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.

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.

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.

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.

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.

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.

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.

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.

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.

The gut bacterial microbiome of Nile tilapia (Oreochromis niloticus) from lakes across an altitudinal gradient.

BACKGROUND: Microorganisms inhabiting the gut play a significant role in supporting fundamental physiological processes of the host, which contributes to their survival in varied environments. Several studies have shown that altitude affects the composition and diversity of intestinal microbial communities in terrestrial animals. However, little is known about the impact of altitude on the gut microbiota of aquatic animals. The current study examined the variations in the gut microbiota of Nile tilapia (Oreochromis niloticus) from four lakes along an altitudinal gradient in Ethiopia by using 16S rDNA Illumina MiSeq high-throughput sequencing. RESULTS: The results indicated that low-altitude samples typically displayed greater alpha diversity. The results of principal coordinate analysis (PCoA) showed significant differences across samples from different lakes. Firmicutes was the most abundant phylum in the Lake Awassa and Lake Chamo samples whereas Fusobacteriota was the dominant phylum in samples from Lake Hashengie and Lake Tana. The ratio of Firmicutes to Bacteroidota in the high-altitude sample (Lake Hashengie, altitude 2440 m) was much higher than the ratio of Firmicutes to Bacteroidota in the low altitude population (Lake Chamo, altitude 1235 m). We found that the relative abundances of Actinobacteriota, Chloroflexi, Cyanobacteria, and Firmicutes were negatively correlated with altitude, while Fusobacteriota showed a positive association with altitude. Despite variability in the abundance of the gut microbiota across the lakes, some shared bacterial communities were detected. CONCLUSIONS: In summary, this study showed the indirect influence of altitude on gut microbiota. Altitude has the potential to modulate the gut microbiota composition and diversity of Nile tilapia. Future work will be needed to elucidate the functional significance of gut microbiota variations based on the geographical environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study determined the composition and diversity of the gut microbiota in Nile tilapia collected from lakes across an altitude gradient. Our findings greatly extend the baseline knowledge of fish gut microbiota in Ethiopian lakes that plays an important role in this species sustainable aquaculture activities and conservation.

Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities.

Edwardsiellosis is a serious bacterial disease affecting Nile tilapia (Oreochromis niloticus), causing septicemia and mortalities. Edwardsiella tarda and Edwardsiella anguillarum were isolated from Nile tilapia summer mortality events in Egypt. Diseased fish showed hemorrhagic septicemia, skin erosions, and eye opacity. A total of 24 Edwardsiella spp. isolates were retrieved from the investigated fish specimens. Phenotypic and biochemical characteristics grouped isolates into typical Ed. tarda (n = 14 strains) and atypical Ed. tarda (n = 10 strains). The BLAST analysis of sodB gene sequencing confirmed the conventional identification of typical Ed. tarda strains (n = 14) and reidentified all the atypical strains (n = 10) as Ed. anguillarum. Isolates showed a combination of virulence factors, including biofilm formation (66.6%), hemolysis (100%), chondroitinase (50%), and proteolytic activity (20.8%). The major part of isolates showed high resistance to ampicillin, amoxicillin, gentamycin antibiotics and harbored tetA, blaCTX-M, and aadA1 resistance genes. Pathogenicity testing of isolates in O. niloticus confirmed their virulence. Challenged fish exhibited septicemic signs similar to naturally diseased fish. Infections in naturally infected tilapia triggered acute and chronic histopathological alterations. Degenerative and necrotic changes were noticed in hematopoietic organs. Granulomas were noticed in between the hepatic parenchyma. The data extracted from the study confirm that accurate identification of the causative agents of edwardsiellosis should be reliant on genetic-based approaches. Analysis of the bacterium virulence properties offers insights into establishing novel therapeutics for edwardsiellosis control. The findings refer to the need for antimicrobial sensitivity testing to minimize antimicrobial resistance and increase therapy efficacy.

