Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells
Introduction
The complement system plays an important role in host innate immune defense. It comprises of large number of plasma proteins that work together to achieve lysis of pathogens. The structural and functional diversity of complement proteins in teleost fish have resulted in extensive innate immune recognition capabilities (Oriol Sunyer et al., 1998). Complement activity has been extensively used as an indicator of innate humoral immune response in various fish species. The three different activation pathways: classical, alternative and lectin-dependent, have been well studied in fish (Forsgren and Quie, 1974; Montero et al., 1998).
Complement proteins in fish exhibit high degree of structural and functional conservation along with isotype diversity of components as a result of gene duplications (Boshra et al., 2006; Nakao et al., 2006; Nakao et al., 2003). Data obtained from studies on the complement system in several fish species have demonstrated the presence of almost all homologues to mammalian complement proteins (Nakao et al., 2011). The alternative pathway comprises of C3 and factors B and D which can be activated by bacterial lipopolysaccharide and β-glucans, leading to formation of C3-convertase. In normal physiological conditions at non-activated state, C3 is suppressed by regulatory proteins, factors H and I, and suppression is released by the various complement activators (Nakao et al., 2003).
Analysis of the alternative complement activity is usually achieved by measuring the ability of serum samples to lyse red blood cells, also known as the ACH50 assay (Alternative Complement pathway Hemolytic activity), in which the volume of serum that provides 50% lysis of sheep or rabbit red blood cells is calculated. These assays have been widely used to measure complement activity, mostly in blood plasma and serum.
Precursor zymogens of complement proteins are distributed in body tissues and synthesized at multiple sites including hepatocytes and cell surfaces (Morgan and Gasque, 1997; Nesargikar et al., 2012). Thus measuring complement activity in tissues and organs should be possible and could be an alternative for employing this analysis in cases where collection of blood is impractical.
This manuscript addresses several topics in complement analysis. (1) The source of red blood cells (RBCs): RBCs from sheep and rabbit have been used interchangeably for complement analysis in fish (Chiu et al., 2008; Matsuyama et al., 1988; Pionnier et al., 2013; van den Berg et al., 1993). In this study we carry out a comparative analysis using RBCs from these two animals. (2) Examining the application of complement analysis to small-sized fish, from which blood cannot be drawn (i.e. small ornamental species and fish larvae). (3) Analyzing the effects of freeze and thaw on complement activity in serum and WBHs.
Section snippets
Fish
Adult guppies (Poecilia reticulate Peters; males only), mollies (Poecilia sphenops) and zebrafish (Danio rerio) were obtained from commercial aquaculture farms in the Arava Valley, Israel. The fish were maintained in 100-L plastic tanks, at 25 ± 1.5 °C with light:dark cycle of 12:12 h, and were fed at 2% of their body weight once a day with a commercial fish feed (Ocean Nutrition, San Diego, USA). Water quality parameters were monitored once or twice a week. Serum samples were obtained from
Results and discussion
Substantial amount of complement activity was recorded in WBH of the different fish species. Hemolytic activity was confirmed by visual observation of the red blood cell pellet, including presence of intact RBCs, absence (suggesting complete lysis) or shrinkage of the pellet (suggesting partial lysis of RBCs; these observations are not shown). The assay was also verified by use of appropriate heat inactivated control samples, in which no RBCs lysis was observed. To rule out lysis by other
Conclusion
To our knowledge, this is the first study where WBHs were used for assaying complement activity. Overall our results support the feasibility of applying this methodology for measuring complement activity in WBHs of fish. This method could be particularly useful as an indicator of innate immunity in small fish or larvae, when blood cannot be obtained. The study also presents a comparative analysis of complement activity using RRBCs and SRBCs, suggesting that RRBCs are preferable for both fish
Acknowledgements
This work was supported by the U.S. Agency for International Development, MERC Projects, M33-034 (application and scale-up of rabbitfish production in Jordan and Israel Arava/Araba Valley: Phase B) and by the Goldinger Trust (the Jewish Federation of Delaware, USA). The postdoctoral fellowship of SN was supported by the Israeli Council for Higher Education, PBC Program.
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These authors contributed equally to this work and thus share first authorship.