MURDOCH RESEARCH REPOSITORY This is the author s final version of the work, as accepted for publication following peer review but without the publisher s layout or pagination. The definitive version is available at http://dx.doi.org/10.1016/j.exppara.2012.11.004 Yang, R., Brice, B., Bennett, M.D., Elliot, A.D. and Ryan, U. (2013) Novel Eimeria sp. isolated from a King s skink (Egernia kingii) in Western Australia. Experimental Parasitology, 133 (2). pp. 162-165 http://researchrepository.murdoch.edu.au/12553/ Copyright: 2012 Elsevier Inc. It is posted here for your personal use. No further distribution is permitted.
Accepted Manuscript Research brief Novel Eimeria sp. isolated from a King s skink (Egernia kingii) in Western Australia Rongchang Yang, Belinda Brice, Mark D. Bennett, Aileen Eliott, Una Ryan PII: S0014-4894(12)00333-5 DOI: http://dx.doi.org/10.1016/j.exppara.2012.11.004 Reference: YEXPR 6550 To appear in: Experimental Parasitology Received Date: 18 September 2012 Revised Date: 5 November 2012 Accepted Date: 7 November 2012 Please cite this article as: Yang, R., Brice, B., Bennett, M.D., Eliott, A., Ryan, U., Novel Eimeria sp. isolated from a King s skink (Egernia kingii) in Western Australia, Experimental Parasitology (2012), doi: http://dx.doi.org/ 10.1016/j.exppara.2012.11.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Research Brief 2 3 4 Novel Eimeria sp. isolated from a King s skink (Egernia kingii) in Western Australia. 5 6 Rongchang Yang a, Belinda Brice b, Mark D. Bennett a, Aileen Eliott a and Una Ryan a* 7 8 9 10 11 a School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia, 6150. b Kanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western Australia 6076. 12 13 14 15 16 17 *Corresponding author. Mailing address: Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia, Australia, 6150. Phone: 61 89360 2482. Fax: 61 89310 4144. E-mail: Una.Ryan@murdoch.edu.au 18
19 20 21 22 23 24 25 26 27 28 29 ABSTRACT A novel Eimeria species was identified in faeces collected from a King's skink (Egernia kingii) housed at the Kanyana Wildlife Rehabilitation Centre in Western Australia. Oocysts measure 17.0 15.0 µm with a length/width ratio (L/W) of 1.13. Phylogenetic analysis of 18S rrna sequences indicated that the novel Eimeria sp. shared the highest genetic similarity to Eimeria antrozoi and Eimeria rioarribaensis from vespertilionid bats from North America ( 98.9%). At the COI locus, bat-derived sequences were not available and phylogenetic analysis placed the novel Eimeria sp. in a clade by itself and shared 98.8% similarity with the rodent-derived species E. falciformis and E. vermiformis. This suggests that the isolate from the King s skink s feces was probably derived from a mammal, possibly a rodent or a bat. 30 31 32 33 34 Keywords: Eimeria; King s skink; morphology; genetic characterization; 18S rrna; mitochrondial cytochrome oxidase gene (COI); phylogeny. 35 2
36 37 38 39 40 41 42 43 44 45 46 1. Introduction Skinks are lizards belonging to the family Scincidae. The King's Skink (Egernia kingii) is a species of skink native to coastal regions of south-western Australia. It is a large, heavy-bodied black skink that can reach a length of 55 centimetres with a mass of up to 220 grams (Arena and Wooller, 2003). More than 17 named species and numerous un-named species of Eimeria have been described in skinks (Duszynski et al., 2000), however relatively little is known about their life cycles, biology and genetic diversity. In the present study, we characterized a novel species of Eimeria from the King s skink, housed at the Kanyana Wildlife Rehabilitation Centre (KWRC) in Western Australia, both morphologically and genetically. 