Resistance of Nile tilapia fed with Padina boergesenii extract to Pseudomonas putida infection.

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1ß), beta-defensin (ß-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection.

Virus-like particles displaying the mature C-terminal domain of filamentous hemagglutinin are immunogenic and protective against Bordetella pertussis respiratory infection in mice.

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA's mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge.

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Immunization with an mRNA DTP vaccine protects against pertussis in rats.

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo. We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.

Nontypeable Haemophilus influenzae challenge during gammaherpesvirus infection enhances viral reactivation and latency.

Gammaherpesviruses are ubiquitous, lifelong pathogens associated with multiple cancers that infect over 95% of the adult population. Increases in viral reactivation, due to stress and other unknown factors impacting the immune response, frequently precedes lymphomagenesis. One potential stressor that could promote viral reactivation and increase viral latency would be the myriad of infections from bacterial and viral pathogens that we experience throughout our lives. Using murine gammaherpesvirus 68 (MHV68), a mouse model of gammaherpesvirus infection, we examined the impact of bacterial challenge on gammaherpesvirus infection. We challenged MHV68 infected mice during the establishment of latency with nontypeable Haemophilus influenzae (NTHi) to determine the impact of bacterial infection on viral reactivation and latency. Mice infected with MHV68 and then challenged with NTHi, saw increases in viral reactivation and viral latency. These data support the hypothesis that bacterial challenge can promote gammaherpesvirus reactivation and latency establishment, with possible consequences for viral lymphomagenesis.

Immunomodulatory effects of single and combined exposure to ZnO and TiO2 nanoparticles on rainbow trout challenged with Aeromonas salmonicida achromogenes.

The increasing release of engineered nanoparticles (ENPs) in aquatic ecosystems stresses the need for stringent investigations of nanoparticle mixture toxicity towards aquatic organisms. Here, the individual and combined immunotoxicity of two of the most consumed ENPs, the ZnO and the TiO2 ones, was investigated on rainbow trout juveniles (Oncorhynchus mykiss). Fish were exposed to environmentally realistic concentrations (21 and 210 µg L-1 for the ZnO and 210 µg L-1 for the TiO2) for 28 days, and then challenged with the pathogenic bacterium, Aeromonas salmonicida achromogenes. Antioxidant and innate immune markers were assessed before and after the bacterial infection. None of the experimental conditions affected the basal activity of the studied innate immune markers and the redox balance. However, following the bacterial infection, the expression of genes coding for pro and anti-inflammatory cytokines (il1ß and il10), as well as innate immune compounds (mpo) were significantly reduced in fish exposed to the mixture. Conversely, exposure to ZnO NPs alone seemed to stimulate the immune response by enhancing the expression of the IgM and c3 genes for instance. Overall, our results suggest that even though the tested ENPs at their environmental concentration do not strongly affect basal immune functions, their mixture may alter the development of the immune response when the organism is exposed to a pathogen by interfering with the inflammatory response.

The Potential of Fish Protein Hydrolysate Supplementation in Nile Tilapia Diets: Effects on Growth and Health Performance, Disease Resistance, and Farm Economic Analysis.

