Introduction
The black tiger shrimp Penaeus monodon is the most widely cultured shrimp species in Asian countries. However, the shrimp culture industry has recently been badly affected by the outbreak of diseases caused by both pathogenic (virus, bacteria, fungi, and parasites) and non-pathogenic agents (nutritional deficiency, algal toxins, and fluctuating environmental parameters), where the pathogenic forms are responsible for most of the major economic losses. Among the bacterial pathogens, Vibrio species cause vibriosis in penaeid shrimp [21], which is mainly caused by Vibrio parahaemolyticus, a Gram-negative halophilic, non-spore forming, curved rod-shaped bacterium that naturally lives in estuarine and marine environments worldwide [26]. Meanwhile, the most problematic virus for shrimp cultures around the world is the white spot syndrome virus (WSSV), which belongs to the genus Whispovirus in the family Nimaviridae [16]. WSSV is found in almost all shrimp-producing countries and is lethal to all commercially cultivated Penaeid shrimp species [8, 22].
As chemicals are no longer able to reduce or control the WSSV, alternative methods using probiotic microbes have been attracting attention. Certain halophilic bacteria produce secondary metabolites, which inhibit or kill other harmful microorganisms [1]. Thus, the use of probiotics to enhance the survival, growth, immunity, and disease resistance of farmed aquatic animals is increasing [19].
Infection by Vibrio and WSSV causes oxidative stress via the release of reactive oxygen species that are toxic to cells. Shrimp produce a high amount of antioxidant enzymes during a pathogenic infection, which are potential indicators of oxidative stress [17]. Catalase is an important antioxidant enzyme and known to be involved in the crustacean’s innate immune reaction [7, 15, 27]. Superoxide dismutase (SOD) is the major O2 scavenger and its enzymatic action results in H2O2 and O2 formation. Thus, since increased enzyme activity indicates higher stress in the animal, while lower enzyme activity indicates less stress [4], the level of antioxidant enzyme activity could be used as a parameter to measure pathogenic infection.
Accordingly, the present study focused on the effect of Bacillus sp. Mk22 (accession number, F794553), a halophilic bacterium previously isolated by the authors [1]. This strain was tested against Vibrio and WSSV infection of P. monodon under laboratory conditions, as well as on the growth improvement of the shrimp, to explore its possible use as a probiotic.
Materials and Methods
Cultivation of probiotic bacteria and pathogen
Bacillus sp. Mk22 isolated from a saltpan (Tuticorin, India) [1] was used for the Vibrio and WSSV infection study. A commercial probiotic (Charoen Pokhpond Aquaculture Pvt. Ltd, India) comprising a Bacillus sp. was used for the comparative study. The Bacillus cells were cultivated in a trypticase soy broth in a rotary shaking incubator at 37℃ and 200 × g. The V. parahaemolyticus (laboratory stock culture) was cultured in a nutrient broth supplemented with 3% NaCl at 37℃ and 200 × g. For the V. parahaemolyticus specific cell counting medium, a thiosulfate citrate bile salts sucrose agar (TCBS; HiMedia, India) was used.
Shrimp rearing
The Penaeus monodon post larvae PL 15 (stage 15) were purchased from the Sona shrimp hatchery, Marakkanam, India and 40 brooders were screened for WSSV infection using the OIE-endorsed nested PCR test [14]. The shrimp were stocked in a 50 L plastic tank containing a 35 L sand-filter and chlorine-treated estuarine water. The postlarvae PL 15 were fed with basic feed at 7.5% body weight per day up to 30 days and then fed at 3.5−5.0% body weight per day. Any unutilized feed was removed 8 hrs after the feeding time.
