Insect Meal in Aquafeeds: A Sustainable Path to Enhanced Mucosal Immunity in Fish.

The mucosal surfaces of fish, including their intestines, gills, and skin, are constantly exposed to various environmental threats, such as water quality fluctuations, pollutants, and pathogens. However, various cells and microbiota closely associated with these surfaces work in tandem to create a functional protective barrier against these conditions. Recent research has shown that incorporating specific feed ingredients into fish diets can significantly boost their mucosal and general immune response. Among the various ingredients being investigated, insect meal has emerged as one of the most promising options, owing to its high protein content and immunomodulatory properties. By positively influencing the structure and function of mucosal surfaces, insect meal has the potential to enhance the overall immune status of fish. This review provides a comprehensive overview of the potential benefits of incorporating insect meal into aquafeed as a feed ingredient for augmenting the mucosal immune response of fish.

Leveraging the biotechnological promise of the hagfish variable lymphocyte receptors: tools for aquatic microbial diseases.

The jawless vertebrates (agnathans or cyclostomes) are ancestral animals comprising lampreys and hagfishes, which are the only extant representatives. They possess an alternative adaptive immune system (AIS) that uses leucine-rich repeats (LRR)-based variable lymphocyte receptors (VLRs) instead of the immunoglobulin (Ig)-based antigen receptors of jawed vertebrates (gnathostomes). The five VLR types (VLRA-VLRE) are expressed on agnathan lymphocytes and functionally resemble lymphocyte antigen receptors. In particular, VLRB is functionally similar to the B cell receptor expressed and secreted by B-like lymphocytes as VLRB antibodies that bind antigens with high affinity and specificity. The potential repertoire scale of VLR-based antigen receptors is believed to be at least comparable to that of Ig-based systems. VLR proteins inherently possess characteristics that render them excellent candidates for biotechnological development, including tractability to recombinant approaches. In recent years, scientists have explored the biotechnological development and utility of VLRB proteins as alternatives to conventional mammalian antibodies. The VLRB antibody platform represents a non-traditional approach to generating a highly diverse repertoire of unique antibodies. In this review, we first describe some aspects of the biology of the AIS of the jawless vertebrates, which recognize antigens by means of unique receptors. We then summarize reports on the development of VLRB-based antibodies and their applications, particularly those from the inshore hagfish (Eptatretus burgeri) and their potential uses to address microbial diseases in aquaculture. Hagfish VLRB mAbs are being developed and improved, while obstacles to the advancement of the VLRB platform are being addressed to utilize VLRBs effectively as tools in immunology. Hence, mAbs for novel antigen targets are expected to emerge to provide new opportunities to tackle various scientific questions. We anticipate a greater interest in the agnathan AIS in general and particularly in the hagfish AIS for greater elucidation of the evolution of adaptive immunity and its applications to tackle microbial pathogens in farmed aquatic animals and beyond.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

Knowns and unknowns of TiLV-associated neuronal disease.

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus's neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field.

Effect of Moringa oleifera Leaf Extract on the Growth Performance, Hematology, Innate Immunity, and Disease Resistance of Nile Tilapia (Oreochromis niloticus) against Streptococcus agalactiae Biotype 2.

The present study aimed to investigate the effects of Moringa oleifera leaf (MLE) extract-supplemented diets on the growth, feed utilization, hematology, innate immune response, and disease resistance of Nile tilapia against Streptococcus agalactiae Biotype 2. Four hundred and fifty Nile tilapia (32.61 ± 0.2 g/fish) were randomly allocated into fifteen tanks (30 fish/tank). Different concentrations of MLE at 0%, 0.5%, 1%, 1.5%, and 2% were fed to the Nile tilapia for 30 days, and the growth, feed utilization, hematology, and innate immune response of the Nile tilapia were determined. After the feeding trial, the Nile tilapia were challenged with a S. agalactiae Biotype 2 infection, and the relative percentage of survival (RPS) was determined. Results revealed the presence of quercetin, kaempferol, and p-coumaric acid in the MLE extract, exhibiting stronger antimicrobial activity against S. agalactiae Biotype 2. The diets supplemented with the MLE-0.5 group showed a significantly higher growth, feed utilization, hematology, and innate immune response in the Nile tilapia compared to the control and other MLE groups. Additionally, the MLE-0.5 group exhibited a significantly higher RPS of the Nile tilapia against S. agalactiae Biotype 2. Therefore, MLE-0.5 can be employed as an alternative feed supplement in sustainable Nile tilapia farming to protect against S. agalactiae Biotype 2.

