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1 1 IDENTIFICATION OF BACTERIAL AGENT(S) FOR ACUTE HEPATOPANCREATIC 2 NECROSIS SYNDROME, A NEW EMERGING SHRIMP DISEASE 3 4 5 Jyoti Joshia,b, Jiraporn Srisalac, Waraporn Sakaewb, Anuphap Prachumwat b,c, Kallaya Sritunyalucksanaa,b,c, Timothy W. Flegelb and Siripong Thitamadeea,b,* 6 7 a Department of Biotechnology and bCenter of Excellence for Shrimp Molecular Biology and 8 Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, 9 Thailand 10 c 11 Biotechnology, National Science and Technology Development Agency, Rama VI Rd. Bangkok, 12 10400, Thailand 13 *Corresponding author: Siripong Thitamadee 14 Email: [email protected]. Tel: 02 201 5867, Fax: 02 354 7344 15 Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, 16 Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand. Tel: +66 2 201 5878, Fax: +66 2 17 3547344. Shrimp-Virus Interaction Laboratory, National Center for Genetic Engineering and 18 19 ABSTRACT 20 21 A new emerging shrimp disease known as acute hepatopancreatic necrosis syndrome 22 (AHPNS) has been reported to cause significant losses among shrimp farms in China (2009), 23 Vietnam (2010), and Malaysia (2011). Recently, it has been reported to affect shrimp (Penaeus 2 24 monodon and P. vannamei) in the eastern Gulf of Thailand (2012). The disease is characterized 25 by mass mortalities (reaching up to 100% in some cases) during the first 20-30 days post- 26 stocking in grow-out ponds. The apparent spread of AHPNS throughout the region suggests that 27 infectious or at least biological agent may be involved. Shrimp suffering from AHPNS show 28 significant atrophy of hepatopancreas (HP), pale to white HP due to pigment loss in the 29 connective tissue capsule and guts with discontinuous or no contents. The purpose of this study 30 was to identify if bacteria could be the cause of AHPNS. DNA samples were prepared either 31 directly from hepatopancreatic tissue or from culturable bacteria isolated from hepatopancreas of 32 the diseased shrimp as templates to specifically amplify 770 bp fragment of 16S rRNA gene for 33 bacterial classification. Nucleotide sequences obtained from the direct DNA extraction of the 34 hepatopancreas revealed majority of Vibrio species, including V. parahaemolyticus, V. harveyi, 35 V. vulnificus, and V. chaqassi. Similarly, the consensus sequences obtained from nine culturable 36 bacterial isolates were identified as V. parahaemolyticus. The healthy shrimps challenged with 37 the selected Vibrio isolate at a dose of 10³ CFU per shrimp showed high mortality within 6 hours 38 after injection, however AHPNS histopathology was not observed. 39 40 Keywords: Penaeid shrimp, acute hepatopancreatic necrosis syndrome (AHPNS), 16S rRNA 41 gene, Vibrio species. 42 43 INTRODUCTION 44 45 The pacific white leg shrimp, Penaeus (Litopenaeus) vannamei is the world’s most 46 extensively cultivated species of penaeid shrimp. The rapid expansion of the shrimp farming 3 47 industry in Thailand has been suffering from many serious infectious diseases such as white spot 48 syndrome virus (WSSV), yellow head virus (YHV), etc. Recently, a new emerging disease 49 known as Early Mortality Syndrome (EMS), descriptively called as Acute Hepatopancreatic 50 Necrosis Syndrome (AHPNS), has been a major issue of concern for economic loss in the shrimp 51 farming industry. EMS was officially reported in Southern China (2009), Vietnam and Malaysia 52 (2011) and reached the eastern coast of Thailand in late 2011. In early 2012, EMS/AHPNS was 53 reported mostly in the coastal provinces of Thailand including Rayong, Chantaburi, 54 Chachoengsao, and Prachuap Khiri Khan. Both Penaeus monodon and Penaeus vannamei are 55 susceptible to this disease. As the name suggested, the disease causes high and rapid mortality in 56 20 to 30 days post-culture shrimp in the grow-out ponds of eastern provinces. Shrimp suffering 57 from EMS/AHPNS show significant atrophy of hepatopancreas (HP), pale to white HP due to 58 pigment loss in the connective tissue capsule, often soft shells and discontinuous or no gut 59 contents. Some pathological features include enlarged hepatopancreatic nuclei, sloughed HP 60 cells—blister-like (B), fibrilla (F), and resorptive (R) cells and frequently followed by secondary 61 bacterial infections (www.enaca.org). However, the potential cause(s) of the disease is still under 62 investigation. 