47 48 2. Materials and methods 49 50 51 52 53 54 2.1 Sample collection Two King's skinks were admitted to the KWRC in Perth, Western Australia between January and August 2012 and a faecal sample was obtained from each skink under the KWRC permit. Samples were collected into sterile containers, labeled and screened for coccidean parasites as described below. 55 56 57 58 59 60 2.2 Morphological analysis Microscopic examination of a wet mount and faecal flotation analysis were performed on both samples. Faecal flotation was done using a saturated sodium chloride and 50% sucrose (w/v) solution. If any sample was found to contain coccidean oocysts, a portion of faeces was placed in 2% (w/v) potassium dichromate 3
61 62 63 64 65 66 67 solution (K 2 Cr 2 O 7 ), mixed well and poured into petri dishes to a depth of less than 1cm and kept at room temperature in the dark to facilitate sporulation. Sporulated oocysts were observed using the oil immersion objective of an Olympus BX51 microscope. Images were captured and measurements made under 0 magnification using an Olympus DP70 digital camera and associated imaging software. Results are presented in micrometers as the mean ± SD, with the observed range in parentheses. 68 69 70 71 72 2.3 DNA isolation Total DNA was extracted from 200mg of each faecal sample using a QIAamp DNA Mini Stool Kit (Qiagen, Hilden, Germany). A negative control (no faecal sample) was included. 73 74 75 76 77 78 79 80 81 82 83 84 2.4 PCR amplification and sequencing Samples were screened at the 18S rrna locus for Eimeria spp. using primers and conditions described by Yang et al., (2012). Amplification at the mitochrondial cytochrome oxidase gene (COI) locus was initially attempted using methods described by Ogedengbe et al., (2011). However as these primers resulted in nonspecific amplification (as determined by sequencing of amplicons), the following Eimeria-specific internal primers were designed using Primer 3 (http://frodo.wi.mit.edu/); COIF2 TAA GTA CAT CCC TAA TGT C and COIR2 GTCATCATATGRTGTGCCCA. The resulting nested PCR reaction amplifies a 465 bp fragment of COI gene and the PCR conditions used were the same as for the external reaction. The specificity of the primers was tested against Isospora ohioensis 4
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 (2 isolates), Giardia duodenalis (2 isolates), Cyclospora sp. (2 isolates), human genomic DNA (Promega), sheep and cattle DNA. PCR contamination controls were used including negative controls and separation of preparation and amplification areas. A spike analysis (addition of 0.5 µl of positive control DNA from Eimeria crandallis into each sample) was conducted on both samples to determine if there was any PCR inhibition present. The amplified DNA fragments from the secondary PCR products were separated by gel electrophoresis and purified using the freeze-squeeze method (Ng et al., 2006). Gel-purified PCR products were sequenced in both directions, using an ABI Prism TM Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, California) according to the manufacturer s instructions with the exception that the annealing temperature was raised to 58 ºC. The results of the sequencing reactions were analysed and edited using Chromas lite version 2.0 (http://www.technelysium.com.au), compared to existing Eimeria spp. 18S rdna and COI sequences on GenBank using BLAST searches and aligned with reference genotypes from GenBank using Clustal W (http://www.clustalw.genome.jp). 101 102 103 104 105 106 107 108 109 2.5 Phylogenetic analysis Phylogenetic trees were constructed for Eimeria spp. at the 18S and COI loci with additional isolates from GenBank. 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 5.1 (MEGA5.1: Molecular Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA). Bootstrap analyses were conducted using 1,000 replicates to assess the 5
