Marine Biotechnology

, Volume 16, Issue 2, pp 181–192 | Cite as

Isolation of Lactic Acid Bacteria from Kuruma Shrimp (Marsupenaeus japonicus) Intestine and Assessment of Immunomodulatory Role of a Selected Strain as Probiotic

  • M. Maeda
  • A. Shibata
  • G. Biswas
  • H. Korenaga
  • T. Kono
  • T. Itami
  • M. SakaiEmail author
Original Article


Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (105 cfu g−1) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P < 0.01) resistance to the bacterial pathogen in term of better post-infection survival (61.7 %) was observed in the shrimps fed with the probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.


Lactic acid bacteria Immunomodulation Shrimp probiotic Innate immune genes Disease resistance 



This work was supported by JSPS Asian CORE Program and Scientific Research (23580257) and grants from the Research and Development Program for New Bio-industry Initiatives, Japan. During this study, GB received a scholarship from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho), Japan.


  1. Ajitha S, Sridhar M, Sridhar N, Singh ISB, Varghese V (2004) Probiotic effects of lactic acid bacteria against Vibrio alginolyticus in Penaeus (Fenneropenaeus) indicus (H. Milne Edwards). Asian Fish Sci 17:71–80Google Scholar
  2. Amparyup P, Kondo H, Hirono I, Aoki T, Tassanakajon A (2008) Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon. Mol Immunol 45:1085–1093CrossRefPubMedGoogle Scholar
  3. Antony SP, Singh ISB, Jose RM, Anil Kumar PR, Philip R (2011a) Antimicrobial peptide gene expression in tiger shrimp, Penaeus monodon in response to gram-positive bacterial probionts and white spot virus challenge. Aquaculture 316:6–12CrossRefGoogle Scholar
  4. Antony SP, Singh ISB, Sudheer NS, Vrinda S, Priyaja P, Philip R (2011b) Molecular characterization and expression profile of a crustin-like antimicrobial peptide in the haemocytes of the giant tiger shrimp, Penaeus monodon, in response to various immunostimulants and challenge with WSSV. Immunobiology 216:184–194CrossRefPubMedGoogle Scholar
  5. Apún-Molina JP, Santamaría-Miranda A, Luna-González A, Martínez-Díaz SF, 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 40:887–894CrossRefGoogle Scholar
  6. Bachère E, Gueguen Y, Gonzalez M, Lorgeril J, Garnier J, Romestand B (2004) Insights into the anti-microbial defense of marine invertebrates: the penaeid shrimps and the oyster Crassostrea gigas. Immunol Rev 198:149–168CrossRefPubMedGoogle Scholar
  7. Buntin N, Chanthachum S, Hongpattarakere T (2008) Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. Songklanakarin J Sci Technol 30(Suppl 1):141–148Google Scholar
  8. Burge EJ, Madigan DJ, Burnett LE, Burnett KG (2007) Lysozyme gene expression by hemocyte of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio. Fish Shellfish Immunol 22:327–339CrossRefPubMedGoogle Scholar
  9. Campa-Córdova AI, Hernández-Saaverdra NY, De Philippis R, Ascencio F (2002) Generation of superoxide anion and SOD activity in haemocytes and muscle of American white shrimp (Litopenaeus vannamei) as a response to β-glucan and sulphated polysaccharide. Fish Shellfish Immunol 12:353–366CrossRefPubMedGoogle Scholar
  10. Castex M, Chim L, Pham D, Lemaire P, Wabete N, Nicolas JL, Schmidely P, Mariojouls C (2008) Probiotic P. acidilactici application in shrimp Litopenaeus stylirostris culture subject to vibriosis in New Caledonia. Aquaculture 275:182–193CrossRefGoogle Scholar
  11. Castex M, Lemairea P, Wabete N, Chim L (2009) Effect of dietary probiotic Pediococcus acidilactici on antioxidant defenses and oxidative stress status of shrimp Litopenaeus stylirostris. Aquaculture 294:306–313CrossRefGoogle Scholar
  12. Chiu CH, Guu YK, Liu CH, Pan TM, Cheng W (2007) Immune response and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol 23:364–377CrossRefPubMedGoogle Scholar
