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Gibson-Kueh, S., Thuy, N.T.N., Elliot, A., Jones, J.B., Nicholls,

P.K. and Thompson, R.C.A. (2011) An intestinal Eimeria infection in juvenile Asian seabass (Lates calcarifer) cultured in Vietnam – A first report. Veterinary Parasitology, 181 (2-4). pp.

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Title: An intestinal Eimeria infection in juvenile Asian seabass(Lates calcarifer) cultured in Vietnam - a first report

Authors: S. Gibson-Kueh, N.T.N. Thuy, A. Elliot, J.B. Jones,P.K. Nicholls, R.C.A. Thompson

PII: S0304-4017(11)00301-3DOI: doi:10.1016/j.vetpar.2011.04.040Reference: VETPAR 5845

To appear in: Veterinary Parasitology

Received date: 11-1-2011Revised date: 27-4-2011Accepted date: 28-4-2011

Please cite this article as: Gibson-Kueh, S., Thuy, N.T.N., Elliot, A., Jones, J.B.,Nicholls, P.K., Thompson, R.C.A., An intestinal Eimeria infection in juvenile Asianseabass (Lates calcarifer) cultured in Vietnam - a first report, Veterinary Parasitology(2010), doi:10.1016/j.vetpar.2011.04.040

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dx.doi.org/doi:10.1016/j.vetpar.2011.04.040

dx.doi.org/10.1016/j.vetpar.2011.04.040

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Eimeria in L. calcarifer in Vietnam Gibson-Kueh et al., 2011 Vetpar-D11-4695 Page 1

An intestinal Eimeria infection in juvenile Asian seabass (Lates calcarifer) cultured in 1

Vietnam - a first report 2

3

S. Gibson-Kueh a*

, N.T.N. Thuy b, A. Elliot

a, J.B. Jones

c, P.K. Nicholls

a, R.C.A. Thompson

a 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

Correspondence: S. Gibson-Kueh, School of Veterinary and Biomedical Sciences, Murdoch 15

University, South St., Murdoch, Western Australia 6150, Australia. E-mail: 16

[email protected] 17

18

mailto:[email protected]

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ABSTRACT 19

This is the first report of an intestinal Eimeria infection in Asian seabass (Lates calcarifer) at 20

the histopathological and ultrastructural levels. The Eimeria infection was often associated 21

with severe pathology and significant mortality in the absence of other pathogens. This 22

showed that it is an important disease of juvenile L. calcarifer in small scale nurseries in 23

Vietnam. Heavy infection and high prevalence levels of the Eimeria infection are suspected to 24

be linked to the low daily water exchange rates practised in these nurseries. Although 25

systemic iridovirus infection was concurrently observed in some of the fish examined, it was 26

not as consistently present in diseased fish as the Eimeria infection. 27

28

Keywords: Lates calcarifer, Eimeria, systemic iridovirus, pathology 29

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1. Introduction 31

Asian seabass or barramundi (Lates calcarifer) is an aquaculture food fish of rapidly 32

growing importance in Australia and Asia. The culture of L. calcarifer is typically divided 33

into specialized operations in hatcheries, nurseries and grow-out farms. Farms are generally 34

small to medium scale though some larger grow out farms recently reported an annual 35

production of 300-400 tonnes (http://www.marineproduce.com/annual_reports.html; 36

http://www.seafoodsource.com/newsarticledetail.aspx?id=4294990500). Thailand and 37

Indonesia are currently the largest producers of cultured L. calcarifer at 15,700 and 4,417 38

tonnes, respectively (http://library.enaca.org/AquacultureAsia/Articles/april-june-2010/8-39

cage-culture-asia.pdf). The emerging L. calcarifer industry in Vietnam depends on the 40

grow-out of juvenile fish from small scale nurseries, from which the samples in this study 41

were taken. 42

Significant diseases limiting the culture of L. calcarifer include viral nervous necrosis 43

(Glazebrook et al., 1990; Munday et al., 1992; Maeno et al., 2004; Parameswaran et al., 44

2008), streptococcosis (Bromage et al., 1999; Creeper and Buller, 2006), flavobacteriosis or 45

tenacibaculosis (Carson et al., 1993; Avendano-Herrera et al., 2006; 46

http://aqua.intervet.com/news/2007-10-10.aspx), pot-belly disease (Gibson-Kueh et al., 2004) 47

and Neobenedenia melleni (Deveney et al., 2001; Ruckert et al., 2008). 48

Piscine apicomplexan parasites exhibiting epicytoplasmic development on host cells 49

may belong to the genera Cryptosporidium, Eimeria, Epieimeria or Goussia (Paperna 1995). 50

