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Yang, R., Brice, B., Al Habsi, K., Elliot, A. and Ryan, U. (2015) Isospora streperae n. sp. (Apicomplexa: Eimeriidae) from a grey (Strepera versicolour plumbea) (Passeriformes: ) in Western . Experimental Parasitology, 151-152. pp. 49-55.

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Title: Isospora streperae n. sp. (Apicomplexa: Eimeriidae) from a grey currawong (Strepera versicolour plumbea) (Passeriformes: Artamidae) in

Author: Rongchang Yang, Belinda Brice, Khalid Al Habsi, Aileen Elliot, Una Ryan

PII: S0014-4894(14)00208-2 DOI: http://dx.doi.org/doi: 10.1016/j.exppara.2014.08.018 Reference: YEXPR 6937

To appear in: Experimental Parasitology

Received date: 3-5-2014 Revised date: 22-7-2014 Accepted date: 31-8-2014

Please cite this article as: Rongchang Yang, Belinda Brice, Khalid Al Habsi, Aileen Elliot, Una Ryan, Isospora streperae n. sp. (Apicomplexa: Eimeriidae) from a grey currawong (Strepera versicolour plumbea) (Passeriformes: Artamidae) in Western Australia, Experimental Parasitology (2015), http://dx.doi.org/doi: 10.1016/j.exppara.2014.08.018.

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1 Isospora streperae n. sp. (Apicomplexa: Eimeriidae) from a grey currawong (Strepera

2 versicolour plumbea) (Passeriformes: Artamidae) in Western Australia

3

4 Rongchang Yanga*, Belinda Briceb, Khalid Al Habsia, Aileen Elliota, Una Ryana

5 aSchool of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, 6150.

6 bKanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076.

7

8 ______

9 *Corresponding author: Rongchang Yang

10 Mailing address: School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western

11 Australia, Australia, 6150. Phone: 61 89360 2495. Fax: 61 89310 4144.

12 E-mail: [email protected]

13

14

15 Highlights

16  Description of a new Isospora species(I. streperae n. sp.) in Grey currawong. 17  Necropsy study: hemorrhaging mostly along the ileum and jejunum. 18  Morphology study: similar but distinct to other Isospora species from . 19  Genetic study: 99.5% similar to Isospora sp. (MS-2003) (AY331571) at 18S locus.

20

21 Graphical Abstract

22

Page 1 of 17 0.1

Isospora streperae n. sp. KJ634021 Isospora gryphoni AF080613

74 Isospora dovati AB757860 84 Isospora MS-2003 AY331571

83 Isospora JM-2013 JX984669 Isospora robini AF080612 Eimeria mitis U67118

Cyclospora sp. U40261

99 Eimeria necatrix U67119 Eimeria tenella U67121 100 100 Eimeria falciformis AF080614 Eimeria nieschulzi U40263

Caryospora bigenetica AF060975

100 100 Sarcocystis fusiformis U03071

98 Sarcocystis gigantea L24384 100 Sarcocystis tenella L24383 Sarcocystis tenella L24382 75 100 Sarcocystis neurona U07812 Sarcocystis sp. U97524 75 100 Cystiosospora suis U97523 100 Cystiosospora belli U94787 100 Toxoplasma gondii M97703 Neospora caninum U03069

Cryptosporidium parvum L16996 23

24

25

26

27

28

29

30

31

32

33

34 Abstract

35 A new species, Isospora streperae n. sp., (Apicomplexa:Eimeriidae) is described from a

36 single grey currawong (Strepera versicolour) (subspecies S. v. plumbea) in Western Australia.

