Characterization of serum complement immune activity in the prairie rattlesnake (Crotalus viridis)

Abstract

Background: Reptile populations face a growing number of threats including global climate change and emerging pathogens. Unfortunately, research investigating the reptile immune system lags behind other taxa groups, hindering our ability to predict or mitigate species' response to threats. Baseline studies are critical for our understanding of reptile immune response and overall health of wild populations. Results: Treatment of unsensitized sheep red blood cells (SRBCs) with plasma collected from captive prairie rattlesnakes (Crotalus viridis) resulted in volume-dependent hemolysis, with a CH 50 of 0.23 ± 0.01 mL. Kinetic analyses at different temperatures revealed that the hemolysis was relatively rapid, with 50% of hemolytic activity occurring within 15 min (25°C), 18 min (35°C), or 23 min (15°C), and near maximum hemolysis within 60-90 min at all three temperatures. A comparative thermal profile revealed that complement activity was low at 5°C, but increased sharply at 10 and 15°C, and was maximal at 20-30°C. A steep decline in activity was noted at temperatures > 30°C. Mild heat treatment of the snake plasma (56°C, 30 min) or treatment with proteases completely abolished the hemolytic activity. In addition, inclusion of 5 mM EDTA inhibited 90% of the hemolysis, but the activity could be reconstituted with the addition of 15 mM Ca 2+ or Mg 2+ , but not Ba 2+ , Cu 2+ , or Fe 2+. Furthermore, the hemolysis was unaffected in the presence of 20 mM methylamine, indicating that the alternative mechanism of complement activation is responsible for the observed activities. Conclusions: Rattlesnakes show a relatively robust innate immune response as measured by hemolysis of SRBCs. However, hemolytic activity is reduced at high temperatures, indicating that rising global temperatures may have immune consequences for snake species, making them more vulnerable to known and emerging pathogens.

Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina)

“…We found that box turtle plasma antimicrobial activity was significantly reduced following treatment with protease, heat, and EDTA, indicating that the underlying mechanism is likely complement‐mediated. The complement cascade can be activated using one of three pathways: classical, alternative, or lectin; each of which has been documented in different reptilian species (Baker & Merchant, 2018b; Baker, Kessler, Darville‐Bowleg, & Merchant, 2019; Merchant & Britton, 2006; Merchant, Determan, Falconi, & Shirley, 2013; Merchant, McFatter, Mead, McAdon, & Wasilewski, 2010; Merchant, Verret, & Elsey, 2005). Further testing is necessary to determine which of the three complement pathways is utilized in eastern and ornate box turtles, and to characterize additional components of innate immunity in these species including natural antibodies, lysozyme, toll‐like receptors, and others.…”

Plasma complement activation mechanisms differ in ornate (Terrapene ornata ornata) and eastern box turtles (Terrapene carolina carolina)

“…Robust SRBC hemolysis and plasma antibacterial activity (Adamovicz et al, 2020), lack of response to classical complement pathway activation inhibitors, absence of detectable circulating mannan‐binding proteins, and attenuation of plasma hemolytic activity in the presence of alternative pathway inhibitors suggest that ornate box turtles use the alternative pathway of complement activation. The alternative pathway is commonly utilized in several species of reptiles (Baker & Merchant, 2018; Merchant & Britton, 2006; Merchant et al, 2005), including chelonians such as common snapping turtles (Baker, Kessler, Darville‐Bowleg, et al, 2019) and red‐eared sliders (Koppenheffer, 1987).…”

“…While direct comparison between volumedependent hemolysis studies is challenging due to differences in methodology, general observations can still be made. For example, the hemolytic capacity of box turtle plasma was more potent than Geoffrey's side-necked turtles (Phrynops geoffroanus) (Ferronato et al, 2009) and less potent than Komodo dragons (Varanus komodoensis), prairie rattlesnakes (Crotalus viridis), and crocodilians (Baker & Merchant, 2018;Merchant & Britton, 2006;Merchant et al, 2005Merchant et al, , 2012Merchant et al, , 2013Merchant et al, , 2014Siroski et al, 2010). Ornate box turtle plasma hemolytic capacity was similar to common snapping turtles (Chelydra serpentina) and superior to alligator snapping turtles (Macrochelys temminckii), while eastern box turtle hemolytic capacity was inferior to both members of the Chelydridae (Baker, Kessler,``Darville-Bowleg, et al, 2019).…”

“…The reptilian immune system is characterized by robust innate immunity and moderate acquired immunity (Rios & Zimmerman, 2015;Zimmerman et al, 2010Zimmerman et al, , 2016. Complement activity has been demonstrated in multiple reptile species (Baker & Merchant, 2018;Merchant et al, 2012;Vogel & Muller-Eberhard, 1985a, 1985b, but is best characterized in crocodilians (Merchant & Britton, 2006;Merchant et al, 2005Merchant et al, , 2010Merchant et al, , 2013Merchant et al, , 2014Siroski et al, 2010). While a handful of studies have employed complement assays to explore immune function in chelonians (Borysenko & Lewis, 1979;Chen et al, 2007;Ferronato et al, 2009;Freedberg et al, 2010;Soltanian, 2016;Soltanian et al, 2018;West & Klukowski, 2018;Yektaseresht et al, 2014Yektaseresht et al, , 2018, very few have provided mechanistic data to determine the pathways of complement activation utilized by this taxon (Baker, Kessler, Darville-Bowleg, et al, 2019;Koppenheffer, 1987).…”

Characterization of plasma secretory phospholipase A2 activity in the prairie rattlesnake (Crotalus viridis)

“…However, our knowledge of the function of reptile immune systems lags behind other vertebrates (Warr et al, 2003). Recent studies have described the broad-acting antibacterial properties of plasma from C. viridis (Baker & Merchant, 2018a) and have assigned much of those properties to an active serum complement system of proteins (Baker & Merchant, 2018b).…”