These parasitic infections have not been reported in L. calcarifer except for a brief mention of 51

Cryptospordium by Glazebrook and Campbell (1987). Cryptosporidium is typically 52

epicytoplasmic while Eimeria and Goussia may be either epicytoplasmic or intracytoplasmic 53

parasites (Molnar and Baska, 1986; Landsberg and Paperna, 1987; Molnar 1989; Lukes and 54

Dykova, 1990; Szekely and Molnar, 1992; Landsberg 1993; Benajiba et al., 1994; Paperna 55

http://aqua.intervet.com/news/2007-10-10.aspx

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and Vilenkin, 1996; Alvarez-Pellitero et al., 1997; Baska 1997; Alvarez-Pellitero and Sitja-56

Bobadilla, 2002; Alvarez-Pellitero et al., 2004; Ryan et al., 2004; Murphy et al., 2009). These 57

apicomplexan parasites are distinguished by the morphology of their oocysts. Oocysts of 58

Eimeria and Epieimeria have four dizoic sporocysts each with a Stieda body or polar plug. In 59

Goussia, oocysts are characterized by four dizoic sporocysts each with a suture line. 60

Cryptosporidium oocysts have four naked sporozoites. (Davies and Ball, 1993; Molnar 2006) 61

Meronts, gamonts and oocysts of piscine Cryptosporidium with a size range of 3-5m 62

are much smaller than corresponding stages of Eimeria and Goussia, in the size range of 5-63

20m. The presence of invaginating feeder organelles at the attachment juncture of 64

Cryptosporidium distinguishes it from the other genera (Valigurova et al., 2008). The 65

attachment organelles of epicytoplasmic Eimeria and Goussia vary ultrastructurally from 66

monopodial to multiple finger-like attachment organelles (Paperna 1991; Benajiba et al. 67

1994; Alvarez-Pellitero et al. 1997; Lukes 1992; Lukes and Stary, 1992). 68

This is the first report of an intestinal Eimeria infection in juveniles of L. calcarifer, at 69

the histopathological and ultrastructural levels. This infection was often associated with 70

severe pathology even in the absence of other significant pathogens, and is therefore a 71

significant disease of L. calcarifer in small scale nurseries in Ca Mau, Vietnam. 72

73

2. Materials and methods 74

2.1 Background information on samples examined 75

Diseased juvenile L. calcarifer 2.5-7cm in body length were sampled from a total of 76

five nurseries in Ca Mau, Vietnam in Jan to Mar 2008, Mar and Dec 2009, and Nov to Dec 77

2010. Fixed tissue samples were sent to Murdoch University, Australia where they were 78

processed for histopathology (181 fish) and transmission electron microscopy (10 fish). 79

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Alcohol fixed oocysts obtained from discharged waste water from culture tanks were also 80

examined. 81

82

2.2 Light microscopy (LM) 83

Tissues were fixed in 10% phosphate buffered formalin for at least 24 h, dehydrated in 84

an ethanol-xylene series and embedded in paraffin wax. Formalin fixed bony tissues were 85

decalcified in 5% nitric acid overnight prior to dehydration and embedded in paraffin wax. 86

5µm tissue sections were dewaxed in xylene, rehydrated in an ethanol series and stained by 87

haematoxylin & eosin (H&E) or Giemsa. 88

89

2.3 Transmission electron microscopy (TEM) 90

Tissues were fixed in 5% glutaraldehyde in phosphate-buffered saline (PBS) at 4oC 91

overnight, washed in several changes of PBS and post-fixed in Dalton’s chrome osmic acid 92

for1 h at 4oC. Fixed tissues were dehydrated through a graded ethanol series to propylene 93

oxide followed by 1 h in 60:40 solution of propylene oxide/epoxy resin, overnight in pure 94

epoxy resin on a rotator and baked in an oven at 60oC for 24 h. Ultra-thin sections were 95

stained with uranyl acetate and lead citrate for viewing on a Philips CM100 Bio TEM. 96

