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Microbial immunostimulants reduce mortality in whiteleg shrimp (Litopenaeus vannamei) challenged with Vibrio sinaloensis strains
Aquaculture 320 (2011) 51–55
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Microbial immunostimulants reduce mortality in whiteleg shrimp (Litopenaeus vannamei) challenged with Vibrio sinaloensis strains Ma. del Carmen Flores-Miranda a, Antonio Luna-González a,⁎, Ángel I. Campa‐Córdova b, Héctor A. González-Ocampo a, Jesús A. Fierro-Coronado a, Blanca O. Partida-Arangure a a b
Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional-Instituto Politécnico Nacional, Unidad Sinaloa, Sinaloa, Mexico Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, B.C.S., Mexico
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 28 July 2011 Accepted 4 August 2011 Available online 10 August 2011 Keywords: Immunostimulants Bacteria Yeast Vibrio Litopenaeus vannamei Pacific white shrimp
a b s t r a c t The effect of microbial immunostimulants on the survival and immune response of juvenile Litopenaeus vannamei challenged with Vibrio sinaloensis strains was evaluated. Dead microorganisms were added to feed with the attractant Dry Oil® and consisted of four lactic acid bacteria (Lta2, Lta6, Lta8, and Lta10) and one yeast (Lt6). V. sinaloensis strains or saline solution were inoculated to shrimp by injection. The bioassay was conducted for 21 days with five treatments in triplicate: (I) shrimp fed with commercial feed + sterile saline solution at 2.5% NaCl (control group I); (II) shrimp feed with commercial feed + LD50 Vibrio (control group II); (III) shrimp fed daily with experimental diet + LD50 Vibrio; (IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; and (V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. Shrimp (8.1 ± 1.4 g) were cultured in 120-L plastic tanks and fed twice a day. The activity of lysosomal enzymes in plasma and hemocytes were determined with the API ZYM kit and lysoplate assay. Survival of shrimp in treatment IV was significantly higher than those of control II. Total hemocyte count in treatment III was significantly higher than control II. The activity of nine hydrolytic enzymes was found in plasma and six in the hemocyte lysate supernatant (HLS). Shrimp fed with immunostimulants every six days were not protected against V. sinaloensis. The results indicate that these microbial immunostimulants administered every three days is a good feed additive against Vibrio spp. in shrimp culture. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture represents one of the main food providers in the world. In shrimp farming, whiteleg shrimp (Litopenaeus vannamei) is the primary penaeid shrimp currently being cultured in Central and South America (Burge et al., 2007; Chang-Che and Jiann-Chu, 2008). However, during the past two decades, worldwide commercial shrimp farming has suffered outbreaks of diseases caused mainly by Vibrio bacteria and viruses due to a deteriorated pond environment (Lo et al., 2003). Species of Vibrio are well known in penaeid shrimp culture as causative agents of vibriosis, which is a serious threat to the aquaculture industry, responsible for massive mortality of cultured penaeids worldwide (Baticados et al., 1990; Lightner and Lewis, 1975). Treatment of shrimp suspected of being infected with Vibrio spp. is mainly based on the use of antibiotics, but the susceptibility of vibrios to antibiotics varies widely among strains of the same species. There is little information about the detailed use of antibiotics; even their use in ⁎ Corresponding author at: Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (Unidad Sinaloa), Boulevard Juan de Dios Bátiz Paredes 250, Guasave, Sinaloa 81101, Mexico. Tel./fax: + 52 687 87 2 96 26. E-mail address: [email protected] (A. Luna-González). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.08.005
aquaculture may develop antibiotic resistance in pathogens that infect those cultured animals and humans (Soto-Rodríguez et al., 2008). The application of immunostimulants in shrimp aquaculture is increasingly gaining interest as an environmentally safe alternative to antibiotics and chemotherapeutics (Song et al., 1997). Shrimp possess an innate immune system, consisting of cellular and humoral elements. Hemocytes play a central role in the non-specific immune response of shrimp, which rely mainly on phagocytosis, melanization, encapsulation, cytotoxicity, and clotting (Sritunyalucksana et al., 1999). Humoral defense factors, such as clotting proteins, agglutinins, hydrolytic enzymes, and antimicrobial peptides are released upon lysis of hemocytes, which is induced by lipopolysaccharides (LPS), peptidoglycans, and β-1,3-glucans (Chisholm and Smith, 1995; Destoumieux et al., 2000; Johansson and Söderhäll, 1989; Muta and Iwanaga 1996; Söderhäll et al., 1994). Diets containing immunostimulants are used in aquaculture in order to increase resistance to stress and diseases of cultured fishes and invertebrates by alerting the immune system (Doñate et al., 2010; Rendón and Balcázar, 2003). They can be extracted from the walls of microorganisms such as Gram-negative bacteria (lipopolysaccharides), Gram-positive bacteria (peptidoglycans), and fungi (β-1, 3-glucans). Furthermore, the whole cell can be used as immunostimulant (Sajeevan et al., 2009a; Partida-Arangure et al., unpublished data). There are
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several methods of stimulation like immersion, injection, and feeding among others (Chen and Ainsworth, 1992; Jorgensen et al., 1993; Rorstad et al., 1993). However, the most practical method of stimulation is incorporating the immunostimulating substances into the feed (Azad et al., 2005). Currently many commercial immunostimulants are available in the shrimp aquaculture industry and are extensively used by shrimp farmers. However, scientific data in support of their function and dose/frequency of application are lacking. Information regarding the dose is essential as overdose leads to immunosuppression, rendering less protection to infection (Chang et al., 2000; Sajeevan et al., 2006, Sajeevan et al., 2009b). This study was undertaken to examine the survival and the immune response of L. vannamei against Vibrio sinaloensis strains when shrimp were treated with whole-cell microbial immunostimulants. 2. Materials and methods 2.1. Microbial immunostimulants Lactic acid bacteria (Lta2, Lta6, Lta8, and Lta10) and yeast (Lt6) used in this work as immunostimulants were originally isolated, characterized, and tested by Apún-Molina et al. (2009) and Peraza-Gómez et al. (2011). 2.2. Preparation of experimental diet with microorganisms A mixture of four selected lactic acid bacteria (LAB) and one heatkilled yeast (74 °C) was sprayed on commercial feed (Purina ®, Ciudad Obregón, Mexico, 40% protein) at 8.4 mg kg feed − 1 (1.68 mg per microbial isolate). The amount of immunostimulants was based on the work of Partida-Arangure et al. (unpublished data) whose work with the same microorganisms at a concentration of 2 × 10 6 CFU g feed − 1 (4 × 10 5 CFU strain − 1). Microorganisms were grown, washed, and count as in Apún-Molina et al. (2009). Cells (2 × 10 6) from each isolate were centrifuged at 12,000 g, dried in an oven (Felisa, Jalisco, Mexico) at 74 °C for 4 h, and weighed. The dried cell pellet was ground in a mortar. Dry Oil ® (Innovaciones Acuícolas, S.A. de C.V., Culiacán, Mexico) was used as adhesive and as feed attractant. Feed was dried at room temperature and stored at 4 °C for 6 days (Peraza-Gómez et al., 2011; Partida-Arangure et al., unpublished data). 2.3. V. sinaloensis strains
the water was changed at day 3 and uneaten food and waste matter were removed daily before feeding. 2.5. Disease resistance trial Shrimp weighing 8.1 ± 1.4 g were fed with experimental diet (Purina ®, 40% protein) with immunostimulants at different frequencies. The bioassay was conducted for 21 days as a completely randomized design with five treatments in triplicate: (I) shrimp fed with commercial feed + sterile saline solution at 2.5% NaCl (control group I); (II) shrimp feed with commercial feed + LD50 Vibrio (control group II); (III) shrimp fed daily with experimental diet + LD50 Vibrio; (IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; and (V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. During the first 6 days, animals in all treatments were fed with their respective diet, on the 7th day the shrimp were injected intramuscularly with 40 μL of either vibrio mixture (treatments II–VI) or saline solution (treatment I). During the bioassay, the water temperature was maintained at 23.3 ± 0.1 °C, oxygen at 6.2 ± 0.2 mg mL − 1, and salinity 35‰. Water parameters measured during the trial period remained within acceptable ranges (Brock and Main, 1994). At the end of the bioassay, the survival percentage was determined and hemolymph samples were collected to determine immunological parameters. 2.6. Hemolymph collection and total hemocytes count Hemolymph was sampled from 12 intermolt shrimp per treatment and total hemocytes count was determined. Hemolymph (100 μL) of individual shrimp was withdrawn from the pleopod base of the first abdominal segment with a sterile 1-mL syringe (25 G × 13 mm needle). Before hemolymph extraction, the syringe was loaded with a precooled (4 °C) solution (SIC-EDTA, Na2) (450 mM NaCl, 10 mM KCl, 10 mM hepes and 10 mM EDTA, Na2 at pH 7.3) used as an anticoagulant (Vargas-Albores et al., 1993). Fifty microliters of the anticoagulant–hemolymph mixture were diluted in 150 μL of formaldehyde (4%) and then 20 μL were placed on a hemocytometer (Neubauer) to determine the total hemocytes count (THC) using a compound microscope. The remainder of the hemolymph was stored individually in Eppendorf tubes and kept on ice for separation of plasma and hemocytes. 2.7. Separation of plasma and hemocytes Samples of hemolymph were immediately centrifuged at 800 g for 10 min at 4 °C and the plasma was frozen at − 80 °C. The hemocyte pellet was re-suspended and washed once in precooled anticoagulant solution by centrifugation at 800 g for 10 min at 4 °C. Finally, the hemocytes were re-suspended in 200–600 μL cacodylate bufer (10 mM, pH 7).
