ARTICLE IN PRESS Experimental Parasitology ■■ (2015) ■■–■■
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Full length article
Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus) Q1 Rongchang Yang a,*, Belinda Brice b, Una Ryan a a b
School of Veterinary and Biomedical 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
• • •
Re-description of Eimeria haematodi in a rainbow lorikeet in Australia. Morphological study: Morphology and morphometry identical to E. haematodi. Phylogeny: 98.1% genetic similarity to E. alabamensis at the 18S locus.
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A R T I C L E
I N F O
Article history: Received 16 October 2014 Received in revised form 3 March 2015 Accepted 6 March 2015 Available online Keywords: Eimeria haematodi Molecular characterization 18S rRNA locus Mitochondrial cytochrome oxidase gene (COI) Phylogeny
G R A P H I C A L
A B S T R A C T
E. cahirinensis JQ993645 E. cahirinensis JQ993646 E. callospermophili JQ993648 E. chobotari AF324214 E. cahirinensis JQ993647 E. cahirinensis JQ993645 E. onychomysis AF307879 E. reedi AF311642 E. peromysci AF339492 E. chaetodipi AF339489 E. albigulae AF307880 E. arizonensis AF307878 E. antrozoi AF307876 E. catronensis AF324213 70, 75, 70 E. pilarensis AF324215 Isospora sp. 1 JM-2013 JX984669 99, 100, 99 Isospora robini AF080612 Isospora sp. Tokyo AB757862 58, 61, 58 Isospora sp. MS-2003 AY331571 Isospora gryphoni AF080613 E. vilasi JQ993653 100, 100, 99 E. sp. 2 JK-2013 JQ993652 E. praecox GQ421692 71, 78, 73 75, 73, _ E. maxima EU025110 100, 100, 99 E. acervulina FJ236372 E. mitis FR775307 96, 95, 89 E.a mivati EMU76748 Cyclospora colobi AF111186 E. sp. ex Apodemus agrarius JQ993656 E. alorani JQ993659 63, 53, _ E. sp. ex Apodemus sylvaticus JQ993661 E. sp. ex Apodemus agrarius JQ993655 E. sp. ex Mastomys natalensis JQ993667 E. myoxi JF304148 59, 61, 70 E. sp. ex Phataginus tricuspis JQ993651 E. sp. ex Gerbillus dasyurus JQ993664 E. chinchillae JQ993650 53, 53, _ 100, 100, 99 E. caviae JQ993649 80, 80, 84 E. nieschulzi U40263 E. sp. ex Gerbillus dasyurus JQ993664 E. haematodi KM884825 E. alabamensis AF291427 E. alabamensis AB769552 99, 99, 99 99, 100, 99 E. alabamensis AB769556 E.vejdovskyi HQ173838 97, 98, 91 E. vejdovskyi HQ173838 100, 100, 100 E. media HQ173834 57, 60, 57 E. perforans HQ173835 E. perforans EF694017 E. auburnensis AB769571 E. zuernii AB769665 99, 100, 100 94, 96, 90 E. bovis AB769589 Eimeria paludosa KJ767187 98, 98, 99 E. gruis AB544336 100, 100, 99 E. gruis AB544335 100, 99, 97 E. reichenowi AB544342 99, 99, 100 E. reichenowi AB544343 T. Gondii EF472967
A B S T R A C T
Eimeria haematodi was first described in 1977 from the rainbow lorikeet (Trichoglossus haematodus) in Papua New Guinea. In the present study, we re-describe this coccidian species morphologically and molecularly from a rainbow lorikeet bird in Western Australia (WA). The oocysts were ovoid to slightly piriform and measured 28.5–37.8 by 25.8–33.0 μm (33.3 by 28.1 μm). Oocyst wall was approximately 1.5 μm thick and bilayered. Micropyle (5–7 μm) and oocyst residuum (8.0–10.0 μm) present; polar granule was absent. Sporocysts ellipsoidal, 11.8–13.6 by 8.0–9.6 μm (12.2 by 8.3 μm), with thin convex Stieda body and granular sporocyst residuum (4.0–5.0 μm). Molecular characterization of E. haematodi was conducted at 18S ribosomal RNA and the mitochondrial cytochrome oxidase gene (COI) loci. At the 18S ribosomal RNA locus, E. haematodi shared 98.1% genetic similarity to E. alabamensis from cattle in New South Wales, Australia. At COI locus, E. haematodi was closest (92.3% similarity) to E. praecox from domestic chickens (Gallus gallus domesticus) from Canada and China. Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.
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* Corresponding author. Fax: +61 89310 4144. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.exppara.2015.03.005 0014-4894/Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.
Please cite this article in press as: Rongchang Yang, Belinda Brice, Una Ryan, Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.005
ARTICLE IN PRESS R. Yang et al./Experimental Parasitology ■■ (2015) ■■–■■
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1. Introduction Coccidia are a complex and diverse group of protozoan parasites with more than 1700 described species (Duszynski et al., 2000), most of which do not cause clinical disease. In birds, most diseasecausing or pathogenic forms of coccidia parasites belong to the genus Eimeria (Friend and Franson, 2012). Traditionally, identification of Eimeria species has relied not only on morphological characters but also on host species, pathology and geographic distribution (Duszynski and Wilber, 1997; Tenter et al., 2002). However, some species of Eimeria are morphologically identical and occur worldwide in several hosts, e.g. Eimeria paludosa has been reported from all continents of the world in a range of host species (Duszynski et al., 2000; McAllister and Upton, 1990; Yang et al., 2014b). It is now recognized that molecular data are essential to accurately delimit species and infer phylogenetic relationships among Eimeria species (Tenter et al., 2002). To date, eight species of Eimeria have been described from psittacine hosts; Eimeria dunsingi and Eimeria psittacina from the budgerigar (Melopsittacus undulates) (Farr, 1960; Gottschalk, 1972), Eimeria haematodi from the rainbow lorikeet (Trichoglossus haematodus) (Varghese, 1977), Eimeria aratinga from the orangefronted conure (Aratinga canicularis) (Upton and Wright, 1994), Eimeria amazonae and Eimeria ochrocephalae from yellow-crowned Amazons/parrots (Amazona ochrocephala) (Hofstatter and Kawazoe, 2011), Eimeria aestivae from the blue-fronted Amazon parrot Q2 (Amazona aestiva) (Hofstatter and Guaraldo, 2011) and Eimeria ararae from the blue-and-yellow macaw (Ara ararauna) (do Bomfim Lopes et al., 2014). In Australia, E. dunsingi has been identified in freeliving musk lorikeets (Glossopsitta concinna) (Gartrell et al., 2000) and E. psittacina has been described in the Australian Faunal DiQ3 rectory (http://bie.ala.org.au/species/urn:lsid:biodiversity.org.au:afd. taxon:9f3bd2a7-1e25-4d12-a595-944771349f20#tab_records, but no reference was found). No other Eimeria species in psittaciform hosts have been described from Australia. Eimeria haematodi was first described morphologically by Varghese (1977) from the rainbow lorikeet (Trichoglossus haematodus) in Papua New Guinea (PNG), but has not been reported since. In the present study, we characterized E. haematodi in a rainbow lorikeet (T. haematodus) from Western Australia (WA), both morphologically and phylogenetically. This is the first molecular characterization of Eimeria from psittaciform hosts. 