Virulence of Vibrio harveyi responsible for the “Bright-red” Syndrome in the Pacific white shrimp Litopenaeus vannamei

Journal of Invertebrate Pathology 109 (2012) 307–317

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Virulence of Vibrio harveyi responsible for the ‘‘Bright-red’’ Syndrome in the Pacific white shrimp Litopenaeus vannamei Sonia A. Soto-Rodriguez a,⇑, Bruno Gomez-Gil a, Rodolfo Lozano a, Rodolfo del Rio-Rodríguez b, Ana L. Diéguez c, Jesús L. Romalde c a b c

CIAD, A.C. Mazatlan Unit for Aquaculture and Environmental Management, A.P. 711 Mazatlan, Sinaloa 82010, Mexico Instituto EPOMEX, Universidad Autonoma de Campeche, Av. Agustin Melgar s/n, Col. Buenavista 24039, Campeche, Mexico Universidad de Santiago de Compostela, Campus Sur s/n, 15782 Santiago de Compostela, Spain

a r t i c l e

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Article history: Received 14 June 2011 Accepted 11 January 2012 Available online 28 January 2012 Keywords: ‘‘Bright-red’’ Syndrome Vibrio harveyi Shrimp Pathogenicity Histopathology Antibiotics Extracellular products

a b s t r a c t Vibrio harveyi (Vh) CAIM 1792 strain was isolated from Litopenaeus vannamei affected with ‘‘Bright-red’’ Syndrome (BRS). The strain grew in 1–10% NaCl, at 15–35 °C and was resistant to ampicillin (10 lg), carbenicillin (100 lg) and oxytetracycline (30 lg). The lowest MIC was for enrofloxacine (0.5 lg ml1). The in vivo and in vitro toxicity of bacterial cells and the extracellular products (ECPs) of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl were evaluated. Adherence ability, enzymatic activities and siderophore production of bacterial cell was tested. The ECPs exhibited several enzymatic activities, such as gelatinase, amylase, lipase, phospholipase and caseinase. These ECPs displayed a strong cytotoxic effect on HELA cell line at 6 and 24 h. Challenges using 103 CFU g1 caused opacity at the site of injection and over 80% shrimp mortality before 24 h p.i. (post-injection). Mortality caused by the ECPs was higher than mortalities with bacteria, especially in the first hours p.i. Bacteria were re-isolated from hemolymph samples of moribund shrimp and identified as Vh CAIM 1792 by rep-PCR. Histological analysis of shrimp L. vannamei injected with Vh CAIM 1792 revealed generalized necrosis involving skeletal muscle (MU) at the injection site, the lymphoid organ (LO), heart and connective tissues. Melanization within the MU at the site of injection was also observed as well as hemocytic nodules within the hearth and MU at 168 h p.i. LO was the target organ of BRS. Necrosis of the MU at the injection site was the main difference in comparison to other shrimp vibriosis. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Mexican shrimp aquaculture began developing in the 1980s, but it did not become a well-established industry until about 10 years ago. Today aquaculture production exceeds the volumes obtained from captures in Mexico, and in 2009 67.84% of shrimp production came from aquaculture, which is produced in 71,000 ha (SAGARPA, 2009). Most of the Mexican shrimp farming occurs in the northwest part of the country, where the Pacific white shrimp Litopenaeus vannamei is grown in semi-intensive earthen pond systems. Although the yearly shrimp production has been showing a clear tendency to increase, the industry has suffered serious economic losses due to infectious diseases. More than 90% of the shrimp farms in Mexico have been seriously affected in the last years by the White Spot Syndrome Virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) and bacterial necrotizing hepatopancreatitis (NHP-B). Vibriosis is another impor-

tant disease affecting the shrimp industry in Mexico, causing recurrent outbreaks (Soto-Rodriguez et al., 2010a). Some Vibrio strains have proved to be pathogenic to penaeid shrimp (AlapideTendencia and Dureza, 1997; Goarant et al., 2006; Jayasree et al., 2006; Soto-Rodriguez et al., 2003, 2010b; Rattanama et al., 2009; Lin et al., 2010). Recently, Mexican farmers have experienced disease problems in cultures of L. vannamei associated with signs of diseases not related to TSV, WSSV, IHHNV and NHP-B. The industry has suffered serious economic losses due a new disease called Bright-red Syndrome (BRS), it has been reported in farms in northwestern Mexico and the causal agent was identified as a Vh CAIM1 1792 strain (Soto-Rodriguez et al., 2010b). Farmed shrimp affected with BRS become lethargic, anorexic, with flaccid body showing multifocal reddish discoloration spots on the abdominal cuticle and sometimes melanized erosions around spots. Vibrio harveyi has been recognized as a serious pathogen for a variety of important aquatic organisms in aquaculture around the world. To date, V. harveyi has been the most

⇑ Corresponding author. Fax: +52 669 989 8701. E-mail address: [email protected] (S.A. Soto-Rodriguez). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2012.01.006

1

CAIM is Collection of Aquatic Important Microorganisms.

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virulent and prevalent pathogen of larval and grow-out shrimp culture (Lavilla-Pitogo et al., 1990; Liu et al., 1996; Soto-Rodriguez et al., 2010b). Most studies on pathogenicity of V. harveyi are challenges using different sizes of fish or shrimp inoculated with a bacterial inoculum or extracellular products (ECPs) because they are considered the most important virulence determinants of some V. harveyi strains. These ECPs contain proteases, phospholipases, lipases, siderophores, chitinases and hemolysins (Liu et al., 1996; Zhang and Austin, 2000; Soto-Rodriguez et al., 2003; Zhong et al., 2006; Won and Park, 2008; Defoirdt et al., 2010). Vibriosis has been experimentally induced in penaeid shrimp by either immersing shrimp in water containing bacteria or by injecting bacteria into the musculature (de la Peña et al., 1993; Esteve and Herrera, 2000; Selvin and Lipton, 2003). These challenge studies report mainly the mortality of test organisms and sometimes the potential virulence factors of ECPs are evaluated. However, to date, the infection process of shrimp vibriosis has been little studied due to the difficulty in reproducing the disease (Roque et al., 1998; Saulnier et al., 2000). Histological techniques afford valuable knowledge regarding the state of health and the physiological processes of shrimp. More specific cytological and cytochemical information may be obtained by special tissue staining methods. Therefore, the histological study of tissue preparations has become one of the routine diagnostic methods in aquaculture for infections caused by viruses, bacteria, and parasites. A few studies have evaluated the histological damage caused by vibrios during experimental infection in the shrimp tissues (Egusa et al., 1988; Esteve and Herrera, 2000). This paper reports the phenotypic characteristic, antibiotic sensibility, adherence ability, cytotoxic and enzymatic activities, and siderophore production of Vh CAIM 1792 and ECPs from Vh CAIM 1792. Histopathology of L. vannamei juveniles shrimp challenged with bacterial cells and ECPs from bacterial cells was also evaluated. 2. Materials and methods 2.1. Phenotypic characterization Phenotypic identification of Vh CAIM 1792 followed the methods as described by Alsina and Blanch (1994) and Holt et al. (1994), which were Gram stain, growth on thiosulphate citrate bile salt sucrose agar (TCBS, Bioxon, Mexico), cell morphology, luminescence, oxidase, sensitivity to the vibriostatic agent O/129, motility, O–F test, swarming on triptych soy agar (TSA, Bioxon, Mexico) + 2.0% NaCl, arginine dehydrolase, ornithine decarboxylase and lysine decarboxylase. Growth at 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% NaCl, growth at 4, 15, 25, 30, 35 and 40 °C, indole production, gelatinase production, Voges–Proskauer, and utilization of citrate, L-arabinose and D-glucosaminic acid were also tested. A further characterization was done with the API 20E system and different carbon sources were tested too with addition of NaCl to give a final concentration of 2.5%.Vh CAIM 1792 was always incubated at 30 °C for 24–48 h. 2.2. Bacterial viability Two bacterial viability tests were done. In the first test, Vh CAIM 1792 was recovered from the cryovials and inoculated in 5.0 ml of tryptic soy broth (TSB, Bioxon, Mexico) + 2.0% NaCl incubated overnight at 30 °C. The bacterial suspension was obtained by inoculation of 100 ll in 10.0 ml of TSB (Bioxon, Mexico) + 2.0% NaCl (Bioxon Ò) and incubated overnight at 30 °C. Tubes with 5.0 ml of peptone broth (DibicoÒ) or 5.0 ml of TSB (BioxonÒ) were adjusted

