Experimental Parasitology 145 (2014) 68–73
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A new Caryospora coccidian species (Apicomplexa: Eimeriidae) from the laughing kookaburra (Dacelo novaeguineae) Rongchang Yang a,⇑, Belinda Brice b, Una Ryan a a b
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia 6150, Australia Kanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076, Australia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Description of a new Caryospora – like
Neospora caninum L24380 Neospora caninum U17345 89 Neospora caninum U17346 52 Neospora caninum AJ271354 Neospora caninum U16159 65 Neospora caninum U03069 Hammondia Triffittae AF096498 Toxoplasma gondii M97703 99 92 ToxoplasmaGondii X68523 Toxoplasma.gondii L37415 Hammondia triffittae GQ984223 68 74 Hammondia triffittae GQ984222 96 |Besnoia besnoitii DQ227419 80 |Besnoia besnoitii FJ797432 |Besnoia besnoitii AF109678 59 Caryospora daceloe n. sp. KJ634019
0.1
coccidian parasite in kookaburra. Morphological characterisation: most similar to Caryospora. Molecular characterisation at 18S and 28S rRNA loci: most close to Besnoitia.
64
96 Isospora bellii DQ060683 100 Cystoisospora spp AY279205 Cystoisospora spp EU200792 Sarcocysti ssp EU443095 Frenkelia glareoli AF009245 100 Sarcocysti ssp EU443095 59 Sarcocysti ssp AF513487 59 Sarcocysti ssp JQ733508 92 100 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 100
a r t i c l e
i n f o
Article history: Received 31 March 2014 Received in revised form 14 July 2014 Accepted 22 July 2014 Available online 1 August 2014 Keywords: 18S rRNA 28S rRNA Besnoitia Caryospora Kookaburra Morphology Phylogenetics
a b s t r a c t A new Caryospora coccidian species is described from the laughing kookaburra (Dacelo novaeguineae). Sporulated oocysts (n = 30) are ovoid in shape with a smooth, colourless, bilayered oocyst wall and measure 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 subspheroidal sporocyst, 21.2 20.6 (20.0–24.0 20.0–21.0) lm. A spheroidal shaped sporocyst residuum is present; micropyle, Stieda, substieda and parastieda bodies are absent. Vermiform sporozoites (n = 8) are arranged either parallel or randomly in the sporocyst, measuring 17.0 4.8 (16.0– 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 middle of the sporozoite. Morphologically, this new species is most similar to Caryospora. The prevalence of this parasite was 6.7% in birds sampled in the morning and 33.3% from those sampled after midday. Further molecular characterisation was conducted at two loci; the 18S and 28S ribosomal RNA (rRNA). At the 18S locus, the new species of Caryospora was most closely related to Besnoitia besnoiti (99.2% similarity) and Hammondia triffittae (98.8% similarity). Although, no 28S partial sequences from Caryospora were available in GenBank, the highest similarity was with B. besnoiti (91.3%). Based on morphological and molecular data, this coccidian parasite is a new species that to date has not been reported. The new coccidian parasite is named Caryospora daceloe n. sp. after its host D. novaeguineae (the laughing kookaburra). Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved.
1. Introduction Caryospora Léger, 1904 (Apicomplexa: Eimeriidae) is a genus of coccidian protozoa in the phylum Apicomplexa and the third ⇑ Corresponding author. Fax: +61 89310 4144. E-mail address:
[email protected] (R. Yang). http://dx.doi.org/10.1016/j.exppara.2014.07.008 0014-4894/Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved.
largest genus in the family Eimeriidae. The species in this genus infect primarily predatory birds and reptiles with the majority of described species infecting snakes ( Levine, 1988; Upton et al., 1992a,b; McAllister et al., 2013a,b; Viana et al., 2013). The parasite may or may not be pathogenic but can result in listlessness, regurgitation, anorexia, mucoid haemorrhagic diarrhoea, muscle cramps or sudden death (Coles, 2007).