The growth rate and feed conversion rate (FCR) of the shrimp were calculated using the following equations:
Growth rate (g) = (final body weight − initial body weight) / number of days
FCR (%) = [Feed taken (dry weight in g) / Weight gain (g)] × 100
Shrimp experimental design
The Penaeus monodon 4−5 g shrimp (25 shrimp/50 L tank × 3 tanks = 75 shrimp per group) were maintained in separate experimental groups. The control group was fed with basic feed containing no probiotic, the commercial probiotic (CP) group was fed with basic feed containing the commercial probiotic Bacillus sp. at a concentration of 1.2 × 108 CFU/ml. The commercial probiotic Bacillus sp, was claimed by the manufacturer to have ability to increase the productivity of Penaeus monodon and to improve the water quality with decreased concentration of ammonia and nitrite and to control the bacterial infections. The probiotics was also claimed to improve the digestibility of nutrients and to increase the stress tolerance. The Mk22 group was fed with basic feed and Bacillus sp. Mk22 at a concentration of 1.2 × 108 CFU/ml. The total numbers of bacteria and Bacillus sp. were quantified from the water and shrimp digestive tracts.
Antioxidant enzyme activities
The haemolymph (200 μl) was withdrawn from the haemal sinuses of each shrimp using a 1 ml syringe fitted with a 26-gauge needle, where each syringe was prefilled with 400 μl of an anticoagulant containing 0.94 mM EDTA in an isotonic NaCl solution [6]. The shrimp hepatopancreas tissue (500 mg) was rinsed in ice-cold saline and homogenized with 1 ml 0.15 M Tris-HCl pH 7.4. The haemolymph and hepatopancreas homogenates were then assayed for catalase activity using a colorimetric method [24], and SOD activity using nitroblue tetrazolium [2].
In vivo study
For the Vibrio infection, the V. parahaemolyticus cells were added to the shrimp culture water at a final concentration of 1.2 × 107 CFU/ml, while the V. parahaemolyticus cells mixed with the basic feed at a final concentration of 1.2 × 107 CFU/g were used for oral feeding infection.
For the WSSV infection, a WSSV-infected tissue extract was used. To prepare the tissue extract, 1 g of infected shrimp tissue was homogenized in 9 ml of a TNE buffer (Tris 50 mM, NaCl 100 mM and EDTA 1 mM, pH 7.4) and centrifuged at 4,000 × g for 10 min. Thereafter, the supernatant was filtered using a 0.25 μm membrane filter and the resulting clear solution used for the WSSV infection study. A dose of 0.01% WSSV-infected tissue extract was directly added to the shrimp culture water. For oral feeding infection, 500 mg of the WSSV-infected tissue was given to 25 cultured shrimp. Two days before infection, the commercial probiotic and Mk22 groups were both fed with a basic feed pellet mixed with the respective Bacillus sp. cells (1.2 × 108 CFU/g). 10% of the water in the shrimp culture tank was exchanged at four-day intervals, and the shrimp behavior was observed every day.
Genomic DNA and PCR amplification
Genomic DNA was isolated from the normal and infected P. monodon. The tissue (500 mg) was homogenized using 4.5 ml of a lysis buffer [10 mM Tris (pH 8), 0.1 M EDTA, 20 μg/ml pancreatic RNAase, 0.5% SDS], treated with proteinase K (0.2 mg/ml) and sarkosyl (1%) at 45℃ for 2−4 h, followed by phenol-chloroform extraction and dialysis against a TE buffer [20]. Two pairs of oligonucleotide primers of 22 bp and 21 bp named as LoF1, LoR1 and LoF2, LoR2 (PROLIGO Primers & Probes, Australia) specific for WSSV DNA sequences [3] were used. The PCR reaction kit (Boehringer Mannheim, Germany) and reaction conditions were initial denaturation at 94℃ for 7 min, followed by 35 cycles of 94℃ for 30 s, 52℃ for 30 s, 68℃ for 1 min, and the final extension was 72℃ for 10 min. The 1.2% agarose gels were visualized using UV transillumination (Gel doc 2000, Japan).
