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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

Gibson-Kueh, S., Yang, R., Thuy, N.T.N., Jones, J.B., Nicholls,

P.K. and Ryan, U. (2011) The molecular characterization of an

Eimeria and Cryptosporidium detected in Asian seabass (Lates

calcarifer) cultured in Vietnam. Veterinary Parasitology, 181

(2-4). pp. 91-96.

Copyright: © 2011 Elsevier B.V.

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Accepted Manuscript

Title: The molecular characterization of anEimeriaand

Cryptosporidiumdetected in Asian seabass (Lates calcarifer) cultured in Vietnam

Authors: S. Gibson-Kueh, R. Yang, N.T.N. Thuy, J.B. Jones, P.K. Nicholls, U. Ryan

PII: S0304-4017(11)00307-4

DOI: doi:10.1016/j.vetpar.2011.05.004

Reference: VETPAR 5851

To appear in: Veterinary Parasitology

Received date: 4-2-2011 Revised date: 2-5-2011 Accepted date: 3-5-2011

Please cite this article as: Gibson-Kueh, S., Yang, R., Thuy, N.T.N., Jones, J.B., Nicholls, P.K., Ryan, U., The molecular characterization of an Eimeria and

Cryptosporidium detected in Asian seabass (Lates calcarifer) cultured in Vietnam,

Veterinary Parasitology(2010), doi:10.1016/j.vetpar.2011.05.004

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.

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Accepted Manuscript

The molecular characterization of an Eimeria and Cryptosporidium detected in Asian 1

seabass (Lates calcarifer) cultured in Vietnam 2

3

S. Gibson-Kueh a*, R. Yang a, N.T.N. Thuy b, J.B. Jones c, P.K. Nicholls a, U. Ryana 4

5 a

School of Veterinary and Biomedical Sciences, Murdoch University, South St., Murdoch, 6

Western Australia, 6150 Australia 7

b

Minh Hai Sub-Institute for Fisheries Research, 21-24 Phan Ngoc Hien, Group 3, Ward 6, Ca 8

Mau City, Ca Mau Province, Vietnam 9

c

Fish Health Laboratory, Department of Fisheries, 3 Baron-Hay Court, South Perth, Western 10 Australia, 6151, Australia 11 12 13 14 15 16 __________________________________ 17

*Corresponding author. Mailing address: School of Veterinary and Biomedical Sciences, 18

Murdoch University, South St., Murdoch, Western Australia, 6150 Australia. E-mail: 19

S.Kueh@murdoch.edu.au 20

21

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ABSTRACT

22

An intestinal Eimeria was previously reported as a significant pathogen of Asian seabass 23

(Lates calcarifer) in nurseries in Vietnam. In the present study, both Eimeria and 24

Cryptosporidium were detected by sequence analyses of fragments of the 18S rRNA gene 25

amplified from these Vietnamese L. calcarifer tissues. Based on these analyses, the Eimeria 26

from the Vietnamese L. calcarifer formed clades with the Eimeria detected in L. calcarifer 27

tissues from Australia, but clustered separately from other known Eimeria and Goussia 28

species. The Cryptosporidium detected in L. calcarifer from Vietnam clustered closest with 29

C. parvum and C. hominis. In situ hybridization using DIG-labeled DNA probes generated 30

from 18S PCR products on the Vietnamese L. calcarifer wax block tissues showed that this 31

method could not be used to distinguish between Eimeria and Cryptosporidium, due to the 32

conserved nature of the 18S locus. A previously published study on the morphology of 33

parasite developmental stages and oocysts in the Vietnamese L. calcarifer tissues showed 34

only an intestinal Eimeria infection. The Cryptosporidium could be present at very low levels 35

undetectable by microscopy in intestines, or being ubiquitous, was a possible contaminant 36

from feed or water. While molecular analysis is a very useful tool in the study of disease and 37

identification of aetiological agents, this study reiterates the importance of demonstrating 38

organisms in situ in tissues. 39

40 41 42

Keywords: Lates calcarifer, Eimeria, Cryptosporidium, 18S rRNA genotyping, in situ 43

hybridization 44

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1.Introduction

46

Cryptosporidium is an apicomplexan parasite that is now recognized as being more 47

closely related to gregarines than to coccidia (Barta and Thompson, 2006). Currently, there 48

are two recognised species of Cryptosporidium in fish: Cryptosporidium molnari in gilthead 49

sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and 50

Cryptosporidium scophthalmi in turbot (Psetta maxima, syn. Scophthalmus maximus) 51