110 111 112 113 reliability of inferred tree topologies. Maximum Likelihood (ML) analyses were conducted using the program 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). 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 3. Results 3.1 Microscopy One of the two King s skinks examined was positive for Eimeria sp. Other parasites identified in the faecal samples of the Eimeria-positive skink included Entamoeba cysts, Giardia cysts, embryonated nematode eggs (Physaloptera sp.) and cestode (Rodentolepis) eggs. Sporulated Eimeria oocysts (n=39) were subspherical, with colorless to yellowbrown trilaminate oocyst wall 0.8±0.04 (0.73-0.89) thick. Four spheroidal to subspheroidal sporocysts, occasionally with polar granule, but without micropyle or oocyst residuum (Fig. 1). Oocyst length 17.0±0.41 (16.2-17.8); oocyst width 15.0±0.38 (14.4-15.7); oocyst length/width (L/W) ratio 1.13±0.02 (1.07-1.16). Sporocysts with globular sporocyst residuum and 2 sporozoites. Stieda, parastieda and substieda bodies not detected. Sporocyst length was 7.0±0.37 (6.3-7.8); sporocyst width 6.2±0.46 (5.0-7.0), sporocyst L/W ratio 1.14±0.08 (1.00-1.40). 129 130 131 132 133 134 3.2 Phylogenetic analysis of Eimeria sp. from the King s skink at the 18S locus A partial 18S sequence (1,300 bp) was obtained from the Eimeria-positive King s skink. Phylogenetic analyses using Distance, Parsimony and ML analyses produced similar results (Fig. 2 NJ tree shown). The Eimeria sp. grouped in a separate clade and shared the highest genetic similarity to Eimeria antrozoi (98.98%) and 6
135 136 137 138 Eimeria rioarribaensis from vespertilionid bats from North America (98.9%) (Duszynski et al., 1999). The partial 18S rrna nucleotide sequences from Eimeria sp. from the King s skink was deposited in GenBank with the accession number of JX 839286. 139 140 141 142 143 144 145 146 3.3 Phylogenetic analysis of Eimeria sp. from the King s skink at the COI locus At the COI locus, bat-derived sequences were not available and phylogenetic analysis placed the novel Eimeria sp. in a clade by itself and shared 98.8% similarity with the rodent-derived species E. falciformis and E. vermiformis (Fig. 3). The partial COI nucleotide sequences and translated amino acid sequence from Eimeria sp. from the King s skink were deposited in GenBank with the accession number of JX 839285. 147 148 149 150 4. Discussion 151 152 153 154 155 156 157 158 159 160 In the present study, sporulated Eimeria oocysts identified in the faeces of a King s skink, measured 17.0 15.0 with a L/W ratio of 1.13. However, the morphological similarity of oocysts, the broad host specificity of some Eimeria spp. and the diversity of Eimeria spp. within one host confound species delimitation (Tenter et al., 2002). Molecular data are therefore essential to accurately delimit species. Phylogenetic analysis at both the 18S and COI loci suggest that the isolate from the King s skink s feces was derived from a mammal, possibly a rodent or a bat. At the 18S locus, the skink isolate was most closely related to E. antrozoi and E. rioarribaensis, which are both from bats. Eimeria antrozoi oocysts are subspheroidal 7
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 and measure 24.8 21.6 (22-27 19-24) µm with a L/W ratio of 1.15 (1.0-1.3) (Duszynski et. al., 1999). The micropyle is absent but an oocyst residuum is present. Eimeria rioarribaensis oocysts measure 24.9 20.1 µm with a L/W ratio of 1.2 and both the micropyle and the oocyst residuum are absent. At the COI locus, the isolate was most closely related to E. falciformis and E. vermiformis from rodents. Oocysts of E. falciformis are spherical to ovoid and measure 14-27 11-24 µm and the sporocysts also spherical to ovoid, and measure 10-12 6-8 µm (Levine and Ivens, 1990). Although descriptions for E. falciformis resemble those of the current isolate, the