A New Caryospora Coccidian Species (Apicomplexa: Eimeriidae) from the Laughing Kookaburra (Dacelo Novaeguineae)

MURDOCH RESEARCH REPOSITORY

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Yang, R., Brice, B. and Ryan, U. (2014) A new Caryospora coccidian species (Apicomplexa: Eimeriidae) from the laughing kookaburra (Dacelo novaeguineae). Experimental Parasitology, 145 . pp. 68-73.

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YEXPR 6913 No. of Pages 6, Model 5G 4 August 2014

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

Experimental Parasitology

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5 6

3 A new Caryospora coccidian species (Apicomplexa: Eimeriidae)

4 from the laughing kookaburra (Dacelo novaeguineae)

a,⇑ b a 7 Q1 Rongchang Yang , Belinda Brice , Una Ryan

8 Q2 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

1110 12 highlights graphical abstract 14 2215

Neospora L24380 16 0.1 caninum Description of a new Caryospora – like Neospora caninum U17345 89 Neospora caninum U17346 17 coccidian parasite in kookaburra. 52 Neospora caninum AJ271354 Neospora caninum U16159 65 Neospora caninum U03069 18 Hammondia Triffittae AF096498 Morphological characterisation: most M97703 99 Toxoplasma gondii 92 ToxoplasmaGondii X68523 19 similar to Caryospora. Toxoplasma.gondii L37415 68 Hammondia triffittae GQ984223 74 Hammondia triffittae GQ984222 20 Molecular characterisation at 18S and 96 |Besnoia besnoitii DQ227419 80 |Besnoia besnoitii FJ797432 |Besnoia AF109678 59 besnoitii 21 28S rRNA loci: most close to Besnoitia. Caryospora daceloe n. sp. KJ634019

64 96 Isospora bellii DQ060683 100 Cystoisospora spp AY279205 100 Cystoisospora spp EU200792 Sarcocysti ssp EU443095 AF009245 100 Frenkelia glareoli EU443095 59 Sarcocysti ssp AF513487 59 Sarcocysti ssp JQ733508 100 92 Sarcocysti ssp 71 Sarcocysti ssp JF975681 88 Cystoisosporasp EU502869 Sarcocysti ssp GQ245670 Sarcocysti ssp JQ73351 55 E falciformis AF080614 100 E macropodis JQ392575 100 Caryospora bigenetica AF060976 Caryospora bigenetica AF060975 C. parvum AF093491

24 article info abstract 4126 27 Article history: A new Caryospora coccidian species is described from the laughing kookaburra (Dacelo novaeguineae). 42 28 Received 31 March 2014 Sporulated oocysts (n = 30) are ovoid in shape with a smooth, colourless, bilayered oocyst wall and mea- 43 29 Received in revised form 14 July 2014 sure 31.4 29.3 (30.0–32.0 28.0–31.0) lm with a shape index of 1.1. Oocysts contain one spheroidal to 44 30 Accepted 22 July 2014 subspheroidal sporocyst, 21.2 20.6 (20.0–24.0 20.0–21.0) lm. A spheroidal shaped sporocyst resid- 45 31 Available online xxxx uum is present; micropyle, Stieda, substieda and parastieda bodies are absent. Vermiform sporozoites 46 (n = 8) are arranged either parallel or randomly in the sporocyst, measuring 17.0 4.8 (16.0– 47 32 Keywords: 18.0 4.0–6.0) lm, with a L/W ratio of 3.5. There is a large spheroidal, posterior refractile body in the 48 33 18S rRNA middle of the sporozoite. Morphologically, this new species is most similar to Caryospora. The prevalence 49 34 28S rRNA 35 Besnoitia of this parasite was 6.7% in birds sampled in the morning and 33.3% from those sampled after midday. 50 36 Caryospora Further molecular characterisation was conducted at two loci; the 18S and 28S ribosomal RNA (rRNA). 51 37 Kookaburra At the 18S locus, the new species of Caryospora was most closely related to Besnoitia besnoiti (99.2% sim- 52 38 Morphology ilarity) and Hammondia trifﬁttae (98.8% similarity). Although, no 28S partial sequences from Caryospora 53 39 Phylogenetics were available in GenBank, the highest similarity was with B. besnoiti (91.3%). Based on morphological 54 40 and molecular data, this coccidian parasite is a new species that to date has not been reported. The 55 new coccidian parasite is named Caryospora daceloe n. sp. after its host D. novaeguineae (the laughing 56 kookaburra). 57 Ó 2014 Published by Elsevier Inc. 58 59

