New vaccination approach using formalin-killed Streptococcus pyogenes vaccine on the liver of Oreochromis niloticus fingerlings.

Newly synthesized vaccines prepared from formalin-killed bacteria Streptococcus pyogenes were investigated in the current study to evaluate the effectiveness of the newly synthesized vaccine as well as their safety by injected intraperitoneal. The study involved several steps 1st step is the preparation of the vaccine followed by the 2nd step: Evaluate the effectiveness and vaccine safety against pathogenic S. pyogenes through 4 different groups including control (Group I). Group II (Bacterial, infected group), Group III (Vaccine), and the Last group was the challenged group after the vaccination (Vacc + Bac). Different Immunological and biochemical parameters were measured in addition to hematological and histopathological examinations. For example, oxidative/antioxidants, inflammatory biomarkers, fragmentation and cell damage, and finally the histopathological study. The current study showed an increase in all oxidative, inflammatory, and cell damage (DNA fragmentation assays), additionally markedly elevation in histopathological cell damage in the infected group (Group II) compared with the control group. The vaccine and challenged after vaccination group (vaccine + Bacteria), showed great improvement in oxidative biomarkers (LPO) and an increase in antioxidants biomarkers (GSH, SOD, GST, DPPH, ABTS, GR and GPx), Also the inflammation and histopathological examination. The newly synthesized vaccine improved the resistance of Oreochromis niloticus and can be used as a preventive therapy agent for pathogenic bacteria S. pyogenes.

Design of an agro-industrial by-products-based media for the production of probiotic bacteria for fish nutrition.

Probiotic production in commercial culture media is expensive, so, it is necessary to design culture media based on "low-cost" components like agro-industrial by-products. Therefore, this study aimed to design an agro-industrial by-product-based culture media using whey, sugarcane molasses, and palm kernel cake as components to produce Lactococcus lactis A12, Priestia megaterium M4, and Priestia sp. M10 isolated from Nile tilapia (Oreochromis niloticus) associated gut microbiota. Higher bacterial concentrations were achieved at high whey concentrations and low concentrations of sugarcane molasses and palm kernel cake (PKC) using agitation. The optimal conditions were whey, 3.84% w/v; sugarcane molasses, 7.39% w/v; PKC, 0.77% w/v; and agitation speed, 75 RPM. Bacterial growth under optimal conditions was compared to that in commercial Brain-Heart Infusion (BHI) broth. L. lactis A12 showed similar growth in the optimal media and BHI. The estimated cost of the culture media based on component prices was USD $ 3.01/L, which is 86.93% lower than BHI broth (USD $ 23.04/L). It was possible to design a "low-cost agro-industrial by-product-based culture media to produce L. lactis A12 and the two Priestia species under monoculture conditions.

Assessment of Bacillus species capacity to protect Nile tilapia from A. hydrophila infection and improve growth performance.

The present study evaluated the capacity of three Bacillus species to improve health status and growth performance of Nile Tilapia fed with high levels of soybean meal and challenged with Aeromonas hydrophila. In vitro experiments showed that ß-hemolysin and metalloprotease enzymes were produced by A. hydrophila throughout the exponential growth phase. In vivo experiments showed that 107 colony-forming units (CFUs)/ml of this pathogen killed 50% of control group fishes in 13 days. To evaluate the influence of Bacillus strains on health status and growth performance in Nile Tilapia, 180 fishes (33.44 + 0.05 g) were distributed in 12 tanks of 200 L each, and animals were fed twice per day until satiety. 1) Control group without Bacillus, 2) Bacillus sp1, 3) Bacillus sp2, and 4) Bacillus sp3 groups were formulated containing 106 CFU/g. After 40 days of feeding, the fishes were intraperitoneally injected with 1 ml of A. hydrophila at 2 × 107 CFU/ml, and mortality was recorded. The results showed that cumulative mortality rate was significantly (p< 0.05) lower in the Bacillus sp1 (25%), sp2 (5%), and sp3 (15%) groups, than the control group (50%). Weight gain was also significantly better (p< 0.05) in the Bacillus sp1 (36%), sp2 (67%), and sp3 (55%) groups with respect to the control group (30%). In conclusion, functional diet formulated with high levels of soybean meal and supplemented with Bacillus sp2 could be an alternative to protect Nile tilapia cultures from A. hydrophila infections and improve fish growth performance.