Statistical analysis
All the statistical data with means were compared using Duncan’s multiple range tests (SPSS 16 software, IBM, USA). A significance level of p ≤ 0.05 was used for all tests. The data are reported as the means ± standard deviation. The different letters on the same patterned/colored bars of the charts indicate a statistical difference among the experiments (p < 0.05).
Results
Growth and FCR
The growth rate (7.1 ± 0.21 g) and total weight (178 ± 4.93 g) were the highest in the Mk22 group (on day 45). The CP group showed a lower growth rate (5.8 ± 0.20 g) and total weight (147.7 ± 5.69 g), and the control group showed the lowest growth rate (3.6 ± 0.20 g) and total weight (90.7 ± 5.51 g). A high FCR of 1.32 ± 0.09% was recorded in the control group, a lower rate of 1.00 ± 0.09% in the CP group, and the lowest of 0.80 ± 0.03% in the Mk22 group. Therefore, Bacillus sp. Mk22 showed the best results for the growth rate and FCR.
Bacterial count
The highest total Vibrio count (TVC) 4.97 ± 0.053 × 102 CFU/ml was in the control group, with a low count of 0.2 ± 0.01 × 102 CFU/ml in the CP group, and the lowest of 0.02 ± 0.01 × 102 CFU/ml in the Mk22 group observed on day 45 of culturing (data not shown). Bacillus sp. Mk22 showed the highest number in both the culture water and the digestive tract of the shrimp (Table 1) (p ≤ 0.05), and seemed to be well colonized in the digestive tract compared to the other groups.
Table 1.aMean ± Standard deviation.
Mortality of P. monodon infected with either V. parahaemolyticus or WSSV
The mortality of the shrimp challenged with V. parahaemolyticus was examined. For the water infection of the control group, 2 out of 25 shrimp had died by day 15, while the maximum mortality of 12 was reached on day 25. For the oral feeding infection of the control group, 1 shrimp had died by day 9, while the maximum mortality of 8 was reached on day 20. Therefore, the water infection produced a higher mortality. For both the CP and Mk22 groups, no mortality was recorded from either the water or the oral feeding infection (data not shown). Therefore, the Bacillus sp. used for the CP and Mk22 groups were significantly effective in reducing the shrimp mortality by V. parahaemolyticus.
For the WSSV water infection, 100% mortality was reached on day 15 in both the control and CP groups (Fig. 1A). In the Mk22 group (water infection), 68% of the shrimp were still alive on day 45 (Fig. 1A). For the oral feeding infection, 100% mortality was reached on day 8 and 10 in the control and CP group, respectively (Fig. 1B). In the Mk22 group (oral feeding infection), 20% of the shrimp were still alive on day 45 (Fig. 1B). The surviving animals were tested using a 2-step PCR and the results revealed they were WSSV positive. Therefore, the above results indicate that the dietary administration of Bacillus sp. Mk22 was strongly effective in reducing the shrimp mortality by WSSV, especially by water infection.
Fig. 1.Mortality of WSSV-infected P. monodon in water (A) and oral (B) infection.
Catalase and SOD activity in either Vibrio or WSSV-infected hepatopancreas and haemolymph
In the V. parahaemolyticus infection experiments, the highest catalase and SOD activities were observed in the control group, median activities in the CP group, and the lowest activities in the Mk 22 group (Fig. 2A, B). The catalase and SOD activities were higher in the haemolymph than in the hepatopancreas for all three groups.
Fig. 2.Catalase (A) and SOD (B) activity of Vibrio-infected hepatopancreas and haemolymph in P. monodon.
The WSSV infection experiments also showed similar results to the experiments with V. parahaemolyticus; for both the hepatopancreas and the haemolymph, the highest catalase and SOD activities were observed in the control group, median activities in the CP group, and the lowest activities in the Mk 22 group (Fig. 3A, B). Therefore, the Mk22 group exhibited the least catalase and SOD activities in both the V. parahaemolyticus-infected and WSSV-infected shrimp when compared to the other experimental groups, indicating that Bacillus sp. Mk22 contributed to reducing the catalase and SOD activities in both the hepatopancreas and the haemolymph of the infected shrimp.
