(2014) Isospora Anthochaerae N. Sp. (Apicomplexa: Eimeriidae) from a Red Wattlebird (Anthochaera Carunculata) (Passeriformes: Meliphagidae) in Western Australia

MURDOCH RESEARCH REPOSITORY

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Yang, R., Brice, B. and Ryan, U. (2014) Isospora anthochaerae n. sp. (Apicomplexa: Eimeriidae) from a Red wattlebird (Anthochaera carunculata) (Passeriformes: Meliphagidae) in Western Australia. Experimental Parasitology, 140. pp. 1-7.

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YEXPR 6817 No. of Pages 7, Model 5G 6 March 2014

Experimental Parasitology xxx (2014) xxx–xxx 1 Contents lists available at ScienceDirect

Experimental Parasitology

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6 7

3 Isospora anthochaerae n. sp. (Apicomplexa: Eimeriidae) from a Red

4 wattlebird (Anthochaera carunculata) (Passeriformes: Meliphagidae)

5 in Western Australia

a b a,⇑ 8 Q1 Rongchang Yang , Belinda Brice , Una Ryan

9 Q2 a School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia 10 b Kanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076, Australia

1211 13 highlights graphical abstract 15 2116

17 Description of a new species of 51 Isospora sp iSAT1 FJ269357 Blackcap (Sylvia atricapila) 78 18 Isospora in Red wattlebirds. Isospora sp iSAT3 FJ269359 Blackcap (Sylvia atricapila) Isospora sp iSAT4 FJ269360 Blackcap (Sylvia atricapila) 19 Morphological characterisation. Isospora sp iSAT2 FJ269358 Blackcap (Sylvia atricapila) 20 94 Isospora sp iSAT6 FJ269362 Blackcap (Sylvia atricapila) Molecular characterisation at 4 loci. Isospora hypoleucae FJ269363 Eimeria ex Apodemus sylvaticus JQ993707

99 Eimeria apoinodes JX464221 Pangolins (Mamm: Pholidota) 72 Eimeria ex Apodemus flavicollis 4 JQ993704 Isospora sp iSAT5 FJ269361 Blackcap (Sylvia atricapila)

95 Eimeria cahirinensis JQ993686 Isospora lesouefi HQ221885 Regent honeyeater (Xanthomyza phrygia) Isospora anthochaerae n. sp. KF766054 Red wattlebird (Anthochaera carunculata)

Eimeria tenella 161-27 JQ993711 Eimeria bruntti HQ702480 Eimeria ex Apodemus flavicollis B13 FJ236458 Toxoplasma gondii HM771690

0.10 0.05 0.00

23 article info abstract 4125 26 Article history: A new species, Isospora anthochaerae n. sp. is described from a Red wattlebird (Anthochaera carunculata). 42 27 Received 29 October 2013 Sporulated oocysts (n = 37) are subspherical, with smooth colourless to pale brown bilayered oocyst wall, 43 28 Received in revised form 13 February 2014 0.8 lm thick (outer layer 06 lm, inner 0.2 lm thick). Oocyst with 2 spheroidal to subspheroidal 44 29 Accepted 18 February 2014 sporocysts. Oocyst length, 23.4 lm (20.0–26.0); oocyst width, 20.7 lm (19.0–22.0); oocyst length/width 45 30 Available online xxxx (L/W) ratio, 1.1. Micropyle, oocyst residuum and polar granule are absent. Sporocysts with compact spo- 46 rocyst residuum and 4 sporozoites. Sporocyst length, 14.5 lm; sporocyst width, 10.1 lm sporocyst L/W 47 31 Keywords: ratio, 1.4. Molecular analysis was conducted at four loci; the ribosomal internal transcribed spacer (ITS), 48 32 Isospora the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase gene (COI). At the COI locus, I. 49 33 Red wattlebird 34 Morphology anthochaerae n. sp. exhibited 98.5% similarity to Isospora lesoueﬁ from a Regent honeyeater (Xanthomyza 50 35 Phylogeny phrygia) and 98% similarity with an Isospora sp. (iSAT5) from a blackcap (Sylvia atricapilla). Based on mor- 51 36 ITS rRNA phological and molecular data, this isolate is a new species of coccidian parasite that to date has only 52 37 18S rRNA been found in Red wattlebirds. 53 38 28S rRNA Ó 2014 Published by Elsevier Inc. 54 39 COI 55 40 56 57 58 1. Introduction morphological differences and paraphylogeny of the genus. In 61 1977, the genus Cystoisospora was proposed because of the pres- 62 59 The genus Isospora was ﬁrst described by Schneider in 1881 and ence of unizoic tissue cysts in lymphoid tissues in rodents which 63 60 its taxonomy has been a source of controversy since due to function as intermediate hosts of Cystoisospora felis and Cystoisos- 64 pora rivolta of cats (Frenkel, 1977). In 2005, Barta et al. assigned 65 all tetrasporozoic, diplosporocystic oocysts from mammals with- 66 ⇑ Corresponding author. Fax: +61 89310 4144. out Stieda bodies in their sporocysts, to the genus Cystoisospora 67 E-mail address: [email protected] (U. Ryan).