Medulla oblongata and NCCs are central defenders against Streptococcus agalactiae infection of the tilapia brain.

Various types of professional immune cells first emerge in fish and likely represent the primordial form and functions. Recent advancements revealed the direct connection between the central nervous system and the immune system in the mammalian brain. However, the specifics of brain-immune networks in the fish and the underlying mechanisms of teleost's brain against pathogen infection have not been fully elucidated. In this study, we investigated the distribution of markers representing cerebral cells associated with protection and professional lymphocytes in the seven major components of the Nile tilapia brain through RNA-Seq assay and observed the most dominant abundance in the medulla oblongata. The subsequent challenge test revealed the non-specific cytotoxic cells (NCCs) exhibited the strongest response against streptococcal infection of the brain. The presence of NCCs in the brain was then confirmed using immunofluorescence and the cytotoxic effects usually induced by NCCs under infection were determined as well. Collectively, these findings contribute significantly to comprehending the mechanism of fish neuroimmune interaction and enhancing our understanding of its evolutionary development.

Pichia kudriavzevii as feed additive in Nile tilapia (Oreochromis niloticus) diet.

Yeasts are unicellular eukaryotic microorganisms extensively employed in various applications, notably as an alternative source of protein in feeds, owing to their nutritional benefits. Despite their potential, marine and mangrove yeast species used in the aquaculture industry have received little attention in the Philippines. Pichia kudriavzevii (A2B R1 ISO 3), sourced from bark samples, was selected and mass-produced due to its high protein content and amino acid profile. The dried biomass of P. kudriavzevii was incorporated into the diets of Nile tilapia (Oreochromis niloticus) juveniles at varying inclusion levels (0, 1, 2, and 4 g/kg diet) and its effect on their growth performance, body composition, and liver and intestinal morphology was assessed after 40 days of feeding. The groups that received P. kudriavzevii at a concentration of 2 g/kg diet exhibited higher final body weight, percent weight gain, and specific growth rate in comparison to the other treatment groups. Whole body proximate composition did not vary among the dietary groups. Intestinal and liver histopathology also indicated no abnormalities. These findings suggest the potential of ascomycetous P. kudriavzevii as a beneficial feed additive in Nile tilapia diets, warranting further investigation into its long-term effects and broader applications in fish culture.

Effects of a multi-strain Bacillus probiotic on the intestinal microbiome, haemato-immunology, and growth performance of Nile tilapia.

The study evaluated dietary supplementation with a feed additive composed of multi-strain Bacillus for Nile tilapia Oreochromis niloticus. In vitro and in vivo assays employing culture-based microbiological methods and metagenomics were performed. Additionally, the study assessed the haemato-immunology, intestinal microbiome, and growth performance of the animals. For this, 30 juvenile Nile tilapia were used in the in vitro assay and 180 (60 + 120) in the in vivo assays. In the in vitro assay, we found evidence of adhesion of the probiotic bacteria to the intestinal mucus of fish, corroborated in the 15-day in vivo assay, in which the count of B. licheniformis was significantly higher in fish fed with probiotic when compared to fish of the control group. Furthermore, in the 50-day in vivo trial, a metagenomic analysis provided evidence for the modulation of the intestine microbiome of Nile tilapia by dietary supplementation of the probiotic. In addition, there was an increase in species richness, higher abundance of potentially probiotic autochthonous species and a lower abundance of Aeromonas sp. when the animals were fed the supplemented diet. Finally, no significant differences were observed in growth performance and haemato-immunological analyses, suggesting no harm to fish health when the product was supplemented for 15 and 50 days. The in vitro results indicate that the multi-strain probiotics were able to adhere to the intestinal mucus of Nile tilapia. Additionally, a modulation of the intestinal microbiome was evidenced in the in vivo assay.

Chlorella vulgaris algae ameliorates chlorpyrifos toxicity in Nile tilapia with special reference to antioxidant enzymes and Streptococcus agalactiae infection.