to salinities of 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0% and 10.0% NaCl, including tubes without NaCl with five replicates, for the first and second test respectively. Afterwards, all tubes were inoculated with 100 ll of the bacterial suspension and incubated for 24 h at 30 °C. Serial dilutions were made with SSE and 100 ll of each dilution was inoculated on TCBS (BioxonÒ) plates and incubated for 24 h at 30 °C and the absorbance was measured at 600 nm for each tube. 2.3. Susceptibility to antibiotics The strain Vh CAIM 1792 was analyzed for its susceptibility to antibiotics in vitro using a multidisc system to amikacin (30 lg), ampicillin (10 lg), carbenicillin (100 lg), cephalothin (30 lg), cefotaxime (30 lg), ceftriaxone (30 lg), chloramphenicol (30 lg), gentamicin (10 lg), netilmicin (30 lg), nitrofurantoin (300 lg), pefloxacin (5 lg), trimethoprim-sulphamethoxazole (25 lg) (Sanofi Diagnostics Pasteur, Mexico). For oxytetracycline (OTC, 30 lg) (Sigma–Aldrich Co., Mexico), norfloxacine (10 lg) (ScheringPlough, Mexico), erythromycin (15 lg) (Sigma–Aldrich Co., Mexico), gentamicin (10 lg) Sigma–Aldrich Co., Mexico, chloramphenicol (30 lg) (Sigma–Aldrich Co., Mexico) the disk diffusion test (Bauer et al., 1966) was modified to allow the growth of Vh CAIM 1792. The inoculum was brought to an optical density of 1.0 at a wavelength of 610 nm, corresponding to a density of 1  108 bacterial cells ml1 according to the McFarland scale. The strain was suspended in sterile 2.5% NaCl solution and spread on Mueller-Hinton agar (MHA; Difco) supplemented with 2.5% NaCl. A 6.0 mm sterile sensidisk (Oxoid, Basingstoke, England) was inoculated with antibiotic according to Hindler (1992). All concentrations refer to the concentration per disk. Vh CAIM 1792 was inoculated in duplicate on the disk, and then the disks were placed on MHA. After incubation (24 h, 30 °C), the inhibition zones were measured in millimeters using a transparent ruler. The minimum inhibitory concentrations (MIC) of enrofloxacine (Cheminova de Mexico, Mexico), fosfomycine (Sigma–Aldrich Co., Mexico), OTC (Sigma–Aldrich Co., Mexico), flumequine (Sigma– Aldrich Co., Mexico), norfloxacine (Cheminova de Mexico, Mexico) and florfenicol (Schering-Plough, Mexico) to Vh CAIM 1792 were estimated following the method of Hindler (1992), using media supplemented with NaCl at a final concentration of 2.5%. 2.4. Adherence ability The cell surface hydrophobicity of Vh CAIM 1792 was determined by bacterial adhesion to hydrocarbons (BATH) method of Rosenberg et al. (1980) using the n-octane (Sigma™). The percentage of partitioning in the hydrocarbon phase was calculated as follows:

PABH ð%Þ ¼

A600 original bacterial suspension  A600 aqueous phase A600 original bacterial suspension  100

Vh CAIM 1792 was inoculated on TSA previously adjusted 1.0%, 2.0% and 4.0% NaCl. The criteria of hydrophobicity proposed by Santos et al. (1990) were used to evaluate the hydrophobicity of each isolate tested in this study. Interpretations for the BATH-test (>50% partitioning = strongly hydrophobic, 20–50% partitioning = moderately hydrophobic, and <20% partitioning = not hydrophobic). A biofilms formation assay protocol was adapted from that of Won and Park (2008). Polystyrene tubes containing 5.0 ml of TSB adjusted 1.0%, 2.0% and 4.0% NaCl were inoculated with overnight cultures of Vh CAIM 1792 and incubated for 48 h at 30 °C without agitation. At regular time intervals, the tubes were rinsed thoroughly with distilled water, and a 1% solution of crystal violet

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was added to stain the attached cells. After 15 min of incubation at room temperature, the tubes were rinsed with distilled water, and the biomass of attached cells (biofilm) was quantified by solubilization of the dye in 3.0 ml of 95% ethanol. The absorbance of the solubilized dye was measured at 600 nm.

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Bradford (1976) using bovine serum albumin (BSA; Sigma) as a standard. To evaluate the total proteolytic activity present in the ECP samples, a multiprotein substrate (Azocoll; Sigma–Aldrich) was used following the manufacturer’s instructions. An absorbance reading of 1.0 at 520 nm, after a 30 min assay at 37 °C, was defined as one unit of proteolytic activity (pa).