http://dx.doi.org/10.1016/j.exppara.2014.02.011 0014-4894/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Yang, R., et al. Isospora anthochaerae n. sp. (Apicomplexa: Eimeriidae) from a Red wattlebird (Anthochaera carunculata) (Passeriformes: Meliphagidae) in Western Australia. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.02.011 YEXPR 6817 No. of Pages 7, Model 5G 6 March 2014

2 R. Yang et al. / Experimental Parasitology xxx (2014) xxx–xxx

68 (Sarcocystidade), and all such oocysts from birds with Stieda bodies boiling water to ensure efficient lysis of oocysts before being 127 69 in their sporocysts to the genus Isospora. Atoxoplasma was deter- processed using the manufacturer’s protocol. A negative control 128 70 mined to be a junior objective synonym for Isospora (Barta et al., (no faecal sample) was used in each extraction group. 129 71 2005). 72 The avian order Passeriformes includes 5000 species worldwide 2.4. PCR amplification of ITS, 18S, 28S and COI loci 130 73 and accounts for 50% of all avian species and Isospora are the most 74 common coccidian parasites infecting passerine birds (Duszynski Amplification of a 404 bp region of the ribosomal internal tran- 131 75 et al., 1999). While numerous species of Isospora infecting birds scribed spacer (ITS) locus from samples was conducted as de- 132 76 have been described (cf. Berto et al., 2011), relatively few species scribed by Johnson et al. (2008). Samples were then amplified at 133 77 Q3 have been characterised genetically (Olson et al., 1998; Carreno the 18S locus for Isospora using a nested PCR with the primers 134 78 et al., 1999; Schrenzel et al., 2005; Dolnik et al., 2009; Morin-Adeline and PCR conditions as described by Pieniazek et al. (1996), which 135 79 et al., 2011) produced a 497 bp product. Samples were also amplified at the 136 80 The Red wattlebird (Anthochaera carunculata) (also known as Isospora 28S ribosomal RNA (28S rRNA) locus using a nested PCR 137 81 Barkingbird or Gillbird) is a passerine species that belongs to the with the external primers: 28SExF: 50-TAC CCG CTG AAC TTA 138 82 family Meliphagidae (honeyeaters), a group of birds found mainly AGC and 28SExR: 50-CMA CCA AGA TCT GCA CTA G as previously 139 83 in Australia and New Guinea. It is amongst the largest of the Aus- described (Schrenzel et al., 2005), which produced a PCR product 140 84 tralian honeyeaters and has a wide range of habitats, which include size of 1495 bp. Internal primers were designed for the present 141 85 woodlands, eucalypt forests, scrubs, heaths, orchards and parks. study using Primer 3 (http://frodo.wi.mit.edu/). The internal prim- 142 86 The Red wattlebird has a fleshy reddish wattle on the side of the ers 28SInF: 50-ACT ATG TTC CCT AGT AAC G and 28SInR 5’-AAC GCT 143 87 neck and its plumage is grey–brown on its body, whilst the middle TCG CCA CGA TCC produced a PCR product size of 1420 bp. The 144 88 of the belly is lemon-yellow in colour. The tail is long and is tipped 25 ll PCR reaction contained 2.5 llof10 Kapa PCR buffer, 2 ll 145

89 in white. It uses its thin curved beak to probe ﬂowers for nectar on of 25 mM MgCl2,1ll of 10 nM dNTP’s, 10 pM of each primer, 146 90 which it feeds, supplemented with insects and berries (Pizzey and 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 ll of DNA and 147

