Morphological and molecular characterization of Eimeria purpureicephali n. sp. (Apicomplexa:Eimeriidae) in a red-capped parrot (Purpureicephalus spurius, Kuhl, 1820) in Western Australia

A new Eimeria species is described from a red-capped parrot (Purpureicephalus spurius). Sporulated oocysts (n = 31) were spherical to subspherical, with a rough bilayered oocyst wall 0.8 μm thick. Oocysts measured 24.0 × 22.8 (20.4–26.4 × 18.3–25.9) μm, oocyst length/width ratio, 1.10. Oocyst residuum, polar granule and micropyle were absent. Sporocysts are elongate-ovoid, 11.0 × 7.3 (12.7–9.2 × 7.9–6.6) μm, sporocyst length/width ratio, 1.51 (1.33–1.71). The thin convex Stieda body and indistinct substieda bodies were present and the sporocyst residuum was composed of numerous small granules less than 1.0 μm in diameter dispersed randomly. Each sporocyst contained 2 sausage-shaped sporozoites in head-to-tail arrangement. The sporozoite nuclei were located centrally surrounded by refractile bodies. Molecular analysis was conducted at two loci; the 18S ribosomal RNA gene and the cytochrome c oxidase subunit I gene. At the18S locus, the new isolate shared 99.0% genetic similarity with Eimeria dispersa and Eimeria innocua from the turkey. At the cytochrome c oxidase subunit I gene locus, this new isolate was most closely related to E. dispersa and E. innocua, presented 99.0% and 98.0% genetic similarity, respectively. This new isolate and E. dispersa grouped together in the same clade. Based on the morphological and molecular data, this isolate is a new species of coccidian parasite, which is named Eimeria purpureicephali n. sp. after its host, the red-capped parrot (Purpureicephalus spurius).


Introduction
The red-capped parrot (Purpureicephalus spurius), also called the pileated parakeet (Alderton, 2003) and king parrot locally in Western Australia (Lendon, 1973), is an Australian species of broadtailed parrot which is related to the rosellas. The colourful redcapped parrot has a specialized long beak, which helps them to remove seeds from gumnuts of marri (Eucalyptus calophylla) as well as seeds from other eucalypts and native plants. These parrots live in eucalypt forests, woodlands, timbered watercourses, parks, orchards and gardens. Red-capped parrots are endemic to the south west of Western Australia. (Pizzey and Knight, 2007).
Eimeria (Coccidia: Eimeriidae), is a genus of apicomplexan parasites that includes various species and is known as the enteric monoxenous coccidian parasite. In birds, pathogenic Eimeria causes enteric disease and major economic losses in the global poultry industry (McDougald and Reid, 1997). Eimeria usually invade the intestinal tract, but some invade other organs, such as the liver and kidney. In recent years, more Eimeria species have been identified from free-range birds globally (Hofstatter and Guaraldo, 2011;Yang et al., 2014).
A total of four Eimeria species have been identified and recorded in the coccidian database (Duszynski et al., 2000) from the family Psittaciformes including E. aratinga (Upton and Wright, 1994), Eimeria dunsingi (Farr, 1960), Eimeria haematodi (Varghese, 1977) and E. psittacina (Gottschalk, 1972). Recently, a new Eimeria species, Eimeria ararae n. sp., from the blue-and-yellow macaw Ara ararauna (Linnaeus) in Brazil was added in the family Psittaciformes. With the exception of E. haematodi, which was molecularly characterized by Yang et al. (2015), the other four Eimeria species were identified by their oocyst morphological features only. To date there have been no reported cases of Eimeria species identified from the redcapped parrot (Purpureicephalus spurius, Kuhl, 1820). This is the first study to characterize Eimeria purpureicephali n. sp. in a red-capped parrot in Western Australia, using both morphological data and molecular techniques.