37 Sporulated oocysts (n = 32) are spherical to subspherical, with smooth colourless bilayered oocyst

38 wall, 1.0 μm thick (outer layer 0·8 μm, inner 0.2 μm thick). Oocyst with a polar granule, an oocyst

2 Page 2 of 17 39 residuum and two spheroidal to subspheroidal sporocysts. Oocyst length, 23.8 (20.4-25.0) µm;

40 oocyst width, 22.5 (20.0-24.6) µm; a shape index of 1.06, with Stieda, substieda bodies. Micropyle

41 is absent. Sporocysts with compressed sporocyst residuum and four sporozoites. Sporocyst length,

42 14.4 (12.5 – 15.2) µm; sporocyst width, 11.2 (10.6 -14.0) µm, sporocyst L/W ratio, 1.29. Necropsy

43 of the bird identified hemorrhaging mostly along the ileum and jejunum, which is where Isospora

44 oocysts were also mostly detected. Molecular analysis was conducted at three loci; the 18S, 28S

45 ribosomal RNA and the mitochondrial cytochrome oxidase (COI) gene. At the 18S locus, I.

46 streperae n. sp. exhibited 99.5% and 99.4% similarity respectively to an Isospora sp. (MS-2003)

47 from a Southern cape sparrow (Passer melanurus melanurus) and Isospora dovati from a domestic

48 pigeon (Columba livia domestica). At the 28S locus, I. streperae n. sp. exhibited 96.9% similarity

49 to an Isospora sp. (MS-2003) from a grosbeak starling (Scissirostrum dubium) and 95.8% similarity

50 with the Isospora sp. (MS-2003) from a Southern cape sparrow. At the COI locus, I. streperae n.

51 sp. exhibited 95.0% similarity to Isospora sp. from a yellow-necked mouse (Apodemus flavicollis)

52 from the Czech Republic. Based on morphological and molecular data, this isolate is a new species

53 of Isospora, which is named Isospora streperae n. sp. after its host, the grey currawong (Strepera

54 versicolour plumbea).

55

56 Key words: Isospora; Grey currawong ; morphology; phylogeny; 18S rRNA; 28S rRNA; COI.

3 Page 3 of 17 57 1. Introduction

58

59 The order Passeriformes is the largest of all avian orders and consists of 63 families

60 comprising 5,206 different species. The order is extremely heterogeneous, containing granivorous,

61 insectivorous, frugivorous, omnivorous and carnivorous species ranging in size from a few grams to

62 over 1 kilogram (Duszynski et al., 1999; Sandmeier and Couttel, 2006).

63 The grey currawong (Strepera versicolor) is a large bird (family Artamidae) that is

64 native to southern Australia and . It is one of three currawong species in the genus

65 Strepera that can be found in Australia. It is possible to confuse the grey currawong with the pied

66 currawong. The is found in east Australia as well as western Victoria. However,

67 the pied currawong in east Australia has a conspicuous white base to the tail feathers. This is not

68 present in the grey currawong. The pied currawong found in western Victoria often has reduced

69 white markings to the base of the tail as well as reduced white in the wings (Pizzey and Knight,

70 2007). As the pied currawong is not found in Western Australia we are confident that the bird

71 admitted to the KWRC was indeed a grey currawong. The third species of currawong, the black

72 currawong, is only found on the Bass Straight islands. Six subspecies of grey currawong are spread

73 around Australia. They vary extensively in the colour of their , from grey to sooty black

74 and the amount of white on their wings, and most were at one time considered separate species

75 (Neville, 1974). Strepera versicolour, subspecies plumbea (S. v. plumbea) is found from western

76 and the south-western corner of the westwards into Western

77 Australia (Higgins et al. 2006).

78 Isospora are the most common coccidian parasites infecting passerine birds (Duszynski et

79 al., 1999). The genus was first described by Schneider in 1881 and its has been a source

80 of controversy since due to morphological differences and paraphylogeny of the genus (Barta et al.,

81 2005; Yang et al., 2014). To date, numerous species of Isospora has been characterised from

4 Page 4 of 17 82 passerine birds worldwide (Schrenzel et al., 2005; Schoener et al., 2013). In Australia, two species

83 of Isospora from honeyeaters (Meliphagidae) have been described; Isospora lesouefi from the

84 endangered regent honeyeater (Xanthomyza phrygia), which is endemic to south-eastern Australia

85 (Morin-Adeline et al., 2011) and Isospora anthochaerae n. sp. from a (Anthochaera

86 carunculata) in Western Australia (Yang et al., 2014). To date however, species of Isospora have

87 not been characterized from the avian family Artamidae. In the present study, we characterized a

88 new species of Isospora from a grey currawong (Strepera versicolour plumbea) in Western

89 Australia, both morphologically and genetically, and propose the species name Isospora streperae

90 n. sp.

