Vet Med - Czech, 2021, 66(7):298-304 | DOI: 10.17221/189/2020-VETMED

Optimisation of phagocytosis assay in rainbow trout (Oncorhynchus mykiss)Original Paper

H Minarova ORCID...1,2, P Ondrackova2, M Palikova1,3, J Mares3, J Blahova4, K Jarova1, M Faldyna2
1 Department of Ecology & Diseases of Zoo Animals, Game, Fish and Bees, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, Czech Republic
2 Department of Infectious Diseases and Preventive Medicine, Veterinary Research Institute, Brno, Czech Republic
3 Department of Zoology, Fisheries, Hydrobiology and Apiculture, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic
4 Department of Animal Protection and Welfare & Veterinary Public Health, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, Czech Republic

A phagocytosis assay is one of the most commonly used functional immunological methods. There are many possible ways of assessing leucocytes and their ability to ingest different particles. The aim of this study was to optimise the phagocytosis assay in rainbow trout (Oncorhynchus mykiss) using labelled zymosan particles (Alexa Fluor 488 and Texas Red conjugate). Whole blood was incubated with the particles under different conditions and leucocytes were subsequently isolated by haemolysis in a hypotonic environment. The effect of the different incubation time, temperature, blood volume and dilution on the phagocytic activity was evaluated by flow cytometry. Our experiments showed that the incubation for at least 2 h at 15 °C provided optimal results, while the blood volume and dilution had no significant effect. The optimised assay will be used for the examination of fish health and in further experimental studies.

