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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.vetpar.2010.08.006 Zanguee, N., Lymbery, A., Lau, J., Suzuki, A., Yang, R., Ng, J. and Ryan, U. (2010) Identification of novel Cryptosporidium species in aquarium fish. Veterinary Parasitology, 174 (1-2). pp. 43-48. http://researchrepository.murdoch.edu.au/3349/ Copyright: © 2010 Elsevier B.V. It is posted here for your personal use. No further distribution is permitted. Accepted Manuscript Title: Identification of novel Cryptosporidium species in aquarium fish. Authors: N. Zanguee, J.A. Lymbery, J. Lau, A. Suzuki, R. Yang, J. Ng, U. Ryan PII: S0304-4017(10)00431-0 DOI: doi:10.1016/j.vetpar.2010.08.006 Reference: VETPAR 5418 To appear in: Veterinary Parasitology Received date: 1-6-2010 Revised date: 29-7-2010 Accepted date: 10-8-2010 Please cite this article as: Zanguee, N., Lymbery, J.A., Lau, J., Suzuki, A., Yang, R., Ng, J., Ryan, U., Identification of novel Cryptosporidium species in aquarium fish., Veterinary Parasitology (2010), doi:10.1016/j.vetpar.2010.08.006 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. *Manuscript 1 Identification of novel Cryptosporidium species in aquarium fish. 2 3 4 N. Zangueea, b, J A. Lymberyb, J. Lauc, A. Suzukic, R. Yangb, J. Ng, U. Ryanc*. 5 6 aDepartment of Fisheries, Faculty of Marine Natural Resources, Khoramshahr Marine 7 Science and Technology, Khoramshahr, Khouzestan, Iran. 8 bFish Health Unit, Centre for Fish and Fisheries Research, Murdoch University, Murdoch, 9 Western Australia, 6150. 10 cDivision of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western 11 Australia, 6150. 12 13 14 __________________________________ 15 *Corresponding author. Mailing address: Division of Health Sciences, School of Veterinary 16 and Biomedical Sciences, Murdoch University, Murdoch, Western Australia, Australia 6150. 17 Phone: 61 89360 2482. Fax: 61 89310 414. E-mail: [email protected] 18 Accepted Manuscript 1 Page 1 of 19 19 ABSTRACT 20 Little is known about the prevalence and genotypes of Cryptosporidium in fish. The 21 present study investigated the prevalence of Cryptosporidium species in 200 aquarium fish of 22 39 different species in Western Australia by PCR amplification at the 18S rRNA locus. A 23 total of twenty-one positives were detected by PCR (10.5% prevalence) from 13 different 24 species of fish. Nineteen of these isolates were successfully sequenced. Of these, 12 were 25 similar or identical to previously described species/genotypes of Cryptosporidium, while the 26 remaining seven isolates appeared to represent three novel species. 27 28 29 30 Keywords: Cryptosporidium, ornamental fish, 18S rRNA, new species. 31 32 Accepted Manuscript 2 Page 2 of 19 33 1. Introduction 34 Currently little is known about the epidemiology, taxonomy, pathology and host 35 specificity of Cryptosporidium species infecting piscine hosts. There are two recognised 36 species of Cryptosporidium in fish: Cryptosporidium molnari in gilthead sea bream (Sparus 37 aurata) and European sea bass (Dicentrarchus labrax) and Cryptosporidium scophthalmi in 38 turbot (Psetta maxima, syn. Scophthalmus maximus) (Alvarez-Pellitero and Sitja-Bobadilla 39 2002; Alvarez-Pellitero et al., 2004). Cryptosporidium molnari primarily infects the 40 epithelium of the stomach and seldom the intestine (Alvarez-Pellitero and Sitja-Bobadilla 41 2002), whereas C. scophthalmi mainly infects the epithelium of the intestine and very seldom 42 the stomach (Alvarez-Pellitero et al., 2004). Currently, genetic sequences are available in 43 GenBank for C. molnari (GenBank accession number HM243547) but not C. scophthalmi. To 44 date only three additional studies have generated genetic sequences from piscine-derived 45 Cryptosporidium spp; an isolate from a guppy (Poecilia reticulate) (hereafter referred to as 46 piscine genotype 1) (Ryan et al., 2004), a freshwater angelfish (Pterophyllum scalare) 47 (hereafter referred to piscine genotype 2) (Murphy et al., 2009) and more recently C. parvum, 48 C. xiaoi and pig genotype II were identified in whiting (Sillago vittata) (Reid et al., 2010) and 49 a novel Cryptosporidium spp. was identified in sea mullet (Mugil cephalus) (hereafter 50 referred to as piscine genotype 3) (Reid et al., 2010). 51 The aim of the present study was to determine the prevalence of different species of 52 Cryptosporidium in ornamental fish in Western Australia (WA). 