97

3. Results 98

3.1 Field observations made on L. calcarifer nurseries sampled in this study 99

The L. calcarifer nurseries in Ca Mau, Vietnam were mainly small scale with less than 100

five ½- to 1-tonne tanks. These nurseries obtained their fry from hatcheries in Vung Tau or 101

Khanh Hoa Province in Vietnam, or as imported fry from Thailand. Fiber glass or cement 102

tanks were mainly used as holding facilities with static or closed recirculation systems. Less 103

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than 20-30% partial daily water exchange rates were practised. In earthen ponds which were 104

less commonly used, the fry were kept in nets suspended in the water column. Salinity of 105

rearing water ranged from 15 to 25 parts per thousand (ppt). Stocking density varied from 280 106

to 350 fish/m3 water. Fish were fed commercial feed pellets supplemented with coarsely 107

chopped trash fish. The trash fish fed consisted of wild caught fish from the sea. 108

Nurseries stocked 1-3 cm L. calcarifer fry obtained from hatcheries, and grow them 109

on to 7-9 cm body length fish to sell to grow-out farmers. Nursery reared 2.5 to 7.0 cm body 110

length L. calcarifer juveniles were reported to suffer low grade clinical disease soon after 111

stocking, with a cumulative mortality of up to 30% of stocked fish. Clinical signs included 112

fish preferentially hanging at water surface, inappetance, lethargy, darkened bodies, tail rot 113

and scales loss. 114

115

3.2 Histopathology 116

An Eimeria infection was observed in greater than 60% of diseased L. calcarifer 117

sampled from the five nurseries, often as early as the first week post stocking. Fish kept in 118

cement or fiber glass tanks or ponds in salinities of both 15 and 25 ppt were found to be 119

infected with this parasite. Concurrent systemic iridoviral disease was observed in 120

approximately 20% of diseased L. calcarifer examined. Low grade to heavy gill trichodinid 121

infection was sometimes observed but not associated with any significant pathology. 122

The primary infection site of the L. calcarifer Eimeria was the small intestine. Both 123

merogony and gamogony were mainly epicytoplasmic and occurred simultaneously (Figure 124

1). Infection levels varied from light to heavy, often with obliteration of the microvillous 125

brush border. Meronts were much smaller in size than gamonts, and had merozoites arranged 126

in rosettes or in parallel (Figures 1 and 2a). Intracytoplasmic meronts or unusually large 127

meronts with at least 18 merozoites were occasionally observed (Figure 2b). Macrogamonts 128

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with foamy cytoplasm due to the presence of amylopectin granules often outnumbered 129

microgamonts. Microgamonts were smaller than macrogamonts and had peripherally arranged 130

nuclei (Figures 1 and 2c). Mature microgamonts had numerous flagellated microgametes 131

(Figure 2c). Meronts measured 4.8 x 3.5 m (n=5), macrogamonts 13.1 x 7.6 m (n=10) and 132

microgamonts 8.1 x 6.0m (n=5). 133

Sporulated oocysts were very rarely observed in histological tissue sections, in fact in 134

only 1 out of 181 fish examined, and measured 18.5 x 12.3 µm (n = 5). These oocysts in 135

faecal materials within the intestinal lumen had four pairs of sporozoites held loosely within a 136

thin membranous oocyst wall (Figure 3a). Both unsporulated and sporulated oocysts were 137

readily observed in faeces collected from tank bottom and in waste water from rearing tanks 138

by wet mount microscopic examinations (Figures 3b and 3c). Nomarski interference 139

microscopy on alcohol fixed sporulated oocysts showed the absence of Stieda bodies and 140

suture lines. Alcohol fixed sporulated oocysts measured 36.6 x 22.8 µm (n=5). A residual 141

body was present in oocysts, and each pair of sporozoites was held together by a thin 142

sporocyst membrane (Figure 3c). 143

Squamous to cuboidal intestinal epithelium and low grade to severe mononuclear 144

inflammatory infiltrates in the lamina propria were frequently observed in association with the 145

Eimeria infection. The inflammation was sometimes extended into the mucosal epithelium. 146

There were often focal to extensive areas of intestinal mucosal degeneration, necrosis and 147

sloughed necrotic cells in intestinal lumen (Figure 4). Extra-intestinal parasite stages were not 148

commonly observed, except for two macrogamonts in renal tubules from 1 fish. Other 149

abnormalities observed included dermatitis in caudal peduncle, renal glomerular 150

degeneration, moderately reactive spleens with white pulps depleted of leucocytes, and 151

reduced levels of hepatic glycogen stores. 152

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3.3 Ultrastructural observations by TEM 154