BACKGROUND: Chlorpyrifos (CPF) is a widely used pesticide in the production of plant crops. Despite rapid CPF biodegradation, fish were exposed to wastewater containing detectable residues. Recently, medicinal plants and algae were intensively used in aquaculture to replace antibiotics and ameliorate stress impacts. METHODS AND RESULTS: An indoor experiment was conducted to evaluate the deleterious impacts of CPF pollution on Nile tilapia health and the potential mitigation role of Chlorella vulgaris algae. Firstly, the median lethal concentration LC50 - 72 h of CPF was determined to be 85.8 µg /L in Nile tilapia (35.6 ± 0.5 g body weight) at a water temperature of 27.5 °C. Secondly, fish were exposed to 10% of LC50 - 72 h for six weeks, and tissue samples were collected and examined every two weeks. Also, Nile tilapia were experimentally infected with Streptococcus agalactiae. Exposed fish were immunosuppressed expressed with a decrease in gene expressions of interleukin (IL) 1ß, IL-10, and tumor necrosis factor (TNF)-α. Also, a decline was recorded in glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) gene expression in the head kidney tissue. A high mortality rate (MR) of 100% was recorded in fish exposed to CPF for six weeks and challenged with S. agalactiae. Fish that received dietary C. vulgaris could restore gene expression cytokines and antioxidants compared to the control. After six weeks of CPF exposure, fish suffered from anemia as red blood cell count (RBCs), hemoglobin (Hb), and packed cell volume (PCV) significantly declined along with downregulation of serum total protein (TP), globulin (GLO), and albumin (ALB). Liver enzymes were significantly upregulated in fish exposed to CPF pollution, alanine aminotransferase (ALT) (42.5, 53.3, and 61.7 IU/L) and aspartate aminotransferase (AST) (30.1, 31.2, and 22.8) after 2, 4, and 6 weeks, respectively. On S. agalactiae challenge, high MR was recorded in Nile tilapia exposed to CPF (G3) 60%, 60%, and 100% in week 2, week 4, and week 6, and C. vulgaris provided a relative protection level (RPL) of 0, 14.29, and 20%, respectively. CONCLUSIONS: It was concluded that CPF pollution induces immunosuppressed status, oxidative stress, and anemic signs in Nile tilapia. In contrast, C. vulgaris at a 50 g/kg fish feed dose could partially ameliorate such withdrawals, restoring normal physiological parameters.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Summer mortality syndrome bacterial pathogens in farmed Nile tilapia (Oreochromis niloticus).

Background: The high summer mortality in many fish farms, which had detrimental economic and social implications, was a serious challenge that the fish industry had to deal with. Aim: With an examination of the most effective antibiotic, the ongoing research was intended to shed light on the identification of the main bacterial pathogens associated with the summer mortality syndrome in the diseased farmed Nile tilapia. Methods: Six hundred dead Nile tilapia samples that had suffered from summer mortality were collected from several fish farms between May and October of 2022. The gathered fish displayed hemorrhagic areas on the skin, scale detachment, fin degeneration, erosions, skin ulcers, and corneal opacity with unilateral and/or bilateral exophthalmia. The most prominent internal appearance was swelling of the internal organs with sanguineous ascetic fluid. Results: There were 225 bacterial isolates found. Six species were identified through phenotypic and biochemical analysis; they were Aeromonas, Vibrio, Streptococcus, Pseudomonas, Enterococcus, and Edwardsiella spp., in descending percentage, respectively. Aeromonas spp., Vibrio spp., and Streptococcus spp. were the three most frequent isolated bacterial pathogens. The identification of Aeromonas hydrophila, Vibrio spp., and Streptococcus iniae, the three most common bacterial isolates, was confirmed by molecular analysis by polymerase chain reaction. Most of the tested strains were found to be responsive to Ciprofloxacin (CIP), Gentamicin (CN), and Chloramphenicol (C) but resistant to Amoxicillin (AMX), according to an antibiotic sensitivity test. Conclusion: The three most dangerous common bacterial infections discovered during mass-farmed tilapia summer mortality are A. hydrophil a, Vibrio sp., and S. iniae. This makes it clear that high water temperatures may raise the possibility of bacterial infections, which could cause widespread tilapia mortality and substantial financial losses. Therefore, it is crucial to maintain a beneficial fish culture, environment, and husbandry practices to enhance the tilapia-rearing environment and lessen the virulence of the disease. Isolated bacterial strains showed low levels of resistance to AMX but were vulnerable to CIP, CN, and C.

Pathogenic bacteria prevalence in cultured Nile tilapia in Southwest Mexico: A real-time PCR analysis.

The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.

