- Isolated single oocysts were placed on a slide and checked under asian coot (Fulica atra L.), and common moorhen (Gallinula chloropus the microscope (Olympus DP71 digital micro-imaging camera). Once L.), at least 5 synonyms of Eimeria paludosa have been reported. In the existence of a single oocyst on the cover slip was confirmed, 1990, McAllister and Upton, re-described E. paludosa in the Amer- photographs were recorded for morphological identification. The ican Coot (Fulica americana) and clarified synonyms of eimerian coverslip was then transferred into a PCR tube containing 10 μl of species from related birds such as E. polucephali from rails (Porphyrio lysis buffer (0.005% SDS in TE solution). After a brief centrifuga- caerulens) in Russia (Yakimoff and Matschoulsky, 1939) and E. dauki tion, the tube was frozen in liquid nitrogen and thawed in a 95 °C in the Eurasian coot (F. atra) and the purple swamp hen (Porphyrio water bath for four rounds to disrupt the oocyst wall. After the ad- porphyrio) in India (Bathia and Pande, 1967). Currently there are only dition of 0.5 μl proteinase K (20 mM), the tube was incubated at 56 °C 6 valid Eimeria species in the family Rallidae: Eimeria bragini, Eimeria for 2 h and then at 95 °C for 15 min. The whole lysate of the single monglica, Eimeria paludosa, Eimeria porphyrulae, Eimeria crecis and oocyst was used directly for PCR. Q2 Eimeria nenei (Duszynski and Upton, 2000; Jeanes et al., 2013). Un- fortunately, no DNA sequence data are available from the first 4 species, as the identifications were based on morphological fea- 2.4. PCR amplification and sequencing tures only. Two Eimeria species, Eimeria gruis and Eimeria reichenowi, derived Generic apicomplexan primers (CRYPTOF 5′-AAC CTG GTT GAT from cranes, have been described in the family Gruidae and both CCT GCC AGT and CRYPTOR 5′-GCT TGA TCC TTC TGC AGG TTC ACC have been extensively characterized genetically (Honma et al., 2007, TAC) were used to amplify the almost full length 18S rRNA gene as 2011; Matsubayashi et al., 2005; Novilla and Carpenter, 2004). described by Eberhard et al. (1999). The expected PCR product was In the present study, we re-characterized the E. paludosa in a ~1584 bp. dusky moorhen (Gallinula tenebrosa) (Gruiformes: Rallidae), both The PCR for the 28S rRNA locus was carried out using a nested morphologically and genetically. This is the first report of E. paludosa PCR with the external primers: 28SExF: 5′-TAC CCG CTG AAC TTA identified in Australia from a new host, the dusky moorhen (Gallinula AGC-3′ and 28SExR: 5′-CMA CCA AGA TCT GCA CTA G-3′ as previ- tenebrosa). ously described (Schrenzel et al., 2005), which produced a PCR product size of ~1495 bp. The internal primers (28InF: 5′-ACT ATG TTC CCT AGT AAC G-3′ and 28SInR 5′-AAC GCT TCG CCA CGA TCC- 2. Materials and methods 3′) were as previously reported (Yang et al., 2014) and produced an amplicon size of 1420 bp. 2.1. Sample collection Amplification of a 465 bp region of the COI locus from this sample was conducted as described by Ogedengbe et al. (2011) and Yang A young dusky moorhen chick came into care at the Kanyana et al. (2013). Wildlife Rehabilitation Centre (KWRC), Perth, after it had been at- The results of the sequencing reactions were analysed and edited tacked by a cat. On admission it had a swollen hock. Substantial using FinchTV (Version 1.4), compared to existing Eimeria spp. 18S weight loss (19% total body weight) was observed over the next five and 28S rRNA and COI sequences on GenBank using BLAST searches