2.5. Enzymatic activities 2.9. Determination of enzymatic activities of ECPs Bacterial cultures that were grown overnight were spot-inoculated onto TSA with 1.0%, 2.0% and 4.0% NaCl that contained 1% gelatin (gelatinase test) or 2.5% skim milk (protease test) as substrate. The plates were incubated for 24 h to 48 h at 30 °C, and the diameter of the lytic halo around each well was measured. Urease activity was examinated in urea broth (DibicoÒ Mexico) supplement with 1.0%, 2.0% and 4.0% NaCl. Tubes were incubated for 24 h at 30 °C. A color change in the medium was indicative of a positive activity. 2.6. Siderophore production The universal method of Schwyn and Neilands (1987) and further modified by Soto-Rodriguez et al. (2003) was employed. Briefly, Vh CAIM 1792 was grown in iron deficient MM9 broth for 48 h at 30 °C under constant agitation at 95 rpm. Treatments with six replicates were plated directly onto chrome azurol S agar (CAS). All media employed were supplemented with 1.0%, 2.0% and 4.0% NaCl. Incubation in CAS agar was at 30 °C for up to 72 h and orange halos around colonies were measured at 48 and 72 h by measuring the diameter of the halo around each colony and subtracting from the colony diameter to give the relative activity (mm). Siderophore production was considered positive when the halo diameter divided by the colony diameter exceeded 1.3 (Amaro et al., 1990). 2.7. Bacterial inoculum The strain Vh CAIM 1792 was previously isolated from hemolymph samples taken from shrimp that were affected by BRS (Soto-Rodriguez et al., 2010b). The strain was recovered from the cryovials and inoculated in 10 ml of tryptic soy broth (TSB has a 0.5% NaCl, Bioxon, Mexico) supplement with 2.0% NaCl incubated and plated on TSA (TSA has a 0.5% NaCl, Bioxon, Mexico) supplement with 2.0% NaCl and incubated overnight at 30 °C. Colonies were suspended in sterile 2.5% NaCl and centrifuged at 5724g for 10 min at 15 °C. The bacterial suspension was adjusted to an optical density of 1.0 at 610 nm, similar to 0.5 MacFarlane Standard (equal to 108 colony forming units (CFU) ml1, tested in previous trials) (Soto-Rodriguez et al., 2003) and serially diluted to achieve densities from 103 to 107 CFU ml1. These suspensions were plated onto TCBS (Bioxon, Mexico), after serially diluting to determine the real density of Vh CAIM 1792 inoculated in the challenges. 2.8. ECPs extraction Bacterial ECPs were obtained by the cellophane plate technique (Zhang and Austin, 2000) by spreading 3 ml of an overnight broth culture of Vh CAIM 1792 over sterilized cellophane sheets placed on TSA plates. TSA agar was previously adjusted to 1.0%, 2.0% and 4.0% NaCl. After incubation for 24 h at 30 °C, bacterial cells were washed off the cellophane sheets with phosphate buffered saline (PBS) at pH 7.0. The bacterial suspensions were centrifuged at 13,000g for 30 min at 4 °C, and the respective supernatants were filtered through 0.22 lm pore-size membrane filters (Millipore™). All the ECP samples were stored at 20 °C until used. Filtered products were subjected to a sterility test by inoculation of 20 ll on TCBS agar (DibicoÒ) and incubation overnight at 30 °C. The protein concentration of the ECPs was determined by the method of

The enzymatic activities presented in the ECPs obtained from Vh CAIM 1792 cultured at 1.0%, 2.0% and 4.0% NaCl were evaluated using the API ZYM system (bioMérieux). Other enzymatic activities, such as caseinase, gelatinase, elastase, amylase, phospholipases, and lipase were determined in plates by a diffusion method. Plate dishes of basal nutrient agar (Difco) were adjusted to 1.0%, 2.0% and 4.0% (NaCl, w/v) and supplemented with 1% (w/v) sodium caseinate (Difco), 1% (w/v) gelatin (Oxoid), 0.4% (w/v) starch (Difco), 2% (v/v) egg yolk emulsion (Sigma-Aldrich) and 1% (v/v) Tween-20/Tween-80 (Sigma–Aldrich), respectively. In all the tests, aliquots of 10 ll of each ECP were placed on the plates and incubated at 25 °C for 48 h. The appearance of clear halos around the ECP spot indicates a positive result, except in lipase, where positive halos are opaque. 2.10. Cytotoxic activity of the ECPs Cytotoxicity assays with ECPs obtained from Vh CAIM 1792 cultured at 1.0%, 2.0% and 4.0% NaCl were basically carried out as described by Toranzo et al. (1983). HELA cell lines (human cervical carcinoma) were employed. Confluent eukaryotic cells monolayers were prepared in 96-wells plates and maintained at 37 °C. Before the test the medium was replaced from the wells with fresh medium. Medium was removed from wells containing eukaryotic cells, replaced with fresh medium and, inoculated directly with 50 ll different concentrations of ECPs. PBS (pH 7.4) being used as negative control. The development of cytotoxic effects was observed at 6 and 24 h post-inoculation. 2.11. Challenges with Vh CAIM 1792 and ECPs from Vh CAIM 1792 Two batches of juvenile shrimp (around 5 and 15 g) were transported from a local farm to the laboratory and acclimatized for 1 week in 400 l round tanks filled with filtered seawater, aerated and held at 27–28 °C with a 400% of daily water exchange. The animals were put in the experimental units and allowed to adapt for another three days before trials began during which the health status of the shrimp was evaluated through observation. The experimental units consisted of 60 l glass aquaria or 54 l fiberglass filled with filtered (10 lm), UV sterilized and aerated seawater at 28–30 °C with 12 h photoperiod. The bacterial water quality was evaluated by plating a sample onto Marine Agar. Before the challenge with bacteria, hemolymph samples were taken from randomly selected shrimp and the total heterotrophic bacteria and vibrios load were quantified on Marine Agar and TCBS respectively. Five trials with shrimp using Vh CAIM 1792 cells and ECPs from Vh CAIM 1792 were done (Table 1). In all trials shrimp were removed from the acclimation tank, weighed, disinfected and injected in the third abdominal segment with 100 ll of each treatment or injected with 100 ll of sterile 2.5% NaCl for the control group. The fifth challenge was done to evaluate the susceptibility to bacteria of shrimp acclimated to different salinities, to bacteria. Shrimp were acclimated to 1.0%, 2.0% and 4.0% NaCl water culture before challenge with bacteria. Conditions were similar for all trials; experimental units were randomly allocated and held for 168 h with a 100% of daily water exchange. Shrimp were fed ad libitum twice a day with a commercial diet (protein 35% Purina™).