A mixture of V. sinaloensis strains (VHPC18, VHPC23, VHPC24, and VIC30), isolated from L. vannamei, was used to challenge white shrimp with a lethal doses 50 value (LD50) of 1.178 × 10 5 CFU g− 1 of body weight. Overnight cultures (TS broth) of the bacterial strains to be tested were washed by centrifugation (10,000 g for 10 min) and suspended in sterile saline solution (2.5% NaCl). The bacterial suspensions were adjusted to an optical density of one. The experimental inoculation of bacteria was performed with a mixture containing isolates VHPC18, VHPC23, VHPC24, and VIC30 at the same proportion. Shrimp were injected into the first abdominal segment with 40 μL of either bacterial mixture or saline solution (2.5% NaCl) using a sterile 1-mL syringe with a 25-gauge needle (Flores-Miranda, unpublished data).
Samples were frozen at −80 °C to break the hemocytes and then thawed; this procedure was carried out twice. Individual samples were centrifuged at 15,000 g for 10 min at 4 °C and the HLS was used immediately to run the immunological analysis or stored at −80 °C.
2.4. Shrimp acclimation to laboratory conditions
2.9. Enzymatic activities in plasma and HLS (The API ZYM system)
The healthy shrimp selection was done for visible features. Shrimp were acclimated to ambient laboratory conditions for 5 days in 120-L indoor plastic tanks containing 80 L of filtered (20 mm) sea water (34–35‰) and constant aeration in groups of 10 organisms per tank. Shrimp were fed twice daily at 09:00 and 17:00 h with commercial feed (Purina®, 40% protein). Ration was 6% of the body weight. Half of
The API ZYM® commercial kit for enzymatic activity detection (BioMerieux, Durham, NC, USA) is a semiquantitative colorimetric micromethod to assess 19 hydrolytic enzymes (proteases: leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin; lipases: lipase esterase (C8), lipase (C14); glycosidases: a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase,
2.8. Preparation of hemocyte lysate supernatant (HLS)
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b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase; esterases: esterase (C1); and phosphatases: alkaline phosphatase, acid phosphatase, naphthol phosphohydrolase) that was used according to the instruction manual of the manufacturer. Samples (one pool, each of six animals) of HLS or plasma were added to the reaction strips, 65 μL well − 1, and incubated at 37 °C for 4 h. Five to 10 min after addition of reagents from the API ZYM ® kit at room temperature, the resulting colors were estimated under natural light and recorded from 0 to 5, according to the color scale provided by the manufacturer, and transformed to the amount of hydrolyzed substrate (nM). The specific activity was expressed as units, where one unit represents the substrate hydrolyzed in nanomoles (nM) per milligram of protein. One-time measurement was performed for each pool (one strip). 2.10. Lysozyme-like activity in the plasma and HLS To detect lysozyme-like activity, the inoculated substrate in Petri dishes (90 mm diameter; 15 mm height) was used as a standard assay. One milliliter was taken from a 4 mg mL− 1 suspension of dried Micrococcus luteus (Sigma) and diluted in 14 mL of 50 mM Tris–HCl buffer-1% agarose at pH 5.2, and then spread on a Petri dish. Once the agarose had solidified, 6.0 mm diameter wells were sunk in the substrate. Human saliva, diluted 1:9 in a 0.1% NaCl solution, was used as a positive control. Tris–HCl buffer was used as a negative control. Wells were filled with 30 μL of sample (HLS) and controls (positive and negative). After 24-h incubation at 37 °C, the diameter of the clear zone surrounding the wells was measured. The diameter of each clearance zone was obtained by measuring the total diameter minus the diameter of the well. Results were expressed in units (0.1 mm= 1 U) per mg protein (U mg− 1 protein). One-time measurement was done for each pool, each with three replicates (Canicatti, 1990; Luna-González et al., 2004). 2.11. Protein determination The protein concentration in plasma and HLS was determined according to the method described by Bradford (1976), with bovine serum albumin (BSA) from Sigma as standard. In plasma, protein concentration ranged from 39.47 ± 0.41 to 122.07 ± 11.07 mg mL − 1. In HLS, protein concentration ranged from 1.25 ± 0.01 to 3.22 ± 0.11 mg mL − 1. 2.12. Statistical analysis
Fig. 1. Percentage of shrimp survival. I) shrimp fed with commercial feed + saline solution at 2.5% NaCl (control group I); II) shrimp feed with commercial feed + LD50 Vibrio (control group II); III) shrimp fed daily with experimental diet + LD50 Vibrio; IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. The survival data represent mean ± SD. The survival data with different letters are significantly different (p b 0.05).
obtained in treatments III and IV suggest that continuous use of immunostimulants led to lowered survival. Total hemocyte count was determined at the end of the bioassay with shrimp fed with microbial immunostimulants. THC of shrimp in treatment I was 19.9 × 10 6 ± 2.0 cells mL − 1; 13.8 ± 1.0 in treatment II; 24.0 × 10 6 ± 3.0 in treatment III; 21.1 × 10 6 ± 3.0 in treatment IV; and 17.6 × 10 6 ± 2.0 in treatment V. THC of shrimp in treatment III was the highest of all treatments and also was significantly higher than treatment II (p = 0.03) (Fig. 2). These data indicated immunostimulation; however, survival was not better than treatment II. At the end of the bioassay, an important activity of nine hydrolytic enzymes, included in the API ZYM kit, were detected in samples (HLS and plasma) of L. vannamei. Nine enzymes were found in plasma and six were found in HLS. The highest levels of enzymatic activity were found in HLS (Table 1). Six enzymes were found in plasma with treatments I, II, III, whereas nine and seven enzymes were found with treatments IV and V, respectively. Some enzymes (alkaline phosphatase, esterase lipase, acid phosphatase, N-acetyl-β-glucosaminidase, α-fucosidase) of treatment II had higher enzymatic activity than treatments I, III, IV, and V. In treatment II, shrimp were challenged with Vibrio and fed only with commercial feed.