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a SC SC PG SB MP PG

POW SB SR 1 2 3 4 5

MP PG b

1 Fig. 1. a. (1–4) Nomarski interference-contrast photomicrographs of E. paludosa oocysts showing 4 spheroidal to subspheroidal sporocysts and (5) showing sporocysts from 2 a ruptured oocyst. POW = pitted oocyst wall; SC = sporocyst; SB = stieda body; PG = polar granule; SR = Sporocyst residuum; MP = micropyle. Note the abundant sporocyst 3 residuum, which occupies much of the sporocyst volume. Scale bar = 10 μm. b. Line Drawing of the sporulated oocyst of E. paludosa. Scale bar = 10 μm. 4 5 and aligned with reference genotypes from GenBank using Clustal Tamura–Nei model and grouped using Neighbour-Joining. Parsi- 18 6 W in BioEdit (V7.2.5). mony analyses were conducted using MEGA version 6 (MEGA6: 19 7 Molecular Evolutionary Genetics Analysis software, Arizona State 20 8 2.5. Phylogenetic analysis University, Tempe, Arizona, USA). Bootstrap analyses were con- 21 9 ducted using 1000 replicates to assess the reliability of inferred tree 22 10 Phylogenetic trees were constructed for Eimeria spp. at the 18S, topologies. Maximum Likelihood (ML) analyses were conducted using 23 11 28S and COI loci with additional isolates from GenBank. Distance the program PhyML (Dereeper et al., 2008) and the reliability of the 24 12 estimation was conducted using TREECON (Van de Peer and De inferred trees was assessed by the approximate likelihood ratio test 25 13 Wachter, 1994), based on evolutionary distances calculated with the (aLRT) (Anisimova and Gascuel, 2006). 26

0.02

57 AF336339 Eimeria crandallis 72 AB769588 Eimeria bovis 100 AF345998 Eimeria faurei 100 AB769654 Eimeria wyomingensis 56 AB769624 Eimeria cylindrica 0.1 a 42 100 AF080612 Isospora robini 14 AF080613 Isospora gryphoni KJ767187 45 99 Eimeria paludosa AF060975 97 HE653904 Eimeria crecis n. sp. Caryospora bigenetica 100 HE653905 Eimeria crecis n. sp. 100 AY613853 Eimeria arnyi 99 HE653906 Eimeria nenei n. sp. KJ000078 Eimeria hermani 97 AB544336 Eimeria gruis 92 96 AB544330 Eimeria gruis KJ767187 Eimeria paludosa. 100 100 AB544342 Eimeria reichenowi AB544336 Eimeria gruis KJ000078 100 Eimeria hermani AY613853 100 AB544330 Eimeria gruis Eimeria arnyi EF472967 Toxoplasma gondii AB544342 Eimeria reichenowi EF472967 Toxoplasma gondii

15 Fig. 2. a. Evolutionary relationships of E. gallinulae n. sp. inferred by distance analysis of 18S rRNA sequences based on 1653 bp of sequence. Percentage support (>50%) 16 from 1000 pseudoreplicates from neighbour-joining analyses is indicated at the left of the supported node. b. Phylogenetic position of E. paludosa, E. crecis, E. nenei, E. gruis, 17 E. reichenowi, E. hermani and E. arnyi (based on ~1142 bp of 18S rDNA only).

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1 3. Results (Porphyrio caerulens), Eurasian coot (F. atra) and purple swamp hen 29 2 (Porphyrio porphyrio). 30 3 3.1. Species description Prepatent period: Unknown. 31 4 Patent period: Unknown. 32 5 3.1.1. Eimeria paludosa (Fig. 1a and 1b) Site of infection: Unknown. 33 6 Diagnosis: Oocysts are ovoid, with a pitted single-layered oocyst Sporulation time: 48–96 hours. 34 7 wall in immature oocysts and a relatively smooth wall in the mature Material deposited: Oocysts in 10% formalin and oocyst 35 8 oocysts. Oocyst wall was 1.0 μm thick. Oocysts measured 17.3 × 13.3 phototypes were deposited in Western Australian Museum under 36 9 (16.3–17.9 × 12.7–13.9) μm, oocyst length/width (L/W) ratio, 1.3. the reference number WAM Z68798. DNA sequences have been de- 37 10 Oocyst residuum was absent. A large polar granule was observed posited in GenBank under accession numbers KJ767187, KJ767188, 38 11 in the centre of the micropyle and many small polar granules were and KJ767189 for the 18S, 28S and COI loci, respectively. 39 12 observed as well when the focus was on the wall. Sporocysts are 40 13 elongate-ovoid, 8.4 × 5.1 (8.0–8.9 × 4.9–5.5) μm, sporocyst L/W ratio, 3.2. Phylogenetic analysis of E. paludosa at the 18S locus 41 14 1.65 (1.47–1.85), sporocyst residuum was present, composed of nu- 42 15 merous granules in a spherical or ovoid mass. A large polar granule Analysis of the four individual oocysts produced identical se- 43 16 was located between the two micropyles and many small polar gran- quences at all loci analysed. At the 18S rRNA locus, a 1653 bp PCR 44 17 ules were present as well. Each sporocyst contained 2 elongate product of E. paludosa was successfully ampliﬁed and sequenced. 45 18 sporozoites, 7.7 × 2.6 (7.0–10.0 × 2.2–3.0) μm, a spherical-ellipsoid Phylogenetic analyses of E. paludosa at this locus using Distance, Par- 46 19 posterior refractile body was found in the sporozoites. A nucleus simony and ML analyses produced similar results (Fig. 2,NJtree 47 20 is located immediately anterior to the posterior refractile body. shown). Eimeria paludosa grouped in a clade with E. gruis (GenBank 48 21 Host: Dusky moorhen (Gallinula tenebrosa). accession number: AB544336) from a hooded crane (Grus monacha) 49 22 Locality: Perth, Western Australia. in Japan (97.3% similarity) (Honma et al., 2011). Eimeria paludosa 50 23 Prevalence: Unknown. also shared 97.0% similarity with E. reichenowi (GenBank acces- 51 24 Other hosts: Eurasian coot (Fulica atra L.), common moorhen sion number: AB544326) from a hooded crane reported by the same 52 25 (Gallinula chloropus L.), American Coot (Fulica americana), rails group. A clade which consisted of E. paludosa E. gruis, E. reichenowi 53