91 Knight, 2007). To date, Isospora lesouefi has been characterised 16.9 llofH2O. Both primary and secondary PCR’s were conducted 148 92 from the endangered Regent honeyeater (Xanthomyza phrygia), with the same cycling conditions; 1 cycle of 94 °C for 3 min, fol- 149 93 which is endemic to south-eastern Australia (Morin-Adeline lowed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 150 94 et al., 2011) and Isospora samoaensis has been described morpho- 90 s and a final extension of 72 °C for 5 min. Finally, samples were 151 95 logically from the Wattled honeyeater (Foulehaio carunculata) from screened at the COI locus for Isospora using primers and conditions 152 96 American Samoa (Adamczyk et al., 2004). In the present study, we described by Dolnik et al. (2009). 153 97 characterized a new species of Isospora from a Red wattlebird (A. 98 carunculata) in Western Australia, both morphologically and genet- 2.5. Sequence and cloning analysis 154 99 ically, and propose the species name Isospora anthochaerae. Secondary PCR products were gel purified using an in house fil- 155 100 2. Materials and methods ter tip method without any further purification for down stream 156 sequencing as previously described (Yang et al., 2013) 157 101 2.1. Sample collection Gel-purified PCR amplicons from the 28S rRNA and ITS loci were 158 cloned in the pGEM-T Easy Vector System II (Promega, USA) due to 159 102 A survey was conducted over a 7-month period (September the low PCR product yield. After transformation into JM109 compe- 160 103 2012–March 2013), to determine the incidence of coccidian parasites tent cells, plasmid DNA was extracted using the QIAprep Spin 161 104 inapopulationofRedwattlebirds(A. carunculata) that had been Miniprep Kit (Qiagen, Germany) from cultured clones grown over- 162 105 admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC) in night and 10 colonies were sequenced with the T7 (50 TAA TAC GAC 163 106 Western Australia. All birds were wild and came into care either as TCA CTA TAG GG) and SP6 (50 ATT TAG GTG ACA CTA TAG) primers 164 107 a result of cat attacks or as nestlings that had fallen out of their nests. in both directions, using v3.1 BigDyeÒ Terminator chemistry at 1/ 165 108 A total of 13 faecal samples were collected from 13 different Red 16 dilution (Life Technologies, Foster City, California). 166 109 wattlebirds at KWRC under the KWRC permit. Samples were stored The results of the sequencing reactions were analysed and edi- 167 110 at 4 °C until parasitological examination and DNA extraction. ted using Finch TVÒ v1.4.0. (http://seq.mc.vanderbilt.edu/dna/ 168 html/SoftDetail.html). Sequences were compared to existing Isos- 169 111 2.2. Morphological analysis pora sp. ITS, 18S and 28S rDNA and COI sequences on GenBank 170 using BLAST searches and aligned with reference sequences from 171 112 The presence of oocysts was determined by direct microscopic GenBank using Clustal W (http://www.clustalw.genome.jp). 172 113 examination of a faecal suspension in saline, as well as faecal flota- 114 tion analysis using a saturated sodium chloride and 50% sucrose 2.6. Phylogenetic analysis 173 115 (w/v) solution. If any sample was found to contain coccidian oo- 116 cysts, a portion of faeces was placed in 2% (w/v) potassium dichro- Phylogenetic trees were constructed for Isospora sp. at the ITS, 174 117 mate solution (K2Cr2O7), mixed well and poured into petri dishes 18S, 28S and COI loci with additional isolates from GenBank. Dis- 175 118 to a depth of less than 1 cm and kept at room temperature in the tance estimation was conducted using TREECON (Van de Peer 176 119 dark to facilitate sporulation. Sporulated oocysts were observed and De Wachter, 1994), based on evolutionary distances calculated 177 120 using the 100 oil immersion objective of an Olympus CH-2 binoc- with the Tamura–Nei model and grouped using Neighbour-Joining. 178 121 ular microscope, in combination with an ocular micrometre. Parsimony analyses were conducted using MEGA version 5.1 179 (MEGA5.1: Molecular Evolutionary Genetics Analysis software, Ari- 180 122 2.3. DNA isolation zona State University, Tempe, Arizona, USA). Bootstrap analyses 181 were conducted using 1000 replicates to assess the reliability of in- 182 123 Total DNA was extracted from 200 mg of each faecal sample ferred tree topologies. Maximum Likelihood (ML) analyses were 183 124 using a Power Soil DNA Kit (MolBio, Carlsbad, California) with conducted using the program PhyML (Dereeper et al., 2008) and 184 125 some modifications. Briefly, the faeces for DNA extraction were the reliability of the inferred trees was assessed by the approxi- 185 126 subjected to four cycles of freeze/thaw by liquid nitrogen and mate likelihood ratio test (aLRT) (Anisimova and Gascuel, 2006). 186

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187 2.7. Statistical analysis Sporocyst length, 14.5 (11–17); sporocyst width, 10.1 (9–11); spo- 203 rocyst L/W ratio, 1.4 (1.1–1.8). The stieda body is broad, hemi- 204 188 Measurements of 37 sporulated oocysts were analysed using dome-like with a rather rectangular-shaped substieda body. Para- 205 189 Statistical Package for the Social Sciences (SPSS v21) and results steidia body is absent (Fig. 1). 206 190 are presented in micrometres as the mean, with the observed Type hosts: Red wattlebird (A. carunculata). 207 191 range in parentheses. Type locality: Leeming, Perth, Western Australia. 208 Prevalence: Isospora sp. was detected in 2/13 samples screened, 209 192 3. Results an estimated prevalence of 15.4% (95% CI 0–35). 210 Other hosts: Unknown. 211 193 3.1. Description Prepatent period: Unknown. 212 Patent period: Unknown. 213 194 3.1.1. I. anthochaerae n. sp. (Alveolata Cavalier-Smith, 1991, Site of infection: Unknown. 214 195 Apicomplexa Levine, 1970, Eimeriidae Minchin, 1903) Sporulation time: Unknown but assumed to be less than 24 h as 215 196 Diagnosis: Sporulated oocysts (n = 37) are spherical to subsp- some of the oocysts were already sporulated in the fresh faecal 216 197 herical with colourless to pale brown bilayered oocyst wall, 0.8 samples. 217 198 (0.6–0.9) thick (outer layer 06 lm, inner 0.2 lm) and measure Material deposited: DNA sequences have been deposited in Gen- 218 199 23.4 (20–26) 20.7 (19–22) lm in size with a width to length ratio Bank under accession numbers KF766052, KF766053, KF766051 219 200 of 1.1 (1.0–1.4). Oocysts with 2 spherical to subspherical spo- and KF766054 for the 18S, 28S, ITS and COI loci, respectively. 220 201 rocysts. Micropyle, oocyst residuum and polar granule are absent. Etymology: This species is named I. anthochaerae n. sp. after its 221 202 Sporocysts with compact sporocyst residuum and 4 sporozoites. host A. carunculata (Red wattlebird). 222

