Study animals

Between 2002 and 2013 we captured 197 (49 female, 148 male) adult (>2 years of age) and sub-adult (>1–2 years of age) free-ranging cheetahs and 36 (19 female, 17 male) adult (>2 years of age) and sub-adult (>1–2 years of age) free-ranging leopards in box traps on farmland in east-central Namibia and immobilized them as previously described68. The animals were captured throughout the year. Once captured, they were kept in the box traps in the shade for several hours or overnight until the research team met at the box trap which was normally in the morning hours. The animals were immobilized with a dart gun and blood samples were taken between 20 min and 35 min after darting. After approximately 45 min to 60 min, the animals were given an antidote and observed until they had fully recovered from anaesthesia. From animals that were captured and sampled more than once (N = 30 cheetahs), one sample was randomly selected in order to avoid pseudo-replication in the analyses. The proportions of males (75.1% in cheetahs, 47.2% in leopards) differed between the species (Fisher’s exact test, P = 0.001), whereas the proportion of sub-adults (15.2% in cheetahs, 16.6% in leopards) did not (Fisher’s exact test, P = 0.61).

Animals in traps often acquired small injuries caused by their behavior in the trap. We recorded injuries and assessed the level of injuries as level 1 if they presented no injuries or only had old scars, as level 2 if they presented bloody claws, a bloody nose or one small abrasion, as level 3 if they presented several abrasions, small wounds or one abrasion and a bloody nose or bloody claws and as level 4 if they presented larger abrasions or larger wounds or injuries.

All experimental procedures described in the material and methods were approved by the Internal Ethics Committee of the Leibniz Institute for Zoo and Wildlife Research (IZW, Berlin, Germany) (permit number: 2002-04-01) and the Ministry of Environment and Tourism of Namibia (permit numbers: 525/2002, 700/2003, 764/2004, 939/2005, 1089/2006, 1194/2007, 1300/2008, 1392/2009, 1514/2010, 1514/2011, 1689/2012, 1813/2013), and all experiments were carried out in accordance with the approved guidelines of the IZW.

Blood sampling and storage

Blood was taken with serum and heparin Vacutainer tubes (Becton Dickinson, Franklin Lakes, USA), transported to the field laboratory in a cool box and centrifuged within 12 hours, very rarely within 24 hours after sampling. Results of immunological tests conducted with serum and plasma from whole blood centrifuged after 2 hours, 4 hours, 6 hours and 12 hours from blood collection and after 2 hours and 4 hours from blood collection, respectively, do not differ100. There was no systematic bias for samples centrifuged later than 12 h after sampling, thus all samples were included for immunological tests. Serum and plasma were aliquoted and stored in liquid nitrogen. Samples were transported to Germany in full compliance with the Convention on International Trade in Endangered Species (CITES) and stored at −80 °C until laboratory analysis.

ELISA for Immunoglobulin G

Immunoglobulin G (IgG) concentration were measured in plasma samples with a protein A enzyme-linked immunosorbent assay (ELISA)101. Plasma samples were diluted 1:20.000 with 50 mM NaHCO 3 . As a standard, we diluted purified cat IgG with a starting concentration of 1 mg/ml (Bethyl Laboratories, Montgomery, USA, Catalog N° P20-105) with 50 mM NaHCO 3 and created standard concentrations of 4.0 μg/ml, 2.0 μg/ml, 1.0 μg/ml, 0.5 μg/ml, 0.25 μg/ml, 0.125 μg/ml and 0.0625 μg/ml.

We pipetted 100 μl of diluted samples or standards in duplicates into each well of 96-well ELISA plates. Plates were incubated for 1 hour at 37 °C and washed twice with Tris-Buffered-Saline-Tween-20 (TSB-T20). Gelatine was added to TSB-T20 solution and 200 μl of 1% of this mixture was pipetted to each well to block non-specific reaction bindings. Plates were incubated for 30 min at 37 °C and washed twice with TSB-T20. Then, 100 μl of 1:12.000 solution of protein-A-horseradish-peroxidase (Protein A- HRP; Invitrogen; Catalog N° 10-1023) in TSB-T20 was added to each well and plates were incubated for 30 min at room temperature. Plates were washed with TSB-T20 and 100 μl of phosphate-citrate-buffer containing a 1% dilution of 3,3′,5,5′-Tetramethylbenzidin (TMB; TMB One Component Microwell Substrate; SouthernBiotech; Catalog N° 0411-01), dimethylsulfoxide (DMSO) and H 2 O 2 was added to each well. The reaction was stopped after 5 min with 100 μl of 1% H 2 SO 4 . We measured the absorbance of the wells in the plates at 450 nm in a photometric microplate reader (Biotek; μQuant Microplate Spectrophotometer). A linear standard curve was calculated for each plate using the standard concentrations. IgG concentrations were then calculated based on the standard curve.