0.02

AF026388 Eimeria tenella 100

GU593705 Goussia desseri 89

EU252544 Cycospora cayetanensis 92 100 GU593706 Eimeria papillata

100 KJ7671878 Eimeria paludosa

26 GU593704 Eimeria anguillae 100 91

GU593705 Goussia desseri 100 GU188426 Sarcocystis rileyi

100 DQ227420 Besnoitia besnoiti

AF101077 Hammondia hammondi 100 X75453 Toxoplasma gondii 53

AF076901 Toxoplasma gondii

AF001946 Neospora caninum

27 Fig. 3. Evolutionary relationships of E. paludosa inferred by distance analysis of 28S rRNA sequences based on 1553 bp of sequence. Percentage support (>50%) from 1000 28 pseudoreplicates from neighbour-joining analyses is indicated at the left of the supported node.

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0.1

JX464221 Eimeria apionodes 93 HM771683 Eimeria vermiformis 74 HM771682 Eimeria falciformis 95 JQ993701 Eimeria alorani isolate 100 JX839285 Eimeria tiliquae 100JQ993708 Eimeria nafuko JQ993709 Eimeria burdai JQ993706 Eimeria apodemus 61 JQ993688 99 Eimeria callospermophili 85 JQ993687 Eimeria cahirinensis JQ993686 Eimeria cahirinensis 100 JQ993692 Eimeria flavescens 93 JQ993698 Eimeria piriformis 64 JQ993694 Eimeria irresidua JQ993693 99 Eimeria intestinalis 95 JQ993699 Eimeria vejdovskyi KF419217 Eimeria magna 100HQ702480 Eimeria brunetti |HM771675 Eimeria brunetti JN596590 Eimeria pavonina 61 FJ236403 Eimeria tenella 100 HM117017 Eimeria meleagris KJ7671879 Eimeria paludosa JQ392580 Eimeria macropodis HM771690 T. gondii

1 Fig. 4. Evolutionary relationships of E. paludosa inferred by distance analysis of mitochrondial cytochrome oxidase gene (COI). Percentage support (>50%) from 1000 2 pseudoreplicates from neighbour-joining analyses is indicated at the left of the supported node.