(Alvarez-Pellitero and Sitja-Bobadilla 2002; Alvarez-Pellitero et al., 2004). Genetic 52

sequences are however only available for C. molnari (GenBank accession number 53

HM243547) (Palenzuela et al., 2010). A total of 9 additional species/genotypes have been 54

identified in fish using molecular tools: piscine genotype 1 from a guppy (Poecilia reticulata) 55

(Ryan et al., 2004), piscine genotype 2 from a freshwater angelfish (Pterophyllum scalare) 56

(Murphy et al., 2009), piscine genotype 3 from sea mullets (Mugil cephalus) (Reid et al., 57

2010), piscine genotypes 4-6 from ornamental fish (Zanguee et al. 2010) and C. parvum, C. 58

xiaoi and pig genotype II in whiting (Sillago vittata) (Reid et al., 2010). 59

A great variety of coccidian parasites belonging to the genera Eimeria, Epieimeria and 60

Goussia in the suborder Eimeriorina have been reported in fish (Upton et al., 1984; Paperna, 61

1991; Davies and Ball, 1993; Landsberg, 1993; Costa and MacKenzie, 1994; Sitja-Bobadilla 62

et al., 1996; Molnar and Ogawa, 2000; Molnar et al., 2004; Molnar, 2006). However, no 63

genetic sequences for piscine-derived Eimeria are available. An intestinal Eimeria was 64

previously reported as a significant pathogen of Asian seabass (Lates calcarifer) in nurseries 65

in Vietnam (Gibson-Kueh et al., 2011). 66

In the present study, analysis of partial 18S rRNA gene sequence detected in tissues of 67

these nursery reared L. calcarifer from Vietnam identified novel species of Eimeria and 68

Cryptosporidium. These are the first piscine-derived Eimeria partial 18S gene sequences to be 69

published. 70

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71

2. Materials and methods 72

2.1 General 73

Fixed tissues from 211 fish were originally sampled from five nurseries in Ca Mau, 74

Vietnam between 2008 and 2010. Of these samples, 181 fish were previously examined by 75

histopathology while 10 fish were examined by electron microcopy, and found to be infected 76

by an intestinal Eimeria. Intestinal tissues from 10 formalin fixed fish sampled in 2008, and 77

10 alcohol fixed fish sampled in 2010 were processed for DNA extraction. Polymerase chain 78

reaction (PCR) amplification of 18S rRNA gene was performed on DNA extracted from both 79

formalin and alcohol fixed Vietnamese L. calcarifer tissues. Included in this study were wax 80

block tissues from one case of cultured barramundi (also Lates calcarifer) from Western 81

Australia (WA). 82

83

2.2 18S Polymerase Chain Reaction (PCR) and sequencing 84

DNA was extracted from formalin and alcohol fixed tissues of L. calcarifer from 85

Vietnam and wax block tissues of L. calcarifer from Australia, using a Qiagen DNeasy tissue 86

kit (Qiagen, Germany). Nested PCRs of a fragment of the 18S locus of Cryptosporidium was 87

conducted as previously described (Ryan et al., 2003). For Eimeria, a hemi-nested PCR was 88

used. The primary amplification was conducted using the primers EIF1 5’- GCT TGT CTC 89