Fig. 3.Catalase (A) and SOD (B) activity of WSSV-infected hepatopancreas and haemolymph in P. monodon.
Discussion
In shrimp cultures, vibriosis causes more than 70% mortality within one day after infection [12]. Plus, WSSV is one of the most serious viral pathogens for shrimp; 100% mortality can be reached within a short span of time (7 to 10 days) [13]. A growing number of studies have demonstrated the use of probiotics in aquaculture and their ability to control potential pathogens, while also increasing the growth rates and welfare of the farmed aquatic animals [25]. Thus, the present study attempted to enhance the growth rate of P. monodon supplemented with the bacterial isolate Bacillus sp. Mk22, while also reducing the infection in the P. monodon culture. The results showed a significantly increased final weight compared with the cultures supplemented with a commercial probiotic or without probiotic supplementation. Plus, Bacillus sp. Mk22 facilitated a high survival rate of 100% during the Vibrio challenge experiment, while Pediococcus acidilactici in another report only exhibited a 67% survival [4].
Some of the reasons for the enhanced shrimp growth could be higher activities of digestive enzymes, like amylase, protease, and lipase, induced by the probiotics [11, 28] and high probiotic bacteria in the digestive tract [28], along with the infection-reducing effect of the probiotics [4]. Enhanced resistance to pathogens occurs by activating both cellular and humoral immune responses in shrimp [19], and Bacillus surface antigens or their metabolites act as immunogens for shrimp by stimulating the phagocytic activity of granulocytes [9]. Halophilic bacteria also produce several secondary metabolites, such as bacteriocins, bacteriocin-like substances, and antibacterial lipopeptides against pathogens [11].
When P. monodon was challenged with WSSV, the results revealed that 68 and 20% of the shrimp survived in the Mk 22 group following water infection and oral feeding infection, respectively, whereas 100% mortality was recorded at a much earlier time in both the control and CP groups. Therefore, Bacillus sp. Mk22 significantly reduced the mortality compared with the other groups.
When shrimp are infected, oxidative stress that produces a high amount of antioxidant enzymes, like catalase and SOD, is also increased [15]. Catalase and SOD are important antioxidant enzymes, present in almost all oxygen-respiring animals. Thus, in this study, the hepatopancreas and haemolymph were used to measure the change in catalase and SOD activities. The hepatopancreas is the main organ for the reserve and detoxification of xenobiotics in crustaceans, and is highly sensitive to physiological and environmental changes [10]. Plus, the catalase and SOD activities were also measured in the haemolymph, since the blood cells of invertebrates are the primary effectors in the host defense and involved in various immune processes, such as phagocytosis [23].
The level of these enzymes in the Mk22 group was significantly decreased in both the V. parahaemolyticus and WSSV-infected P. monodon hemolymph and hepatopancreas when compared with the other groups. In the Mk22 group, the lowered catalase and SOD activities seemed to be due to less stress caused by reduced infection as a result of the administration of Bacillus sp. Mk22. These results concur with the finding of Chang et al. [5], where SOD activity decreased in WSSV-infected P. monodon with probiotic administration. Castex et al. [4] also reported that in shrimp exposed to Vibrio, the antioxidant response was characterized by higher antioxidant enzyme activities (catalase and SOD) and a higher oxidative stress level compared to the levels found in the control without Vibrio. We also found that the shrimp fed with the probiotic diet exhibited a lower prevalence of Vibrio throughout the test in the digestive tract, plus the antioxidant response and oxidative stress level recorded in the digestive gland of the shrimp fed with the probiotic diet were lower.