Systemic and mucosal immune responses in red tilapia (Oreochromis sp.) following immersion vaccination with a chitosan polymer-based nanovaccine against Aeromonas veronii.

A mucoadhesive chitosan polymer-based nanoplatform has been increasingly recognized as an effective mucosal vaccine delivery system for fish. The present study aimed to investigate the effectiveness of immersion vaccination with a chitosan polymer-based nanovaccine to elicit an immune response in serum and mucus of red tilapia and evaluate its protective efficacy after immersion challenge with a heterogenous strain of Aeromonas veronii UDRT09. Six hundred red tilapia (22 ± 1.8 g) were randomly allocated into four experimental groups: control, empty-polymeric nanoparticle (PC), formalin-killed vaccine (FKV), and chitosan polymer-based nanovaccine (CS-NV) in triplicate. The specific IgM antibody levels and their bactericidal activity were assessed in serum and mucus for 28 days after immersion vaccination and followed by immersion challenge with A. veronii. The immersion vaccine was found to be safe for red tilapia, with no mortalities occurring during the vaccination procedure. The specific IgM antibody levels and bactericidal activity against A. veronii in both serum and mucus were significantly higher in red tilapia vaccinated with CS-NV compared to the FKV and control groups at all time points. Furthermore, the serum lysozyme activity, ACH50, and total Ig levels demonstrated a significant elevation in the groups vaccinated with CS-NV compared to the FKV and control groups. Importantly, the Relative Percentage Survival (RPS) value of the CS-NV group (71 %) was significantly higher than that of the FKV (15.12 %) and PC (2.33 %) groups, respectively. This indicates that the chitosan polymer-based nanovaccine platform is an effective delivery system for the immersion vaccination of tilapia.

Modification of Physiochemical and Techno-Functional Properties of Stink Bean (Parkia speciosa) by Germination and Hydrothermal Cooking Treatment.

Stink bean, Parkia speciosa, is recognized as a significantly underutilized legume with versatile utility and diverse benefits. However, information on the impact of different processing methods, such as germination and hydrothermal cooking, is scarce on stink beans (SBs). Therefore, the current research aimed to explore the efficacy of germination (G) and hydrothermal cooking (HTC) on the physiochemical properties, proximate composition, techno-functional properties, and antioxidant potential of SB flour. Furthermore, Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) were employed to assess structural and morphological changes. The results revealed that the physiochemical properties of SB were significantly enhanced through processing, with more pronounced improvements observed during germination. Additionally, SBG exhibited a significantly higher protein content and lower fat content compared to SBHTC and stink bean raw (SBR). Moreover, techno-functional properties such as color intensity, least gelation concentration, and pasting properties were significantly improved in SBG compared to SBHTC and SBR. FTIR analysis of SBG and SBHTC indicated structural modifications in the lipid, protein, and carbohydrate molecules. FESEM examination revealed morphological changes in SBG and SBHTC when compared to SBR. Importantly, SBG exhibited higher antioxidant activity and total phenolic content in comparison to SBHTC and SBR. Therefore, processed SB flour can be incorporated and utilized in product development, highlighting its potential as a plant-based protein source for protein-rich breakfast bars and cookies.

CD4+ T lymphocyte responses to viruses and virus-relevant stimuli in teleost fish.