Fig. 1. (A) Nomarski interference-contrast photomicrographs of Isospora anthochaerae n. sp. showing sporulated oocysts. Scale bar = 10 lm. (B) Composite line drawing of Isospora anthochaerae n. sp. sporulated oocyst. Scale bar = 10 lm.

Cystiosospora belli DQ060660 Cystiosospora belli DQ060658 Cystiosospora belli DQ060659 99 Cystiosospora belli DQ060663 Cystiosospora belli DQ060661 Cystiosospora belli HM630352 99 Cystiosospora belli HM630353 Cystiosospora belli EU124687 Cystiosospora rivolta EU124686

97 Cystiosospora ohioensis GU292308 99 Cystiosospora ohioensis GU292307 99 Cystiosospora ohioensis GU292306 Cystiosospora ohioensis EU124688 Cystiosospora suis EU124685 Isospora anthochaerae n. sp. KF766051 Toxoplasma gondii X75453

0.1

Fig. 2. Evolutionary relationships of Isospora anthochaerae n. sp. inferred by distance analysis of ITS rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from Maximum Likelihood (ML) analyses is indicated at the left of the supported node.

4 R. Yang et al. / Experimental Parasitology xxx (2014) xxx–xxx

223 3.2. Phylogenetic analysis of I. anthochaerae n. sp. at the ITS locus et al., 2005) and Isospora robini – GenBank accession number: 241 AF080612 (Carreno and Barta, 1999) and 3 mammalian Cystiosos- 242 224 Two partial ITS sequences (404 bp) were obtained from cloned pora sp. (C. suis – GenBank accession number: U97523, C. felis – 243 225 PCR products of I. anthochaerae n. sp., which exhibited 1 single GenBank accession number: L76471 and Cystiosospora belli – Gen- 244 226 nucleotide polymorphism (SNP), when compared to each other. Bank accession number: U94787), as well as other apicomplexan 245 227 Phylogenetic analyses of the partial nucleotide sequences from I. 18S rRNA sequences. Cryptosporidium muris was used as an 246 228 anthochaerae n. sp. at the ITS locus using Distance, Parsimony outgroup. 247 229 and ML analyses produced similar results (Fig. 2 – ML tree shown). Phylogenetic analysis using distance, parsimony and ML re- 248 230 Unfortunately bird-derived Isospora sequences were not available vealed that I. anthochaerae n. sp. exhibited 98% similarity with I. 249 231 at the ITS locus and phylogenetic analysis placed I. anthochaerae gryphoni and Isospora sp. MS-2003, which were identiﬁed from 250 232 n. sp. in a clade by itself but grouping closest (93% similarity) with American goldﬁnches (Carduelis tristis L.) and Southern cape spar- 251 233 Cystioisospora suis. The two sequences from I. anthochaerae n. sp. rows (Passer melanurus melanurus), respectively (Fig. 3). 252 234 were 99.9% similar to each other.

3.4. Phylogenetic analysis of I. anthochaerae n. sp. at the 28S locus 253 235 3.3. Phylogenetic analysis of I. anthochaerae n. sp. at the 18S locus Four identical 28S cloned PCR amplicons from I. anthochaerae 254 236 Two identical 18S Isospora sequences were obtained from the were obtained. In Genbank, 32 28S Isospora sequences were avail- 255 237 Red wattlebirds faecal samples and were aligned with 3 other Isos- able, of which 31 were sequences and genotypes from a unique 256 238 pora sp. sequences from passerine birds; Isospora gryphoni – Gen- species of Isospora (MS-2003), from passerine birds (Schrenzel 257 239 Bank accession number: AF080613 (Olson et al., 1998), Isospora et al., 2005), as well as a 28S sequence of C. felis from a cat. Phylo- 258 240 sp. MS-2003 – GenBank accession number: AY331571 (Schrenzel genetic analysis at this locus showed that I. anthochaerae was most 259

4 I. robini AF080612 14 E. bovis U77084 I. sp. MS-2003 AY331571

9 I. anthochaerae n. sp. I. gryphoni AF080613 E. mitis U67118 Cyclospora sp. U40261 22 98 21 Lankesterella minima AF080611 32 E. tenella U67121 94 E. necatrix U67119 Caryospora bigenetica AF060975

23 E. nieschulzi U40263 78 E. falciformis AF080614

64 I. belli U94787 88 I. suis U97523 74 I. felis L76471 T. gondii M97703 99 N. caninum U03069 99 S. neurona U07812 Sarcocystis sp. U97524 92 S. fusiformis U03071 65 98 S. gigantea L24384 76 L24382 87 S. tenella L24383 Perkinsus marinus X75762

74 Crypthecodinium cohnii M64245 61 Cryptosporidium parvum X64341 100 Cryptosporidium muris X64342

0.05

Fig. 3. Evolutionary relationships of Isospora anthochaerae n. sp. inferred by distance analysis of 18S rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from maximum likelihood analyses is indicated at the left of the supported node.

