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

Sarah J Baker,Mark E Merchant

doi:10.1186/s41936-018-0050-6

Abstract

BackgroundReptile 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.ResultsTreatment of unsensitized sheep red blood cells (SRBCs) with plasma collected from captive prairie rattlesnakes (Crotalus viridis) resulted in volume-dependent hemolysis, with a CH50 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 Ca2+ or Mg2+, but not Ba2+, Cu2+, or Fe2+. 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.ConclusionsRattlesnakes 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.

Highlights

Reptile populations face a growing number of threats including global climate change and emerging pathogens
The relationship between C. viridis plasma dilution and Sheep red blood cells (SRBC) hemolysis was biphasic, with a sharp initial increase in activity at low dilutions followed by a slow linear increase at higher dilutions (Fig. 1)
Addition of only 6 μL of plasma to a 400 μL reaction resulted in 31% of maximal hemolytic activity

Summary

Introduction

Reptile populations face a growing number of threats including global climate change and emerging pathogens. Host defense mechanisms can be divided into two broad divisions, namely innate and adaptive immunity. Innate immunity is generally nonspecific, does not require previous exposure, and is very rapid in response. Adaptive immunity requires previous exposure and contains components of “immunological memory,” is very specific, but can take several days to develop and launch a coordinated response. One important mechanism of innate host defense is the serum complement system. Serum complement is an ancient mechanism of innate defense characterized by an activation cascade that culminates in damage to the outer envelope and nonspecific destruction of microbes (Ember & Hugli, 1997). Serum complement components are crucial for coordinating some important events in adaptive immune response (Nielsen, Fischer, & Leslie, 2000)

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