Aquaculture 470 (2017) 190–195 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture The impact of catecholamine sensing on the virulence of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) Nguyen Thao Suong a, Nguyen Van Hao a, Nguyen Van Sang a, Nguyen Dinh Hung a, Nguyen Thi Ngoc Tinh a, Le Hong Phuoc a, Doan Van Cuong a, Nguyen Thanh Luan a, Do Viet Phuong b, To Thi Thom c, Phan Hong Thao c, Peter Bossier d, Patrick Sorgeloos d, Tom Defoirdt d,e,⁎ a Research Institute for Aquaculture No.2, 160 Nguyen Dinh Chieu, District 1, Ho Chi Minh City, Viet Nam Virbac Regional RDL Center Asia, 17 Ba Huyen Thanh Quan, District 3, Ho Chi Minh City, Viet Nam c Faculty of Bio-Food Technology and Environment, Ho Chi Minh University of Technology, 144/24, Dien Bien Phu, Binh Thanh District, Ho Chi Minh City, Viet Nam d Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Coupure Links 653, 9000 Gent, Belgium e Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links 653, 9000 Gent, Belgium b a r t i c l e i n f o Article history: Received 17 September 2016 Received in revised form 20 December 2016 Accepted 22 December 2016 Available online 02 January 2017 Keywords: Early mortality syndrome (EMS) Host-microbe interaction Microbial endocrinology Vibrio parahaemolyticus a b s t r a c t Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease that causes significant losses in the shrimp industry worldwide In 2013, specific strains of Vibrio parahaemolyticus were found to be responsible for AHPND Recently, inhibiting the detection of catecholamines has been reported to decrease the virulence of various pathogenic bacteria, including Vibrio anguillarum, Vibrio campbellii and human pathogenic Vibrio parahaemolyticus Thus, in this study we investigated whether catecholamine sensing has any effect on the virulence of an AHPND-causing V parahaemolyticus strain isolated from outbreaks in Vietnam We found that the catecholamines norepinephrine and dopamine (50 μM) increased motility of V parahaemolyticus (P b 0.05) Further, the catecholamine-induced motility could be neutralized by the prokaryotic catecholamine receptor antagonist LED209 Finally, pre-treatment of V parahaemolyticus with catecholamines significantly increased its virulence to whiteleg shrimp (Penaeus vannamei), and pretreatment with the antagonist LED209 neutralized this effect (P b 0.05) LED209 increased the survival of shrimp challenged with catecholamine-pretreated V parahaemolyticus to levels that were even higher than those observed in shrimp challenged with untreated V parahaemolyticus, suggesting that this type of compounds might be useful to decrease losses due to AHPND in shrimp © 2017 Elsevier B.V All rights reserved Introduction Outbreaks of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus, have been particularly devastating in the cultivation of shrimp in a number of countries, with global losses of more than $ billion per year (Tran et al., 2013; Han et al., 2015; Thitamadee et al., 2016) The disease has been reported to be responsible for an 80% decrease in shrimp production in Hainan, Guangdong, Fujian and Guangxi (China) during the first half of 2011 (Panakorn, 2012), a 33% decrease in Thai shrimp production during the first quarter of 2013 (Joshi et al., 2014), and about $ 118 million lost in Mexico in 2013 (Schryver et al., 2014) It has been estimated that 39,000 of shrimp ponds in the Vietnamese provinces Tra Vinh, Soc Trang, Bac ⁎ Corresponding author at: Center for Microbial Ecology and Technology (CMET), Coupure Links 653, B-9000 Gent, Belgium E-mail address: Tom.Defoirdt@UGent.be (T Defoirdt) http://dx.doi.org/10.1016/j.aquaculture.2016.12.030 0044-8486/© 2017 Elsevier B.V All rights reserved Lieu and Ca Mau in the Mekong delta were affected by AHPND in 2011, with mortality rates of up to 100% (FAO, 2013) Strategies to control AHPND mainly focus on prevention by pond renovation and disinfection This approach is not capable of stopping the epidemiological situation once the disease has started and has been argued not to be the most effective strategy to prevent AHPND (Schryver et al., 2014) Furthermore, the use of antibiotics against V parahaemolyticus to treat the disease has been documented to result in antibiotic resistance in the pathogen (Tran et al., 2015) Consequently, new approaches to tackle this problem (both preventive and curative) are urgently needed One option is to disrupt the mechanisms the pathogen needs to infect its host, a strategy that has been termed antivirulence therapy (Defoirdt, 2014) Catecholamines such as dopamine and norepinephrine are highly conserved in animals (both vertebrates and invertebrates) Elevated levels of these compounds have for a long time been associated with a decreased activity of the defense system of animals (including shrimp) N.T Suong et al / Aquaculture 470 (2017) 190–195 (Cheng et al., 2006) More recent evidence showed that catecholamines are also used as host cues to enhance the virulence of pathogenic bacteria (Lyte, 2004) In various Vibrio species, including V anguillarum, V harveyi and human pathogenic V parahaemolyticus, catecholamines have been reported to increase growth in media containing serum (which limits iron availability, thereby mimicking the in vivo situation), motility, biofilm formation and other virulence-related phenotypes (Nakano et al., 2007; Yang et al., 2014; Pande et al., 2015) Hence, catecholamine sensing might be an interesting target for the development of novel virulence inhibitors A specific inhibitor of bacterial catecholamine receptors, N-phenyl-4-{[(phenylamino)thioxomethyl]amino}benzenesulphonamide (LED209), has recently been reported (Rasko et al., 2008) Interestingly, LED209 also inhibited catecholamine-induced virulence in V harveyi, both in vitro and in vivo in a challenge test with gnotobiotic brine shrimp larvae (Yang et al., 2014) In this study, we investigated the impact of the catecholamines dopamine and norepinephrine and the bacterial catecholamine receptor antagonist LED209 on swimming motility (a virulence-related phenotype that is induced by catecholamines in many bacteria) in the Vietnamese AHPND-causing V parahaemolyticus isolate CM1 We further determined the impact of these compounds on the virulence of strain CM1 towards white leg shrimp (Penaeus vannamei) This work will lay the foundation for a better understanding of the factors that are involved in the AHPND epidemiology and provide a first step towards novel and innovative therapies to control the disease 191 India) and incubated at 28 °C under constant agitation (100 min−1) overnight Bacterial density was measured spectrophotometrically at 600 nm 2.3 Catecholamines and the inhibitor of bacterial catecholamine receptors LED209 The catecholamines dopamine and norepinephrine were obtained from Sigma (Bornem, Belgium) and the receptor inhibitor LED209 were obtained from Cayman Chemicals (Michigan, USA) Stocks of dopamine and norepinephrine (10 mM) were prepared in distilled water and distilled water containing 0.1 N HCl, respectively LED209 was dissolved in dimethyl sulfoxide at mM Dopamine and norepinephrine were tested at 50 μM and 100 μM each LED 209 was tested at 0.05 μM and 0.1 μM All stocks solutions were stored at −20 °C 2.4 Swimming motility assays Soft LB + plates containing 0.3% agar were used for performing swimming motility as described previously by Yang et al (2014) and Pande et al (2015) Overnight grown V parahaemolyticus was diluted to OD600 = 0.5, and 10 μl aliquots were spotted in the center of the soft agar plates Plates were incubated overnight, after which the diameters of the motility halos were measured 2.5 Preparation of experimental shrimp Materials and methods 2.1 Isolation and identification of V parahaemolyticus One hundred samples of AHPND-positive P vannamei shrimp were collected from September 2014 to December 2014 from the provinces Ca Mau, Tien Giang and Ben Tre in the Mekong delta, Vietnam At the collection time, aseptically excised tissue of the hepatopancreas (HP) was disaggregated and streaked on CHROMagar Vibrio agar (CHROMagar, Paris, France) plates After incubation at 28 °C overnight, violet colonies on CHROMagar