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.

Liver vs. spleen: Time course of organ-dependent immune gene expression in an LPS-stimulated toad (Rhinella diptycha).

The inflammatory response comprises highly orchestrated events that are conserved amongst vertebrate groups. Hepatic and splenic cytokines are major mediators of the systemic inflammatory processes. However, the liver is still neglected as an immune organ in amphibians. This study reports organ-dependent gene expression using an anuran model. We tracked mRNA levels of immune proteins [C1s (subcomponent S of the complement protein 1), IFN-γ, IL-1ß, IL-6, and IL-10] at four time-points (1 h, 3 h, 6 h, and 18 h post-injection) in spleens and livers of intraperitoneal LPS-challenged (2 mg/kg) adult male toads (Rhinella diptycha) using independent samples. We found acute C1s up-regulation in the liver 1 h post-injection, with no treatment effect in the spleen. The LPS injection did not show any effect in splenic IFN-γ gene expression while eliciting only a marginal effect in the hepatic tissue. IL-1ß was up-regulated in both organs, with the liver initially displaying early expression (1 h and 3 h) and the spleen taking over late expression (18 h). Both organs exhibited similar patterns for IL-6, with early up-regulation (1 h and 3 h) and late peak (18 h). Although IL-10 was early detected and up-regulated only in the liver, both organs showed up-regulation in 6 h and 18 h post-injection. Our results show an exclusive hepatic prominence in complement protein expression during the acute-phase response. Furthermore, hepatic pro-inflammatory cytokine expression was more pronounced in earliest time-points, while the spleen offers a slower and more consistent response overall. Our data provide an organ-integrative outlook into the initial hours of the inflammation in amphibians, confirming the liver's pivotal role as a regulator in the acute-phase of the inflammatory response in amphibians.

Systemic hormonal and immune regulation induced by intraperitoneal LPS injection in bullfrogs (Lithobates catesbeianus).

Host's defense against external challenges activates an inflammatory response regulated by a set of chemical signals, including hormones. These immunomodulatory hormones, such as corticosterone, testosterone, and melatonin, trigger the systemic immune responses responsible for inflammatory assembly and resolution. This study aimed to investigate the effects of an immune challenge on endocrine and innate immune responses in the bullfrog (Lithobates catesbeianus). Adult males were intraperitoneally injected with lipopolysaccharide (LPS; 2 mg/kg) or saline, and blood samples were collected 6 and 24 h after injection for measurement of neutrophil/lymphocyte ratio, blood leukocyte phagocytosis, plasma bacterial killing ability, and plasma levels of corticosterone, melatonin, and testosterone. Our results showed LPS-induced increased neutrophil/lymphocyte ratio and leukocyte phagocytosis, and decreased melatonin and testosterone plasma levels, which were more pronounced 24 h after injection. Overall, we conclude that LPS intraperitoneal injection can activate the innate immune response and modulate the hormonal profile of the bullfrogs, with effects more pronounced 24 h than 6 h after treatment.