Sample collection and examination
A juvenile red-capped parrot came into care at the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth in November, 2014. On admission it was observed that this bird had labored breathing. Radiographs revealed multiple fractures of the keel bone and congestion of the air sacs. Reduced breast muscle mass was also noted. No clinical signs of coccidiosis were observed. Treatment was implemented but a decision was made to euthanize the bird a few days later. A faecal sample was taken soon after admission to look for evidence of avian gastric yeast (AGY). Microscopic examination of the faeces found no AGY in the sample, however unsporulated coccidian oocysts were seen.
Faecal flotation was conducted using a saturated sodium chloride and 50% sucrose (w/v) solution. A portion of faeces was placed in 2% (w/v) potassium dichromate solution (K 2 Cr 2 O 7 ), 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 microimaging camera and images were taken using Nomarski contrast with a 100Â oil immersion objective. Faecal samples from another 23 red-capped parrots were screened for AGY (by wet mount) during the period January to December 2014. None of these 23 samples were found to be positive for coccidia.
A 3 axis hydraulic micromanipulator (MO-102, Nirashige, Japan) was used to isolate four separate single oocysts for DNA extraction and PCR.

DNA isolation
Oocyst DNA extraction was as described by Yang et al. (2014). Briefly, isolated single oocysts were placed on a slide and checked under the microscope (Olympus DP71 digital micro-imaging camera). Once the existence of a single oocyst on the cover slip was confirmed, photographs were recorded for morphological identification. The coverslip was then transferred into a PCR tube containing 10 ml of lysis buffer (0.005% SDS in TE solution). After a brief centrifugation, the tube was frozen in liquid nitrogen and thawed in a 95 C water bath for four rounds to disrupt the oocyst wall. After the addition of 0.5 ml proteinase K (20 mM), the tube was incubated at 56 C for 2 h and then at 95 C for 15 min. The entire lysate from the single oocyst was used for three separate PCRs as described below.

PCR amplification and sequencing
A nested PCR with the primers EiGTF1 and EIGTR1 was used for the external amplification of the 18S rRNA gene. The expected PCR product was~1510 bp. The primers EiGTF2 and EiGTR2 (Yang et al., 2015) were used for the internal reaction.
The amplicons from the second round PCRs were gel purified using an in house filter tip method as previously described (Yang et al., 2013b). All the PCR products were sequenced using forward and reverse primers in duplicate using amplicons from different PCR runs. An ABI Prism™ Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California) was used for Sanger sequencing according to the manufacturer's instructions. The results of the sequencing reactions were analysed and edited using FinchTV (Version 1.4), compared to existing Eimeria spp. 18S rRNA and COI sequences on GenBank using BLAST searches and aligned with reference genotypes from GenBank using Clustal W in BioEdit (V7.2.5).

Phylogenetic analysis
Phylogenetic trees were constructed for Eimeria spp. at the 18S, and COI loci with additional isolates from GenBank. Parsimony analyses were conducted using MEGA (Molecular Evolutionary Genetics Analysis software, version 6, Arizona State University, Tempe, Arizona, USA). Maximum likelihood (ML) and Neighborjoining (NJ) analyses were conducted using Tamura-Nei based on the most appropriate model selection using ModelTest in MEGA 6.
Bootstrap analyses were conducted using 1000 replicates to assess the reliability of inferred tree topologies. residuum was composed of numerous small granules less than 1.0 mm in diameter dispersed randomly. Each sporocyst contained 2 sausage-shaped sporozoites in head-to-tail arrangement. The sporozoite nuclei were located centrally surrounded by refractile bodies (Fig. 1).

Material deposited
Oocysts in 10% formalin and oocyst phototypes were deposited in Western Australian Museum under the reference number WAMZ68782. DNA sequences have been deposited in GenBank under accession numbers KU140597 and KU140598 for the 18S and COI loci, respectively.

