Laboratory-controlled challenges of streptococcosis in Nile tilapia using the oral route (infected-feed) for infection.

The study aimed to evaluate the oral infected-feed, intragastric-gavage, and intraperitoneal routes of the Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus). For this purpose, 270 juveniles of Nile tilapia, with an average weight of 2 g, were distributed in 18 experimental units of 90 L, acclimatized, and raised for 55 days, until reaching 50 g (median weight). The experimental design was entirely randomized in six treatments, three of which were composed by bacterial infection routes: intraperitoneal 100 µL fish-1 [108 CFU], intragastric 100 µL fish-1 [108 CFU], feed inoculum 100 µL g feed-1 [109 CFU], and three respective control groups. Clinical signs were observed, and mortalities monitored until reaching 50% in the infected groups. Then, tissue samples from the spleen, liver, intestine, brain, and blood were collected from 20 fish per treatment for histopathological and hemato-immunological analyses. In addition, a related mortality curve was established at the end of the experimental challenge. The intraperitoneal and intragastric routes were more aggressive than the oral inoculum, causing greater brain damage, acute hemato-immunological response, and early mortality. While the orally fed inoculum, fish presented brain lesions with less intensity, and a chronic haemato-immunological response, the mortalities occurred twice as long as the other routes. The present research demonstrated that the S. agalactiae oral (feed inoculum) administration can be an innovative methodology to future experimental challenges in aquaculture research.

Toxic effects in Aphanomyces brasiliensis and zebrafish embryos caused by oomyceticides.

In fish farming, high losses occur during egg incubation and larviculture due to diseases caused by oomycetes. This study aimed to identify the oomycete species that occurs in zebrafish Danio rerio eggs and to evaluate the oomyceticidal effect of copper sulfate, bronopol and methylene blue on the mycelial growth of this organism, as well as to determine the lethal and sublethal toxicity of these compounds in embryos of D. rerio. The isolates were cultivated in yeast-starch medium to determine the concentration necessary to inhibit mycelial growth by 50% (IC50) and 100% (minimum oomyceticidal concentration) after a 96 h exposure to these compounds. In addition, tests with D. rerio eggs were conducted to determine the lethal concentrations for 50% of the organisms (96h-LC50), and the concentrations that inhibited 17% of the eggs hatching (96h-IC17) after 96 h. The organism responsible for the mortality of D. rerio eggs was classified by classical and molecular methods as Aphanomyces brasiliensis, representing the first report of this pathogen in zebrafish eggs. IC50 values could be determined for both bronopol and copper sulfate, whereas methylene blue had low effectiveness against the oomycete. Copper showed high toxicity to D. rerio eggs at low concentrations, while methylene blue and bronopol toxicity was low and similar to each other. The use of bronopol at a concentration of 4.8 mg l-1 for the treatment of zebrafish eggs allows controlling the pathology without causing deleterious effects to the treated organisms.

Two epizootic Perkinsus spp. events in commercial oyster farms at Santa Catarina, Brazil.

Perkinsus spp. have been detected in various bivalve species from north-east Brazil. Santa Catarina is a South Brasil state with the highest national oyster production. Considering the pathogenicity of some Perkinsus spp., a study was carried out to survey perkinsosis in two oyster species cultured in this State, the mangrove oyster Crassostrea gasar and the Pacific oyster Crassostrea gigas. Sampling involved eight sites along the state coast, and oyster sampling was collected during the period between January 2013 and December 2014. For the detection of Perkinsus, Ray's fluid thioglycollate medium (RFTM) and histology were used, and for the identification of the species, PCR and DNA sequencing were used. Perkinsus spp. was found by RFTM in C. gigas and C. gasar from São Francisco do Sul. This pathology was also detected in C. gasar from Balneário Barra do Sul both, by RFTM and histology. Perkinsus marinus was identified in C. gigas and C. gasar from São Francisco do Sul and Perkinsus beihaiensis in C. gasar from Balneário Barra do Sul. This is the first report of P. marinus in C. gigas from South America. Results of this preliminary study suggest that both oyster species tolerate the species of Perkinsus identified, without suffering heavy lesions.