Vibrio agar plates were selected (HaraKudo et al., 2001) Individual colonies were re-streaked onto Luria Bertani agar plates (Himedia, India) containing 2% NaCl (LB+) to obtain pure isolates These isolates were stored in glycerol at −80 °C For bacterial identification, pure isolates on LB+ plates were sent to Macrogen, Biochemistry Company, Vietnam to conduct 16S rDNA sequencing Acquired 16S rDNA sequences were aligned and compared with a collection of 16S rDNA sequences in GenBank by using the NCBI Basic Local Alignment Search Tools, nucleotide (BLASTn) program in order to verify high identity with Vibrio parahaemolyticus sequences PCR detection of AHPND bacteria was performed according to Joshi et al (2014), based on the amplification of unique DNA sequences of AHPND-causing V parahaemolyticus isolates that are not present in non-AHPND V parahaemolyticus isolates The forward (F) and reverse (R) primer sets that were used are AP2F: 5′ TCACCCGAATGCTCGCTTGTGG - 3′; and AP2R: 5′ - CGTCGCTACTGTCTAGCTGAAG - 3′ The cycling conditions were at 94 °C, followed by 30 cycles of 30 s denaturation at 94 °C, 30 s annealing at 60 °C and 60 s extension at 72 °C, plus a final 10 extension at 72 °C The amplified PCR products were analysed on 2% agarose gels, stained with ethidium bromide, and visualized under UV transillumination Isolate CM1 (16S rDNA sequence submitted to GenBank under accession number KX619616) was retained for further experiments This isolate gave positive PCR test results (bands at approximately 700 bp), while non-AHPND V parahaemolyticus gave a negative result 2.2 Growth conditions of AHPND-causing V parahaemolyticus CM1 Isolate CM1 was stored in glycerol at −80 °C and re-streaked on an LB + plate A single colony was inoculated into LB + broth (Himedia, Specific-pathogen free (SPF) P vannamei shrimp (2–5 g body weight) were purchased from commercial hatcheries (Vung Tau province, Vietnam) The animals were maintained in composite tanks containing aerated filtered seawater at 20 ppt salinity for week before the start of the experiments The shrimp health status was checked by monitoring swimming activity, luminescence, survival rate, muscle opaqueness, deformities, size variation, gut content, and colour of the hepatopancreas Formulated feed for shrimp weighing 1–5 g (Monotech, Cargill) was used for feeding the shrimp and airlifts were used to supply oxygen 2.6 Pathogenicity of V parahaemolyticus CM1 to P vannamei Shrimp pathogenicity experiments were performed according to the method described by Joshi et al (2014) with some modifications Briefly, groups of ten randomly selected healthy shrimp were transferred to tanks containing 15 l of 20 ppt filtered seawater at 28 ± 0.5 °C The shrimp were then challenged by inoculation of 5.107, 1.107, 6.106, or 2.106 CFU/ml V parahaemolyticus cells in the rearing water, respectively The control animals were maintained in fresh filtered seawater supplemented with a volume of LB broth equal to the V parahaemolyticus inoculum Gross signs of disease and cumulative mortality were recorded after 96 h Histopathological examination of moribund shrimp was performed using the methods described by Tran (2013) The average lethal dose (LD50) values were calculated as described by Reed and Muench (1938) Each experiment was conducted in triplicate 2.7 Impact of catecholamines and the inhibitor of the bacterial catecholamine receptor (LED209) on the virulence of V parahaemolyticus CM1 towards P vannamei Groups of 16, 20 or 30 healthy white leg shrimp were distributed to glass tanks containing 30 l of filtered sea water (20 g l− salinity) in experiment 1, and 3, respectively Shrimp were fed twice daily with a pelleted shrimp feed (Monotech, Cargill) In order to avoid a direct effect of catecholamines (50 μM) and LED209 (0.05 μM and 0.1 μM) on the animals, V parahaemolyticus was grown overnight in LB + with the compounds, after which the cultures were washed with autoclaved sea water (20 ppt) supplemented with 10% (v/v) 192 N.T Suong et al / Aquaculture 470 (2017) 190–195 of LB+ V parahaemolyticus cultures grown in