2.7.1. Haptoglobin

Using a commercially available assay kit (Tridelta PHASE Haptoglobin Assay, TP-801; Tridelta Development Limited, Maynooth, County Kildare, Ireland), we measured circulating concentrations of haptoglobin, which is an acute phase protein that increases in concentration in response to infection, inflammation, or trauma [7,10]. We followed the manufacturer's instructions, measuring absorbance at 630 nm 5 min after initiation of the color change reaction. We calculated concentrations in our samples using a linear standard curve that we generated from six standards ranging from 0.039 to 1.25 mg Ml−1. Most serum samples were analyzed in singlicate, but samples from four individuals were run in duplicate to allow for the quantification of within assay (i.e., within plate) variability. The average coefficient of variation of the four duplicated samples was 5.6%. For each duplicated sample, we used the mean concentration in further analyses.

#### 2.7.2. Hemolysis and Hemagglutination

We measured titers of both complement-driven hemolysis and natural antibody-driven hemagglutination following standard protocols [7,9]. Since the test serum was from mammals, we used blood from a bird (*Columba livia domestica*) to make the required 1% suspension of exogenous erythrocytes. All test samples were analyzed in singlicate, but each of the six assay plates included a duplicate (and in one case, triplicate) sample from a brown rat (*Rattus norvegicus*) to allow for the quantification of within assay (i.e., within plate) and among assay (i.e., among plate) variability. On average, the rat standard exhibited lysis of 4.4 titers and agglutination of 6.1 titers, and the variability (coefficient of variation) was as follows: hemolysis, within 12.4%; hemolysis, among 12.5%; hemagglutination, within 8.3%; and hemagglutination, among 15.5%.

#### 2.7.3. Neutrophil to Lymphocyte Ratio

One author (E.B.) analyzed all of the stained blood smears using a light microscope (at 1000×). Per slide, she counted the neutrophils and lymphocytes until a combined total of up to 100 cells was achieved (*<sup>x</sup>total* = 83 cells). This process was repeated twice per slide, and the average count of each cell type was used in further analyses of their ratio (neutrophil to lymphocyte ratio).