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Table 1 Description of the conditions of trials with L. vannamei using Vh CAIM 1792 cells and ECPs from Vh CAIM 1792. Trial

Treatment

Dose

Weight of shrimp (g)

Replicates/no. of shrimp

1

Vh CAIM 1792 cells

13.66, SD = 0.77

4/6

2 3

Vh CAIM 1792 cells Vh CAIM 1792 grown in TSA supplemented with 1.0%, 2.0% and 4.0% NaCl. ECPs from Vh CAIM 1792 cultured at 1.0%, 2.0% and 4.0% NaCl Vh CAIM 1792 cells

8.0  102, 8.0  103 or 8.0  104 CFU g1 2.0  102 or 2.0  103 CFU g1 2.8  103 CFU g1

14.90, SD = 0.82 6.12, SD = 0.77

3/10 4/10

100 ll per shrimp 2.73  103 CFU g1

4.60, SD = 0.76 7.3, SD = 1.10

3/10 3/10

4 5

Temperature, nitrates, total ammonia, salinity, and pH were recorded daily. A record of mortality and moribund organisms for each aquarium was kept. 2.12. Re-isolation of bacteria and molecular identification Hemolymph samples from moribund organisms within a period of 24 h p.i. were inoculated on TCBS agar and incubated at 30 °C for 24 h. The shrimp were preserved immediately in Davidson’s fixative for 48 h. Dominant yellow colonies were DNA fingerprinted by rep-PCR (Cabanillas-Beltran et al., 2006). Fixed samples were processed for histological analysis, following standard procedures (Bell and Lightner, 1988). Tissue sections were prepared for hematoxylin and eosin (H&E) and Gram-Humberstone methods and analyzed under optical microscopy (Olympus CX31). 3. Results 3.1. Phenotypic characterization and bacterial viability of Vh CAIM 1792 Cells were Gram-negative rods that were motile when they were grown in liquid media. The cells did not swarm on TSA. Colonies on marine agar (Difco Laboratories, Detroit, Mich.) were smooth, circular, and off-white and the strain was not luminescent. Colonies on TCBS agar (Bioxon, Mexico) were yellow (sucrose fermenting), circular with undulate borders and granular surface. The strain Vh CAIM 1792 was facultatively anaerobic, fermented glucose, Kovacs oxidase positive, and was sensitive to the vibriostatic agent O/129 at 150 mg, Voges–Proskauer and gelatinase positive, arginine dehydrolase negative, lysine decarboxylase and ornithine decarboxylase positive (Table 2). These characteristics placed it within the genus Vibrio (Alsina and Blanch, 1994). Sodium chloride was required for growth in a wide range; the only salinity at which Vh CAIM 1792 was not able to grow was at 0% NaCl (Fig. 1). 3.2. Susceptibility to antibiotics The largest inhibition zone (30.0 mm) occurred with chloramphenicol at 30 lg and the smallest with OTC at 30 lg (7 mm, Table 3). Vh CAIM 1792 was sensitive to most of the tested antibiotics and was resistant only to ampicillin (10 lg) and carbenicillin (100 lg). MICs were determined for the antibiotics commonly used in the Mexican shrimp culture; enrofloxacine MIC 0.50 lg ml1, OTC 2.0 lg ml1, norfloxacine and florfenicol 4.0 lg ml1. In addition MICs for flumequine was 2.0 lg ml1 and fosfomycine >512 lg ml1. 3.3. Adherence ability, enzymatic activities and siderophore production of Vh CAIM 1792 Cell surface hydrophobicity was examined using BATH test (Table 4). Vh CAIM 1792 strain grown at 1.0%, 2.0% and 4.0% NaCl

exhibited strong hydrophobicity using n-octane as a solvent. The biofilm formation assay produced absorbance in the range of 0.99–1.30 after 48 h. Both tests were not statistically different (p > 0.05) among three salinities. Only a weak caseinase activity was observed for 1.0 and 2.0% NaCl conditions; meanwhile siderophore production was observed in Vh CAIM 1972 grown at three NaCl concentrations. 3.4. Characterization of ECPs The enzyme activity, proteolytic activity, and cytotoxic activities of Vh CAIM 1792 ECPs are shown in Table 5. The highest enzyme activity from API ZYM was observed in the ECPS of Vh CAIM 1792 grown at 2.0% NaCl. Hydrolytic activity of the other tested enzymes was weak without differences among three treatments, only gelatinase, amylase and lipase (tween 20) activities were detected in all salinities. ECPs obtained from Vh CAIM 1792 grown at 1.0% and 2.0% NaCl had stronger cytotoxic activity. The observed morphological changes consisted mainly of vacuolization, elongation, cellular clumps, detachment and finally monolayer destruction. 3.5. Challenges with bacterial cells (trial 1 and 2) The water quality of the experimental units registered was: temperature = 27–29 °C, salinity = 34 g l1, pH = 8.1 ± 0.2, total ammonium 60.4 mg l1, nitrites 6 0.2 mg l1. No vibrios were registered in the water intake nor in the hemolymph samples before the challenges. The gross signs of challenged shrimp were lethargy, erratic swimming (whirling movement), anorexia, shell discoloration and flaccid body at first hours p.i. The mortality of the shrimp challenged with 103 CFU g1 (Fig. 2) was almost 100% before 24 h, with the shrimp showing opacity at site of injection. Bacterial density in hemolymph of moribund shrimp before 24 h p.i. was 4.80  103 for shrimp challenged with 104 CFU g1. All colonies in TCBS were yellow. At the end of the trial no vibrios were found in hemolymph samples from the control and challenged groups. In the second trial with Vh CAIM 1792 cells, cumulative mortalities were lower than in the first trial with Vh CAIM 1792 cells (Table A1 in Appendix), however we used lower doses. 3.6. Challenges with Vh CAIM 1792 grown at different salinities (trial 3) and ECPs from Vh CAIM 1792 (trial 4) Challenges were done to evaluate the pathogenicity of Vh CAIM 1792 and ECPs from Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl. A wide variability in mortality with bacteria challenge compared with ECPs was observed. Mortalities caused by the ECPs in the first hours were higher than mortalities with bacteria (Fig. 3). Treatment of 4.0% NaCl was more pathogenic before 24 h p.i. for both bacteria and ECPs with mortalities of 70% and 100% respectively. After 48 h p.i., no statistically differences among three salinities (p > 0.05) were observed in challenge with bacteria (see

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S.A. Soto-Rodriguez et al. / Journal of Invertebrate Pathology 109 (2012) 307–317 Table 2 Morphological and physiological characteristics of Vh CAIM 1792 isolated from L. vannamei with ‘‘Bright-red’’ Syndrome. +: positive; : negative; Y: yellow colonies. Characteristic

CAIM 1792

Gram staining Cell morphology Motility Swarming on TSA (2% NaCl) Pigment Growth on TCBS Luminescence Sensitivity to vibriostatic 0/129 Oxidase Arginine dehydrolase Ornithine decarboxilase Lysine decarboxilase O–F

– Rod + – – Y – + + – + + +

Production of H2S Nitrate reduction Indole Methyl red Gas from D-glucose Voges–Proskauer b-Galactosidase (OPNG test) Tryptophan deaminase Gelatinase Urease Triple sugar iron (TSI)

– + – + – + – – + – +

Temperature tolerance 4 °C 15 °C 25 °C 30 °C 35 °C 40 °C

– + + + + –

Utilization of Acetate L-Alanine

Amygdalin c-Aminobutyrate

+++ ++

L-Arabinose

+ ++ –

L-Arginine

+

L-Aspartate

++

Cellobiose Citrate Fructose D-Galactose

– ++ + –

D-Galacturonate

–

D-Gluconate

–

D-Glucose

+

D-Glucosamine

–

D-Glucuronate

+

L-Glutamate

+++

Glutarate

+ +++

L-Glutamine

Glycerol Glycine L-Histidine p-Hydroxybenzoate b-Hydroxybutirate myo-Inositol a-Ketoglutarate DL-Lactate Lactose L-Leucine