Fish protein hydrolysate (FPH) has shown immense potential as a dietary protein supplement and immunostimulant in aquaculture, especially in Nile tilapia production. Four isoproteic diets (30% crude protein) were prepared by including FPH at varying percentages (0%, 0.5%, 1%, and 2%). Nile tilapia fed with FPH diets for 90 days, and their growth performance, feed utilization, blood biochemistry, liver and gut morphology, and resistance against Streptococcus iniae were investigated. The findings revealed that diets physical attributes such as pellet durability index and water stability were remarkably (p < 0.05) varied between experimental diet groups. Furthermore, the test diets were more palatable when FPH was included at 1% and 2%. Fish that were fed with a 2% FPH-treated diet had significantly (p < 0.05) greater growth indices than other treatments. Additionally, their feed utilization was significantly (p < 0.05) improved. The experimental diets and intestinal total bacteria count (TBC) exhibited a rising trend with FPH levels, where the 2% FPH-treated diet recorded the highest TBC. Neutrophil (109/L), lymphocyte (109/L), eosinophil (109/L), and red blood cell(1012/L) counts were significantly (p < 0.05) higher in the 2% FPH-treated group, while the white blood cell (109/L), and basophil (109/L) counts were not influenced by the FPH inclusion. Moreover, the FPH-treated groups displayed lower creatinine, bilirubin, and urea levels than the control. The histological examination demonstrated that themid-intestine of 2% FPH-fed Nile tilapia had an unbroken epithelial wall, more villi with frequent distribution of goblet cells, wider tunica muscularis, and stronger stratum compactum bonding than other treatments. Additionally, this group exhibited more nuclei and erythrocytes and less vacuolar cytoplasm in liver than their counterparts. Nile tilapia that were given a diet containing 2% FPH had significantly (p < 0.05) higher resistance (83.33%) to S. iniae during the bacterial challenge test. A significant (p < 0.05) enhancement in farm economic efficiency was observed in the higher inclusion of FPH in diets. In summary, 2% FPH supplementation in Nile tilapia diets improved their growth performance, feed utilization, health status, disease resistance, and farm economic efficiency.

BECC438b TLR4 agonist supports unique immune response profiles from nasal and muscular DTaP pertussis vaccines in murine challenge models.

The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.

Susceptibility of the cultured Amazonian fish, Colossoma macropomum, to experimental infection with Aeromonas species from ornamental fish.

The global ornamental fish trade carries important risk factors for spreading pathogens between different countries and regions, not only for ornamental fish but also for cultured fish and even other animal species. In the current study, we reported the capacity of Aeromonas veronii and A. hydrophila isolated from ornamental fish to experimentally infect the reared Amazonian fish Colossoma macropomum. For this, those bacteria were identified, and a primary characterization was performed. Fish were inoculated with 0.1 mL of increasing concentrations of A. hydrophila or A. veronii (C1 = 1 × 102; C2 = 1.8 × 104; C3 = 2.1 × 106; C4 = 2.4 × 108 bacterial cells per mL) in the coelomic cavity. In the control group, fish received the same volume of sterile saline solution (0.9 %). Fish presented petechiae, skin suffusions, and mortality rates up to 100 % according to the inoculum concentration. Histopathologically, fish presented necrosis with karyolysis, loss of the cytoplasmic delimitation of cells of the renal tubules and hepatocytes, hemorrhage, cellular edema, and the presence of bacterial cells. The LD50-96h of A. veronii on C. macropomum was estimated at 2.4 × 106 CFU mL-1 and of A. hydrophila at 1.408 × 105 CFU mL-1. The results demonstrated that it is possible that Aeromonas species isolated from ornamental fish affect C. macropomum, causing similar clinical signs and lesions. This shows the importance of promoting risk control measures worldwide regarding the trade of ornamental fish.

Dietary ARA, DHA, and Carbohydrate Ratios Affect the Immune Status of Gilthead Sea Bream Juveniles upon Bacterial Challenge.

This study aims to assess the effects of different dietary n-6/n-3 long-chain polyunsaturated fatty acid ratios and CHO content in the immune response of gilthead seabream. For that purpose, gilthead sea bream juveniles (initial body weight = 47.5 g) were fed for 84 days with four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets with high (20%) or low (5%) level of gelatinized starch (HS or LS diets, respectively) and included approximately 2.4% ARA or DHA. At the end of the trial, the DHA-enriched groups presented increased red blood cell (RBC) count, hemoglobin, plasmatic nitric oxide (NO) content, and antiprotease and alternative complement activities. The ARA groups had increased thrombocyte count, and plasmatic bactericidal activity against Vibrio anguillarum was lower in the fish fed the ARA/LS diet. After the feeding trial, the fish were challenged with an intraperitoneal injection (i.p.) of killed Photobacterium damselae subsp. piscicida (Phdp) and sampled at 4 and 24 h after the challenge. At 4 h after i.p., the ARA groups presented increased plasma total immunoglobulins (Ig) and bactericidal activity against V. anguillarum. In addition, the fish fed the ARA/LS diet presented lower white blood cell (WBC) and alternative complement activity. At 24 h after i.p., the ARA groups presented increased RBC, WBC, and thrombocyte numbers, total IG, plasma peroxidase activity, and casp3 expression in the distal intestine. The HS groups presented increased plasma NO content and bactericidal activity against Phdp and decreased protease, antiprotease activity, and bactericidal activity against V. anguillarum. In conclusion, high dietary DHA levels seemed to improve the immune status of unchallenged gilthead sea bream juveniles, while high dietary ARA levels improved the fish immune response to a bacterial challenge. The energy provided by dietary starch seems to be important to promote a fast response by the fish immune system after a challenge.