Therefore, based on the above observations, it was concluded that the strain of halophilic Bacillus sp. Mk 22 was effective in inhibiting infection by shrimp pathogens, such as V. paraheamolyticus and WSSV. The bacterium significantly reduced the mortality and did not have any pathogenic effect on the shrimp. Indeed, the P. monodon exhibited a significantly higher weight gain and survival ratio than the control group. Therefore, Bacillus sp. Mk 22 could be used effectively to control such shrimp pathogens and enhance shrimp production, thereby substituting for the use of antibiotics in aquaculture.
References
- Ashokkumar S, Mayavu P. 2014. Screening, identification and antagonistic activity of halo stable Bacillus sp. Mk22 used as probiotic in Penaeus monodon Fabricius, 1798. Afr. J. Food. Sci. 8: 48-53. https://doi.org/10.5897/AJFS2013.1048
- Beauchamp C, Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287. https://doi.org/10.1016/0003-2697(71)90370-8
- Bell TA, Lightner DV. 1988. A handbook of normal penaeid shrimp histology, pp. 114. World Aquaculture Society, Baton Rough, L.A.
- Castex M, Lemaire P, Wabete N, Chim L. 2009. Effect of dietary probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress status of shrimp Litopenaeus stylirostris. Aquaculture 294: 306-313. https://doi.org/10.1016/j.aquaculture.2009.06.016
- Chang CF, Su MS, Chen HY, Liao IC. 2003. Dietary β-1.3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus. Fish. Shellfish. Immunol. 15: 297-310. https://doi.org/10.1016/S1050-4648(02)00167-5
- Chih HP, Yew HC, Brian H. 2003. Alterations of antioxidant capacity and hepatopancreatic enzymes in Penaeus monodon (Fabricius) juveniles fed diets supplemented with astaxanthin and exposed to Vibrio damsela challenge. J. Fish Soc. Taiwan 30: 279-290.
- Chongsatja PO, Bourchookarn A, Lo CF, Thongboonkerd V, Krittanai C. 2007. Proteomic analysis of differentially expressed proteins in Penaeusvannameihemocytes upon Taura syndrome virus infection. Proteomics 7: 3592-3601. https://doi.org/10.1002/pmic.200700281
- Escobedo-Bonilla CM, Alday-Sanz V, Wille M, Sorgeloos P, Pensaert MB, Nauwynck HJ. 2008. A review on the morphology, molecular characterization, morphogenesis and pathogenesis of white spot syndrome virus. J. Fish. Dis. 31: 1-18.
- Itami T, Asano M, Tokushige K, Kubono K, Nakagawa A, Takeno N, Nishimura H, Maeda M, Kondo M, Takahashi Y. 1998. Enhancement of disease resistance of Kuruma shrimp, Penaeusjaponicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 164: 277-288. https://doi.org/10.1016/S0044-8486(98)00193-8
- Johnston DJ, Alexander CG, Yellowhees D. 1998. Epithelial cytology and function in the digestive gland of Thenus orientalis (Decapoda, Scyllaridae). J. Crust. Biol. 18: 271-278. https://doi.org/10.2307/1549320
- Kamarudin MS, Jones DA, le Vay L, Abidin AZ. 1994. Ontogenetic change in digestive enzyme activity during larval development of Macrobrachiumrosenbergii. Aquaculture 123: 323-333. https://doi.org/10.1016/0044-8486(94)90068-X
- Lavilla-Pitogo CR, Leano EM, Paner MG. 1998. Mortalities of pond-cultured juvenile shrimp, Penaeus monodon, associated with dominance of luminescent vibrios in the rearing environment. Aquaculture 164: 337-349. https://doi.org/10.1016/S0044-8486(98)00198-7
- Lightner DV. 1996. A handbook of shrimp pathology and diagnostic procedures for disease of cultured penaeid shrimp, pp. 304. The world Aquaculture Society, Baton Rouge, USA, LA.