4 and Eimeria hermani was separated from the other Eimeria species locus, there were no gruiform-derived Eimeria COI sequences avail- 34 5 by a clade which consisted of Isospora, Caryospora and Eimeria arnyi able in GenBank. Phylogenetic analysis placed E. paludosa in a clade 35 6 (Fig. 2a). Two slightly shorter 18S sequences from Eimeria crecis by itself. It had the highest similarity (87.2% at the nucleotide level) 36 7 (GenBank accession numbers: HE653904 and HE653905) and one with E. irresidua from a European rabbit (Oryctolagus cuniculus)from 37 8 18S sequence from Eimeria nenei (GenBank accession number: the Czech Republic (GenBank accession number JQ993694) (Fig. 4). 38 9 HE653906) were available, and a phylogenetic analysis including Eimeria paludosa shared 88.0% identity at amino acid level with 39 10 these three sequences was also conducted (Fig. 2b). This analysis E. alorani from a striped field mouse (Apodemus agrarius) and an 40 11 showed that Eimeria paludosa shared 99.2% similarity with E. crecis Eimeria sp. ex Apodemus flavicollis. 41 12 and 98.5% with E. nenei. 42 13 4. Discussion 43 14 3.3. Phylogenetic analysis of E. paludosa at the 28S locus 44 15 The dusky moorhen or waterhen is a medium-sized, dark grey- 45 16 A 1553 bp sequence of 28S DNA from E. paludosa was used for black water bird. It has a yellow-tipped scarlet bill and a scarlet bill 46 17 phylogenetic analysis. There were few 28S rRNA sequences from shield. Its legs are red above the “knee” and red or greenish below 47 18 Eimeria species available in GenBank and no sequences from Eimeria with long slender toes. It is found from Indonesia through New 48 19 derived from gruiform birds; therefore phylogenetic analysis could Guinea to Australia. It is widespread in south-western and eastern 49 20 only be conducted using available Eimeria 28S rRNA sequences and Australia. This bird inhabits well-vegetated wetlands such as rivers, 50 21 other coccidian 28S sequences including Cycospora spp., Goussi spp., swamps and artificial waterways. It is omnivorous and feeds on algae 51 22 Sarcocystis spp., Besnoitia besnoiti, Hammondia hammondi and Toxo- and water plants in the water but also eats grasses, seeds, fruits, 52 23 plasma gondii. Neospora caninum was used as an outgroup. molluscs and other invertebrates (Pizzey and Knight, 2007). 53 24 Phylogenetic analysis grouped E. paludosa in a clade by itself (Fig. 3). In 1990, McAllister and Upton, re-described E. paludosa from the 54 25 It shared 91.0% and of 89.0% genetic similarity with E. papillata American coot (Fulica americana), with oocyst measurements of 55 26 (GenBank accession number: GU593706) and E. tenella (GenBank 16.5 × 12.6 (15.0–23.0 × 11.0–14.0) μm and with elongate-ovoid spo- 56 27 accession number: AF026388) respectively from chickens (Gallus rocysts measuring 10.8 × 6.2 (10.0–12.0 × 5.0–7.0) μm and a L/W ratio 57 28 gallus). of 1.7 (1.5–1.9) (McAllister and Upton, 1990). Both the L/W ratio of 58 29 the oocysts and sporocysts from the present study were similar to 59 30 3.4. Phylogenetic analysis of E. paludosa at the COI locus those from E. paludosa (Table 1). Three other species of Eimeria de- 60 31 scribed from the Rallidae family; Eimeria polucephali, Eimeria crecis 61 32 Direct sequencing of the COI gene fragment produced a clean and Eimeria nenei (Jeanes et al., 2013; Lainson, 1994) are morpho- Q3 62 33 chromatogram and resulted in 467 bp of sequence. As with the 28S logically different from E. paludosa (Table 1). A remarkable 63

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morphological feature from E. paludosa is that there is a large polar 6 granule in the middle of the two micropyles, which was also present 7 in the oocysts isolated from the dusky moorhen in the present study. 8 Another recognizable feature of E. paludosa is that the young oocysts 9 have a rough wall surface, which was also detected in some of the 10 young oocysts in the dusky moorhen. This rough oocyst wall surface 11 was also observed in some mature oocysts in the present study. On 12 compact mass Residuum faint granules faint granules the basis of all these morphological findings, the Eimeria oocysts 13 isolated from the dusky moorhen were confirmed as E. paludosa. 14 Molecular analysis was also used for the first time to characterize 15 2.0 Composed of very fine

× E. paludosa and it shared its closest genetic similarity (97.3%) with 16

Substida body E. gruis at the 18S locus. 17 Eimeria paludosa appears to be highly dispersed as it has now 18

1.5 1.2 been reported from all continents of the world (McAllister and Upton, 19 × 1990; Duszynski et al., 2000; this study). In addition, the white- 20 present Present Scattered granules or present Present Large, central, compact 0.6 present Present Small, compact body dome Rectangular Composed of very ﬁne breasted waterhen (Amaurornis phoenicurus) that is widely 21 distributed across Southeast Asia and the Indian Subcontinent, and 22 the common moorhen (Gallinula chloropus), which is distributed 23 4.9–5.5) 8–10) 6–8) 5–7)

