Phage therapy against Vibrio parahaemolyticus infection in the whiteleg shrimp (Litopenaeus vannamei) larvae Carlos O. Lomel´ı-Ortega, Sergio F. Mart´ınez-D´ıaz PII: DOI: Reference:

S0044-8486(14)00410-4 doi: 10.1016/j.aquaculture.2014.08.018 AQUA 631302

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

14 July 2014 8 August 2014 12 August 2014

Please cite this article as: Lomel´ı-Ortega, Carlos O., Mart´ınez-D´ıaz, Sergio F., Phage therapy against Vibrio parahaemolyticus infection in the whiteleg shrimp (Litopenaeus vannamei) larvae, Aquaculture (2014), doi: 10.1016/j.aquaculture.2014.08.018

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ACCEPTED MANUSCRIPT Title: Phage therapy against Vibrio parahaemolyticus infection in the whiteleg

Running title Phage therapy for VP in whiteleg shrimp

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Authors:

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shrimp (Litopenaeus vannamei) larvae

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Carlos O. Lomelí-Ortega, Sergio F. Martínez-Díaz*

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* Corresponding author

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Author Affiliation:

Instituto Politécnico Nacional. Microbiology and Molecular Biology Lab.

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CICIMAR, Av. Instituto Politécnico Nacional sn. Col Playa Palo de Sta Rita. La Paz, Baja California Sur, Mexico CP. 23090 Tel. (+52) 61225344, Fax. (+52)

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61225322 e-mail [email protected]

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ACCEPTED MANUSCRIPT Abstract Vibrio parahaemolyticus is an important cause of disease, mortality, and

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economical losses in the shrimp aquaculture industry. Bacteriophages are natural bio-controlling agents, broadly recognized for their ability to reduce

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pathogen populations. Hence, in the present study, we evaluated the effectiveness of phage therapy in the prevention and control of vibriosis in Litopenaeus vannamei. Vibriosis was induced in shrimp larvae with 2 • 106 CFU

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• mL-1 of V. parahaemolyticus. The infected larvae were treated with different

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doses of selected phages and their efficacy was evaluated at different times after their application. Results revealed that selected lytic phages (A3S and Vpms1) are effective to reduce mortality caused by V. parahaemolyticus. In

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both cases, the early application (at 6 hours post-infection) was effective to

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avoid mortality. Low multiplicity of infection (MOI) values (< 0.1) were enough to counteract V. parahaemolyticus infection. Delayed phage applications (> 6

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hours post-infection) hindered mortality and the progress of infection. This study

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provides the basis for the use of bacteriophages in the prevention and control of V. parahaemolyticus in shrimps.

Keywords Phage therapy, Litopenaeus vannamei, Vibrio parahaemolyticus, Shrimp larvae

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ACCEPTED MANUSCRIPT 1. Introduction Vibrio spp. are the putative cause of strong economic losses in the shrimp

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industry; they can infect all life stages (from eggs to broodstock); generating in most cases 100% mortality (Prayitno & Latchford, 1995; Harris & Owens, 1999;

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Aguirre-Guzmán et al., 2010).

Vibrio parahaemolyticus (VP) is a Gram-negative bacterium that has been commonly associated with infections in aquatic organisms. In addition, it is a

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major concern for human health because it is a leading cause of seafood-borne

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bacterial gastroenteritis worldwide (DePaola et al., 2003; Gopal et al., 2005; Zimmerman et al., 2007, Turner et al., 2013). In Mexican shrimp hatcheries, the presence of VP is monitored frequently and has been associated with necrosis,

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slow growth, muscle opacity, anorexia, and mortality during seed production

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(Balcázar et al., 2007; Aguirre-Guzmán et al., 2010). During 2013, some strains of VP were reported as the etiological agent of the acute hepatopancreatic

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necrosis syndrome (AHPNS/ESM) that caused the collapse of the shrimp

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aquaculture in Asia (Tran et al., 2013) and Mexico. Currently there are scarce alternatives to control vibriosis during shrimp rearing, including some disinfectants and few legally allowed antibiotics (Santiago et al., 2009; Labreuche, 2012). However, new promising approaches are under development and, apparently, some of them can provide an acceptable level of control of pathogenic vibrios with little or null environmental damage. The potential of phage therapy (use of bacteriophages to control bacterial infections) in aquaculture is gaining the interest of the scientific community and, during the last 5 years, different opinions and multiple reviews have been published. However, few efforts have been made to validate their efficacy or the

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ACCEPTED MANUSCRIPT possible impacts associated with the massive releasing of phages to the environment.