Fish pathogen bacteria: Adhesion, parameters influencing virulence and interaction with host cells.

Wild fisheries are declining due to over-fishing, climate change, pollution and marine habitat destructions among other factors, and, concomitantly, aquaculture is increasing significantly around the world. Fish infections caused by pathogenic bacteria are quite common in aquaculture, although their seriousness depends on the season. Drug-supplemented feeds are often used to keep farmed fish free from the diseases caused by such bacteria. However, given that bacteria can survive well in aquatic environments independently of their hosts, bacterial diseases have become major impediments to aquaculture development. On the other hand, the indiscriminate uses of antimicrobial agents has led to resistant strains and the need to switch to other antibiotics, although it seems that an integrated approach that considers not only the pathogen but also the host and the environment will be the most effective method in the long-term to improve aquatic animal health. This review covers the mechanisms of bacterial pathogenicity and details the foundations underlying the interactions occurring between pathogenic bacteria and the fish host in the aquatic environment, as well as the factors that influence virulence. Understanding and linking the different phenomena that occur from adhesion to colonization of the host will offer novel and useful means to help design suitable therapeutic strategies for disease prevention and treatment.

Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities.

An 84-day feeding trial was conducted to evaluate the effect of the dietary administration of Bacillus subtilis on the growth performance, body composition, intestinal probiotic recovery, hematology, and non-specific immunity of Nile tilapia (Oreochromis niloticus) raised at two stocking densities. Five hundred twenty male Nile tilapias (32.63 ± 1.25 g) were distributed in 16,800-L tanks. The experimental design was completely randomized using four replications and a 2 × 2 factorial scheme with two stocking densities (18.75 fish m(-3) 62.50 fish m(-3)) and two diets (control and with probiotic). The probiotic-supplemented diet included 5 × 10(6) CFU g feed(-1). There were no significant differences (P > 0.05) in the growth performance, body composition, and levels of cortisol and glucose between the animals fed with the control diet and the animals fed with the probiotic-supplemented diet. Differences in the growth performance were observed between the fish reared at different stocking densities; in particular, the fish raised at the high stocking density exhibited reduced weight gain, feed intake, and specific growth rate compared with those raised at the low stocking density. The B. subtilis remained viable after its inclusion in the feed, storage, and passage through the stomach, which demonstrations the feasibility of using this bacteria as a probiotic. Higher values (P < 0.05) in the plasma lysozyme levels and phagocytic activity were observed in the fish that received the probiotic-supplemented diet and reared at the high stocking density, but this difference was not observed in the fish raised at the low stocking density and fed the different diets. The administration of the probiotic caused decreases in the number of erythrocytes and the hematocrit level in the fish reared at the high stocking density, but these erythrocytes showed higher values of mean corpuscular hemoglobin. The stocking density was shown to be a stressor agent that causes a lower fish growth rate. The fish fed the control diet and raised at the high stocking density had lower lysozyme levels than the fish subjected to the other treatments. The inclusion of the probiotic bacteria B. subtilis at a concentration of 5 × 10(6) CFU g feed(-1) benefited the innate immune system of Nile tilapia by decreasing the stress associated with exposure to a high stocking density, increasing the mean corpuscular hemoglobin, and improving the innate immune system (lysozyme and phagocytic activities of macrophages).

Symbiotic microparticles produced through spray-drying-induced in situ alginate crosslinking for the preservation of Pediococcus pentosaceus viability.