ELISA for serum amyloid A (SAA)

We measured and calculated SAA concentrations with a commercial solid phase sandwich SAA Multispecies ELISA kit (Tridelta, Phase Range; Multispecies SAA ELISA kit; Catalog N° TP-802) following the instructions of the manufacturer. Cheetah and leopard plasma samples were diluted 1:1,500 and 1:2,000, respectively, with sample diluent (provided with the ELISA kit) prior to the assay. A linear standard curve was calculated using the calibrator standard for cats (100.0 ng/ml, 50.0 ng/ml, 25.0 ng/ml, 12.5 ng/ml, 6.25 ng/ml and 0.0 ng/ml). SAA concentrations were calculated according to the standard curve on each plate. Sixty-two samples had a higher absorbance than measurable with our spectrophotometer (Biotek; μQuant Microplate Spectrophotometer). These samples were either rerun (N = 42) with a higher dilution of 1:5,000 for cheetahs or 1:8,000 for leopards (samples were thawed again) or the entire plate was re-measured directly after discarding half of the volume in each well (N = 20).

Bacterial killing assay (BKA)

We measured the in vitro bacterial killing ability of serum against Escherichia coli. The method was previously described in detail in ref. 102. Briefly, serum samples were serially diluted with phosphate-buffered-saline (PBS), resulting in eight dilutions from 1:2 to 1:265. Each well of a 96-well-plate was filled with 44 μl and mixed with 10 μl of a bacterial working solution of ~1.5 × 105 colony-forming units (CFU)/ml. After incubation for 30 min at 37 °C, tryptic soy broth was added to each well. Absorbance was measured with a spectrophotometer (Biotek; μQuant Microplate Spectrophotometer) to determine background absorbance and again after the plates had been incubated for 12 hours at 37 °C. Bacterial killing capacity was calculated for each dilution of serum against a positive control (wells that contain only bacteria without serum). Ranks were assigned to each dilution before killing capacity dropped from 100% to 0%, such that dilution 1:2 corresponded to rank 1, the usually lowest rank, dilution 1:4 to rank 2, etc. If bacterial killing did not reach 100% even at dilution 1:2, rank 0 was assigned.

Lysoplate assay

To measure the concentration of lysozyme we used the lysoplate assay method103. We prepared 1% noble agar (Sigma Aldrich; St. Louis, USA, Catalog N° A5431-250G) with PBS at pH = 6.3 and added the required amount of lysozyme-sensitive bacteria Micrococcus lysodeikticus (Sigma Aldrich; M3770) to reach a bacterial concentration of 25 mg/100 ml in the agar for cheetahs and 12.5 mg/100 ml in the agar for leopards. Plates had a diameter of 14.2 cm and were put on a pre-heated surface (50 °C), horizontally leveled with a water spirit to avoid quick and uneven cooling of the 30 ml noble agar on the plates. After cooling, 25 holes with diameters of 4.5 mm were punched into the agar and filled with 25 μl of serum samples (18 holes) or standards (7 holes). Standards with concentrations of 10.0 μg/ml, 7.5 μg/ml, 5.0 μg/ml, 2.5 μg/ml, 2.0 μg/ml, 1.25 μg/ml and 1.0 μg/ml were prepared using lysozyme form chicken egg white (Sigma Aldrich; St. Louis, USA, Catalog N° L6876). Plates were incubated at room temperature for 18 hours.

Micrococcus lysodeikticus is particularly sensitive to lysozyme, thus the bacterial lysis of the samples and standards creates a clear zone around the inoculated wells. The diameter of this clear zone is proportional to the logarithmic (basis of 10) lysozyme concentration in the samples and standards103. We photographed each plate in a photobox (Imaging system; peqlab) with a ruler next to it as a reference scale. The diameter of the lytic areas was measured digitally using the software ImageJ (version 1.48, http://imagej.nih.gov/ij/). Each lytic area was measured three times and the mean was used for calculations. The measurements of the lysis standards were plotted as a linear function of the log lysozyme concentration. This regression line was then used to infer the lysozyme concentrations of the cheetah and leopard samples.

