orcid , M Palikova, J Mares, E Syrova, J Blahova, M Faldyna, P Ondrackova | Agricultural Journals" />

Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss)

https://doi.org/10.17221/98/2019-VETMEDCitation:Minarova H., Palikova M., Mares J., Syrova E., Blahova J., Faldyna M., Ondrackova P. (2019): Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). Veterinarni Medicina, 64: 547-557.
download PDF

The lymphocyte proliferation assay is a valuable method used for the evaluation of the fish immune system. However, there are many variations and optimal results are not always obtained. Unification is necessary to ensure the comparability between different studies. The aim of this study was to optimise the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). This goal included the determination of the optimal incubation length, serum type, incubation temperature, type of mitogen and its concentration, and anticoagulant. The peripheral blood and head kidney lymphocytes were isolated by density gradient centrifugation. Subsequently, the cells were incubated for 3–8 days with different mitogens (pokeweed mitogen 5, 10 and 50 µg/ml, concanavalin A 1, 10 and 20 µg/ml, phytohaemagglutinin 25, 50 and 100 µg/ml, lipopolysaccharide 1, 50 and 100 µg/ml). The use of the different serum types (foetal bovine serum, trout serum), incubation temperatures (10–20 °C) and anticoagulants (heparin, EDTA) was compared. Labelled thymidine was used to evaluate the assay. The best results were obtained after seven days of incubation at 15 °C with foetal bovine serum (FBS). The head kidney lymphocytes showed the highest proliferative response with 50 µg/ml phytohaemagglutinin. With the peripheral blood lymphocytes (heparin and EDTA), the best results were obtained with 50 µg/ml pokeweed mitogen. The highest proliferation levels were detected with heparinised blood. In conclusion, optimisation of this assay contributes to the improved assessment of the rainbow trout immune function.