2. Materials and methods 2.1. Sample collection A wild rainbow lorikeet (T. haematodus) came into care at the Native Animal Rescue Centre (ARC), Perth, WA after it had been struck by a motor vehicle. On admission it was noted that the bird was emaciated and the feathers were dirty. A fecal sample was taken 3 days later. Direct microscopic examination of a fecal suspension in saline, as well as fecal flotation analysis using a saturated sodium chloride and 50% sucrose (w/v) solution were applied to identify coccidian oocysts in the feces. The bird was negative for avian gastric yeast (AGY). Unfortunately, this bird died before antiprotozoal treatment was implemented. It is not known if this bird died as a result of the coccidian infection or from another cause. A further 49 fecal samples from another 49 wild rainbow lorikeets were analyzed over a 15 month period (June 2013–August 2014). These rainbow lorikeets were admitted to the Kanyana Wildlife Rehabilitation Centre in Perth, WA. None of the rainbow lorikeets tested in the present study was released back into the wild. If a sample was found to contain coccidian oocysts, a portion of feces was placed in 2% (w/v) potassium dichromate solution (K2Cr2 O7), 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. 2.2. DNA isolation Total DNA was extracted from 200 mg of fecal sample using a Power Soil DNA Kit (MolBio, Carlsbad, California) with some modifications as described by Yang et al. (2012). Briefly, the feces 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 fecal sample) was used in each extraction group. 2.3. PCR amplification and sequencing The PCR for the 18S rRNA locus was carried out using a heminested PCR as described by Yang et al. (2012) with the external primers: EiF1 5′-GCT TGT CTC AAA GAT TAA GCC-3′, EiR3 5′-ATG CAT ACT CAA AAG ATT ACC-3′ and internal primers: EiF3 5′-CTA TGG CTA ATA CAT GCG CAA TC-3′, and EiR3, which amplifies a 1320 bp product. The PCR reaction contained 2.5 μL of 10 × Kapa PCR buffer, 2 μL of 25 mM MgCl2, 1.5 μL of 10 nM dNTPs, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 μL of DNA (about 50 ng) and 15.9 μL 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. Amplification of a 496 bp region of the COI locus from this sample was conducted as described by Ogedengbe et al. (2011) and Yang et al. (2013). The results of the sequencing reactions were analyzed and edited using FinchTV (version 1.4 – http://finchtv.software.informer.com/1.4/), compared to existing Eimeria spp. 18S rDNA and COI sequences in the GenBank database using BLAST searches and aligned with reference genotypes from the GenBank using the Clustal W algorithm in BioEdit (version 7.2.5 – http://www.mbio.ncsu.edu/bioedit/ bioedit.html). 2.4. Phylogenetic analysis Phylogenetic trees were constructed for Eimeria spp. at the 18S rRNA and COI loci with additional isolates from the GenBank database. Phylogenies were conducted using MEGA (Molecular Evolutionary Genetics Analysis software, version 6, Arizona State University, Tempe, Arizona, USA). Neighbor-joining (NJ) and maximum likelihood (ML) analyses were conducted 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. 3. Results 3.1. Description of E. haematodi Morphology: Oocysts are ovoid and some were piriform shaped with bilayered oocyst wall, 1.5 μm thick. Outer layer light brown, 1.0 μm thick and the inner layer colorless, about 0.5 μm thick. Forty oocysts measured 33.3 × 28.1 (28.5–37.8 × 25.8–33.0) μm in size with a length to width ratio of 1.19. Sporocysts are elongate– ovoid, 12.2 × 8.3 (11.8–13.6 × 8.0–9.6) μm, sporocyst L/W ratio, 1.47. Oocyst residuum round, granular, membrane-bound and 8–10 μm. Sporocyst residuum was present about 4.0–5.0 μm at the broader pole, Stieda body present, substieda body absent (Fig. 1 and Table 1).
Please cite this article in press as: Rongchang Yang, Belinda Brice, Una Ryan, Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.005
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
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N/A N/A 20.6 × 10.1 Ovoidal Single polar granule, rounded Absent 1.55 43.8 × 27.7 Hofstatter and Guaraldo (2011)
1.19 Present study
33.3 × 28.1
N/A
Granular Absent 12.2 × 8.3 Spherical to ovoid Absent Present
Present 1.17 32.3 × 27.6
Ovoid to slightly piriform Ovoid to slightly piriform Ellipsoidal
Bi-layered
Absent 1.56 35.8 × 23.0 Ovoid
Bi-layered
Absent 1.35 35.0 × 25.9 Ellipsoidal
N/A
Absent 1.42 28.7 × 20.2 Ovoidal
N/A
Absent N/A 1.35 48.9 × 36.2 Ellipsoidal
Bi-layered
Granular Absent 13.3 × 8.4 Ellipsoidal
Granular Absent 11.4 × 8.5 Ovoid to pyriform
N/A N/A 17.0 × 8.3 Elongate–ovoidal
N/A N/A 17.0 × 8.3 Elongate–ovoidal
N/A N/A 22.2 × 11.9 Ellipsoidal
N/A N/A 19.8 × 9.3 Ovoidal
Single rounded polar granule Single rounded polar granule, Present, 2 to 4 granules Present and fragmented Single polar granule Absent Absent 1.55 36.8 × 23.7 Ovoidal
Wall Shape index
E. ochrocephalae
E. haematodi
E. haematodi
Q10
A 1320 bp PCR product of 18S rDNA of E. haematodi was successfully amplified. Phylogenetic analyses of the nucleotide sequences from E. haematodi at the 18S rRNA locus using Distance (NJ), ML and Parsimony analyses produced essentially the same trees (Fig. 2, ML tree, bootstrap support (>50%) from NJ, ML and Parsimony trees are shown on each node). Unfortunately no psittaciform-derived sequences are available in the GenBank for the 18S rRNA locus. Eimeria haematodi grouped in a separate clade, but most closely
E. dunsingi
3.3. Phylogenetic analysis of E. haematodi at the 18S rRNA locus
E. aratinga
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E. ararae
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E. amazonae
Only 2 of the 11 coccidia positive birds in the present study were found to be underweight. No other clinical signs of coccidian infection were noted. All of the coccidia positive birds were treated with Toltrazuril (50 mg/mL) at a dose rate of 15 mg/kg SID for 3 days. None of these 11 rainbow lorikeets were positive for AGY.