AAA GAT TAA GCC (Power et al., 2009) and EIR3 5’ – ATG CAT ACT CAA AAG ATT 90

ACC (this study). Diluted amplicons (1:10) of the primary PCR were used as template for a 91

secondary amplification using the primers EIF3 5’- CTA TGG CTA ATA CAT GCG CAA 92

TC (this study) and EIR3. The PCR was performed in a 25 µl reaction mixture that contained 93

approximately 15 ng of DNA, 1 x PCR buffer (FisherBiotech Perth, Western Australia), 0.2 94

mM deoxynucleoside triphosphates, 2.5 mM MgCl2, 5% (wt/vol) dimethyl sulfoxide, 0.2 95

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µmol of each primer and 1 U of Tth+ DNA polymerase (FisherBiotech Perth, Western 96

Australia). Reactions were initially denatured at 94°C for 3 min. and then subjected to 45 97

cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 90 sec. The final extension was 72°C 98

for 7 min. The reaction mixture and cycling program were identical for both the primary and 99

secondary PCRs but the cycle number was lowered to 35 for the primary PCR. 100

PCR products were purified using QIAquick PCR purification spin columns (Qiagen, 101

Germany), and sequenced using an ABI Prism Dye Terminator Cycle Sequencing kit 102

(Applied Biosystems, Foster City, Calif.). All commercial kits were used according to 103

manufacturer’s instructions unless otherwise specified. PCR products were sequenced in both 104

directions and analyzed using SeqEd version 1.0.3 (Applied Biosystems). For all DNA 105

extracts from fish tissue samples that were positive at the 18S locus for Cryptosporidium by 106

PCR, attempts were also made to amplify the actin locus as previously described (Ng et al., 107 2006). 108 109 2.3 Phylogenetic analysis 110

Nucleotide sequences were analyzed using Chromas lite version 2.0 111

(http://www.technelysium.com.au) and aligned with reference genotypes from GenBank 112

using Clustal W (http://www.clustalw.genome.jp). Phylogenetic trees were constructed using 113

additional isolates from Genbank. Distance estimation was conducted using TREECON (Van 114

de Peer and De Wachter, 1994), based on evolutionary distances calculated with the Tamura-115

Nei model and grouped using Neighbour-Joining. Parsimony analyses were conducted using 116

MEGA version 3.1 (MEGA3.1: Molecular Evolutionary Genetics Analyses software, Arizona 117

State University, Tempe, Arizona, USA). Bootstrap analyses were conducted using 1,000 118

replicates to assess the reliability of inferred tree topologies. Maximum Likelihood (ML) 119

analyses were conducted using the program PhyML (Dereeper et al., 2008) and the reliability 120

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of the inferred trees was assessed by the approximate likelihood ratio test (aLRT) (Anisimova 121

and Gascuel, 2006) 122

123

2.4 In situ hybridization (ISH) using DIG labeled 18S PCR products 124

The in-situ hybridization steps were adapted from published literature (Bearham et al., 125

2007; Bennett et al., 2008). QIAquick PCR purification spin column (Qiagen, Germany) 126

purified products from both Eimeria and Cryptosporidium 18S PCR described above were 127

used to generate dioxygenin (DIG) labeled DNA probes using DIG-nick translation mix 128

(Roche, Germany) according to manufacturer’s instructions. DIG labeled DNA probes made 129

from 1 g of PCR products were used to make up 1 ml of DNA probe mixture that contained 130

50% formamide, 10% dextran sulphate and 2x Saline Sodium Citrate (SSC) buffer. The DNA 131

probe mixture was stored at 4oC until required. A negative control DIG labeled DNA probe 132

generated from 18S PCR products to detect Trypanosoma irwini was kindly provided by 133

Linda McInnes, School of Veterinary and Biomedical Sciences, Murdoch University. All 134

incubations were at room temperature unless otherwise stated. 135

5µm formalin fixed paraffin wax embedded (FFPE) tissue sections on silanised slides 136

were dewaxed in two changes of xylene and rehydrated through an ethanol series to tap water. 137