– ++ – + ++ – +++ +++ – –

L-Lisina

+

DL-Malate

–

D-Mannitol

+

D-Mannose

+

Melibiose Methanol

– ++

Table 2 (continued) Characteristic

CAIM 1792

L-Ornithine

–

Propionate Putresine Pyruvate L-Rhamnose

– + +++ –

D-Ribose

+

Salicin

– –

D-Sorbitol

Sucrose Succinate Trehalose L-Threonine

+ +++ ++ +

D-Xylose

–

Table A2 in Appendix). In contrast, challenge with ECPs obtained at 1.0% NaCl was statistically different compared with 2.0% and 4.0% NaCl. In the fifth trial, shrimp were acclimated to different water salinity conditions; the highest mortality was observed at 4.0% and 1.0% (see Table A3 in Appendix). Before the challenges with bacterial cells, bacteria were re-isolated from hemolymph samples of moribund shrimp and fingerprinted by rep-PCR (Cabanillas-Beltran et al., 2006) showing the same electrophoretic band pattern corresponding to Vh CAIM 1792. 3.7. Histopathological analysis (trial 1 and 2) In the first trial (168 h of exposure) Vh CAIM 1792 caused a general necrosis in the lymphoid organ (LO), striated muscle, heart, antennal gland and gills of L. vannamei with the LO as the main affected organ. Around the site of injection, the bacteria caused a severe necrosis of the muscle fibers with melanin deposits after 168 h p.i. (Fig. 4a and b). During the first hours p.i. the hemocytes showed a tendency to agglutinate within the heart chambers, probably as a first response to the infection (Fig. 4c); at 168 h hemocytic nodules could be observed within the heart muscle (Fig. 4d). LO tubules gradually lost their structure during the first hours p.i. (Fig. 5a), associated with the presence of Gram-negative bacteria clumps dispersed in the stromal matrix (Fig. 5d). A week later, the LO was severely necrotic and bacterial clumps could still be observed within the tubules (Fig. 5c). The antennal gland epithelium and gill lamellae were necrotic in the first hours p.i. with Vh CAIM 1792 (Fig. 6a–c); meanwhile after 168 h p.i., formation of hemocytic nodules was evident (Fig. 6d). Histopathological observations were similar in the second trial. Histopathological analysis of L. vannamei challenged with V. harveyi CAIM 1792 (trial 3) and ECPs from Vh CAIM 1792 (trial 4) at different salinities. No significant histological differences were observed between L. vannamei challenged with Vh CAIM 1792 or ECPs grown 1.0%, 2.0% and 4.0% NaCl. Damage was mainly observed at the site of injection, LO, heart and connective tissue (Fig. 7). From 9 to 13 h p.i., severe necrosis of the skeletal muscle (MU) with bacterial masses was observed. At 48 h p.i. there was a decrease of bacterial masses within MU, showing melanizated hemocytic nodules and hemocytic infiltration. As infection progressed, hemocytic infiltration and formation of multifocal hemocytic nodules was observed (Fig. 7a) culminating with the formation of granulomas within the MU (Fig. 7c). ECPs caused severe acute necrosis at the site of injection site, without

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A

Table 5 Characterization of the ECPs of Vh CAIM 1792 grown at three salinities. NaCl (%)

API ZYM Alkaline phosphatase Esterase Esterase lipase Lipase Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin a-chymotrypsin Acid phosphatase Naphthol-AS-BI-phosphohydrolase a-galactosidase b-galactosidase b-glucuronidase a-glucosidase b-glucosidase N-Acetyl-b-glucosaminidase a-mannosidase a-fucosidase Gelatinase Amylase Esculin Lipase Tween 80 Lipase Tween 20 Caseinase DNase Elastase Urease Total protein (lg ml1) Proteolytic activity (ua ml1) Cytotoxic activity (HELA cells) 6/24 h Crude ECPs 1:2 Dilution 1:5 Dilution

B

Fig. 1. Bacterial viability of Vh CAIM 1792 grown in (A) peptone broth and (B) TSB at different salinity conditions. Bars indicate standard deviation.

Table 3 Sensitivity of Vh CAIM 1792 to antimicrobial agents. Antibiotic

Concentration (lg)

Chloranphenicol Ceftriaxone Cefotaxime Trimethoprimsulphamethoxazole Pefloxacin Norfloxacine Netilmicin Nitrofurantoin Erythromycin Gentamicin Amikacin Cephalothin Oxytetracycline Ampicillin Carbenicillin

30 30 30 25 5 10 30 300 15 10 30 30 30 10 100

Halo (mm)

Sensitivity*

30 28 27 26

S S S S

23 21.5 21 20 18.5 18 17 16 7 0 0

S n.d. S S S S S I n.d. R R

*

1.0

2.0

4.0

+++ – – – – – – ++ – +++ ++ – – – – – + – – +(17 mm) +(7 mm) – +(5 mm) +(5 mm) +(10 mm) – – – 90 4.72

+++ ++ ++ – + – – +++ + +++ ++ – – – – – + – – +(15 mm) +(7 mm) – +(9 mm) +(9 mm) – – – – 119 2.70

++ + – – – – – ++ ++ +++ – – – – – – – – – +(14 mm) +(5 mm) – – +(5 mm) – – – – 27.5 2.49

++/++ ++/++ +/+

++/++ ++/++ (+)/(+)

++/++ +/+ /

hemocytic infiltration (Fig. 7b). Accumulation of hemocytic nodules, bacterial masses and melanized hemocytic nodules were also found within the LO and the heart which are lesions typically observed as consequence of systemic vibriosis in these animals (see Fig. A1a and b in Appendix).

4. Discussion

The sensitivity criterion used was taken from human or veterinary medicine, since there are no criteria defined for shrimp bacterial pathogens (Giono Cerezo, 1983; Lynch and Raphael, 1983; Barry and Thornsberry, 1985). S: sensitive, R: resistant; n.d. no data.

Variable phenotypic results, e.g., for arginine dihydrolase, nitrate reduction, Voges–Proskauer, indol, cellobiose, citrate, sucrose, lactose, luminescence of V. harveyi strains isolates from shrimp, lobster and coral, have been reported (Karunasagar et al., 1996; Diggles et al., 2000; Stabili et al., 2006; Najiah et al., 2008). For instance, Vibrio campbellii, V. harveyi and Vibrio rotiferianus have nearly indistinguishable phenotypes (Gomez-Gil et al., 2003, 2004). Due to the economic importance of V. harveyi infections,

Table 4 Cell surface hydrophobicity, biofilm formation, hydrolytic activities, and siderophore production of Vh CAIM 1792 grown at different salinities. Cas: caseianse; Gel: gelatinase; Ure: urease; Sid: siderophores. NaCl (%)

% Part. BATH (S.D.)

