Transmission of Francisella noatuensis subsp. orientalis from subclinically infected hybrid red tilapia broodstock (Oreochromis sp.) to their offspring

Journal Pre-proof Transmission of Francisella noatuensis subsp. orientalis from subclinically infected hybrid red tilapia broodstock (Oreochromis sp.) to their offspring Vuong Viet Nguyen, Ha Thanh Dong, Saengchan Senapin, Warachin Gangnonngiw, Nopadon Pirarat, Pattanapon Kayansamruaj, Tilladit Rung-ruangkijkrai, Channarong Rodkhum PII:

S0882-4010(19)30120-2

DOI:

https://doi.org/10.1016/j.micpath.2019.103670

Reference:

YMPAT 103670

To appear in:

Microbial Pathogenesis

Received Date: 20 January 2019 Revised Date:

27 May 2019

Accepted Date: 13 August 2019

Please cite this article as: Nguyen VV, Dong HT, Senapin S, Gangnonngiw W, Pirarat N, Kayansamruaj P, Rung-ruangkijkrai T, Rodkhum C, Transmission of Francisella noatuensis subsp. orientalis from subclinically infected hybrid red tilapia broodstock (Oreochromis sp.) to their offspring, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.103670. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

1

Transmission of Francisella noatuensis subsp. orientalis from subclinically infected

2

hybrid red tilapia broodstock (Oreochromis sp.) to their offspring

3 4

Vuong Viet Nguyena,b, Ha Thanh Dongc*, Saengchan Senapind,e, Warachin Gangnonngiwd,e

5

Nopadon Piraratf, Pattanapon Kayansamruajg, Tilladit Rung-ruangkijkraih, Channarong

6

Rodkhuma,i*

7 8

a

9

Bangkok, Thailand

Department of Microbiology, Faculty of Veterinary Science, Chulalongkorn University,

10

b

11

c

12

d

13

Technology Development Agency (NSTDA), Pathumthani, Thailand

14

e

15

Faculty of Science, Mahidol University, Bangkok, Thailand

16

f

17

10330, Thailand

18

g

19

h

20

Bangkok, THAILAND

21

i

22

University, Bangkok, Thailand

Research Institute of Aquaculture No. 1 (RIA1), Dinh Bang, Tu Son, Bac Ninh, Vietnam

Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and

Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp),

Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok,

Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand Department of Veterinary Anatomy, Faculty of Veterinary Science, Chulalongkorn University,

Fish Infectious Diseases Research Unit (FID RU), Faculty of Veterinary Science, Chulalongkorn

23 24

*

25

C. Rodkhum, E-mail: [email protected]

26

H. T Dong, E-mail: [email protected]

Corresponding authors:

27

1

28

ABSTRACT

29

Francisella noatunensis subsp. orientalis (Fno) has been reported as an important bacterial

30

pathogen causing significant mortality (30-95%) in farmed tilapia in broad geographic areas.

31

However, we found that there was a proportion of broodfish in our laboratory that appeared to be

32

healthy but which tested positive for Fno. We therefore hypothesized that Fno might be able to

33

be transmitted from subclinically infected tilapia mouthbrooders to their offspring through the

34

current practice of fry production in tilapia hatcheries. To prove this, experimentally infected

35

hybrid red tilapia broodstock were mated and their offspring were examined for the presence of

36

Fno. In this study, three pairs of infected broodfish were mated for natural spawning and

37

fertilized eggs from each couple were then collected from the female mouths for artificial

38

incubation. The newly hatched larvae were cultured for 30 days and sample collection was

39

performed at different developmental stages i.e. yolk-sac larvae, 5 and 30-day old fry. The

40

results showed that the ovary and testis of all 3 pairs of the broodstock, as well as their fertilized

41

eggs and offspring were Fno positive by Fno-specific PCR and in situ DNA hybridization. In

42

summary, this study revealed that with the current practice in tilapia hatcheries, Fno might be

43

able to transmit from subclinically infected tilapia mouthbrooders to their offspring. Therefore,

44

using Fno-free broodfish in tilapia hatcheries should be considered in order to produce Fno-free

45

tilapia fry.