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260 closely related to Isospora sp. MS-2003 isolated from a Grosbeak 4. Discussion 279 261 starling (Scissirostrum dubium) (95.3% similarity) (Fig. 4). In the present study, the prevalence of I. anthochaerae n. sp. in 280 281 262 3.5. Phylogenetic analysis of I. anthochaerae n. sp. at the COI locus Red wattlebirds was 15.4% (2/13). The prevalence rate of coccidian oocysts in our samples is lower than the 91% reported by 282 283 263 Direct sequencing of the COI gene fragment from 2 isolates pro- Morin-Adeline et al., 2011 for I. lesouefi in the Regent honeyeater 284 264 duced clean and identical chromatograms, indicating that only one (X. phrygia). In that study, the authors compared the rate of 285 265 sequence was present. These were then aligned with 8 other Isospora I. lesouefi oocyst shedding in faeces in the morning (AM) to the 286 266 sp. sequences from passerine birds (Isospora hypoleucae – GenBank afternoon (PM). Significant diurnal periodicity was revealed in 287 267 accession number: FJ269363, I. lesouefi – GenBank accession number: oocyst shedding, as the AM prevalence was 21% (18/84) 1 288 268 HQ221885, Isospora sp. iSAT1 – GenBank accession number: (mean = 499 oocysts/g ) compared to a PM prevalence of 91% 1 289 269 FJ269357, Isospora sp. iSAT2 – GenBank accession number: (82/90) (mean = 129,723 oocysts/g ). In the present study, all 13 290 270 FJ269358, Isospora sp. iSAT3 – GenBank accession number: samples were collected in the morning and the prevalence of 291 271 FJ269359, Isospora sp. iSAT4 – GenBank accession number: 15.4% identified for I. anthochaerae is similar to the morning 292 272 FJ269360, Isospora sp. iSAT5 – GenBank accession number: FJ269361, prevalence for I. lesouefi (21%). The Regent honeyeaters tested in 293 273 Isospora sp. iSAT6 – GenBank accession number: FJ269362), as well the study by Morin-Adeline et al. (2011) were captive birds 294 274 as 7 Eimeria COI gene sequences. Toxoplasma gondii was used as whereas the Red wattlebirds in the present study were wild- 295 275 the outgroup. I. anthochaerae n. sp. exhibited 98.5% similarity with caught and had only been in care for a few days. 296 276 I. lesouefi and 98% similarity with Isospora sp. iSAT5, which were Sporulated oocysts of I. anthochaerae n. sp. were spherical to 297 277 identified from a Regent honeyeater (X. phrygia) and a blackcap subspherical and measured 23.4 (20.0–26.0) 20.7 (19.0–22.0) 278 warbler (Sylvia atricapilla), respectively (Fig. 5). lm in size with a width to length ratio of 1.12. Oocysts of I. lesouefi 298