91

92 2. Materials and methods

93 2.1 Sample collection

94 An adult grey currawong with a suspected spinal injury was admitted to the Kanyana

95 Wildlife Rehabilitation Centre (KWRC) on the 29th of January, 2014 and was euthanized the

96 following day. Faecal samples were collected on the day of admittance to KWRC and were stored

97 at 4 °C until parasitological examination and DNA extraction. Necropsy and examination of the

98 intestines was conducted under the KWRC permit.

99

100 2.2 Morphological analysis

101 The presence of oocysts was determined by direct microscopic examination of a faecal

102 suspension in saline, as well as faecal flotation analysis using a saturated sodium chloride and 50%

103 sucrose (w/v) solution. A portion of faeces was placed in 2% (w/v) potassium dichromate solution

104 (K2Cr2 O7), mixed well and poured into petri dishes to a depth of less than 1cm and kept at room

105 temperature in the dark to facilitate sporulation. Sporulated oocysts were observed using the 100x

5 Page 5 of 17 106 oil immersion objective of an Olympus CH-2 binocular microscope, in combination with an ocular

107 micrometre.

108

109 2.3 DNA isolation

110 Total DNA was extracted from 200 mg of each faecal sample using a Power Soil DNA Kit

111 (MolBio, Carlsbad, California) with some modifications. Briefly, samples were subjected to four

112 cycles of freeze/thaw by liquid nitrogen and boiling water to ensure efficient lysis of oocysts before

113 being processed using the manufacturer’s protocol. A negative control (no faecal sample) was

114 included.

115

116 2.4 PCR amplification of 18S and 28S ribosomal sequences and the mitochondrial cytochrome

117 oxidase (COI) gene

118 Generic apicomplexan primers (CRYPTOF 5’-AAC CTG GTT GAT CCT GCC AGT and

119 CRYPTOR 5’-GCT TGA TCC TTC TGC AGG TTC ACC TAC) were used to amplify the almost

120 full length 18S rRNA gene as described by Eberhard et al., (1999). The expected PCR product was

121 ~1,584 bp. The PCR reaction contained 2.5 µl of 10 × Kapa PCR buffer, 3 µl of 25 mM MgCl2, 1.5

122 µl of 10nM dNTP’s, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 µl of

123 DNA (~50 ng) and 14.9 µl of H2O. PCR cycling conditions were 1 cycle of 94 ºC for 3 min,

124 followed by 45 cycles of 94 ºC for 30 sec, 55 ºC for 30 sec and 72 ºC for 2 min and a final

125 extension of 72 ºC for 5 min.

126 The PCR for the 28S rRNA locus was carried out using a nested PCR with the external

127 primers: 28SExF: 5’-TAC CCG CTG AAC TTA AGC and 28SExR: 5’- CMA CCA AGA TCT

128 GCA CTA G as previously described (Schrenzel et al., 2005), which produced a PCR product size

129 of ~1,495 bp. The internal primers (28InF: 5’ – ACT ATG TTC CCT AGT AAC G and 28SInR 5’-

6 Page 6 of 17 130 AAC GCT TCG CCA CGA TCC) were designed for the present study using Primer 3

131 (http://frodo.wi.mit.edu/) and produced an amplicon size of 1,420 bp. The PCR reaction contained

132 2.5 µl of 10 × Kapa PCR buffer, 2 µl of 25mM MgCl2, 1 µl of 10mM dNTP’s, 10 pM of each

133 primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 µl of DNA (~50 ng) and 16.9 µl of H2O.