Parasitic stages were observed within complete parasitophorous envelopes at 155

extracytoplasmic positions on the microvillous brush border of intestinal epithelium. 156

Shortening to loss of microvilli and necrosis of affected intestinal epithelium were often 157

observed. Both meronts and gamonts had finger-like attachment organelles that extended into 158

host cells but were limited to the extracytoplasmic cellular boundaries (Figures 5, 7a, 7b and 159

8). A large number of rodlet cells were often seen in association with these parasitic 160

infections, and sometimes within blood vessels in the intestine (Figure 6). 161

Meronts had merozoites with apical complexes in various stages of formation and up 162

to eight merozoites (Figures 7a and 7b). Macrogamonts had abundant amylopectin granules 163

(Figure 8) while microgamonts had flagellated microgametes with a large residual body 164

(Figure 5). 165

4. Discussions 166

This is the first report of a natural Eimeria infection in L. calcarifer. While it does not 167

supply all the answers, the high prevalence of the Eimeria infection in association with severe 168

pathology showed that it is a significant disease under nursery culture conditions in Ca Mau, 169

one which will need to be managed. Significant pathology was frequently reported in fish 170

with apicomplexan infections (Benajiba et al. 1994; Molnar 2006; Gjurcevic et al., 2008; 171

Morrison et al., 1993; Jendrysek et al., 1994; Hemmer et al., 1998). Although systemic 172

iridoviral disease was also observed, it was not as consistently observed as the Eimeria 173

infection in diseased fish. Nonetheless, iridovirus is a serious pathogen which can co-174

contribute to losses during the culture cycle (Gibson-Kueh et al. 2003). 175

The L. calcarifer Eimeria did not possess the feeder organelles typical of 176

Cryptosporidium but had finger-like attachment organelles very similar to epicytoplasmic 177

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species of Eimeria and Goussia. Although the sporocysts in oocysts examined in this study 178

lack Steida bodies, this was also the case in some previously described piscine Eimeria 179

(Upton et al., 1984; Landsberg and Paperna 1987; Paperna 1995; Molnar 2006). Therefore, 180

we will refer to this parasite as Eimeria until molecular analysis can be conducted. Since 181

sporulated oocysts were rarely observed in tissue sections of Eimeria infected L. calcarifer, it 182

is presumed that sporulation was mainly exogenous. Both unsporulated and sporulated 183

oocysts were readily observed in faecal materials collected from tank bottoms. The L. 184

calcarifer Eimeria oocysts in histological tissue sections were almost half the size of alcohol 185

fixed oocysts obtained from waste water, likely due to the dehydration process used in 186

histology. There is also the possibility that more than one species of Eimeria were involved. 187

A study on Goussia carpelli in common carp suggested the correlation of infection 188

rates to stress and immunosupression (Steinhagen et al., 1998). Depletion of splenic white 189

pulp of leucocytes in diseased L. calcarifer examined in this study is expected to have an 190

impact on their immunity, and may explain the heavy Eimeria infection often observed. The 191

diseased fish examined in this study were sampled during the initial post-stocking period 192

when the fish would be recovering from transport and acclimatization stress. 193

The origin of the Eimeria infection in L. calcarifer from nurseries in Ca Mau is 194

unknown, and warrants further study. The feeding of trash fish is a possible source of 195

infection. The stocking of fish in static or closed recirculation aquaculture systems with 196

relatively low daily water exchange rates (20-30%) would encourage the level of Eimeria 197

infection to build up to the high prevalence observed. Large scale L. calcarifer hatcheries and 198

nurseries in Indonesia, Singapore and Australia practised much higher water exchange rates 199

of 100 to 300% an hour (Schipp et al., 2007; personal observations). Whether the Eimeria 200

infection will persist in older fish as a chronic infection or were present in fish before being 201

stocked in nurseries in Vietnam remains to be elucidated and is vital information for its 202

