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Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.)


Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.


  1. Lundqvist C, Hammarström M-L, Athlin L, Hammarström S. Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J Immunol Methods 1992; 152:253–263.

    Article  PubMed  CAS  Google Scholar 

  2. Di Sabatino A, Ciccocioppo R, DrsAlò S, Parroni R, Millimaggi D, Cifone MG, Corazza GR. Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease. Gut 2007; 49:380–386.

    Article  Google Scholar 

  3. Solano-Aguilar GI, Vengroski KG, Beshah E, Lunney JK. Isolation and purification of lymphocyte subsets from gut-associated lymphoid tissue in neonatal swine. J Immunol Methods 2000; 241:185–199.

    Article  PubMed  CAS  Google Scholar 

  4. Medina F, Segundo C, Salcedo I, García-Poley A, Brieva JA. Purification of human lamina propria plasma cells by an immunomagnetic selection method. J Immunol Methods 2004; 285:129–135.

    Article  PubMed  CAS  Google Scholar 

  5. Press CM, Evensen O. The morphology of the immune system in teleost fishes. Fish Shellfish Immunol 1999; 9:309–318.

    Article  Google Scholar 

  6. Hart S, Wrathmell AB, Harris JE, Grayson TH. Gut immunology in fish: a review. Dev Com Immunol 1998; 12:453–480.

    Article  Google Scholar 

  7. Fournier-Betz V, Quentel C, Lamour F, LeVen A. Immunocytochemical detection of Ig-positive cells in blood, lymphoid organs and the gut associated lymphoid tissue of the turbot (Scophtalmus maximus). Fish Shellfish Immunol 2000; 10:187–202.

    Article  PubMed  CAS  Google Scholar 

  8. Hébert P, Ainsworth AJ, Boyd B. Histological enzyme and flow cytometry analysis of channel catfish intestinal tract immune cells. Dev Comp Immunol 2002; 26:53–62.

    Article  PubMed  Google Scholar 

  9. Rombout JH, Taverne-Tiele AJ, Villena MI. The gutassociated lymphoid tissue (GALT) of carp (Cyprinus carpio L.): an immunocytochemical analysis. Dev Comp Immunol 1993; 17:55–66.

    Article  PubMed  CAS  Google Scholar 

  10. Clerton P, Troutaud D, Verlhac V, Gabadaudan J, Deschaux P. Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocytes functions: effect on gut and head kidney leucocytes. Fish Shellfish Immunol 2001; 11:1–13.

    Article  PubMed  CAS  Google Scholar 

  11. Bernard D, Six A, Rigottier-Gois L, Messiaen S, Chilmonczyk S, Quillet E, Boudinot P, Benmansour A. Phenotypic and functional similarity of gut intraepithelial and systemic T cells in a teleost fish. J Immunol 2006; 176:3942–3949.

    PubMed  CAS  Google Scholar 

  12. Buddington RK, Hilton JW. Intestinal adaptations of rainbow trout to changes in dietary carbohydrate. Am J Physiol Gastrointest Liver Physiol 1987; 253:G489-G496.

    CAS  Google Scholar 

  13. Evans DH. The physiology of fishes. 2nd ed: CRC Press; 1998. p. 43–64.

  14. Fontaine M. Nutrition des poissons. Paris: Editions du CNRS, 1981. p. 1–20.

  15. Salinas I, Cuesta A, Esteban MA, Meseguer J. Dietary administration of Lactobacillus delbrüeckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses. Fish Shellfish Immunol 2005; 19:66–76.

    Article  CAS  Google Scholar 

  16. Rodrigues PNS, Hermsen TT, Rombout JH, Egberts E, Stet RJM. Detection of MHC class II transcripts in lymphoid tissues of the common carp (Cyprinus carpio L.). Dev Comp Immunol 1995; 19:483–496.

    Article  PubMed  CAS  Google Scholar 

  17. McMillan DN, Secombes CJ. Isolation of rainbow trout (Oncorhynchis mykiss) intestinal intraepithelial lymphocytes (IEL) and measurement of their cytotoxic activity. Fish Shellfish Immunol 1997; 7:527- 541.

    Article  Google Scholar 

  18. Schwarz E, Saalmüller A, Gerner W, Claus R. Intraepithelial but not lamina propria lymphocytes in the porcine gut are affected by dexamethasone treatment. Vet Immunol Immunopathol 2005; 105:125- 139.

    Article  PubMed  CAS  Google Scholar 

  19. Muñoz P, Alvarez-Pellitero P, Sitja-Bobadilla A. Modulation of the in vitro activity of European sea bass (Dicentrarchus labrax L.) phagocytes by the myxosporean parasite Sphaerospora dicentrarchi (Myxosporea: Bivalvulida). Fish Shellfish Immunol 2000; 10:567–581.

    Article  PubMed  Google Scholar 

  20. Schley PD, Field CJ. The immune-enhancing effects of dietary fibres and prebiotics. Br J Nutr 2002; 87 Suppl S221-S230.

    Article  PubMed  CAS  Google Scholar 

  21. Faure M, Mettraux C, Moennoz D, Godin JP, Vuichoud J, Rochat F, Breuille D, Obled C, Corthesy-Theulaz I. Specific amino acids increase mucin synthesis and microbiota in dextran sulfate sodiumtreated rats. J Nutr 2006; 136:1558–1564.

    PubMed  CAS  Google Scholar 

  22. Brown DC, Maxwell CV, Erf GF, Davis ME, Singh S, Johnson ZB. The influence of different management systems and age on intestinal morphology, immune cell numbers and mucin production from goblet cells in post-weaning pigs. Vet Immunol Immunopathol 2006; 111:187–198.

    Article  PubMed  CAS  Google Scholar 

  23. Hathcock KS. T-cell enrichment by nonoadherence to nylon. In: Coligan JE, Kruisbeek AM, David H. Margulies DH, Shevach EM, Strober W, editors. Current Protocols in Immunology. New York, 1992.

  24. Picchieti S, Terribili FR, Mastrolia L, Scapigliati G, Abelli L. Expression of lymphocyte antigenic determinants in developing gut-associated lymphoid tissue of the seabass Dicentrarchus labrax (L.). Anat Embryol 1997; 196:457–463.

    Article  Google Scholar 

  25. Rombout JH, Joosten PH, Engelsma MY, Vos AP, Taverne N, Taverne-Thiele JJ. Indications for a distinct putative T cell population in mucosal tissue of carp (Cyprinus carpio L.). Dev Comp Immunol 1998; 22:63–77.

    Article  PubMed  Google Scholar 

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Correspondence to Maria Ángeles Esteban.

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Salinas, I., Meseguer, J. & Esteban, M.Á. Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (Sparus aurata L.). Biol. Proced. Online 9, 43–55 (2007).

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Indexing terms

  • Sea Bream
  • Flow Cytometry
  • Respiratory Burst