Genetic and morphological characterization of a new genotype of nervous necrosis virus circulating among Nile tilapia in the south of Egypt.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis in freshwater and marine fishes. In this study, NNV circulating among wild and farmed Nile tilapia (Oreochromis niloticus) was genetically and morphologically characterized using reverse transcription polymerase chain reaction (RT-PCR), sequencing analysis, and transmission electron microscopy (TEM). Brain, eye, and other organ (spleen, kidney, heart, and liver) specimens were collected from 87 wild (66) and farmed (21) Nile tilapia fish during their adult or juvenile stage at different localities in Qena and Sohag governorates in southern Egypt. Among them, 57/87 fish showed suspected NNV clinical signs, and 30/87 were healthy. The results revealed that NNV was detected in 66 out of 87 fish (58.62% in the wild and 17.24% in farmed Nile tilapia by RT-PCR), and the prevalence was higher among diseased (55.17%) than in healthy (20.69%) fish. NNV was detected in the brain, eye, and other organs. Using TEM, virion size variations based on the infected organs were observed. Nucleotide sequence similarity indicated that NNVs had a divergence of 75% from other fish nodaviruses sequenced in Egypt and worldwide. Phylogenetic analysis distinguished them from other NNV genotypes, revealing the emergence of a new NNV genotype in southern Egypt. In conclusion, NNV is circulating among diseased and healthy Nile tilapia, and a new NNV genotype has emerged in southern Egypt.

Selenium Nanoparticles Ameliorate Adverse Impacts of Aflatoxin in Nile Tilapia with Special Reference to Streptococcus agalactiae Infection.

Aflatoxin B1 (AFB1) is a plant-origin toxin that could induce oxidative stress in fish. The micromineral selenium (Se) possesses well-documented antioxidant properties. To assess the ameliorative effects of SeNPs (1 mg/kg fish feed) on oxidative stress induced by AFB1 (500 µg/kg fish feed), Nile tilapia (32.2±1.7 g body weight) were distributed randomly and even in six groups for 8-week feeding trial. Live enzymes, AST, ALT, and ALP levels were increased in the serum of fish fed AFB1-contaminated diet, and the addition of SeNPs could restore normal values compared to the control. The gene expression of antioxidant enzymes, superoxide dismutase (SOD) enzyme and catalase (CAT) enzyme, and DNA fragmentation were significantly increased in response to aflatoxin exposure, while dietary SeNPs could mitigate the generated oxidative stress. The innate immunity, serum antibacterial activity (SAA), oxidative burst activity (OBA), phagocytic activities (PA and PI), and gene expression of cytokines (interleukin (IL)-1ß, heat shock protein70 (Hsp), and tumor necrosis factor (TNF)-α) revealed a status of immunosuppression in Nile tilapia fed on AFB1-contaminated diet. These findings showed that fish became more vulnerable to Streptococcus agalactiae infection with a high mortality rate while dietary SeNPs provided a high relative protection level (RPL). From the obtained findings, SeNPs could mitigate the oxidative stress induced by feeding the AFB1 diet and could boost the immunity of stressed Nile tilapia.

Study of the Antioxidant, Antimicrobial, and Wound Healing Properties of Raw Hydrolyzed Extract from Nile Tilapia Skin (Oreochromis niloticus).

Oreochromis niloticus (Nile tilapia) skin is a by-product of Brazilian fish farming, rich in collagen. The present study aims to evaluate the wound healing, antioxidant, and antimicrobial potential of the raw hydrolyzed extract of Nile tilapia skin, as well as the identification of the main compounds. The in vitro activity was performed using antioxidant, antimicrobial and scratch wound healing assays. An in vivo experiment was performed to evaluate the wound healing potential. On days 1, 7, 14 and 21, the lesions were photographed to assess wound retraction and on the 7th , 14th and 21st  days the skins were removed for histological evaluation and the blood of the animals was collected for glutamic oxaloacetic transaminase and glutamic pyruvic transaminase determination. The chemical study was carried out through liquid chromatography-tandem mass spectrometry and de novo sequencing of peptides. The in vitro assays showed a reduction of the gap area in 24 h, dose-dependent antimicrobial activity for both bacteria, and antioxidant activity. The chemical analysis highlighted the presence of active biopeptides. The histological evaluation showed that the raw hydrolyzed extract of Nile tilapia skin has a healing potential, and does not present toxicological effects; therefore, is promising for the treatment of wounds.