Biofilm Abs600 (S.D.)

Hydrolytic activities (diam., mm) Cas (48 h)

Gel (48 h)

Ure (24 h)

Sid (72 h)

1.0 2.0 4.0

93.9 (3.1)a 84.6 (10.7)a 93.0 (3.3)a

0.99 (0.06)a 1.30 (0.15)a 1.00 (0.13)a

1.7 (w) 1.5 (w) –

– – –

– – –

+ + +

BATH: bacterial adhesion to hydrocarbons test: >50% partitioning = strong hydrophobicity. Hydrolytic activities: w = weak; – = negative activity. Siderophore production: + = positive activity. a Treatments with the same letter are not statistically different (p > 0.05, for BATH and biofilms test n = 3; for hydrolytic activities and siderophores, n = 6).

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Fig. 2. Cumulative mortality in the first trial of L. vannamei challenged with three doses of Vh CAIM 1792 during 168 h. Contl: control group. Bars indicate standard deviation.

A

B

Fig. 3. Cumulative mortality of L. vannamei challenged with 2.8  103 CFU g1 of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl. Live cells (A) and ECPs (B). Contl: control group. Bars indicate standard deviation.

there is considerable interest in methods not only to phenotyping V. harveyi-related populations associated with marine reared animals; it is also important to study the mechanisms of virulence. Phenotypic differences were also registered in NaCl tolerance, where Vh CAIM 1792 grew from 1% to 10% NaCl. More than 80% of V. harveyi isolates from Penaeus monodon grew only at 0% and 5% NaCl (Najiah et al., 2008). This viability in a wide range of salinity means that Vh CAIM 1792 could proliferate in extreme environmental conditions that occur during the shrimp farming season in Mexico, which include brackish water during the rainy season and hyper saline water during the dry season. Regarding the sensibility tests, Vh CAIM 1792 was resistant to ampicillin and carbenicillin, the latter used by farmers in Thailand to combat shrimp vibriosis (Nakayama et al., 2006). This result coincides with other works where 70–100% of V. harveyi strains isolated from farmed shrimp were resistant to both antibiotics (Baticados et al., 1990; Abraham et al., 1997; Roque et al., 2001;

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Najiah et al., 2008). The resistance against b-lactam antibiotics might be due to the natural occurrence of b-lactamases in V. harveyi strains (Teo et al., 2002). This work found that Vh CAIM 1792 was also resistant to OTC such as most of V. harveyi strains isolated from diseased shrimp (Baticados et al., 1990; Chanratchakool et al., 1995; Soto-Rodriguez et al., 2006; Manilal et al., 2010). The MIC for OTC against Vibrio sp. isolated from diseased shrimp was the highest reported (Soto-Rodriguez et al., 2008). Even so, currently, OTC is amongst the most commonly used antibiotic by shrimp farmers worldwide, including Mexico (Soto-Rodriguez et al., 2010a). Interestingly, Vh CAIM 1792 was found to be sensitive to most of the antibiotics tested; in contrast, other works have found multiple resistances of V. harveyi strains to antibiotics (Tendencia and de la Pena, 2001). In this work, the lowest MIC value was for enrofloxacine, currently one of the most used antibiotics in Mexico (Soto-Rodriguez et al., 2010a). Bacterial adhesion to host surfaces has been described as one of the initial steps in microbial pathogenesis. It has been suggested that hydrophobicity and biofilm formation are the determining factors in the adhesive process and in the survival of pathogens in cells (Daly and Stevenson, 1987). Vh CAIM 1792 grown at three salinities exhibited strong hydrophobicity in the BATH assay. Biofilm formation is known as one of the virulent factors of V. harveyi (Karunasagar et al., 1996; Won and Park, 2008). We observed that biofilm formation of Vh CAIM 1792 grown at three salinities were not statistically different (p > 0.05). Other virulent factors associated with the pathogenicity of this bacterium were identified by Soto-Rodriguez et al. (2003) and Owens et al. (1996), who demonstrated the role of siderophore production in V. harveyi virulence. We found siderophore production by Vh CAIM 1792 grown at three salinities, but they did not show differences among treatments. The production of extracellular enzymes by bacterial fish pathogens has been widely observed (Amaro et al., 1992; Alcaide et al., 1999); however, the role of these products in pathogenesis is still not clear. ECPs are considered to be one of the most important determinants of virulence in V. harveyi (Austin and Zhang, 2006; Won and Park, 2008; Manilal et al., 2010). In our results, ECPs of Vh CAIM 1792 grown at 2.0% NaCl exhibited the highest enzyme activity compared with ECPs obtained from 1.0% and 4.0% NaCl by API ZYM test. Only gelatinase, amylase and lipase activities were detected in the three salinities, the hydrolytic activity of the other enzymes was weak without differences among treatments. Several studies consider the hemolytic activity as a major virulence factor for some V. harveyi strains (Zhong et al., 2006; Haldar et al., 2010; Rattanama et al., 2009) but Vh CAIM 1792 does not have it (data not shown). There was no correlation between Artemia mortality and hemolytic activity of V. harveyi strains (Soto-Rodriguez et al., 2003). However, we found serine and Zn proteases in the Vh CAIM 1792 genome by analysis in silico (data not shown) which coincide with Won and Park (2008) who revealed that the major protease of V. harveyi ECPs isolated from marine fishes was serine protease, meanwhile Liu and Lee (1999) and Liu et al. (1997) considered a cysteine protease as the major exotoxin. Based on these observations, we suggest that the major protease of V. harveyi ECPs is strain-dependent. Tissue cultures have played a crucial role in investigating bacterial–host interactions due to the easy manipulation of cells and their maintenance under controlled conditions. Ideally, we should use crustacean cell lines to test cytotoxic activities of pathogenic vibrios, but to date we have not that methodology. In the present study, severe damage to HELA cell line for 6 and 24 h was shown in ECPs samples obtained from three salinities, although ECPs from Vh CAIM 1792 grown at 1.0% and 2.0% NaCl had stronger cytotoxic activity. Montero and Austin (1999) revealed that V. harveyi isolated from infected shrimp, L. vannamei exhibited a strong cytotoxic activity 1 h after inoculation.

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Fig. 4. (a and b) Microphotographs of skeletal muscle (MU) of L. vannamei 168 h p.i. with Vh CAIM 1792 at 102 CFU g1; general necrosis of the muscle bands (arrows) with hemocytic infiltration (H) and melanization (M); (b) high magnification of (a) showing severely necrotic tissue (arrows) with hemocytic infiltration (H) and melanization; (c), longitudinal section of myocardium of L. vannamei at 5-7.5 h p.i. with Vh CAIM 1792 at 103 CFU g1 showing agglutinated hemocytes (arrows) inside the heart lumen, normal muscle bands (MB) and lumen (L) are observed; (d), 168 h p.i. with Vh CAIM 1792 at 102 CFU g1 showing a severe necrosis with multifocal hemocytic nodules (arrow), bacterial clumps (asterisks) and hemocytic infiltration (H). H&E bar, 50 lm (a); 20 lm (b and d); 100 lm (c).

