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.

Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and has been a key driver in the catastrophic decline of amphibians globally. While many strategies have been proposed to mitigate Bd outbreaks, few have been successful. In recent years, the use of probiotic formulations that protect an amphibian host by killing or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy. The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics (hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates. We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically, 90.3% of LABs exhibited hydrophilic properties that may promote adhesion to the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus, Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus. Compatibility assays performed with cLAB and the 16 species described above indicate that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation.

Use of polyclonal antibodies, electron microscopy and histopathology to detect iridovirus-like particles in bullfrogs / Uso de anticorpos policlonais, microscopia eletrônica e histologia para detecção de partículas semelhantes ao iridovírus em rãs-touro

Iridoviruses of the Ranavirus genus have been implicated in the decline in amphibians worldwide, capable of affecting animals both in the wild and in captivity. This study aimed to detect iridovirus-like particles from three frog farms in southeastern Brazil using primary polyclonal antibodies, transmission electron microscopy (TEM) and histologic findings. The target organs were liver and kidneys. Sixty adults and sixty tadpoles of bullfrogs (Lithobates catesbeianus) were used in the study. TEM revealed the presence of iridovirus-like particles in hepatic tissue using the negative staining technique. Positive results were also observed by immunoelectron microscopy and immunocytochemistry (ICC). The histological analysis of the samples showed liver hemorrhage and corpuscles inclusion in hepatocytes as well as glomerulotubular degeneration and necrosis in the kidneys. The methods used in this study were highly efficient to detect the presence of iridovirus-like particles and possible infection of ranavirus.

Os iridovirus do gênero Ranavirus têm sido implicados no declínio dos anfíbios em todo o mundo afetando animais de vida livre e aqueles em cativeiro. O objetivo deste estudo foi detectar a presença de partículas semelhantes ao iridovírus em três ranários na região sudeste do Brasil, utilizando anticorpos policlonais primários, microscopia eletrônica de transmissão (MET) e achados histológicos. Os órgãos alvo foram o fígado e os rins. Sessenta rãs-touro adultas (Lithobates catesbeianus) e sessenta girinos da mesma espécie foram usados para o estudo. A MET revelou a presença de partículas semelhantes ao iridovírus em tecido hepático utilizando a técnica de contrastação negativa. Os resultados positivos foram também observados por imunomicroscopia eletrônica e imunocitoquímica. As análises histológicas nas mesmas amostras evidenciaram hemorragia no fígado e corpúsculos de inclusão em hepatócitos, e degeneração glomerulotubular e necrose nos rins. Os métodos usados neste estudo foram altamente eficientes na detecção de partículas semelhantes ao iridovírus e possivel infecção por ranavirus.

Use of polyclonal antibodies, electron microscopy and histopathology to detect iridovirus-like particles in bullfrogs / Uso de anticorpos policlonais, microscopia eletrônica e histologia para detecção de partículas semelhantes ao iridovírus em rãs-touro

Iridoviruses of the Ranavirus genus have been implicated in the decline in amphibians worldwide, capable of affecting animals both in the wild and in captivity. This study aimed to detect iridovirus-like particles from three frog farms in southeastern Brazil using primary polyclonal antibodies, transmission electron microscopy (TEM) and histologic findings. The target organs were liver and kidneys. Sixty adults and sixty tadpoles of bullfrogs (Lithobates catesbeianus) were used in the study. TEM revealed the presence of iridovirus-like particles in hepatic tissue using the negative staining technique. Positive results were also observed by immunoelectron microscopy and immunocytochemistry (ICC). The histological analysis of the samples showed liver hemorrhage and corpuscles inclusion in hepatocytes as well as glomerulotubular degeneration and necrosis in the kidneys. The methods used in this study were highly efficient to detect the presence of iridovirus-like particles and possible infection of ranavirus.(AU)

Os iridovirus do gênero Ranavirus têm sido implicados no declínio dos anfíbios em todo o mundo afetando animais de vida livre e aqueles em cativeiro. O objetivo deste estudo foi detectar a presença de partículas semelhantes ao iridovírus em três ranários na região sudeste do Brasil, utilizando anticorpos policlonais primários, microscopia eletrônica de transmissão (MET) e achados histológicos. Os órgãos alvo foram o fígado e os rins. Sessenta rãs-touro adultas (Lithobates catesbeianus) e sessenta girinos da mesma espécie foram usados para o estudo. A MET revelou a presença de partículas semelhantes ao iridovírus em tecido hepático utilizando a técnica de contrastação negativa. Os resultados positivos foram também observados por imunomicroscopia eletrônica e imunocitoquímica. As análises histológicas nas mesmas amostras evidenciaram hemorragia no fígado e corpúsculos de inclusão em hepatócitos, e degeneração glomerulotubular e necrose nos rins. Os métodos usados neste estudo foram altamente eficientes na detecção de partículas semelhantes ao iridovírus e possivel infecção por ranavirus.(AU)

Vitamin C supplementation has no effect on American bullfrog's immune response.

American bullfrog (Lithobates catesbeianus) is the only species raised in Brazilian commercial frog farms, and the intensive culture of these animals has gained great popularity in Brazil over the past several years. Stress is one of the major obstacles in frog culture. To minimise this problem, the aim of the present study was to investigate the role of added vitamin C in the diet of American bullfrogs as an antistress factor through the assessment of plasma corticosterone, leucocyte levels and macrophage activation in animals raised in confinement. The experimental design was entirely randomised, with six treatments (supplementation of 0, 250, 500, 750, 1000 and 2000 mg of vitamin C/kg of feed) and four replications. The plasma corticosterone level, leucocyte level and macrophage activation were evaluated. It was concluded that vitamin C had no influence on the evaluated parameters due to the possible adaptation of the animals to life in captivity (domestication). The results of this research indicate that farmers should not increase the levels of vitamin C in commercial feeds because this would only enhance production costs.

Somatostatin-, calcitonin gene-related peptide, and gamma-aminobutyric acid-like immunoreactivitity in the frog lumbosacral spinal cord: distribution and effects of sciatic nerve transection.

Using immunohistochemistry and optical densitometry, somatostatin (SOM), calcitonin gene-related peptide (CGRP), and gamma-aminobutyric acid (GABA) were investigated in the lumbosacral spinal cord of the frog Rana catesbeiana after sciatic nerve transection. In control animals, the densest network of the SOM-, CGRP- and GABA-like immunoreactive fibers was located in the dorsal part of the lateral funiculus. SOM and GABA-like fibers were found in the dorsal terminal field and in the mediolateral band. The latter region showed CGRP and SOM-like immunoreactive cell bodies. SOM- and GABA-like immunoreactive neurons also occurred around the cavity of the central canal, and other GABA-like fibers were found in the ventral terminal field. While the ventral horn showed scarce somatostatin-like fibers, the putative motoneurons were immunoreactive for the two peptides investigated and GABA, but only a few SOM- and GABA-like fibers occurred in the ventral funiculus. After axotomy, GABA-like immunoreactivity decreased in the dorsal part of the lateral funiculus on the same side of the lesion. The other regions remained labeled. These changes were observed at 3 days following axonal injury and persisted at 5, 8 and 15 days. There was no significant difference in the pattern of CGRP- and SOM- immunoreactivity between the axotomized and the control sides. These results are discussed in relation to the effects of the peripheral axotomy on GABA, SOM, and CGRP expression in vertebrates, emphasizing the use of frogs as a model to study the effects of peripheral nerve injury.