Hofstatter and Guaraldo (2011) Hofstatter and Kawazoe (2011) do Bomfim Lopes et al. (2014) Upton and Wright (1994) Gartrell et al. (2000) Varghese (1977)
3.2. Clinical information
Blue-fronted parrot (Amazona aestiva) Yellow-crowned parrot (Amazona ochrocephala) Blue-fronted parrot (Amazona aestiva) Orange-fronted conure (Eupsittula canicularis) Musk lorikeet (Glossopsitta concinna) Rainbow lorikeet (PNG) (Trichoglossus haematodus) Rainbow lorikeet (WA) (Trichoglossus haematodus) Yellow-crowned parrot (Amazona ochrocephala)
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E. aestivae
18
Size
Type hosts: rainbow lorikeet (Trichoglossus haematodus). Type locality: Perth, Western Australia. Other locality: Port Moresby (Papua) and Wau (New Guinea Highlands) in Papua New Guinea Prevalence: 43.3% (Varghese, 1977), 22.0% (11/50) (this study). Other hosts: Unknown. Prepatent period: Unknown. Patent period: Unknown. Site of infection: Unknown. Sporulation time: 48–96 hours. Material deposited: DNA sequences have been deposited in GenBank under accession numbers KM884825 and KM891727 for the 18S rRNA, and COI locus, respectively.
N/A
Polar granule Shape
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References
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Hosts
Fig. 1. (A) Nomarski interference-contrast photomicrographs of E. haematodi oocysts showing spheroidal to subspheroidal sporocysts (scale bar = 10 μm). (B) Line drawing of the sporulated oocyst of E. haematodi (scale bar = 10 μm).
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Species
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Table 1 Comparative morphology of E. haematodi from rainbow lorikeets in Perth, WA with other species recorded from psittacine birds.
Oocyst Residuum
Sporocysts Oocysts
Shape
Size
Substieda body
Residuum
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Please cite this article in press as: Rongchang Yang, Belinda Brice, Una Ryan, Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.005
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R. Yang et al./Experimental Parasitology ■■ (2015) ■■–■■
E. cahirinensis JQ993645 E. cahirinensis JQ993646 E. callospermophili JQ993648 E. chobotari AF324214 E. cahirinensis JQ993647 E. cahirinensis JQ993645 E. onychomysis AF307879 E. reedi AF311642 E. peromysci AF339492 E. chaetodipi AF339489 E. albigulae AF307880 E. arizonensis AF307878 E. antrozoi AF307876 E. catronensis AF324213 70, 75, 70 E. pilarensis AF324215 99, 100, 99 Isospora sp. 1 JM-2013 JX984669 Isospora robini AF080612 Isospora sp. Tokyo AB757862 58, 61, 58 Isospora sp. MS-2003 AY331571 Isospora gryphoni AF080613 E. vilasi JQ993653 100, 100, 99 E. sp. 2 JK-2013 JQ993652 E. praecox GQ421692 71, 78, 73 75, 73, _ E. maxima EU025110 100, 100, 99 E. acervulina FJ236372 E. mitis FR775307 96, 95, 89 E. mivati EMU76748 Cyclospora colobi AF111186 E. sp. ex Apodemus agrarius JQ993656 E. alorani JQ993659 63, 53, _ E. sp. ex Apodemus sylvaticus JQ993661 E. sp. ex Apodemus agrarius JQ993655 E. sp. ex Mastomys natalensis JQ993667 59, 61, 70 E. myoxi JF304148 E. sp. ex Phataginus tricuspis JQ993651 E. sp. ex Gerbillus dasyurus JQ993664 E. chinchillae JQ993650 53, 53, _ 100, 100, 99 E. caviae JQ993649 80, 80, 84 E. nieschulzi U40263 E. sp. ex Gerbillus dasyurus JQ993664 E. haematodi KM884825 E. alabamensis AF291427 E. alabamensis AB769552 99, 99, 99 99, 100, 99 E. alabamensis AB769556 E.vejdovskyi HQ173838 97, 98, 91 E. vejdovskyi HQ173838 100, 100, 100 E. media HQ173834 57, 60, 57 E. perforans HQ173835 E. perforans EF694017 E. auburnensis AB769571 E. zuernii AB769665 99, 100, 100 94, 96, 90 E. bovis AB769589 Eimeria paludosa KJ767187 98, 98, 99 E. gruis AB544336 100, 100, 99 E. gruis AB544335 100, 99, 97 E. reichenowi AB544342 99, 99, 100 E. reichenowi AB544343 T. gondii EF472967
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Fig. 2. Evolutionary relationship of E. haematodi inferred by distance, ML and parsimony analysis of the 18S rRNA gene (ML tree shown). Percentage support (>50%) from 1000 psudoreplicates from distance, ML and parsimony analysis, respectively, is indicated at the left of the support node (‘_’ = Not available).
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(98.1% similarity) with E. alabamensis (GenBank accession number: AF291427) from cattle in New South Wales, Australia.
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3.4. Phylogenetic analysis of E. haematodi at the COI locus A 496 bp PCR product of COI of E. haematodi was successfully amplified. There were fewer Eimeria COI sequences available in the GenBank database compared to the 18S rRNA locus and no psittaciform-derived sequences. Phylogenetic analysis grouped E. haematodi closest (92.3% similarity) with E. praecox from domestic chickens (Gallus gallus domesticus) from Canada (GenBank accession
number: JQ65930) and China (GenBank accession number: HQ702483) (Fig. 3).
