Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 hours post-injection (hpi), all the other genes followed 72h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanismpublishedVersio

First report on the toxicity of SARS-CoV-2, alone and in combination with polyethylene microplastics in neotropical fish

The COVID-19 pandemic has caused unprecedented negative impacts in the modern era, including economic, social, and public health losses. On the other hand, the potential effects that the input of SARS-CoV-2 in the aquatic environment from sewage may represent on non-target organisms are not well known. In addition, it is not yet known whether the association of SARS-CoV-2 with other pollutants, such as microplastics (MPs), may further impact the aquatic biota. Thus, we aimed to evaluate the possible ecotoxicological effects of exposure of male adults Poecilia reticulata, for 15 days, to inactivated SARS-CoV-2 (0.742 pg/L; isolated SARS.CoV2/SP02.2020.HIAE.Br) and polyethylene MP (PE MPs) (7.1 × 104 particles/L), alone and in combination, from multiple biomarkers. Our data suggest that exposure to SARS-CoV-2 induced behavioral changes (in the open field test), nephrotoxic effect (inferred by the increase in creatinine), hepatotoxic effect (inferred by the increase in bilirubin production), imbalance in the homeostasis of Fe, Ca, and Mg, as well as an anticholinesterase effect in the animals [marked by the reduction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity]. On the other hand, exposure to PE MPs induced a genotoxic effect (assessed by the comet assay), as well as an increase in enzyme activity alpha-amylase, alkaline phosphatase, and carboxylesterases. However, we did not show synergistic, antagonistic, or additive effects caused by the combined exposure of P. reticulata to SARS-CoV-2 and PE MPs. Principal component analysis (PCA) and values from the "Integrated Biomarker Response" index indicate that exposure to SARS-CoV-2 was determinant for a more prominent effect in the evaluated animals. Therefore, our study sheds light on the ecotoxicity of the new coronavirus in non-target organisms and ratifies the need for more attention to the impacts of COVID-19 on aquatic biota.The authors are grateful to the Goiano Federal Institute (IF Goiano/Brazil), National Council for Scientific and Technological Development (CNPq/Brazil), Foundation for Research Support of the State of Goiás (FAPEG, Brazil), and Foundation for Research Support (FUNAPE, Brazil) for the financial support needed to conduct this research. Nós agradecemos a todos os estudantes do Laboratório de Toxicologia Aplicada ao Meio Ambiente (IF Goiano/Brazil) que contribuíram na coleta de dados ao longo do experimento. Malafaia G. holds a productivity scholarship from CNPq (Proc. No. 308854/2021-7).Peer reviewe

Photobiomodulation reduces the cytokine storm syndrome associated with Covid-19 in the zebrafish model

Although the exact mechanism of the pathogenesis of COVID-19 is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red PBM as an attractive therapy to downregulate the cytokine storm caused by COVID-19 from a zebrafish model. RT-PCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that rSpike was responsible for generating systemic inflammatory processes with significantly increased pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a, coa1) mRNA markers, with a pattern like those observed in COVID-19 cases in humans. On the other hand, PBM treatment decreased the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most impacted metabolic pathways between PBM and the rSpike-treated groups were related to steroid metabolism, immune system, and lipids metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19, and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials.publishedVersio

Avaliação da atividade antiparasitária de Artemisia annua sobre Rhipicephalus (Boophilus) microplus e Haemonchus contortus, in vivo, por meio do fornecimento do material vegetal seco na alimentação animal