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effective management. Experimental trials will complement what has been learnt from 203

examination of the naturally infected fish in this study. 204

There are currently no treatment options. The sequestering of these epicytoplasmic 205

parasites in parasitophorus envelopes away from the intestinal lumen and host cell cytoplasm 206

makes it resistant to currently available therapeutic drugs (Sterling 2000). Recent research 207

revealed that addition of proteins produced by Cryptosporidium competitively inhibited their 208

attachment to intestinal epithelium (Tzipori and Ward 2002). A similar approach could be 209

applied for this epicytoplasmic Eimeria. 210

211

Acknowledgement 212

Andrea Valigurova, Masaryk University, and Miloslav Jirku, Institute of Parasitology, Czech 213

Republic, for expert advice. Peter Fallon, EM unit, and Michael Slaven and Gerard Spoelstra, 214

Histology Laboratory, School of Veterinary Science and Biomedical Sciences, Murdoch 215

University, for technical help. 216

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Ruckert, S., Palm, H.W., Klimpel, S., 2008. Parasite fauna of seabass (Lates calcarifer) under 317

mariculture conditions in Lampung Bay, Indonesia. J. Appl. Ichthyol. 24, 321-327 318

Ryan, U., O’Hara, A., Xiao, L-.H., 2004. Molecular and biological characterization of a 319

Cryptosporidium molnari-like isolate from a guppy (Poecilia reticulata). Appl. 320

Environ. Microbiol., 3761–3765 321

Schipp, G., Bosmans, J., Humphrey, J., 2007. Barramundi Farming Handbook (3rd

Ed.). 322

Department of Primary Industry, Fisheries and Mines, Northern Territory Government, 323

Australia, pp. 23-24 324

Steinhagen, D., Hespe, K., Ellmer, B., Korting, W., 1998. Goussia carpelli (Portozoa: 325

Coccidia) infection in stressed and immunosuppressed common carp Cyprinus carpio. 326

Dis. Aquat. Organ. 34, 199-204 327

Sterling, C.S., 2000. Cryptosporidiosis: the treatment dilemma. Journal of Medical 328

Microbiology. 49, 207-208 329

Szekely, C., Molnar, K., 1992. Goussia trichogaster n. sp. (Apicomplexa: Eimeriidae) 330

infecting the aquarium-cultured golden gourami Trichogaster trichopterus trichopterus. 331

Dis. Aquat. Organ. 13, 79-81. 332

Tzipori, S., Ward, H., 2002. Cryptosporidiosis: biology, pathogenesis and disease. Microbes 333

Infect. 4, 1047-1058. 334

Upton, S.J., Reduker, D.W., Current, W.L., Duszynski, D.W., 1984. Taxonomy of North 335

American fish Eimeriidae. In: National Oceanic and Atmospheric Administration 336

(NOAA) Technical Report NMFS 11, U.S. Department of Commerce, 18 pp 337

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Valigurova, A., Jirku, M., Koudela, B., Gelnar, M., Modry, D., Slapeta, J., 2008. 338

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Parasitol. 38, 913-922 340

341

342

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Legends for Figures 343

Figure 1. Eimeria infection on the brush border of intestinal mucosa in L. calcarifer from 344

Vietnam showed increased mononuclear infiltrate in lamina propria (Inf). Meronts (Me) were 345

smaller than gamonts (Ma, Mi), and commonly occurred simultaneously. Meronts often had 346

merozoites arranged in rosettes (Me). Macrogamonts (Ma) with foamy cytoplasm, 347

microgamonts (Mi) with peripherally arranged nuclei and darker basophilic stained 348

trophozoites (T). (H&E) 349

350

Figure 2a-c. Different developmental forms of meronts and microgamonts. (2a) Meronts with 351

merozoites arranged in parallel (Me) and merozoites apparently still within parasitophorus 352

envelopes (z). (Giemsa). (2b) An unusually large meront with at least 18 merozoites (arrows). 353

(H&E) (2c) Microgamonts with peripherally arranged nuclei (*) and microgametes (arrows). 354

(Giemsa) 355

356

Figures 3a-c. (3a) Sporulated oocysts were very rarely seen in histological tissue sections. 357

Sporulating oocysts (*) had four sporocysts. Sporulated oocysts (arrows) in faecal materials 358

within intestinal lumen had four pairs of sporozoites and a thin membranous wall. (H&E) (3b) 359