134 Both primary and secondary PCR’s were conducted using the same cycling conditions; 1 cycle of

135 94 ºC for 3 min, followed by 35 cycles of 94 ºC for 30 sec, 60 ºC for 30 sec and 72 ºC for 90 sec

136 and a final extension of 72 ºC for 5 min.

137 The PCR for the COI locus was carried out using a nested PCR as previously described

138 (Yang et al., 2014).

139 2.5 Sequence analysis

140 The amplicons from primary18S PCR, the secondary 28S and COI PCRs were gel purified

141 using an in house filter tip method as previously described (Yang et al., 2013). All the PCR

142 products were sequenced using forward and reverse primers in duplicate using amplicons from

143 different PCR runs. Any mismatches were confirmed by re-amplifying with pfu (Promega, Sydney,

144 Australia) and repeating the sequencing. An ABI PrismTM Dye Terminator Cycle Sequencing kit

145 (Applied Biosystems, Foster City, California) was used for Sanger sequencing according to the

146 manufacturer’s instructions (with the exception that the annealing temperature was at 60ºC).

147 The results of the sequencing reactions were analysed and edited using Finch TV® v1.4.0.

148 (http://seq.mc.vanderbilt.edu/dna/html/SoftDetail.html). Sequences were compared to existing

149 Isospora and other coccidian parasite sequences available on GenBank using BLAST searches and

150 aligned with reference sequences with BioEditor (http://bioeditor.sdsc.edu/download.shtml).

151

7 Page 7 of 17 152 2.6 Phylogenetic analysis

153 Phylogenetic trees were constructed for Isospora spp. at the 18S, 28S and COI loci with

154 additional isolates from GenBank. Distance estimation was conducted using TREECON (Van de

155 Peer and De Wachter, 1994), based on evolutionary distances calculated with the Tamura-Nei

156 model and grouped using Neighbour-Joining. Parsimony analyses were conducted using MEGA

157 version 6 (MEGA6: Molecular Evolutionary Genetics Analysis software, Arizona State University,

158 Tempe, Arizona, USA). Bootstrap analyses were conducted using 1,000 replicates to assess the

159 reliability of inferred tree topologies. Maximum Likelihood (ML) analyses were conducted using

160 the program PhyML (Dereeper et al., 2008) and the reliability of the inferred trees was assessed by

161 the approximate likelihood ratio test (aLRT) (Anisimova and Gascuel, 2006).

162

163 2.5 Statistical Analysis

164 Measurements of 32 sporulated oocysts were analysed using SPSS (Version 21), and results are

165 presented in micrometers as the mean ± SD, with the observed range in parentheses.

166

167 2.6 Line drawing

168 Oocyst line drawings were conducted using Inkscape (http://www.inkscape.org/en/).

169 3. Results

170 3.1 Description

171 3.1.1 Isospora streperae n. sp. (Alveolata Cavalier-Smith, 1991, Apicomplexa Levine, 1970,

172 Eimeriidae Minchin, 1903)

173 Diagnosis: Sporulated oocysts (n = 32) are spherical to subspherical with colorless bilayered oocyst 174 wall, 1.0 (0.8-1.1) thick (outer layer 0·8 μm, inner 0.2 μm) and measure 23.8 (20.4-25.0) × 22.5 175 (20.0-24.6) µm in size with a width to length ratio of 1.06 (1.0-1.2). Oocysts with a polar granule,

8 Page 8 of 17 176 an oocyst residuum and two spheroidal to subspheroidal sporocysts, with stieda, substieda bodies, 177 polar granule and oocyst residuum. Micropyle is absent. Sporocysts with compact sporocyst 178 residuum and four sporozoites. Sporocyst length, 14.4 (12.5-15.2); sporocyst width, 11.2 (10.6- 179 14.0); sporocyst L/W ratio, 1.29 (1.22-1.35). Compact sporocyst residuum present. The stieda body 180 is dark in colour, hemi-dome-like with flat substieda body. Parasteidia body is absent (Fig. 1a and 181 1b and Table 1).