R. Yang et al. / Experimental Parasitology 145 (2014) 68–73
There are over 25 species of Caryospora identified from birds worldwide (http://www.k-state.edu/parasitology/worldcoccidia/ CARYOSPORA), mainly in captive birds of prey (Upton et al., 1992a; Papazahariadou et al., 2001; McAllister et al., 2013a,b). Relatively little is known about the distribution and significance of Caryospora in free-living birds. In Australia, Caryospora has been identified in 2 native Australian bird species, one of which is the tawny frogmouth (Podargus strigoides), in which it caused severe disease (Montali et al., 2005). The laughing kookaburra is also known as the laughing jackass. This carnivorous bird is the largest member of the Kingfisher family (Halcyonidae). It has a dark brown crown and ‘‘ear patch’’. The eye is brown. It has a brown wing mottled with pale blue. Older males have a centre rump that is blue-green in colour. The tail is reddish brown and is patterned with black stripes. The stout beak is used to catch mainly mice, small snakes and lizards, small birds and insects. It inhabits woodland, forest clearings, farmland, orchards, parks and gardens. The laughing kookaburra is native to Eastern Australia from the Cape York Peninsula to the Eastern Eyre Peninsula in South Australia. It was introduced into Tasmania and Western Australia and is now widespread throughout the South West (Pizzey and Knight, 2007). In the present study, we describe a new species of Caryospora coccidian parasite from the laughing kookaburra (Dacelo novaeguineae), both morphologically and genetically, and propose the species name Caryospora daceloe n. sp. 2. Materials and methods 2.1. Sample collection A survey was conducted from January to October 2012 (n = 30 faecal samples) to determine the incidence of coccidian parasites in a population of laughing kookaburras (D. novaeguineae) that were admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC) in Perth, Western Australia. All faecal samples were collected from individual birds in the mornings. During December 2013, an additional 9 faecal samples were taken from another 9 individual birds but these samples were collected after midday. All the birds were wild and came into care for a variety of reasons such as victims of motor vehicle accidents, dog bites, falling into suburban swimming pools or having fallen out of the nest. All the faecal samples were collected under the KWRC permit. Samples were stored at 4 °C until parasitological examination and DNA extraction. Three positive samples were used for molecular analysis. The first positive sample was obtained from a fledgling. On admission it was seen to have malalignment of the beak as well as a muco-purulent discharge from a nare. Depigmentation of the area around this nare was noted. No diarrhoea was noted. Scoliosis was seen on X-ray. The second positive sample was from an adult bird that had been a victim of a dog attack. This kookaburra was underweight on admission. The third positive sample was from a nestling that was dehydrated and underweight. Its lower abdomen was distended and rigid on palpation. It was listless and had no appetite. No diarrhoea was noted. This young bird died suddenly 5 days later despite being treated with Toltrazuril (50 mg/ml) at a dose rate of 15 mg/kg, in a single daily dose for a period of 3 days. 2.2. Morphological analysis Microscopic examination of a wet mount and faecal flotation analysis were performed on all samples. Faecal flotation was done using a saturated sodium chloride and 50% sucrose (w/v) solution. A portion of faeces, from all samples containing coccidian oocysts, was placed in 2% (w/v) potassium dichromate solution (K2Cr2O7),