unsupplemented LB + and treated in the same way as the other cultures, were used as controls The bacteria were inoculated into the rearing water at × 106 cells/ml The survival of the shrimp was counted daily Each treatment was carried out in triplicate 2.8 Statistical analyses All statistical analyses were done using the Statistical Package for Social Sciences (SPSS), version 16.0 Collected data were analysed using one-way ANOVA Significant differences between treatments were identified using Tukey's post hoc test Results 3.1 Pathogenicity of V parahaemolyticus isolate CM1 towards white leg shrimp (Penaeus vannamei) White leg shrimp were challenged with isolate CM1 by inoculating the strain into the rearing water The cumulative mortality of shrimp at 96 h after challenge was proportional to the concentration of bacteria added to the rearing water (Table 1), and the median lethal dose (LD50) was × 106 CFU/ml The infected shrimp died within 48 h after the appearance of clinical signs of disease The signs of disease included lethargy, whitish appearance of the stomach, pale, atrophied hepatopancreas and soft shells Histopathological examination indicated the presence of large numbers of bacteria in the hepatopancreas of the infected shrimp (Fig 1) The basal laminae of the hepatopancreatic tubules were ruptured and severe necrosis, loss of structure of the tubular epithelial cells were observed along with significant bacterial colonisation and remnants of hepatopancreatic tubules surrounded by hemocytic infiltration In contrast, the hepatopancreata of nonchallenged shrimp were normal, with a normal structure of tubules and epithelial cells 3.2 The impact of catecholamines on swimming motility of V parahaemolyticus CM1 Swimming motility of V parahaemolyticus CM1 was determined using soft LB + agar (0.3% agar) Both norepinephrine and dopamine significantly increased the swimming motility of the isolate (Fig 2) Remarkably, the effect was slightly stronger at 50 μM than at 100 μM for both catecholamines (data not shown) Furthermore, the bacterial catecholamine receptor antagonist LED 209 blocked norepinephrine- and dopamine-induced motility of V parahaemolyticus CM1 (Fig 2) 3.3 The impact of catecholamines and the bacterial catecholamine receptor antagonist LED209 on the virulence of V parahaemolyticus CM1 towards white leg shrimp In a last series of experiments, we investigated the impact of dopamine and norepinephrine (with or without the bacterial catecholamine receptor antagonist LED209) on the virulence of V parahaemolyticus CM1 towards white leg shrimp To avoid any direct impact of the Table Survival of P vannamei challenged with V parahaemolyticus isolate CM1, 96 h after addition of the strain into the rearing water (average ± standard deviation of three shrimp cultures) CM1 density (cells/ml) × × × × 10 107 106 106 Survival (%) 0±0 30 ± 10 53 ± 63 ± 100 ± Fig Histological sections of the hepatopancreas of Penaeus vannamei (10× magnification) (A) Hepatopancreas of nonchallenged shrimp showing a normal structure (B) Hepatopancreas of shrimp challenged to V parahaemolyticus isolate CM1 showing lesions A: normal tubule, B: tubule displaying atrophy, C: hemocytic infiltration, D: granuloma compounds on the host, V parahaemolyticus CM1 was pre-treated with the catecholamines (with or without LED209), after which the compounds were removed prior to inoculation into the shrimp rearing water Pre-treatment of V parahaemolyticus CM1 with catecholamines resulted in lower survival rates of challenged white leg shrimp when compared to the survival of shrimp challenged with untreated CM1 (Table 2) Interestingly, significant increases in survival rates of white leg shrimp were observed in the treatments in which V parahaemolyticus CM1 was pretreated with the bacterial receptor antagonist LED209, with survival rates that were even higher than those observed in shrimp challenged with untreated V parahaemolyticus CM1 (Table 2) Discussion In this study, we isolated a V parahaemolyticus strain, CM1, from AHPND outbreaks in Vietnam The isolate caused rapid, high mortality