6261 1. Introduction est genus in the family Eimeriidae. The species in this genus infect 65 primarily predatory birds and reptiles with the majority of 66 63 Caryospora Léger, 1904 (Apicomplexa: Eimeriidae) is a genus of described species infecting snakes (Levine, 1988; Upton et al., 67 64 coccidian protozoa in the phylum Apicomplexa and the third larg- 1992a,b; McAllister et al., 2013a,b; Viana et al., 2013). The parasite Q3 68 may or may not be pathogenic but can result in listlessness, regur- 69 ⇑ Corresponding author. Fax: +61 89310 4144. gitation, anorexia, mucoid haemorrhagic diarrhoea, muscle cramps 70 E-mail address: [email protected] (R. Yang). or sudden death (Coles, 2007). 71

http://dx.doi.org/10.1016/j.exppara.2014.07.008 0014-4894/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Yang, R., et al. A new Caryospora coccidian species (Apicomplexa: Eimeriidae) from the laughing kookaburra (Dacelo novaeguineae). Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.07.008 YEXPR 6913 No. of Pages 6, Model 5G 4 August 2014

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72 There are over 25 species of Caryospora identified from birds was placed in 2% (w/v) potassium dichromate solution (K2Cr2O7), 132 73 worldwide (http://www.k-state.edu/parasitology/worldcoccidia/ mixed well and poured into petri dishes to a depth of less than 133 74 CARYOSPORA), mainly in captive birds of prey (Upton et al., 1 cm and kept at room temperature in the dark to facilitate sporu- 134 75 1992a; Papazahariadou et al., 2001; McAllister et al., 2013a,b). Rel- lation. Sporulated oocysts were observed using an Olympus DP71 135 76 atively little is known about the distribution and significance of digital micro-imaging camera and images were taken using Nomar- 136 77 Caryospora in free-living birds. ski contrast imaging system with a 100 oil immersion objective. 137 78 In Australia, Caryospora has been identified in 2 native Austra- 79 lian bird species, one of which is the tawny frogmouth (Podargus 2.3. DNA isolation 138 80 strigoides), in which it caused severe disease (Montali et al., 2005). 81 The laughing kookaburra is also known as the laughing jackass. Total DNA was extracted from 200 mg of each faecal sample 139 82 This carnivorous bird is the largest member of the Kingfisher fam- using a Power Soil DNA Kit (MolBio, Carlsbad, California) with 140 83 ily (Halcyonidae). It has a dark brown crown and ‘‘ear patch’’. The some modifications as described by Yang et al. (2013). Briefly, 141 84 eye is brown. It has a brown wing mottled with pale blue. Older the faeces for DNA extraction were subjected to four cycles of 142 85 males have a centre rump that is blue-green in colour. The tail is freeze/thaw (liquid nitrogen followed by boiling water) to ensure 143 86 reddish brown and is patterned with black stripes. The stout beak efficient lysis of oocysts before being processed using the manufac- 144 87 is used to catch mainly mice, small snakes and lizards, small birds turer’s protocol. A negative control (no faecal sample) was used in 145 88 and insects. It inhabits woodland, forest clearings, farmland, orch- each extraction group. 146 89 ards, parks and gardens. The laughing kookaburra is native to East- 90 ern Australia from the Cape York Peninsula to the Eastern Eyre 2.4. PCR amplification of 18S rRNA and 28S rRNA 147 91 Peninsula in South Australia. It was introduced into Tasmania 92 and Western Australia and is now widespread throughout the Generic apicomplexan primers (CRYPTOF 50-AAC CTG GTT GAT 148 93 South West (Pizzey and Knight, 2007). CCT GCC AGT and CRYPTOR 50-GCT TGA TCC TTC TGC AGG TTC ACC 149 94 In the present study, we describe a new species of Caryospora TAC) were used to amplify the almost full length 18S rRNA gene as 150 95 coccidian parasite from the laughing kookaburra (Dacelo novaegui- described by Eberhard et al. (1999). The expected PCR product was 151 96 neae), both morphologically and genetically, and propose the spe- about 1584 bp. The PCR reaction contained 2.5 llof10 Kapa PCR 152