Molecular cloning and functional characterization of peroxiredoxin 4 (prx 4) in freshwater crayfish, Procambarus clarkii.

Peroxiredoxin (Prx), which is a newly discovered member of the antioxidant protein family, performs important biological functions in intracellular signal transduction. In the present study, a peroxiredoxin 4 gene was cloned from crayfish for the first time and named Pc-prx 4. According to the amino acid sequence signature, Pc-Prx 4 was identified as the typical 2-Cys Prx molecule, which possessed two conserved cysteines (Cys98 and Cys219). Time-course expression patterns post V. harveyi infection revealed that Pc-prx 4 was likely related to crayfish innate immune defense responses. In particular, the highest fold upregulation of the Pc-prx 4 mRNA transcript reached approximately 170 post V. harveyi infection in the crayfish hepatopancreas. The results of the mixed functional oxidase assay showed that rPc-Prx 4△ could resist the damaging effect of reactive oxygen species generated from the thiol/Fe3+/O2- reaction system to some extent. In addition, the results of the RNAi assay revealed that the crayfish survival rate was obviously increased post injection of V. harveyi when Pc-prx 4 was knocked down. Further study revealed that both hemolymph melanization and PO activity were strengthened to different degrees in the RNAi assay. Therefore, we speculated that the increase in the crayfish survival rate was likely due to the increase in hemolymph melanization. The obviously reinforced hemolymph melanization was directly caused by the upregulation of hemolymph PO activity, which was induced by the knockdown of Pc-prx 4. However, further studies are still indispensable for illuminating the molecular mechanism of Pc-prx 4 in the crayfish innate immune defense system.

The role of Nrf2 signaling pathway in the mud crab (Scylla paramamosain) in response to Vibrio parahaemolyticus infection.

The transcription factor Nrf2 plays vital roles in detoxification and antioxidant enzymes against oxidative stress. However, the function of Nrf2 in crustaceans is not well studied. In this study, a novel Nrf2 gene from the mud crab (Sp-Nrf2) was identified. It was encoded 245 amino acids. Sp-Nrf2 expression was ubiquitously expressed in all tested tissues, with the highest expression level in the gill. Sp-Nrf2 protein was mainly located in the nucleus. The expression levels of Sp-Nrf2, and antioxidant-related genes (HO-1 and NQO-1) were induced after Vibrio parahaemolyticus infection, indicating that Nrf2 signaling pathway was involved in the responses to bacterial infection. Over-expression of Sp-Nrf2 could improve cell viability after H2O2 exposure, indicating that Sp-Nrf2 might relieve oxidative stress. Silencing of Sp-Nrf2 in vivo decreased HO-1 and NQO-1 expression. Moreover, knocking down Sp-Nrf2 in vivo can increase malondialdehyde content and the mortality of mud crabs after V. parahaemolyticus infection. Our results indicated that Nrf2 signaling pathway played a significant role in immune response against bacterial infection.