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mixed well and poured into petri dishes to a depth of less than 1 cm and kept at room temperature in the dark to facilitate sporulation. Sporulated oocysts were observed using an Olympus DP71 digital micro-imaging camera and images were taken using Nomarski contrast imaging system with a 100 oil immersion objective. 2.3. DNA isolation Total DNA was extracted from 200 mg of each faecal sample using a Power Soil DNA Kit (MolBio, Carlsbad, California) with some modifications as described by Yang et al. (2013). Briefly, the faeces for DNA extraction were subjected to four cycles of freeze/thaw (liquid nitrogen followed by boiling water) to ensure efficient lysis of oocysts before being processed using the manufacturer’s protocol. A negative control (no faecal sample) was used in each extraction group. 2.4. PCR amplification of 18S rRNA and 28S rRNA Generic apicomplexan primers (CRYPTOF 50 -AAC CTG GTT GAT CCT GCC AGT and CRYPTOR 50 -GCT TGA TCC TTC TGC AGG TTC ACC TAC) were used to amplify the almost full length 18S rRNA gene as described by Eberhard et al. (1999). The expected PCR product was about 1584 bp. The PCR reaction contained 2.5 ll of 10 Kapa PCR buffer, 3 ll of 25 mM MgCl2, 1.5 ll of 10 nM dNTP’s, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 ll of DNA (about 50 ng) and 14.9 ll of H2O. PCR cycling conditions were 1 cycle of 94 °C for 3 min, followed by 45 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min and a final extension of 72 °C for 5 min. The PCR for the 28S rRNA locus was carried out using a nested PCR with the external primers: 28SExF: 50 -TAC CCG CTG AAC TTA AGC and 28SExR: 50 -CMA CCA AGA TCT GCA CTA G as previously described (Schrenzel et al., 2005), which produced a PCR product size of 1495 bp. The internal primers (28InF: 50 -ACT ATG TTC CCT AGT AAC G and 28SInR 50 -AAC GCT TCG CCA CGA TCC) were designed for the present study using Primer 3 (http://frodo. wi.mit.edu/) and produced an amplicon size of 1420 bp. The PCR reaction contained 2.5 ll of 10 Kapa PCR buffer, 2 ll of 25 mM MgCl2, 1 ll of 10 mM dNTP’s, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 ll of DNA (about 50 ng) and 16.9 ll of H2O. Both primary and secondary PCR’s were conducted with the same cycling conditions; 1 cycle of 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 90 s and a final extension of 72 °C for 5 min. 2.5. Sequence analysis The primary amplicons from the 18S PCR and the secondary amplicons from the 28S PCR were gel purified using an in house filter tip method as previously described (Yang et al., 2013). Amplicons were sequenced using an ABI Prism™ Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California) according to the manufacturer’s instructions (with the exception that the annealing temperature was at 58 °C). The results of the sequencing reactions were analysed and edited using Finch TVÒ v1.4.0. (http:// seq.mc.vanderbilt.edu/dna/html/SoftDetail.html). Sequences were compared to existing Caryospora and other coccidian parasite 18S and 28S rRNA sequences on GenBank using BLAST searches and aligned with reference sequences with BioEditor (http://bioeditor. sdsc.edu/download.shtml). 2.6. Phylogenetic analysis Phylogenetic tree was constructed for Caryospora spp. and other coccidian parasites at the 18S locus. No Caryospora spp. sequences
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R. Yang et al. / Experimental Parasitology 145 (2014) 68–73
were available in GenBank for the 28S locus. Distance estimation was conducted using TREECON (Van de Peer and De Wachter, 1994), based on evolutionary distances calculated with the Tamura–Nei model and grouped using Neighbour-Joining. Parsimony analyses were conducted using MEGA version 6.06 (MEGA6.06: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA). Bootstrap analyses were conducted using 1,000 replicates to assess the reliability of inferred tree topologies. Maximum Likelihood (ML) analyses were conducted using the programme PhyML (Dereeper et al., 2008) and the reliability of the inferred trees was assessed by the approximate likelihood ratio test (aLRT) (Anisimova and Gascuel, 2006).
Fig. 1b. Composite line drawing of Caryospora daceloe n. sp. sporulated oocyst. Scale bar = 10 lm.