63 The purpose of this study was to identify if the bacteria could be the causative agent(s) of 64 EMS/AHPNS through microscopic examination, sequence analysis of bacterial isolates, and 65 challenge tests. 66 67 MATERIALS AND METHODS 68 69 Sample collection 4 70 P. vannamei suffering from EMS were collected from KO shrimp farm (KO represents 71 name code for the particular farm) at Sam Roi Yod, Prachuap Khiri Khan province. Shrimps 72 were transported to the laboratory in plastic containers with proper aeration and temporarily 73 reared indoor using 48x70x41 cm plastic tanks. Upon arrival to the laboratory, some shrimp 74 specimens were immediately fixed in Davidson’s fixative (32% EtOH, 22% formalin and 11% 75 acetic acid in distilled water) and processed for histological examination. 76 77 Bacterial identification by 16S rDNA analysis 78 79 The HP of each individual lived EMS shrimps were removed by using sterile scissors and 80 subsequently excised into two pieces. One piece of the tissue was ground in lysis buffer for DNA 81 extraction. The second piece of hepatopancreatic tissue was used for bacterial isolation on the 82 agar plate (section 2.3). DNA was extracted from the frozen HP tissue and used as templates for 83 PCR amplification with16S rRNA gene-specific primers 40F: gCCTAACACATgCAAgTCgA 84 and 802R: gACTACCAgggTATCTAATCC. The amplification condition was performed by pre- 85 denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 86 seconds, 72°C for 1 minute, and a final elongation step at 72°C for 10 minutes. Amplified PCR 87 products were cloned into pGEM-T EASY Vector (Promega) and transformed into E. coli 88 JM109. The transformed cells were plated on LB agar containing 50 µg/ml ampicillin. The 89 positive clones were confirmed by colony PCR and 50 clones were picked for sequencing 90 analysis (Macrogen, Korea). The nucleotide sequences data were subjected to BLAST sequence 91 analysis against NCBI databases for bacterial identification. 92 5 93 Culturable bacterial identification by 16S rDNA analysis 94 95 The excised HP was disaggregated and streaked on tryptic soy agar plates (TSA 96 supplemented with 1.5% NaCl) using sterile loop. The streaked plates were incubated at 30°C 97 for 16 hours to allow the culturable bacteria to grow. Each individual colony obtained were re- 98 streaked to obtain the pure isolate before storing in glycerol stocks at -80°C for later use. The 99 culturable bacteria obtained were identified by 16S rDNA analysis. DNA was extracted from the 100 pure bacterial isolates using phenol-chloroform extraction method. The DNA template was 101 amplified by PCR reaction according to the conditions mentioned elsewhere. The amplified PCR 102 products were analysed for their nucleotide sequences by Macrogen, Korea and subjecting to 103 BLAST sequence analysis against NCBI databases for bacterial identification. 104 105 Bacterial challenge test 106 107 For bacterial preparation, the selected V. parahaemolyticus isolate, KO HP03 (HP03) was 108 cultured in tryptic soy broth (TSB) containing 1.5% NaCl at 30°C with vigorous shaking until 109 reaching OD600 of 0.6-0.8. The bacterial cells were collected at 3,000 rpm for 10 min prior to 110 diluting with 0.1 M PBS (pH 7.4) at the concentration of 10³ CFU per 50 µl. 111 The specific pathogen free (SPF) P. vannamei shrimps with body weight of 12-15 g 112 (Syaqua, Thailand) were transported to the lab and reared temporarily in plastic containers for 4 113 days before challenge test. For challenge trials, 20 shrimps each were used for either injection 114 with 1) HP03 bacteria or 2) phosphate buffer saline (PBS) control, with two replicates of 10 115 shrimps in each tank. Shrimps were intramuscularly injected with HP03 into the third abdominal 6 116 segment at concentration of 10³ CFU per shrimp. The equal volume of PBS (50 µl) was injected 117 into control shrimps at the same position. The shrimps were observed for the histopathological 118 signs of EMS/AHPNS and recorded for mortality rate after injection. 