Alford RH (1970): Metal cation requirements for phytohemagglutinin-induced transformation of human peripheral blood lymphocytes. Journal of Immunology (Baltimore, MD: 1950) 104, 698–703.
Bly JE, Clem LW (1992): Temperature and teleost immune functions. Fish & Shellfish Immunology 2, 159–171.
Chilmonczyk S (1978): In vitro stimulation by mitogens of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri). Annales d’Immunologie 129, 3–12.
Daly JG, Moore AR, Olivier G (1995): A colorimetric assay for the quantification of brook trout (Salvelinus fontinalis) lymphocyte mitogenesis. Fish & Shellfish Immunology 5, 265–273.
DeKoning J, Kaattari S (1991): Mitogenesis of rainbow trout peripheral blood lymphocytes requires homologous plasma for optimal responsiveness. In Vitro Cellular & Developmental Biology: Journal of the Tissue Culture Association 27A, 381–386.
Divya KB, Sathish MHK, Kapila S, Sabikhi L (2015): Immunosuppressive potential of low fat buffalo milk supplemented with omega-3 fatty acids. Food and Agricultural Immunology 26, 558–565. https://doi.org/10.1080/09540105.2014.998633
Espelid S, Lokken GB, Steiro K, Bogwald J (1996): Effects of cortisol and stress on the immune system in Atlantic Salmon (Salmo salar L.). Fish & Shellfish Immunology 6, 95–110.
Etlinger HM, Hodgins HO, Chiller JM (1976): Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. Journal of Immunology (Baltimore, MD: 1950) 116, 1547–1553.
Finkelstein ME, Grasman KA, Croll DA, Tershy BR, Keitt BS, Jarman WM, Smith DR (2007): Contaminant-associated alteration of immune function in black-footed albatross (Phoebastria nigripes), a North Pacific predator. Environmental Toxicology and Chemistry 26, 1896–1903. https://doi.org/10.1897/06-505R.1
Flory CM, Bayne CJ (1991): The influence of adrenergic and cholinergic agents on the chemiluminescent and mitogenic responses of leukocytes from the rainbow trout, Oncorhynchus mykiss. Developmental & Comparative Immunology 15, 135–142.
Gauthier DT, Cartwright DD, Densmore CL, Blazer VS, Ottinger CA (2003): Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5 ‘-bromo-2 ‘-deoxyuridine. Fish & Shellfish Immunology 14, 279–288.
Grasman KA, Echols KR, May TM, Peterman PH, Gale RW, Orazio CE (2013): Immunological and reproductive health assessment in herring gulls and black-crowned night herons in the Hudson-Raritan Estuary. Environmental Toxicology and Chemistry 32, 548–561. https://doi.org/10.1002/etc.2089
Hardie LJ, Fletcher TC, Secombes CJ (1994): Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout (Oncorhynchus mykiss) leucocytes. Developmental & Comparative Immunology 18, 57–66.
Harford AJ, O’Halloran K, Wright PFA (2007): Effect of in vitro and in vivo organotin exposures on the immune functions of murray cod (Maccullochella peelii peelii). Environmental Toxicology and Chemistry 26, 1649–1656. https://doi.org/10.1897/06-449R.1
Hebert N, Gagne F, Cejka P, Cyr D, Marcogliese DJ, Blaise C, Pellerin J, Fournier M (2008): The effects of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss): Exposure duration and contribution of suspended particles. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 148, 258–264.
Janossy G, Greaves MF (1971): Lymphocyte activation. I. Response of T and B lymphocytes to phytomitogens. Clinical & Experimental Immunology 9, 483–498.
Karami Z, Mesdaghi M, Karimzadeh P, Mansouri M, Taghdiri MM, Kayhanidoost Z, Jebelli B, Shekarriz Foumani R, Babaie D, Chavoshzadeh Z (2016): Evaluation of Lymphocyte Transformation Test Results in Patients with Delayed Hypersensitivity Reactions following the Use of Anticonvulsant Drugs. International Archives of Allergy and Immunology 170, 158–162. https://doi.org/10.1159/000448284
Kearns RJ, Hayek MG, Turek JJ, Meydani M, Burr JR, Greene RJ, Marshall CA, Adams SM, Borgert RC, Reinhart GA (1999): Effect of age, breed and dietary omega-6 (n-6): Omega-3 (n-3) fatty acid ratio on immune function, eicosanoid production, and lipid peroxidation in young and aged dogs. Veterinary Immunology and Immunopathology 69, 165–183. https://doi.org/10.1016/S0165-2427(99)00052-5
Kehrer SR, Hannan CM, Raison RL (1998): Identification of a subpopulation of leucocytes from the rainbow trout (Oncorhynchus mykiss) responsive to pokeweed mitogen. Fish & Shellfish Immunology 8, 477–487.
Kollner B, Wasserrab B, Kotterba G, Fischer U (2002): Evaluation of immune functions of rainbow trout (Oncorhynchus mykiss) – how can environmental influences be detected? Toxicology Letters 131, 83–95. https://doi.org/10.1016/S0378-4274(02)00044-9
Kowalska A, Siwicki AK, Kowalski RK (2017): Dietary resveratrol improves immunity but reduces reproduction of broodstock medaka Oryzias latipes (Temminck & Schlegel). Fish Physiology and Biochemistry 43, 27–37.
Kralova-Kovarikova S, Leva L, Knotek Z, Toman M (2016): Changes in lymphocyte function and subset counts in cats with spontaneous chronic kidney disease. Veterinarni Medicina 61, 553–559. https://doi.org/10.17221/282/2015-VETMED
Leonardi MO, Klempau AE (2003): Artificial photoperiod influence on the immune system of juvenile rainbow trout (Oncorhynchus mykiss) in the Southern Hemisphere. Aquaculture 221, 581–591. https://doi.org/10.1016/S0044-8486(03)00032-2