17 18 19
4. Discussion The rainbow lorikeet (T. haematodus) is a species of Australasian parrot found in Australia, eastern Indonesia (Maluku and Western New Guinea), Papua New Guinea, New Caledonia, Solomon Islands and Vanuatu (BirdLife International, 2012). In Australia, it is common along the eastern seaboard, from Queensland to South Australia and Tasmania. Its habitat is rainforest, coastal bush and woodland areas (Sampson and Day, 1999). The rainbow lorikeet was Q4 accidentally released into the southwest of the state of WA near the University of Western Australia as an ornamental bird in the 1960s and since then has been classified as a pest (Manley, 2012). Rainbow lorikeets are considered as a threat to WA’s agricultural stone fruit and viticulture industry and are also hosts for transferable parrot diseases such as psittacine beak and feather disease – a viral disease attacking the immune system causing beak abnormalities and loss of feathers (Manley, 2012). The prevalence of E. haematodi in wild rainbow lorikeets screened in the present study was 22.0% (11/50). This is lower than the prevalence of 43.3% previously reported by Varghese (1977). This could be due to the fact that some of the fecal samples (n = 18) were collected before midday, as previous studies have shown that coccidian shedding is much higher in the afternoon in birds (Morin-Adeline et al., 2011; Yang et al., 2014a). Compared with other Eimeria species from psittacine hosts (Table 1), the Eimeria oocysts isolated from the rainbow lorikeet in this study corresponded to E. haematodi, which was first described by Varghese (1977). For example, the oocysts shape was similar, as both were ovoid to slightly piriform in shape, oocyst and sporocyst measurements were also similar, the polar granule was absent in both and the sporocyst residuum was rounded and granular in both (Table 1). Phylogenetic analysis of 18S rRNA and COI sequences based on Distance, ML and Parsimony analyses produced similar results and placed E. haematodi in a separate clade but most closely related to E. alabamensis (98.1% similarity). The genetic similarity between E. haematodi and E. alabamensis is similar to the genetic differences between accepted species of Eimeria. For example, the genetic similarity between E. arnyi and E. ranae is 97.5% and the similarity between E. tenella and E. necatrix and between E. bovis and E. crandallis is 99.1% and 99.5%, respectively, across the same length of sequence. This supports the species status of E. haematodi. Previous studies have reported that Eimeria species have a coevolutionary relationship with their host species (Power et al., 2009; Vermeulen, 2004). In the present study however, the 18S rRNA sequence of E. gruis (GenBank accession number: AB544336) from a hooded crane (Grus monacha) was genetically more distant from E. haematodi (5.2% distance) than E. alabamensis from cattle (1.9% distance), even though both E. haematodi and E. gruis are parasites of birds. This supports a previous study, which reported that while the clustering of eimerian species is influenced by their host specificity, it does not arise from a co-phylogenetic/co-speciation process but from adaptive processes (Kvicˇerová and Hypša, 2013). This is the first molecular characterization of Eimeria species from psittacine hosts. Due to the absence of Eimeria sequences from psittacine hosts in GenBank, the current phylogenetic analysis may not accurately reflect the genetic relationship between E. haematodi and other Eimeria species. For example, E. haematodi exhibited the highest similarity (92.3%) at the COI locus with E. praecox from domestic chickens (Gallus gallus domesticus) from Canada (GenBank accession number: JQ65930) and China (GenBank accession number: HQ702483). As more sequences from psittacine hosts become
Please cite this article in press as: Rongchang Yang, Belinda Brice, Una Ryan, Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.005
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91, 85, 74 E. piriformis JQ993698 77, 84, _ E. flavescens JQ993692 84, 93, 82 E. exigua JQ993691 E.irresidua JQ993694 E. intestinalis JQ993693 96, 99, 97 E. magna KF419217 89, 91, _ E.coecicola JQ993690 Isospora sp. ex Apodemus flavicollis isolate B13 JQ993711 E. tiliquae JX839284 Isospora gryphoni KC346355 95, 98, 84 Isospora sp. 1 JRB-2013 KC346356 E. burdai JQ993709 100, 100, 100 E.nafuko JQ993708 100, 100, 100 E. sp. ex Alectoris chukar KF499078 E. sp. Alectoris graeca HM117020 E. pavonina JN596590 E. necatrix HQ702482 E. sp. Phasianus colchicus HM117019 E. adenoeides FR846201 74, 77, 81 E. paludosa KJ7671879 100, 100, 100 E. praecox JQ659301 E. praecox HQ702483 E. haematodi KM891727 E. dispersa HG793048 89,90, 91 E. innocua HG793049 E. acervulina HQ702479 100, 100, 100 E. acervulina FJ236428 E. acervulina FJ236443 E. brunetti HM771675 E. sp. ex Phataginus tricuspis JQ993697 E. cahirinensis isolate NFS JQ993686 E. sp. ex Apodemus flavicollis isolate 12 JQ993705 99, 99, 99 E. sp. ex Apodemus sylvaticus isolate 08-50 JQ993706 53,59, _ E. sp. ex Sorex araneus JQ993710 I. sp. ex Talpa europaea JQ993714 E. trichosuri JN192136 77, 87, 82 E. setonicis KF225638 E. macropodis JQ392578 75, 60, 70 100, 100, 100 E. macropodis JQ392579 E. cf. mivati FJ236441 E. mitis KC409031 92, 94, 92 E. mitis JN864949 100, 100, 99 E. mivati FJ236433 E. cf. tenggilingi JX464222 Toxoplasma gondii HM771690 0.1
1 2
Fig. 3. Evolutionary relationship of E. haematodi inferred by distance, ML and parsimony analysis of the COI gene (ML tree shown). Percentage support (>50%) from 1000 psudoreplicates from distance, ML and parsimony analysis respectively, is indicated at the left of the support node (‘_’ = Not available).
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
available in the GenBank, phylogenetic analysis will be able to provide more meaningful relationships. Acknowledgements The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife Rehabilitation Centre and Native ARC for their commitment and dedication in caring for all the animals admitted into their centers. 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. References
Q5
BirdLife International, 2012. “Trichoglossus rubiginosus”. IUCN Red List of Threatened Species. Version 2013.2. International Union for Conservation of Nature. (accessed 26.11.13). do Bomfim Lopes, B., Berto, B.P., de Carvalho Balthazar, L.M., Coelho, C.D., Neves, D.M., Lopes, C.W., 2014. Coccidia of New World psittaciform birds (Aves: Psittaciformes): Eimeria ararae n. sp. (Apicomplexa: Eimeriidae) from the blue-and-yellow macaw Ara ararauna (Linnaeus). Syst. Parasitol. 88, 175–180. Duszynski, D.W., Wilber, P.G., 1997. A guideline for the preparation of species descriptions in the Eimeriidae. J. Parasitol. 83, 333–336.