  13. de-la-Re-Vega E, García-Galaz A, Díaz-Cinco ME, Sotelo-Mundo RR (2006) White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram-negative bacteria: Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio cholerae. Fish Shellfish Immunol 20:405–408CrossRefPubMedGoogle Scholar
  14. Deng D, Mei C, Mai K, Tan BP, Ai Q, Ma H (2012) Effects of a yeast-based additive on growth and immune responses of white shrimp, Litopenaeus vannamei (Boone, 1931), and aquaculture environment. Aquac Res. doi: 10.1111/j.1365-2109.2012.03139.x Google Scholar
  15. Fall J, Kono T, Tanekhy M, Itami T, Sakai M (2010) Expression of innate immune-related genes of kuruma shrimp, Marsupenaeus japonicus, after challenge with Vibrio nigripulchritudo. Afr J Microbiol Res 4:2426–2433Google Scholar
  16. Farzanfar A (2006) The use of probiotics in shrimp aquaculture. FEMS Immunol Med Microbiol 48:149–158CrossRefPubMedGoogle Scholar
  17. Ghosh S, Sinha A, Sahu C (2007) Isolation of putative probionts from the intestines of Indian major carps. Isr J Aquacult Bamidgeh 59:127–132Google Scholar
  18. Holmblad T, Söderhäll K (1999) Cell adhesion molecules and antioxidative enzymes in a crustacean, possible role in immunity. Aquaculture 172:111–213CrossRefGoogle Scholar
  19. 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, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum. Aquaculture 164:277–288CrossRefGoogle Scholar
  20. Khouiti Z, Simon JP (1997) Detection and partial characterization of a bacteriocin produced by Carnobacterium piscicola 213. J Industrial Microbiol Biotechnol 19:28–33Google Scholar
  21. Kimoto H, Mizumachi K, Okamoto T, Kurisaki J (2004) New Lactococcus strain with immunomodulatory activity: enhancement of Th1-type immune response. Microbiol Immunol 48:75–82CrossRefPubMedGoogle Scholar
  22. Kimura M, Danno K, Yasui H (2006) Immunomodulatory function and probiotic properties of lactic acid bacteria isolated from Mongolian fermented milk. Biosci Microflora 25:147–155CrossRefGoogle Scholar
  23. Kongnum K, Hongpattarakere T (2012) Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi. Fish Shellfish Immunol 32:170–177CrossRefPubMedGoogle Scholar
  24. Kumagai Y, Takeuchi O, Akira S (2008) Pathogen recognition by innate receptors. J Infect Chemother 14:86–92CrossRefPubMedGoogle Scholar
  25. Lara-Flores M, Olvera-Novoa MA, Guzmán-Méndez BE, López-Madrid W (2003) Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus). Aquaculture 216:193–201CrossRefGoogle Scholar
  26. Leyva-Madrigal KY, Luna-González A, Escobedo-Bonilla CM, Fierro-Coronado JA, Maldonado-Mendoza IE (2011) Screening for potential probiotic bacteria to reduce prevalence of WSSV and IHHNV in whiteleg shrimp (Litopenaeus vannamei) under experimental conditions. Aquaculture 322–323:16–22CrossRefGoogle Scholar
  27. Li F, Xiang J (2012) Recent advances in researches on the innate immunity of shrimp in China. Dev Comp Immunol. doi: 10.1016/j.dci.2012.03.016 Google Scholar
  28. Lin YC, Lee FF, Wu CL, Chen JC (2010) Molecular cloning and characterization of a cytosolic manganese superoxide dismutase (cytMnSOD) and mitochondrial manganese superoxide dismutase (mtMnSOD) from the kuruma shrimp Marsupenaeus japonicas. Fish Shellfish Immunol 28:143–150CrossRefPubMedGoogle Scholar
  29. Liu H, Liu M, Wang B, Jiang K, Jiang S, Sun S, Wang L (2010) PCR-DGGE analysis of intestinal bacteria and effect of Bacillus spp. on intestinal microbial diversity in kuruma shrimp (Marsupenaeus japonicus). Chin J Oceanol Limnol 28:808–814CrossRefGoogle Scholar
  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 ─ΔΔC T method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  31. Mekata T, Kono T, Yoshida T, Sakai M, Itami T (2008) Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus. Fish Shellfish Immunol 24:122–133CrossRefPubMedGoogle Scholar