in white leg shrimp, and this was accompanied by histopathological signs of acute hepatopancreatic necrosis consistent with the case definition of AHPND (i.e., massive sloughing of HP tubule epithelial cells) (Tran et al., 2013; Joshi et al., 2014) The LD50 value of the isolate to white leg shrimp using an immersion challenge test was × 106 CFU/ml In another study, Joshi et al (2014) reported that the LD50 of another AHPND isolate, V parahaemolyticus 5HP, was 105 CFU/ml and that there was variation in virulence between different isolates of AHPND-causing V parahaemolyticus N.T Suong et al / Aquaculture 470 (2017) 190–195 193 Fig The impact of the catecholamines (A) norepinephrine (NE) and (B) dopamine (Dopa), with and without the bacterial catecholamine receptor antagonist LED209 on the swimming motility of V parahaemolyticus CM1 The error bars indicate the standard deviation of five replicates For each panel, different letters indicate significant differences (One way ANOVA with Tukey's post hoc test; P b 0.05) Upon entrance into a host, a pathogen encounters a new environment, and environmental changes can act as cues to initiate regulatory cascades that in turn affect (virulence) gene expression Molecules produced by the host, such as catecholamines, can also trigger virulence production in bacteria (Alksne and Projan, 2000; Burton et al., 2002; Karavolos et al., 2008; Yang et al., 2014; Pande et al., 2015) In this study, we demonstrated that the catecholamines norepinephrine and dopamine significantly induced the swimming motility of V parahaemolyticus CM1 Swimming motility has been considered as an important virulence factor in bacterial pathogens It is essential for pathogenic bacteria during the initial phases of infection as it helps them to overcome repulsive forces between the bacterial cell and the host tissues and hence, facilitates attachment to the host (McCarter, 2001) Furthermore, motility may increase the efficiency of nutrient acquisition, avoidance of toxic substances, and translocation to preferred host Table Survival of P vannamei after 96 h challenge with V parahaemolyticus CM1, either or not pre-treated with the catecholamines norepinephrine (NE) or dopamine (Dopa), and with or without the bacterial catecholamine receptor antagonist LED209 (average ± standard deviation of three replicate shrimp cultures) V parahaemolyticus CM1 was added to the shrimp rearing water at × 106cells/ml Norepineprhine and dopamine were used at 50 μM Treatment CM1 CM1 + NE CM1 + NE + LED 209 (0.05 μM) CM1 + NE + LED 209 (0.1 μM) CM1 + Dopa CM1 + Dopa + LED209 (0.05 μM) CM1 + Dopa + LED209 (0.1 μM) Survival (%)a Statisticsb Experiment Experiment Experiment 58 ± 15 38 ± 94 ± 15 54 ± 11 36 ± 82 ± 20 60 ± 14 35 ± 74 ± 12 B A C 83 ± 19 84 ± 17 93 ± 28 C 29 ± 73 ± 13 27 ± 84 ± 11 36 ± 12 76 ± 12 A C 71 ± 16 74 ± 11 81 ± C a No mortality was observed in non-challenged shrimp that were otherwise treated in the same way as challenged shrimp b Treatments with a different letter are significantly different from each other (One way ANOVA with Tukey's post hoc test; P b 0.05) sites (Ottemann and Miller, 1997) Hence, sensing of catecholamines might be an important cue enhancing the colonisation efficiency of V parahaemolyticus Our finding is in agreement with previous work in which catecholamines were found to increase the motility of other Vibrio species, including V harveyi (Yang et al., 2014), V anguillarum (Pande et al., 2015), as well as motility of human pathogens such as Escherichia coli (Kendall et al., 2007), Salmonella typhimurium (Bearson and Bearson, 2008) and Campylobacter jejuni (Cogan et al., 2007) Catecholamines have been detected in representatives of various crustacean taxa (Fingerman and Kulkarni, 1993) In shrimp and prawn, catecholamines in the hemolymph have been reported to range between 10 nM and μM (Chen et al., 2003; Hsieh et al., 2006), which is at least an order of magnitude lower than the concentrations that were found to affect motility in this study (50 μM) However, as indicated by Pande et al (2015) and Yang et al (2014), local