Os extratos vegetais têm sido importantes fontes de substâncias com diferentes estruturas químicas expressando diversas atividades deletérias contra os parasitas. A presente pesquisa teve como objetivo avaliar a ação antiparasitária de Artemisia annua in vivo. Para tal, foram propostos dois estudos: um utilizando bovinos naturalmente infestados e outro usando ovinos artificialmente infectados. Experimento I: foram selecionadas vinte novilhas com peso médio de 280 kg para avaliar a eficácia carrapaticida sobre R. (Boophilus) microplus. As mesmas foram divididas em dois grupos experimentais, um grupo que recebeu por via oral 200g/dia de A. annua em cocho coletivo e outro não recebeu. As contagens foram realizadas nos dias 0, 3, 7, 21, 35, 42, 49, 56, pós tratamento.Foi realizada a quantificação de artemisinina por cromatografia líquida de alta eficiência. Os resultados das três contagens de carrapatos com o valor referente às médias do dia foi 96,55. Nas contagens posteriores não observou-se diferença significativa entre ambos os grupos. Experimento II: Para avaliar a atividade antihelmíntica, foi conduzido um estudo controlado utilizando-se vinte e quatro ovinos machos Santa Inês com peso médio de 20 Kg artificialmente infectados com H. contortus e randomizados em quatro grupos homogêneos com seis animais cada, sendo, controle não tratado (Grupo I), Controle positivo tratado com levamizol 10 mg/kg (GII), A. annua a 10% (GIII) e A. annua a 20% (GIV). Contagens de OPG foram realizadas nos dias -3, -2, - 1, 0 (dia do tratamento), 3, 7, 10, 14, 17, 21, 24 e 28 pós-tratamento. De acordo com as quantificações estatísticas, foram observadas eficácias persistentes de 92,57% e de 85,00% no grupo controle positivo tratado com levamizol. Nota-se não houve diferença significativa entre ambos os grupos. Portanto, os resultados obtidos possibilitam inferir que A. annua administrada via...Vegetal extracts has been important sources for substances with different chemical structures that express several deleterious activities against parasites. The present study focused on evaluating the anti-parasitic action of Artemisia annua in vivo. For such, two experiments were proposed: one using naturally infested animals and a second one with artificially infected sheep. Experiment I: twenty heifers weighing around 280 kg were selected in order to evaluate the acaricidal efficacy of the compound against Rhipicephalus (Boophilus) microplus. These animals were divided in two experimental groups: one being held as a control group and the second one were animals received, orally, 200 g/day of A. annua in a collective trough on days 0, 3, 7, 21, 35, 42, 49 and 56, post-treatment. Artemisinine was quantified through high performance liquid chromatography. Results of the three tick counts, regarding the average values, was of 96.55. In subsequent counts, no significant difference was observed between both groups. Experiment II: To evaluate the anthelmintic efficacy of the compound, a controlled trial was conducted using 24 male sheep, from the “Santa Inês” breed, with an average weight of 20 Kg, artificially infected with H. contortus. These animals were randomized in four homogeneous groups with six animals each, being: untreated control (Group I); positive control, treated with levamisole at 10 mg/kg (Group II); A. annua at 10% (Group III) and A. annua at 20% (Group IV). EPG counts were conducted on days -3, -2, -1, 0 (treatment day), 3, 7, 10, 14, 17, 21, 24 and 28 post-treatment. According to statistical quantifications, persistent efficacies of 92.57% and 85.00% were observed in the positive controls, treated with levamisole. It is possible to note that no significant difference was observed between the other groups. Therefore, the obtained results allow inferring that Artemisia annua ..

Efficacy of acaricides against larvae of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) and Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae)

Parasitism by mites has always been of concern with regard to losses in animal production and to the transmission of etiologic agents of important diseases as well, and about 95% of the developing forms of ticks in the non-parasitic phase are in the environment. In view of the importance of controlling larval stages of ticks in the environment, this study evaluated the efficacy of acaricides in vitro against larvae of Rhipicephalus (B.) microplus and R. sanguineus. Accordingly, we tested: 15% cypermethrin (T1); 25% deltamethrin (T2); combination of cypermethrin 5% + 45% dichlorvos + 25% piperonyl butoxide (T3); combination of 15% cypermethrin + 25% chlorpyrifos + 1% citronella (T4); and 12.5% amitraz (T5). In each treatment, there were nine replicates with 20 larvae each. The larvae were observed for motility at: 1, 5, 10, 20 and 30 minutes post-treatment (MPT); 1, 2, 6, 12 and 24 hours post-treatment (HPT); and 5 days post-treatment. Tests in R. (B.) microplus and R. sanguineus larvae with 25% deltamethrin served as a positive control and demonstrated 100% loss of motility at 2 HPT for both tick species. R. (B.) microplus larvae were most sensitive to treatments containing 15% cypermethrin or 5% cypermethrin combined with dichlorvos and piperonyl butoxide. Larvae of R. sanguineus were most sensitive to treatment with 12.5% amitraz. However, despite the differences in acaricidal effect during the observation time, 100% efficacy was obtained for all treatments against larvae of R. sanguineus and R. (B.) microplus at 12 HPT, demonstrating that appropriate doses of commercial acaricides can be effective in environmental control strategies against tick larvae