  32. Michel C, Pelletier C, Boussaha M, Douet DG, Lautraite A, Tailliez P (2007) Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis. Appl Environ Microbiol 73:2947–2955PubMedCentralCrossRefPubMedGoogle Scholar
  33. Moss SM, LeaMaster BR, Sweeney JN (2000) Relative abundance and species composition of gram-negative, aerobic bacteria associated with the gut of juvenile white shrimp Litopenaeus vannamei reared in oligotrophic well water and eutrophic pond water. J World Aquacult Soc 31:255–263CrossRefGoogle Scholar
  34. Mota R, Moreira JL, Souza M, Horta F, Teixeira S, Neumann E, Nicoli J, Nunes A (2006) Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines. BMC Biotechnol 6:1–11CrossRefGoogle Scholar
  35. Muta T, Miyata T, Tokunaga F, Nakamura T, Iwanaga S (1987) Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus. J Biochem 101:1321–1330PubMedGoogle Scholar
  36. Ouwehand AC, Vesterlund S (2004) Antimicrobial components from lactic acid bacteria. In: Salminen S, von Wright A, Ouwehand AC (eds) Lactic acid bacteria: microbiological and functional aspects. Marcel Dekker, New York, pp 375–396Google Scholar
  37. Oxley APA, Shipton W, Owens L, McKay D (2002) Bacterial flora from the gut of the wild and cultured banana prawn, Penaeus merguiensis. J Appl Microbiol 93:214–233CrossRefPubMedGoogle Scholar
  38. Pan D, He N, Yang Z, Liu H, Xu X (2005) Differential gene expression profile in hepatopancreas of WSSV-resistant shrimp (Penaeus japonicus) by suppression subtractive hybridization. Dev Comp Immunol 29:103–112CrossRefPubMedGoogle Scholar
  39. Panigrahi A, Kiron V, Satoh S, Hirono I, Kobayashi T, Sugita H, Puangkaewa J, Aoki T (2007) Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding. Dev Comp Immunol 31:372–382CrossRefPubMedGoogle Scholar
  40. Panigrahi A, Kiron V, Satoh S (2011) Real-time quantification of immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus. Aquac Res 42:906–917CrossRefGoogle Scholar
  41. Peraza-Gómez V, Luna-González A, Campa-Córdova A, López-Meyer M, Fierro-Coronado JA, 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 40:1481–1489CrossRefGoogle Scholar
  42. Rattanachai A, Hirono I, Ohira T, Takahashi Y, Aoki T (2004) Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression. Fish Sci 70:765–771CrossRefGoogle Scholar
  43. Ringø E (1993) Does dietary linoleic acid affect intestinal microflora in Arctic charr, Salelinus alpinus? Aquacult Fish Manage 24:133–135Google Scholar
  44. Ringø E, Gatesoupe FJ (1998) Lactic acid bacteria in fish: a review. Aquaculture 160:177–203CrossRefGoogle Scholar
  45. Ringø E, Olsen RE, Vecino JLG, Wadsworth S, Song SK (2012) Use of immunostimulants and nucleotides in aquaculture: a review. J Marine Sci Res Dev 1:104. doi: 10.4172/2155-9910.1000104 Google Scholar
  46. Rosenberry B (2001) World shrimp farming 2001. Shrimp News International, San DiegoGoogle Scholar
  47. Salinas I, Myklebust R, Esteban MA, Olsen RE, Meseguer J, Ringø E (2008) In vitro studies of Lactobacillus delbrueckii subsp. lactis in Atlantic salmon (Salmo salar L.) foregut: tissue responses and evidence of protection against Aeromonas salmonicida subsp. salmonicida epithelial damage. Vet Microbiol 128:167–177CrossRefPubMedGoogle Scholar
  48. Schoenborn JR, Wilson CB (2007) Regulation of interferon-gamma during innate and adaptive immune responses. Adv Immunol 96:41–101CrossRefPubMedGoogle Scholar
  49. Selsted ME, Ouellette AJ (2005) Mammalian defensins in the antimicrobial immune response. Nat Immunol 6:551–557CrossRefPubMedGoogle Scholar
  50. Shida K, Takahashi R, Iwadate E, Takamizawa K, Yasui H, Sato T, Habu S, Hachimura S, Kaminogawa S (2002) Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model. Clin Exp Allergy 32:563–570CrossRefPubMedGoogle Scholar
  51. Shockey JE, O'Leary NA, de la Vega E, Browdy CL, Baatz JE, Gross PS (2009) The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: an in vivo approach. Dev Comp Immunol 33:668–673CrossRefPubMedGoogle Scholar