concentrations can be considerably higher For instance, the intrasynaptic concentration of norepinephrine in the central nervous system of mammals is as high as 10 mM (vs nM levels in serum) Hence, upon infection, pathogens can come into contact with local concentrations that are several orders of magnitude higher than those that are found in hemolymph (or serum in case of vertebrates) This probably is also the case when tissues and/or hemocytes are damaged during infection, and catecholamines leach from these damaged cells Hence, elevated catecholamine levels might be a cue informing the pathogen of tissue damage (Yang et al., 2014) LED209, N-phenyl-4-{[(phenylamino)thioxomethyl]amino}benzenesulphonamide, is the first antagonist of bacterial catecholamine receptors that has been reported The compound has been found to block the binding of epinephrine and norepinephrine to the bacterial receptor QseC (Rasko et al., 2008) QseC homologues are present in the genomes of at least 25 important pathogens, including V parahaemolyticus (Hughes and Sperandio, 2008) LED209 prevents the autophosphorylation of QseC and consequently inhibits QseC-mediated activation of the expression of virulence genes (Rasko et al., 2008) In this study, we found that LED209 could neutralize the motility-inducing effect of dopamine and norepinephrine in the AHPND isolate V parahaemolyticus CM1 at very low concentrations (50–100 pM) Our finding is consistent with the work of Yang et al (2014), who reported that LED209 neutralized norepinephrine- and dopamine-induced motility in V harveyi LED209 has also been reported to decrease the expression of several virulence genes (including the motility-related operon flhDC) in other 194 N.T Suong et al / Aquaculture 470 (2017) 190–195 pathogens, such as S typhimurium and Francisella tularensis (Rasko et al., 2008) The catecholamines and LED209 were further evaluated in in vivo experiments with white leg shrimp The lowest survival was recorded for shrimp that were challenged with V parahaemolyticus isolate CM1 that was pretreated with catecholamines Pretreatment of V parahaemolyticus with catecholamines and LED209 significantly increased the survival of challenged white leg shrimp This finding is consistent with the work of Rasko et al (2008) who have established that LED209 was able to improve the survival of mice challenged with S typhimurium or F tularensis in the presence of norepinephrine In addition, Yang et al (2014) also indicated that LED209 protected brine shrimp (Artemia franciscana) from the increased virulence induced by norepinephrine and dopamine in V harveyi Interestingly, in this study, we found that pretreatment of V parahaemolyticus with LED209 resulted in survival levels that were even higher than those observed for shrimp challenged with untreated V parahaemolyticus, indicating that the effect of LED209 was stronger than only neutralising the effect of the catecholamine pretreatment This might be due to the fact that during infection of white leg shrimp, V parahaemolyticus comes into contact with endogenous catecholamines produced by the shrimp, (and thus that LED209 also to some extent neutralises the virulence-inducing effect of these endogenous catecholamines) Another explanation might be that in addition to swimming motility (and thus colonisation efficiency), LED209 also inhibits another important virulence factor in V parahaemolyticus that is not affected by the catecholamines Recently, Lee et al (2015) reported that two plasmid-encoded toxins are responsible for the hepatopancreatic necrosis symptoms induced by V parahaemolyticus Hence, it would be highly interesting to investigate whether the production of these toxins is affected by catecholamines and/or LED209 Conclusions In conclusion, this study showed that catecholamines significantly increase the motility of AHPND-causing V parahaemolyticus As swimming motility is necessary during the early stages of infection in many pathogens, catecholamine-induced swimming motility might be an important factor affecting the colonisation of shrimp by V parahaemolyticus