- Lo CF, Leu JH, Chen CH, Peng SE, Chen YT, Chou CM, et al. 1996. Detection of Baculovirus associated with White Spot Syndrome (WSBV) in penaeid shrimp using polymerase chain reaction. Dis. Aquat. Org. 25: 133-141. https://doi.org/10.3354/dao025133
- Mathew S, Ashok Kumar K, Anandan R, Viswanathan Nair PG, Devadasan K. 2007. Changes in tissue defense system in white spot syndrome virus (WSSV) infected Penaeus monodon. Comp. Biochem. Physiol C Pharmacol. Toxicol. Endocrinol. 145: 315-320. https://doi.org/10.1016/j.cbpc.2007.01.001
- Mayo MA. 2002. A summary of taxonomic changes recently approved by ICTV. Arch Virol. 147: 1655-1656. https://doi.org/10.1007/s007050200039
- Mohanakumar K, Ramasamy P. 2006. White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus. Viru. Res. 115: 69-75. https://doi.org/10.1016/j.virusres.2005.07.006
- Pazir MK, Afsharnasab M, JalaliJafari B, Sharifpour I, Motalebi AA, Dashtiannasab A. 2011. Detection and identification of white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) of Litopenaus vannamei from Bushehr and Sistan and Baloochestan provinces, Iran, during 2009-2010. Iran. J. Fish. Sci. 10: 708-726.
- Rengpipat S, Rukpratanporn S, Piyatiratitivorakul S, Menasaveta P. 2000. Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). Aquaculture 191: 271-288. https://doi.org/10.1016/S0044-8486(00)00440-3
- Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: A Laboratory Manual. Vol. 1, 2, 3. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
- Saulnier D, Haffner P, Goarant C, Levy P, Ansquer D. 2000. Experimental infection models for shrimp vibriosis studies: a review. Aquaculture 191: 133-144. https://doi.org/10.1016/S0044-8486(00)00423-3
- Shin EJ, Park JH, Lee YH. 2001. White spot syndrome virus in penaeid shrimp culture in Korea. J. Microbiol. Biotechnol. 11: 394-398.
- Söderhäll K, Cerenius L. 1998. Role of the prophenoloxidase-activating system in invertebrate immunity. Curr. Opin. Immunol. 10: 23-28. https://doi.org/10.1016/S0952-7915(98)80026-5
- Takahara S, Hamilton BH, Nell JV, Kobra TY, Ogura Y, Nishimura ET. 1960. Hypocatalasemia, a new genetic carrier state. J. Clin. Invest. 29: 610-619.
- Wang YB, Xu ZR. 2006. Effect of probiotics for common carp (Cyprinuscarpio) based on growth performance and digestive enzyme activities. Anim. Feed. Sci. Technol. 127: 283-292. https://doi.org/10.1016/j.anifeedsci.2005.09.003
- Wu Y, Wen J, Ma Y, Ma X, Chen Y. 2014. Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus, China, 2003-2008. Food. Control 46: 197-202. https://doi.org/10.1016/j.foodcont.2014.05.023
- Zhang Q, Li F, Zhang X, Dong B, Zhang J, Xie Y. 2008. cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis. Fish. Shellfish. Immunol. 24: 584-591. https://doi.org/10.1016/j.fsi.2008.01.008
- Ziaei-Nejad S, HabibiRezaei M, Azari Takami G, Lovett D, Mirvaghefi AR, Shakouri M. 2006. The effect of Bacillus sp. bacteria used as probiotics on digestive enzymes activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus. Aquaculture 252: 516-524. https://doi.org/10.1016/j.aquaculture.2005.07.021
Cited by
- Probiotics at War Against Viruses: What Is Missing From the Picture? vol.11, pp.None, 2020, https://doi.org/10.3389/fmicb.2020.01877
- Mechanisms and the role of probiotic Bacillus in mitigating fish pathogens in aquaculture vol.46, pp.3, 2016, https://doi.org/10.1007/s10695-019-00754-y
- Probiotics and competitive exclusion of pathogens in shrimp aquaculture vol.13, pp.1, 2021, https://doi.org/10.1111/raq.12477