46 47

Keywords: Francisella noatunensis subsp. orientalis; francisellosis; transmission; hybrid red

48

tilapia

49

1. Introduction

50

Francisellosis is a systemic disease that is caused by Gram-negative, small coccobacillus

51

bacterium, Francisella noatunensis subsp. orientalis (Fno). It has been reported worldwide and

52

is responsible for considerable economic losses in various warm water fish species [1], and

53

tilapia in particular, has been considered as the most susceptible host resulting in mortality levels

54

of up to 95% and as little as 23 colony forming units (CFU) can be lethal for tilapia fingerlings

55

[1, 2]. Since the first case of Fno was reported in Thailand in 2013, the disease has been reported

56

in farmed tilapia in several provinces [3, 4]. According to the private sector, francisellosis is

2

57

presently considered as one of the top three most important infectious diseases of farmed tilapia

58

in Thailand.

59

Currently, the horizontal transmission of Fno has been proven by experimental challenge using

60

different infection routes e.g. injection, immersion, cohabitation between infected and healthy

61

fish or direct exposure to contaminated water [2, 5, 6]. Previous studies found that reproductive

62

organs (ovary and testis) of infected tilapia showed multiple white nodules that formed

63

granulomatous inflammation in histological analysis. Thus, vertical transmission of Fno is

64

potentially suspected [7, 8]. So far only one published work by Pradeep et al. (2017) supported

65

the potential of Fno vertical transmission by performing artificial fertilization using naturally

66

infected broodfish and examining the presence of Fno from their reproductive organs and

67

offspring. However, Fno detection was carried out by a single technique called loop mediated

68

isothermal amplification (LAMP). In the current study, eggs were collected from natural

69

mouthbrooding fish in an experiment set up similar to current practices in tilapia hatchery. The

70

hybrid red tilapia (Oreochromis sp.) broodfish were subclinically infected by pre-exposure to

71

Fno. Confirmation of Fno in the fish reproductive organs and their progeny was performed by a

72

combination of PCR, histology and in situ hybridization (ISH) assays.

73

2. Materials and methods

74

2.1. Experimental fish

75

This project has been reviewed and approved by the Biosafety Committee (approval no. IBC

76

1831055) and Animal Ethics Committee (approval no. CU-ACUP 1931007) from Chulalongkorn

77

University. Clinically healthy four-month-old hybrid red tilapia (initial body weight 30 ± 6 g)

78

were kindly provided by Kamphaengsaen Fisheries Research Station, Faculty of Fisheries,

79

Kasetsart University, Thailand. The fish were acclimatized in two 1-m3 fiber glass tanks

80

containing chlorine-free water at a temperature of 26.5 ± 0.5 oC for two weeks. The fish were

81

fed with commercial tilapia pellet feed (CP) containing ~30% crude protein at the rate of 5%

82

biomass twice per day. The tank contained air stones and cotton filters. The water and cotton

83

filters were replaced two times per week and water parameters (pH, nitrite, total ammonia)

84

were checked daily during the experimental period. Ten fish were randomly selected for

85

bacterial and parasitic examination to verify that the fish were healthy prior to the challenge

86

experiment. It should be noted that for Fno examination, species-specific PCR [14] and 3

87

bacterial culture using cysteine heart agar (CHA) [2] were performed with the spleen and

88

reproductive organs to ensure that the fish were not infected with Fno.

89

2.2. Bacterial preparation

90

Francisella noatunensis subsp. orientalis (Fno) strain VMCU-FNO131 originally isolated from

91

farmed hybrid red tilapia suffering the piscine francisellosis disease in Thailand [3] was used

92

in this study. The bacteria was recovered from glycerol stock and prepared as described

93

previously [2, 3]. The actual number of Fno used in challenge tests was evaluated through

94

tenfold serial dilution using a standard plate count method.

95

2.3. Experimental design

96

An experimental design for investigating the Fno transmission in the present study is illustrated

97

in Fig. 1. In order to obtain subclinically infected broodstock, a sub-lethal dose of the Fno isolate

98

VMCU-FNO131 (2.88 x 105 CFU mL-1) previously identified from a median lethal dose (LD50)

99

was used [9]. Using this dose, 18 male and 18 female fish were immersed for 30 min in two 20-L

100

tanks containing the bacterium before being transferred to two 1-m3 tanks. At 10-day post

101

challenge (dpc), 4 males and 4 females were randomly collected for confirmation of presence of

102

the Fno infection. The remaining broodstock were observed and maintained for use in the

103

mating experiment.