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The early evaluations of phage therapy to prevent vibriosis produced very encouraging results; for example, during experimental shrimp larval production,

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Vinod et al. (2006) demonstrated that the application of V. harveyi phages improves survival rate, even when compared with antibiotic-treated organisms. However, we can expect that, under commercial conditions, their actual

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effectiveness or apparent beneficial effect will be linked to the presence of

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specific pathogens, because phages have a narrow hosts range, and their ability to control ongoing infections is still unknown. Therefore, at this time, it is crucial to generate models to assess the conditions under which the phage

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therapy is effective and the factors that affect their efficacy. In the present study,

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we evaluated phage therapy as an alternative to prevent and control the

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2. Methods

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damage produced by VP in the whiteleg shrimp larvae.

2.1 Bacteria and phages V. parahaemolyticus ATCC 17802 was obtained directly from the American Type Culture Collection (ATCC). The stocks were maintained at -50 ºC with 50% glycerol. For experiments, bacteria were cultured in marine agar (MA) and the cells were harvested at 24 h and adjusted at an optic density of 1 at 585 nm (OD585 = 1) (corresponding at ca. 108 CFU ml−1) in artificial sea water (ASW) (Instant Ocean®).The phages A3S and Vpms1 used in this study were previously isolated from healthy shrimp cultures (Makarov, 2008) and clams (Martínez-Díaz and Hipólito-Morales, 2013), respectively. Phage stocks were

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ACCEPTED MANUSCRIPT produced at 30 ºC in fresh VP cultures; the lysates were centrifuged at 3500 rpm to eliminate bacterial debris and then filtered through 0.02-µm membranes.

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The number of viable phages was quantified by standard PFU counts and

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stored at 4 ºC until use.

2.2 Shrimp larvae

Shrimp larvae at nauplius III stage were obtained from two commercial

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hatcheries (Acuacultura Mahr SA de CV, La Paz, BCS, Mexico, and

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BIOGEMAR SA, Salinas, Ecuador). For each experiment, a group of apparently healthy larvae (obtained from the collective spawn of at least 10 females) were transported to the laboratory and maintained in the carrying boxes until

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reaching the nauplius IV-V stage (N IV-V), then they were disinfected with 0.3

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ppm chlorine dioxide (ClO2) during 5 min, washed with sterile sea water and distributed in sterile containers with 100 mL artificial seawater (ASW) (Instant

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Ocean at 35 ppt) at a density of 1 larva • mL-1. At 24 h after disinfection,

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different treatments were applied (including infection or phage therapy) and maintained during 96 h at 30 ºC. A gnotobiotic culture of Chaetoceros calcitrans was provided as the sole food source during experiments: at an initial dose of 1 • 104 cell·mL-1 and, successively, adjusting to 1 • 105 cell • mL-1.

2.3 V. parahamolyticus challenge and phage therapy Groups of 100 larvae (previously disinfected with ClO2 and acclimatized as described in the section 2.2) were infected in the same container with a single dose of VP at 2 • 106 CFU • mL-1 and treated with 100 μL of Vpms1 or A3S phage suspensions. Groups of VP-infected larvae that were no treated with

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ACCEPTED MANUSCRIPT phages were used as positive controls and uninfected larvae were used as negative controls or blanks. Each treatment was assayed in triplicate and the

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survival rate and vibriosis signs were recorded at 96 hpi (hours post infection).

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2.4 Minimal effective dose of phages to control VP effects

Eighteen groups of 100 larvae at 24 h (previously disinfected with ClO2 and acclimatized as described in the section 2.2) were infected with VP (at a 2 • 106

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CFU·mL-1 dose). The containers were randomly selected in groups of three and

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treated with different volumes of A3S or Vpms1 phages, to reach MOI values of 0.1, 1, and 10. Positive and negative controls (as previously described) were

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simultaneously maintained and all groups were analyzed at 96 hpi.

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2.5 Effect of delayed application of phage therapy Twenty-four groups of 100 larvae (previously disinfected with ClO2 and

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acclimatized as described in the section 2.2) were simultaneously infected with

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VP (at a 2 • 106 CFU • mL-1 dose), triplicate groups were randomly selected to be treated with phages at different times (0, 6, 12, 24, and 36 hpi). Treatments comprised a single dose of 100 μL of Vpms1 or A3S phage suspension (reaching MOI values of 1 and 2, respectively). Positive and negative controls (as previously described) were maintained simultaneously and all groups were analyzed at 96 hpi.

