1. Few studies have examined host-pathogen interactions in wildlife from an immunological perspective, particularly in the context of seasonal and longitudinal dynamics. In addition, though most ecological immunology studies employ serological antibody assays, endpoint titer determination is usually based on subjective criteria and needs to be made more objective. 2. Despite the fact that anthrax is an ancient and emerging zoonotic infectious disease found worldwide, its natural ecology is not well understood. In particular, little is known about the adaptive immune responses of wild herbivore hosts against Bacillus anthracis. 3. Working in the natural anthrax system of Etosha National Park, Namibia, we collected 154 serum samples from plains zebra (Equus quagga), 21 from springbok (Antidorcas marsupialis), and 45 from African elephants (Loxodonta africana) over 2-3 years, resampling individuals when possible for seasonal and longitudinal comparisons. We used enzyme-linked immunosorbent assays to measure anti-anthrax antibody titers and developed three increasingly conservative models to determine endpoint titers with more rigorous, objective mensuration. 4. Between 52-87% of zebra, 0-15% of springbok, and 3-52% of elephants had measurable anti-anthrax antibody titers, depending on the model used. While the ability of elephants and springbok to mount anti-anthrax adaptive immune responses is still equivocal, our results indicate that zebra in ENP often survive sublethal anthrax infections, encounter most B. anthracis in the wet season, and can partially booster their immunity to B. anthracis. 5. Thus, rather than being solely a lethal disease, anthrax often occurs as a sublethal infection in some susceptible hosts. Though we found that adaptive immunity to anthrax wanes rapidly, subsequent and frequent sublethal B. anthracis infections cause maturation of anti-anthrax immunity. By triggering host immune responses, these common sublethal infections may act as immunomodulators and affect population dynamics through indirect immunological and co-infection effects. 6. In addition, with our three endpoint titer models, we introduce more mensuration rigor into serological antibody assays, even under the often-restrictive conditions that come with adapting laboratory immunology methods to wild systems. With these methods we identified significantly more zebras responding immunologically to anthrax than have previous studies using less comprehensive titer analyses.
Anti-PA Titers for Equus quagga
Anti-anthrax (anti-protective antigen toxin) antibody titers for wild plains zebra immobilized and sampled over 1-5 seasons in Etosha National Park, Namibia. id=animal capture ID. titer=anti-PA titer; the titer is the highest dilution in the titration series for which the mean sample optical density (OD) for that dilution is greater than the mean negative control OD titration curve from all plates (dilution is as 1:dilution; e.g. 1024 is a 1:1024 dilution). titerDUP=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-duplicate error at that dilution, across all samples. titerNCONT=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-negative control error at that dilution, across all samples. plate=plate identifier (date and plate number on that day). ncont=identifier of the negative control sample used.
EQtitersPA.csv
Anti-PA Titers for Antidorcas marsupialis
Anti-anthrax (anti-protective antigen toxin) antibody titers for wild springbok immobilized and sampled over 1-2 seasons in Etosha National Park, Namibia. id=animal capture ID. titer=anti-PA titer; the titer is the highest dilution in the titration series for which the mean sample optical density (OD) for that dilution is greater than the mean negative control OD titration curve from all plates (dilution is as 1:dilution; e.g. 1024 is a 1:1024 dilution). titerDUP=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-duplicate error at that dilution, across all samples. titerNCONT=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-negative control error at that dilution, across all samples. plate=plate identifier (date and plate number on that day). ncont=identifier of the negative control sample used.
AMtitersPA.csv
Anti-PA Titers for Loxodonta africana
Anti-anthrax (anti-protective antigen toxin) antibody titers for wild African elephants immobilized and sampled over 1-2 seasons in Etosha National Park, Namibia. id=animal capture ID. titer=anti-PA titer; the titer is the highest dilution in the titration series for which the mean sample optical density (OD) for that dilution is greater than the mean negative control OD titration curve from all plates (dilution is as 1:dilution; e.g. 1024 is a 1:1024 dilution). titerDUP=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-duplicate error at that dilution, across all samples. titerNCONT=anti-PA titer for which the titer is the last dilution at which the mean sample OD is greater than the mean negative control OD buffered by a 95% confidence interval determined by the inter-negative control error at that dilution, across all samples. plate=plate identifier (date and plate number on that day). ncont=identifier of the negative control sample used.
LAtitersPA.csv