104

To investigate fish maturity, the broodstock were starved for one day before being checked

105

individually. The males that showed reddish color of protruded papilla and the females that

106

released eggs after wiping their abdomen were selected for breeding [10]. Each pair of a total

107

of three pairs of the broodstock were then transferred to a 50-L glass aquarium tank in a flow

108

water system with a water temperature of 26.5 ± 0.5 oC. To encourage breeding, 50% of the

109

water in the tanks was changed daily. Behavior of the fish was monitored continuously until

110

eggs were spawned, fertilized, and scooped into female mouths naturally in the tank. These

111

events occurred approximately 5 to 6-week post Fno challenge. The fertilized eggs were then

112

collected from the female’s mouth and washed with water that was treated by ultraviolet light

113

(UV) one week before using. Subsequently, the eggs were artificially incubated in round-

114

bottomed hatching chamber as previously described. [11]. After hatching period, the larvae of

115

each family were cultured in a 50-L aquarium tank with filtered chlorine treated water for 30

4

116

days. Water parameters were checked daily during the experiment period. The larvae were fed

117

with powdered feed (28% protein, CP) twice a day.

118

After mating and fertilized eggs were collected from the mouth of female broodfish, the parental

119

fish were humanly terminated for Fno diagnosis. The collected samples in this task included

120

spleen tissues (50 mg) and reproductive organs (50 mg) of individual broodfish, pool of 10

121

fertilized eggs, 10 yolk-sac larvae, 10 five-day old fry, and 10 thirty-day old fry from each

122

family. Three sets of the samples were prepared and used for i) bacterial isolation, ii)

123

preservation in 95% ethanol for PCR detection and iii) preservation in 10% buffered formalin for

124

histology and in situ hybridization (ISH) assay (see below).

125

In this experiment, a non-infected control family of hybrid red tilapia was treated in the same

126

manner and respective samples of reproductive organs, fertilized eggs, yolk-sac larvae and fry

127

were preserved for PCR analysis.

128

2.4. Bacterial isolation

129

Spleen of the unchallenged fish as well as spleen, ovary and testis of broodstock, pooled

130

fertilized eggs, larvae, and fry fish from the 3 breeding families were aseptically collected and

131

washed carefully in distilled water three times. The samples were then homogenized in 100 µL

132

of normal saline solution. The obtained suspension was streaked on selective cysteine heart agar

133

(CHA) plates supplemented with 10% sheep blood, polymyxin B 100 IU mL-1 and ampicillin 50

134

µg mL-1 [2]. Plates were incubated at 28 oC and observed for Fno growth daily for 5 days.

135

2.5. DNA extraction and Fno PCR detection

136

The sample set preserved in alcohol described above was individually ground in 60 µL of Tris-

137

EDTA (TE) buffer and heated at 65 oC for 10 min. After a brief centrifugation, the upper layer

138

was subjected to DNA extraction using the Wizard® Genomic DNA Purification kit (Promega,

139

USA) according to the manufacturer’s instructions (Suppl. Fig.1). The DNA was then eluted

140

with nuclease-free water, quantified using the NanoDrop spectrophotometer (Thermo

141

Scientific), and tested for the presence of Fno using an improved PCR detection protocol.

142

One-tube semi-nested PCR assay was developed in this study to increase the Fno detection

143

sensitivity. The target sequence was a unique hypothetical protein gene sequence (GenBank

144

accession no. JQ780323) described to be specific for Fno strains [12]. A published primer pairs 5

145

FnoF1/FnoR1 (203 bp) [13] in combination with a newly designed primer FnoRev2 externally

146

targeting a larger fragment (375 bp) were used. A 25 µL of PCR reaction was composed of 12.5

147

µL of Master Mix (Go-Taq®Green, Promega USA); 4 µL of DNA template (150–200 ng); and

148

0.6, 0.4 and 0.4 µM of primer FnoF1 (5’- GGC GTA ACT CCT TTT AGC TTC C-3’), FnoR1