182 Type hosts: Grey Currawong (Strepera versicolour plumbea)

183 Type locality: Perth (31.9522°S, 115.8589°E), Western Australia.

184 Prevalence: Unknown

185 Other hosts: Unknown.

186 Prepatent period: Unknown.

187 Patent period: Unknown.

188 Site of infection: Ileum and middle of the small intestine.

189 Sporulation time: 48 to 72 hours.

190 Material deposited: An oocyst slide and photosyntypes were deposited into the Western Australian 191 Museum under the reference number: WAM Z68800. DNA sequences have been deposited in 192 GenBank under the accession numbers KJ634021, KJ634022 and KM206142 for the 18S, 28S and 193 COI loci respectively.

194 Etymology: This species is named I. streperae n. sp. after its host (Strepera versicolour plumbea) 195 (Grey Currawong). 196

197 3.2 Pathogenic infection in the small intestine

198 Necropsy was performed and the small intestines were examined. In general, the bird

199 exhibited signs of diarrhoea and body emaciation and severe intestinal lesions were noted.

200 Hemorrhaging was observed mostly along the ileum and jejunum. Histological analysis identified

201 oocysts mostly in the ileum and some at the jejunum region. No other life-cycle stages were

202 identified from this study.

9 Page 9 of 17 203 3.3 Phylogenetic analysis of I. streperae n. sp. at the 18S locus

204 A 908 bp 18S Isospora sequence was aligned with four other Isospora spp. sequences from

205 passerine birds; Isospora gryphoni – GenBank accession number: AF080613 (Olson et al., 1998),

206 Isospora sp. MS-2003 – GenBank accession number: AY331571 (Schrenzel et al., 2005), Isospora

207 robini– GenBank accession number: AF080612 (Carreno and Barta, 1999) and Isospora JM-2013 -

208 GenBank accession number: JX984669, Isospora dovati from a domestic pigeon (GenBank

209 accession number: AB757860) and two mammalian Cystiosospora spp. (Cystiosospora suis –

210 GenBank accession number: U97523; Cystiosospora belli – GenBank accession number: U94787),

211 as well as other apicomplexan 18S rRNA sequences. Cryptosporidium parvum was used as an

212 outgroup.

213 Phylogenetic analysis using distance, parsimony and ML revealed that I. streperae n. sp.

214 exhibited 99.5%, 99.4%, 99.3%, 99.2% and 98.9% similarity with an Isospora sp. (MS-2003) from

215 a Southern cape sparrow (Passer melanurus melanurus), Isospora dovati sp. n. from a domestic

216 pigeon (Columba livia domestica), I. gryphoni from an American goldfinch (Carduelis tristis), I.

217 robini from the American robin (Turdus migratorius) and an Isospora sp. (JM-2013) from an

218 Austral thrush (Turdus falcklandii) (Fig. 2). Isospora anthochaerae was not included in the 18S tree

219 as only 497 bp of sequence was available for this species compared to the 908 bp used for the 18S

220 analysis in the present study. However, phylogenetic analysis with this species revealed that the

221 genetic similarity between I. streperae n. sp. and I. anthochaerae was 98.3%.

222

223 3.4 Phylogenetic analysis of I. streperae n. sp. at the 28S locus

224 A 944 bp 28S PCR amplicon from I. streperae n. sp. were obtained. In Genbank, 32 Isospora

225 sequences at the 28S locus were available; thirty-one sequences from Isospora species from

226 passerine birds (Schrenzel et al., 2005) and one Cystioisospora felis 28S sequence from a cat.

227 Phylogenetic analysis at this locus showed that I. streperae n. sp. was most closely related to

10 Page 10 of 17 228 Isospora sp. MS-2003 isolated from a Grosbeak starling (Scissirostrum dubium) (96.9% similarity)

229 and an Isospora sp. (MS-2003) isolated from a Sichuan white-browed laughing thrush (Garrulax

230 sannio oblectans) (96.8%) (Fig. 3). Isospora streperae n. sp. exhibited 95.8% and 93.9% similarity

231 with the Isospora sp. (MS-2003) from a Southern cape sparrow and I. anthochaerae from a red

232 wattlebird respectively.