2.7. Statistical analysis Measurements of 30 sporulated oocysts were analysed using Statistical Package for the Social Sciences (SPSS Version 21) and results are presented in micrometres as the mean, with the observed range in parentheses. 2.8. Line drawing The oocyst line drawing was constructed using the software of Inkscape (http://www.inkscape.org/en/). 3. Results 3.1. Description 3.1.1. C. daceloe n. sp. (Caryospora Léger, 1904, Apicomplexa Levine, 1970, Eimeriidae Minchin, 1903) Diagnosis: Sporulated oocysts (n = 30) are ovoid in shape with smooth, colourless, bilayered oocyst wall and measure 31.4 29.3 (30–32 28–31) lm in size with a width to length ratio of 1.05 (1.01–1.1). Oocysts with one spherical to subspherical sporocyst. Micropyle, oocyst residuum and polar granule are absent. Sporocyst with compact sporocyst residuum. Sporocyst length, 21.2 (20.0–24.0); sporocyst width, 20.6 (20.0–21.0); sporocyst L/W ratio, 1.03 (1.0–1.14). A spheroidal shaped sporocyst residuum is present; micropyle, Stieda, substieda and parastieda bodies are absent. Eight vermiform sporozoites are arranged in either parallel or randomly in the sporocyst, measuring 17.0 4.8 (16–18 4.0–6.0) lm, with a L/W ratio of 3.54. There is a large spheroidal posterior refractile body in the middle of the sporozoite (Figs. 1a and b, Table 1). Type hosts: Laughing kookaburra (D. novaeguineae). Type locality: Leeming, Perth, Western Australia (32.0820° S, 115.8510° E).
Prevalence: Caryospora sp. were detected in 2/30 samples screened from January to October 2012 from the morning samplings, an estimated prevalence of 6.7% (95% CI 1.7 to 20.2). It was detected in 3/9 samples screened in December 2013 from the afternoon samplings, an estimated prevalence of 33.3% (95% CI 12.1 to 64.6). Other hosts: None. Prepatent period: Unknown. Patent period: Unknown. Site of infection: Unknown. Sporulation time: Unknown but assumed to be 48–72 h. Material deposited: The oocyst slide and photosyntypes were deposited into Western Australian Museum with the reference number as: WAM Z68799. DNA sequences have been deposited in GenBank under accession numbers KJ634019 and KJ634020 for the 18S and 28S locus respectively. Etymology: This species is named C. daceloe n. sp. after its host D. novaeguineae (laughing kookaburra). 3.2. Phylogenetic analysis of C. daceloe n. sp. at the 18S locus Three identical nearly full length 18S Caryospora sequences were obtained from the kookaburra faecal samples and were aligned with 2 Caryospora bigenetica sequences (Clone 1 and Clone 2) (GenBank accession numbers: AF060975 and AF060976); and 3 Besnoitia besnoiti sequences (Bb-GER1, Bb Spain-1, Bb-Portugal – GenBank accession numbers FJ797432, EU789637 and AY833646 respectively), as well as other apicomplexan 18S rRNA sequences. Cryptosporidium parvum was used as an outgroup. Phylogenetic analysis using distance, parsimony and ML revealed that C. daceloe n. sp. exhibited 99.2% similarity with B. besnoiti and 98.8% to Hammondia triffittae (GenBank accession numbers: Q984222 and GQ984223), followed by 98.6% to Neospora caninum (GenBank accession number, L24380). There was a genetic
SZ
SR
SZ
SZ Sporocyst with large residuum (SR)
SC Eight sporozoites (SZ) packed inside sporocyst (SC)
Sporozoites (SZ)
Fig. 1a. Nomarski interference-contrast photomicrographs of Caryospora daceloe n. sp. showing one spheroidal to subspheroidal sporocyst. Scale bar = 10 lm.