One-way analysis of variance (ANOVA) using the F test was applied to examine the differences in total hemocyte count and survival (%) among treatments. Survival data were arcsine transformed according to Daniel (1997). Where significant ANOVA differences were found, a Tukey's HSD test was used to identify the nature of these differences at p b 0.05. 3. Results Fig. 1 summarizes the results obtained in the experiment. The survival of shrimp in treatment I was 100 ± 0.0%, which was significantly different from treatment II (70 ± 0.0%) (p = 0.003). In treatment III, the survival of shrimp (90 ± 0.0%) was not significantly different from the treatments I, II, IV, and V. Treatment IV showed a survival (93.3 ± 11.5%) significantly different as compared to treatment II (p = 0.02). Treatment V showed a survival (86.7 ± 5.7%) significantly different as compared with treatment I (p = 0.03). There were no significant differences among the treatments with the experimental diet (III, IV, and V). Shrimp fed with immunostimulants every six days were not protected against V. sinaloensis. Results
Fig. 2. Total hemocyte count of Litopenaeus vannamei. I) shrimp fed with commercial feed + saline solution at 2.5% NaCl (control group I); II) shrimp feed with commercial feed + LD50 Vibrio (control group II); III) shrimp fed daily with experimental diet + LD50 Vibrio; IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. Error bars = mean ± SD. Different letters indicate significant difference (p b 0.05).
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Table 1 Enzymatic activity using the API ZYM kit (nM of hydrolyzed substrate) and the lysoplate (U mg− 1 protein) detection assay in the plasma and hemocyte lysate of Litopenaeus vannamei. Plasma
HLS
Enzyme
I
II
III
IV
V
I
II
III
IV
V
Alkaline phosphatase Esterase (C4) Esterase lipase (C8) Acid phosphatase Naphthol-AS-Bi-phosphohydrolase β-Galactosidase α-Glucosidase N-acetyl-β-glucosaminidase α-Fucosidase Lysozyme
6.1 0 0.9 5.2 0.9 0 0 1.7 5.2 20.8
13.6 0 3.9 11.7 1.9 0 0 5.8 9.7 31.0
3.8 0 1.3 3.8 1.3 0 0 1.3 3.8 0
6.3 0.8 4.7 6.3 0.8 3.9 2.4 3.1 3.9 0
4.9 1.2 1.6 6.6 2.5 0 0 3.3 5.8 0
37.5 150.1 187.7 300.3 75.1 0 0 56.3 0 0
23.9 191.2 71.7 95.6 71.7 0 0 95.6 0 241.7
30.5 91.5 152.5 244.0 30.5 0 0 30.5 0 0
61.8 61.8 92.6 154.4 61.8 0 0 0 0 0
38.2 95.5 133.7 152.8 38.2 0 0 38.2 0 0
In HLS, six enzymes were found with treatments I, II, III, and V. With treatment IV, five enzymes were found. Alkaline phosphatase showed higher activity with treatment IV (61.8 U) than treatments I, II, III, and V. Acid phosphatase with treatment I showed higher activity than treatments with vibrios and IM. With treatment II, N-acetyl-βglucosaminidase showed higher activity than with treatments I, III, and V, but it was not found with treatment IV. Table 1 shows the lysozyme activity in plasma and HLS. Enzymatic activity in plasma was detected only with treatments I and II. In the HLS, lysozyme showed activity only with treatment II where shrimp were challenged with vibrios and fed commercial diet. Results indicated that some enzymes (β-galactosidase, α-glucosidase, α-fucosidase) were found in plasma but not in HLS, suggesting that some enzymes present in plasma have a different origin from those in hemocytes. 4. Discussion Infectious diseases have become a major constraint and the most limiting factor for the shrimp culture industry. This situation indicates that there is an urgent need to explore every possible avenue for developing novel control strategies against shrimp diseases (Li-Shi et al., 2007). According to the above, this study was conducted to investigate whether the oral administration of microbial immunostimulants is capable of protecting L. vannamei against V. sinaloensis strains. The effect of several immunostimulants on disease resistance has been widely studied in shrimp culture. In this work, the level of survival in response to bacterial challenge in L. vannamei was significantly higher in shrimp fed with the commercial feed plus immunostimulants every 3 days, whereas the survival declined in the control group II fed only with commercial feed. These results are consistent with those of Itami et al. (1998) who fed peptidoglycan to Penaeus monodon at 0.2% of diet. Their results showed improved resistance to Vibrio penaeicida as compared to controls. Similarly, Burgents et al. (2004) observed enhanced resistance of juvenile L. vannamei fed with XP Yeast Culture® and challenged with Vibrio sp. 90-69B3. In Fenneropenaeus indicus, Sajeevan et al. (2006) observed that adults fed with Candida sake S165 in the diet (10%) showed enhanced resistance to WSSV infection. In contrast, shrimp fed with immunostimulants (treatment III) daily showed a declined survival similar to control II. This negative effect associated with daily administration of immunostimulants is consistent with results obtained in P. monodon (Chang et al., 2000; Song and Hsieh, 1994) and F. indicus (Sajeevan et al., 2006). The high survival of shrimp obtained in this work with a known dose and discontinuous administration of immunostimulants confirms the results of other authors, as Itami et al. (1998) who fed peptidoglycan to P. monodon at 0.2% of the diet for seven consecutive days alternated with 7 days without the immunostimulant. These
authors reported improved resistance to V. penaeicida as compared to controls. Recently, Sajeevan et al. (2009b) observed that F. indicus fed with 0.2% glucan once every seven days had maximum survival when challenged orally with WSSV. In agreement with Saajevan and coworkers, we suggest that continuous use of immunostimulants even at an optimal dose may suppress the immunity in shrimp and led to lowered survival. In this work, the highest total hemocyte count was found in shrimp fed with dead microorganisms. The protective effect of the yeast and LAB supplement might be attributed to its glucan and peptidoglycan content. THC in control I and treatments (III, IV, V) with immunostimulants was high despite the bacterial challenge. Even more, THC in treatment III was significantly higher than control II although survival was similar. These results are consistent with those obtained by Peraza-Gómez et al. (2009) with the same live microorganisms but at a concentration of 1 × 10 6 CFU g − 1 of feed. They found that THC (40 × 10 6 cells mL − 1) was significantly higher in L. vannamei fed daily with microorganisms during 20 days as compared with shrimp fed with commercial feed only (25 × 10 6 cells mL − 1). Similar results were also found in Penaeus chinensis (Kim et al., 1999) and Penaeus japonicus (Sequeira et al., 1996). In contrast to our study, Chiu et al. (2007) reported a decreased THC in L. vannamei fed a diet containing live cells of Lactobacillus plantarum. Such higher THC numbers may provide improved immunity during periods of higher activity or increased pathogen loads. Hemocytes are the first line of defense in invertebrates (Johansson and Söderhäll, 1985). Therefore, hemocyte number is very important because individuals with a high amount of hemocytes in circulation resist better the presence of a pathogen (Le Moullac et al., 1997). Lysosomal hydrolytic enzymes have been scarcely studied in crustaceans, but as in mollusks, the hemocytes of shrimp produce several such enzymes (protease, esterase, phosphatase, lipase, and phosphatase) (Carajaraville et al., 1995; Peraza-Gómez et al., 2011). In plasma, shrimp challenged with vibrios and fed commercial feed (control II) showed the activity of seven enzymes (alkaline phosphatase, esterase lipase, acid phosphatase, N-acetyl-β-glucosaminidase, Naphthol-AS-Bi-phosphohydrolase, α-fucosidase, and lysozyme) with the highest activity as compared with treatments I, III, IV, and V. However, in treatment IV, we found activity of nine enzymes. Remarkably, no lysozyme was found in treatments III, IV, and V (plasma, HLS) challenged with vibrios and fed with immunostimulants. The lysozymes of penaeid are well characterized and shown to have lytic activity against several species of Gram positive and Gram negative bacteria, including pathogenic species of Vibrio (De la Re-Vega et al., 2006; Hikima et al., 2003). Peraza-Gómez et al. (2011) found that L. vannamei fed commercial feed with probiotics and challenged with WSSV showed high activity of acid phosphatase and N-acetyl-β-glucosaminidase. In bivalve mollusks, lysosomal enzymes are released into plasma during physiological events and pathological stress. Lysosomal enzymes are involved in the death and degradation