Fish diseases caused by viruses are a major threat to aquaculture. Development of disease protection strategies for sustainable fish aquaculture requires a better understanding of the immune mechanisms involved in antiviral defence. The innate and adaptive arms of the vertebrate immune system collaborate to mount an effective defence against viral pathogens. The T lymphocyte components of the adaptive immune system, comprising two major classes (helper T, Th or CD4+ and cytotoxic T lymphocytes, CTLs or CD8+ T cells), are responsible for cell-mediated immune responses. In particular, CD4+ T cells and their different subsets orchestrate the actions of various other immune cells during immune responses, making CD4+ T cells central drivers of responses to pathogens and vaccines. CD4+ T cells are also present in teleost fish. Here we review the literature that reported the use of antibodies against CD4 in a few teleost fish species and transcription profiling of Th cell-relevant genes in the context of viral infections and virus-relevant immunomodulation. Studies reveal massive CD4+ T cell proliferation and expression of key cytokines, transcription factors, and effector molecules that evoke mammalian Th cell responses. We also discuss gaps in the current understanding and evaluation of teleost CD4+ T cell responses and how development and application of novel tools and approaches to interrogate such responses could bridge these gaps. A greater understanding of fish Th cell responses will further illuminate the evolution of vertebrate adaptive immunity, inform strategies to address viral infections in aquaculture, and could further foster fish as model organisms.

Addressing Nanovaccine Strategies for Tilapia.

Tilapia is the world's most extensively farmed species after carp. It is an attractive species for aquaculture as it grows quickly, reaching harvest size within six to seven months of production, and provides an important source of food and revenue for many low-income families, especially in low- to middle-income countries. The expansion of tilapia aquaculture has resulted in an intensification of farming systems, and this has been associated with increased disease outbreaks caused by various pathogens, mostly bacterial and viral agents. Vaccination is routinely used to control disease in higher-value finfish species, such as Atlantic salmon. At the same time, many tilapia farmers are often unwilling to vaccinate their fish by injection once the fish have been moved to their grow-out site. Alternative vaccination strategies are needed to help tilapia farmers accept and use vaccines. There is increasing interest in nanoparticle-based vaccines as alternative methods for delivering vaccines to fish, especially for oral and immersion administration. They can potentially improve vaccine efficacy through the controlled release of antigens, protecting antigens from premature proteolytic degradation in the gastric tract, and facilitating antigen uptake and processing by antigen-presenting cells. They can also allow targeted delivery of the vaccine at mucosal sites. This review provides a brief overview of the bacterial and viral diseases affecting tilapia aquaculture and vaccine strategies for farmed tilapia. It focuses on the use of nanovaccines to improve the acceptance and uptake of vaccines by tilapia farmers.

Immune responses to Tilapia lake virus infection: what we know and what we don't know.

Tilapia lake virus (TiLV) is a novel contagious pathogen associated with a lethal disease affecting and decimating tilapia populations on several continents across the globe. Fish viral diseases, such as Tilapia lake virus disease (TiLVD), represent a serious threat to tilapia aquaculture. Therefore, a better understanding of the innate immune responses involved in establishing an antiviral state can help shed light on TiLV disease pathogenesis. Moreover, understanding the adaptive immune mechanisms involved in mounting protection against TiLV could greatly assist in the development of vaccination strategies aimed at controlling TiLVD. This review summarizes the current state of knowledge on the immune responses following TiLV infection. After describing the main pathological findings associated with TiLVD, both the innate and adaptive immune responses and mechanisms to TiLV infection are discussed, in both disease infection models and in vitro studies. In addition, our work, highlights research questions, knowledge gaps and research areas in the immunology of TiLV infection where further studies are needed to better understand how disease protection against TiLV is established.

Oral delivery of a Streptococcus agalactiae vaccine to Nile tilapia (Oreochromis niloticus) using a novel cationic-based nanoemulsion containing bile salts.

