The effects of probiotic-based additives on aflatoxin intoxication in Piaractus mesopotamicus: a study of liver histology and metabolic performance.

Mycotoxins, produced by fungi, can contaminate fish food and harm their health. Probiotics enhance immune balance and primarily function in the animal intestine. This study aimed to assess aflatoxin's impact on Piaractus mesopotamicus and explore probiotic-based additive (PBA) benefits in mitigating these effects, focusing on antioxidant activity, biochemical indices, and hepatic histopathology. Two experiments were conducted using P. mesopotamicus fry. The first experimental assay tested various levels of aflatoxin B1 (0.0, 25.0, 50.0, 100.0, 200.0, and 400.0 µg kg-1) over a 10-day period. The second experimental assay examined the efficacy of the probiotic (supplemented at 0.20%) in diets with different levels of aflatoxin B1 (0.0, 25.0, and 400.0 µg kg-1) for 15 days. At the end of each assay, the fish underwent a 24-hour fasting period, and the survival rate was recorded. Six liver specimens from each treatment group were randomly selected for metabolic indicator assays, including superoxide dismutase, catalase, alanine aminotransferase, aspartate aminotransferase, and albumin. Additionally, histopathological analysis was performed on six specimens. The initial study discovered that inclusion rates above 25.0 µg kg-1 resulted in decreased activity of AST (aspartate aminotransferase), ALT (alanine aminotransferase), ALB (albumin), CAT (catalase), and SOD (superoxide dismutase), accompanied by liver histopathological lesions. In the second study, the inclusion of PBA in diets contaminated with AFB1 improved the activity of AST and ALT up to 25.0 µg kg-1 of AFB1, with no histopathological lesions observed. The study demonstrated the hepatoprotective effects of PBA in diets contaminated with AFB1. The enzyme activity and hepatic histopathology were maintained, indicating a reduction in damage caused by high concentrations of AFB1 (400.0 µg kg-1 of AFB1). The adverse effects of AFB1 on biochemical and histopathological parameters were observed from 25.0 µg kg-1 onwards. Notably, PBA supplementation enhanced enzymatic activity at a concentration of 25 µg kg-1 of AFB1 and mitigated the effects at 400.0 µg kg-1 of AFB1. The use of PBAs in pacu diets is highly recommended as they effectively neutralize the toxic effects of AFB1 when added to diets containing 25.0 µg kg-1 AFB1. Dietary inclusion of aflatoxin B1 at a concentration of 25.0 µg kg-1 adversely affects the liver of Piaractus mesopotamicus (Pacu). However, the addition of a probiotic-based additive (PBA) to the diets containing this concentration of aflatoxin neutralized its toxic effects. Therefore, the study recommends the use of PBAs in Pacu diets to mitigate the adverse effects of aflatoxin contamination.

Antifungal activity and genomic characterization of the biocontrol agent Bacillus velezensis CMRP 4489.

The development of bio-based products has increased in recent years, and species of the Bacillus genus have been widely used for product development due to their elevated production of antimicrobial molecules and resistance to extreme environmental conditions through endospore formation. In this context, the antifungal potential of Bacillus velezensis CMRP 4489 was investigated using in silico predictions of secondary metabolites in its genome and in vitro tests against the following phytopathogenic fungi: Sclerotinia sclerotiorum, Macrophomina phaseolina, and Botrytis cinerea. The in-silico predictions indicated that CMRP 4489 possesses several Biosynthetic Gene Clusters (BGCs) capable of producing molecules with antifungal properties and other non-identified BGCs. The in vitro assay results evidenced strong antifungal activity, inhibiting more than 60% of the tested fungi, and the isolate's molecules were stable under diverse physicochemical conditions. The in vitro assay evidenced significant antifungal activity, deformation of the hyphal structure in SS, biofilm formation capacity, and swarming motility. In the colonization assay, we observed attachment, colonization, and net-shaped biofilm formation, with the strain transitioning from the seeds to nearby structures. Therefore, CMRP 4489 showed to be a potential biocontrol agent against various diseases with agronomic importance and can be used under adverse environmental conditions.

Presence of antibodies against Mycobacterium avium subspecies paratuberculosis in Brazilian high-producing dairy herds.

This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Komagataeibacter intermedius V-05: An Acetic Acid Bacterium Isolated from Vinegar Industry, with High Capacity for Bacterial Cellulose Production in Soybean Molasses Medium.