233

234 3.5 Phylogenetic analysis of I. streperae n. sp. at the COI locus

235 A 258 bp amplicon at the COI locus from I. streperae n. sp. was obtained. Ten sequences

236 from various Isospora sp. were available at this locus in GenBank and an additional twenty-one

237 Eimeria COI gene sequences were included in the phylogenetic analysis. Toxoplasma gondii was

238 used as the outgroup. Isospora streperae n. sp. exhibited 95.0% similarity with Isospora lesouefi

239 (GenBank accession number: HQ221885) from a Regent honeyeater in New South Wales, Australia

240 and Isospora sp. from a from a yellow-necked mouse (Apodemus flavicollis) (GenBank accession

241 number: JQ993711), 94.6% similiarity with Isospora anthochaerae from a Red wattlebird

242 (Anthochaera carunculata) in Western Australia (GenBank accession number: KF766054), and

243 94.5% with Isospora sp. iSAT5 (GenBank accession number: FJ269361) (Fig. 4). The genetic

244 similarity between Isospora streperae n. sp. and the Isospora sp. (MS-2003) from the Southern cape

245 sparrow was 95.9%.

246

247 4. Discussion

248 In the present study, Isospora oocysts were identified in the faeces of a grey currawong

249 admitted to a wildlife centre in Western Australia in January 2014. Following the euthanasia of the

250 bird the next day, histological analysis of the intestines identified a coccidian infection.

11 Page 11 of 17 251 Morphological and molecular analysis was performed and data indicated that the parasite was a

252 novel species of Isospora (I. streperae n. sp.).

253 Sporulated oocysts of I. streperae n. sp. are morphologically similar but distinct from other

254 Isospora species from birds and did not match any other existing documented Isospora species

255 from Passeriformes (http://biology.unm.edu/biology/coccidia/passer1.html. Accessed 30 Apr.

256 2014). For example oocysts of I. streperae n. sp. are spherical to subspherical and measured 23.8

257 (20.4-25.0) × 22.5 (20.0-24.6) µm in size with a width to length ratio of 1.06. Oocysts of Isospora

258 gryphoni n. sp are also spherical and measured 30.7 (28-34) x 29.2 (25 -33) µm with a width to

259 length ratio of 1.05, with 2-4 rice-grain-shaped polar bodies (Olson et al., 1998) (Table 1). Oocysts

260 of I. anthochaerae measured 23.4 (20.0-26.0) x 20.7 x (19.0-22.0) μm with a width to length ratio

261 of 1.12 (Yang et al., 2014). There is however a polar granule presents in I. streperae n. sp. oocysts

262 but not in I. anthochaerae. All three species have two ovoid shaped sporocysts, but sporocysts of I.

263 streperae n. sp. measured 14.5 (11.0-17.0) x 10.1 (9.0-11.0) µm, sporocysts of I. gryphoni

264 measured 22.2 (15-25) x 13.4 (12-14.5) µm and sporocysts of I. anthochaerae measured 14.5 (11-

265 17) x10.1 (9-11) μm.

266 Delimiting avian species of Isospora is problematic due to (i) ambiguities in the morphology

267 and (ii) unknown host specificity (Grul et et al., 1982; Levine, 1982). Molecular data are therefore

268 essential to accurately delimit species. In the present study, a molecular characterization of I.

269 streperae n. sp. was conducted at three loci; (18S, 28S rRNA and COI). Due to the limited

270 availability of sequences for avian Isospora species at these loci, the phylogenetic trees were

271 generated with different data sets. The 18S rRNA is the most common locus for phylogenetic

272 analysis of coccidian parasites and is widely used for Eimeria and Isospora phylogenetic analysis.