Streptococcus agalactiae is one of Thailand's most important pathogens in tilapia aquaculture. Vaccination is a very effective method for protecting fish against disease in aquaculture. Oral vaccination is an interesting route for vaccine delivery as it mimics the pathogenesis of S. agalactiae and provides convenient administration for mass vaccination of fish. Moreover, gut mucosal immunity is associated with a mucus layer on the gastrointestinal tract. Therefore, this study aimed to develop a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS) and determined its physicochemical characterization, morphology, in vitro mucoadhesive property, permeability, and acid-base tolerance. In addition, the efficacy of NEB-CS as an oral vaccination for Nile tilapia was evaluated in order to investigate the innate immune response and protection against S. agalactiae. The groups of fish consisted of: (1) deionized water as a non-vaccinated control (Control); (2) an inactivated vaccine formulated from formalin-killed bacteria (IB); and (3) a novel cationic-based nanoemulsion vaccine containing bile salts (NEB) coated by chitosan (CS). The control, IB, and NEB-CS were incorporated into commercial feed pellets and fed to Nile tilapia. In addition, we evaluated the serum bactericidal activity (SBA) for 14 days post-vaccination (dpv) and protective efficacy for 10 days post-challenge, respectively. The mucoadhesiveness, permeability, and absorption within the tilapia intestine were also assessed in vivo. The NEB-CS vaccine appeared spherical, with the nanoparticles having a size of 454.37 nm and a positive charge (+47.6 mV). The NEB-CS vaccine had higher levels of mucoadhesiveness and permeability than the NEB (p < 0.05). The relative percent survival (RPS) of IB and NEB-CS, when administered orally to fish, was 48% and 96%, respectively. Enhanced SBA was noted in the NEB-CS and IB vaccine groups compared to the control group. The results demonstrate that a feed-based NEB-CS can improve the mucoadhesiveness, permeability, and protective efficacy of the vaccine, and appear to be a promising approach to protecting tilapia in aquaculture against streptococcosis.

Development of a bivalent mucoadhesive nanovaccine to prevent francisellosis and columnaris diseases in Nile tilapia (Oreochromis niloticus).

The occurrence of francisellosis caused by Francisella orientalis sp. nov. (Fo) and columnaris disease caused by Flavobacterium oreochromis (For) is negatively impacting Nile tilapia (Oreochromis niloticus) production, especially when high stocking densities are used. A new and innovative bivalent mucoadhesive nanovaccine was developed in this study for immersion vaccination of tilapia against francisellosis and columnaris disease. It was shown to have the potential to improve both innate and adaptive immunity in vaccinated Nile tilapia. It increased innate immune parameters, such as lysozyme activity, bactericidal activity, phagocytosis, phagocytic index, and total serum IgM antibody levels. Additionally, the vaccine was effective in elevating specific adaptive immune responses, including IgM antibody levels against Fo and For vaccine antigens and upregulating immune-related genes IgM, IgT, CD4+, MHCIIα, and TCRß in the head kidney, spleen, peripheral blood leukocytes, and gills of vaccinated fish. Furthermore, fish vaccinated with the mucoadhesive nanovaccine showed higher survival rates and relative percent survival after being challenged with either single or combined infections of Fo and For. This vaccine is anticipated to be beneficial for large-scale immersion vaccination of tilapia and may be a strategy for shortening vaccination times and increasing immune protection against francisellosis and columnaris diseases in tilapia aquaculture.

Masculinization of Red Tilapia (Oreochromis spp.) Using 17α-Methyltestosterone-Loaded Alkyl Polyglucosides Integrated into Nanostructured Lipid Carriers.

The aim of the present study was to optimize a masculinization platform for the production of all-male red tilapia fry by oral administration of 30 and 60 ppm of MT and alkyl polyglucoside nanostructured lipid carriers (APG-NLC) loaded with MT, respectively, for 14 and 21 days. The characterization, encapsulation efficiency and release kinetics of MT in lipid-based nanoparticles were assessed in vitro. The results showed that the MT-loaded nanoparticles were spherical, ranging from 80 to 125 nm in size, and had a negative charge with a narrow particle distribution. The APG-NLC loaded with MT provided higher physical stability and encapsulation efficacy than the NLC. The release rate constants of MT from MT-NLC and MT-APG-NLC were higher than those of free MT, which is insoluble in aqueous media. There was no significant difference in survival between the fish administered MT or the those fed orally with MT-APG-NLC fish. According to the logistic regression analysis, the sex reversal efficacy of MT-APG-NLC (30 ppm) and MT (60 ppm), resulted in significantly higher numbers of males after 21 days of treatment compared with the controls. The production cost of MT-APG-NLC (30 ppm) after 21 days of treatment was reduced by 32.9% compared with the conventional MT treatment group (60 ppm). In all the treatments, the length-weight relationship (LWR) showed negatively allomeric growth behavior (b < 3), with a relative condition factor (Kn) of more than 1. Therefore, MT-APG-NLC (30 ppm) would seem to be a promising, cost-effective way to reduce the dose of MT used for the masculinization of farmed red tilapia.