97 cies name Caryospora daceloe n. sp. buffer, 3 ll of 25 mM MgCl2, 1.5 ll of 10 nM dNTP’s, 10 pM of each 153 primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 ll of DNA 154

(about 50 ng) and 14.9 llofH2O. PCR cycling conditions were 1 cycle 155 98 2. Materials and methods of 94 °C for 3 min, followed by 45 cycles of 94 °C for 30 s, 55 °C for 156 30 s and 72 °C for 2 min and a final extension of 72 °C for 5 min. 157 99 2.1. Sample collection The PCR for the 28S rRNA locus was carried out using a nested 158 PCR with the external primers: 28SExF: 50-TAC CCG CTG AAC TTA 159 100 A survey was conducted from January to October 2012 (n =30 AGC and 28SExR: 50-CMA CCA AGA TCT GCA CTA G as previously 160 101 faecal samples) to determine the incidence of coccidian parasites described (Schrenzel et al., 2005), which produced a PCR product 161 102 in a population of laughing kookaburras (D. novaeguineae) that size of 1495 bp. The internal primers (28InF: 50-ACT ATG TTC 162 103 were admitted to the Kanyana Wildlife Rehabilitation Centre CCT AGT AAC G and 28SInR 50-AAC GCT TCG CCA CGA TCC) were 163 104 (KWRC) in Perth, Western Australia. All faecal samples were col- designed for the present study using Primer 3 (http://frodo.wi. 164 105 lected from individual birds in the mornings. During December mit.edu/) and produced an amplicon size of 1420 bp. The PCR 165 106 2013, an additional 9 faecal samples were taken from another 9 reaction contained 2.5 llof10 Kapa PCR buffer, 2 llof25mM 166 107 individual birds but these samples were collected after midday. MgCl2,1ll of 10 mM dNTP’s, 10 pM of each primer, 1 unit of 167 108 All the birds were wild and came into care for a variety of reasons KapaTaq (Geneworks, Adelaide, SA), 1 ll of DNA (about 50 ng) 168 109 such as victims of motor vehicle accidents, dog bites, falling into and 16.9 llofH2O. Both primary and secondary PCR’s were con- 169 110 suburban swimming pools or having fallen out of the nest. All ducted with the same cycling conditions; 1 cycle of 94 °C for 170 111 the faecal samples were collected under the KWRC permit. Sam- 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 171 112 ples were stored at 4 °C until parasitological examination and 72 °C for 90 s and a final extension of 72 °C for 5 min. 172 113 DNA extraction. Three positive samples were used for molecular 114 analysis. 2.5. Sequence analysis 173 115 The first positive sample was obtained from a fledgling. On 116 admission it was seen to have malalignment of the beak as well The primary amplicons from the 18S PCR and the secondary 174 117 as a muco-purulent discharge from a nare. Depigmentation of the amplicons from the 28S PCR were gel purified using an in house fil- 175 118 area around this nare was noted. No diarrhoea was noted. Scoliosis ter tip method as previously described (Yang et al., 2013). Ampli- 176 119 was seen on X-ray. The second positive sample was from an adult cons were sequenced using an ABI Prism™ Dye Terminator Cycle 177 120 bird that had been a victim of a dog attack. This kookaburra was Sequencing kit (Applied Biosystems, Foster City, California) accord- 178 121 underweight on admission. The third positive sample was from a ing to the manufacturer’s instructions (with the exception that the 179 122 nestling that was dehydrated and underweight. Its lower abdomen annealing temperature was at 58 °C). The results of the sequencing 180 123 was distended and rigid on palpation. It was listless and had no reactions were analysed and edited using Finch TVÒ v1.4.0. (http:// 181 124 appetite. No diarrhoea was noted. This young bird died suddenly seq.mc.vanderbilt.edu/dna/html/SoftDetail.html). Sequences were 182 125 5 days later despite being treated with Toltrazuril (50 mg/ml) at compared to existing Caryospora and other coccidian parasite 18S 183 126 a dose rate of 15 mg/kg, in a single daily dose for a period of 3 days. and 28S rRNA sequences on GenBank using BLAST searches and 184 aligned with reference sequences with BioEditor (http://bioeditor. 185 127 2.2. Morphological analysis sdsc.edu/download.shtml). 186