Antigen retrieval was performed on tissue sections in Tris EDTA buffer (pH 9) bath in a 138

domestic microwave (Kambrook Model KER-686LE) on ‘reheat’ for 4 minutes and ‘low 139

heat’ function for 4 minutes, followed by cooling in running tap water. Tissue sections were 140

incubated in Tris-buffered saline (TBS) 0.05% Tween 20 for 5 minutes before tapping off. 30-141

50µl of DIG labeled DNA probe mixture were added to each tissue section which were then 142

cover-slipped, and incubated at 95oC for 15 minutes. The hybridization step was carried out at 143

42oC overnight (approximately 16-20 hours) in a moist chamber. Cover-slips were removed 144

and tissue sections washed for several seconds with 2x SSC buffer containing 0.05% Tween 145

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20. A blocking solution (0.1% bovine serum albumin in TBS 0.05% Tween 20) was added to 146

tissue sections for 10 minutes before tapping off. Alkaline phosphatase (AP) conjugated anti-147

dioxygenin (anti-DIG) (Roche, Germany) antibody diluted 1:600 in blocking solution was 148

added to each tissue section for a 60-minute incubation. The AP conjugated anti-DIG was 149

rinsed off followed by a 5-minute bath in TBS 0.05% Tween 20. Liquid Permanent Red 150

(Dako, USA) with 300 µg/ml levamisole to block endogenous alkaline phosphatase was 151

added to each slide for 20 minutes, and rinsed off with tap water. Tissue sections were 152

counterstained with haematoxylin for 30 seconds and rinsed in tap water followed by a few 153

short dips in Scott’s solution. Slides were flicked dry prior to cover-slipping with Faramount 154

Mounting Media (Dako, USA) for visualization on an Olympus BX51 fluorescent microscope 155

using the U-MWIBA2 filter. 156

157

3. Results 158

3.1Sequence and phylogenetic analysis of Eimeria 159

Partial 18S rRNA gene sequences were obtained from two alcohol fixed Vietnamese 160

L. calcarifer tissue samples (VTASB1 and VTASB2) and two Australian L. calcarifer wax 161

block tissue samples (AUBara1 and AUBara2). Neighbour-joining, parsimony and ML 162

analysis of the 18S rRNA partial sequences from these four samples and a range of Eimeria, 163

Goussia and other species obtained from GenBank produced similar results and showed that 164

these isolates grouped separately from known Eimeria and Goussia species (Figure 1-NJ tree 165

shown). VTASB1 & 2 and AUBarra1 & 2 shared 99.93% - 98.1% similarity to each other and 166

formed a distinct clade, but shared only 92.8- 88.7% similarity with all other species. 167

The unique partial 18S rRNA gene sequences of the Vietnamese L. calcarifer and 168

Australian L. calcarifer Eimeria genotypes have been deposited in the GenBank database 169

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under accession numbers JF261140 (VTASB1), JF261139 (VTASB2), JF261138 (AUBarra1) 170

and JF261137 (AUBarra2), respectively. 171

172

3.2 Sequence and phylogenetic analysis of Cryptosporidium 173

Partial 18S rRNA gene sequences were obtained from two formalin fixed Vietnamese 174

L. calcarifer tissue samples (H08384 and H08391). Neighbour-joining, parsimony and ML 175

analysis of the 18S rRNA sequences from these two samples and a range of Cryptosporidium 176

species and genotypes obtained from GenBank produced similar results and showed that these 177

DNA sequences grouped most closely with C. parvum and C. hominis (Figure 2-NJ tree 178

shown). H08384 and H08391 had 3 single nucleotide polymorphisms (SNPs) from each 179

other, and between 7 and 12 SNPs from C. parvum KSU isolate type B (GenBank accession 180

no. AF308600). H08384 and H08391 had between 11-17 SNPs from C. parvum (GQ121019) 181

and between 14-17 SNPs from C. hominis. H08384 and H08391 shared 98.5% genetic 182

similarity with each other, 97-98% similarity with the C. parvum KSU type B isolate, and 96-183