Isospora sp. MS-2003 AY283842 Northern house sparrow (Passer domesticus) Isospora sp. MS-2003 AY283847 Southern cape sparrow (Passer melanurus) Isospora sp. MS-2003 AY283841Northern house sparrow (Passer domesticus) Isospora sp. MS-2003 AY283848 Southern cape sparrow (Passer melanurus) Isospora sp. MS-2003 AY283869 Eastern golden-breasted starling (Cosmopsarus regius magnificus) Isospora sp. MS-2003 AY283843 Northern house sparrow (Passer domesticus) Isospora sp. MS-2003 AY283840 Northern house sparrow (Passer domesticus) Isospora sp. MS-2003 AY283870 Eastern golden-breasted starling (Cosmopsarus regius magnificus) Isospora sp. MS-2003 AY283849 Southern cape sparrow (Passer melanurus) 75 Isospora sp. MS-2003 AY283856 Wattled starling (Creatophora cinerea) Isospora sp. MS-2003 AY283867 Emerald starling (Lamprotornis iris) Isospora sp. MS-2003 AY283860 Cowbird (Molothrus ater) 50 Isospora sp. MS-2003 AY283859 Cowbird (Molothrus ater) Isospora sp. MS-2003 AY283864 Surinam crested oropendola (Psarocolius decumanus) Isospora sp. MS-2003 AY283846 Northern white-headed buffalo weaver (Dinemellia dinemelli) Isospora sp. MS-2003 AY283858 Cowbird (Molothrus ater) Isospora sp. MS-2003 AY283855 Canary (Serinus canaria) Isospora sp. MS-2003 AY283853 Guianan turquoise tanager (Tangara mexicana) Isospora sp. MS-2003 AY283854 Canary (Serinus canaria) Isospora sp. MS-2003 AY283868 California towhee (Pipilo crissalis) 55 Isospora sp. MS-2003 AY283862 Costa rican orange-billed sparrow (Arremon aurantiirostris rufidorsalis) Isospora sp. MS-2003 AY283844 Himalayan grey-headed bullfinch (Pyrrhula erythaca) 99 Isospora sp. MS-2003 AY283845 Himalayan grey-headed bullfinch (Pyrrhula erythaca) 52 Isospora sp. MS-2003 AY283851 Reichenow's weaver (Ploceus baglafecht reichenowi) Isospora sp. MS-2003 AY283839 Northern house sparrow (Passer domesticus) Isospora sp. MS-2003 AY283857 Eastern white-starred bush robin (Pogonocichla stellata orientalis) 97 98 Isospora sp. MS-2003 AY283861 Eastern blue-winged sivia (Minla cyanouroptera) Isospora sp. MS-2003 AY283852 Sichuan white-browed laughing thrush (Garrulax sannio oblectans) 95 Isospora sp. MS-2003 AY283850 Javan ruby-throated bulbul (Pycnonotus melanicterus dispar) 80 99 Isospora sp. MS-2003 AY283863 Chinese collared finchbill (Spizixos semitorques semitorques) Isospora sp. MS-2003 AY283866 Grosbeak starling (Scissirostrum dubium) 63 Isospora anthochaerae n. sp. KF766053 Red wattlebird (Anthochaera carunculata) 100 72 Eimeria papillata GU593706 Chicken (Gallus gallus) Eimeria tenella AF026388 Chicken (Gallus gallus) Cyclospora cayetanensis EU252544 Goussia balatonica GU593717 100 Goussia desseri GU593705 100 Sarcocystis rieyi GU188426 82 Frenkelia glareoli AF044251 Cystiosospora felis U85705 100 Besnoitia besnoiti DQ227420 Neospora caninum 100 AF001946 100 Hammondia hmmaondi AF101077 87 Toxoplasma gondii L25635

0.02

Fig. 4. Evolutionary relationships of Isospora anthochaerae n. sp. inferred by distance analysis of 28S rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from maximum likelihood analyses is indicated at the left of the supported node.

6 R. Yang et al. / Experimental Parasitology xxx (2014) xxx–xxx

51 Isospora sp iSAT1 FJ269357 Blackcap (Sylvia atricapila) 78 Isospora sp iSAT3 FJ269359 Blackcap (Sylvia atricapila) Isospora sp iSAT4 FJ269360 Blackcap (Sylvia atricapila) Isospora sp iSAT2 FJ269358 Blackcap (Sylvia atricapila) 94 Isospora sp iSAT6 FJ269362 Blackcap (Sylvia atricapila) Isospora hypoleucae FJ269363 Eimeria ex Apodemus sylvaticus JQ993707

99 Eimeria apoinodes JX464221 Pangolins (Mamm: Pholidota) 72 Eimeria ex Apodemus flavicollis 4 JQ993704 Isospora sp iSAT5 FJ269361 Blackcap (Sylvia atricapila)

95 Eimeria cahirinensis JQ993686 Isospora lesouefi HQ221885 Regent honeyeater (Xanthomyza phrygia) Isospora anthochaerae n. sp. KF766054 Red wattlebird (Anthochaera carunculata) Eimeria tenella 161-27 JQ993711 Eimeria bruntti HQ702480 Eimeria ex Apodemus flavicollis B13 FJ236458 Toxoplasma gondii HM771690

0.10 0.05 0.00

Fig. 5. Evolutionary relationships of Isospora anthochaerae n. sp. inferred by distance analysis of mitochrondial cytochrome oxidase gene (COI). Percentage support (>50%) Q6 from 1000 pseudoreplicates from maximum likelihood analyses is indicated at the left of the supported node.