Phenotypic, molecular detection and antibiogram analysis of Aeromonas Hydrophila from Oreochromis Niloticus (Nile Tilapia) and Ready-To- eat fish products in selected Rift Valley lakes of Ethiopia.

BACKGROUND: Aeromonas hydrophila is a zoonotic bacterial pathogen that frequently causes disease and mass mortalities among cultured and feral fishes worldwide. In Ethiopia, A. hydrophila outbreak was reported in Sebeta fish ponds and in Lake Tana fishery. However, there is no to little information on the molecular, and phenotypical characteristics of A. hydrophila in Ethiopian fisheries. Therefore, a cross-sectional study was conducted from November 2020 to May 2021 in selected Ethiopian Rift valley lakes. RESULTS: A total of 140 samples were collected aseptically from fish (Muscle, Gill, Intestine, Spleen and Kidney) from fish landing sites, market and restaurants with purposive sampling methods. Aeromonas selective media (AMB), morphological and biochemical tests were used to isolate and identify A. hydrophila. Accordingly, the pathogen was isolated from 81 (60.45%) of samples. Among the isolates 92.59% expressed virulence trait through ß hemolysis on blood agar media with 5% sheep blood. Moreover, 54 strains (66.67%) were further confirmed with Real-Time PCR (qPCR) using ahaI gene specific primers and optimized protocol. The highest (68.51%) were detected from live fish, (24.07%) were from market fish and the lowest (7.4%%) were from ready-to-eat products. Antibiogram analysis was conducted on ten representative isolates. Accordingly, A. hydrophila isolates were susceptible to ciprofloxacin (100%), chloramphenicol (100%) and ceftriaxone (100%). However, all ten isolates were resistant to Amoxicillin and Penicillin. CONCLUSIONS: The study indicates A. hydrophila strains carrying virulence ahaI gene that were ß-hemolytic and resistant to antibiotics commonly used in human and veterinary medicine are circulating in the fishery. The detection of the pathogen in 140 of the sampled fish population is alarming for potential outbreaks and zoonosis. Therefore, further molecular epidemiology of the disease should be studied to establish potential inter host transmission and antibiotic resistance traits. Therefore, raising the public awareness on risk associated with consuming undercooked or raw fish meat is pertinent.

In vivo antibacterial activity of medicinal plant Sophora flavescens against Streptococcus agalactiae infection.

Streptococcosis disease caused by Streptococcus agalactiae (Group B Streptococcus, GBS) results in a huge economic loss of tilapia culture. It is urgent to find new antimicrobial agents against streptococcosis. In this study, 20 medicinal plants were evaluated in vitro and in vivo to obtain medicinal plants and potential bioactive compounds against GBS infection. The results showed that the ethanol extracts of 20 medicinal plants had low or no antibacterial properties in vitro, with a minimal inhibitory concentration ≥256 mg/L. Interestingly, in vivo tests showed that 7 medicinal plants could significantly inhibit GBS infection in tilapia, and Sophora flavescens (SF) had the strongest anti-GBS activity in tilapia, reaching 92.68%. SF could significantly reduce the bacterial loads of GBS in different tissues (liver, spleen and brain) of tilapia after treated with different tested concentrations (12.5, 25.0, 50.0 and 100.0 mg/kg) for 24 h. Moreover, 50 mg/kg SF could significantly improve the survival rate of GBS-infected tilapia by inhibiting GBS replication. Furthermore, the expression of antioxidant gene cat, immune-related gene c-type lysozyme and anti-inflammatory cytokine il-10 in liver tissue of GBS-infected tilapia significantly increased after treated with SF for 24 h. Meanwhile, SF significantly reduced the expression of immune-related gene myd88 and pro-inflammatory cytokines il-8 and il-1ß in liver tissue of GBS-infected tilapia. The negative and positive models of UPLC-QE-MS, respectively, identified 27 and 57 components of SF. The major components of SF extract in the negative model were α, α-trehalose, DL-malic acid, D- (-)-fructose and xanthohumol, while in the positive model were oxymatrine, formononetin, (-)-maackiain and xanthohumol. Interestingly, oxymatrine and xanthohumol could significantly inhibit GBS infection in tilapia. Taken together, these results suggest that SF can inhibit GBS infection in tilapia, and it has potential for the development of anti-GBS agents.