Unsporulated oocyst observed in discharged tank waste water. (3c) Sporulated oocyst from 360

waste water with oocyst residual body (R) and sporozoites in pairs, bounded by thin sporocyst 361

membrane. Sporocysts (S) had no stieda bodies or suture lines. (Nomarski interference 362

microscopy) 363

364

Figure 4. The intestinal epithelium was denuded in some areas (arrows) with corresponding 365

intense inflammatory response (Inf) in the lamina propria, and a significant amount of 366

sloughed cellular debri (D) in intestinal lumen. (Giemsa) 367

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Figure 5. Macrogamonts (Ma) with amylopectin granules and microgamonts with 368

microgametes (MiG) at the microvillous brush border of intestines. ‘X’ was presumably a 369

microgamont from which microgametes had been released, thus giving a crenate appearance. 370

Parasitophorous envelopes (PE), residual body (Re) in microgamont. Figure 6. Rodlet cells 371

(Ro) often associated with response to parasitism in fish were observed within blood vessels 372

(bv) in intestines. One of the rodlet cells appeared to be in the process of exiting the blood 373

vessel (*). Vascular endothelial cells (E), fibroblast cells (F) that produced the collagen (C) of 374

blood vessel wall. 375

376

Figure 7a. Meront (Me) with finger-like attachment organelles (fao) and residual body (Re). 377

Merozoites (z) had apical complexes (A) at various stages of formation. Trophozoites (T) and 378

developing meront (dMe) in epicytoplasmic position. Figure 7b. Meront with at least 8 379

merozoites (z) and finger-like attachment organelles (fao). 380

381

Figure 8. Macrogamont with abundant amylopectin (A) granules and finger-like attachment 382

organelles (fao) extended into host cell but limited to the epicytoplasmic boundary. 383

Parasitophorus envelope (PE). 384

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Figs Eimeria in L. calcarifer Gibson-Kueh et al., 2011 Vetpar-D11-4695 Page 1

Figure 1. Eimeria infection on the brush border of intestinal mucosa in L.

calcarifer from Vietnam showed increased mononuclear infiltrate in lamina

propria (Inf). Meronts (Me) were smaller than gamonts (Ma, Mi), and

commonly occurred simultaneously. Meronts often had merozoites arranged in

rosettes (Me). Macrogamonts (Ma) with foamy cytoplasm, microgamonts (Mi)

with peripherally arranged nuclei and darker basophilic stained trophozoites

(T). (H&E)

Ma

Mi

Me

T Inf

2a

Me

z

2c

*

10m

2b

10m

Figure 2a-c. Different developmental forms of meronts and microgamonts. (2a)

Meronts with merozoites arranged in parallel (Me) and merozoites apparently

still within parasitophorus envelopes (z). (Giemsa). (2b) An unusually large

meront with at least 18 merozoites (arrows). (H&E) (2c) Microgamonts with

peripherally arranged nuclei (*) and microgametes (arrows). (Giemsa)

B&W Figures Vetpar-D11-4695

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Figures 3a-c. (3a) Sporulated oocysts were very rarely seen in histological tissue

sections. Sporulating oocysts (*) had four sporocysts. Sporulated oocysts (arrows) in

faecal materials within intestinal lumen had four pairs of sporozoites and a thin

membranous wall. (H&E) (3b) Unsporulated oocyst observed in discharged tank waste

water. (3c) Sporulated oocyst from waste water with oocyst residual body (R) and

sporozoites in pairs, bounded by thin sporocyst membrane. Sporocysts (S) had no

stieda bodies or suture lines. (Nomarski interference microscopy)

3b

3a

*

*

3c

R

S

Figure 4. The intestinal epithelium was denuded in some areas (arrows)

with corresponding intense inflammatory response (Inf) in the lamina

propria, and a significant amount of sloughed cellular debri (D) in

intestinal lumen. (Giemsa)

Inf Inf

D

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Figure 5. Macrogamonts (Ma) with amylopectin granules and microgamonts with microgametes (MiG)

at the microvillous brush border of intestines. ‘X’ was presumably a microgamont from which

microgametes had been released, thus giving a crenate appearance. Parasitophorous envelopes (PE),

residual body (Re) in microgamont. Figure 6. Rodlet cells (Ro) often associated with response to

parasitism in fish were observed within blood vessels (bv) in intestines. One of the rodlet cells appeared

to be in the process of exiting the blood vessel (*). Vascular endothelial cells (E), fibroblast cells (F)

that produced the collagen (C) of blood vessel wall.