299 Q4 are also spherical and measured 25.8 (22.5–28.7) 23.8 (20–26.2) different data sets. Initial characterisation at the ITS locus was only 321 300 lm with a width to length ratio of 1.08 (Morin-Adeline et al., 2011) able to group I. anthochaerae n. sp. with C. suis (93% similarity) as no 322 301 (Table 1). Oocysts of I. samoaensis measured 28.9 26.1 (25–32 reference sequence from avian Isospora species were available at 323 302 23–30) lm with a width to length ratio of 1.1 (Adamczyk et al., the ITS locus. The 18S rRNA is the most common locus for phyloge- 324 303 2004). All three species have two ovoid shaped sporocysts, but spo- netic analysis of coccidia and is widely used for Eimeria and Isospora 325 304 rocysts of I. anthochaerae n. sp. measured 14.5 (11.0–17.0) 10.1 phylogenetic analysis. At this locus, distance, ML and parsimony 326 305 (9.0–11.0) lm, sporocysts of I. lesouefi measured 18.7 (17–19) analysis grouped I. anthochaerae n. sp. most closely (98% similarity) 327 306 9.5 (9–10) lm and sporocysts of I. samoaensis measured with I. gryphoni from American goldfinches (C. tristis L.), whose 328 307 17.1 10.9 (16–18 10–11) lm. A polar granule is present in I. oocysts are considerably larger (29.2–30.7 lm) than I. anthochaerae 329 308 lesouefi and I. samoaensis but is absent in I. anthochaerae n. sp. In n. sp. (Olson et al., 1998) and Isospora sp. MS-2003 from a Southern 330 309 addition, the morphological dimensions of I. anthochaerae n. did cape sparrow (P. melanurus melanurus)(Schrenzel et al., 2005). At 331 310 not match any other existing Isospora species from Passeriformes the 28S rRNA locus, I. anthochaerae n. sp. exhibited 95.3% similarity 332 311 listed online (http://biology.unm.edu/biology/coccidia/passer1. with Isospora sp. MS-2003 (GenBank accession number – 333 312 html. Accessed 21 Oct 2013). AY283866) from a Grosbeak starling (S. dubium). Interestingly, 334 313 Delimiting avian species of Isospora is problematic due to (i) I. anthochaerae n. sp. also grouped with Eimeria papillata (GenBank 335 314 ambiguities in the morphology and (ii) unknown host specificity accession number – GU593706), which was isolated from a chicken 336 315 (Grulet et al., 1982; Levine, 1982). Molecular data are therefore (Gallus gallus) but exhibited 94.9% similarity with this isolate. At 337 316 essential to accurately delimit species. In the present study, a the COI locus, I. anthochaerae n. sp. exhibited 98.5% similarity to 338 317 comprehensive molecular characterization of I. anthochaerae n. sp. I. lesouefi (Morin-Adeline et al., 2011) and 98% similarity with an 339 318 was conducted at 4 different loci; the ITS, 18S, 28S and COI loci. Isospora sp. (iSAT5) from a blackcap (S. atricapilla)(Dolnik et al., 340 319 Due to the very limited availability of sequences for avian Isospora 2009). The genetic differences at the COI locus are phylogenetically 341 320 species at the 4 loci, the phylogenetic trees were generated with significant as the COI gene is highly conserved (Barta, 2001) and has 342

Table 1 Comparative morphology of Isospora anthochaerae n. sp. and Isospora sp. recorded from the family Meliphagidae (honeyeaters).

Species Hosts References Oocysts Sporocysts Shape Measurements Shape Wall (lm) Polar Shape Measurements Stieda Substida Residuum (lm) index granule body body Isospora Wattled Adamczyk Ovoid 28.9 26.1 1.1 Bi-layered Present Ovoid 17.1 10.9 Broad Dome-like Compact samoaensis honeyeater et al. (25–32 23–30) c. 0.8 (16–18 (Foulehaio (2004) 10–11) carunculata) Isospora lesoueﬁ Regent Morin- Spherical 25.8 23.8 1.08 Bi-layered Present Ovoid 18.67 9.45 Flat Spherical Compact Honeyeater Adeline (22.5–28.7 c.1.0 (17–19 9–10) (Xanthomyza et al. 20–26.2) phrygia) (2011) Isospora Red wattlebird Current Subspherical 23.4 20.7 1.1 Bi-layered Absent Ovoid 14.5 10.1 Hemi- Rectangular- Compact anthochaerae (Anthochaera study (20.0–26.0 c. 0.8 (11–17 dome shaped carunculata) 19.0–22.0) 9–11)