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R. Yang et al. / Experimental Parasitology 145 (2014) 68–73 Table 1 Comparative morphology of C. daceloe n. sp. and other Caryospora species. Species
Hosts
References
Oocysts
Measurements Shape Shape index (lm)
Shape C. hanebrink
Bald eagle (Haliaeetus McAllister leucocephalus) et al. (2013a)
C. aquilae
Golden eagle (Aquila chrysaetos) Marsh harrier (Circus aeruginosus)
Volf et al. (2000) Volf et al. (2000)
Sharp-shinned hawk (Accipiter striatus) Great horned owl (Bubo virginianus)
McAllister et al. (2013b) Cawthorn and Stockdale (1981) Lindsay et al. (1994) Lindsay and Blagburn (1986) Montali et al. (2005) This study
C. circi
C. petersoni C. bubonis n.
C. tremula C. uptoni
Caryospora sp. C. daceloe n. sp.
Turkey vultures (Cathartes aura) Red-Tailed Hawks (Buteo jamaicensis borealis) Tawny Frogmouth (Podargus strigoides) Kookaburra (Dacelo novaeguineae)
Sporocyst
Ellipsoidal
48.1 3 42.1 (42–54 3 37–50) Subspherical 43 37.5 (40– 49 34–39) Widely oval 24.5 21.8 (23–25 21– 24) Subspherical 43.1 39.8
Sporozoite Measurements (lm)
Shape
Measurement (lm)
Shape index
1.2
Spheroidal
24.8 (23–28)
Stout
18.6 5.6 (16– 20 4–6)
3.3
1.1
Spherical to subspherical Spherical to subspherical
23.8 23.3 (23– 25 22 25) 16.2 15.6 (15– 17 5–17)
Vermiform 13.5 4.5 (13– 14 4–5) Elongate 10.4 4.3 (9– oval 11 4–5)
3.0
Subspherical to spherical Subspherical
23.4 23.3
Stout
3.7
1.1
1.1
1.1 Subspherical 43.9 40.2 (38–52 33– 47) Subspherical 33.4 28.0 1.1
26.6 25.6 (20– 33 20–32)
15.6 4.2 (15– 16 4–5) Elongated 15.5 2.5 (13– 20.8 2.3–3)
2.4
6.2
Spheroidal
20.4 20.1
16.3 5.3
3.1
Spherical or 28.1 26.4 subspherical
1.1
Spheroidal
18.2 17.9
Elongated 12.6 4.2
3.0
Spherical or subspherical Spherical or subspherical
N/A
Ovoid
19–24 18–23
N/A
N/A
1.1
Spheroidal to 21.2 20.6 (20.0– Vermiform 17.0 4.8 (16– subspheroidal 24.0 20.0–21.0) 18 4.0–6.0)
28–34 28– 32 31.4 29.3 (30–32 28– 31)
0.1
N/A
3.5
Neospora caninum L24380 Neospora caninum U17345 89 Neospora caninum U17346 52 Neospora caninum AJ271354 Neospora caninum U16159 65 Neospora caninum U03069 Hammondia Triffittae AF096498 Toxoplasma gondii M97703 99 92 ToxoplasmaGondii X68523 Toxoplasma.gondii L37415 68 Hammondia triffittae GQ984223 74 Hammondia triffittae GQ984222 96 |Besnoia besnoitii DQ227419 80 |Besnoia besnoitii FJ797432 |Besnoia besnoitii AF109678 59 Caryospora daceloe n. sp KJ634019 64 100
100
96 Isospora bellii DQ060683 100 Cystoisospora spp AY279205 Cystoisospora spp EU200792 Sarcocysti ssp EU443095 Frenkelia glareoli AF009245 100 Sarcocysti ssp EU443095 59 Sarcocysti ssp AF513487 59 Sarcocysti ssp JQ733508 92 71 Sarcocysti ssp JF975681 88 Cystoisosporasp EU502869 Sarcocysti ssp GQ245670 Sarcocysti ssp JQ73351 E falciformis AF080614 55 100 E macropodis 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.
similarity of only 89.0% between C. daceloe n. sp. and C. bigenetica (Fig. 2). A partial 18S sequence (565 bp) from a Caryospora isolate (DB2236) from a snake (Psammophis schokari). (GenBank number: KC696572) exhibited 85.7% similarity with C. daceloe n. sp.