RESEARCH BACKGROUND: Despite the great properties of bacterial cellulose, its manufacture is still limited due to difficulties in large-scale production. These problems are mainly related to low production yields and high overall costs of the conventional culture media normally used. To surpass these problems, it is necessary to identify new cheap and sustainable carbon sources. Thus, this work aims to isolate and select a high cellulose-producing Komagataeibacter strain from vinegar industry, and study its potential for bacterial cellulose synthesis in an industrial soybean co-product, known as soybean molasses, used as fermentation medium. EXPERIMENTAL APPROACH: One isolated strain was able to produce high amount of cellulose in the standard Hestrin-Schramm medium, so we tested its ability to produce this biopolymer in a soybean molasses medium. The characteristics and properties of the produced bacterial cellulose membranes were analyzed by thermogravimetric analysis, X-ray diffraction, infrared spectroscopy, water-holding capacity and rehydration ratio. Genetic analysis of the selected strain served to determine its genus and species. RESULTS AND CONCLUSIONS: An isolated strain that produced the highest amount of cellulose in Hestrin-Schramm medium (3.7 g/L) was genetically identified as Komagataeibacter intermedius V-05. This strain produced 10.0 g/L of cellulose in soybean molasses medium. Membranes from both substrates had similar chemical structure, crystallinity and thermal degradation. Soybean molasses proved to be a suitable alternative medium for biosynthesis of cellulose in comparison with the standard medium. In addition to providing higher production yield, the membranes showed great structural characteristics, similar to those obtained from standard medium. NOVELTY AND SCIENTIFIC CONTRIBUTION: In this research, we have isolated and identified a Komagataeibacter strain which exhibits a high capacity for cellulose production in soybean molasses. The isolation and selection of strains with high capacity for microbial metabolite production is important for decreasing bioprocess costs. Furthermore, as there is a necessity today to find cheaper carbon sources to obtain microbial products at a lower cost, soybean molasses represents an interesting alternative medium to produce bacterial cellulose for its industrial application.

Phenotypic characteristics and transcriptome profile of Cryptococcus gattii biofilm.

In this study, we characterized Cryptococcus gattii biofilm formation in vitro. There was an increase in the density of metabolically active sessile cells up to 72 h of biofilm formation on polystyrene and glass surfaces. Scanning electron microscopy and confocal laser scanning microscopy analysis revealed that in the early stage of biofilm formation, yeast cells adhered to the abiotic surface as a monolayer. After 12 h, extracellular fibrils were observed projecting from C. gattii cells, connecting the yeast cells to each other and to the abiotic surface; mature biofilm consisted of a dense network of cells deeply encased in an extracellular polymeric matrix. These features were also observed in biofilms formed on polyvinyl chloride and silicone catheter surfaces. We used RNA-Seq-based transcriptome analysis to identify changes in gene expression associated with C. gattii biofilm at 48 h compared to the free-floating planktonic cells. Differential expression analysis showed that 97 and 224 transcripts were up-regulated and down-regulated in biofilm, respectively. Among the biological processes, the highest enriched term showed that the transcripts were associated with cellular metabolic processes, macromolecule biosynthetic processes and translation.

Sylimarin as hepatic protector and immunomodulator in Nile tilapia during Streptococcus agalactiae infection.

This study investigated the use of silymarin, an extract obtained from the milk thistle (Silybum marianum) and its effects as a possible hepatoprotector in Nile tilapia (Oreochromis niloticus). Silymarin was used as feed additive to the diet at a concentration of 0.1% (1 kg per ton of dry ration) with the commercial product named Di-Heptarine S® (16% silymarin phosphatide). A total of 90 juvenile tilapia with approximately 45 days old and mean weight of 0.72 ±â€¯0.04 g were distributed in two groups, one fed with a diet with the hepatoprotector and the other without the additive. At the end of the assay (55 days after feeding), samples of blood were collected for hematological, immunological, histological (liver, spleen and intestine) and enzymatic analysis such as superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). After 55 days all fish were challenged with Streptococcus agalactiae serotype Ib to verify the sylimarin effects on the immunological parameters and its protection effect while challenged. During the challenge period another biological material sample was collected for hematological, immunological and histopathological analysis (liver, spleen and intestine). Before the challenge, an increase on the count of thrombocyte was found in the supplemented fish. In the liver, dilation of the sinusoids was observed in unsupplemented fish while supplemented fish the alteration was less severe. No significant alteration was found in SOD, CAT and GST between the groups. Histological changes after the challenge were provoked by bacterial toxins as a result of inflammatory processes. Periacinar degeneration was less intense in unsupplemented fish when compared to supplemented fish. On the other hand, eosinophilic and lymphocytic infiltrate did occur in unsupplemented fish differently from supplemented fish which did not show the alteration. The survival was 28% higher in silymarin supplemented fish when compared to unsupplemented fish that presented no survival. Silymarin supplementation in the diet provided a hepatoprotective and immunomodulatory effect on Nile tilapia.