2.6 Statistical Analysis

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ACCEPTED MANUSCRIPT Normality and homoscedasticity were evaluated using the Kolmogorov-Smirnov and Bartlett test, respectively, according to Zar, (1999); data were analyzed by

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one-way ANOVA and Tukey multiple comparisons using Statistica 8.0 software.

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3. Results

3.1 V. parahamolyticus challenge and phage therapy

VP infected larvae developed the typical signs of vibriosis, including empty

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digestive tract, red chromatophores, appendage deformations, and lethargy;

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mortality and changes in behavior were apparent at 48 hpi, and survival was significantly reduced as a result of infection (p < 0.05). The uninfected group had an average survival of 90 ± 6.8%, and no signs of vibriosis were recorded.

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In the VP-infected groups and treated with phages, most larvae appeared very

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active. The digestive tract was filled with food, few larvae showed signs of vibriosis, including loss of spines from the anterior appendages, and survival

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(Table 1).

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was not significantly reduced as compared to uninfected controls (p > 0.05)

3.2 Minimal effective dose of phages to control VP effects The beneficial effects observed during the application of Vpms1 or A3S phages were not affected by the reduction in doses (Table 2). Doses corresponding to 0.1 MOI were effective to control the adverse effects caused by VP in shrimp larvae. The survivals recorded in shrimps treated with different doses of Vpms1 were not statistically different among them or compared with uninfected controls (p > 0.05). The highest survival rate (75 ± 4.5%) was recorded in organisms treated with A3S at 0.1 MOI, however, no significant differences were recorded

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ACCEPTED MANUSCRIPT between 0.1 and 1 MOI of A3S or when compared to uninfected organisms (p >

3.3 Effect of delayed application of phage therapy

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0.05).

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A single dose of Vpms1 or A3S, applied at any time, was enough to control the infection and mortality in shrimp larvae challenged with VP. Compared with uninfected controls, no significant reduction in survival was recorded in the

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groups challenged with VP and treated with Vpms1 at 0 to 12 hpi (p > 0.05),

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although minor signs of vibriosis were recorded. When Vpms1 phages were applied at 24 hpi or later, the survival was reduced, however, the effect caused by VP was lower than that recorded in challenged larvae without phage

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treatment (Fig. 1a). Larvae treated with A3S phage between 0 and 6 hpi did not

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show negative effects, and mortality was not significantly different from that of the control group (p > 0.05). Whenever A3S phage inoculation was delayed

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more than 6 hpi, survival rate decreased with time, however all treated groups

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showed significant differences (p < 0.05) as compared to larvae without phage treatment (Fig. 1b). In all cases, signs of vibriosis were apparently reduced by the application of A3S.

4. Discussion Infections caused by Vibrio spp. are responsible for mass mortality in shrimp aquaculture and exert strong impacts on the economy of producing countries. In the past, their control was achieved mainly through antibiotic therapy, but the restriction in the use of antibiotics in aquaculture left a gap that needs to be compensated with new strategies to control the effects of infections, preferably

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ACCEPTED MANUSCRIPT environmentally friendly and biologically safe ones. At prima facie, the use of phage therapy to control vibriosis in shrimp production looks very promising,

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however, phage therapy technology for aquaculture is currently in the early

of prevention or control of specific infections.

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developmental stage and requires studies to validate their scope as a method

In this study, we examined the ability of two lytic phages to prevent and control the detrimental effects caused by Vibrio parahaemolyticus in whiteleg shrimp

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larvae. We found that a single dose of Vpms1 or A3S phages is enough to

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reduce the signs of infection and mortality in shrimp larvae caused by VP. In previous studies, it has been shown that the application of repeated doses of phages exerts benefits on the performance of the culture (measured as the

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number of surviving larvae) (Karunasagar et al., 2005; Vinod et al., 2006); but it

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was applied under blind conditions, i.e., without considering the presence or absence of the targeted bacteria, which can radically influence the apparent

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advantages obtained with the phage therapy. Unlike broad spectrum treatments

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(v.g., antibiotics or disinfectants), the effect of phages is very selective (some phages even recognize and infect only a specific strain); this feature can be decisive for the success or failure of their use as control or preventive treatment (Calendar, 2006), since mixed infections are common in cultured organisms. Blind application of phage therapy will be the ultimate evidence of its therapeutic potential; hence, at this time, it is very important to establish, under controlled conditions, the parameters that determine its success and factors that compromise its optimal function to avoid false expectations or disprove its value by lack of consistent results.