128 Microscopic examination of a wet mount and faecal ﬂotation 2.6. Phylogenetic analysis 187 129 analysis were performed on all samples. Faecal ﬂotation was done 130 using a saturated sodium chloride and 50% sucrose (w/v) solution. Phylogenetic tree was constructed for Caryospora spp. and other 188 131 A portion of faeces, from all samples containing coccidian oocysts, coccidian parasites at the 18S locus. No Caryospora spp. sequences 189

R. Yang et al. / Experimental Parasitology xxx (2014) xxx–xxx 3

190 were available in GenBank for the 28S locus. Distance estimation 191 was conducted using TREECON (Van de Peer and De Wachter, 192 1994), based on evolutionary distances calculated with the Tam- 193 ura–Nei model and grouped using Neighbour-Joining. Parsimony 194 analyses were conducted using MEGA version 6.06 (MEGA6.06: 195 Molecular Evolutionary Genetics Analysis software, Arizona State 196 University, Tempe, Arizona, USA). Bootstrap analyses were con- 197 ducted using 1,000 replicates to assess the reliability of inferred 198 tree topologies. Maximum Likelihood (ML) analyses were con- 199 ducted using the programme PhyML (Dereeper et al., 2008) and 200 the reliability of the inferred trees was assessed by the approxi- 201 mate likelihood ratio test (aLRT) (Anisimova and Gascuel, 2006). Fig. 1b. Composite line drawing of Caryospora daceloe n. sp. sporulated oocyst. Scale bar = 10 lm. 202 2.7. Statistical analysis Prevalence: Caryospora sp. were detected in 2/30 samples 232 203 Q4 Measurements of 30 sporulated oocysts were analysed using screened from January to October 2012 from the morning sam- 233 204 Statistical Package for the Social Sciences (SPSS Version 21) and plings, an estimated prevalence of 6.7% (95% CI 1.7 to 20.2). It 234 205 results are presented in micrometres as the mean, with the was detected in 3/9 samples screened in December 2013 from 235 206 observed range in parentheses. the afternoon samplings, an estimated prevalence of 33.3% 236 (95% CI 12.1 to 64.6). 237 207 2.8. Line drawing Other hosts: None. 238 Prepatent period: Unknown. 239 208 The oocyst line drawing was constructed using the software of Patent period: Unknown. 240 209 Inkscape (http://www.inkscape.org/en/). Site of infection: Unknown. 241 Sporulation time: Unknown but assumed to be 48–72 h. 242 Material deposited: The oocyst slide and photosyntypes were 243 210 3. Results deposited into Western Australian Museum with the reference 244 number as: WAM Z68799. DNA sequences have been deposited 245 211 3.1. Description in GenBank under accession numbers KJ634019 and KJ634020 246 for the 18S and 28S locus respectively. 247 212 3.1.1. C. daceloe n. sp. (Caryospora Léger, 1904, Apicomplexa Levine, Etymology: This species is named C. daceloe n. sp. after its host 248 213 1970, Eimeriidae Minchin, 1903) D. novaeguineae (laughing kookaburra). 249 214 250 215 Diagnosis: Sporulated oocysts (n = 30) are ovoid in shape with 216 smooth, colourless, bilayered oocyst wall and measure 3.2. Phylogenetic analysis of C. daceloe n. sp. at the 18S locus 251 217 31.4 29.3 (30–32 28–31) lm in size with a width to length 218 ratio of 1.05 (1.01–1.1). Oocysts with one spherical to subspher- Three identical nearly full length 18S Caryospora sequences 252 219 ical sporocyst. Micropyle, oocyst residuum and polar granule were obtained from the kookaburra faecal samples and were 253 220 are absent. Sporocyst with compact sporocyst residuum. Sporo- aligned with 2 Caryospora bigenetica sequences (Clone 1 and Clone 254 221 cyst length, 21.2 (20.0–24.0); sporocyst width, 20.6 (20.0–21.0); 2) (GenBank accession numbers: AF060975 and AF060976); and 3 255 222 sporocyst L/W ratio, 1.03 (1.0–1.14). A spheroidal shaped sporo- Besnoitia besnoiti sequences (Bb-GER1, Bb Spain-1, Bb-Portugal – 256 223 cyst residuum is present; micropyle, Stieda, substieda and par- GenBank accession numbers FJ797432, EU789637 and AY833646 257 224 astieda bodies are absent. Eight vermiform sporozoites are respectively), as well as other apicomplexan 18S rRNA sequences. 258 225 arranged in either parallel or randomly in the sporocyst, mea- Cryptosporidium parvum was used as an outgroup. 259 226 suring 17.0 4.8 (16–18 4.0–6.0) lm, with a L/W ratio of Phylogenetic analysis using distance, parsimony and ML 260 227 3.54. There is a large spheroidal posterior refractile body in revealed that C. daceloe n. sp. exhibited 99.2% similarity with B. bes- 261 228 Q5 the middle of the sporozoite (Figs. 1a and b). noiti and 98.8% to Hammondia trifﬁttae (GenBank accession num- 262 229 Type hosts: Laughing kookaburra (D. novaeguineae). bers: Q984222 and GQ984223), followed by 98.6% to Neospora 263 230 Type locality: Leeming, Perth, Western Australia (32.0820° S, caninum (GenBank accession number, L24380). There was a genetic 264 231 115.8510° E). similarity of only 89.0% between C. daceloe n. sp. and C. bigenetica 265