149

(5’- TTA GAG GAG CTT GGA AAA GCA-3’) and FnoRev2 (5’-AGG TAT GCA GTC TAC

150

TTC TAA TG-3’), respectively. PCR conditions consisted of initial denaturation at 94°C for 3

151

min; 40 cycles of amplification at 94°C for 30 s, annealing at 58°C for 30 min, and extension at

152

72°C for 30 min; final extension at 72°C for 5 min. Expected PCR products of 375 and 203 bp

153

were generated by FnoF1/FnoRev2 and FnoF1/FnoR1 primers, respectively. Amplified products

154

were electrophoresed with 1% agarose gel and visualized under UV light. The newly established

155

one-tube semi-nested PCR has the limit detection of 20 fg genomic DNA that is 100-fold more

156

sensitive than a 203 bp-single PCR (Suppl. Fig. 1). This protocol exhibited no cross-

157

amplification to DNA extracted from a healthy hybrid red tilapia and 9 common fish bacterial

158

pathogens (Streptococcus agalactiae, S. iniae, Flavobacterium columnare, Aeromonas veronii,

159

A. hydrophila, A. schubertii, Edwardsiella ictaluri, E. tarda and Hahella chejuensis) recovered

160

from diseased fish (Suppl. Fig. 2).

161

2.6. Histology and in situ hybridization

162

The sample set of spleen, ovary, testis of the broodfish, and their offspring from each family

163

preserved in 10% neutral buffered formalin was used for histological assessment. The samples

164

were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined

165

under a light microscope. Representative samples from 2 families were further subjected to ISH

166

assay. ISH was performed as previously described [14]. The 203 bp Fno-specific probe was

167

prepared using Fno VMCU-FNO131 extracted DNA as template in a PCR DIG labelling

168

reaction according to a manufacturer’s protocol (Roche Molecular Biochemicals). Negative

169

control were sections processed using same manner without adding the DIG-labeled probe.

170

3. RESULTS

171

3.1. Establishment of subclinically Fno-infected broodstock in the laboratory

172

Using a sub-lethal dose of Fno for immersion challenge, only two fish died at 10 dpc during 6

173

weeks-period while the majority of the experimental broodfish appeared to be unaffected

174

externally. Eight out of the 34 remaining fish were then randomly selected for histopathological 6

175

and PCR examination. The internal organs of these fish were abnormally enlarged with the

176

presence of white nodules in the spleen and head kidney, a typical sign of francisellosis.

177

Additionally, the spleens of all examined fish were positive for Fno by specific PCR test (Suppl.

178

Fig. 3). The results indicated that a population of subclinically Fno-infected broodfish was

179

successfully established in the laboratory.

180

3.2. Evidence of Fno in the gonad tissues of broodfish

181

Externally, all 3 pairs of the infected broodfish still showed normal appearance post challenge.

182

Internally, the spleen, liver, and head kidney were enlarged. Presence of white nodules-like

183

granulomas was noticed on the ovaries of all 3 female broodfish but were not seen on the testes

184

(Suppl. Fig. 4). Using a newly established one-tube semi-nested PCR assay, it was shown that

185

the spleen and gonad tissues of 6 broodfish from 3 infected families tested positive for Fno (Fig.

186

2, lanes 1-4). DNA sequences of representative 203 bands were sequenced and exhibited 100%

187

identity to Fno sequences in the GenBank database. Respective samples from non-infected

188

control broodfish tested negative for TiLV (Fig. 2, lane 1-4).

189

Histopathologically, presence of granulomas forming feature, the typical feature of francisellosis

190

was not observed in the testis and ovary but clearly presented in the spleen of all broodfish

191

(Table 1, Suppl. Fig 5). ISH results shown in Fig. 3 using an Fno-specific probe confirmed the

192

results obtained with the PCR assay. Reactive signals were detected in oocyte cytoplasm and

193

their membranes as well as various locations in the testis of broodfish (Fig. 3). With respect to

194

bacterial isolation, Fno was not successfully cultured from the spleen, ovary or testis of the

195

broodfish using CHA medium, a selective medium for Fno.