Characterization of the core gut microbiota of Nile tilapia (Oreochromis niloticus): indication of a putative novel Cetobacterium species and analysis of its potential function on nutrition.

The genus Cetobacterium has been considered a dominant group of gut bacteria in many freshwater fish, and members of this genus contribute to anaerobic metabolism. Because of its significant place in the gut of freshwater fish, many studies on Cetobacterium were performed. Those studies mostly focused on the temporal and spatial changes of its abundance in fish intestine, which were affected by food or other environmental conditions. However, only a few studies isolated strains from genus Cetobacterium and reported their characteristics. In the present study, we performed 16S rRNA sequencing of the intestinal mucosa of Nile tilapia (Oreochromis niloticus) and found that Cetobacterium sp. existed widely in the foregut, midgut and hindgut mucosa, and a strain of Cetobacterium was successfully isolated from the gut of tilapia. We sequenced its whole genome and predicted it to be a novel candidate species of Cetobacterium sp. and named it NK01. The size of its genome was 3,095,946 bp, with a guanine + cytosine content of 28.8%. Among the identified genes, 2855 were predicted to be coding DNA sequences, 84 were tRNA and 34 were rRNA. We found that NK01 produced amino acids, including leucine, isoleucine, valine, glycine, alanine, phenylalanine and proline. Strain NK01 could use starch, sucrose, maltose, glucose, and mannose and synthesize and utilize glycogen. INV, GPI, malQ, malZ, sacA, scrK, glgC, glgA and glk, which were related to carbohydrate metabolism, were detected. yiaY and adhE, which oxidize ethanol to acetaldehyde and participate in a variety of metabolic pathways, were also present in the genome. No coding genes directly involved in acetate or butyrate production were detected. NK01 could also catabolize a variety of vitamins, and all genes involved in folate synthesis were detected, including folP, folC, folA and eutT, which converted vitamin B12s into vitamin B12 coenzyme. Here, we investigated the draft genome and in vitro function of Cetobacterium isolated from the intestinal tract of Nile tilapia. The results provided a preliminary understanding of the core microbiota of fish gut.

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.

The synergistic effects of plant polysaccharide and Pediococcus acidilactici as a synbiotic additive on growth, antioxidant status, immune response, and resistance of Nile tilapia (Oreochromis niloticus) against Aeromonas hydrophila.

This study evaluated the growth performance, immune responses, and disease resistance of Nile tilapia upon pistachio hulls derived polysaccharide (PHDP) and Pediococcus acidilactici (PA) separately or as synbiotic. Fish received four types of diets: T1, control; T2, PHDP (0.1%); T3, PA (0.2%); T4, PHDP (0.1%) +PA (0.2%) for 56 days. The results showed that final weight and weight gain were markedly higher in fish fed T4 diet than that given T1 and T2 diets (P ≤ 0.05). In addition, a significantly greater specific growth rate was obtained by the T4 diet compared to the control. Fish survival was significantly improved in all supplemented diets compared to the control. On the other hand, the activities of lipase, protease, and amylase showed significant increases in the T4 group compared with other feeding groups. The total leucocytes and lymphocytes proportion significantly elevated in T3 and T4 than remaining groups (P ≤ 0.05). Further, fish fed T3 diet presented significantly higher serum total protein, total immunoglobulin, lysozyme activity (LYZ), alternative complement activity (ACH50), and alkaline phosphatase activity compared to fish fed T1 and T2 diets, while the mentioned indices were found significantly highest in T4 group than others. Fish received T3 and T4 diets had higher skin mucus LYZ and ACH50 than those fed T1 and T2 diets (P ≤ 0.05). The malondialdehyde levels were significantly declined in T3 and T4 when compared to the control. Fish fed T3 and T4 diets demonstrated significantly enhanced superoxide dismutase, catalase, and glutathione peroxidase activities compared to the control. The intestinal propionic acid significantly increased by T2 and T4 diets, while the highest levels of acetic acid detected in fish given T4 diet. The expression levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) were significantly affected by T3 and T4 supplements. The efficacy of T4 diet against Aeromonas hydrophila infection was documented by a significantly lower mortality rate. In conclusion, the combination of PHDP and PA presented promising results as a synbiotic feed additive for Nile tilapia.