3.3. Phylogenetic analysis of C. daceloe n. sp. at the 28S rRNA locus Three identical 28S rRNA PCR amplicons from three separate kookaburra faecal DNA samples were obtained. Unfortunately, there were no 28S rRNA Caryospora sequences available from
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R. Yang et al. / Experimental Parasitology 145 (2014) 68–73 0.1
|Besnoia besnoi AY833646 |Besnoia tarandi AY616164 84
|Besnoia besnoi DQ227419
|Besnoia besnoi DQ227420 96 |Besnoia besnoi DQ227418 100 |Besnoia besnoi AY778965 100 |Besnoia besnoi AY827838 100
|Besnoia besnoi AF076900 Neospora caninum AF001946
100 99 53
100
Toxoplasma Gondii L25635 Toxoplasma Gondii X75430 Hammondia hammondi AF101077
100
Caryospora daceloe n. sp KJ634020
Isospora felis U85705
52
100
100
Sarcocystis singaporensis AF237617 100
Frenkelia micro AF044252 Sarcocystis albifronsi EF079885
100
93
Eimeria anguillae GU593704 Goussia balatonica GU593717
91
Eimeria papillata GU593706 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.
GenBank and therefore phylogenetic analysis could only be conducted using other apicomplexan 28S rRNA sequences including B. besnoiti (GenBank accession number: AY833646). Cyclospora cayetanensis (GenBank accession number: EU252544) was used as an outgroup. Similar to the 18S rRNA locus, phylogenetic analysis at 28S rRNA locus showed that C. daceloe n. sp. was most closely related to B. besnoiti (91.3%) (GenBank accession number: AY833646) followed by Hammondia hammondia (89.3%) (GenBank accession number of AF101077) and N. caninum (89.0%) (GenBank accession number of AF001946) (Fig. 3).
4. Discussion In the present study, a novel species of Caryospora was detected in the faeces of the laughing kookaburra (D. novaeguineae). The prevalence of C. daceloe n. sp. in laughing kookaburras was 6.7% (2/30) from faecal samples collected in the morning and 33.3% (6/9) from faecal samples collected in the afternoon. Differences in coccidian oocyst prevalence rates between faecal samples collected in the morning and afternoon was also reported for Isospora lesouefi from the regent honeyeater (Xanthomyza phrygia) (MorinAdeline et al., 2011). In that study, the authors noted a prevalence of 21.0% in samples collected during the morning and 91.0% for afternoon samples. The regent honeyeaters tested in the study by Morin-Adeline et al. (2011) were captive birds, whereas the kookaburras in the present study were wild-caught and had only been in care for 1–2 weeks. Sporulated oocysts of C. daceloe n. sp. were spherical to subspherical and measured 31.4 29.3 (30–32 28–31) lm with a shape index of 1.1. The oocyst has one spheroidal to subspheroidal sporocyst, 21.2 20.6 (20.0–24.0 20.0–21.0) lm. A previous study reported similar oocysts from a tawny frogmouth (P. strigoides) in Sydney (Montali et al., 2005). In that study the oocysts were spherical or subspherical and measured 28.0–34.0 28.0–32.0 lm and the ovoid sporocyst measured 19.0–24.0 18.0–23.0 lm
(Montali et al., 2005). There are however morphological differences between C. daceloe n. sp. and the oocysts described by Montali et al. (2005), as a large spheroidal posterior refractile body is present in the middle of C. daceloe n. sp. sporozoites, which were not described in the Caryospora spp. in the tawny frogmouth (Montali et al., 2005). Unfortunately genetic sequences from this isolate were not available. A study by Forbes et al. (1997) found that 22% of captive-bred Merlins in the United Kingdom were shedding Caryospora. Lindsay and Blagburn (1989) examined the faeces of red-tailed hawks (Buteo borealis) and found Caryospora uptoni in 31%. Recent work by McAllister et al. (2013b) also identified a new species of Caryospora from a free-ranging sharp-shinned hawk, Accipiter striatus. Most of the Caryospora-infected kookaburras in the present study appeared to be healthy and displayed no