reported range of sporocyst lengths (SLs) for E. falciformis exceeds that of all the oocysts examined from the King s skink sample. Oocysts of E. vermiformis measure 18-26 15-21 µm, with sporocysts 11-14 6-10 µm (Levine and Ivens, 1990). Again, the SL range of E. vermiformis exceeds that of the current isolate. The data suggest that the skink acquired the isolate through eating feces or intestinal material from an unknown mammalian host, possibly a rodent or bat. During the five days that the skink was held at KWRC, it was fed a diet of mealworms and beef mince. The King's skink is however also known to occasionally eat carrion (Wilson, 2012). As the skink was in care for only a few days it is likely that it ingested the parasite before it was admitted to the KWRC. Whether the skink was actually infected or simply passing oocysts is unknown. In the present study, molecular data were used to describe a novel Eimeria sp. found in the faeces of the King s skink in Western Australia. This is also the first study to design Eimeria-specific primers for the COI locus as the primers published by Ogedengbe et al., (2011) resulted in non-specific amplification when applied to total DNA extracted from faecal samples. This limits their usefulness as a screening tool. The COI internal primers described in the present study were shown to be very 8
186 187 188 189 190 191 192 193 194 195 specific and did not amplify other enteric parasites, human, sheep and cattle DNA. Studies comparing the utility of the 18S and COI genes indicate the latter has higher resolving power for Eimeria sp., especially with respect to recent speciation events (Ogedengbe et al., 2011). COI has become the target gene for the Barcode of Life project that aims to use the marker for rapid identification of animals, including parasites (Ratnasingham and Hebert, 2007). Future studies need to concentrate on obtaining morphologically characterized Eimeria species derived from lizard hosts and generating sequence data that are directly related to described species. Analyzing the isolates at multiple loci will also provide a more in-depth analysis of the evolution of lizard-derived Eimeria spp. 196 197 Acknowledgements 198 199 200 201 The authors wish to thank June Butcher and the volunteers at the Kanyana Wildlife Rehabilitation Centre for their dedication in caring for all the animals admitted to the centre. 202 9
203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 References Anisimova, M., Gascuel, O., 2006. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst. Biol. 55, 539-552. Arena, P.C., Wooller, R.D. 2003. The reproduction and diet of Egernia kingii (Reptilia : Scincidae) on Penguin Island, Western Australia. Aust. J. Zool. 51, 495-504. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J. F., Guindon, S., Lefort, V., Lescot, M., Claverie, J. M. and Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucl. Acids Res. 36, W465-469. Duszynski, D.W., Scott, D.T., Aragon, J., Leach, A., Perry, T., 1999. Six new Eimeria species from vespertilionid bats of North America. J. Parasitol. 85, 496-503. Duszynski, D.W., Couch, L., Upton, S.J., 2000. Coccidia of the world. Available at: http://biology.unm.edu/biology/coccidia/home.html Levine, N. D., Ivens, V., 1990. The coccidian parasites of rodents. CRC Press, Boca Raton, Florida, pp. 228. Ng, J., Pavlasek, I., Ryan, U., 2006. Identification of novel Cryptosporidium genotypes from avian hosts. Appl. Environ. Microbiol. 72, 7548-7553. Ogedengbe, J.D., Hanner, R.H., Barta, J.R., 2011. DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata). Int. J. Parasitol. 41, 843 850. Ratnasingham, S., Hebert, P.D., (2007). BOLD: the barcode of life data system. (http://www. barcodinglife.org). Mol. Ecol. Notes 7, 355 64. 10