273 At this locus, distance, ML and parsimony analysis grouped I. streperae n. sp. most closely (99.5%

274 similarity) with an Isospora sp. (MS-2003) from a Southern cape sparrow (Schrenzel et al., 2005). It

275 exhibited 99.4%, 99.3%, 99.2% similarity with Isospora dovati sp. n. from a domestic pigeon

12 Page 12 of 17 276 (Columba livia domestica), I. gryphoni from an American goldfinch (Carduelis tristis) and I. robini

277 from the American robin (Turdus migratorius) respectively. At the 28S rRNA locus, I. streperae n.

278 sp. exhibited 96.9% similarity with the Isospora sp. (MS-2003) (from a Grosbeak starling), 95.8%

279 similarity with the Isospora sp. (MS-2003) from a Southern cape sparrow and 93.9% similarity with

280 I. anthochaerae. At the COI locus, I. streperae n. sp. exhibited 95.0% similarity with the Isospora

281 lesouefi from a Regent honeyeater in New South Wales, Australia and an Isospora sp. from a

282 yellow-necked mouse from the Czech Republic.

283 In the present study, morphological and molecular data were used to describe I. streperae n.

284 sp. found in the faeces of a grey currawong in Western Australia. Future studies need to concentrate

285 on pathogenic studies and genetic characterisation of more isolates and loci to better understand the

286 epidemiology of Isospora sp. infecting grey .

287

288 Acknowledgments 289 290 291 The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife

292 Rehabilitation Centre for their commitment and dedication in caring for all the admitted to

293 the centre. We are also grateful to the staff at the Wattle Grove Veterinary Hospital, Perth for their

294 expert treatment and care of the wildlife seen at their clinic. We thanks Mr Michael Slaven for

295 making the histology slides.

296

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348

349 Fig. 1a. Nomarski interference-contrast photomicrographs of Isospora streperae n. sp. showing two

350 spheroidal to subspheroidal sporocysts. Scale bar = 20 µm. Fig. 1b. Composite line drawing of

351 Isospora streperae n. sp. sporulated oocyst. Scale bar = 10 µm.

352 Fig. 2. Evolutionary relationships of Isospora streperae n. sp. inferred by distance analysis of 18S

353 rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from maximum

354 likelihood analyses is indicated at the left of the supported node.

355 Fig. 3. Evolutionary relationships of Isospora streperae n. sp. inferred by distance analysis of 28S

356 rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from maximum

357 likelihood analyses is indicated at the left of the supported node.

358 Fig. 4. Evolutionary relationships of Isospora streperae n. sp. inferred by distance analysis of COI

359 sequences. Percentage support (>50%) from 1000 pseudoreplicates from maximum likelihood

360 analyses is indicated at the left of the supported node.

361

362

363 Table 1 Comparative morphology of Isopora streperae n. sp and Isospora spp. recorded from passerine birds

Oocysts Sproscysts Sha S Stie Coccidi Refer Polar oocyst Substi Res Hosts Sha Measureme pe Wall h Measureme da a ences granu residu eda idu pe nts (um) inde (um) a nts bod le um body um x pe y American bi- I. Olson 29.2 x ov 22.2 x 13.4 pro goldfinches sphe layere pres smal indistin gryphon et al., 30.7(25- 1.05 absent oi (15-25 x12- min (Carduelis tristis rical d c. ent l ct i n. sp 1998 33x28-34) d 14.5) ent L.) 0.8 I. 23.4x20.7(2 bi- hem Red wattlebird Yang subs ov rectang co anthoch 0.0- layere abse 14.5x10.1(1 i- (Anthochaera et al., pher 1.12 absent oi ular- mp aerae n. 26.0x19.0- d c. nt 1-17x9-11) dom carunculata) 2014 ical d shaped act sp. 22.0) 0.8 e bi- 14.4x11.2(1 hem I. Grey currawong 23.8x22.5(2 ov rectang co This sphe layere abse presen 1.5- i- strepera (Strepera 2-24.5 x 1.06 oi ular- mp study rical d c. nt t 15.8x(10.4- dom e n. sp versicolor) 21.8x 24.5) d shaped act 1.0 12.5) e 364

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