97.5% similarity with C. parvum and C. hominis. Unfortunately all attempts to amplify at the 184

actin locus were unsuccessful. 185

The unique partial 18S rRNA sequences of the Cryptosporidium genotypes detected in 186

Vietnamese L. calcarifer have been deposited in the GenBank database under accession 187

numbers JF285332 (H08384) and JF285333 (H08391). 188

189

3.3 In-situ hybridization (ISH) 190

The epicytoplasmic intestinal Eimeria in tissue sections of L. calcarifer from Vietnam 191

showed positive red fluorescence in ISH carried out using DIG-labeled Eimeria 18S PCR 192

products (Figure 3). Negative controls using DIG-labeled 18S PCR products generated to 193

detect Trypanosoma irwini showed negative fluorescence (inset of Figure 3). Low to 194

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moderate level red fluorescence was observed on the same organisms in tissue sections using 195

DIG-labeled Cryptosporidium 18S PCR products (not shown). 196

197

4. Discussion 198

Vietnamese and Australian L. calcarifer are the same fish species, but are from 199

distinct geographically separated populations. Analysis of Eimeria from both Vietnamese and 200

Australian L. calcarifer revealed that they are unique and form a clade basal to the rest of 201

Eimeriidae, which is clearly distinct from previously described clades including anuran 202

Eimeria and Goussia (Jirku et al., 2009a and b). They represent the first published sequences 203

of piscine Eimeria and are most probably conspecific due to their high sequence similarity. 204

Cryptosporidium is known to have four type A and one type B ribososmal units which 205

differ genetically (Le Blancq et al., 1997). Analysis of the two Cryptosporidium partial 18S 206

rRNA sequences detected in Vietnamese L. calcarifer in the present study revealed that they 207

clustered closest with C. parvum type B. Analysis at a second locus is therefore essential to 208

determine the true identity and phylogenetic placement of this Cryptosporidium. 209

Unfortunately all attempts to amplify at the actin locus for sequencing were unsuccessful. 210

Previous studies in fish for which morphological and genetic data are available have 211

identified piscine-derived Cryptosporidium species as genetically very distinct. For example, 212

C. molnari, and piscine genotype 1 and 2, which were all identified in the stomach, clustered 213

separately in a clade basal to other gastric Cryptosporidium species (Ryan et al. 2004; 214

Murphy et al., 2009; Palenzuela et al., 2010). Conversely, C. scophthalmi has been identified 215

in turbot intestines (Alvarez-Pellitero et al., 2004), but no molecular data is available. In 216

addition, morphological but not molecular descriptions have been reported in the intestinal 217

villi of L. calcarifer (Glazebrook and Campbell, 1987) and carp (Cyprinus carpio) (Pavlasek, 218

1983). Although both Eimeria and Cryptosporidium were detected at the molecular level in 219

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the L. calcarifer tissues from Vietnam, only the former but not the latter could be identified at 220

the light and electron microscopic levels (Gibson-Kueh et al., 2011). This could possibly be 221

due to a low rate of infection by Cryptosporidium. Being ubiquitous organisms, 222

Cryptosporidium might be a contaminant in the L. calcarifer intestines from feed or culture 223

water, and not a true colonizing infection. This was further supported by the clustering of this 224

Cryptosporidium closest to mammalian genotypes. 225

Positive fluorescence was obtained for Vietnamese L. calcariferEimeria in tissues by 226

ISH using DIG labeled DNA probes generated from Eimeria and Cryptosporidium but not the 227

negative control (Trypanosoma irwini) 18S PCR products. This showed that ISH using DIG 228

labeled 18S PCR generated products could not be used to distinguish between Eimeria and 229