Identification and Functional Analysis of Tartrate-Resistant Acid Phosphatase Type 5b (TRAP5b) in Oreochromis niloticus.

Tartrate-resistant acid phosphatase type 5 (TRAP5) is an enzyme that is highly expressed in activated macrophages and osteoclasts and plays important biological functions in mammalian immune defense systems. In the study, we investigated the functions of tartrate-resistant acid phosphatase type 5b from Oreochromis niloticus (OnTRAP5b). The OnTRAP5b gene has an open reading frame of 975 bp, which encodes a mature peptide consisting of 302 amino acids with a molecular weight of 33.448 kDa. The OnTRAP5b protein contains a metallophosphatase domain with metal binding and active sites. Phylogenetic analysis revealed that OnTRAP5b is clustered with TRAP5b of teleost fish and shares a high amino acid sequence similarity with other TRAP5b in teleost fish (61.73-98.15%). Tissues expression analysis showed that OnTRAP5b was most abundant in the liver and was also widely expressed in other tissues. Upon challenge with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro, the expression of OnTRAP5b was significantly up-regulated. Additionally, the purified recombinant OnTRAP5b ((r)OnTRAP5) protein exhibited optimal phosphatase activity at pH 5.0 and an ideal temperature of 50 °C. The Vmax, Km, and kcat of purified (r)OnTRAP5b were found to be 0.484 µmol × min-1 × mg-1, 2.112 mM, and 0.27 s-1 with respect to pNPP as a substrate, respectively. Its phosphatase activity was differentially affected by metal ions (K+, Na+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Fe3+) and inhibitors (sodium tartrate, sodium fluoride, and EDTA). Furthermore, (r)OnTRAP5b was found to promote the expression of inflammatory-related genes in head kidney macrophages and induce reactive oxygen expression and phagocytosis. Moreover, OnTRAP5b overexpression and knockdown had a significant effect on bacterial proliferation in vivo. When taken together, our findings suggest that OnTRAP5b plays a significant role in the immune response against bacterial infection in Nile tilapia.

Multidrug-resistant Aeromonas bacteria prevalence in Nile tilapia broodstock.

BACKGROUND: Aeromonas hydrophila is an opportunistic pathogen. Thus, it has received significant attention mainly in the fish sectors with high production scales. Nile tilapia broodstock confined in the environment of fish hatcheries can be stressed. Hence, they are vulnerable to A. hydrophila. RESULTS: Sequencing of the gyr B gene revealed the presence of 18 different A. hydrophila strains (kdy 10,620-10,637), which were deposited in the NCBI under accession numbers ON745861-ON745878. The median lethal doses of the isolates ranged from 2.62 × 104 to 3.02 × 106 CFU/mL. Antibiotic resistant genes, sulfonamide (sul1) and tetracycline (tetA) were found in the eighteen isolates. Approximately 83.3% of A. hydrophila strains were sensitive to ciprofloxacin and florfenicol. Further, eight A. hydrophila strains had high MDR indices at 0.27-0.45. All isolates presented with hemolysin activity. However, only 72.22% of them had proteolytic activity, and only 61.11% could form biofilms. Bacterial isolates harbored different pattern virulence genes, the heat-stable cytotonic enterotoxin (ast), cytotoxic enterotoxin (act), and hemolysin (hly) genes were the most prevalent. Also, a trial to inhibit bacterial growth was conducted using titanium dioxide nanoparticles (TiO2 NPs) with three sizes (13, 32, and 123 nm). If A. hydrophila strains with a high MDR index were tested against TiO2 NPs (20 µg/mL) for 1, 12, and 24 h, those with a small size had a greater bactericidal action than large ones. Bacterial strains were inhibited at different percentages in response to TiO2 NP treatment. CONCLUSIONS: Nile tilapia broodstock, mortality is associated with different A. hydrophila strains, which harbored virulent and MDR genes. Furthermore, TiO2 NPs had bactericidal activity, thereby resulting in a considerable reduction in bacterial load.

Hybrid of Metapenaeus dobsoni lectin and platinum nanoparticles exert antimicrobial and immunostimulatory effects to reduce bacterial bioburden in infected Nile tilapia.

A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential "green" application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics.

Cyathocotylidae spp and motile aeromonads co-infections in farmed Nile tilapia (Oreochromis niloticus) causing mass mortality.