119 120 Construction of phylogenetic trees 121 122 Multiple sequences alignment by MUSCLE was used for phylogenetic tree construction 123 in MEGA 5 with Neighbor-Joining method (5000 bootstrap replicas). For the culturable bacteria 124 samples, consensus sequences used for multiple sequences alignment were generated by CAP3. 125 The average read-length of 16S rRNA amplified gene product was 770 bp. 126 127 RESULTS 128 129 Histopathological Analysis 130 131 Histological analysis of the samples obtained from the KO farm at Sam Roi Yot, 132 Prachuap Khiri Khan province showed obvious histological lesions of typical AHPNS symptoms 133 including sloughed hepatopancreatic cells (Fig. 1C) and followed by haemocytic infiltration (Fig. 134 1D) as compared to the normal HP (Fig. 1A and B). In addition to the histological analysis, the 135 diseased shrimps harvested from KO farm were parallelly processed for further bacterial 136 isolation and identification. 137 138 7 139 Identification of the bacterial species in diseased HP by sequence analysis of 16S rRNA 140 amplified PCR products 141 142 Among 50 clones, 37 clones were identified as V. parahaemolyticus, while 3 clones were 143 as V. vulnificus, V. harveyi, and V. chaqassi. The remaining 10 clones were excluded from 144 BLAST analysis due to non-specific sequences and short-read length. Phylogenetic tree was 145 generated and shown in Fig. 2A. 146 147 Identification of culturable bacteria isolated from diseased HP 148 149 After 16 hours of incubation at 30°C, the bacterial colonies with white and opaque 150 appearance appeared on non-selective TSA plates were randomly picked and cultured on 151 thiosulfate citrate bile salt sucrose (TCBS) for selective screen of the Vibrio species. All tested 152 bacterial colonies were able to grow on TCBS and identified as gram-negative rod-shaped 153 bacteria (data not shown). A consensus sequence of nine bacterial isolates was produced from 154 the sequence information of 3-4 clones per each isolate. The sequence homology of each isolate 155 was identified as V. parahaemolyticus. The phylogenetic construction revealed a possible sub- 156 grouping of V. parahaemolyticus among the nine isolates (Fig. 2B). 157 158 Confirmation of the causative pathogen 159 160 In order to identify whether the V. parahaemolyticus isolates obtained form KO farm was 161 the causative pathogen, SPF shrimps were collected and divided into two groups. The SPF 8 162 shrimps were randomly injected by V. parahaemolyticus isolate HP03 (Fig. 2B) and PBS for the 163 HP03 treatment and the control, respectively. The shrimps injected with HP03 were died within 164 6 hours after injection (19/20), whereas no mortality was observed in the shrimps injected with 165 PBS. The HP of moribund (dying) shrimps was prepared for histological analysis. Pathological 166 lesions found in the shrimp injected with V. parahaemolyticus showed partial collapse of the cell 167 types which normally appeared in hepatopancreatic lobes (Fig. 3B) as compared to control (Fig. 168 3A). The histopathological results suggested that, by intramuscular injection, the selected isolate 169 of V. parahaemolyticus is pathogenic to shrimp but unlikely to be the direct cause of 170 EMS/AHPNS since there was no typical cellular defects unique to the disease. 171 172 DISCUSSION 173 174 Vibrio species are the most frequent bacterial pathogens detected in the cultivated shrimp 175 (Zhou et al., 2012). Our results showed that V. parahaemolyticus was the major bacterial 176 population found in the diseased shrimps with the signs of EMS/AHPNS. However, due to 177 highly conserved sequences of 16S rRNA gene among the Vibrio species, to gain a precise 178 classification of the isolated bacteria at species level would be problematic. According to the 179 phylogenetic construction of the culturable Vibrio isolates, we are currently examining the 180 correlation between each phylogenetic sub-group and its ability to cause EMS/AHPNS in 181 challenged shrimps. We initially examined the virulence of the selected HP03, which was able to 182 cause high mortality at a dose of 10³ CFU per shrimp within 6 hours post injection, indicating 183 that the isolates were highly virulent and lethal. Although these Vibrios were isolated from 9 184 shrimp with AHPNS, the injection of the selected bacteria did not induce EMS/AHPNS in the 185 challenged shrimps. 