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Streptococcus agalactiae (GBS) are Gram-positive cocci responsible for substantial losses in tilapia fish farms in Brazil and worldwide. It causes septicemia, meningoencephalitis and mortality of whole shoals that can occur within 72 h. Thus, diagnostic methods are needed that are rapid, specific and sensitive. In this study, a pair of specific primers for GBS was generated based on the cfb gene sequence and initially evaluated by conventional PCR. The protocols for absolute quantitative real-time PCR (qPCR) were then adapted to validate the technique for the identification and quantification of GBS isolated by real-time detection of amplicons using fluorescence measurements. Finally, an infectivity test was conducted in tilapia infected with GBS strains. Total DNA from the host brain was subjected to the same technique, and the strains were re-isolated to validate Koch's postulates. The assay showed 100% specificity for the other bacterial species evaluated and a sensitivity of 367 gene copies per 20 mg of brain tissue within 4 h, making this test a valuable tool for health monitoring programs. [ABSTRACT FROM AUTHOR]

Abstract Due to the lack of regulated drugs for aquaculture, the present study considered specific issues relating to environmental and food safety aspects concerning the potential use of emamectin benzoate (EMA) in freshwater fishes such as tilapia (Oreochromis niloticus) – an important commercial fish species worldwide. The residual depletion of EMA (EMA-B 1a) in fillet (muscle plus skin in natural proportions) of tilapia treated with a daily dose of 50 μg/kg BW during seven consecutive days was evaluated. To facilitate this, analytical methods for quantitation of EMA in fish feed and in fish fillet employing LC-MS/MS were developed and validated. To eliminate the risk of EMA leaching from feed into the aquatic environment during fish medication via oral administration, a promising procedure for drug incorporation into feed involving the coating of feed pellets with ethyl cellulose polymer containing EMA was evaluated. The medicated feed exhibited good homogeneity (CV < 2.1%) with negligible EMA release (< LOQ) when the medicated feed remained in the water for up to 20 min. Depletion study analysis revealed the highest EMA concentration obtained in fish fillet to be 13.3 ng/g. Therefore, under the employed rearing conditions of this study, the obtained results did not evidence requirement for a minimum withdrawal period to be proposed considering the maximum residue limit of 100 μg/g for fish muscle. In response to the well-recognized demands and need for new alternative veterinary drugs for use within aquaculture, this study offers impetus for consideration of EMA use in tilapia taking into account environmental contamination and food safety issues. Graphical abstract Unlabelled Image Highlights • Alternative procedure is proposed for drug incorporation in fish feed. • Simple and feasible analytical methods were developed for analysis in feed and in fish fillet. • Depletion study of EMA residues in tilapia fillets was executed. • Evaluation and discussion on the need to propose a minimum withdrawal period was considered taking account food safety issues. [ABSTRACT FROM AUTHOR]

• An original bioanalytical method for erythromycin residues analysis in fish was developed, optimized, and validated. • The combination of two modern extraction techniques promoted high detectability. • The method was successfully applied in real samples to evaluate residual depletion of erythromycin in fish. An analytical method for the determination of erythromycin A (ERY) residues in fish fillet was developed, optimized, and validated employing a modified QuEChERS procedure associated to DLLME technique as a preconcentration step. The obtained LOD and the LOQ were 0.1 µg kg−1 and 1 µg kg−1, respectively. The validated method provides linearity in the range of 1 to 20 µg kg−1, precision (CV < 6.3 %) and accuracy (recovery ranging from 103 to 110 %). The procedure was applied in an experimental study to evaluate the residual depletion profile of ERY in fish (Piaractus mesopotamicus) after oral administration. The treatment was carried out at a daily dose of 100 mg (kg BW)-1 of ERY, for 7 consecutive days and with an average water temperature of 30 °C. A withdrawal time of 240°-day was estimated for eliminating ERY residues at concentration levels below the maximum residue limit considered (MRL 100 µg kg−1). [ABSTRACT FROM AUTHOR]