Duszynski, D.W., Couch, L., Upton, S.J., 2000. Coccidia of the world. . Farr, M.M., 1960. Eimeria dunsingi n.sp. (Protozoa: Eimeriidae) from the Intestine of a Parakeet, Melopsittacus undulatus (Shaw). Mexico, pp. 31–35. Sobretiro del Libro Homenaje al Dr Eduardo Caballero y Caballero. Friend, M., Franson, J.C., 2012. Field Manual of Wildlife Diseases - General Field Procedures and Diseases of Birds (Information and Technology Report). CreateSpace Independent Publishing Platform, p. 207. Gartrell, B.D., O’Donoghue, P., Raidal, S.R., 2000. Eimeria dunsingi in free living musk lorikeets (Glossopsitta concinna). Aust. Vet. J. 78, 717–718. Gottschalk, C., 1972. Beitrag zur Faunistik der Vogelkokzidien Thuringens und Sachsens. Beitr Vogelkd. 18, 61–69. Hofstatter, P.G., Guaraldo, A.M.A., 2011. A new eimerian species (Apicomplexa: Eimeriidae) from the blue-fronted Amazon parrot Amazona aestiva L. (Aves: Psittacidae) in Brazil. J. Parasitol. 97, 1140–1141. Hofstatter, P.G., Kawazoe, U., 2011. Two new Eimeria species (Apicomplexa: Eimeriidae) from the yellow-crowned Amazon Amazona ochrocephala (Aves: Psittacidae) in Brazil. J. Parasitol. 97, 503–505. Kvicˇerová, J., Hypša, V., 2013. Host-parasite incongruences in rodent Eimeria suggest significant role of adaptation rather than cophylogeny in maintenance of host specificity. PLoS ONE 8, e63601. Manley, V., 2012. Rainbow lorikeet population under control. SeienceNet WA. . McAllister, C.T., Upton, S.J., 1990. Description of the oocysts of Eimeria paludosa (Apicomplexa: Eimeriidae) from Fulica americana (Aves: Gruiformes), with comments on synonyms of eimerian species from related birds. J. Parasitol. 76, 27–29.
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Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
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Full length article
Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus) Q1 Rongchang Yang a,*, Belinda Brice b, Una Ryan a a b
School of Veterinary and Biomedical 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
• • •
Re-description of Eimeria haematodi in a rainbow lorikeet in Australia. Morphological study: Morphology and morphometry identical to E. haematodi. Phylogeny: 98.1% genetic similarity to E. alabamensis at the 18S locus.
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A R T I C L E
I N F O
Article history: Received 16 October 2014 Received in revised form 3 March 2015 Accepted 6 March 2015 Available online Keywords: Eimeria haematodi Molecular characterization 18S rRNA locus Mitochondrial cytochrome oxidase gene (COI) Phylogeny
G R A P H I C A L
A B S T R A C T
E. cahirinensis JQ993645 E. cahirinensis JQ993646 E. callospermophili JQ993648 E. chobotari AF324214 E. cahirinensis JQ993647 E. cahirinensis JQ993645 E. onychomysis AF307879 E. reedi AF311642 E. peromysci AF339492 E. chaetodipi AF339489 E. albigulae AF307880 E. arizonensis AF307878 E. antrozoi AF307876 E. catronensis AF324213 70, 75, 70 E. pilarensis AF324215 Isospora sp. 1 JM-2013 JX984669 99, 100, 99 Isospora robini AF080612 Isospora sp. Tokyo AB757862 58, 61, 58 Isospora sp. MS-2003 AY331571 Isospora gryphoni AF080613 E. vilasi JQ993653 100, 100, 99 E. sp. 2 JK-2013 JQ993652 E. praecox GQ421692 71, 78, 73 75, 73, _ E. maxima EU025110 100, 100, 99 E. acervulina FJ236372 E. mitis FR775307 96, 95, 89 E.a mivati EMU76748 Cyclospora colobi AF111186 E. sp. ex Apodemus agrarius JQ993656 E. alorani JQ993659 63, 53, _ E. sp. ex Apodemus sylvaticus JQ993661 E. sp. ex Apodemus agrarius JQ993655 E. sp. ex Mastomys natalensis JQ993667 E. myoxi JF304148 59, 61, 70 E. sp. ex Phataginus tricuspis JQ993651 E. sp. ex Gerbillus dasyurus JQ993664 E. chinchillae JQ993650 53, 53, _ 100, 100, 99 E. caviae JQ993649 80, 80, 84 E. nieschulzi U40263 E. sp. ex Gerbillus dasyurus JQ993664 E. haematodi KM884825 E. alabamensis AF291427 E. alabamensis AB769552 99, 99, 99 99, 100, 99 E. alabamensis AB769556 E.vejdovskyi HQ173838 97, 98, 91 E. vejdovskyi HQ173838 100, 100, 100 E. media HQ173834 57, 60, 57 E. perforans HQ173835 E. perforans EF694017 E. auburnensis AB769571 E. zuernii AB769665 99, 100, 100 94, 96, 90 E. bovis AB769589 Eimeria paludosa KJ767187 98, 98, 99 E. gruis AB544336 100, 100, 99 E. gruis AB544335 100, 99, 97 E. reichenowi AB544342 99, 99, 100 E. reichenowi AB544343 T. Gondii EF472967
A B S T R A C T
Eimeria haematodi was first described in 1977 from the rainbow lorikeet (Trichoglossus haematodus) in Papua New Guinea. In the present study, we re-describe this coccidian species morphologically and molecularly from a rainbow lorikeet bird in Western Australia (WA). The oocysts were ovoid to slightly piriform and measured 28.5–37.8 by 25.8–33.0 μm (33.3 by 28.1 μm). Oocyst wall was approximately 1.5 μm thick and bilayered. Micropyle (5–7 μm) and oocyst residuum (8.0–10.0 μm) present; polar granule was absent. Sporocysts ellipsoidal, 11.8–13.6 by 8.0–9.6 μm (12.2 by 8.3 μm), with thin convex Stieda body and granular sporocyst residuum (4.0–5.0 μm). Molecular characterization of E. haematodi was conducted at 18S ribosomal RNA and the mitochondrial cytochrome oxidase gene (COI) loci. At the 18S ribosomal RNA locus, E. haematodi shared 98.1% genetic similarity to E. alabamensis from cattle in New South Wales, Australia. At COI locus, E. haematodi was closest (92.3% similarity) to E. praecox from domestic chickens (Gallus gallus domesticus) from Canada and China. Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.
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* Corresponding author. Fax: +61 89310 4144. E-mail address: [email protected] (R. Yang). http://dx.doi.org/10.1016/j.exppara.2015.03.005 0014-4894/Crown Copyright © 2015 Published by Elsevier Inc. All rights reserved.