196

3.3. Detection of Fno in different developmental stages of the infected broodfish’s offspring

197

The samples derived from Fno-infected broodfish’s offspring including fertilized egg, yolk-sac

198

larvae, 5-day old fry, and 30-day old fry were also tested for the presence of Fno by bacterial

199

isolation, PCR and ISH. Similar to broodfish samples, Fno was unable to be cultured from the

200

offspring samples using CHA medium. Despite no visually abnormal signs being noticed in all

201

development stages of the offspring, all of them tested positive for Fno using specific PCR

202

(Table 1, Fig. 2). All tested samples yielded 203 bp-nested products (Fig. 2, lanes 5-8),

203

indicating low bacterial loads in the tissues. Sequencing of representative PCR products revealed

204

100% identity to the target sequence of PCR assay (supplementary data). Consistent with the 7

205

PCR results, the ISH using Fno-specific probe revealed weak reactive signals in the larvae and

206

fry samples compared to no signals in sections from the control. Representative ISH staining of

207

the offspring samples are shown in Fig. 3.

208

4. DISCUSSION

209

In nature, as a mouth brooder fish, female tilapia incubate eggs in their mouth until hatching.

210

During the intensive aquaculture practices, artificial incubation and hatching of fish embryos in

211

water recirculation systems significantly supports large scale production of tilapia fry.

212

Subclinical infection with infectious agent(s) is of concern not only for the health of the

213

broodstock themselves but also for possible pathogen transmission to their fry. Potential vertical

214

transmission of Fno and other tilapia bacterial pathogens including Shewanella putrefaciens,

215

Streptococcus agalactiae, and S. iniae was previously reported from healthy red tilapia

216

broodstock without clinical symptoms [16, 17]. In the mentioned studies, broodfish from

217

hatcheries with history of bacterial infections were used for in vitro fertilization. Interesting,

218

even though not all pairs of the parents were Fno positive, all their progeny at late stage i.e. 30-

219

day old fry were tested positive for Fno by LAMP detection. Additionally, concurrent

220

transmission of S. putrefaciens was co-investigated together with Fno in the same sample sets

221

[16]. The present study investigated a single transmission of Fno using experimentally infected

222

broodfish. Consequently, the presence of Fno in the reproductive organs of the brooders and

223

their offspring was confirmed by a combination of PCR and ISH assays. Note that this

224

experiment was set up to mimic current practice in tilapia hatcheries where fertilized eggs were

225

collected from the mouth of female broodfish for incubation. Therefore, transmission of Fno

226

from the infected broodfish to their offspring might take place in either reproductive organ

227

(direct vertical transmission) or during incubation in the mouth of broodfish (indirect vertical

228

transmission). Despite the fact that truly vertical transmission requires further investigation, this

229

study suggests that by using Fno subclinically infected tilapia mouthbrooders for fry production,

230

their offspring will be likely infected with this pathogen through either direct or indirect vertical

231

transmission.

232

This work also supported other studies [5, 7, 8, 17] that showed that Fno could be detected in

233

reproductive organs and/or gametes of infected tilapia, apart from spleen, kidney, and liver, the

234

main target organs [3, 14, 17]. Thus, non-lethal sampling of eggs and semen from broodstock 8

235

might be practical for monitoring this pathogen in tilapia hatcheries thereby allowing selection of

236

the specific pathogen free (SPF) broodfish for fry production.

237

In conclusion, this study revealed that with the current practice in tilapia hatcheries, Fno is likely

238

transmitted from subclinically infected broodstock of hybrid red tilapia to their progeny. Fno

239

could be found in reproductive organs of broodfish and different development stages including

240

embryo, yolk-sac larvae and fry fish. The results also implied that SPF broodfish should be

241

considered for production of Fno-free fry.

242

Acknowledgements

243

The research was supported by the 90th anniversary of Chulalongkorn University fund

244

(Ratchadaphiseksomphot Endowment Fund) and the 100th anniversary of Chulalongkorn

245

University, THAILAND, fund for doctoral scholarship to V.V. Nguyen. Additionally, some part

246

of research was supported by Grant from Fish Infectious Diseases Research Unit (FID RU),

247

faculty of Veterinary Science, Chulalongkorn University, THAILAND.