× across Africa, Europe, and Asia (which are known hosts of E. paludosa) 24 5–7) × × × 6.5 6.2 × 9 5.4 5.1 × × (Duszynski et al., 2000), are sympatric with the dusky moorhen and, 25 × × × potentially, can cross-transmit this coccidium in the wild. 26 (17–19 (11–14 10.8 (10–12 (8–10 8.4 (8.0–8.9 Future studies need to concentrate on obtaining morphologi- 27 cally characterized Eimeria species derived from gruiform birds and 28 generate sequence data that are directly related to described species. 29 Analysing these isolates at multiple gene loci will also provide a more 30 ovoid Shape Measurements Stieda Sporoscysts ovoid in-depth analysis of the evolution of gruiform-derived Eimeria spp. 31 32 Acknowledgments 33 Present Ellipsoidal 12.6 Present Elongate- Present Ovoid 9.2 granule Present Elongate- 34

) Polar The authors wish to thank June Butcher and the volunteers at 35

μm the Kanyana Wildlife Rehabilitation Centre for their commitment 36 and dedication in caring for all the animals admitted to the centre. 37 c. 0.8 c. 0.8 c.1.0 Wall ( c. 1.0 We are also grateful to the staff at the Wattle Grove Veterinary Hos- 38 pital, Perth, for their expert treatment and care of the wildlife treated 39 at their clinic. 40 1.31.3 bi-layered bi-layered Present Ellipsoidal 17.5 1.31 one-layered 1.07 bi -layered Shape index 1.3 one-layered 41 References Q4 42 18.7)

× 43

12.7–13.9) Aarthi, S., Dhinakar Raj, G., Raman, M., Gomathinayagam, S., Kumanan, K., 2010. 44 17–20) 16.2 × Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil 45 × × 11–14) 12–16)

× × Nadu, India. Parasitol. Res. 107, 1013–1017. 46 12.6 14.3 18.1 17.7 13.3 × × × ×

× Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: a 47 ) fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552. 48 μm ( (15–23 (13–18 (21–26.0 (20–23.7 (16.3–17.9 Atkinson, C.T., Thomas, N.T., Hunter, D.B., 2008. Parasitic Diseases of Wild Birds, ﬁrst 49 ed. Wiley-Blackwell., Iowa, USA, p. 169. 50 Bathia, B.B., Pande, B.P., 1967. On eimerian oocysts from the purple moorhen 51 (Porphyrio porphyrio L.) and the coot (Fulica atra atra): a preliminary study. Acta 52 Vet. Acad. Sci. Hung. 17, 347–349. 53

Shapeovoid Measurements Spherical 16.5 15.3 ellipsoidal 23.6 ellipsoidal 22.4 Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., et al., 2008. 54 Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids 55 ) ) Res. 36, W465–W469. 56

) Duszynski, D.W., Wilber, P.G., 1997. A guideline for the preparation of species 57 ) descriptions in the Eimeriidae. J. Parasitol. 83, 333–336. 58

spp. recorded from Rallidae. Duszynski, D.W., Couch, L., Upton, S.J., 2000. Coccidia of the world. (accessed 26.04.14.). Q5 60 Eberhard, M.L., da Silva, A.J., Lilley, B.G., Pieniazek, N.J., 1999. Morphologic and 61

Eimeria molecular characterization of new Cyclospora species from Ethiopian monkeys: 62 McAllister and Upton (1990 Jeanes et al. (2013 Jeanes et al. (2013 Lainson (1994 This study Ovoid 17.3 C. cercopitheci sp.n., C. colobi sp.n., and C. papionis sp.n. Emerg. Infect. Dis. 5, 63 and ) ) 651–658. 64 Fitzgerald, P.R., 1980. The economic impact of coccidiosis in domestic animals. Adv. 65 Vet. Sci. Comp. Med. 24, 121–143. 66 (Fulica Honma, H., Yokoyama, T., Inoue, M., Uebayashi, A., Matsumoto, F., Watanabe, Y., et al., Crex Crex Crex Crex 67

E. paludosa 2007. Genetical identiﬁcation of coccidia in red-crowned crane, Grus japonensis. 68 Parasitol. Res. 100, 637–640. 69 Honma, H., Suyama, Y., Watanabe, Y., Matsumoto, F., Nakai, Y., 2011. Accurate analysis 70 of prevalence of coccidiosis in individually identiﬁed wild cranes in inhabiting 71 Gallinula tenebrosa) Dusky moorhen ( Purple gallinule (Porphyrula martinica) Corncrakes ( American coot americana) Corncrakes ( and migrating populations in Japan. Environ. Microbiol. 13, 2876–2887. 72 Jeanes, C., Vaughan-Higgins, R., Green, R.E., Sainsbury, A.W., Marshall, R.N., Blake, 73 D.P., 2013. Two new Eimeria species parasitic in corncrakes (Crex crex) 74 (Gruiformes: Rallidae) in the United Kingdom. J. Parasitol. 99, 634–638. 75 Lainson, R., 1994. Observations on some avian coccidia (Apicomplexa: Eimeriidae) 76 in Amazonian Brazil. Mem. Inst. Oswaldo Cruz 89, 303–311. 77 porphyrulae Leger, L., Hesse, E., 1922. Coccidie d’oiseaux palustres, le genre Jarrina n. g. C. R. Hebd. 78 Coccidia Hosts References Oocysts E. paludosa Eimeria Eimeria nenei E. paludosa Eimeria crecis