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of microorganisms and particles inside the hemocytes and in some cases are released from the cell to plasma or other tissues where they change the molecular conformation of the cellular surface pathogens, encouraging recognition and phagocytosis (Carajaraville et al., 1995; Cheng, 1983; Cheng, 1992; López et al., 1997). In this work, some enzymes (β-galactosidase, α-glucosidase, α-fucosidase) were found in plasma but not in HLS, suggesting that some enzymes present in plasma have a different origin from those in hemocytes. 5. Conclusion The mixture of four lactic acid bacteria and one yeast at 8.4 mg kg feed − 1 administered every 3 days increased the survival of shrimp after their challenge with V. sinaloensis as compared to the control group fed with commercial feed and a posterior bacterial infection. Shrimp fed daily with microbial immunostimulants enhanced THC. This study demonstrates the positive effect of microbial immunostimulants in juvenile L. vannamei. Acknowledgments Authors are grateful to Consejo Estatal de Ciencia y Tecnología del Estado de Sinaloa (CECyT-Sinaloa) and Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional (SIP-IPN) for financial support. Ma. del Carmen Flores Miranda acknowledges CONACYTMexico and SIP-IPN for the M.Sc. grants. References Apún-Molina, J.P., Santamaría-Miranda, A., Luna-González, A., Martínez-Díaz, S.F., Rojas-Contreras, M., 2009. Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature. Aquac. Res. 40, 887–894. Azad, I.S., Panigrahi, A., Gopal, C., Paulpandi, S., Mahima, C., Ravichandran, P., 2005. Routes of immunostimulation vis-a-vis survival and growth of Penaeus monodon postlarvae. Aquaculture 248, 227–234. 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. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brock, J., Main, K.L., 1994. A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. World Aquaculture Society, Baton Rouge, Louisiana, USA. 242 pp. Burge, E.J., Madigan, D.J., Burnett, L.E., Burnett, K.G., 2007. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio. Fish Shellfish Immunol. 22, 327–339. Burgents, J.E., Burnett, K.G., Burnett, L.E., 2004. Disease resistance of Pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 231, 1–8. Canicatti, C., 1990. Distribution d'une activite lysozymiale dans un echinoderme holothuroide, Holothuria polii, et dans les oeufs et les larves d'un echinoderme echinoide, Paracentrotus lividus. Eur. Arch. Biol. 101, 309–318. Carajaraville, M.P., Pal, S.G., Robledo, Y., 1995. Light and electron microscopical localization of lysosomal acid hydrolases in bivalve haemocytes by enzyme cytochemistry. Acta Histochem. Cytoc. 28, 409–416. Chang, C.F., Chen, H.Y., Su, M.S., Liao, I.C., 2000. Immunomodulation by dietary β-1,3 glucan in the brooders of the black tiger shrimp, Penaeus monodon. Fish Shellfish Immunol. 10, 505–514. Chang-Che, L., Jiann-Chu, C., 2008. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress. Fish Shellfish Immunol. 25 (6), 701–709. Chen, D., Ainsworth, A.J., 1992. Glucan administration potentiates immune defence mechanisms of channel catfish, Ictalurus punctatus Rafinesque. J. Fish Dis. 15, 295–304. Cheng, T.C., 1983. The role of lysosomes in molluscan inflammation. Am. Zool. 23, 129–144. Cheng, T.C., 1992. Selective induction of release of hydrolases from Crassostrea virginica hemocytes by certain bacteria. J. Invertebr. Pathol. 59, 197–200. Chisholm, J.R.S., Smith, V.J., 1995. Comparison of antibacterial activity in the hemocytes of different crustacean species. Comp. Biochem. Physiol. 110A, 39–45. Chiu, C.H., Guu, Y.K., Liu, C.H., Pan, T.M., Cheng, W., 2007. Immune responses and gene expression inwhite shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol. 23, 364–377. Daniel, W.W., 1997. Bioestadística. Base para el análisis de las ciencias de la salud. D. F. Editorial Limusa, Mexico. 639–693pp. De la Re-Vega, E., García-Galaz, A., Díaz-Cinco, M.E., Sotelo-Mundo, R.R., 2006. White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity
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against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae. Fish Shellfish Immunol. 20, 405–408. Destoumieux, D., Munñoz, M., Cosseau, C., Rodríguez, J., Bulet, P., Comps, M., Bachère, E., 2000. Penaedins, antimicrobial peptides with chitin binding activity, are produced and stored in shrimp granulocytes and released after microbial challenge. J. Cell Sci. 113, 461–469. Doñate, C., Balasch, J.C., Callol, A., Bobe, J., Tort, L., MacKenzie, S., 2010. The effects of immunostimulation through dietary manipulation in the rainbow trout; evaluation of mucosal immunity. Mar. Biotechnol. 12, 88–99. Hikima, S., Hikima, J., Rojtinnakorn, J., Hirono, I., Aoki, T., 2003. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene 316, 187–195. Itami, T., Kubono, K., Asano, M., Tokushige, K., Takeno, N., Nishimura, H., Kondo, M., Takahashi, Y., 1998. Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 164, 277–288. Johansson, M.W., Söderhäll, K., 1985. Cellular immunity in crustaceans and the proPO system. Parasitol. Today 5, 171–176. Johansson, M.W., Söderhäll, K., 1989. A cell adhesion factor from crayfish haemocyte has degranulating activity towards crayfish granular cells. Insect Biochem. 19, 183–190. Jorgensen, J.B., Lunde, H., Robertsen, B., 1993. Peritoneal and head kidney cell response to intraperitoneally injected glucans in Atlantic salmon, Salmo salar L. J. Fish Dis. 16, 313–325. Kim, Y.J., Choi, W.Ch., Kim, H.R., Jung, S.J., Oh, M.J., 1999. Changes in Penaeus chinensis haemocytes during whitespot baculovirus (WSBV) infections. Bull. Eur. Ass. Fish Pathol. 19, 213–215. Le Moullac, G., Le Groumellec, M., Ansquer, D., Froissard, S., Levy, P., 1997. Haematological and phenoloxidase activity changes in the shrimp Penaeus stylirostris in relation with the moult cycle: protection against vibriosis. Fish Shellfish Immunol. 7, 227–234. Lightner, D.V., Lewis, D.H., 1975. A septicemia bacterial disease syndrome in penaeid shrimp. Mar. Fish. Rev. 37, 25–28. Li-Shi, Y., Zhi-Xin, Y., Ji-Xiang, L., Xian-De, H., Chang-Jun, G., Shao-Ping, W., Siu-Ming, Ch., Xiao-Qiang, Y., Jian-Guo, H., 2007. A Toll receptor in shrimp. Mol. Immunol. 44, 1999–2008. Lo, C.F., Chang, Y.S., Peng, S.E., Kou, G.H., 2003. Major viral diseases of Penaeus monodon in Taiwan. J. Fish Soc. Taiwan 30, 1–13. López, C., Carballal, M.J., Azevedo, C., Villalba, A., 1997. Enzyme characterization of the circulating haemocytes of the carpet shell clam, Ruditapes decissatus (Mollusca: Bivalvia). Fish Shellfish Inmunol. 7, 595–608. Luna-González, A., Maeda-Martínez, A.N., Ascencio-Valle, F., Robles-Mungaray, M., 2004. Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas. Fish Shellfish Immunol. 16, 287–294. Muta, T., Iwanaga, S., 1996. The role of hemolymph coagulation in innate immunity. Curr. Opin. Immunol. 8, 41–47. Peraza-Gómez, V., Luna-González, A., Campa-Córdova, A.I., López-Meyer, M., FierroCoronado, J.A., Álvarez–Ruiz, P., 2009. Probiotic microorganisms and antiviral plants reduce mortality and prevalence of WSSV in shrimp (Litopenaeus vannamei) cultured under laboratory conditions. Aquac. Res. 40, 1481–1489. Peraza-Gómez, V., Luna-González, A., Campa-Córdova, Á.I., Fierro-Coronado, J.A., González Ocampo, H.A., Sainz-Hernández, J.C., 2011. Dietary microorganism and plant effects on the survival and immune response of Litopenaeus vannamei challenged with the white spot syndrome virus. Aquac. Res. 42, 559–570. Rendón, L., Balcázar, J.L., 2003. Inmunología de camarones: conceptos básicos y recientes avances. Revista AquaTIC 19, 27–33 http://www.revistaaquatic.com/ aquatic/art.asp?t=p&c=158. Rorstad, G., Aasjord, P.M., Robertsen, B., 1993. Adjuvant effect of a yeast glucan in vaccines against furunculosis in Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol 3, 179–190. Sajeevan, T.P., Rosamma, P., Bright Singh, I.S., 2006. Immunostimulatory effect of amarine yeast Candida sake S165 in Fenneropenaeus indicus. Aquaculture 257, 150–155. Sajeevan, T.P., Lowman, D.W., Williams, D.L., Selven, S., Anas, A., Rosamma, P., 2009a. Marine yeast diet confers better protection than its cell wall component (1–3)-β-D-glucan as an immunostimulant in Fenneropenaeus indicus. Aquac. Res. 40, 1723–1730. Sajeevan, T.P., Rosamma, P., Bright Singh, I.S., 2009b. Dose/frequency: a critical factor in the administration of glucan as immunostimulant to Indian white shrimp Fenneropenaeus indicus. Aquaculture 287, 248–252. Sequeira, T., Tavarest, D., Arala-Chaves, M., 1996. Evidence for circulating hemocyte proliferation in the shrimp Penaeus japonicus. Dev. Comp. Immunol. 