Motile aeromonads, and Cyathocotylidaespp.co-infections were identified in farmed Nile tilapia(Oreochromis niloticus) which suffering from mortalities. Moribund fish showed signs of septicemia, skin irritations, and respiratory distress. A total of 150 O. niloticus specimens showing signs of disease were collected from the affected earthmen ponds and examined. Bacteriological examination of fish samples revealed infections with motile aeromonads species. Phenotypic characteristics and phylogenetic analysis of gyrB gene sequences of aeromonads isolates identified them as Aeromonas hydrophila (12.6%), A.sobria (12.6%), and A. caviae (30.4%). Aeromonads strains harbored some virulence genes: Aer (78.62%); Hyl (60.86%); laf-A (52.17%); and Act (47.82%). The antibiogram of aeromonads showed high resistance against tetracycline (73.9%), and gentamycin (65.2%), while a high sensitivity was noticed to ciprofloxacin (82.6%),and trimethoprim/sulfamethoxazole (60.86%). Parasitological examination of fish revealed the presence of Cyathocotylidae spp. encysted metacercaria (EMC). High levels of interleukin 6 (IL-6) and cluster of differentiation 4 (CD4) were noticed in fish with parasitic and bacterial co-infection compared to those with a single infection or non-infected. Experimentally infected fish with Aeromonas spp. showed septicemic signs similar to that noticed in naturally infected tilapia with variable cumulative mortality. The study is one of the earlier reports identifying as Cyathocotylidae spp., and motile aeromonads co-infections, and their link with the exaggerated tilapia mortality which will be of value for incorporating these pathogens in the necessary management strategies to protect fish health.

Suppression of Streptococcosis and Modulation of the Gut Bacteriome in Nile Tilapia (Oreochromis niloticus) by the Marine Sediment Bacteria Bacillus haynesii and Advenella mimigardefordensis.

Streptococcosis is one of the major threats to Nile tilapia (Oreochromis niloticus) in most regions of the world. Recently, Enterococcus faecalis has been widely reported to be involved in streptococcosis in O. niloticus in Asia and Africa. This study aimed to isolate beneficial marine bacteria to evaluate their effects on growth, hematological parameters, nonspecific immunity, the gut bacteriome, and streptococcosis prevention efficacy in O. niloticus. A total of 36 marine soil bacteria were isolated, and in vitro screening was conducted to determine their antibacterial activities against fish pathogens. Two antagonistic bacteria were identified based on 16S rRNA gene sequencing, Bacillus haynesii CD223 and Advenella mimigardefordensis SM421. These bacteria were incorporated into fish feed and fed to O. niloticus for 90 days. The application of these strains via incorporation into fish feed significantly promoted growth, improved hematological parameters and immunoglobulin M (IgM) levels, modulated the gut bacteriome by reducing the load of pathogenic Enterococcus spp., and developed disease prevention efficacy in O. niloticus. Furthermore, in vivo assays revealed that the inclusion of extracellular products (ECPs) (at 250 µg mL-1) of CD223 and SM421 with feed significantly enhanced the rate of survival (100%) of O. niloticus from streptococcosis compared to the controls (only 30%). The ECPs of these bacteria also prevented 90 to 100% of fish from developing streptococcosis. These strains could be promising for safe use in O. niloticus farming to prevent and control the emergence of streptococcosis caused by E. faecalis. IMPORTANCE Nile tilapia (Oreochromis niloticus) is one of the most economically important cultured fish species throughout the world. Streptococcosis is a significant threat to global Nile tilapia farming. Enterococcus faecalis has recently emerged as an important pathogen of streptococcosis in Asia and Africa. The application of antibiotics and probiotics and vaccination are the major ways to combat streptococcosis. However, the extensive use of antibiotics leads to the development of antibiotic resistance in pathogenic as well as environmental bacteria, which is a great threat to public health. There is no study on preventing streptococcosis caused by E. faecalis using beneficial bacteria. For the first time, the present study demonstrated that two marine bacteria, Bacillus haynesii strain CD223 and Advenella mimigardefordensis strain SM421, have great potential for controlling streptococcosis in Nile tilapia. These bacteria also enhanced the growth, improved hematological parameters and IgM levels, and positively modulated the gut bacteriome of Nile tilapia.