symptoms, with the exception of a very young bird. Clinical coccidiosis has occasionally been reported in free-ranging birds but it is mostly a disease of birds in captivity. It may be brought on by the stress of captivity and/or a concurrent disease (Ladds, 2009). With the advent of molecular methods, the suite of characters available for inferring relationships among protistan parasites of vertebrates has expanded greatly (Sogin and Silberman, 1998). These molecular characters have been used to augment classification schemes that were originally erected based on morphological and life history criteria. In some cases, the phylogenetic hypotheses have been in serious conflict with taxonomic systems erected using other means. Examples include Blastocystis species as stramenopiles (instead of flagellates, amoebae, yeasts or sporozoas (Silberman et al., 1996; Barta, 2001). In the present study, phylogenetic analysis of C. daceloe n. sp. at the 18S locus revealed that this coccidian parasite was more closely to Besnoitia and Hammondia than Caryospora or Eimeria. Although Caryospora sequences were not available for the 28S rRNA locus, phylogenetic analysis also showed a close relationship with Besnoitia and Hammondia. It is possible that this new Caryospora-like coccidian parasite may be a new genus, which has similar
R. Yang et al. / Experimental Parasitology 145 (2014) 68–73
morphological features as Caryospora. Future studies which provide more sequence data on Caryospora isolates and analysis of these isolates at multiple gene loci will provide a more in-depth analysis of the phylogenetic relationships and evolution of this novel sp. Further studies are also required to investigate the lifecycle and pathological development of this coccidian parasite in kookaburras and other birds. Acknowledgments The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife Rehabilitation Centre for their commitment and dedication in caring for all the animals admitted to the centre. We are also grateful to the staff at the Wattle Grove Veterinary Hospital, Perth for their expert treatment and care of the wildlife treated at their clinic. We also appreciate the assistance of Ms. Aileen Elliot for producing the oocyst images and Mr. Michael Slaven for making the histological Slides. References Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552. Barta, J.R., 2001. Molecular approaches for inferring evolutionary relationships among protistan parasites. Vet. Parasitol. 101, 175–186. Cawthorn, R.J., Stockdale, P.H.G., 1981. Description of Eimeria bubonis sp.n. (Protozoa: Eimeriidae) and Caryospora bubonis sp.n. (Protozoa: Eimeriidae) in the great horned owl, Bubo virginianus (Gmelin), of Saskatchewan. Can. J. Zoolog. 59, 170–173. Coles, B., 2007. Essentials of Avian Medicine and Surgery, third ed. Blackwell Publishing Ltd., Milton, Queensland, Australia. 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: robust phylogenetic analysis for the non-specialist. Nucl. Acids Res. 36, W465– 469. Eberhard, M.L., da Silva, A.J., Lilley, B.G., Pieniazek, N.J., 1999. Morphologic and molecular characterization of new Cyclospora species from Ethiopian monkeys: C. cercopitheci sp.n., C. colobi sp.n., and C. papionis sp.n.. Emerg. Infect. Dis. 5, 651–658. Forbes et al., 1997. Caryospora neofalconis: an emerging threat to captive-bred raptors in the United Kingdom. J. Avian Med. Surg. 11 (2), 110–114. Ladds, P., 2009. Pathology of Australian Native Wildlife. CSIRO Publishing, 150 Oxford Street, Collingwood Victoria, Australia. Levine, N.D., 1988. The Protozoan Phylum Apicomplexa, Vol. 1. CRC Press Inc, Boca Raton, Florida, U.S.A., pp. 178–179. Lindsay, D.S., Blagburn, B.L., 1986. Caryospora uptoni n. sp. (Apicomplexa: Eimeriidae) from red-tailed hawks (Buteo jamaicensis borealis). J. Parasitol. 72, 762–765.
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