20, 97–104. Söderhäll, K., Cerenius, L., Johnson, M.W., 1994. The prophenoloxidase activity system and its role in invertebrate defense: foundations for the invertebrate immune system. Ann. Ny. Acad. Sci. 712, 155–166. Song, Y.L., Hsieh, Y.T., 1994. Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Dev. Comp. Immunol. 18, 102–209. Song, Y.L., Liu, J.J., Chan, L.C., Sung, H.H., 1997. Glucan induced disease resistance in tiger shrimp (Penaeus monodon). Dev. Biol. Stand. 90, 413–421. Soto-Rodríguez, S., Gómez-Gil, B., Lozano, R., 2008. MICs de antibióticos de Vibrio spp. aislados del L. vannamei cultivado en México. Panorama Acuícola 14 (1), 52–57. Sritunyalucksana, K., Cerenius, L., Söderhäll, K., 1999. Molecular cloning and characterisation of prophenoloxidase in the black tiger shrimp, Penaeus monodon. Dev. Comp. Immunol. 23, 179–186. Vargas-Albores, F., Guzmán-Murillo, M.A., Ochoa, J.L., 1993. An anticoagulant solution for haemolymph collectionand prophenoloxidase studies of Penaeid shrimp (Penaeus californiensis). Comp. Biochem. Phys. A 106, 299–303.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Microbial immunostimulants reduce mortality in whiteleg shrimp (Litopenaeus vannamei) challenged with Vibrio sinaloensis strains Ma. del Carmen Flores-Miranda a, Antonio Luna-González a,⁎, Ángel I. Campa‐Córdova b, Héctor A. González-Ocampo a, Jesús A. Fierro-Coronado a, Blanca O. Partida-Arangure a a b
Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional-Instituto Politécnico Nacional, Unidad Sinaloa, Sinaloa, Mexico Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, B.C.S., Mexico
a r t i c l e
i n f o
Article history: Received 24 June 2011 Received in revised form 28 July 2011 Accepted 4 August 2011 Available online 10 August 2011 Keywords: Immunostimulants Bacteria Yeast Vibrio Litopenaeus vannamei Pacific white shrimp
a b s t r a c t The effect of microbial immunostimulants on the survival and immune response of juvenile Litopenaeus vannamei challenged with Vibrio sinaloensis strains was evaluated. Dead microorganisms were added to feed with the attractant Dry Oil® and consisted of four lactic acid bacteria (Lta2, Lta6, Lta8, and Lta10) and one yeast (Lt6). V. sinaloensis strains or saline solution were inoculated to shrimp by injection. The bioassay was conducted for 21 days with five treatments in triplicate: (I) shrimp fed with commercial feed + sterile saline solution at 2.5% NaCl (control group I); (II) shrimp feed with commercial feed + LD50 Vibrio (control group II); (III) shrimp fed daily with experimental diet + LD50 Vibrio; (IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; and (V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. Shrimp (8.1 ± 1.4 g) were cultured in 120-L plastic tanks and fed twice a day. The activity of lysosomal enzymes in plasma and hemocytes were determined with the API ZYM kit and lysoplate assay. Survival of shrimp in treatment IV was significantly higher than those of control II. Total hemocyte count in treatment III was significantly higher than control II. The activity of nine hydrolytic enzymes was found in plasma and six in the hemocyte lysate supernatant (HLS). Shrimp fed with immunostimulants every six days were not protected against V. sinaloensis. The results indicate that these microbial immunostimulants administered every three days is a good feed additive against Vibrio spp. in shrimp culture. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Aquaculture represents one of the main food providers in the world. In shrimp farming, whiteleg shrimp (Litopenaeus vannamei) is the primary penaeid shrimp currently being cultured in Central and South America (Burge et al., 2007; Chang-Che and Jiann-Chu, 2008). However, during the past two decades, worldwide commercial shrimp farming has suffered outbreaks of diseases caused mainly by Vibrio bacteria and viruses due to a deteriorated pond environment (Lo et al., 2003). Species of Vibrio are well known in penaeid shrimp culture as causative agents of vibriosis, which is a serious threat to the aquaculture industry, responsible for massive mortality of cultured penaeids worldwide (Baticados et al., 1990; Lightner and Lewis, 1975). Treatment of shrimp suspected of being infected with Vibrio spp. is mainly based on the use of antibiotics, but the susceptibility of vibrios to antibiotics varies widely among strains of the same species. There is little information about the detailed use of antibiotics; even their use in ⁎ Corresponding author at: Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional (Unidad Sinaloa), Boulevard Juan de Dios Bátiz Paredes 250, Guasave, Sinaloa 81101, Mexico. Tel./fax: + 52 687 87 2 96 26. E-mail address: [email protected] (A. Luna-González). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.08.005
aquaculture may develop antibiotic resistance in pathogens that infect those cultured animals and humans (Soto-Rodríguez et al., 2008). The application of immunostimulants in shrimp aquaculture is increasingly gaining interest as an environmentally safe alternative to antibiotics and chemotherapeutics (Song et al., 1997). Shrimp possess an innate immune system, consisting of cellular and humoral elements. Hemocytes play a central role in the non-specific immune response of shrimp, which rely mainly on phagocytosis, melanization, encapsulation, cytotoxicity, and clotting (Sritunyalucksana et al., 1999). Humoral defense factors, such as clotting proteins, agglutinins, hydrolytic enzymes, and antimicrobial peptides are released upon lysis of hemocytes, which is induced by lipopolysaccharides (LPS), peptidoglycans, and β-1,3-glucans (Chisholm and Smith, 1995; Destoumieux et al., 2000; Johansson and Söderhäll, 1989; Muta and Iwanaga 1996; Söderhäll et al., 1994). Diets containing immunostimulants are used in aquaculture in order to increase resistance to stress and diseases of cultured fishes and invertebrates by alerting the immune system (Doñate et al., 2010; Rendón and Balcázar, 2003). They can be extracted from the walls of microorganisms such as Gram-negative bacteria (lipopolysaccharides), Gram-positive bacteria (peptidoglycans), and fungi (β-1, 3-glucans). Furthermore, the whole cell can be used as immunostimulant (Sajeevan et al., 2009a; Partida-Arangure et al., unpublished data). There are
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several methods of stimulation like immersion, injection, and feeding among others (Chen and Ainsworth, 1992; Jorgensen et al., 1993; Rorstad et al., 1993). However, the most practical method of stimulation is incorporating the immunostimulating substances into the feed (Azad et al., 2005). Currently many commercial immunostimulants are available in the shrimp aquaculture industry and are extensively used by shrimp farmers. However, scientific data in support of their function and dose/frequency of application are lacking. Information regarding the dose is essential as overdose leads to immunosuppression, rendering less protection to infection (Chang et al., 2000; Sajeevan et al., 2006, Sajeevan et al., 2009b). This study was undertaken to examine the survival and the immune response of L. vannamei against Vibrio sinaloensis strains when shrimp were treated with whole-cell microbial immunostimulants. 2. Materials and methods 2.1. Microbial immunostimulants Lactic acid bacteria (Lta2, Lta6, Lta8, and Lta10) and yeast (Lt6) used in this work as immunostimulants were originally isolated, characterized, and tested by Apún-Molina et al. (2009) and Peraza-Gómez et al. (2011). 2.2. Preparation of experimental diet with microorganisms A mixture of four selected lactic acid bacteria (LAB) and one heatkilled yeast (74 °C) was sprayed on commercial feed (Purina ®, Ciudad Obregón, Mexico, 40% protein) at 8.4 mg kg feed − 1 (1.68 mg per microbial isolate). The amount of immunostimulants was based on the work of Partida-Arangure et al. (unpublished data) whose work with the same microorganisms at a concentration of 2 × 10 6 CFU g feed − 1 (4 × 10 5 CFU strain − 1). Microorganisms were grown, washed, and count as in Apún-Molina et al. (2009). Cells (2 × 10 6) from each isolate were centrifuged at 12,000 g, dried in an oven (Felisa, Jalisco, Mexico) at 74 °C for 4 h, and weighed. The dried cell pellet was ground in a mortar. Dry Oil ® (Innovaciones Acuícolas, S.A. de C.V., Culiacán, Mexico) was used as adhesive and as feed attractant. Feed was dried at room temperature and stored at 4 °C for 6 days (Peraza-Gómez et al., 2011; Partida-Arangure et al., unpublished data). 2.3. V. sinaloensis strains