Lin SH, Ellis AE, Davidson GA, Secombes CJ (1999): Migratory, respiratory burst and mitogenic responses of leucocytes isolated from the gills of rainbow trout (Oncorhynchus mykiss). Fish & Shellfish Immunology 9, 211–226.
Lunden T, Bylund G (2000): The influence of in vitro and in vivo exposure to antibiotics on mitogen-induced proliferation of lymphoid cells in rainbow trout (Oncorhynchus mykiss). Fish & Shellfish Immunology 10, 395–404.
Mosmann T (1983): Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55–63. https://doi.org/10.1016/0022-1759(83)90303-4
Muller C, Ruby S, Brousseau P, Cyr D, Fournier M, Gagné F (2009): Immunotoxicological effects of an activated-sludge-treated effluent on rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 150, 390–394.
Nowell PC (1960): Phytohemagglutinin: An Initiator of Mitosis in Cultures of Normal Human Leukocytes. Cancer Research 20, 462–466.
O’Halloran K, Ahokas JT, Wright PFA (1998): Response of fish immune cells to in vitro organotin exposures. Aquatic Toxicology 40, 141–156. https://doi.org/10.1016/S0166-445X(97)00054-4
Ottinger CA, Kaattari SL (2000): Long-term immune dysfunction in rainbow trout (Oncorhynchus mykiss) exposed as embryos to aflatoxin B1. Fish & Shellfish Immunology 10, 101–106.
Reitan L, Thuvander A (1991): In vitro stimulation of salmonid leucocytes with mitogens and with Aeromonas salmonicida. Fish & Shellfish Immunology 1, 297–307.
Rycyzyn MA, Wilson MR, Bengten E, Warr GW, Clem LW, Miller NW (1998): Mitogen and growth factor-induced activation of a STAT-like molecule in channel catfish lymphoid cells. Molecular Immunology 35, 127–136. https://doi.org/10.1016/S0161-5890(98)00006-6
Sakai DK (1981): Heat inactivation of complements and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, goldfish and tilapia. Bulletin of the Japanese Society of Scientific Fisheries 47, 565–571. https://doi.org/10.2331/suisan.47.565
Scharsack JP, Steinhagen D, Leibold W, Rabe U, Korting W, Schuberth H-J (2001): Flow cytometric analysis of mitogen-induced activation of rainbow trout (Oncorhynchus mykiss) peripheral blood leucocytes. Journal of Veterinary Medicine Series B 48, 331–339. https://doi.org/10.1046/j.1439-0450.2001.00454.x
Shelley LK, Ross PS, Kennedy CJ (2012): Immunotoxic and cytotoxic effects of atrazine, permethrin and piperonyl butoxide to rainbow trout following in vitro exposure. Fish & Shellfish Immunology 33, 455–458.
Siwicki AK, Miyazaki T, Komatsu I, Matsusato T (1996): In vitro influence of heat extract from firefly squid Watasenia scintillans on the phagocyte and lymphocyte activities in rainbow trout Oncorhynchus mykiss. Fish Pathology 31, 1–7. https://doi.org/10.3147/jsfp.31.1
Siwicki AK, Morand M, Fuller J, Nissen S, Goryczko K, Ostaszewski P, Kazun K, Glombski E (2003): Influence of feeding the leucine metabolite β-hydroxy-β-meth-ylbutyrate (HMB) on the non-specific cellular and humoral defence mechanisms of rainbow trout (On corhynchus mykiss). Journal of Applied Ichthyology 19, 44–48. https://doi.org/10.1046/j.1439-0426.2003.00348.x
Tahir A, Secombes CJ (1995): The effects of diesel oil-based drilling mud extracts on immune responses of rainbow trout. Archives of Environmental Contamination and Toxicology 29, 27–32. https://doi.org/10.1007/BF00213083
Tillitt DE, Giesy JP, Fromm PO (1988): In vitro mitogenesis of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri). Comparative Biochemistry and Physiology Part A: Comparative Physiology 89, 25–35. https://doi.org/10.1016/0300-9629(88)91134-6
van der Heijden MHT, Booms GHR, Tanck MWT, Rombout JHWM, Boon JH (1995): Influence of flumequine on in vivo mitogen responses of European eel (Anguilla anguilla L., 1758) lymphoid cells. Veterinary Immunology and Immunopathology 47, 143–152. https://doi.org/10.1016/0165-2427(94)05397-B
Verlhac V, Obach A, Gabaudan J, Schuep W, Hole R (1998): Immunomodulation by dietary vitamin C and glucan in rainbow trout (Oncorhynchus mykiss). Fish & Shellfish Immunology 8, 409–424.
Wagner U, Burkhardt E, Failing K (1999): Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the 3H-thymidine incorporation assay. Veterinary Immunology and Immunopathology 70, 151–159. https://doi.org/10.1016/S0165-2427(99)00041-0
Walsh CJ, Luer CA, Noyes DR (2005): Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris. Veterinary Immunology and Immunopathology 103, 247–256. https://doi.org/10.1016/j.vetimm.2004.09.026
Wang C, Noga EJ, Avtalion R, Levy MG (1997): Whole blood assay for examining lymphocyte blastogenesis of percichthyid bass (Morone) mrr01. Veterinary Immunology and Immunopathology 58, 355–362. https://doi.org/10.1016/S0165-2427(97)00049-4
Wendelaar Bonga SE (1997): The stress response in fish. Physiological Reviews 77, 591–625. https://doi.org/10.1152/physrev.1997.77.3.591
Yaqoob P, Newsholme EA, Calder PC (1994): The effect of dietary lipid manipulation on rat lymphocyte subsets and proliferation. Immunology 82, 603–610.
Yun C-H, Son CG, Jung U, Han SH (2006): Immunosuppressive effect of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through the inhibition of T-lymphocyte proliferation and IL-2 production. Toxicology 217, 31–38. https://doi.org/10.1016/j.tox.2005.08.011
download PDF

© 2020 Czech Academy of Agricultural Sciences