Exploring the Effect of Functional Diets Containing Phytobiotic Compounds in Whiteleg Shrimp Health: Resistance to Acute Hepatopancreatic Necrotic Disease Caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis (AHPND) is an emerging severe disease caused by strains of Vibrio parahaemolyticus (VpAHPND) in whiteleg shrimp (Litopenaeus vannamei). Mitigating its negative impact, and at the same time minimizing antibiotics treatments, is the major challenge in shrimp aquaculture. A sustainable strategy could be to include immunostimulants in diet. Phytobiotics, harmless plant extracts with immunostimulatory and biocidal activities, are promising candidates. In this study, we evaluated the effectiveness of two diets (E and F) supplemented with phytobiotics (functional diets) in terms of protecting shrimp against AHPND. For this purpose, groups of animals were fed functional or control diets for 4 and 5 weeks and, subsequently, they were challenged with VpAHPND by immersion. We compared the mortality in infected groups and estimated the percentage of carriers by using a specific qPCR in hepatopancreas tissue. The results showed that mortality was significantly lower in the group fed functional diet E and, after a 5-week feeding schedule. This group also showed the lowest percentage of carriers. The pathological effects were also reduced with diet F. Thus, feeding shrimp with phytobiotic-enriched diets in critical periods will be highly beneficial because it increases the host's resistance to AHPND pathology.

Antioxidant, Immunostimulant, and Growth-Promoting Effects of Dietary Annona squamosa Leaf Extract on Nile Tilapia, Oreochromis niloticus, and Its Tolerance to Thermal Stress and Aeromonas sobria Infection.

Plant extracts are a phytochemically-rich alternative to antibiotic and synthetic feed additives, with high systemic bioactivity in animals. The present study aimed to evaluate the effect of a hydroalcoholic extract of custard apple (Annona squamosa) leaf (ASLE) on the growth, hematobiochemical parameters, digestive enzyme activities, redox status, nonspecific immune response, and cold and bacterial infection tolerance in Nile tilapia (Oreochromis niloticus). A total of 300 Nile tilapia fingerlings (11.87 ± 0.48 g) were fed ASLE-supplemented diets at increasing levels of 0, 5, 10, 15, and 20 g/kg for 60 days. At the end of the feeding period, the fish were experimentally challenged with cold water stress or Aeromonas sobria, and mortalities were recorded for 10 days. The results revealed that the growth performance and feed conversion ratio were significantly improved with an increasing level of ASLE supplementation. The hematologic profile and hepato-renal functions were retained within a healthy range in the various groups supplemented with an ASLE diet. Antioxidant status was significantly improved in the serum of fish fed ASLE-supplemented diets, in terms of superoxide dismutase (SOD), catalase (CAT) activities, reduced glutathione, and total antioxidant capacity. Meanwhile, the myeloperoxidase (MPO) and malondialdehyde (MDA) levels decreased significantly. Similarly, there was a noticeable improvement in the hepatic CAT and SOD activities and a reduction of hepatic MDA. Marked improvements in lysozyme activity, nitric oxide production, complement3 level, and phagocytic activity were recorded in groups fed ASLE-supplemented diets, which peaked with the 20 g ASLE/kg diet. Moreover, the serum glucose and cortisol levels significantly declined in groups fed ASLE at levels of 15-20 g/kg compared to the other groups. Supplementation with ASLE increased the activities of protease, lipase, and α-amylase. ASLE supplementation at a concentration of 10-20 g/kg diet enhanced the resistance of Nile tilapia to A. sobria infection. According to this study, ASLE supplementation enhanced the antioxidant balance, non-specific immune response, physiological status, resistance against infection, and growth performance of Nile tilapia at supplementation levels of 10-20 g/kg diet.

The Pathogen Aeromonas salmonicida achromogenes Induces Fast Immune and Microbiota Modifications in Rainbow Trout.