the water was changed at day 3 and uneaten food and waste matter were removed daily before feeding. 2.5. Disease resistance trial Shrimp weighing 8.1 ± 1.4 g were fed with experimental diet (Purina ®, 40% protein) with immunostimulants at different frequencies. The bioassay was conducted for 21 days as a completely randomized design with five treatments in triplicate: (I) shrimp fed with commercial feed + sterile saline solution at 2.5% NaCl (control group I); (II) shrimp feed with commercial feed + LD50 Vibrio (control group II); (III) shrimp fed daily with experimental diet + LD50 Vibrio; (IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; and (V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. During the first 6 days, animals in all treatments were fed with their respective diet, on the 7th day the shrimp were injected intramuscularly with 40 μL of either vibrio mixture (treatments II–VI) or saline solution (treatment I). During the bioassay, the water temperature was maintained at 23.3 ± 0.1 °C, oxygen at 6.2 ± 0.2 mg mL − 1, and salinity 35‰. Water parameters measured during the trial period remained within acceptable ranges (Brock and Main, 1994). At the end of the bioassay, the survival percentage was determined and hemolymph samples were collected to determine immunological parameters. 2.6. Hemolymph collection and total hemocytes count Hemolymph was sampled from 12 intermolt shrimp per treatment and total hemocytes count was determined. Hemolymph (100 μL) of individual shrimp was withdrawn from the pleopod base of the first abdominal segment with a sterile 1-mL syringe (25 G × 13 mm needle). Before hemolymph extraction, the syringe was loaded with a precooled (4 °C) solution (SIC-EDTA, Na2) (450 mM NaCl, 10 mM KCl, 10 mM hepes and 10 mM EDTA, Na2 at pH 7.3) used as an anticoagulant (Vargas-Albores et al., 1993). Fifty microliters of the anticoagulant–hemolymph mixture were diluted in 150 μL of formaldehyde (4%) and then 20 μL were placed on a hemocytometer (Neubauer) to determine the total hemocytes count (THC) using a compound microscope. The remainder of the hemolymph was stored individually in Eppendorf tubes and kept on ice for separation of plasma and hemocytes. 2.7. Separation of plasma and hemocytes Samples of hemolymph were immediately centrifuged at 800 g for 10 min at 4 °C and the plasma was frozen at − 80 °C. The hemocyte pellet was re-suspended and washed once in precooled anticoagulant solution by centrifugation at 800 g for 10 min at 4 °C. Finally, the hemocytes were re-suspended in 200–600 μL cacodylate bufer (10 mM, pH 7).
A mixture of V. sinaloensis strains (VHPC18, VHPC23, VHPC24, and VIC30), isolated from L. vannamei, was used to challenge white shrimp with a lethal doses 50 value (LD50) of 1.178 × 10 5 CFU g− 1 of body weight. Overnight cultures (TS broth) of the bacterial strains to be tested were washed by centrifugation (10,000 g for 10 min) and suspended in sterile saline solution (2.5% NaCl). The bacterial suspensions were adjusted to an optical density of one. The experimental inoculation of bacteria was performed with a mixture containing isolates VHPC18, VHPC23, VHPC24, and VIC30 at the same proportion. Shrimp were injected into the first abdominal segment with 40 μL of either bacterial mixture or saline solution (2.5% NaCl) using a sterile 1-mL syringe with a 25-gauge needle (Flores-Miranda, unpublished data).
Samples were frozen at −80 °C to break the hemocytes and then thawed; this procedure was carried out twice. Individual samples were centrifuged at 15,000 g for 10 min at 4 °C and the HLS was used immediately to run the immunological analysis or stored at −80 °C.
2.4. Shrimp acclimation to laboratory conditions
2.9. Enzymatic activities in plasma and HLS (The API ZYM system)
The healthy shrimp selection was done for visible features. Shrimp were acclimated to ambient laboratory conditions for 5 days in 120-L indoor plastic tanks containing 80 L of filtered (20 mm) sea water (34–35‰) and constant aeration in groups of 10 organisms per tank. Shrimp were fed twice daily at 09:00 and 17:00 h with commercial feed (Purina®, 40% protein). Ration was 6% of the body weight. Half of
The API ZYM® commercial kit for enzymatic activity detection (BioMerieux, Durham, NC, USA) is a semiquantitative colorimetric micromethod to assess 19 hydrolytic enzymes (proteases: leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin; lipases: lipase esterase (C8), lipase (C14); glycosidases: a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase,
2.8. Preparation of hemocyte lysate supernatant (HLS)
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b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase; esterases: esterase (C1); and phosphatases: alkaline phosphatase, acid phosphatase, naphthol phosphohydrolase) that was used according to the instruction manual of the manufacturer. Samples (one pool, each of six animals) of HLS or plasma were added to the reaction strips, 65 μL well − 1, and incubated at 37 °C for 4 h. Five to 10 min after addition of reagents from the API ZYM ® kit at room temperature, the resulting colors were estimated under natural light and recorded from 0 to 5, according to the color scale provided by the manufacturer, and transformed to the amount of hydrolyzed substrate (nM). The specific activity was expressed as units, where one unit represents the substrate hydrolyzed in nanomoles (nM) per milligram of protein. One-time measurement was performed for each pool (one strip). 2.10. Lysozyme-like activity in the plasma and HLS To detect lysozyme-like activity, the inoculated substrate in Petri dishes (90 mm diameter; 15 mm height) was used as a standard assay. One milliliter was taken from a 4 mg mL− 1 suspension of dried Micrococcus luteus (Sigma) and diluted in 14 mL of 50 mM Tris–HCl buffer-1% agarose at pH 5.2, and then spread on a Petri dish. Once the agarose had solidified, 6.0 mm diameter wells were sunk in the substrate. Human saliva, diluted 1:9 in a 0.1% NaCl solution, was used as a positive control. Tris–HCl buffer was used as a negative control. Wells were filled with 30 μL of sample (HLS) and controls (positive and negative). After 24-h incubation at 37 °C, the diameter of the clear zone surrounding the wells was measured. The diameter of each clearance zone was obtained by measuring the total diameter minus the diameter of the well. Results were expressed in units (0.1 mm= 1 U) per mg protein (U mg− 1 protein). One-time measurement was done for each pool, each with three replicates (Canicatti, 1990; Luna-González et al., 2004). 2.11. Protein determination The protein concentration in plasma and HLS was determined according to the method described by Bradford (1976), with bovine serum albumin (BSA) from Sigma as standard. In plasma, protein concentration ranged from 39.47 ± 0.41 to 122.07 ± 11.07 mg mL − 1. In HLS, protein concentration ranged from 1.25 ± 0.01 to 3.22 ± 0.11 mg mL − 1. 2.12. Statistical analysis
Fig. 1. Percentage of shrimp survival. I) shrimp fed with commercial feed + saline solution at 2.5% NaCl (control group I); II) shrimp feed with commercial feed + LD50 Vibrio (control group II); III) shrimp fed daily with experimental diet + LD50 Vibrio; IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. The survival data represent mean ± SD. The survival data with different letters are significantly different (p b 0.05).