Catecholamine-induced swimming motility could be neutralized by LED209, an antagonist of the bacterial catecholamine receptor QseC, suggesting that a receptor with a similar specificity is involved in the response of the V parahaemolyticus to catecholamines We furthermore found that pretreatment with catecholamines increased the virulence of V parahaemolyticus towards white leg shrimp, and that co-pretreatment with LED209 could neutralize this effect Interestingly, co-pretreatment of V parahaemolyticus with LED209 increased the survival of challenged shrimp to levels that were even higher than those observed in shrimp challenged with untreated V parahaemolyticus Although further research is needed in order to fully elucidate the (molecular) mode of action of antagonists of bacterial catecholamine receptors (such as LED209) in V parahaemolyticus, the results obtained in this study suggest that this type of compounds might be useful to control AHPND in shrimp Given the fact that catecholamine sensing controls virulence factors that are important at the early stage of infection (motility), interfering with catecholamine sensing might lead to a decreased transmission of AHPND between animals Further research is needed in order to establish an active, cost-effective and safe treatment (a chemical to be used as a drug or a micro-organism to be used as probiotic) Acknowledgements This work was funded by the International Foundation for Science (IFS Grant Agreement No A/5533-1) and the Fund for Scientific Research Flanders (FWO project no 1.5.013.12N) The authors have no conflict of interests to declare References Alksne, L.E., Projan, S.J., 2000 Bacterial virulence as a target for antimicrobial chemotherapy Curr Opin Biotechnol 11 (6), 625–636 Bearson, B.L., Bearson, S.M., 2008 The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium Microb Pathog 44, 271–278 Burton, C.L., Chhabra, S.R., Swift, S., Baldwin, T.J., Withers, H., Hill, S.J., Williams, P., 2002 The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system Infect Immun 70 (11), 5913–5923 Chen, Y.N., Fan, H.F., Hsieh, S.L., Kuo, C.M., 2003 Physiological involvement of DA in ovarian development of the freshwater giant prawn, Macrobrachium rosenbergii Aquaculture 228, 383–395 Cheng, W., Chieu, H.T., Ho, M.C., Chen, J.C., 2006 Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei Fish Shellfish Immunol 21, 11–19 Cogan, T.A., Thomas, A.O., Rees, L.E., Taylor, A.H., Jepson, M.A., Williams, P.H., Ketley, J., Humphrey, T.J., 2007 Norepinephrine increases the pathogenic potential of Campylobacter jejuni Gut 56, 1060–1065 Defoirdt, T., 2014 Virulence mechanisms of bacterial aquaculture pathogens and antivirulence therapy for aquaculture Rev Aquac 6, 100–114 FAO, 2013 Report of the FAO/MARD technical workshop on Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPND) of cultured shrimp (under TCP/VIE/3304) Hanoi, Viet Nam, 25–27 June 2013 FAO Fisheries and Aquaculture Report No 1053 Rome 54 pages Fingerman, M., Kulkarni, G.K., 1993 Quantitative measurement by reverse phase high performance liquid chromatography of norepinephrine in the central nervous system of the red swamp crayfish, Procambarus clarkia, and physiologically and pharmacologically induced alterations Comp Biochem Physiol 104, 117–123 Han, J.E., Tang, K.F., Lightner, D.V., 2015 Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp Dis Aquat Org 115 (3), 245–251 Hara-Kudo, Y., Nishina, T., Nakagawa, H., Konuma, H., Hasegawa, J., Kumagai, S., 2001 Improved method for detection of Vibrio parahaemolyticus in seafood Appl Environ Microbiol 67, 5819–5823 Hsieh, S.L., Chen, S.M., Yang, Y.H., Kuo, C.M., 2006 Involvement of norepinephrine in the hyperglycemic response of the freshwater giant prawn, Macrobrachium rosenbergii, under cold shock Comp Biochem Physiol 143, 254–263 Hughes, D.T., Sperandio, V., 2008 Inter-kingdom signalling: communication between bacteria and their hosts Nat Rev Microbiol (2), 111–120 Joshi, J., Srisala, J., Truong, V.H., Chen, I.T., Nuangsaeng, B., Suthienkul, O., Lo, C.F., Flegel, T.W., Sritunyalucksana, K., Thitamadee, S., 