Environmental stressors can disrupt the relationship between the microbiota and the host and lead to the loss of its functions. Among them, bacterial infection caused by Aeromonas salmonicida, the causative agent of furunculosis, results in high mortality in salmonid aquaculture. Here, rainbow trout were exposed to A. salmonicida achromogenes and its effects on the taxonomic composition and structure of the microbiota was assessed on different epithelia (gills, skin, and caudal fin) at 6 and 72 h post-infection (hpi) using the V1-V3 region of the 16S rRNA sequencing. Moreover, the infection by the pathogen and immune gene responses were evaluated in the head kidney by qPCR. Our results suggested that α-diversity was highly diverse but predominated by a few taxa while ß-diversity was affected very early by infection in the gills after 6 h, subsequently affecting the microbiota of the skin and caudal fin. A dysbiosis of the microbiota and an increase in genera known to be opportunistic pathogens (Aeromonas, Pseudomonas) were also identified. Furthermore, an increase in pro-inflammatory cytokines and virulence protein array (vapa) was observed in trout head kidney as soon as 6 hpi and remained elevated until 72 hpi, while the anti-inflammatory genes seemed repressed. This study suggests that the infection by A. salmonicida achromogenes can alter fish microbiota of gills in the few hours post-infection. This result can be useful to develop a non-invasive technique to prevent disease outbreak in aquaculture.

Silencing of the immune gene BmPGRP-L4 in the midgut affects the growth of silkworm (Bombyx mori) larvae.

Peptidoglycan recognition proteins (PGRPs) are one of the receptors in insects' immune pathways, essential for insects to recognize the exogenous pathogens in order to activate the Toll and immune deficiency (IMD) pathway. In the silkworm Bombyx mori, previous studies focused on the short PGRPs and less is known about the long PGRPs. In this study, a long PGRP in silkworm BmPGRP-L4 was cloned and its expression and function were analysed. The results showed that BmPGRP-L4 contains a transmembrane region, a conserved PGRP domain, and an amidase-2 domain. The expression profile demonstrated that BmPGRP-L4 existed in diverse tissues including epidermis, fat body, midgut, and silk glands, with remarkably high expression in the midgut in the 5th instar. Oral infection with Escherichia coli and Staphylococcus aureus significantly induced BmPGRP-L4 in the midgut and epidermis, as well as in the fat body and silk glands. Peptidoglycan also induced the expression of BmPGRP-L4 in midgut tissue ex vivo and BmN4 cells in vitro. RNAi of BmPGRP-L4 was effective in the midgut and epidermis, while the efficiency in the fat body was transient. RNAi-mediated knock-down of BmPGRP-L4 reduced the weight and growth of the silkworm, possibly due to its participation in the immune response and the regulation of the microbiota in the midgut lumen of the silkworm larvae.

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5' upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Physiological Performance, Antioxidant and Immune Status, Columnaris Resistance, and Growth of Nile Tilapia That Received Alchemilla vulgaris-Supplemented Diets.

The current perspective is a pioneering trial to assess the efficacy of the dietary supplementation of Alchemilla vulgaris powder (AVP) in the diet of Nile tilapia (Oreochromis niloticus) on growth performance, blood picture, hepatic and renal biomarkers, immune status, and serum and tissue antioxidant capacity and to investigate the resistance against Flavobacterium columnare challenge. Fish (n = 360) were distributed into six groups (three replicates each) and received increasing AVP supplementation levels (0, 2, 4, 6, 8, and 10 g kg-1) for 60 days. Furthermore, fish were exposed to the bacterial challenge of a virulent F. columnare strain and maintained under observation for 12 days. During the observation period, clinical signs and the cumulative mortality percentage were recorded. The results demonstrated that the growth performance, feed conversion ratio, and hematological profile were noticeably enhanced in the AVP-supplemented groups compared to the control. The most promising results of weight gain and feed conversion ratio were recorded in the groups with 6, 8, and 10 g AVP kg-1 diets in a linear regression trend. The levels of hepatorenal function indicators were maintained in a healthy range in the different dietary AVP-supplemented groups. In a dose-dependent manner, fish fed AVP dietary supplements displayed significant augmented serum levels of innate immune indicators (lysozyme, nitric oxide, and complement 3) and antioxidant biomarkers (Catalase (CAT), superoxide dismutase (SOD), total antioxidant (TAC), and reduced glutathione (GSH) with a marked decrease in myeloperoxidase (MPO) and malondialdehyde (MDA) levels). Likewise, hepatic CAT and SOD activities were significantly improved, and the opposite trend was recorded with hepatic MDA. The highest AVP-supplemented dose (10 g/kg) recorded the highest immune-antioxidant status. Based on the study findings, we highlight the efficacy of AVP as a nutraceutical dietary supplementation for aquaculture to enhance growth, physiological performance, and immune-antioxidant status and as a natural economic antibacterial agent in O. niloticus for sustaining aquaculture. It could be concluded that the dietary supplementation of 10 g AVP/kg enhanced O. niloticus growth, physiological performance, immune-antioxidant status, and resistance against F. columnare.