obtained in treatments III and IV suggest that continuous use of immunostimulants led to lowered survival. Total hemocyte count was determined at the end of the bioassay with shrimp fed with microbial immunostimulants. THC of shrimp in treatment I was 19.9 × 10 6 ± 2.0 cells mL − 1; 13.8 ± 1.0 in treatment II; 24.0 × 10 6 ± 3.0 in treatment III; 21.1 × 10 6 ± 3.0 in treatment IV; and 17.6 × 10 6 ± 2.0 in treatment V. THC of shrimp in treatment III was the highest of all treatments and also was significantly higher than treatment II (p = 0.03) (Fig. 2). These data indicated immunostimulation; however, survival was not better than treatment II. At the end of the bioassay, an important activity of nine hydrolytic enzymes, included in the API ZYM kit, were detected in samples (HLS and plasma) of L. vannamei. Nine enzymes were found in plasma and six were found in HLS. The highest levels of enzymatic activity were found in HLS (Table 1). Six enzymes were found in plasma with treatments I, II, III, whereas nine and seven enzymes were found with treatments IV and V, respectively. Some enzymes (alkaline phosphatase, esterase lipase, acid phosphatase, N-acetyl-β-glucosaminidase, α-fucosidase) of treatment II had higher enzymatic activity than treatments I, III, IV, and V. In treatment II, shrimp were challenged with Vibrio and fed only with commercial feed.
One-way analysis of variance (ANOVA) using the F test was applied to examine the differences in total hemocyte count and survival (%) among treatments. Survival data were arcsine transformed according to Daniel (1997). Where significant ANOVA differences were found, a Tukey's HSD test was used to identify the nature of these differences at p b 0.05. 3. Results Fig. 1 summarizes the results obtained in the experiment. The survival of shrimp in treatment I was 100 ± 0.0%, which was significantly different from treatment II (70 ± 0.0%) (p = 0.003). In treatment III, the survival of shrimp (90 ± 0.0%) was not significantly different from the treatments I, II, IV, and V. Treatment IV showed a survival (93.3 ± 11.5%) significantly different as compared to treatment II (p = 0.02). Treatment V showed a survival (86.7 ± 5.7%) significantly different as compared with treatment I (p = 0.03). There were no significant differences among the treatments with the experimental diet (III, IV, and V). Shrimp fed with immunostimulants every six days were not protected against V. sinaloensis. Results
Fig. 2. Total hemocyte count of Litopenaeus vannamei. I) shrimp fed with commercial feed + saline solution at 2.5% NaCl (control group I); II) shrimp feed with commercial feed + LD50 Vibrio (control group II); III) shrimp fed daily with experimental diet + LD50 Vibrio; IV) shrimp fed every 3 days with experimental diet + LD50 Vibrio; V) shrimp fed every 6 days with experimental diet + LD50 Vibrio. Error bars = mean ± SD. Different letters indicate significant difference (p b 0.05).
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Table 1 Enzymatic activity using the API ZYM kit (nM of hydrolyzed substrate) and the lysoplate (U mg− 1 protein) detection assay in the plasma and hemocyte lysate of Litopenaeus vannamei. Plasma
HLS
Enzyme
I
II
III
IV
V
I
II
III
IV
V
Alkaline phosphatase Esterase (C4) Esterase lipase (C8) Acid phosphatase Naphthol-AS-Bi-phosphohydrolase β-Galactosidase α-Glucosidase N-acetyl-β-glucosaminidase α-Fucosidase Lysozyme
6.1 0 0.9 5.2 0.9 0 0 1.7 5.2 20.8
13.6 0 3.9 11.7 1.9 0 0 5.8 9.7 31.0
3.8 0 1.3 3.8 1.3 0 0 1.3 3.8 0
6.3 0.8 4.7 6.3 0.8 3.9 2.4 3.1 3.9 0
4.9 1.2 1.6 6.6 2.5 0 0 3.3 5.8 0
37.5 150.1 187.7 300.3 75.1 0 0 56.3 0 0
23.9 191.2 71.7 95.6 71.7 0 0 95.6 0 241.7
30.5 91.5 152.5 244.0 30.5 0 0 30.5 0 0
61.8 61.8 92.6 154.4 61.8 0 0 0 0 0
38.2 95.5 133.7 152.8 38.2 0 0 38.2 0 0
In HLS, six enzymes were found with treatments I, II, III, and V. With treatment IV, five enzymes were found. Alkaline phosphatase showed higher activity with treatment IV (61.8 U) than treatments I, II, III, and V. Acid phosphatase with treatment I showed higher activity than treatments with vibrios and IM. With treatment II, N-acetyl-βglucosaminidase showed higher activity than with treatments I, III, and V, but it was not found with treatment IV. Table 1 shows the lysozyme activity in plasma and HLS. Enzymatic activity in plasma was detected only with treatments I and II. In the HLS, lysozyme showed activity only with treatment II where shrimp were challenged with vibrios and fed commercial diet. Results indicated that some enzymes (β-galactosidase, α-glucosidase, α-fucosidase) were found in plasma but not in HLS, suggesting that some enzymes present in plasma have a different origin from those in hemocytes. 4. Discussion Infectious diseases have become a major constraint and the most limiting factor for the shrimp culture industry. This situation indicates that there is an urgent need to explore every possible avenue for developing novel control strategies against shrimp diseases (Li-Shi et al., 2007). According to the above, this study was conducted to investigate whether the oral administration of microbial immunostimulants is capable of protecting L. vannamei against V. sinaloensis strains. The effect of several immunostimulants on disease resistance has been widely studied in shrimp culture. In this work, the level of survival in response to bacterial challenge in L. vannamei was significantly higher in shrimp fed with the commercial feed plus immunostimulants every 3 days, whereas the survival declined in the control group II fed only with commercial feed. These results are consistent with those of Itami et al. (1998) who fed peptidoglycan to Penaeus monodon at 0.2% of diet. Their results showed improved resistance to Vibrio penaeicida as compared to controls. Similarly, Burgents et al. (2004) observed enhanced resistance of juvenile L. vannamei fed with XP Yeast Culture® and challenged with Vibrio sp. 90-69B3. In Fenneropenaeus indicus, Sajeevan et al. (2006) observed that adults fed with Candida sake S165 in the diet (10%) showed enhanced resistance to WSSV infection. In contrast, shrimp fed with immunostimulants (treatment III) daily showed a declined survival similar to control II. This negative effect associated with daily administration of immunostimulants is consistent with results obtained in P. monodon (Chang et al., 2000; Song and Hsieh, 1994) and F. indicus (Sajeevan et al., 2006). The high survival of shrimp obtained in this work with a known dose and discontinuous administration of immunostimulants confirms the results of other authors, as Itami et al. (1998) who fed peptidoglycan to P. monodon at 0.2% of the diet for seven consecutive days alternated with 7 days without the immunostimulant. These