Keywords: flow cytometry; haemolysis; ingestion; particle; zymosan

Received: September 19, 2020; Accepted: March 24, 2021; Prepublished online: June 1, 2021; Published: July 31, 2021  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
Minarova H, Ondrackova P, Palikova M, Mares J, Blahova J, Jarova K, Faldyna M. Optimisation of phagocytosis assay in rainbow trout (Oncorhynchus mykiss). Vet Med - Czech. 2021;66(7):298-304. doi: 10.17221/189/2020-VETMED.
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Bear EA, McMahon TE, Zale AV. Comparative thermal requirements of Westslope Cutthroat Trout and Rainbow Trout: Implications for species interactions and development of thermal protection standards. Trans Am Fish Soc. 2007 Jul;136(4):1113-21. Go to original source...
    2. Chilmonczyk S, Monge D. Flow cytometry as a tool for assessment of the fish cellular immune response to pathogens. Fish Shellfish Immunol. 1999 May;9(4):319-33. Go to original source...
    3. Collazos ME, Barriga C, Ortega E. Effect of high summer temperatures upon granulocyte phagocytic function of the tench (Tinca Tinca, L.). Comp Immunol Microbiol Infect Dis. 1995 Feb;18(2):115-21. Go to original source... Go to PubMed...
    4. Crippen TL, Bootland LM, Leong JAC, Fitzpatrick MS, Schreck CB, Vella AT. Analysis of salmonid leukocytes purified by hypotonic lysis of erythrocytes. J Aquat Anim Health. 2001 Sep;13(3):234-45. Go to original source...
    5. Dibble KL, Yackulic CB, Kennedy TA. Warm water temperatures and shifts in seasonality increase trout recruitment but only moderately decrease adult size in western North American tailwaters. Environ Biol Fish. 2018 May; 101(8):1269-83. Go to original source...
    6. Haugland GT, Jakobsen RA, Vestvik N, Ulven K, Stokka L, Wergeland HI. Phagocytosis and respiratory burst activity in lumpsucker (Cyclopterus lumpus L.) leucocytes analysed by flow cytometry. PLoS One. 2012;7(10):e47909. Go to original source... Go to PubMed...
    7. Heger T, Zukal J, Seidlova V, Nemcova M, Necas D, Papezikova I, Piacek V, Zajickova R, Bandouchova H, Pikula J. Measurement of phagocyte activity in heterotherms. Acta Vet Brno. 2020 Jan;89(1):79-87. Go to original source...
    8. Hu Y, Maisey K, Subramani PA, Liu F, Flores-Kossack C, Imarai M, Secombes CJ, Wang T. Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines. Dev Comp Immunol. 2018 Nov;88(1):104-13. Go to original source... Go to PubMed...
    9. Korytar T, Thi HD, Takizawa F, Kollner B. A multicolour flow cytometry identifying defined leukocyte subsets of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2013 Dec;35(6):2017-9. Go to original source... Go to PubMed...
    10. Lawrence MJ, Raby GD, Teffer AK, Jeffries KM, Danylchuk AJ, Eliason EJ, Hasler CT, Clark TD, Cooke SJ. Best practices for non-lethal blood sampling of fish via the caudal vasculature. J Fish Biol. 2020 Jul;97(1):4-15. Go to original source... Go to PubMed...
    11. Le Morvan C, Deschaux P, Troutaud D. Effects and mechanisms of environmental temperature on carp (Cyprinus carpio) anti-DNP antibody response and non-specific cytotoxic cell activity: A kinetic study. Dev Comp Immunol. 1996 Sep-Oct;20(5):331-40. Go to original source... Go to PubMed...
    12. Li J, Peters R, Lapatra S, Vazzana M, Sunyer JO. Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes. Dev Comp Immunol. 2004 Aug;28(10):1005-21. Go to original source... Go to PubMed...
    13. Lulijwa R, Alfaro AC, Merien F, Meyer J, Young T. Advances in salmonid fish immunology: A review of methods and techniques for lymphoid tissue and peripheral blood leucocyte isolation and application. Fish Shellfish Immunol. 2019 Dec;95(1):44-80. Go to original source... Go to PubMed...
    14. Magnadottir B. Innate immunity of fish (overview). Fish Shellfish Immunol. 2006 Feb;20(2):137-51. Go to original source... Go to PubMed...
    15. Minarova H, Palikova M, Mares J, Syrova E, Blahova J, Faldyna M, Ondrackova P. Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). Vet Med-Czech. 2019 Dec;64(12):547-57. Go to original source...
    16. Nagasawa T, Nakayasu C, Rieger AM, Barreda DR, Somamoto T, Nakao M. Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates. Front Immunol. 2014 Sep 16;5:445. Go to original source... Go to PubMed...
    17. Overland HS, Pettersen EF, Ronneseth A, Wergeland HI. Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol. 2010 Jan;28(1):193-204. Go to original source... Go to PubMed...
    18. Park Y, Abihssira-Garcia IS, Thalmann S, Wiegertjes GF, Barreda DR, Olsvik PA, Kiron V. Imaging flow cytometry protocols for examining phagocytosis of microplastics and bioparticles by immune cells of aquatic animals. Front Immunol. 2020 Feb 18;11:203. Go to original source... Go to PubMed...
    19. Pikula J, Heger T, Bandouchova H, Kovacova V, Nemcova M, Papezikova I, Piacek V, Zajickova R, Zukal J. Phagocyte activity reflects mammalian homeo- and hetero-thermic physiological states. BMC Vet Res. 2020 Jul 6;16(1):232. Go to original source... Go to PubMed...
    20. Secombes CJ, Fletcher TC. The role of phagocytes in the protective mechanisms of fish. Annu Rev Fish Dis. 1992; 2:53-71. Go to original source...
    21. Sieroslawska A, Rymuszka A, Bownik A, Skowronski T. The influence of microcystin-LR on fish phagocytic cells. Hum Exp Toxicol. 2007 Jul;26(7):603-7. Go to original source... Go to PubMed...
    22. Thiele L, Rothen-Rutishauser B, Jilek S, Wunderli-Allenspach H, Merkle HP, Walter E. Evaluation of particle uptake in human blood monocyte-derived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages? J Control Release. 2001 Sep 11;76 (1-2):59-71. Go to original source... Go to PubMed...
    23. Thuvander A, Norrgren L, Fossum C. Phagocytic cells in blood from rainbow trout, Salmo gairdneri (Richardson), characterized by flow cytometry and electron microscopy. J Fish Biol. 1987 Aug;31(2):197-208. Go to original source...
    24. Uribe C, Folch H, Enriquez R, Moran G. Innate and adaptive immunity in teleost fish: A review. Vet Med-Czech. 2011 Oct;56(10):486-503. Go to original source...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content