186 Based on the histological analysis of EMS/AHPNS pathology, a series of infectivity 187 studies were designed to identify the source and mode of infection (Tran et al., 2013). Since the 188 challenge test by intramuscular injection could not reproduce EMS/AHPNS, natural transmission 189 routes might be crucial for induction of the typical EMS/AHPNS pathology in hepatopancreas. 190 Recently, Lightner et al. (2012) has identified the EMS/AHPNS pathogen as a unique strain of a 191 relatively common bacterium, V. parahaemolyticus, by immersion route. Therefore, immersion 192 assay could be a promising way to reproduce EMS/AHPNS with our Vibrio isolates as well. 193 194 CONCLUSION 195 The causative bacterial agent(s) of EMS/AHPNS is still under investigation. Sequencing 196 results of 16S rRNA gene from the diseased shrimp revealed majority of V. parahaemolyticus. 197 Intramuscularly injection of the selected V. parahaemolyticus isolate could not induce 198 EMS/AHPNS in naïve shrimp. 199 200 ACKNOWLEDGEMENTS 201 This work was supported by grants from National Research Council of Thailand and Department 202 of Biotechnology, Mahidol University. We also thank Syaqua Thailand for the experimental 203 materials. The authors would like to convey special appreciation to the academic committee of 204 25th Annual Meeting of Thai Society for Biotechnology and International Conference (TSB2013) 205 for providing opportunity for this work to be published in this journal. 10 206 LITERATURE CITED 207 Flegel, T.W. (2012). Historic emergence, Impact and current status of shrimp pathogens in Asia. 208 209 210 J Invertebr Pathol. 110: 166-173. Lightner, D.V., R.M., Redman, C.R., Pantoja, B.I., Noble and L. Tran. (2012). Early mortality syndrome affects shrimp in Asia. Global Aquaculture Advocate. 15: 40. 211 Tran, L., L., Nunan, R.M., Redman, L.L., Mohney, C.R., Pantoja, K., Fitzsimmons and D.V. 212 Lightner, (2013). Determination of the infectious nature of the agent of acute 213 hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis Aquat Organ. 105: 214 45-55. 215 Zhou J, Fang W, Yang X, Zhou S, Hu L, et al. (2012). A Nonluminescent and Highly Virulent 216 Vibrio harveyi Strain Is Associated with ‘‘Bacterial White Tail Disease’’ of Litopenaeus 217 vannamei Shrimp. PLOS ONE. 7(2): e29961. 218 219 220 221 222 223 224 225 226 227 228 11 229 230 Figure1. Hepatopancreatic tissues obtained from P. vannamei showing an intact morphology of 231 B, F, and R cells in hepatopancreatic lobes (A and B). Histological changes in the 232 hepatopancreas of shrimp affected by EMS/AHPNS (C and D). Hepatopancreatic defects 233 showing sloughed B, F and R cells (C). Haemocytic infiltration (D). Hepatopancreatic sections in 234 A-B and C-D were obtained from SPF and EMS/AHPNS shrimps harvested from KO farm, 235 respectively. Scale bars, 200 µm (A, C and D), 100 µm (B). 236 237 238 239 12 240 241 242 243 244 245 246 247 248 13 249 250 251 Figure2. Phylogenetic tree of bacterial species in diseased HP by sequence analysis of 16S 252 rRNA gene amplified PCR products (A). Phylogenetic tree of culturable bacteria isolated from 253 diseased HP (B). Numbers at the branching point indicate percent bootstrap value. Numbers in 254 bold indicate arbitrary bacterial grouping. The capital letters V, H, C, and P represent V. 255 vulnificus, V. harveyi, V. Chaqassi, and V. parahaemolyticus, respectively. Scale bars denote 256 number of nucleotide differences. 257 258 259 260 261 262 263 264 265 14 266 267 Figure3. A typical histology of shrimp HP injected with PBS, the control (A) and with selected 268 V. parahaemolyticus, the HP03 treatment (B). Scale bars, 200 µm. 269