Immune and endocrine alterations at the early stage of inflammatory assemblage in toads after stimulation with heat-killed bacteria (Aeromonas hydrophila).

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 µl of saline or heat-killed bacteria: groups A1 (3 × 107 cells), A2 (3 × 108 cells), and A3 (3 × 109 cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-6, and IL-10. We found higher expression of IL-6, IL-1ß, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila.

Day vs. night variation in the LPS effects on toad's immunity and endocrine mediators.

The immune-endocrine interactions following an immune challenge have been demonstrated in amphibians. When considering immune challenges, the immune-endocrine implications can vary with the injection time (day or night), a pattern not explored in amphibians. We investigated the immune response following a lipopolysaccharide - LPS injection, measured as plasma bacterial killing ability - BKA, phagocytosis of blood cells - PP, and neutrophil to lymphocyte ratio - NLR, splenic proinflammatory cytokines mRNA (IL-1ß and IL-6), and also endocrine mediators (corticosterone - CORT and melatonin - MEL plasma levels) in Rhinella icterica adult male toads injected at day (10 am) or night (10 pm). LPS induced increases in CORT, NLR, PP, and IL-1ß mRNA compared with amphibian phosphate-buffer saline-injected individuals. For plasma CORT, the response was more pronounced during the night. While for the PP and IL-1ß mRNA, the effect was more evident during the day. For NLR, the increase happened at both times, day and night, in the LPS-injected toads. Meanwhile, no changes were observed in BKA, IL-6 mRNA, and MEL levels. Overall, our results demonstrated an LPS-induced inflammatory response in R. icterica toads, characterized by higher PP, NLR, and IL-1ß mRNA, followed by activation of the hypothalamic-pituitary-interrenal axis (higher CORT levels). The time in which the toads received the LPS injection affected the endocrine and immune mediators. The higher CORT and lower inflammatory response at night suggested a potential functional interaction between CORT and immune reactivity associated with the differences in night vs. day in R. icterica toads. These results highlight the relevance of investigating different injection times and mechanistic pathways to understand LPS-induced immunomodulation in anurans.

Thermal sensitivity of Bullfrog's immune response kept at different temperatures.

Climate change and emerging infectious diseases are often described as the main factors associated with the worldwide amphibian population decline. In this context, rising temperatures due to global warming might act as a chronic stressor for many amphibians, leading to immunosuppression. This study aimed to characterize the thermal sensitivity of the Bullfrog's (Lithobates catesbeianus) immune response and the effect of acclimation at different temperatures on it. Plasma bacterial killing ability (BKA) and phagocytosis activity of blood leukocytes were measured at different incubation temperatures (5-40°C) in individuals kept at 28°C and 34°C. First, all individuals were held under 28°C and sampled on the 16th day. Subsequently, one group was kept at 28°, and the other one was transferred to 34°C. Both groups were sampled at 83 and 106 days of maintenance. Plasma corticosterone (CORT) and testosterone (T) were assessed to evidence thermal stress and possible endocrine correlates of immune changes over time. The incubation temperature affected BKA both on animals kept at 28°C and 34°C, with maximum values at lower temperatures (5-20°C). Phagocytosis activity was constant over the range of assay temperatures. Immune and endocrine variables decreased over time in both thermal regimes, but frogs maintained at 34°C showed lower T and immunosuppression, evidencing stress response. Therefore, exposure to high temperatures might decrease immune function in bullfrogs due to chronic stress response and by exposition to temperatures of lower performance according to the thermal sensitivity curve, which might increase vulnerability to diseases in this anuran species.