Eficácia de diferentes formulações de acaricidas sobre larvas de Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) e Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae)

Parasitism by mites has always been of concern with regard to losses in animal production and to the transmission of etiologic agents of important diseases as well, and about 95% of the developing forms of ticks in the non-parasitic phase are in the environment. In view of the importance of controlling larval stages of ticks in the environment, this study evaluated the efficacy of acaricides in vitro against larvae of Rhipicephalus (B.) microplus and R. sanguineus. Accordingly, we tested: 15% cypermethrin (T1); 25% deltamethrin (T2); combination of cypermethrin 5% + 45% dichlorvos + 25% piperonyl butoxide (T3); combination of 15% cypermethrin + 25% chlorpyrifos + 1% citronella (T4); and 12.5% amitraz (T5). In each treatment, there were nine replicates with 20 larvae each. The larvae were observed for motility at: 1, 5, 10, 20 and 30 minutes post-treatment (MPT); 1, 2, 6, 12 and 24 hours post-treatment (HPT); and 5 days post-treatment. Tests in R. (B.) microplus and R. sanguineus larvae with 25% deltamethrin served as a positive control and demonstrated 100% loss of motility at 2 HPT for both tick species. R. (B.) microplus larvae were most sensitive to treatments containing 15% cypermethrin or 5% cypermethrin combined with dichlorvos and piperonyl butoxide. Larvae of R. sanguineus were most sensitive to treatment with 12.5% amitraz. However, despite the differences in acaricidal effect during the observation time, 100% efficacy was obtained for all treatments against larvae of R. sanguineus and R. (B.) microplus at 12 HPT, demonstrating that appropriate doses of commercial acaricides can be effective in environmental control strategies against tick larvae.O parasitismo por ácaros sempre foi objeto de preocupações e perdas relacionadas à produção animal, assim como à transmissibilidade de agentes etiológicos de importantes doenças; e aproximadamente 95% das formas evolutivas dos carrapatos em fase não parasitária encontram-se no ambiente. Com base na importância de se controlar estágios larvares dos ixodídeos no ambiente, este estudo avaliou a eficácia de acaricidas in vitro sobre larvas de Rhipicephalus (Boophilus) microplus e Rhipicephalus sanguineus. Foram testadas: Cipermetrina 15% (T1); Deltametrina 25% (T2); associação de Cipermetrina 5% + Diclorvós 45% + Butóxido de piperolina 25% (T3); associação de Cipermetrina 15% + Clorpiriphos 25% + Citronela 1% (T4) e Amitraz 12,5% (T5). Para cada tratamento foram realizadas nove repetições contendo 20 larvas. As observações de motilidade foram realizadas: 1, 5, 10, 20 e 30 minutos após o tratamento (MPT); 1, 2, 6, 12 e 24 h após o tratamento (HPT); 5 dias após o tratamento (DPT). Os testes das larvas de R. (B.) microplus e R. sanguineus com deltametrina 25% serviram de controle positivo e demonstraram perda de motilidade de 100% das larvas com duas HPT para ambas de ixodídeos. O R. (B.) microplus apresentou maior sensibilidade aos desafios contendo a cipermetrina 15% e 5% associada ao diclorvós e butóxido de piperonila. Larvas de R. sanguineus apresentaram maior sensibilidade em menor tempo ao tratamento com amitraz a 12,5%. Contudo, apesar das diferenças no efeito acaricida observadas no decorrer dos tempos, obteve-se eficácia de 100% para todos os compostos inseticidas sobre os instares das duas espécies 12 HPT, demonstrando que, quando utilizados nas doses recomendadas pelos fabricantes, os produtos comerciais podem ser eficazes em estratégias de controle ambiental das larvas