α-MSH is partially involved in the immunomodulation of Nile tilapia (Oreochromis niloticus) antibacterial immunity.

α-Melanocyte-stimulating hormone (α-MSH) is a well-studied neuropeptide controlling skin and hair color. Besides, numerous immunomodulation roles of α-MSH were recorded in humans and mice. However, the regulatory effects of α-MSH in teleost immunity haven't been well elucidated. In this study, several precursor molecules of α-MSH (POMCs) and its receptors (MCRs) in Nile tilapia (Oreochromis niloticus) were characterized, and their expression characteristics and specific functions on antibacterial immunity were determined. Overall, POMCs and MCRs were principally detected in the brain, skin, and liver, and were remarkably promoted post Streptococcus agalactiae infection. However, tiny POMCs and MCRs were observed in tilapia immune organs (head kidney and spleen) or lymphocytes, and no evident immunomodulation effect was detected in vitro. Moreover, the in vivo challenge experiments revealed that α-MSH protects tilapia from bacterial infection by regulating responses in the brain and intestine. This study lays theoretical data for a deeper comprehension of the immunomodulation mechanisms of teleost α-MSH and the evolutional process of the vertebrate melanocortin system.

Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate.

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

Genome-Wide DNA Methylation and RNA Analysis Reveal Potential Mechanism of Resistance to Streptococcus agalactiae in GIFT Strain of Nile Tilapia (Oreochromis niloticus ).

Streptococcus agalactiae is an important pathogenic bacterium causing great economic loss in Nile tilapia (Oreochromis niloticus) culture. Resistant and susceptible groups sharing the same genome showed significantly different resistance to S. agalactiae in the genetically improved farmed tilapia strain of Nile tilapia. The resistance mechanism is unclear. We determined genome-wide DNA methylation profiles in spleen of resistant and susceptible O. niloticus at 5 h postinfection with S. agalactiae using whole-genome bisulfite sequencing. The methylation status was higher in the spleen samples from resistant fish than in the susceptible group. A total of 10,177 differentially methylated regions were identified in the two groups, including 3725 differentially methylated genes (DMGs) (3129 hyper-DMGs and 596 hypo-DMGs). The RNA sequencing showed 2374 differentially expressed genes (DEGs), including 1483 upregulated and 891 downregulated. Integrated analysis showed 337 overlapping DEGs and DMGs and 82 overlapping DEGs and differentially methylated region promoters. By integrating promoter DNA methylation with gene expression, we revealed four immune-related genes (Arnt2, Nhr38, Pcdh10, and Ccdc158) as key factors in epigenetic mechanisms contributing to pathogen resistance. Our study provided systematic methylome maps to explore the epigenetic mechanism and reveal the methylation loci of pathogen resistance and identified methylation-regulated genes that are potentially involved in defense against pathogens.

Streptococcus agalactiae infection caused spinal deformity in juvenile red tilapia (Oreochromis sp.).

A case of juvenile red tilapia (Oreochromis sp.) showing body deformity due to spinal curvature was investigated. Approximately 20% of the crop (4,000 fish/crop) was affected. Bacterial isolation from the kidney and tissue surrounding the spinal lesion of the affected fish was negative. Histopathology revealed granulomatous inflammation and Gram-positive cocci in connective tissues around the bone and notochord. PCR assay confirmed the presence of S. agalactiae in the spinal tissue lesion. Spinal deformity in red tilapia observed in our study may be associated with the inflammatory process and granuloma that compress the skeleton structure. The present study highlights chronic streptococcosis in tilapia culture that may be unnoticed.

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus.

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.