Probiotic microorganisms are a promising alternative to antibiotics in preventing and treating bacterial infections. Within the probiotic group, the lactic acid bacteria (LAB)stand out for their health benefits and for being recognized as safe by regulatory agencies. However, these microorganisms are sensitive to various environmental conditions, including the acidic environment of the stomach. Faced with these obstacles, this work aimed to promote the symbiotic microencapsulation of LAB in a composite matrix of alginate and prebiotics to enhance their survival and improve their probiotic activity during gastrointestinal transit. We evaluated the effect of inulin, fructo-oligosaccharides (FOS) and mannan-oligosaccharides (MOS) as prebiotic sources on the growth of Pediococcus pentosaceus LBM34 strain, finding that MOS favored LAB growth and maintenance of microencapsulated cell viability. The symbiotic microparticles were produced using the spray-drying technique with an average size of 10 µm, a smooth surface, and a composition that favored the stabilization of live cells according to the FTIR and the thermal analysis of the material. The best formulation was composed of 1 % of alginate, 10 % MOS and 1 % M10 (% w/v), which presented notable increases in the survival rates of the probiotic strain in both alkaline and acidic conditions. Therefore, this industrially scalable approach to symbiotic LAB microencapsulation can facilitate their growth and colonization within the host. This effort aims to contribute to reducing antibiotic reliance and mitigating the emergence of new zoonotic diseases, which pose significant challenges to public health.

Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Probiotic supplementation causes hematological changes and improves non-specific immunity in Brycon amazonicus

A commercialprobioticcontaining Bacillussubtilis(109 CFU g-1) was evaluated incaged matrinxã,Bryconamazonicus,by measuring hematological parametersand macrophage activity after 42 and 84 days after feeding. The product wasadded tocommercial feed using 2%soybean oil as a protectant. A randomized three-treatmentexperiment was performed using fourreplicates per treatment. The groups included: (a) control without probiotic, (b) 5 g kg-1probiotic, and (c) 10 g kg-1probiotic. Forhematological analysis,eightfishper treatmentwere used to determinetotal cell count (RBC); thrombocytes, differential, and total leukocyte count (TLC); hematocrit (Htc); hemoglobin tax; mean corpuscular volume (MCV); and mean corpuscular hemoglobin concentration (MCHC). Furthermore, plasma cortisol and glucose levels were measured in blood samples. Macrophage phagocytic activity was evaluated by injecting Saccharomyces cerevisiae(11,000 cells in a 3 mLvolume) into the coelomic cavity incubating for 8hours.Addition of probiotics to the diet of caged matrinxã altered the Htc, RBC, MCV, MCHC, TLC, lymphocyte, and eosinophil values. We observed increased cortisol and glucose levels and phagocytic activity, but no increase in the phagocytic index. We thus conclude that supplementing caged Brycon amazonicuswith probiotics improves their non-specific immunity and alters blood profiles.

Tempo de migração dos macrófagos em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae / Macrophagic migration time in matrinxã, Brycon amazonicus, through inoculation of yeast, Saccharomyces cerevisiae, technique

Na aquicultura são utilizados análises da ativação e incremento da migração de macrófagos, com intuito de verificar a capacidade imunológica inespecífica dos peixes frente a um desafio. Neste sentido, o objetivo deste estudo foi determinar o tempo de migração de monócitos/macrófagos para a cavidade peritoneal em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae, e verificar as possíveis alterações dos parâmetros hematológicos após o estímulo. Foram utilizados 30 matrinxãs com peso médio de 101,55 ± 24,50 g e comprimento médio de 19,75 ± 1,72 cm. Os tempos de inoculação utilizados foram 2, 4, 8 e 12 horas, sendo utilizados 6 animais por tempo. Após os períodos de incubação (2, 4, 8 e 12 horas), os exemplares foram anestesiados e alíquotas de sangue foram coletadas por punção do vaso caudal, para a análise: número total de células, contagem diferencial e total dos leucócitos e contagem total de trombócitos, hematócrito, taxa de hemoglobina e índices hematimétricos (VCM, HCM e CHCM). Os resultados mostram que a capacidade fagocítica do macrófago não apresentou diferenças significativas entre os tempos experimentais. Com relação ao índice fagocítico, o tempo de 2 horas representa o tempo em que os macrófagos fagocitaram maior número de leveduras com diferenças significativas em relação aos outros tempos experimentais, indicando que este tempo (2 horas) de incubação foi suficiente para a migração e ativação máxima dos macrófagos da cavidade peritoneal, da espécie estudada. Os valores do número de eritrócitos apresentaram diferenças entre os tempos de incubação. Entretanto, os valores dos outros parâmetros hematológicos não apresentaram diferenças significativas.