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ACCEPTED MANUSCRIPT The ability of phages to reverse vibriosis when the infection is in progress may be limited by their ability to reach the organs or tissues where the infection is

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occurring, i.e., hemolymph, hepatopancreas, intestines or muscles. In this work, we found that the application of phages in shrimp larvae can serve as a strategy

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to control infections caused by VP at any stage of the infection; whenever phage treatment was applied, the infection was controlled and mortality was stopped. The latter is a very important finding because previous studies had

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shown that phage therapy could be effective only as a preventive mechanism

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(Nakai et al., 1999; Nakai and Park, 2002, Martínez-Díaz and Hipólito-Morales, 2013).

During this study, we included treatments with small numbers of phages in

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relation to the amount of the target bacterium in the system. Under these

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conditions, the success of therapy could depend on the ability of phages to infect target cells to generate new viral particles, and the new generations of

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phages will then infect the remnant bacteria in order to gain control over the

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entire population of target bacteria (this process is known as active therapy). A small dose (at MOI = 0.1) was sufficient to prevent and control the effects of VP. Considering that the use of phage therapy for commercial aquaculture may involve treatment of large volumes of water, the successful effect of an active therapy may have significant economic implications for the effective doses required for pathogen control. This study was conducted under gnotobiotic conditions to avoid potential interferences of external factors, however, we cannot discard that the success of phage therapy to control infections, under the actual production conditions, could be affected by other factors that compromise the viability of phages, i.e.,

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ACCEPTED MANUSCRIPT water quality, the presence of bacteria that produce antiviral substances, or the amount of organic matter. We consider that the model on which the

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experiments were conducted during our study will be very useful in future studies to evaluate the effect of those external factors on the efficacy of phage

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therapy.

the

stit to

o it

i o

a io a (project SIP-

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his resear h was s pported

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5. Acknowledgements

20100865) and by the National Council of Science and Technology CONACyT

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(Project 85033 and Grant 37176).

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6. References

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Aguirre-Guzmán, G., Sánchez-Martínez, R., Pérez-Castañeda, R., Palacios-

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Monzón A., Trujillo-Rodríguez, T., de la Cruz-Hernández, N.I., 2010. Pathogenicity and infection route of Vibrio parahaemolyticus in American white shrimp, Litopenaeus vannamei. J World Aquacult Soc 48, 464–470. Balcázar, J.L., Rojas-Luna, T., Cunningham, D.P., 2007. Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp (Litopenaeus

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following

immersion

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with

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parahaemolyticus. J Invertebr Pathol 96, 147–150. DePaola, A., J.L. Nordstrom, J.C. Bowers, J.G. Wells, D.W. Cook., 2003. Seasonal abundance of total and pathogenic Vibrio parahaemolyticus. Appl Environ Microbiol 69 (3), 1521–1526.

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ACCEPTED MANUSCRIPT Gopal, S., Otta, S., Kumar, S., Karunasagar, I., Nishibuchi, M., Karunasagar, I., 2005. The occurrence of Vibrio species in tropical shrimp culture environments;

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implications for food safety. Int J Food Microbiol 102, 151–159. Harris, L.J., Owens, L., 1999. Production of exotoxins by two luminous Vibrio

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harveyi strains known to be primary pathogens of Penaeus monodon larvae. Dis Aquat Org 38, 11–22.

Karunasagar, I., Vinod, M., Kennedy, B., Vijay, A., Deepanjali, A., Umesh, K.,

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Karunasagar I., 2005. Biocontrol of bacterial pathogens in aquaculture with

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emphasis on phage therapy, in: Walker, P., Lester, R., Bondad-Reantaso, M.G. (Eds.), Diseases in Asian Aquaculture V, Fish Health Section, Asian Fisheries Society, Manila, Philiphinas, pp 535-542.

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Labreuche, Y., Pallandre, L., Ansquer, D., Herlin, J., Wapotro, B., Le Roux, F.,

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2012. Pathotyping of Vibrio isolates by multiplex PCR reveals a risk of virulent strain spreading in new caledonian shrimp farms. Microb Ecol 63: 127-138.