The utilization of probiotic bacteria have been widely tested and applied in intestinal microflora modulation, through competitive exclusion of the pathogenic bacteria. Aeromonas hydrophila is a gram-negative bacterium, responsible for fish outbreaks in farms around the world, and is one of the major loss causes for neotropical fish farmers. 660 pacus (67 ± 7 g) were distributed in 20 tanks ( n = 33), constituting five groups (four tanks for each treatment): four groups were fed with different levels (2, 4, 8 and 16 g kg −1 ) of Bacillus cereus and Bacillus subtilis (1:1, 10 8 CFU g −1 ), and the fifth group was fed with a control diet (without probiotic). Pacus fed with probiotic showed increment in the ROS production associated to elevated neutrophil and monocyte counts and increased phagocytic activity without affecting the growth parameters. Probiotic fed fish presented higher survival rates, subjected to an i.p. challenge with 10 8 CFU mL −1 of A. hydrophila . The results demonstrated a dose response effect and the ideal level of the probiotic ( Bacillus cereus and Bacillus subtilis , 1:1, 10 8 CFU g −1 ) in P. mesopotamicus diets was around 8 g kg −1 , as the highest survival rates and immunological responses were found in groups of fish fed with this diet. In addition, too much probiotic should be avoided, as pacus fed diet with 16 g kg −1 showed partial suppression of these responses. [ABSTRACT FROM AUTHOR]

A 60-day feeding trial was conducted to assess the effects of dietary Saccharomyces cerevisiae -originated free nucleotides on growth, innate immunity, intestinal histomorphometry and disease resistance in Nile tilapia, Oreochromis niloticus (average initial weight of 7.85 g). Five diets (treatments) were prepared: negative control diet (NCD) - basal diet without additives; positive control diet (PCD) - basal diet supplemented with a commercial nucleotide-based additive at 170 ppm; and three experimental diets, N170, N320 and N470, supplemented with S. cerevisiae -originated free nucleotides at 170, 320 and 470 ppm, respectively. There were no significant differences in production parameters after the trial period. Nevertheless, the complement hemolytic activity was higher in fish subjected to the three experimental diets, while serum lysozyme concentration was higher in the N470 group compared with both controls. Intestinal villi height and density were greater in fish fed the N470 diet compared with the NCD group. In addition, fish receiving the three experimental diets showed higher absorption surface area magnification ratio, with the greatest values being observed in the N470 group. Lastly, the highest survival rate following Aeromonas hydrophila challenge was recorded in the N470 group. Taken together, the present findings indicate that supplementation of feed with S. cerevisiae -originated free nucleotides at 470 ppm improves non-specific immune responses and intestinal microstructure morphology as well as increases survival rate after bacterial infection in Nile tilapia. Therefore, this blend of natural nucleotides has the potential of being used as an alternative to antimicrobials when included as an additive in aquaculture feeds. • Diets supplemented with Saccharomyces cerevisiae -originated free nucleotides were examined in Nile tilapia. • S. cerevisiae -originated free nucleotides promoted improvements in immunity and intestinal morphology in Nile tilapia. • Diets supplemented with S. cerevisiae -originated free nucleotides increased the survival rate after bacterial infection. [ABSTRACT FROM AUTHOR]