2014 Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease (AHPND) Aquaculture 428–429, 297–302 Karavolos, M.H., Spencer, H., Bulmer, D.M., Thompson, A., Winzer, K., Williams, P., Hinton, J.C.D., Khan, C.M.A., 2008 Adrenaline modulates the global transcriptional profile of Salmonella revealing a role in the antimicrobial peptide and oxidative stress resistance responses BMC Genomics 9, 458 Kendall, M.M., Rasko, D.A., Sperandio, V., 2007 Global effects of the cell-to-cell signaling molecules autoinducer-2, autoinducer-3, and epinephrine in a luxS mutant of enterohemorrhagic Escherichia coli Infect Immun 75, 4875–4884 Lee, C.T., Chen, I.T., Yang, Y.T., Ko, T.P., Huang, Y.T., Huang, J.Y., Lo, C.F., 2015 The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin Proc Natl Acad Sci U S A 112 (34), 10798–10803 Lyte, M., 2004 Microbial endocrinology and infectious disease in the 21st century Trends Microbiol 12, 14–20 McCarter, L.L., 2001 Polar flagellar motility of the Vibrionaceae Microbiol Mol Biol Rev 65, 445–462 Nakano, M., Takahashi, A., Sakai, Y., Kawano, M., Harada, N., Mawatari, K., Nakaya, Y., 2007 Catecholamine-induced stimulation of growth in Vibrio species Lett Appl Microbiol 44 (6), 649–653 Ottemann, K.M., Miller, J.F., 1997 Roles for motility in bacterial–host interactions Mol Microbiol 24, 1109–1117 Panakorn, S., 2012 Opinion article: more on early mortality syndrome in shrimp Aquacult Asia Pac (1), 8–10 Pande, G.S.J., Suong, N.T., Bossier, P., Defoirdt, T., 2015 The catecholamine stress hormones norepinephrine and dopamine increase the virulence of pathogenic Vibrio anguillarum and Vibrio campbellii FEMS Microbiol Ecol 90 (3), 761–769 Rasko, D.A., Moreira, C.G., Li de, R., Reading, N.C., Ritchie, J.M., Waldor, M.K., Williams, N., Taussig, R., Wei, S., Roth, M., Hughes, D.T., Huntley, J.F., Fina, 57.M.W., Falck, J.R., Sperandio, V., 2008 Targeting QseC signaling and virulence for antibiotic development Science 321, 1078–1080 Reed, L.J., Muench, H., 1938 A simple method of estimating fifty percent endpoints Am J Hyg 27, 493–497 Schryver, P., Defoirdt, T., Sorgeloos, P., 2014 Early mortality syndrome outbreaks: a microbial management issue in shrimp farming? PLOS Pathogens Magazines 10 (4), e1003919 Thitamadee, S., Prachumwat, A., Srisala, J., Jaroenlak, P., Salachan, P.V., Sritunyalucksana, K., Flegel, T.W., Itsathitphaisarn, O., 2016 Review of current disease threats for cultivated penaeid shrimp in Asia Aquaculture 452, 69–87 Tran, H.L., 2013 Determination, characterization, and control measures of the agent causing Early Mortality Syndrome (EMS) also known as Acute Hepatopancreatic Necrosis Syndrome (AHPNS) in farmed penaeid shrimp Doctor of Philosophy Thesis The Univeristy of Arizona, Arizona, USA, p 103 N.T Suong et al / Aquaculture 470 (2017) 190–195 Tran, L., Nunan, L., Redman, R.M., Mohney, L.L., Pantoja, C.R., Fitzsimmons, K., Lightner, D.V., 2013 Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp Dis Aquat Org 105 (1), 45–55 Tran, H.L., Reantaso, M., Fitzsimmons, M.K., Lightner, V.D., Hoang, N.P., 2015 Antibiotics use intended to reduce losses due to the early mortality syndrome (EMS/AHPNS) 195 in shrimp farms in Viet Nam Abstract from APA meeting (Retrieved 20 July, 2016, from https://www.was.org/meetings/mobile/MG_Paper.aspx?i=30688) Yang, Q., Anh, N.D., Bossier, P., Defoirdt, T., 2014 Norepinephrine and dopamine increase motility, biofilm formation, and virulence of Vibrio harveyi Front Microbiol (584) ... isolate CM1 by inoculating the strain into the rearing water The cumulative mortality of shrimp at 96 h after challenge was proportional to the concentration of bacteria added to the rearing water... investigate whether the production of these toxins is affected by catecholamines and/or LED209 Conclusions In conclusion, this study showed that catecholamines significantly increase the motility of. .. CFU/ml and that there was variation in virulence between different isolates of AHPND -causing V parahaemolyticus N.T Suong et al / Aquaculture 470 (2017) 190–195 193 Fig The impact of the catecholamines