AIF-1 and RNASET2 are involved in the inflammatory response in the Mediterranean mussel Mytilus galloprovincialis following Vibrio infection.

Filter-feeding bivalves, such as the Mytilus species, are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. Mussels lack an adaptive immune system and hemocytes can recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to activate intracellular signaling pathways to trigger the antimicrobial effectors synthesis. Among the areas of bivalve immunity that deserve study include the role of hemocyte subpopulations. Since little information are available on immune responses at the tissue level to human pathogenic vibrios commonly detected in coastal waters involved in seafood-borne diseases, in this work, immunological parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vivo challenge with Vibrio splendidus. The histological approach has been first used in order to identify the hemocytes recruitment at the infection site and the morphological change of muscular fibers. In addition, using immunolabeling with specific antibody we detected the production of molecules involved in the inflammatory activated cascade: Toll-like receptors 4 (TLR4), the myeloid differentiation factor 88 (MyD88), the Allograft inflammatory factor-1 (AIF-1) and the ribonucleases RNASET2, belonging to the T2 family, that in vertebrates are involved in the recruitment and activation of macrophages. Our results indicate the activation of TLR4 during bacterial infection preparatory to the recruitment of the MyD88 adapter with a putative role in recognition and intracellular signalling. Furthermore, the data presented in this work suggest that challenging with Gram-negative bacteria causes a massive migration of AIF-1+ hemocytes and that the ribonuclease RNASET2 could play a key role in the recruitment of these activated hemocytes. Our approach is useful for further understanding the complex molecular defence mechanisms of the host in invertebrates, especially in relation to the need to develop methods to evaluate the immunological response of bivalve molluscs used in aquaculture.

Molecular Characterization of MyD88 in Anodonta woodiana and Its Involvement in the Innate Immune Response to Bacterial Infection.

Myeloid differentiation factor 88 (MyD88) is a key adapter molecule in Toll-like receptor signal transduction that triggers downstream immune cascades involved in the host defense response to exogenous pathogens. However, the function of MyD88s in mollusks, especially in freshwater shellfish, remains poorly understood. In this study, a novel freshwater shellfish MyD88 (denoted AwMyD88) was characterized from Anodonta woodiana. The present AwMyD88 protein consists of 474 amino acids and contains a conserved a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain with three typical boxes. Quantitative real-time PCR (qRT-PCR) analysis showed that AwMyD88 was broadly expressed in all the examined tissues, and the highest expression level was observed in hemocytes of A. woodiana. When challenged with Aeromonas hydrophila and lipopolysaccharide (LPS), the mRNA expression levels of AwMyD88 were significantly induced in hemocytes of A. woodiana in vivo and in vitro. In addition, in vivo injection experiments revealed that MyD88 signaling pathway genes showed strong responsiveness to A. hydrophila challenge, and their expression levels were significantly upregulated in hemocytes. Knockdown of AwMyD88 reduced the transcript levels of immune related transcription factors (AwNF-κB and AwAP-1) and effectors (AwTNF, AwLYZ, AwDefense and AwAIF) during A. hydrophila infection. Moreover, subcellular localization analysis indicated that AwMyD88 was mainly localized to the cytoplasm in HEK293T cells. Finally, luciferase reporter assays revealed that AwMyD88 associates with AwTLR to activate the NF-κB and AP-1 signaling pathways in HEK293T cells. These results suggested that AwMyD88 might be involved in the host defense response to bacterial challenge, providing new insight into the immune function of the MyD88 signaling pathway in freshwater shellfish.