Table 1 Comparative morphology of Seances Acad. Sci. 174, 74–77. 79 1 2

3 Please cite this article in press as: Rongchang Yang, Belinda Brice, Aileen Elloit, Elvina Lee, Una Ryan, Morphological and molecular characterization of Eimeria paludosa coccid- 4 ian parasite (Apicomplexa:Eimeriidae) in a dusky moorhen (Gallinula tenebrosa, Gould, 1846) in Australia, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.10.010 5 ARTICLE IN PRESS

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1 Matsubayashi, M., Takami, K., Abe, N., Kimata, I., Tani, H., Sasai, K., et al., 2005. Tenter, A.M., Barta, J.R., Beveridge, I., Duszynski, D.W., Mehlhorn, H., Morrison, D.A., 23 2 Molecular characterization of crane Coccidia, Eimeria gruis and E. reichenowi, found et al., 2002. The conceptual basis for a new classification of the coccidia. Int. 24 3 in feces of migratory cranes. Parasitol. Res. 97, 80–83. J. Parasitol. 32, 595–616. 25 4 McAllister, C.T., Upton, S.J., 1990. Description of the oocysts of Eimeria paludosa Van de Peer, Y., De Wachter, R., 1994. TREECON for Windows: a software package 26 5 (Apicomplexa: Eimeriidae) from Fulica americana (Aves: Gruiformes), with for the construction and drawing of evolutionary trees for the Microsoft Windows 27 6 comments on synonyms of eimerian species from related birds. J. Parasitol. 76, environment. Comput. Appl. Biosci. 10, 569–570. 28 7 27–29. Yakimoff, W.L., Matschoulsky, S.N., 1939. A coccidium from rails (Porphyrios 29 8 McDonald, V., Shirley, M.W., 2009. Past and future: vaccination against Eimeria. Polycephalus caerulens). J. R. Microscop. Soc 59, 80–81. 30 9 Parasitology 136, 1477–1489. Yang, R., Brice, B., Bennett, M.D., Ryan, U., 2013. Novel Eimeria sp. isolated from a 31 10 Novilla, M.N., Carpenter, J.W., 2004. Pathology and pathogenesis of disseminated King’s skink (Egernia kingii) in Western Australia. Exp. Parasitol. 133, 162– 32 11 visceral coccidiosis in cranes. Avian Pathol. 33, 275–280. 165. 33 12 Ogedengbe, J.D., Hanner, R.H., Barta, J.R., 2011. DNA barcoding identifies Eimeria Yang, R., Brice, B., Ryan, U., 2014. A new Caryospora coccidian species (Apicomplexa: 34 13 species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Eimeriidae) from the Laughing Kookaburra (Dacelo novaeguineae). Exp. Parasitol. 35 14 Apicomplexa, Alveolata). Int. J. Parasitol. 41, 843–850. 145, 68–73. 36 15 Pizzey, G., Knight, F., 2007. The Field Guide to the Birds of Australia, eighth ed. Harper 16 Collins Publishers, NSW, Australia. 37 17 Schrenzel, M.D., Maalouf, G.A., Gaffney, P.M., Tokarz, D., Keener, L.L., McClure, D., et al., Uncited reference Q6 38 18 2005. Molecular characterization of isosporoid coccidia (Isospora and Atoxoplasma 19 spp.) in passerine birds. J. Parasitol. 91, 635–647. 39 20 Taubert, A., Wimmers, K., Ponsuksili, S., Jimenez, C.A., Zahner, H., Hermosilla, C., 2010. Yang, R., Fenwick, S., Potter, A., Elliot, A., Power, M., Beveridge, I., et al., 2012. Molecular 40 21 Microarray-based transcriptional profiling of Eimeria bovis-infected bovine characterisation of Eimeria species in Macropods. Exp. Parasitol. 132, 216– 41 22 endothelial host cells. Vet. Res. 41, 70. 221. 42