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1. Introduction Coccidia are a complex and diverse group of protozoan parasites with more than 1700 described species (Duszynski et al., 2000), most of which do not cause clinical disease. In birds, most diseasecausing or pathogenic forms of coccidia parasites belong to the genus Eimeria (Friend and Franson, 2012). Traditionally, identification of Eimeria species has relied not only on morphological characters but also on host species, pathology and geographic distribution (Duszynski and Wilber, 1997; Tenter et al., 2002). However, some species of Eimeria are morphologically identical and occur worldwide in several hosts, e.g. Eimeria paludosa has been reported from all continents of the world in a range of host species (Duszynski et al., 2000; McAllister and Upton, 1990; Yang et al., 2014b). It is now recognized that molecular data are essential to accurately delimit species and infer phylogenetic relationships among Eimeria species (Tenter et al., 2002). To date, eight species of Eimeria have been described from psittacine hosts; Eimeria dunsingi and Eimeria psittacina from the budgerigar (Melopsittacus undulates) (Farr, 1960; Gottschalk, 1972), Eimeria haematodi from the rainbow lorikeet (Trichoglossus haematodus) (Varghese, 1977), Eimeria aratinga from the orangefronted conure (Aratinga canicularis) (Upton and Wright, 1994), Eimeria amazonae and Eimeria ochrocephalae from yellow-crowned Amazons/parrots (Amazona ochrocephala) (Hofstatter and Kawazoe, 2011), Eimeria aestivae from the blue-fronted Amazon parrot Q2 (Amazona aestiva) (Hofstatter and Guaraldo, 2011) and Eimeria ararae from the blue-and-yellow macaw (Ara ararauna) (do Bomfim Lopes et al., 2014). In Australia, E. dunsingi has been identified in freeliving musk lorikeets (Glossopsitta concinna) (Gartrell et al., 2000) and E. psittacina has been described in the Australian Faunal DiQ3 rectory (http://bie.ala.org.au/species/urn:lsid:biodiversity.org.au:afd. taxon:9f3bd2a7-1e25-4d12-a595-944771349f20#tab_records, but no reference was found). No other Eimeria species in psittaciform hosts have been described from Australia. Eimeria haematodi was first described morphologically by Varghese (1977) from the rainbow lorikeet (Trichoglossus haematodus) in Papua New Guinea (PNG), but has not been reported since. In the present study, we characterized E. haematodi in a rainbow lorikeet (T. haematodus) from Western Australia (WA), both morphologically and phylogenetically. This is the first molecular characterization of Eimeria from psittaciform hosts. 2. Materials and methods 2.1. Sample collection A wild rainbow lorikeet (T. haematodus) came into care at the Native Animal Rescue Centre (ARC), Perth, WA after it had been struck by a motor vehicle. On admission it was noted that the bird was emaciated and the feathers were dirty. A fecal sample was taken 3 days later. Direct microscopic examination of a fecal suspension in saline, as well as fecal flotation analysis using a saturated sodium chloride and 50% sucrose (w/v) solution were applied to identify coccidian oocysts in the feces. The bird was negative for avian gastric yeast (AGY). Unfortunately, this bird died before antiprotozoal treatment was implemented. It is not known if this bird died as a result of the coccidian infection or from another cause. A further 49 fecal samples from another 49 wild rainbow lorikeets were analyzed over a 15 month period (June 2013–August 2014). These rainbow lorikeets were admitted to the Kanyana Wildlife Rehabilitation Centre in Perth, WA. None of the rainbow lorikeets tested in the present study was released back into the wild. If a sample was found to contain coccidian oocysts, a portion of feces was placed in 2% (w/v) potassium dichromate solution (K2Cr2 O7), 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. 2.2. DNA isolation Total DNA was extracted from 200 mg of fecal sample using a Power Soil DNA Kit (MolBio, Carlsbad, California) with some modifications as described by Yang et al. (2012). Briefly, the feces 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 fecal sample) was used in each extraction group. 2.3. PCR amplification and sequencing The PCR for the 18S rRNA locus was carried out using a heminested PCR as described by Yang et al. (2012) with the external primers: EiF1 5′-GCT TGT CTC AAA GAT TAA GCC-3′, EiR3 5′-ATG CAT ACT CAA AAG ATT ACC-3′ and internal primers: EiF3 5′-CTA TGG CTA ATA CAT GCG CAA TC-3′, and EiR3, which amplifies a 1320 bp product. The PCR reaction contained 2.5 μL of 10 × Kapa PCR buffer, 2 μL of 25 mM MgCl2, 1.5 μL of 10 nM dNTPs, 10 pM of each primer, 1 unit of KapaTaq (Geneworks, Adelaide, SA), 1 μL of DNA (about 50 ng) and 15.9 μL 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. Amplification of a 496 bp region of the COI locus from this sample was conducted as described by Ogedengbe et al. (2011) and Yang et al. (2013). The results of the sequencing reactions were analyzed and edited using FinchTV (version 1.4 – http://finchtv.software.informer.com/1.4/), compared to existing Eimeria spp. 18S rDNA and COI sequences in the GenBank database using BLAST searches and aligned with reference genotypes from the GenBank using the Clustal W algorithm in BioEdit (version 7.2.5 – http://www.mbio.ncsu.edu/bioedit/ bioedit.html). 2.4. Phylogenetic analysis Phylogenetic trees were constructed for Eimeria spp. at the 18S rRNA and COI loci with additional isolates from the GenBank database. Phylogenies were conducted using MEGA (Molecular Evolutionary Genetics Analysis software, version 6, Arizona State University, Tempe, Arizona, USA). Neighbor-joining (NJ) and maximum likelihood (ML) analyses were conducted 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. 3. Results 3.1. Description of E. haematodi Morphology: Oocysts are ovoid and some were piriform shaped with bilayered oocyst wall, 1.5 μm thick. Outer layer light brown, 1.0 μm thick and the inner layer colorless, about 0.5 μm thick. Forty oocysts measured 33.3 × 28.1 (28.5–37.8 × 25.8–33.0) μm in size with a length to width ratio of 1.19. Sporocysts are elongate– ovoid, 12.2 × 8.3 (11.8–13.6 × 8.0–9.6) μm, sporocyst L/W ratio, 1.47. Oocyst residuum round, granular, membrane-bound and 8–10 μm. Sporocyst residuum was present about 4.0–5.0 μm at the broader pole, Stieda body present, substieda body absent (Fig. 1 and Table 1).
