We conducted serologic assays for 8 wildlife and/or zoonotic pathogens: Toxoplasma gondii, Neospora caninum, Francisella tularensis, Coxiella. burnetii, Brucella canis, Brucella abortus/suis, and canine distemper virus (CDV). We analyzed fecal samples for presence of Leptospira and Giardia spp. We tested for C. burnetii, Leptospira spp., N. caninum, and CDV at the Colorado State University Veterinary Diagnostic Laboratory. For detection of Coxiella antibodies, we used a commercially-available enzyme-linked immunosorbent assay (ELISA) kit (IDEXX, Westbrook, ME, USA). The positive result cutoff was set at ≥1:128 and we combined results from Coxiella Phase I and Phase II antibodies. To test for the presence of Leptospira spp. antibodies, we performed a leptospirosis microscopic agglutination test (MAT) using antigen from 6 serovars obtained from the National Veterinary Services Laboratories (Ames, IA, USA), with sera diluted at 1:100, 1:200, and 1:400 and added to serovar-specific antigen using 96-well test plates. For N. caninum, we used a competitive ELISA (cELISA) for detection of IgG antibodies; results are reported as antibodies detected or not detected, which correlates to positive or negative, respectively. For CDV, we used a CDV antibody serum neutralization assay with positive titer cutoff at ≥1:16. Most investigations of morbilliviruses in polar bears have detected CDV rather than phocine distemper or other marine mammal morbilliviruses; thus, we focused on CDV in this study.
Testing for antibodies to B. abortus/suis and B. canis, was conducted at the Colorado Department of Agriculture Rocky Mountain Regional Animal Health Laboratory. Although multiple species of Brucella occur in marine mammals, we selected standard Brucella tests targeting smooth biovariants (B. abortus/suis) based on prior findings in polar bears (Atwood et al. 2017) as well as B. canis, which is occasionally detected in northern canids. Initial screening for B. abortus/suis was conducted by buffered acidified plate antigen (BAPA), followed by fluorescence polarization assay (FPA) for samples positive by BAPA. Only detections that were confirmed by FPA with mP≥20 were considered positive. We used the B. canis 2-Mercaptoethanol Tube Agglutination Test to screen for antibodies to B. canis as described in (CIT TK), with titer values ≥100 considered positive. Antibodies to T. gondii were evaluated using a previously validated MAT at the Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center (Beltsville, MD, USA) with a cutoff titer of 1:25.
We screened sera for F. tularensis antibodies at the USGS Alaska Science Center using a commercially available febrile antigen agglutination test per the manufacturer’s recommended protocol (Becton, Dickinson and Company, Sparks, MS, USA). We ran serial dilutions of sera ranging from 1:20 - 1:320 alongside F. tularensis antisera to serve as positive controls and scored titers for each dilution based on agglutination present during a visual scan 60 seconds after mixing. Titers ≥1:20 compared to positive controls were considered positive.
We analyzed fecal samples for Giardia sp. and Cryptospiridium sp. by immunofluorescence assay at the Colorado State University Veterinary Diagnostic Laboratory. We considered a sample to be positive if any Giardia cysts or Cryptosporidium oocysts were detected. We employed the MERIFLUOR Cryptosporidium/Giardia direct immunofluorescent assay (Meridian Bioscience Inc, Cincinnati, Ohio) per manufacturer's directions, with positive and negative controls for each batch. We examined slides at 100×, and if cysts or oocysts were not detected, then also at 200× magnification using a fluorescence microscope.
Hair isotopes (δ15N and δ13C) were measured for bears in which hair samples were available as an indicator of potential dietary differences Hairs were prepared for isotopic analysis by cleaning with 2:1 chloroform and methanol solution and air drying overnight. Dried hair was loaded into 4 x 6 mm silver capsules (Costech Analytical Technologies, Inc.). Elemental and isotopic composition were measured via conventional continuous flow isotope ratio mass spectrometry. Further details on normalization and data quality and control are provided in Stricker et al. (2022).
Hematology data, including cell counts for white blood cells, neutrophils, lymphocytes, monocytes, and eosinophils, were available for a subset of bears sampled 2009–2011, including five dependent cubs. Within 12 hours of capture, whole blood samples collected in EDTA were analyzed using a veterinary hematology analyzer (HM5, Abaxis, Union City, CA, USA).