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343 been shown to have a higher resolving power than the 18S gene in Eimeria Schneider, 1875 and Isospora Schneider, 1881 (Apicomplexa: 382 Eimeriidae). Syst. Parasitol. 80, 159–204. 383 344 delineating recent speciation events (Ogedengbe et al., 2011). COI Carreno, R.A., Barta, J.R., 1999. An eimeriid origin of isosporoid coccidia with Stieda 384 345 has become the target gene for the ‘‘Barcode of Life’’ project that bodies as shown by phylogenetic analysis of small subunit ribosomal RNA gene 385 346 uses the marker for rapid identification of a range of species includ- sequences. J. Parasitol. 85, 77–83. 386 387 347 ing parasites (Ratnasingham and Hebert, 2007). One drawback of Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr: 388 348 using this gene is the paucity of avian Isospora sequences at this robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, 389 349 locus but for example the genetic similarity between the accepted W465–W469. 390 391 350 Eimeria species, E. tenella and E. necatrix at this locus is 98.4% which Dolnik, O.V., Palinauskas, V., Bensch, S., 2009. Individual oocysts of Isospora (Apicomplexa: Coccidia) parasites from avian faeces: from photo to sequence. 392 351 is very similar to the genetic similarity between I. anthochaerae J. Parasitol. 95, 169–174. 393 352 n. sp. and I. lesouefi (98.5%). Based on the morphological and Duszynski, D.W., Upton, S.J., Couch, L., 1999. The coccidia of Passeriformes (Isospora 394 353 molecular differences, I. anthochaerae n. sp. is a separate species. spp.). (accessed 21 Oct 395 2013). 396 354 In the present study, morphological and molecular data were Duszynski, D.W., Wilber, P.G., 1997. A guideline for the preparation of species 397 355 used to describe I. anthochaerae n. sp. found in the faeces of Red descriptions in the Eimeriidae. J. Parasitol. 83, 333–336. 398 356 wattlebirds in Western Australia. Future studies need to concen- Frenkel, J.K., 1977. Besnoitia wallacei of cats and rodents: with a reclassification of 399 other cyst-forming isosporoid coccidia. J. Parasitol. 63, 611–628. 400 357 trate on obtaining afternoon faecal samples from a variety of Grulet, O., Landau, I., Baccam, D., 1982. Les Isospora du moineau domestique: 401 358 wattlebird species and conducting morphological and genetic multiplicite des especes. Ann. Parasitol. Hum. Comp. 57, 209–235. 402 359 characterisation to understand the extent of diversity within Levine, N.D., 1982. The genus Atoxoplasma (Protozoa, Apicomplexa). J. Parasitol. 68, 403 719–723. 404 360 Isospora sp. in wattlebirds. Johnson, J., Samarasinghe, B., Buddle, R., Armson, A., Ryan, U., 2008. Molecular 405 identification and prevalence of Isospora sp. in pigs in Western Australia using a 406 361 5. Uncited reference PCR–RFLP assay. Exp. Parasitol. 120, 191–193. 407 Morin-Adeline, V., Vogelnest, L., Dhand, N.K., Shiels, M., Angus, W., Šlapeta, J., 2011. 408 Afternoon shedding of a new species of Isospora (Apicomplexa) in the 409 362 Q5 Duszynski and Wilber (1997). endangered regent honeyeater (Xanthomyza phrygia). Parasitology 138, 713– 410 724. 411 Ogedengbe, J.D., Hanner, R.H., Barta, J.R., 2011. DNA barcoding identifies Eimeria 412 363 Acknowledgments species and contributes to the phylogenetics of coccidian parasites 413 (Eimeriorina, Apicomplexa, Alveolata). Int. J. Parasitol. 41, 843–850. 414 364 The authors wish to thank June Butcher and the volunteers at Olson, V.A., Gissing, G.J., Barta, J.R., Middleton, A.L., 1998. A new Isospora sp. from 415 Carduelis tristis (Aves: Fringillidae) from Ontario, Canada. J. Parasitol. 84, 153– 416 365 the Kanyana Wildlife Rehabilitation Centre for their commitment 156. 417 366 and dedication in caring for all the animals admitted to the centre. Pieniazek, N.J., Slemenda, S.B., da Silva, A.J., Alfano, E.M., Arrowood, A.J., 1996. PCR 418 confirmation of infections with Cyclospora cayetanensis. Emerg. Infect. Dis. 2, 419 420 367 References 357–359. Pizzey, G., Knight, F., 2007. The Field Guide to the Birds of Australia. Harper Collins 421 Publishers Pty Limited. 422 368 Adamczyk, K.J., McQuistion, T.E., LaPointe, D.A., 2004. A new coccidian parasite, Ratnasingham, S., Hebert, P.D., 2007. BOLD: the barcode of life data system. (http:// 423 369 Isospora samoaensis, from the Wattled Honeyeater (Foulehaio carunculata) from www.barcodinglife.org). Mol. Ecol. Notes 7, 355–364. 424 370 American Samoa. Acta Protozool. 43, 1–3. Schrenzel, M.D., Maalouf, G.A., Gaffney, P.M., Tokarz, D., Keener, L.L., McClure, D., 425 371 Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: a Griffey, S., McAloose, D., Rideout, B.A., 2005. Molecular characterization of 426 372 fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552. isosporoid coccidia (Isospora and Atoxoplasma spp.) in passerine birds. J. 427 373 Barta, J.R., 2001. Molecular approaches for inferring evolutionary relationships Parasitol. 91, 635–647. 428 374 among protistan parasites. Vet. Parasitol. 101, 175–186. Van de Peer, Y., De Wachter, R., 1994. TREECON for Windows: a software package 429 375 Barta, J.R., Schrenzel, M.D., Carreno, R., Rideout, B.A., 2005. The genus Atoxoplasma for the construction and drawing of evolutionary trees for the Microsoft 430 376 (Garnham 1950) as a junior objective synonym of the genus Isospora (Schneider Windows environment. Comp. Appl. Biosci. 10, 569–570. 431 377 1881) species infecting birds and resurrection of Cystoisospora (Frenkel 1977) as Yang, R., Murphy, C., Song, Y., Ng-Hublin, J., Estcourt, A., Hijjawi, N., Chalmers, R., 432 378 the correct genus for Isospora species infecting mammals. J. Parasitol. 91, 726– Hadfield, S., Bath, A., Gordon, C., Ryan, U.M., 2013. Specific and quantitative 433 379 727. detection and identification of Cryptosporidium hominis and C. parvum in clinical 434 380 Berto, B.P., Flausino, W., McIntosh, D., Teixeira-Filho, W.L., Lopes, C.W.G., 2011. and environmental samples. Exp. Parasitol. 135, 142–147. 435 381 Coccidia of New World passerine birds (Aves:Passeriformes): a review of 436