53 Accepted Manuscript 3 Page 3 of 19 54 2. Materials and methods 55 56 2.1. Sample collection 57 58 A total of 200 ornamental fish from 39 different species (see Table 1) were collected 59 from local aquariums and pet shops in metropolitan WA. These fish included both marine and 60 freshwater, and tropical and temperate species. On arrival in the laboratory, fish were 61 measured for length and weight and dissected. Stomach and intestinal epithelial cells were 62 scraped off using a scalpel blade and placed into a 1.5 mL eppendorf tube. Remaining 63 stomach and intestinal tissue were stored separately in 10% formalin. 64 65 2.2 DNA Extraction and PCR amplification 66 67 DNA was extracted from ~ 250 mg of pooled intestinal and stomach tissue scrapings 68 from each fish sample using a Qiagen DNeasy tissue kit (Qiagen, Germany). DNA was eluted 69 in 50 µL of AE buffer to concentrate the DNA. All extracted DNA samples were stored at - 70 20˚C until required for screening. 71 All samples were screened at the 18S rRNA locus and positives were genotyped by 72 sequencing. A two-step nested PCR protocol was used to amplify the 18S rDNA gene of 73 Cryptosporidium as previously described (Ryan et al., 2003). For all isolates that were 74 positive at the 18SAccepted locus by PCR, attempts were also Manuscript made to amplify the actin locus as 75 previously described (Ng et al., 2006). PCR contamination controls were used including 76 negative controls and separation of preparation and amplification areas. The amplified DNA 77 fragments from the secondary PCR products were separated by gel electrophoresis and 78 purified using the freeze-squeeze method (Ng et al., 2006). A spike analysis (addition of 4 Page 4 of 19 79 0.5µL of Cryptosporidium positive control into each sample) was conducted on randomly 80 selected Cryptosporidium negative samples from each group of DNA extractions to determine 81 if negative results were due to PCR inhibition. 82 83 2.3. Sequence and phylogenetic analysis 84 85 Purified PCR products were sequenced using an ABI PrismTM Dye Terminator cycle 86 sequencing kit (Applied Biosystems, Foster City, California) according to the manufacturer’s 87 instructions with the exception that the annealing temperature was raised to 58ºC. Nucleotide 88 sequences were analyzed using Chromas lite version 2.0 (http://www.technelysium.com.au) 89 and aligned with reference genotypes from GenBank using Clustal W 90 (http://www.clustalw.genome.jp). 91 Phylogenetic trees were constructed using additional isolates from GenBank. Distance 92 estimation was conducted using TREECON (Van de Peer and De Wachter, 1994), based on 93 evolutionary distances calculated with the Kimura’s distance and grouped using Neighbour- 94 Joining. Parsimony analyses were conducted using MEGA version 3.1 (MEGA3.1: Molecular 95 Evolutionary Genetics Analysis software, Arizona State University, Tempe, Arizona, USA). 96 Bootstrap analyses were conducted using 1,000 replicates to assess the reliability of inferred 97 tree topologies. Maximum Likelihood (ML) analyses were conducted using the program 98 PhyML (Dereeper et al., 2008) and the reliability of the inferred trees was assessed by the 99 approximate likelihoodAccepted ratio test (aLRT) (Anisimova Manuscript and Gascuel, 2006). 100 5 Page 5 of 19 101 2.4. Calculation of prevalences 102 103 Prevalences were expressed as percentage of positive samples, with 95% confidence 104 intervals calculated assuming a binomial distribution, using the software Quantitative 105 Parasitology 3.0 (Rozsa et al., 2000). 106 107 108 3. Results 109 110 3.1. Prevalence of Cryptosporidium in fish hosts 111 112 Of the 200 fish sampled during this study from 39 different species, twenty-one 113 positives were detected in 13 different species by PCR (10.5% prevalence, with 95% CI 6.3- 114 14.7) (Table 1). Infected hosts were angelfish (n=1), butter-bream (n=2), dispar anthias (n=1), 115 false gramma (n=1), golden algae eater (n=3), green chromis (n=2), guppy (n=1), kupang 116 damsel (n=1), madder seaperch (1), neon tetra (n=2), oscar (n=4), upside down cat fish (n=1), 117 a bristle tooth tang (n = 1) and a wedgetailed blue tang (n=1). No PCR inhibition was 118 detected. 119 120 3.2. Identification of Cryptosporidium species in fish 121 Accepted Manuscript 122 Partial sequences were obtained for nineteen of the twenty-one positive isolates at the 123 18S rRNA locus. Unfortunately no isolates were successfully amplified at the actin locus 124 despite numerous attempts. Neighbour-joining, parsimony and ML analysis of the 18S rRNA 125 sequences from these 19 isolates and a range of Cryptosporidium species and genotypes 6 Page 6 of 19 126 obtained from GenBank produced similar results (Figure 1-ML tree shown). Twelve of the 19 127 isolates were similar or identical to previously described species/genotypes of 128 Cryptosporidium. Isolate 101 from a neon tetra was 100% identical to piscine genotype 1 129 (GenBank accession no.

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