Virulence of V. harveyi strains, identified by phenotypical methods and isolated from diseased shrimp, has been proven by challenging shrimp larvae and juveniles. However, the results may vary from one trial to another because it is very difficult to reproduce an experimental infection using shrimp (Soto-Rodriguez et al., 2006). Thus, so far, there is no phenotypic or genotypic feature to distinguish pathogenic from non-pathogenic strains, and the only manner to determine the virulence of a given strain

Fig. 5. Microphotographs of lymphoid organ (LO) of L. vannamei. (a and b) Transversal view of LO before 5 h p.i. with Vh CAIM 1792 at 104 CFU g1; LO shows a total loss of structure and hemocytic infiltration in antennal gland (AG) and hepatopancreas (HP); (b) enlarged view of LO with severe necrosis of the tubules (arrows), cells of the stromal matrix (SM) are absent, and tissue shows bacterial clumps (asterisks) around the lumen (L); (c) 168 h p.i. with Vh CAIM 1792 at 102 CFU g1 LO is severely necrotized and melanizated (M) with hemocytic infiltration (H) and bacterial masses (asterisks) surrounding the connective tissue; (d) transversal view of LO from shrimp 5–7 h p.i. with Vh CAIM 1792 at 103 CFU g1. SM shows dispersed Gram negative bacteria (arrows) and bacterial clumps (asterisk). H&E (a–c); Gram-Humberstone (d). Bar, 100 lm (a); 20 lm (b–d).

is through experimental infection because many environmental parameters, the bacterial strain and biological factors may influence the results. Especially because the acquisition of virulence genes by horizontal gene transfer might increase the ability of V. harveyi to infect aquatic organisms, by increasing virulence towards a specific host and/or by broadening its host range (Ruwandeepika et al., 2010). In the present study, BRS was reproduced easily and

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Fig. 6. Microphotographs of antennal gland (AG) of L. vannamei. (a) Before 5 h p.i. with Vh CAIM 1792 at 104 CFU g1 antennal glandular epithelium (GE) shows necrosis and pyknotic hemocytes in the hemolymph sinuses as a response to bacterial masses (asterisk); (b) AG 5–7.5 h p.i. with Vh CAIM 1792 at 103 CFU g1 GE shows necrosis with hemocytic infiltration (H) inside haemal spaces. L: lumen of the glandular tubules; (c) necrosis of gill lamellae of L. vannamei before 5 h p.i. with Vh CAIM 1792 at 104 CFU g1 where bacterial masses were observed (asterisk); (d) necrosis (asterisk) of gill lamellae 168 h p.i. with Vh CAIM 1792 at 102 CFU g1 with formation of hemocytic nodules (arrow). H&E bar, 20 lm (a, c, and d); 50 lm (b).

the gross signs in challenged shrimp were similar to shrimp vibriosis (Alapide-Tendencia and Dureza, 1997; Manilal et al., 2010) but the opacity at the site of injection has never been reported. Muscle necrosis in cultured shrimp is commonly observed and, often results in the appearance of white and opaque lesions in the abdominal MU in response to environmental factors, such as sudden changes in temperature, salinity, or other stress. During the past few years, virus infections also have been found to cause muscle necrosis in cultured shrimp. A nodavirus (tentatively named PvNV,

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Fig. 7. Microphotography of skeletal muscle (MU) of L. vannamei on the site of injection. (a and c) Shrimp challenge with Vh CAIM 1792 at 2.8  103 CFU g1 grown at 4.0% and 1.0% NaCl respectively. (a) 19 h p.i. showed necrosis (N) of the skeletal muscle with an increased inflammatory response (H), starting the formation of hemocytic nodules; (b) L. vannamei injected with ECPs of Vh CAIM 1792 grown to 1.0% NaCl at 8 h p.i. showing a severe necrosis of muscle fibers (N) around the site of injection; (c) 168 h p.i. showing a chronic lesion with a decreased inflammatory response (H). A melanizated (M) granuloma with necrotic material inside was too observed in the tissue. H&E bar, 100 lm.

Penaeus vannamei nodavirus) that causes MU necrosis in P. vannamei was found in Belize in 2004 (Tang et al., 2007). Infectious myonecrosis virus (IMNV) is another virus that causes muscle necrosis in L. vannamei cultured in Brazil and Indonesia (Poulos et al., 2006). The virus causes abdominal muscle necrosis with particularly emphasis on the sixth abdominal segment. Similarly, the main gross signs of BRS are opacity on the abdominal muscle and red discoloration spots on the abdominal cuticle of L. vannamei; however the difference with virus infections is the presence of melanization around the red spots (Soto-Rodriguez et al., 2010b). The similarity between gross signs observed during these viral infections and BRS could be confusing, therefore it is very important to confirm the findings by using histology and/or molecular methods.