Phylogenetic analysis of E. purpureicephali n. sp. at the 18S locus
A 1229 bp 18S rRNA PCR product of E. purpureicephali n. sp. was successfully amplified and sequenced. Analysis of three individual oocysts produced identical 18S rRNA sequences. Phylogenetic analyses of E. purpureicephali n. sp. at this locus using Parsimony, ML and NJ analyses produced similar results (Fig. 2, ML tree shown). Eimeria purpureicephali n. sp. grouped in a clade with Eimeria dispersa (HG793041) from a turkey in the Czech Republic and shared 99.0% genetic similarity (Fig. 2). It exhibited 98.0% genetic similarity to Eimeria innocua (HG793045), which was also identified from a turkey from the Czech Republic (Vrba and Pakandl, 2014). It shared 96.5% similarity with E. haematodi (KM884825) from a rainbow lorikeet (Trichoglossus haematodus: Psittaciformes) in Western Australia (Yang et al., 2015).  Fig. 3. Evolutionary relationships of E. purpureicephali n. sp. inferred by distance analysis of COI sequences (670 bp). Percentage support (>50%) from 1000 pseudoreplicates from distance, ML and parsimony analysis, respectively, is indicated at the left of the support node ('_' ¼ value was <50%). similarity), a turkey-derived Eimeria (Fig. 3). It also exhibited 94.8% similarity with Eimeria columbadomestica n. sp. (KT305929), which was identified from a domestic pigeon. These three sequences grouped in a separate clade from other Eimeria species (Fig. 3). As only short COI sequences (220e311 bp) were available from E. haematodi and other Eimeria species, they were not included in the phylogenetic analysis. Pair-wise comparison of the partial COI gene sequence (311bp) between E. purpureicephali n. sp. and E. haematodi was conducted and they shared 93.6% genetic similarity.

Discussion
Sporulated oocysts of E. purpureicephali n. sp. are morphologically distinct from other characterized Eimeria species and did not match any other existing documented Eimeria species (n ¼ 11) from Psittaciformes (http://biology.unm.edu/biology/coccidia/ Psittaciformes.html (Accessed on 28th Dec. 2015) ( Table 1). The dimensions of the oocysts from E. purpureicephali n. sp. were smaller than those of the Eimeria species from the hosts in the family Psittaciformes (Table 1).
Phylogenetic analysis of 18S rRNA and COI sequences based on ML, NJ and Parsimony analyses produced similar results and placed E. purpureicephali n. sp. in a clade with E. dispersa (both at 99.0% genetic similarity). The genetic similarity between E. dispersa and E. haematodi was 95.5% at the 18S rRNA locus based on 1235 bp of common sequence and 93.6% at the COI locus based on 311 bp of common sequence.
As E. purpureicephali n. sp. exhibited a high genetic similarity with E. dispersa, at the 18S rRNA and COI loci, oocyst morphological features between these two species were compared in Table 1 and the dimensions of the oocysts and sporocysts were different between the two species. For example, oocysts of E. dispersa (22.4 Â 18.0 mm) are smaller than those of E. purpureicephali n. sp (24.0 Â 22.8 mm) (Table 1). Eimeria dispersa was originally described from a turkey (Tyzzer, 1929) and based on cross-transfection studies, it infected chickens and other gallinacerous birds (Tyzzer, 1929;Hansen, 1974;Doran, 1978). To date, there has been no report about E. dispersa in parrots. Therefore, E. purpureicephali n. sp from this study is different from E. dispersa even though they shared 99.0% genetic similarity at the 18S rRNA and COI loci. This is yet another example demonstrating the importance of utilising both traditional morphological data and molecular tools for analysis of coccidian taxonomy, as morphological or molecular data used on their own may lead to incorrect species identification. As more sequence data from avian-derived Eimeria species becomes available on GenBank, particularly from passerine birds, a more robust molecular taxonomy will be developed.
The incidence rate of coccidia in red-capped parrots in the Perth area was 4.2% (1/24). The true incidence rate is probably higher as the faecal samples were only screened by wet mount and not by faecal float. AGY was seen in 20.8% (5/24) of the samples tested. Two of the five red-capped parrots that were positive for AGY, were also exhibiting clinical signs of Psittacine beak and feather disease.
In conclusion, this is the first report of the morphological and molecular characterization of an Eimeria species in the red-capped parrot from Australia. Further studies of the life cycle and pathogenicity of E. purpureicephali n. sp as well as multi-locus sequencing are necessary to further characterize this Eimeria species.

Conflicts of interest
The authors declare that there is no conflict of interest.