227 228 229 230 231 232 233 234 235 236 237 Tenter, A.M., Barta, J.R., Beveridge, I., Duszynski, D.W., Mehlhorn, H., Morrison, D.A., Thompson, R.C, Conrad, P.A., 2002. The conceptual basis for a new classification of the coccidia. Int. J. Parasitol. 32, 595-616. Van de Peer, Y., R., De Wachter., 1994. TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comp. Appl. Biosci. 10, 569 570. Wilson, S., 2012. Australian lizards: a natural history. CSIRO Publishing, 150 Oxford Street, Collingwood, Victoria, 3066, Australia. Yang, R., Fenwick, S., Potter, A., Elliot, A., Power, M., Beveridge, I., Ryan, U., 2012. Molecular characterisation of Eimeria species in Macropods. Exp. Parasitol. 132, 216-21. 238 11
239 240 241 Fig. 1. Nomarski interference-contrast photomicrographs of Eimeria sp. from King s skink (Egernia kingii). Scale bar = 10 µm. 242 243 244 245 246 Fig. 2. Evolutionary relationships of Eimeria sp. from King s skink (Egernia kingii) inferred by distance analysis of 18S rrna sequences. Percentage support (>50%) from 0 pseudoreplicates from neighbor-joining analyses is indicated at the left of the supported node. 247 248 249 250 251 Fig. 3. Evolutionary relationships of Eimeria sp. from King s skink (Egernia kingii) inferred by distance analysis of mitochrondial cytochrome oxidase gene (COI). Percentage support (>50%) from 0 pseudoreplicates from neighbor-joining analyses is indicated at the left of the supported node. 252 253 12
254 Research Highlights 255 256 257 258 259 Morphological characterization of a novel Eimeria Characterisation at 18SrRNA locus Characterisation at the mitochrondial cytochrome oxidase gene (COI) First study to design Eimeria-specific primers to the COI locus 260 261 13
Figure
Figure 0.1 65 53 T. gondii EF472967 99 98 62 54 99 72 54 86 52 E.arizonensis AF3077878 E.reedi AF311642 E.rioarribaensis AF307878 98 E.antrozoi AF307876 92 61 68 E.media HQ173834 E.bovis U77084 King s skink JX839286 E. trichosuri FJ829320 E.magna HQ173833 E.peromysci AF339492 E.albigulae AF307880 83 99 E.perforans HQ173835 E.chobotari AF324214 E.tropidura FJ829323 E.mivati FJ236374 E.chaetodipi AF339489 E.telekii AF246717 E.acervulina EF210324 E.weybridgensis AY028972 E.crandallis AF336339 E.scholtysecki AF324216 E.falciformis AF080614 E.nieschulzi U40263 E.arnyi AY613853 E.necatrix DQ136185 E.tenella DQ136179 E.mitis FR775306 E.brunetti U67116 E.maxima FJ236333 E.praecox FJ236365 E.separata JF419348 E. macropodis JQ392575 E. macropodis JQ392576 E. macropodis JQ392574 E.reichenowi AB544329 E.gruis AB243082 E.ranae EU717219
Figure 0.1 E. mivati EF174185 E. mivati FJ236433 E. mivati HM771681 E. acervulina FJ236419 83 E. acervulina FJ236443 E. acervulina HM771674 82 E. brunetti HM771675 E. praecox HQ702483 E. sp. Alectoris graeca HM117020 E. necatrix HQ702482 95 E. necatrix HM771680 E. necatrix EU025108 68 E. tenella EF174186 85 E. tenella HM771679.E. sp. Meleagris gallopavo HM117018 78 Eimeria sp. Phasianus colchicus HM117019 75 E. sp. Meleagris gallopavo HM117017 E. adenoeides FR846202 E. pavonina JN596590 King s skink JX839285 95 E. falciformis HM771682 E. vermiformis HM771683 E. macropodis JQ392579 E. trichosuri JN192136 Neospora caninum HM771688
*Graphical Abstract (for review) 0.1 E. mivati EF174185 E. mivati FJ236433 E. mivati HM771681 E. acervulina FJ236419 83 E. acervulina FJ236443 E. acervulina HM771674 82 E. brunetti HM771675 E. praecox HQ702483 E. sp. Alectoris graeca HM117020 E. necatrix HQ702482 95 E. necatrix HM771680 E. necatrix EU025108 68 E. tenella EF174186 85 E. tenella HM771679 Eimeria sp. Meleagris gallopavo HM117018 78 75 Eimeria sp. Phasianus colchicus HM117019 E. sp. Meleagris gallopavo HM117017 E. adenoeides FR846202 E. pavonina JN596590 King s skink JX839285 95 E. falciformis HM771682 E. vermiformis HM771683 E. macropodis JQ392579 E. trichosuri JN192136 Neospora caninum HM771688