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67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
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N/A N/A 20.6 × 10.1 Ovoidal Single polar granule, rounded Absent 1.55 43.8 × 27.7 Hofstatter and Guaraldo (2011)
1.19 Present study
33.3 × 28.1
N/A
Granular Absent 12.2 × 8.3 Spherical to ovoid Absent Present
Present 1.17 32.3 × 27.6
Ovoid to slightly piriform Ovoid to slightly piriform Ellipsoidal
Bi-layered
Absent 1.56 35.8 × 23.0 Ovoid
Bi-layered
Absent 1.35 35.0 × 25.9 Ellipsoidal
N/A
Absent 1.42 28.7 × 20.2 Ovoidal
N/A
Absent N/A 1.35 48.9 × 36.2 Ellipsoidal
Bi-layered
Granular Absent 13.3 × 8.4 Ellipsoidal
Granular Absent 11.4 × 8.5 Ovoid to pyriform
N/A N/A 17.0 × 8.3 Elongate–ovoidal
N/A N/A 17.0 × 8.3 Elongate–ovoidal
N/A N/A 22.2 × 11.9 Ellipsoidal
N/A N/A 19.8 × 9.3 Ovoidal
Single rounded polar granule Single rounded polar granule, Present, 2 to 4 granules Present and fragmented Single polar granule Absent Absent 1.55 36.8 × 23.7 Ovoidal
Wall Shape index
E. ochrocephalae
E. haematodi
E. haematodi
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A 1320 bp PCR product of 18S rDNA of E. haematodi was successfully amplified. Phylogenetic analyses of the nucleotide sequences from E. haematodi at the 18S rRNA locus using Distance (NJ), ML and Parsimony analyses produced essentially the same trees (Fig. 2, ML tree, bootstrap support (>50%) from NJ, ML and Parsimony trees are shown on each node). Unfortunately no psittaciform-derived sequences are available in the GenBank for the 18S rRNA locus. Eimeria haematodi grouped in a separate clade, but most closely
E. dunsingi
3.3. Phylogenetic analysis of E. haematodi at the 18S rRNA locus
E. aratinga
27 28 29 30 31 32 33 34 35 36
E. ararae
26
E. amazonae
Only 2 of the 11 coccidia positive birds in the present study were found to be underweight. No other clinical signs of coccidian infection were noted. All of the coccidia positive birds were treated with Toltrazuril (50 mg/mL) at a dose rate of 15 mg/kg SID for 3 days. None of these 11 rainbow lorikeets were positive for AGY.
Hofstatter and Guaraldo (2011) Hofstatter and Kawazoe (2011) do Bomfim Lopes et al. (2014) Upton and Wright (1994) Gartrell et al. (2000) Varghese (1977)
3.2. Clinical information
Blue-fronted parrot (Amazona aestiva) Yellow-crowned parrot (Amazona ochrocephala) Blue-fronted parrot (Amazona aestiva) Orange-fronted conure (Eupsittula canicularis) Musk lorikeet (Glossopsitta concinna) Rainbow lorikeet (PNG) (Trichoglossus haematodus) Rainbow lorikeet (WA) (Trichoglossus haematodus) Yellow-crowned parrot (Amazona ochrocephala)
19 20 21 22 23 24 25
E. aestivae
18
Size
Type hosts: rainbow lorikeet (Trichoglossus haematodus). Type locality: Perth, Western Australia. Other locality: Port Moresby (Papua) and Wau (New Guinea Highlands) in Papua New Guinea Prevalence: 43.3% (Varghese, 1977), 22.0% (11/50) (this study). Other hosts: Unknown. Prepatent period: Unknown. Patent period: Unknown. Site of infection: Unknown. Sporulation time: 48–96 hours. Material deposited: DNA sequences have been deposited in GenBank under accession numbers KM884825 and KM891727 for the 18S rRNA, and COI locus, respectively.
N/A
Polar granule Shape
5 6 7 8 9 10 11 12 13 14 15 16 17
References
4
Hosts
Fig. 1. (A) Nomarski interference-contrast photomicrographs of E. haematodi oocysts showing spheroidal to subspheroidal sporocysts (scale bar = 10 μm). (B) Line drawing of the sporulated oocyst of E. haematodi (scale bar = 10 μm).
3
Species
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Table 1 Comparative morphology of E. haematodi from rainbow lorikeets in Perth, WA with other species recorded from psittacine birds.
Oocyst Residuum
Sporocysts Oocysts
Shape
Size
Substieda body
Residuum
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E. cahirinensis JQ993645 E. cahirinensis JQ993646 E. callospermophili JQ993648 E. chobotari AF324214 E. cahirinensis JQ993647 E. cahirinensis JQ993645 E. onychomysis AF307879 E. reedi AF311642 E. peromysci AF339492 E. chaetodipi AF339489 E. albigulae AF307880 E. arizonensis AF307878 E. antrozoi AF307876 E. catronensis AF324213 70, 75, 70 E. pilarensis AF324215 99, 100, 99 Isospora sp. 1 JM-2013 JX984669 Isospora robini AF080612 Isospora sp. Tokyo AB757862 58, 61, 58 Isospora sp. MS-2003 AY331571 Isospora gryphoni AF080613 E. vilasi JQ993653 100, 100, 99 E. sp. 2 JK-2013 JQ993652 E. praecox GQ421692 71, 78, 73 75, 73, _ E. maxima EU025110 100, 100, 99 E. acervulina FJ236372 E. mitis FR775307 96, 95, 89 E. mivati EMU76748 Cyclospora colobi AF111186 E. sp. ex Apodemus agrarius JQ993656 E. alorani JQ993659 63, 53, _ E. sp. ex Apodemus sylvaticus JQ993661 E. sp. ex Apodemus agrarius JQ993655 E. sp. ex Mastomys natalensis JQ993667 59, 61, 70 E. myoxi JF304148 E. sp. ex Phataginus tricuspis JQ993651 E. sp. ex Gerbillus dasyurus JQ993664 E. chinchillae JQ993650 53, 53, _ 100, 100, 99 E. caviae JQ993649 80, 80, 84 E. nieschulzi U40263 E. sp. ex Gerbillus dasyurus JQ993664 E. haematodi KM884825 E. alabamensis AF291427 E. alabamensis AB769552 99, 99, 99 99, 100, 99 E. alabamensis AB769556 E.vejdovskyi HQ173838 97, 98, 91 E. vejdovskyi HQ173838 100, 100, 100 E. media HQ173834 57, 60, 57 E. perforans HQ173835 E. perforans EF694017 E. auburnensis AB769571 E. zuernii AB769665 99, 100, 100 94, 96, 90 E. bovis AB769589 Eimeria paludosa KJ767187 98, 98, 99 E. gruis AB544336 100, 100, 99 E. gruis AB544335 100, 99, 97 E. reichenowi AB544342 99, 99, 100 E. reichenowi AB544343 T. gondii EF472967
1 2 3 4
Fig. 2. Evolutionary relationship of E. haematodi inferred by distance, ML and parsimony analysis of the 18S rRNA gene (ML tree shown). Percentage support (>50%) from 1000 psudoreplicates from distance, ML and parsimony analysis, respectively, is indicated at the left of the support node (‘_’ = Not available).
5 6 7
(98.1% similarity) with E. alabamensis (GenBank accession number: AF291427) from cattle in New South Wales, Australia.