Prospective Longitudinal Study of Putative Agents Involved in Complex Gill Disorder in Atlantic salmon (Salmo salar).

Complex gill disorder (CGD) is an important condition in Atlantic salmon aquaculture, but the roles of the putative aetiological agents in the pathogenesis are uncertain. A longitudinal study was undertaken on two salmon farms in Scotland to determine the variations in loads of CGD-associated pathogens (Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, salmon gill pox virus (SGPV) and Neoparamoeba perurans) estimated by quantitative PCR. In freshwater, Ca. B. cysticola and SGPV were detected in both populations, but all four pathogens were detected on both farms during the marine stage. Candidatus B. cysticola and D. lepeophtherii were detected frequently, with SGPV detected sporadically. In the marine phase, increased N. perurans loads associated significantly (p < 0.05) with increases in semi-quantitative histological gill-score (HGS). Increased Ca. B. cysticola load associated significantly (p < 0.05) with increased HGS when only Farm B was analysed. Higher loads of D. lepeophtherii were associated significantly (p < 0.05) with increased HGS on Farm B despite the absence of D. lepeophtherii-type microvesicles. Variations in SGPV were not associated significantly (p > 0.05) with changes in HSG. This study also showed that water temperature (season) and certain management factors were associated with higher HGS. This increase in histological gill lesions will have a deleterious impact on fish health and welfare, and production performance.

Antimicrobial Resistance, Pathogenic, and Molecular Characterization of Escherichia coli from Diarrheal Patients in South Korea.

Diarrheal diseases due to foodborne Escherichia coli are the leading cause of illness in humans. Here, we performed pathogenic typing, molecular typing, and antimicrobial susceptibility tests on seventy-five isolates of E. coli isolated from stool samples of patients suffering from foodborne diseases in Busan, South Korea. All the isolates were identified as E. coli by both biochemical analysis (API 20E system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The bacteria displayed entero-pathogenic E. coli (EPEC) (47.0%), entero-aggregative E. coli (EAEC) (33.3%), entero-toxigenic E. coli (ETEC) (6.6%), ETEC and EPEC (6.6%), EPEC and EAEC (4%), and ETEC and EAEC (2.7%) characteristics. The E. coli isolates were highly resistant to nalidixic acid (44.0%), tetracycline (41.3%), ampicillin (40%), ticarcillin (38.7%), and trimethoprim/sulfamethoxazole (34.7%); however, they were highly susceptible to imipenem (98.6%), cefotetan (98.6%), cefepime (94.6%), and chloramphenicol (94.6%). Although 52 strains (69.3%) showed resistance against at least 1 of the 16 antibiotics tested, 23 strains (30.7%) were susceptible to all the antibiotics. Nine different serotypes (O166, O8, O20, O25, O119, O159, O28ac, O127a, and O18), five genotypes (I to V, random-amplified polymorphic DNA), and four phenotypes (A to D, MALDI-TOF MS) were identified, showing the high level of heterogeneity between the E. coli isolates recovered from diarrheal patients in South Korea.

Novel DNA-based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues.

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D. lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D. lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D. lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D. lepeophtherii spores were significantly associated (p < .05) with the development of D. lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

Immunization of Nile Tilapia (Oreochromis niloticus) Broodstock with Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer.