In aquaculture, analysis of activation and increased macrophages migration are used in order to verify the ability of the nonspecific immune fish exposed to a challenge. This study aimed to determine the time of macrophages migration in matrinxã, Brycon amazonicus, through the technique of yeast Saccharomyces cerevisiae inoculation, verifying possible changes in hematological parameters. Thirty animals with average weight of 101.55 ± 24.50 g and average length of 19.75 ± 1.72 cm were employed. The experimental inoculation periods were 2, 4, 8 and 12 hours. Thereafter, animals were anesthetized and blood was withdrawn through a caudal puncture for the determination of total erythrocytes number, differential and total leukocyte counts and total thrombocytes count, hematocrit, hemoglobin concentration and calculation of the erythrocytes index. The results for the phagocytic capacity were not significantly different between experimental periods. In the phagocytic index, the period of 2 hours presented the highest rate of phagocytized cells, indicating that 2 hours of incubation was sufficient for the macrophages migration in B. amazonicus. The number of erythrocyte was the only parameter that presented significant difference among periods.

Tempo de migração dos macrófagos em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae

In aquaculture, analysis of activation and increased macrophages migration are used in order to verify the ability of the nonspecific immune fish exposed to a challenge. This study aimed to determine the time of macrophages migration in matrinxã, Brycon amazonicus, through the technique of yeast Saccharomyces cerevisiae inoculation, verifying possible changes in hematological parameters. Thirty animals with average weight of 101.55 ± 24.50 g and average length of 19.75 ± 1.72 cm were employed. The experimental inoculation periods were 2, 4, 8 and 12 hours. Thereafter, animals were anesthetized and blood was withdrawn through a caudal puncture for the determination of total erythrocytes number, differential and total leukocyte counts and total thrombocytes count, hematocrit, hemoglobin concentration and calculation of the erythrocytes index. The results for the phagocytic capacity were not significantly different between experimental periods. In the phagocytic index, the period of 2 hours presented the highest rate of phagocytized cells, indicating that 2 hours of incubation was sufficient for the macrophages migration in B. amazonicus. The number of erythrocyte was the only parameter that presented significant difference among periods.

Na aquicultura são utilizados análises da ativação e incremento da migração de macrófagos, com intuito de verificar a capacidade imunológica inespecífica dos peixes frente a um desafio. Neste sentido, o objetivo deste estudo foi determinar o tempo de migração de monócitos/macrófagos para a cavidade peritoneal em matrinxã, Brycon amazonicus, por meio da técnica de inoculação de leveduras Saccharomyces cerevisiae, e verificar as possíveis alterações dos parâmetros hematológicos após o estímulo. Foram utilizados 30 matrinxãs com peso médio de 101,55 ± 24,50 g e comprimento médio de 19,75 ± 1,72 cm. Os tempos de inoculação utilizados foram 2, 4, 8 e 12 horas, sendo utilizados 6 animais por tempo. Após os períodos de incubação (2, 4, 8 e 12 horas), os exemplares foram anestesiados e alíquotas de sangue foram coletadas por punção do vaso caudal, para a análise: número total de células, contagem diferencial e total dos leucócitos e contagem total de trombócitos, hematócrito, taxa de hemoglobina e índices hematimétricos (VCM, HCM e CHCM). Os resultados mostram que a capacidade fagocítica do macrófago não apresentou diferenças significativas entre os tempos experimentais. Com relação ao índice fagocítico, o tempo de 2 horas representa o tempo em que os macrófagos fagocitaram maior número de leveduras com diferenças significativas em relação aos outros tempos experimentais, indicando que este tempo (2 horas) de incubação foi suficiente para a migração e ativação máxima dos macrófagos da cavidade peritoneal, da espécie estudada. Os valores do número de eritrócitos apresentaram diferenças entre os tempos de incubação. Entretanto, os valores dos outros parâmetros hematológicos não apresentaram diferenças significativas.