SR SZ

SZ SZ SC

Eight sporozoites (SZ) packed inside Sporozoites (SZ) Sporocyst with large residuum (SR) sporocyst (SC)

Fig. 1a. Nomarski interference-contrast photomicrographs of Caryospora daceloe n. sp. showing one spheroidal to subspheroidal sporocyst. Scale bar = 10 lm.

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

0.1 Neospora caninum L24380 Neospora caninum U17345 89 Neospora caninum U17346 52 Neospora caninum AJ271354 Neospora caninum U16159 65 Neospora caninum U03069 Hammondia Triffittae AF096498 M97703 99 Toxoplasma gondii 92 ToxoplasmaGondii X68523 Toxoplasma.gondii L37415 68 Hammondia triffittae GQ984223 74 Hammondia triffittae GQ984222 96 |Besnoia besnoitii DQ227419 80 |Besnoia besnoitii FJ797432 |Besnoia AF109678 59 besnoitii Caryospora daceloe n. sp KJ634019

64 96 Isospora bellii DQ060683 100 Cystoisospora spp AY279205 100 Cystoisospora spp EU200792 Sarcocysti ssp EU443095 AF009245 100 Frenkelia glareoli EU443095 59 Sarcocysti ssp AF513487 59 Sarcocysti ssp JQ733508 100 92 Sarcocysti ssp 71 Sarcocysti ssp JF975681 88 Cystoisosporasp EU502869 Sarcocysti ssp GQ245670 Sarcocysti ssp JQ73351 55 E falciformis AF080614 100 Emacropodis JQ392575 100 Caryospora bigenetica AF060976 Caryospora bigenetica AF060975 C. parvum AF093491

Fig. 2. Evolutionary relationships of Caryospora daceloe n. sp. inferred by distance analysis of 18 rRNA sequences. Percentage support (>50%) from 1000 pseudoreplicates from Maximum Likelihood (ML) analyses is indicated at the left of the supported node.