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In the present work, Vh CAIM 1792 was pathogenic to shrimp causing more than 80% mortality before 24 h p.i. at dose of 103 CFU g1. Challenges with V. harveyi strains reported mortalities of about 50% at doses exceeding 104 CFU g1 (Liu et al., 1996; Alapide-Tendencia and Dureza, 1997; Jayasree et al., 2006; Rattanama et al., 2009). These findings clearly demonstrate differences in the virulence of V. harveyi strains. In general, challenges with ECPs were more toxic than live cells, especially in the first hours. It has been suggested that salinity concentration of bacteriological media of V. harveyi strains might affect their virulence (Prayitno and Latchford, 1995; Shivappa, 1997). ECPs obtained at 2.0% and 4.0% NaCl caused the highest mortalities. Before 24 h p.i., live cells grown at 4.0% NaCl and corresponding ECPs were more pathogenic causing mortalities that ranged from 70% to 100%. In contrast, after 48 h p.i., no differences were found in mortality between challenges with bacteria at the same salinities (see Table A2 in Appendix). Our results suggest no clear relationship between NaCl concentration and virulence of Vh CAIM 1792. There have been numerous studies describing morphological changes of shrimp tissues infected with Vibrio, but information on experimentally induced infection with V. harveyi is scarce. Histopathology of affected shrimp with Vibrio sp., including V. harveyi, has always reported the hepatopancreas as the target organ of infection (Martin et al., 2004). Histological analysis carried out on shrimp infected with Vh CAIM 1792 revealed the LO as the target organ of infection; in the first hours p.i., a total loss of structure occurred. At 168 h p.i., severe necrosis of the LO, with formation of hemocyitic nodules was observed. Egusa et al. (1988) reported similar pathological changes in the LO of Penaeus japonicus experimentally infected with Vibrio sp. where no extensive necrotic lesions were found in other organs. It is believed that the LO is essentially a filtering organ that clears the hemolymph of infectious agents as well as for homeostatic improvement of the hemolymph filtrate (Van de Braak et al., 2002; Duangsuwan et al., 2008). In the present study, severe necrosis was also observed within muscle fibers at the site of injection and surrounding connective tissue as well as antennal gland and gill filaments. Bacteremia was evident throughout the hemocele. No histopathological differences were observed among L. vannamei challenged with Vh CAIM 1792 or injected with ECPs and that were grown at 1.0%, 2.0% and 4.0% NaCl. Damage again, was mainly observed in the site of injection at the first hours p.i., LO and heart that could be attributed to the enzyme activity of the ECPs. Later, hemocytic inflammation with formation of hemocytic nodules and granulomas was observed within the muscle. Additionally, ECPs also caused acute severe necrosis at the site of injection, without hemocytic infiltration. In conclusion, phenotypic characteristics of Vh CAIM 1792 were different to others V. harveyi strains. These include tolerance to a wide range of salinities, such as extreme environments, where the viability of the bacteria could be retained and remain infectious. Fortunately, Vh CAIM 1792 was sensitive to most antibiotics used in shrimp culture, so if an outbreak by BRS occurs, it can be controlled. Virulence of Vh CAIM 1792 reflects the synergistic interactions among virulence factors mainly the hydrophobicity, biofilm formation, siderophores production, and the proteolytic, enzymatic causing degradation of the shrimp tissue. No clear relationship was found between salinity growth condition of bacteria or ECPs and virulence. Further studies will be required to determine the relationship, if any, between the pathogenicity of Vh CAIM 1792 and natural infection conditions such as water temperature, host species, infection route, and the existence of stress. At present, the authors are looking for, in silico, virulence genes of Vh CAIM 1792. Future work is necessary to prove if those potential virulence genes are responsible for virulence in its natural host, the penaeid shrimp, considering that the acquisition of virulence genes

by horizontal gene transfer might increase the ability of Vh CAIM 1792 to infect aquatic organisms, by increasing its virulence towards a specific host and/or by broadening its host range. Acknowledgments This study was in part supported by shrimp farmer’s contributions. We thank C. Bolan and F. Marrujo for technical assistance, S. Abad for the microphotographs, N. Duncan for reviewing the grammar, and C. Cabrera for donating L. vannamei. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jip.2012.01.006. References Abraham, T.J., Manley, R., Palaniappan, R., Dhevendaran, K., 1997. Pathogenicity and antibiotic sensitivity of luminous Vibrio harveyi isolated from diseased penaeid shrimp. J. Aquat. Tropics 121, 1–8. Alapide-Tendencia, E.V., Dureza, L.A., 1997. Isolation of Vibrio spp. from Penaeus monodon (Fabricius) with red disease syndrome. Aquaculture 154, 107–114. Alcaide, E., Amaro, C., Todolí, R., Oltra, R., 1999. Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus. Dis. Aquat. Org. 35, 77–90. Alsina, M., Blanch, A.R., 1994. Improvement and update of set of keys for biochemical identification of environmental Vibrio species. J. Appl. Bacteriol. 77, 719–721. Amaro, C., Aznar, R., Alcaide, E., Lemos, M.L., 1990. Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments. Appl. Environ. Microbiol. 56, 2410–2416. Amaro, C., Biosca, E.F., Esteve, C., Fouz, B., Toranzo, A.E., 1992. Comparative study of phenotypic and virulence properties in Vibrio vulnificus biotypes 1 y 2 obtained from an European eel farm experiencing mortalities. Dis. Aquat. Org. 13, 29–32. Austin, B., Zhang, X.H., 2006. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Lett. Appl. Microbiol. 43, 119–124. Barry, A., Thornsberry, C., 1985. Susceptibility test diffusion test procedures. In: Lennette, E. (ed.), Manual of Clinical microbiology, 4th. Washington D.C., American Society for Microbiology, pp. 978–987. Baticados, M.C.L., Lavilla-Pitogo, C.R., Cruz-Lacierda, E.R., de la Pena, L.D., Sunaz, N.A., 1990. Studies on the chemical control of luminous bacteria Vibrio harveyi and V. splendidus isolated from diseased Penaeus monodon larvae and rearing water. Dis. Aquat. Org. 9, 133–139. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turck, M., 1966. Antibiotics susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 45, 493–496. Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. World Aquaculture Society, Baton Rouge, LA, USA. Bradford, M.M., 1976. A rapid a sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cabanillas-Beltran, H., LLausas-Magaña, E., Romero, R., Espinoza, A., Garcia-Gasca, A., Nishibuchi, M., Ishibashi, M., Gomez-Gil, B., 2006. Outbreak of gastroenteritis caused by the pandemicVibrio parahaemolyticus O3: K6 in Mexico. FEMS Microbiol. Lett. 265, 76–80. Chanratchakool, P., Pearson, M., Limsuwan, C., Roberts, R.J., 1995. Oxytetracycline sensitivity of Vibrio species isolated from diseased black tiger shrimp, Penaeus monodon Fabricius. J. Fish Dis. 18, 79–82. de la Peña, L., Tamaki, T., Monoyama, K., Nakai, T., Muroga, K., 1993. Characteristics of the causative bacterium of vibriosis in the kuruma prawn, Penaeus japonicus. Aquaculture 115, 1–12. Daly, J.G., Stevenson, R.M.W., 1987. Hydrophobic and haemagglutination properties of Renibacterium salmoninarum. J. Gen. Microbiol. 133, 3575–3580. Defoirdt, T., Darshanee Ruwandeepika, H.A., Karunasagar, I., Boon, N., Bossier, P., 2010. Quorum sensing negatively regulates chitinase in Vibrio harveyi. Environ. Microbiol. Rep. 2, 44–49. Diggles, B.K., Moss, G.A., Carson, J., Anderson, C.D., 2000. Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harveyi. Dis. Aquat. Org. 43, 127–137. Duangsuwan, P., Phoungpetchara, I., Tinikul, Y., Poljaroen, J., Wanichanon, C., Sobhon, P., 2008. Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon. Fish Shellfish Immunol. 24, 426–435. Egusa, S., Takahashi, Y., Itami, T., Momoyama, K., 1988. Histopathology of vibriosis in kuruma prawn Penaeus japonicus Bate. Fish Pathol. 23, 59–65. Esteve, C., Herrera, F.C., 2000. Hepatopancreatic alterations in Litopenaeus vannamei (Boone, 1931) (crustacea: decapoda: penaeidae) experimentally infected with Vibrio alginolyticus strain. J. Invertebr. Pathol. 76, 1–5. Giono Cerezo, S., 1983. Prueba de Kirby-Bauer para sensibilidad a los antimicrobianos. Infectol. 3, 325.

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