Haemagglutination/haemolysis assay

The haemagglutination/haemolysis titers represent the levels of natural antibodies and complement61. Although the method was originally developed for avian species, it has recently been modified for mammals by using chicken erythrocytes as target cells62. After pipetting 25 μl of plasma in the wells of the first two columns of a U-shaped 96-well microtitre plate, 25 μl sterile PBS was added to the 2nd–12th columns. Using a multi-channel pipette, the content of the second column wells was serially diluted until the 11th column, resulting in a dilution series for each sample from 1:2 to 1:1024. We used the last column of the plate as negative controls containing only PBS. We then added 25 μl of 1% chicken red blood cells suspension to all wells, covered them with Parafilm M (Pechiney Plastic Packaging, Chicago, USA), vortexed gently and incubated at 37 °C for 90 min. After incubation the plates were tilted at a 45° angle to increase the visualization of agglutination and kept at room temperature until analyses.

Agglutination and lysis, which reflect the activity of natural antibodies and the interaction between natural antibodies and complement61,104, were recorded after 20 min (haemagglutination titre) and 90 min (haemolysis titre), respectively. Haemagglutination is characterized by the appearance of clumped red blood cells as a result of antibodies binding multiple antigens, whereas during haemolysis red blood cells are destroyed by complement. Haemagglutination/haemolysis titers were given as the log 2 of the reciprocal of the highest dilution (i.e. lowest concentration) of plasma showing positive haemagglutination or haemolysis, respectively62,104.

Measurement of cortisol concentration

Although cheetahs and leopards were captured in the same type of traps and therefore exposed to the same capture conditions, the two species might respond differently to these short-term challenges. Such challenges increase the allostastic load (‘stress’) and thereby may influence various immune parameters, as has been shown for SAA concentrations in rats53. To rule out the possibility that differences in immune parameters between the two species were caused by differences in allostatic load induced by different responses to the capture procedure, we measured the concentration of native cortisol, an indicator of allostatic load which rapidly increases after a stressful stimulus105, in blood samples of cheetahs and leopards. Cortisol (hydrocortisone) was quantified as described earlier106 by an enzyme immunoassay (EIA) using a polyclonal antibody (rabbit) against hydrocortisone-21-hemisuccinate-BSA and hydrocortisone-21-hemisuccinate-peroxidase as label. The inter-assay coefficient of variation of two biological samples was 7.3 and 8.1% (n = 14), respectively.

Statistical analyses

The dataset consisted of 251 captures and sampling events for 197 cheetahs and 36 captures and sampling events for 36 leopards. Sample sizes varied slightly for different immunological measurements because the bacterial killing assay and the hemagglutination/hemolysis assay should be limited to samples which are thawed for the first time only, otherwise they become unreliable. Some samples had been previously thawed for other studies and been frozen again and therefore could not be used. Other samples were too small to provide material for all analyses. We therefore expect that there was no systematic bias in the sets of available samples for the analysis of various immune parameters of cheetahs and leopards. The highest number of missing data was for SAA concentration with 68 missing measurements (26.8%). For all other immunological parameters the percentage of missing measurements was below 20%. We used principal component analysis (PCA), a statistical procedure that uses an orthogonal transformation to convert a set of possibly correlated variables into a set of linearly uncorrelated variables. PCA reduces the dimensionality of data and through the loadings of the original variables on the principal components helps to identify the true sources of variation in the data. Because PCA requires the removal of all samples with missing data, we used the R package Amelia II version 1.7.3, which allows for the imputation of missing values in the dataset107. Amelia II uses the expectation-maximization algorithm to perform multiple imputations for incomplete data. An examination of the quality of imputation is presented in the supplementary information (Figure S1, Table S1 and Supplementary Results).

To test whether both species differ in their immune characteristics, we used the dataset complemented by the imputed values and reduced the dimensionality of immune parameters by performing a mean-centered, scaled PCA with the R package ade4 version 1.7-2108. We then fitted a logistic regression model predicting species identity (a binary variable) as a function of the first two principal components. We tested the effect of each covariate by performing a likelihood ratio test between this model and a model that only contained the intercept and the other principal component. We ruled out the possibility that differences between species were a consequence of possible differences in the proportion of males and females sampled in the two species. To do so, we fitted one logistic regression per species predicting the sex (binary variable) as a function of the two principal components of the PCA, and compared the likelihood of the fit to a model with an intercept only. A non-significant result of this analysis would suggest that sex does not influence the outcome of the PCA analysis which uses the immune components to distinguish species. To control for the possible effects of allostatic load we fitted linear models predicting each immune variable by cortisol concentrations to extract the residuals of these models to be used in another PCA. We again predicted the species identity (binary variable) as a function of the two principal components from this new PCA with a logistic regression model and compared the results obtained with those from the first PCA. All statistical analyses were performed using R version 3.0.3109.

Following the PCA analyses, we compared the median of each immune parameter separately with non-parametric Mann-Whitney-U-tests for not normally distributed data. This allowed us to use the original non-imputed dataset. Levels of injuries caused by the behavior of the animals in the box traps were analyzed with a chi-square-test for independence.