266 (Fig. 2). A partial 18S sequence (565 bp) from a Caryospora isolate 91.0% for afternoon samples. The regent honeyeaters tested in 295 267 (DB2236) from a snake (Psammophis schokari). the study by Morin-Adeline et al. (2011) were captive birds, 296 268 (GenBank number: KC696572) exhibited 85.7% similarity with whereas the kookaburras in the present study were wild-caught 297 269 C. daceloe n. sp. and had only been in care for 1–2 weeks. 298 Sporulated oocysts of C. daceloe n. sp. were spherical to sub- 299 spherical and measured 31.4 29.3 (30–32 28–31) lm with a 300 270 3.3. Phylogenetic analysis of C. daceloe n. sp. at the 28S rRNA locus shape index of 1.1. The oocyst has one spheroidal to subspheroidal 301 sporocyst, 21.2 20.6 (20.0–24.0 20.0–21.0) lm. A previous 302 271 Three identical 28S rRNA PCR amplicons from three separate study reported similar oocysts from a tawny frogmouth (P. strigo- 303 272 kookaburra faecal DNA samples were obtained. Unfortunately, ides) in Sydney (Montali et al., 2005). In that study the oocysts 304 273 there were no 28S rRNA Caryospora sequences available from Gen- were spherical or subspherical and measured 28.0–34.0 28.0– 305 274 Bank and therefore phylogenetic analysis could only be conducted 32.0 lm and the ovoid sporocyst measured 19.0–24.0 18.0– 306 275 using other apicomplexan 28S rRNA sequences including B. besnoiti 23.0 lm(Montali et al., 2005). There are however morphological 307 276 (GenBank accession number: AY833646). Cyclospora cayetanensis differences between C. daceloe n. sp. and the oocysts described by 308 277 (GenBank accession number: EU252544) was used as an outgroup. Montali et al. (2005), as a large spheroidal posterior refractile body 309 278 Similar to the 18S rRNA locus, phylogenetic analysis at 28S is present in the middle of C. daceloe n. sp. sporozoites, which were 310 279 rRNA locus showed that C. daceloe n. sp. was most closely related not described in the Caryospora spp. in the tawny frogmouth 311 280 to B. besnoiti (91.3%) (GenBank accession number: AY833646) fol- (Montali et al., 2005). Unfortunately genetic sequences from this 312 281 Q6 lowed by H. hammondia (89.3%) (GenBank accession number of isolate were not available Table 1. Q7 313 282 AF101077) and N. caninum (89.0%) (GenBank accession number A study by Forbes et al. (1997) found that 22% of captive-bred 314 283 of AF001946) (Fig. 3). Merlins in the United Kingdom were shedding Caryospora. 315 Lindsay and Blagburn (1989) examined the faeces of red-tailed Q8 316 284 4. Discussion hawks (Buteo borealis) and found Caryospora uptoni in 31%. 317 Recent work by McAllister et al. (2013b) also identiﬁed a new 318 285 In the present study, a novel species of Caryospora was detected species of Caryospora from a free-ranging sharp-shinned hawk, 319 286 in the faeces of the laughing kookaburra (D. novaeguineae). The Accipiter striatus. Most of the Caryospora-infected kookaburras 320 287 prevalence of C. daceloe n. sp. in laughing kookaburras was 6.7% in the present study appeared to be healthy and displayed no 321 288 (2/30) from faecal samples collected in the morning and 33.3% symptoms, with the exception of a very young bird. Clinical 322 289 (6/9) from faecal samples collected in the afternoon. Differences coccidiosis has occasionally been reported in free-ranging birds 323 290 in coccidian oocyst prevalence rates between faecal samples col- but it is mostly a disease of birds in captivity. It may be brought 324 291 lected in the morning and afternoon was also reported for Isospora on by the stress of captivity and/or a concurrent disease (Ladds, 325 292 lesoueﬁ from the regent honeyeater (Xanthomyza phrygia) 2009). 326 293 (Morin-Adeline et al., 2011). In that study, the authors noted a With the advent of molecular methods, the suite of characters 327 294 prevalence of 21.0% in samples collected during the morning and available for inferring relationships among protistan parasites of 328

R. Yang et al. / Experimental Parasitology xxx (2014) xxx–xxx 5

100100 |Besnoia besnoi AY778965 |Besnoia besnoi AY827838 100 |Besnoia besnoi AF076900 Neospora caninum AF001946 100 Toxoplasma Gondii L25635 100 99 53 Toxoplasma Gondii X75430 Hammondia hammondi AF101077

100 Caryospora daceloe n. sp KJ634020

Isospora felis U85705 52 100 Sarcocystis singaporensis AF237617 100 100 Frenkelia micro AF044252 Sarcocystis albifronsi EF079885 100 93 Eimeria anguillae GU593704 Goussia balatonica GU593717 Eimeria papillata GU593706 91 100 Isospora sp. MS-2003 AY283868 Isospora sp. MS-2003 AY283869 Cyclospora cayetanensis EU252544

Fig. 3. Evolutionary relationships of Caryospora daceloe 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.