  52. Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28CrossRefPubMedGoogle Scholar
  53. Soonthornchai W, Rungrassamee W, Karoonuthaisiri N, Jarayabhand P, Klinbunga S, Söderhäll K, Jiravanichpaisal P (2010) Expression of immune-related genes in the digestive organ of shrimp, Penaeus monodon, after an oral infection by Vibrio harveyi. Dev Comp Immunol 34:19–28CrossRefPubMedGoogle Scholar
  54. Takahashi Y, Itami T, Maeda M, Kondo M (1998) Bacterial and viral diseases of kuruma shrimp (Penaeus japonicus) in Japan. Fish Path 33:357–364CrossRefGoogle Scholar
  55. Takeda S, Takeshita M, Kikuchi Y, Dashnyam B, Kawahara S, Yoshida H, Watanabe W, Muguruma M, Kurokawa M (2011) Efficacy of oral administration of heat-killed probiotics from Mongolian dairy products against influenza infection in mice: alleviation of influenza infection by its immunomodulatory activity through intestinal immunity. Int Immunopharmacol 11:1976–1983CrossRefPubMedGoogle Scholar
  56. Tseng DY, Ho PL, Huang SY, Cheng SC, Shiu YL, Chiu CS, Liu CH (2009) Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20. Fish Shellfish Immunol 26:339–344CrossRefPubMedGoogle Scholar
  57. Tyagi A, Khushiramani R, Karunasagar I, Karunasagar I (2007) Antivibrio activity of recombinant lysozyme expressed from black tiger shrimp, Penaeus monodon. Aquaculture 272:246–253CrossRefGoogle Scholar
  58. Uma A, Abraham TJ, Sundararaj V (1999) Effect of a probiotic bacterium, Lactobacillus plantarum on disease resistance of Penaeus indicus larvae. Indian J Fish 46:367–373Google Scholar
  59. Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biocontrol agents in aquaculture. Microbiol Mol Biol Rev 64:655–671PubMedCentralCrossRefPubMedGoogle Scholar
  60. Vieira FN, Pedrotti FS, Neto CCB, Mouriño JLP, Beltrame E, Martins ML, Ramirez C, Arana LAV (2007) Lactic-acid bacteria increase the survival of marine shrimp, Litopenaeus vannamei, after infection with Vibrio harveyi. Braz J Oceanogr 55:251–255CrossRefGoogle Scholar
  61. Vijayabaskar P, Somasundaram ST (2008) Isolation of bacteriocin producing lactic acid bacteria from fish gut and probiotic activity against common fresh water fish pathogen Aeromonas hydrophila. Biogeosciences 7:124–128Google Scholar
  62. Wang YC, Chang PS, Chen HY (2008) Differential time-series expression of immune-related genes of Pacific white shrimp Litopenaeus vannamei in response to dietary inclusion of β-1,3-glucan. Fish Shellfish Immunol 24:113–121CrossRefPubMedGoogle Scholar
  63. Yao CL, Wu CG, Xiang JH, Li FH, Wang ZY, Han XZ (2008) The lysosome and lysozyme response in Chinese shrimp Fenneropenaeus chinensis to Vibrio anguillarum and laminarin stimulation. J Exp Marine Biol Ecol 363:124–129CrossRefGoogle Scholar
  64. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395CrossRefPubMedGoogle Scholar
  65. Zhang JQ, Li FH, Wang ZZ, Xiang JH (2007) Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis. J Biotechnol 127:605–614CrossRefPubMedGoogle Scholar
  66. Zhao JM, Song LS, Li CH, Zou HB, Ni DJ, Wang W (2007) Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein. Mol Immunol 44:1198–1208CrossRefPubMedGoogle Scholar
  67. Zokaeifar H, Balcázar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A, Nejat N (2012) Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol 33:683–689CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • M. Maeda
    • 1
  • A. Shibata
    • 1
  • G. Biswas
    • 2
  • H. Korenaga
    • 2
  • T. Kono
    • 4
  • T. Itami
    • 3
  • M. Sakai
    • 3
    Email author
  1. 1.Kyushu Medical Co., Ltd.KitakyushuJapan
  2. 2.Interdisciplinary Graduate School of Agriculture and EngineeringUniversity of MiyazakiMiyazakiJapan
  3. 3.Faculty of AgricultureUniversity of MiyazakiMiyazakiJapan
  4. 4.Interdisciplinary Research OrganizationUniversity of MiyazakiMiyazakiJapan

Personalised recommendations