Aeromonas jandaei is an emerging fish pathogen associated with massive mortalities in cultured freshwater fish. This study investigated the morphological, biochemical, molecular, virulence, pathogenicity, and antimicrobial susceptibility characteristics of four strains of A. jandaei involved in the occurrence of mass mortalities of cultured Nile tilapia in an earthen pond farm and laboratory fibreglass tanks in Brazil. Isolates were identified by morphological, biochemical, and molecular analyses. Bacteria morphology was assessed under light and scanning electron microscopes, biochemical profile by conventional biochemical tests, and molecular identification by nucleotide sequencing and phylogenetic analysis of the highly conserved 16S rRNA gene, as well as the housekeeping genes, gyrB , and rpoB. The virulence and pathogenicity were confirmed by screening for 12 virulence genes and induced experimental infection. Broth microdilution method was used to assess susceptibility to enrofloxacin, florfenicol, oxytetracycline, and thiamphenicol. All isolates were confirmed as A. jandaei. Haemolysin, temperature-sensitive protease, haemolysin-aerolysin, and nuclease were the most predominant virulence genes. Lateral flagella B and cytolytic enterotoxin/cytotonic enterotoxin were only detected in strains isolated from fish from fibreglass tanks. In the experimental challenge, doses above 4.3 × 107 CFU mL−1 resulted in mass mortality (100%) in a short period (less than 12 h) without remarkable external clinical signs. Typical clinical signs of disease, including lethargy, inappetence, surface swimming, exophthalmia, cloudy eyes, haemorrhagic patches, and redness of the skin, below the opercula, and at the base of all fins, fin rot, and pale body surface were observed in fish challenged with doses below 4.0 × 107 CFU mL−1 (10–100% mortality). The most predominant histopathological changes in the internal organs of diseased fish were melano-macrophage centres, vascular congestion with thrombus, haemorrhage, and necrosis. Strains isolated from fish from the earthen pond farm were resistant to oxytetracycline, while the strains from fibreglass tanks were sensitive to all antimicrobials. This study provides pertinent data on A. jandaei host-pathogen interactions, susceptibility to antimicrobials, morphological, biochemical, and molecular characteristics, and geographical distribution, which may serve as a guideline for A. jandaei isolation and identification in aquaculture. • The multilocus phylogenetic analysis allowed to clarify the phylogenetic relationships of the clinical isolates. • This is the first detailed report of mass mortality caused by Aeromonas jandaei in Brazil. • Florfenicol (10 mg kg−1 bw) and Oxytetracycline (120 mg kg−1 bw) were ineffective to control the outbreak in the fish farm. • A. jandaei killed 100% of fish in less than 12 h without remarkable clinical signs. • Haemolysin, temperature-sensitive protease, haemolysin-aerolysin and nuclease were detected in all clinical isolates. [ABSTRACT FROM AUTHOR]

Among the antimicrobial peptides (AMPs), which act as natural antibiotics in preventing the colonization of pathogens, LL-37 is noteworthy. It is a human cathelicidin characterized as a broad-spectrum antibiotic, with chemotactic and immunomodulatory properties. This study aimed to evaluate the antimicrobial activity and therapeutic effectiveness of peptide LL-37 against streptococcosis caused by Streptococcus agalactiae in Nile tilapia and to assess some of its potential adverse effects (in vivo haematological, biochemical, and immunological profiles) in the fish. After in vitro cytotoxicity studies of LL-37 against Nile tilapia red blood cells (RBCs) and antimicrobial susceptibility testing (minimum inhibitory concentration and minimum bactericidal concentration, MIC and MBC, respectively) against Aeromonas hydrophila and S. agalactiae , fish were divided into six experimental groups. G1 and G2 were challenged by intraperitoneal injection with S. agalactiae and received a single oral dose of LL-37 (15 mg/kg bw) and florfenicol (FFC) (15 mg/kg bw), respectively. G3 was challenged with S. agalactiae and received a single intraperitoneal dose of LL-37 (15 mg/kg bw). The other groups were controls for infection (G4) and antimicrobial administration (G5 and G6). LL-37 showed weak hemolytic activity against Nile tilapia RBCs (24.4% lysis at 625 μg/mL) and strong in vitro antimicrobial activity against S. agalactiae (MIC and MBC = 31.25 μg/mL). No significant effects were observed in the haematological, biochemical, and immunological profiles of fish medicated with LL-37 or FFC, compared to the control fish. Twenty days after the challenge, survival rates were 33.38% (G1), 38.51% (G2), 23.12% (G3), 25% (G4), and 100% (G5 and G6). Despite the strong in vitro antimicrobial activity, both antimicrobials were ineffective to control streptococcosis caused by S. agalactiae in Nile tilapia. These findings suggest that administration of LL-37 (15 mg/kg bw) is safe for Nile tilapia but not effective to control streptococcosis. • This is the first report of antimicrobial activity of LL-37 against S. agalactiae and its effects in Nile tilapia. • LL-37 showed strong antimicrobial activity against S. agalactiae and weak hemolytic activity against Nile tilapia RBCs. • The pathogen does not persist in surviving fish after administration of LL-37 (15 mg/kg). • Administration of LL-37 (15 mg/kg) is safe for Nile tilapia but not effective to control streptococose. [ABSTRACT FROM AUTHOR]