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Makarov, R., 2008. Aislamiento

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almeja chocolata Megapitaria squalida

de i riofa os de m estras de amar

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o Litopenaeus

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vannamei. Bachelor thesis

Martínez-Díaz, S.F., Hipólito-Morales, A., 2013. Efficacy of phage therapy to prevent mortality during the Vibriosis of brine shrimp. Aquaculture 400–401, 120–124. Nakai, T., Park, S.C., 2002. Bacteriophage therapy of infectious diseases in aquaculture. Res Microbiol 153, 13-18. Nakai T, Sugimoto R, Park K, Matsuoka S, Mori K, Nishioka T, Maruyama K., 1999. Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail. Dis Aquat Org 37, 33-41.

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ACCEPTED MANUSCRIPT Prayitno, S.B., Latchford, J.W., 1995. Experimental infections of crustaceans with luminous bacteria related to Photobacterium and Vibrio – effect of salinity

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spi o a

amaro i

t ra

e ista

erm de e i a a de

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so de a ti i ti os e

a

ias arma e ti as 40 (3), 22-32.

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a tia o

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and pH on infectiosity. Aquaculture 132, 105–112.

Tran, L., Nunan, J., Redman, R.M., Mohney, L.L., Pantoja, C.R., Fitzsimmons, K., Lightner, D.V., 2013. Determination of the infectious nature of the agent of

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acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis Aquat

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Org 105, 45-55.

Turner, J.W., Paranjpye, R.N., Landis, E.D., Biryukov, S.V., GonzálezEscalona, N., Nilsson, W.B. Strom, M.S., 2013. Population structure of clinical

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and environmental Vibrio parahaemolyticus from the pacific northwest coast of

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the United States. PLoS One 8(2): e55726. Vinod, M.G., Shivu, M.M., Umesha, K.R., Rajeeva, B.C., Krohne, G.,

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Karunasagar, I., Karunasagar, I., 2006. Isolation of Vibrio harveyi bacteriophage

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with a potential for biocontrol of luminous vibriosis in hatchery environments. Aquaculture 255, 117–124. Zar, J.H., 1999. Biostatistical Analysis (4th Edition). Prentice Hall, Englewood Clifs, New Jersey. Zimmerman, A.M., A. DePaola, J.C. Bowers, J.A. Krantz, J.L. Nordstrom, C.N. Johnson,

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of

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parahaemolyticus densities in northern gulf of Mexico water and oysters. Appl Environ Microbiol 73 (23), 7589–7596.

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ACCEPTED MANUSCRIPT Figures

AB

AB

B

B

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80

A

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a

A

C

60

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40 20

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Mean Survival (%)

100

0 Control 0 h

6h

12 h 24 h 36 h

NT

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Treatment

60

A

40

b

AB B

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80

A

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A

AC

Mean Survival (%)

100

BC C

A

20 0 Control 0 h

6h

12 h 24 h 36 h

NT

Treatment Fig. 1. Effect of different times in the application of phage therapy using Vpms1 (A) and A3S (B) phages, during a V. parahaemolyticus challenge in whiteleg shrimp L. vannamei larvae. 14

ACCEPTED MANUSCRIPT Tables Table 1. Effect of phage therapy on survival of whiteleg L. vannamei shrimp

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larvae during a V. parahaemolyticus (VP) challenge. Survival (%) ± SD

Axenic control

87 ± 6.8a

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Treatment

59 ± 4.9b

VP

80 ± 3.5a

VP + A3S phage

77 ± 3.0a

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MA

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VP + Vpms1 phage

Table 2. Effect of different dosages of phages during a V. parahaemolyticus

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challenge in the whiteleg shrimp L. vannamei larvae. MOI

Survival (%) ± SD

Axenic control

-

79 ± 5.0 a

VP

-

53 ± 3.6 b

VP + A3S

0.10

75 ± 4.5 a

VP + A3S

1.00

72 ± 1.0 ac

VP + A3S

10.0

60 ± 2.6 b

VP + Vpms1

0.10

65 ± 5.3 bc

VP + Vpms1

1.00

58 ± 2.9 b

VP + Vpms1

10.0

62 ± 5.9 b

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Treatment

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ACCEPTED MANUSCRIPT Title: Phage therapy against Vibrio parahaemolyticus infection in the whiteleg shrimp (Litopenaeus vannamei) larvae

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

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

In the present study, we evaluated the effectiveness of phage therapy in the prevention and control of vibriosis in Litopenaeus vannamei Vibriosis was induced in shrimp larvae with 2·106 CFU·mL-1 of V. parahaemolyticus and treated with different dosages of phages We found that low doses of selected lytic phages are effective to prevent and control the mortality caused by V. parahaemolyticus in shrimp larvae.

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

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Highlights of the manuscript

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