Tilapia lake virus (TiLV), a major pathogen of farmed tilapia, is known to be vertically transmitted. Here, we hypothesize that Nile tilapia (Oreochromis niloticus) broodstock immunized with a TiLV inactivated vaccine can mount a protective antibody response and passively transfer maternal antibodies to their fertilized eggs and larvae. To test this hypothesis, three groups of tilapia broodstock, each containing four males and eight females, were immunized with either a heat-killed TiLV vaccine (HKV), a formalin-killed TiLV vaccine (FKV) (both administered at 3.6 × 106 TCID50 per fish), or with L15 medium. Booster vaccination with the same vaccines was given 3 weeks later, and mating took place 1 week thereafter. Broodstock blood sera, fertilized eggs and larvae were collected from 6-14 weeks post-primary vaccination for measurement of TiLV-specific antibody (anti-TiLV IgM) levels. In parallel, passive immunization using sera from the immunized female broodstock was administered to naïve tilapia juveniles to assess if antibodies induced in immunized broodstock were protective. The results showed that anti-TiLV IgM was produced in the majority of both male and female broodstock vaccinated with either the HKV or FKV and that these antibodies could be detected in the fertilized eggs and larvae from vaccinated broodstock. Higher levels of maternal antibody were observed in fertilized eggs from broodstock vaccinated with HKV than those vaccinated with FKV. Low levels of TiLV-IgM were detected in some of the 1-3 day old larvae but were undetectable in 7-14 day old larvae from the vaccinated broodstock, indicating a short persistence of TiLV-IgM in larvae. Moreover, passive immunization proved that antibodies elicited by TiLV vaccination were able to confer 85% to 90% protection against TiLV challenge in naïve juvenile tilapia. In conclusion, immunization of tilapia broodstock with TiLV vaccines could be a potential strategy for the prevention of TiLV in tilapia fertilized eggs and larvae, with HKV appearing to be more promising than FKV for maternal vaccination.

Membrane vesicles from antibiotic-resistant Staphylococcus aureus transfer antibiotic-resistance to antibiotic-susceptible Escherichia coli.

AIM: Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi-drug resistant bacteria (MDR) by delivering antibiotic-resistant substances to antibiotic-susceptible bacteria. Here, we aim to show that MVs from Gram-positive bacteria are capable of transferring ß-lactam antibiotic-resistant substances to antibiotic-sensitive Gram-negative bacteria. MATERIALS AND METHODS: MVs were collected from a methicillin-resistant strain of Staphylococcus aureus (MRSA) and vesicle-mediated fusion with antimicrobial-sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial-resistant E. coli (RC85-T). RESULTS: The RC85-T exhibited a higher resistance to ß-lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85-T and the parent strain were nearly equal, the ß-lactamase activity of the MVs from RC85-T was 12-times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85-T were able to protect ß-lactam-susceptible E. coli from ß-lactam antibiotic-induced growth inhibition in a dose-dependent manner. CONCLUSION: MVs play a role in transferring substances from Gram-positive to Gram-negative bacteria, shown by the release of MVs from RC85-T that were able to protect ß-lactam-susceptible bacteria from ß-lactam antibiotics. SIGNIFICANCE AND IMPACT OF STUDY: MVs are involved in the emergence of antibiotic-resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug-resistant bacteria could be reduced.

Immersion Vaccination by a Biomimetic-Mucoadhesive Nanovaccine Induces Humoral Immune Response of Red Tilapia (Oreochromis sp.) against Flavobacterium columnare Challenge.

Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-ß genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish.

Mediterranean Aquaculture in a Changing Climate: Temperature Effects on Pathogens and Diseases of Three Farmed Fish Species.

Climate change is expected to have a drastic effect on aquaculture worldwide. As we move forward with the agenda to increase and diversify aquaculture production, rising temperatures will have a progressively relevant impact on fish farming, linked to a multitude of issues associated with fish welfare. Temperature affects the physiology of both fish and pathogens, and has the potential to lead to significant increases in disease outbreaks within aquaculture systems, resulting in severe financial impacts. Significant shifts in future temperature regimes are projected for the Mediterranean Sea. We therefore aim to review and discuss the existing knowledge relating to disease outbreaks in the context of climate change in Mediterranean finfish aquaculture. The objective is to describe the effects of temperature on the physiology of both fish and pathogens, and moreover to list and discuss the principal diseases of the three main fish species farmed in the Mediterranean, namely gilthead seabream (Sparus aurata), European seabass (Dicentrarchus labrax), and meagre (Argyrosomus regius). We will attempt to link the pathology of each disease to a specific temperature range, while discussing potential future disease threats associated with the available climate change trends for the Mediterranean Sea.