Efficacy of acaricides against larvae of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) and Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae)

Parasitism by mites has always been of concern with regard to losses in animal production and to the transmission of etiologic agents of important diseases as well, and about 95% of the developing forms of ticks in the non-parasitic phase are in the environment. In view of the importance of controlling larval stages of ticks in the environment, this study evaluated the efficacy of acaricides in vitro against larvae of Rhipicephalus (B.) microplus and R. sanguineus. Accordingly, we tested: 15% cypermethrin (T1); 25% deltamethrin (T2); combination of cypermethrin 5% + 45% dichlorvos + 25% piperonyl butoxide (T3); combination of 15% cypermethrin + 25% chlorpyrifos + 1% citronella (T4); and 12.5% amitraz (T5). In each treatment, there were nine replicates with 20 larvae each. The larvae were observed for motility at: 1, 5, 10, 20 and 30 minutes post-treatment (MPT); 1, 2, 6, 12 and 24 hours post-treatment (HPT); and 5 days post-treatment. Tests in R. (B.) microplus and R. sanguineus larvae with 25% deltamethrin served as a positive control and demonstrated 100% loss of motility at 2 HPT for both tick species. R. (B.) microplus larvae were most sensitive to treatments containing 15% cypermethrin or 5% cypermethrin combined with dichlorvos and piperonyl butoxide. Larvae of R. sanguineus were most sensitive to treatment with 12.5% amitraz. However, despite the differences in acaricidal effect during the observation time, 100% efficacy was obtained for all treatments against larvae of R. sanguineus and R. (B.) microplus at 12 HPT, demonstrating that appropriate doses of commercial acaricides can be effective in environmental control strategies against tick larvae

Dietary supplementation of Bacillus velezensis improves Vibrio anguillarum clearance in European sea bass by activating essential innate immune mechanisms

Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 hours post-injection (hpi), all the other genes followed 72h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanis

Environmental impacts of COVID-19 treatment: Toxicological evaluation of azithromycin and hydroxychloroquine in adult zebrafish.

One of the most impact issues in recent years refers to the COVID-19 pandemic, the consequences of which thousands of deaths recorded worldwide, are still inferior understood. Its impacts on the environment and aquatic biota constitute a fertile field of investigation. Thus, to predict the impact of the indiscriminate use of azithromycin (AZT) and hydroxychloroquine (HCQ) in this pandemic context, we aim to assess their toxicological risks when isolated or in combination, using zebrafish (Danio rerio) as a model system. In summary, we observed that 72 h of exposure to AZT and HCQ (alone or in binary combination, both at 2.5 μg/L) induced the reduction of total protein levels, accompanied by increased levels of thiobarbituric acid reactive substances, hydrogen peroxide, reactive oxygen species and nitrite, suggesting a REDOX imbalance and possible oxidative stress. Molecular docking analysis further supported this data by demonstrating a strong affinity of AZT and HCQ with their potential antioxidant targets (catalase and superoxide dismutase). In the protein-protein interaction network analysis, AZT showed a putative interaction with different cytochrome P450 molecules, while HCQ demonstrated interaction with caspase-3. The functional enrichment analysis also demonstrated diverse biological processes and molecular mechanisms related to the maintenance of REDOX homeostasis. Moreover, we also demonstrated an increase in the AChE activity followed by a reduction in the neuromasts of the head when zebrafish were exposed to the mixture AZT + HCQ. These data suggest a neurotoxic effect of the drugs. Altogether, our study demonstrated that short exposure to AZT, HCQ or their mixture induced physiological alterations in adult zebrafish. These effects can compromise the health of these animals, suggesting that the increase of AZT and HCQ due to COVID-19 pandemic can negatively impact freshwater ecosystems