8 9 10 11 12 13 14 15 16
3.4. Phylogenetic analysis of E. haematodi at the COI locus A 496 bp PCR product of COI of E. haematodi was successfully amplified. There were fewer Eimeria COI sequences available in the GenBank database compared to the 18S rRNA locus and no psittaciform-derived sequences. Phylogenetic analysis grouped E. haematodi closest (92.3% similarity) with E. praecox from domestic chickens (Gallus gallus domesticus) from Canada (GenBank accession
number: JQ65930) and China (GenBank accession number: HQ702483) (Fig. 3).
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4. Discussion The rainbow lorikeet (T. haematodus) is a species of Australasian parrot found in Australia, eastern Indonesia (Maluku and Western New Guinea), Papua New Guinea, New Caledonia, Solomon Islands and Vanuatu (BirdLife International, 2012). In Australia, it is common along the eastern seaboard, from Queensland to South Australia and Tasmania. Its habitat is rainforest, coastal bush and woodland areas (Sampson and Day, 1999). The rainbow lorikeet was Q4 accidentally released into the southwest of the state of WA near the University of Western Australia as an ornamental bird in the 1960s and since then has been classified as a pest (Manley, 2012). Rainbow lorikeets are considered as a threat to WA’s agricultural stone fruit and viticulture industry and are also hosts for transferable parrot diseases such as psittacine beak and feather disease – a viral disease attacking the immune system causing beak abnormalities and loss of feathers (Manley, 2012). The prevalence of E. haematodi in wild rainbow lorikeets screened in the present study was 22.0% (11/50). This is lower than the prevalence of 43.3% previously reported by Varghese (1977). This could be due to the fact that some of the fecal samples (n = 18) were collected before midday, as previous studies have shown that coccidian shedding is much higher in the afternoon in birds (Morin-Adeline et al., 2011; Yang et al., 2014a). Compared with other Eimeria species from psittacine hosts (Table 1), the Eimeria oocysts isolated from the rainbow lorikeet in this study corresponded to E. haematodi, which was first described by Varghese (1977). For example, the oocysts shape was similar, as both were ovoid to slightly piriform in shape, oocyst and sporocyst measurements were also similar, the polar granule was absent in both and the sporocyst residuum was rounded and granular in both (Table 1). Phylogenetic analysis of 18S rRNA and COI sequences based on Distance, ML and Parsimony analyses produced similar results and placed E. haematodi in a separate clade but most closely related to E. alabamensis (98.1% similarity). The genetic similarity between E. haematodi and E. alabamensis is similar to the genetic differences between accepted species of Eimeria. For example, the genetic similarity between E. arnyi and E. ranae is 97.5% and the similarity between E. tenella and E. necatrix and between E. bovis and E. crandallis is 99.1% and 99.5%, respectively, across the same length of sequence. This supports the species status of E. haematodi. Previous studies have reported that Eimeria species have a coevolutionary relationship with their host species (Power et al., 2009; Vermeulen, 2004). In the present study however, the 18S rRNA sequence of E. gruis (GenBank accession number: AB544336) from a hooded crane (Grus monacha) was genetically more distant from E. haematodi (5.2% distance) than E. alabamensis from cattle (1.9% distance), even though both E. haematodi and E. gruis are parasites of birds. This supports a previous study, which reported that while the clustering of eimerian species is influenced by their host specificity, it does not arise from a co-phylogenetic/co-speciation process but from adaptive processes (Kvicˇerová and Hypša, 2013). This is the first molecular characterization of Eimeria species from psittacine hosts. Due to the absence of Eimeria sequences from psittacine hosts in GenBank, the current phylogenetic analysis may not accurately reflect the genetic relationship between E. haematodi and other Eimeria species. For example, E. haematodi exhibited the highest similarity (92.3%) at the COI locus with E. praecox from domestic chickens (Gallus gallus domesticus) from Canada (GenBank accession number: JQ65930) and China (GenBank accession number: HQ702483). As more sequences from psittacine hosts become
Please cite this article in press as: Rongchang Yang, Belinda Brice, Una Ryan, Morphological and molecular characterization of Eimeria haematodi, coccidian parasite (Apicomplexa: Eimeriidae) in a rainbow lorikeet (Trichoglossus haematodus), Experimental Parasitology (2015), doi: 10.1016/j.exppara.2015.03.005
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91, 85, 74 E. piriformis JQ993698 77, 84, _ E. flavescens JQ993692 84, 93, 82 E. exigua JQ993691 E.irresidua JQ993694 E. intestinalis JQ993693 96, 99, 97 E. magna KF419217 89, 91, _ E.coecicola JQ993690 Isospora sp. ex Apodemus flavicollis isolate B13 JQ993711 E. tiliquae JX839284 Isospora gryphoni KC346355 95, 98, 84 Isospora sp. 1 JRB-2013 KC346356 E. burdai JQ993709 100, 100, 100 E.nafuko JQ993708 100, 100, 100 E. sp. ex Alectoris chukar KF499078 E. sp. Alectoris graeca HM117020 E. pavonina JN596590 E. necatrix HQ702482 E. sp. Phasianus colchicus HM117019 E. adenoeides FR846201 74, 77, 81 E. paludosa KJ7671879 100, 100, 100 E. praecox JQ659301 E. praecox HQ702483 E. haematodi KM891727 E. dispersa HG793048 89,90, 91 E. innocua HG793049 E. acervulina HQ702479 100, 100, 100 E. acervulina FJ236428 E. acervulina FJ236443 E. brunetti HM771675 E. sp. ex Phataginus tricuspis JQ993697 E. cahirinensis isolate NFS JQ993686 E. sp. ex Apodemus flavicollis isolate 12 JQ993705 99, 99, 99 E. sp. ex Apodemus sylvaticus isolate 08-50 JQ993706 53,59, _ E. sp. ex Sorex araneus JQ993710 I. sp. ex Talpa europaea JQ993714 E. trichosuri JN192136 77, 87, 82 E. setonicis KF225638 E. macropodis JQ392578 75, 60, 70 100, 100, 100 E. macropodis JQ392579 E. cf. mivati FJ236441 E. mitis KC409031 92, 94, 92 E. mitis JN864949 100, 100, 99 E. mivati FJ236433 E. cf. tenggilingi JX464222 Toxoplasma gondii HM771690 0.1
1 2
Fig. 3. Evolutionary relationship of E. haematodi inferred by distance, ML and parsimony analysis of the COI gene (ML tree shown). Percentage support (>50%) from 1000 psudoreplicates from distance, ML and parsimony analysis respectively, is indicated at the left of the support node (‘_’ = Not available).
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available in the GenBank, phylogenetic analysis will be able to provide more meaningful relationships. Acknowledgements The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife Rehabilitation Centre and Native ARC for their commitment and dedication in caring for all the animals admitted into their centers. 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. References
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