248

Conflict of interest

249

The authors declare no conflict of interest.

9

250

References

251

[1] Colquhoun DJ, Duodu S. Francisella infections in farmed and wild aquatic organisms. Vet

252

Res. 42 (2011) 47, https://doi.org/10.1186/1297-9716-42-47.

253

[2] Soto E, Hawke JP, Fernandez D, Morales JA. Francisella sp., an emerging pathogen of

254

tilapia, Oreochromis niloticus (L.), in Costa Rica. J Fish Dis. 32 (2009) 713-22,

255

https://doi.org/10.1111/j.1365-2761.2009.01070.x.

256

[3] Nguyen VV, Dong HT, Senapin S, Pirarat N, Rodkhum C. Francisella noatunensis subsp

257

orientalis, an emerging bacterial pathogen affecting cultured red tilapia (Oreochromis sp.) in

258

Thailand. Aquac Res. 47 (2016) 3697-702, https://doi.org/10.1111/are.12802.

259

[4] Jantrakajorn S, Wongtavatchai J. Francisella Infection in Cultured Tilapia in Thailand and the

260

Inflammatory

261

https://doi.org/10.1080/08997659.2015.1135198.

262

[5] Soto E, Kidd S, Mendez S, Marancik D, Revan F, Hiltchie D, et al. Francisella noatunensis

263

subsp. orientalis pathogenesis analyzed by experimental immersion challenge in Nile tilapia,

264

Oreochromis

265

https://doi.org/10.1016/j.vetmic.2013.01.024.

266

[6] Soto E, Abrams SB, Revan F. Effects of temperature and salt concentration on Francisella

267

noatunensis subsp orientalis infections in Nile tilapia Oreochromis niloticus. Dis Aquat Organ.

268

101 (2012) 217-23, https://doi.org/10.3354/dao02533.

269

[7] Mauel MJ, Soto E, Moralis JA, Hawke J. A piscirickettsiosis-like syndrome in cultured Nile

270

tilapia in Latin America with Francisella spp. as the pathogenic agent. J Aquat Anim Health. 19

271

(2007) 27-34, https://doi.org/10.1577/H06-025.1.

272

[8] Ortega C, Mancera G, Enriquez R, Vargas A, Martinez S, Fajardo R, et al. First identification

273

of Francisella noatunensis subsp orientalis causing mortality in Mexican tilapia Oreochromis

274

spp. Dis Aquat Organ. 120 (2016) 205-15, https://doi.org/10.3354/dao02999.

275

[9] Nguyen VV. Experimental infection of Francisella noatunensis subsp. orientalis strain

276

VMCU-FNO131 in red tilapia (Oreochromis sp.), MSc Thesis, Chulalongkorn University, 2015.

277

http://cuir.car.chula.ac.th/handle/123456789/45772.

Cytokine

niloticus

Response.

(L.).

J

Aquat

Vet

Anim

Microbiol.

10

Health.

28

164

(2016)

(2013)

97-106,

77-84,

278

[10] Rothbard S, Pruginin Y. Induced spawning and artificial incubation of Tilapia. Aquaculture.

279

5 (1975) 315-21, https://doi.org/https://doi.org/10.1016/0044-8486(75)90052-6.

280

[11] P.J P, Srijaya T, Mithun S, Shaharom F, Chatterji A. Seed production and hatchery

281

management techniques in tilapia2011.

282

[12] Duodu S, Larsson P, Sjodin A, Soto E, Forsman M, Colquhoun DJ. Real-time PCR assays

283

targeting unique DNA sequences of fish-pathogenic Francisella noatunensis subspecies

284

noatunensis

285

https://doi.org/10.3354/dao02514.

286

[13] Dong HT, Nguyen VV, Kayansamruaj P, Gangnonngiw W, Senapin S, Pirarat N, et al.

287

Francisella noatunensis subsp orientalis infects striped catfish (Pangasianodon hypophthalmus)

288

and common carp (Cyprinus carpio) but does not kill the hosts. Aquaculture. 464 (2016) 190-5,

289

https://doi.org/10.1016/j.aquaculture.2016.06.033.