Cryptosporidium due to the conserved nature of the 18S locus. 230

Future studies on piscine Eimeria and Cryptosporidium should include both molecular 231

and ultrastructural characterizations. Descriptions of natural infections at the histopathological 232

and ultrastructural levels are grossly inadequate for the identification of these parasites, which 233

undergo complex life cycles. While molecular analysis is a very useful tool in the 234

identification of aetiological agents in disease, this study reiterates the importance of 235

demonstrating organisms in situ in tissues, particularly for ubiquitous organisms which can 236

easily be contaminants in test samples. 237

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Acknowledgements: John Jardine, Vetpath Laboratory Services, Western Australiafor 239

the barramundi wax blocks, and from Murdoch University: Linda McInnes for providing the 240

DIG labeled Trypanosoma irwini DNA probes, Mark Bennett and Masa Wayan for advice on 241

in situ hybridization, and Michael Slaven and Gerard Spoelstra for technical help on 242

histology. 243

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336

Legends for Figures 337

Fig. 1. Phylogenetic tree of Eimeria detected in L. calcarifer from Vietnam and Australia, 338

with GenBank database accession numbers JF261140 (VTASB1), JF261139 (VTASB2), and 339

JF261138 (AUBarra1) and JF261137 (AUBarra2), respectively. 340

341

Fig. 2. Phylogenetic tree of Cryptosporidium detected in L. calcarifer from Vietnam, with 342

GenBank database accession numbers JF285332 (H08384) and JF285333 (H08391). 343

344

Figure 3. Epicytoplasmic Asian seabass Eimeria in intestinesshowed positive red 345

fluorescence in ISH using DIG-labeled Eimeria 18S PCR products and Permanent Red 346

(Dako). Inset (to same scale) shows no fluorescence in ISH using negative control DIG-347

labeled Trypanosoma 18S PCR products. 348

(19)

Accepted Manuscript

0.10 C. bigenetica E.catronensis E.rioarribaensis E. alabamensis E.antrozoi VT ASB1 E.faurei C. cayetanensis E.chaetodipi VT ASB2 G.neglecta E.tenella E.bovis E. scholtysecki E. arizonensis E.chobotari E.peromyscus E.albigulae E.onychomysis E.reedi E.praecox E. acervuline E.weybridge E.ahsata E.separata E.telekii E.falciformis E.nieschulz E.Trichosuri A E.Trichosuri I E.ranae (anuran) E.arnyi (snake) E.reichenowi AB205175 E.reichenowi AB205179 E.reichenowi AB205173 E.reichenowi AB205174 E.gruis AB205169 E.gruis AB205167 G.Bufo G.noelleri AU Barra2 AU Barra1 Sarcocystis neurona Sarcocystis aucheniae 100 99 100 100 78 72 100 100 100 100 85 100 100 99 78 83 98 100 97 100 100 100 100 100 98

Eimeria

and

Caryospora

Anuran

Goussia

This study

Figure 1 Vetpar-D11-4768

(20)

Accepted Manuscript

0.1 C. baileyi C. saurophilum C. fayeri C. suis C.meleagridis C. wrairi C. fragile Piscine genotype 6 HM991857 Piscine genotype 5 HM989834 C. ryanae C. serpentis C. hominis PigII Whiting7 C. galli

C. parvum type B (AF308600) H08384 H08391 C.parvum Whiting1 C. ubiquitum C. macropodum C. canis C. felis Whiting5 Whiting4 C. bovis C. xiaoi C. andersoni C. muris

Piscine genotype 1 AY524773

C. molnari_HM243547 100 70 100 82 96 54 66 59 61 82 77 50 75 100 91 75 100 84 79 61 100 62 63 91 100 93 82

This study

Figure 2 Vetpar-D11-4768

(21)

Accepted Manuscript

Figure 3. Epicytoplasmic Asian seabass Eimeriain intestines showed positive red fluorescence in ISH using DIG-labeled Eimeria18S PCR products and Permanent Red (Dako). Inset (to same scale) shows no fluorescence in ISH using negative control DIG-labeled Trypanosoma 18S PCR products.

References

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