Table 1 Comparative morphology of C. daceloe n. sp. and other Caryospora species.

Species Hosts References Oocysts Sporocyst Sporozoite Shape Measurements Shape Shape Measurements Shape Measurement Shape (lm) index (lm) (lm) index C. hanebrink Bald eagle McAllister Ellipsoidal 48.1 3 42.1 (42– 1.2 Spheroidal 24.8 (23–28) Stout 18.6 5.6 (16– 3.3 (Haliaeetus et al. (2013a) 54 3 37–50) 20 4–6) leucocephalus) C. aquilae Golden eagle (Aquila Volf et al. Subspherical 43 37.5 (40– 1.1 Spherical to 23.8 23.3 (23– Vermiform 13.5 4.5 (13– 3.0 chrysaetos) (2000) 49 34–39) subspherical 25 22 25) 14 4–5) C. circi Marsh harrier (Circus Volf et al. Widely oval 24.5 21.8 1.1 Spherical to 16.2 15.6 (15– Elongate 10.4 4.3 2.4 aeruginosus) (2000) (23–25 21– subspherical 17 5–17) oval (9–11 4–5) 24) C. petersoni Sharp-shinned hawk McAllister Subspherical 43.1 39.8 1.1 Subspherical 23.4 23.3 Stout 15.6 4.2 (15– 3.7 (Accipiter striatus) et al. (2013b) to spherical 16 4–5 Q11 C. bubonis n. Great horned owl Cawthorn and Subspherical 43.9 40.2 1.1 Subspherical 26.6 25.6 (20– Elongated 15.5 2.5 (13– 6.2 (Bubo virginianus) Stockdale (38–52 33– 33 20–32) 20.8 2.3–3) (1981) 47) C. tremula Turkey vultures Lindsay et al. Subspherical 33.4 28.0 1.1 Spheroidal 20.4 20.1 16.3 5.3 3.1 (Cathartes aura) (1994) C. uptoni Red-Tailed Hawks Lindsay and Spherical or 28.1 26.4 1.1 Spheroidal 18.2 17.9 Elongated 12.6 4.2 3.0 (Buteo jamaicensis Blagburn subspherical borealis) (1986) Caryospora sp. Tawny Frogmouth Montali et al. Spherical or 28–34 28–32 N/A Ovoid 19–24 18–23 N/A N/A N/A (Podargus strigoides) (2005) subspherical C. daceloe n. sp. Kookaburra (Dacelo This study Spherical or 31.4 29.3 1.1 Spheroidal to 21.2 20.6 (20.0– Vermiform 17.0 4.8 (16– 3.5 novaeguineae) subspherical (30–32 28– subspheroidal 24.0 20.0–21.0) 18 4.0–6.0) 31)

329 vertebrates has expanded greatly (Sogin and Silberman, 1998). Although Caryospora sequences were not available for the 28S 340 330 These molecular characters have been used to augment classifica- rRNA locus, phylogenetic analysis also showed a close relationship 341 331 tion schemes that were originally erected based on morphological with Besnoitia and Hammondia. It is possible that this new Caryos- 342 332 and life history criteria. In some cases, the phylogenetic hypotheses pora-like coccidian parasite may be a new genus, which has similar 343 333 have been in serious conflict with taxonomic systems erected using morphological features as Caryospora. Future studies which pro- 344 334 other means. Examples include Blastocystis species as stramenopiles vide more sequence data on Caryospora isolates and analysis of 345 335 (instead of flagellates, amoebae, yeasts or sporozoas (Silberman these isolates at multiple gene loci will provide a more in-depth 346 336 et al., 1996; Barta, 2001). analysis of the phylogenetic relationships and evolution of this 347 337 In the present study, phylogenetic analysis of C. daceloe n. sp. at novel sp. Further studies are also required to investigate the life- 348 338 the 18S locus revealed that this coccidian parasite was more cycle and pathological development of this coccidian parasite in 349 339 closely to Besnoitia and Hammondia than Caryospora or Eimeria. kookaburras and other birds. 350

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