290

[14] Dong HT, Gangnonngiw W, Phiwsaiya K, Charoensapsri W, Nguyen VV, Nilsen P, et al.

291

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella

292

noatunensis subsp orientalis in red tilapia. Dis Aquat Organ. 120 (2016) 39-47,

293

https://doi.org/10.3354/dao03021.

294

[15] Soto E, Fernandez D, Hawke JP. Attenuation of the Fish Pathogen Francisella sp. by

295

Mutation of the iglC* Gene. Journal of Aquatic Animal Health. 2009;21:140-9.

296

[16] Pradeep PJ, Suebsing R, Sirthammajak S, Kampeera J, Jitrakorn S, Saksmerprome V, et al.

297

Evidence of vertical transmission and tissue tropism of Streptococcosis from naturally infected

298

red

299

https://doi.org/10.1016/j.aqrep.2015.12.002.

300

[17] Pradeep PJ, Suebsing R, Sirithammajak S, Kampeera J, Turner W, Jeffs A, et al. Vertical

301

transmission and concurrent infection of multiple bacterial pathogens in naturally infected red

302

tilapia (Oreochromis spp.). Aquac Res. 48 (2017) 2706-17, https://doi.org/10.1111/are.13102.

tilapia

and

orientalis.

(Oreochromis

Dis

spp.).

Aquat

Aquacult

11

Organ.

Rep.

101

3

(2012)

(2016)

225-34,

58-66,

303

Table and Figures

304

Table 1. Detection of Fno from broodstock and different developmental stages of their offspring

305

using specific PCR, in situ hybridization (ISH), and granulomas pathology (G). Reproductive organs

Family

Ovary

Development stages

Testes

Fertilized eggs

5 and 30-day old fry

PCR

ISH

G

PCR

ISH

G

PCR

ISH

G

PCR

ISH

G

PCR

ISH

G

1

+

+

-

+

+

-

+

+

-

+

+

-

+

+

-

2

+

+

-

+

+

-

+

+

-

+

+

-

+

+

-

3

+

ND

ND

+

ND

ND

+

ND

ND

+

ND

ND

+

ND

ND

Control

-

ND

ND

-

ND

ND

-

ND

ND

-

ND

ND

-

ND

ND

306 307

Yolk-sac larvae

(+), positive; (-), negative; ND, not determined.

12

308 309 310 311 312 313 314 315 316 317 318 319

Figure 1: Experimental design for investigating the transmission of F. noatunensis subsp.

320

orientalis (Fno) from hybrid red tilapia broodstock (Oreochromis sp.) to their offspring. The

321

broodstock were immersed with an under-lethal dose of Fno before being selected to mate and

322

produce fry. The fertilized eggs were collected from females’ mouth for artificial incubation until

323

the late fry stage. The samples of each family including spleen, ovary, testis of the broodfish,

324

fertilized eggs, yolk sac, 5-day old fry and 30-day old fry were analyzed for the presence of Fno

325

using bacterial culture, PCR and ISH assay.

13

326 327

Figure 2: Detection of Fno in different life stages of three infected families and one non-infected

328

control family of hybrid red tilapia using specific PCR. M, DNA Marker; 1, ovary; 2, testis; 3,

329

spleen of female; 4, spleen of male; 5, fertilized eggs; 6, yolk-sac larvae; 7, 5-day old fry; 8, 30-

330

day old fry; +ve, positive control using Fno extracted DNA as template; -ve, no template control.

331

Note that ~700 bp band derived from cross hybridization of amplified products.

14

332

15

333

Figure 3: Photomicrographs of ISH results of the reproductive organs (A-D) of broodfish and

334

representative different development stages of their progeny (E-H). Arrows indicated reactive

335

signals of ISH in oocyte membrane (B), in different locations of the testis (C), yolk-sac larvae

336

(F) and gill filaments of 30-day old fry (H). Consecutive sections without probe are shown on the

337

left panel.

16

Highlights: •

Subclinically infected hybrid red tilapia broodstock were experimentally established

•

The presence of Fno in the gonad tissues of the broodfish was confirmed by PCR and ISH

•

Transmission of Fno from infected broodstock to their progeny was experimentally proven

Running title: Transmission of F. noatunensis subsp. orientalis