authors reported improved resistance to V. penaeicida as compared to controls. Recently, Sajeevan et al. (2009b) observed that F. indicus fed with 0.2% glucan once every seven days had maximum survival when challenged orally with WSSV. In agreement with Saajevan and coworkers, we suggest that continuous use of immunostimulants even at an optimal dose may suppress the immunity in shrimp and led to lowered survival. In this work, the highest total hemocyte count was found in shrimp fed with dead microorganisms. The protective effect of the yeast and LAB supplement might be attributed to its glucan and peptidoglycan content. THC in control I and treatments (III, IV, V) with immunostimulants was high despite the bacterial challenge. Even more, THC in treatment III was significantly higher than control II although survival was similar. These results are consistent with those obtained by Peraza-Gómez et al. (2009) with the same live microorganisms but at a concentration of 1 × 10 6 CFU g − 1 of feed. They found that THC (40 × 10 6 cells mL − 1) was significantly higher in L. vannamei fed daily with microorganisms during 20 days as compared with shrimp fed with commercial feed only (25 × 10 6 cells mL − 1). Similar results were also found in Penaeus chinensis (Kim et al., 1999) and Penaeus japonicus (Sequeira et al., 1996). In contrast to our study, Chiu et al. (2007) reported a decreased THC in L. vannamei fed a diet containing live cells of Lactobacillus plantarum. Such higher THC numbers may provide improved immunity during periods of higher activity or increased pathogen loads. Hemocytes are the first line of defense in invertebrates (Johansson and Söderhäll, 1985). Therefore, hemocyte number is very important because individuals with a high amount of hemocytes in circulation resist better the presence of a pathogen (Le Moullac et al., 1997). Lysosomal hydrolytic enzymes have been scarcely studied in crustaceans, but as in mollusks, the hemocytes of shrimp produce several such enzymes (protease, esterase, phosphatase, lipase, and phosphatase) (Carajaraville et al., 1995; Peraza-Gómez et al., 2011). In plasma, shrimp challenged with vibrios and fed commercial feed (control II) showed the activity of seven enzymes (alkaline phosphatase, esterase lipase, acid phosphatase, N-acetyl-β-glucosaminidase, Naphthol-AS-Bi-phosphohydrolase, α-fucosidase, and lysozyme) with the highest activity as compared with treatments I, III, IV, and V. However, in treatment IV, we found activity of nine enzymes. Remarkably, no lysozyme was found in treatments III, IV, and V (plasma, HLS) challenged with vibrios and fed with immunostimulants. The lysozymes of penaeid are well characterized and shown to have lytic activity against several species of Gram positive and Gram negative bacteria, including pathogenic species of Vibrio (De la Re-Vega et al., 2006; Hikima et al., 2003). Peraza-Gómez et al. (2011) found that L. vannamei fed commercial feed with probiotics and challenged with WSSV showed high activity of acid phosphatase and N-acetyl-β-glucosaminidase. In bivalve mollusks, lysosomal enzymes are released into plasma during physiological events and pathological stress. Lysosomal enzymes are involved in the death and degradation
M.C. Flores-Miranda et al. / Aquaculture 320 (2011) 51–55
of microorganisms and particles inside the hemocytes and in some cases are released from the cell to plasma or other tissues where they change the molecular conformation of the cellular surface pathogens, encouraging recognition and phagocytosis (Carajaraville et al., 1995; Cheng, 1983; Cheng, 1992; López et al., 1997). In this work, some enzymes (β-galactosidase, α-glucosidase, α-fucosidase) were found in plasma but not in HLS, suggesting that some enzymes present in plasma have a different origin from those in hemocytes. 5. Conclusion The mixture of four lactic acid bacteria and one yeast at 8.4 mg kg feed − 1 administered every 3 days increased the survival of shrimp after their challenge with V. sinaloensis as compared to the control group fed with commercial feed and a posterior bacterial infection. Shrimp fed daily with microbial immunostimulants enhanced THC. This study demonstrates the positive effect of microbial immunostimulants in juvenile L. vannamei. Acknowledgments Authors are grateful to Consejo Estatal de Ciencia y Tecnología del Estado de Sinaloa (CECyT-Sinaloa) and Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional (SIP-IPN) for financial support. Ma. del Carmen Flores Miranda acknowledges CONACYTMexico and SIP-IPN for the M.Sc. grants. References Apún-Molina, J.P., Santamaría-Miranda, A., Luna-González, A., Martínez-Díaz, S.F., Rojas-Contreras, M., 2009. Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature. Aquac. Res. 40, 887–894. Azad, I.S., Panigrahi, A., Gopal, C., Paulpandi, S., Mahima, C., Ravichandran, P., 2005. Routes of immunostimulation vis-a-vis survival and growth of Penaeus monodon postlarvae. Aquaculture 248, 227–234. 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. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brock, J., Main, K.L., 1994. A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. World Aquaculture Society, Baton Rouge, Louisiana, USA. 242 pp. Burge, E.J., Madigan, D.J., Burnett, L.E., Burnett, K.G., 2007. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio. Fish Shellfish Immunol. 22, 327–339. Burgents, J.E., Burnett, K.G., Burnett, L.E., 2004. Disease resistance of Pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 231, 1–8. Canicatti, C., 1990. Distribution d'une activite lysozymiale dans un echinoderme holothuroide, Holothuria polii, et dans les oeufs et les larves d'un echinoderme echinoide, Paracentrotus lividus. Eur. Arch. Biol. 101, 309–318. Carajaraville, M.P., Pal, S.G., Robledo, Y., 1995. Light and electron microscopical localization of lysosomal acid hydrolases in bivalve haemocytes by enzyme cytochemistry. Acta Histochem. Cytoc. 28, 409–416. Chang, C.F., Chen, H.Y., Su, M.S., Liao, I.C., 2000. Immunomodulation by dietary β-1,3 glucan in the brooders of the black tiger shrimp, Penaeus monodon. Fish Shellfish Immunol. 10, 505–514. Chang-Che, L., Jiann-Chu, C., 2008. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress. Fish Shellfish Immunol. 25 (6), 701–709. Chen, D., Ainsworth, A.J., 1992. Glucan administration potentiates immune defence mechanisms of channel catfish, Ictalurus punctatus Rafinesque. J. Fish Dis. 15, 295–304. Cheng, T.C., 1983. The role of lysosomes in molluscan inflammation. Am. Zool. 23, 129–144. Cheng, T.C., 1992. Selective induction of release of hydrolases from Crassostrea virginica hemocytes by certain bacteria. J. Invertebr. Pathol. 59, 197–200. Chisholm, J.R.S., Smith, V.J., 1995. Comparison of antibacterial activity in the hemocytes of different crustacean species. Comp. Biochem. Physiol. 110A, 39–45. Chiu, C.H., Guu, Y.K., Liu, C.H., Pan, T.M., Cheng, W., 2007. Immune responses and gene expression inwhite shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol. 23, 364–377. Daniel, W.W., 1997. Bioestadística. Base para el análisis de las ciencias de la salud. D. F. Editorial Limusa, Mexico. 639–693pp. De la Re-Vega, E., García-Galaz, A., Díaz-Cinco, M.E., Sotelo-Mundo, R.R., 2006. White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity
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