Ma

X

MiG

PE

10m

Re

5

*

Ro

E

C C

E

F

bv

bv

C

5 µm

6

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2 µm

z

z

fao

7b

Figure 7a. Meront (Me) with finger-like attachment organelles (fao) and residual body (Re).

Merozoites (z) had apical complexes (A) at various stages of formation. Trophozoites (T) and

developing meront (dMe) in epicytoplasmic position. Figure 7b. Meront with at least 8 merozoites (z)

and finger-like attachment organelles (fao).

A

A

Re

T

fao Me

z

z

z

z

T dMe

2m

A 7a

Figure 8. Macrogamont with abundant amylopectin (A) granules

and finger-like attachment organelles (fao) extended into host cell

but limited to the epicytoplasmic boundary. Parasitophorus envelope

(PE).

fao

A

A

2 µm

PE

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Figs Eimeria in L.calcarifer Gibson-Kueh et al., 2011 Vetpar-D11-4695 Page 1

Figure 1. Eimeria infection on the brush border of intestinal mucosa in L.

calcarifer from Vietnam showed increased mononuclear infiltrate in lamina

propria (Inf). Meronts (Me) were smaller than gamonts (Ma, Mi), and

commonly occurred simultaneously. Meronts often had merozoites arranged in

rosettes (Me). Macrogamonts (Ma) with foamy cytoplasm, microgamonts (Mi)

with peripherally arranged nuclei and darker basophilic stained trophozoites (T).

(H&E)

Mi

Me

Ma

T Inf

Figure 2a-c. Different developmental forms of meronts and microgamonts. (2a)

Meronts with merozoites arranged in parallel (Me) and merozoites apparently

still within parasitophorus envelopes (z). (Giemsa). (2b) An unusually large

meront with at least 18 merozoites (arrows). (H&E) (2c) Microgamonts with

peripherally arranged nuclei (*) and microgametes (arrows). (Giemsa)

Me

z

2a

2b

10 m

2c

10 m

*

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Figures 3a-c. (3a) Sporulated oocysts were very rarely seen in histological tissue

sections. Sporulating oocysts (*) had four sporocysts. Sporulated oocysts (arrows) in

faecal materials within intestinal lumen had four pairs of sporozoites and a thin

membranous wall. (H&E) (3b) Unsporulated oocyst observed in discharged tank

waste water. (3c) Sporulated oocyst from waste water with oocyst residual body (R)

and sporozoites in pairs, bounded by thin sporocyst membrane. Sporocysts (S) had

no stieda bodies or suture lines. (Nomarski interference microscopy)

3b

*

*

3a

3c

R

S

Figure 4. The intestinal epithelium was denuded in some areas (arrows) with

corresponding intense inflammatory response (Inf) in the lamina propria, and a

significant amount of sloughed cellular debri (D) in intestinal lumen. (Giemsa)

Inf Inf

D

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bv

Figure 5. Macrogamonts (Ma) with amylopectin granules and microgamonts with microgametes (MiG)

at the microvillous brush border of intestines. ‘X’ was presumably a microgamont from which

microgametes had been released, thus giving a crenate appearance. Parasitophorous envelopes (PE),

residual body (Re) in microgamont. Figure 6. Rodlet cells (Ro) often associated with response to

parasitism in fish were observed within blood vessels (bv) in intestines. One of the rodlet cells appeared

to be in the process of exiting the blood vessel (*). Vascular endothelial cells (E), fibroblast cells (F)

that produced the collagen (C) of blood vessel wall.

Ma

X

MiG

PE

10m

Re

5

*

Ro

E

C C

E

F

bv

bv

C

5 µm

6

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Figure 8. Macrogamont with abundant amylopectin (A) granules

and finger-like attachment organelles (fao) extended into host cell

but limited to the epicytoplasmic boundary. Parasitophorus envelope

(PE).

fao

A

A

2 µm

PE

2 µm

z

z

fao

7b

Figure 7a. Meront (Me) with finger-like attachment organelles (fao) and residual body (Re).

Merozoites (z) had apical complexes (A) at various stages of formation. Trophozoites (T) and

developing meront (dMe) in epicytoplasmic position. Figure 7b. Meront with at least 8 merozoites (z)

and finger-like attachment organelles (fao).

A

A

Re

T

fao Me

z

z

z

z

T dMe

2m

A 7a

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