Immunostimulant effects of diet supplementation with yellow (Pouteria campechiana), white (Casimiroa edulis), and black (Diospyros digyna) sapote nanocapsules on laying hens: in vitro and in vivo study.

Poultry is commonly infected by different bacteria and parasites in the environment, resulting in increased morbidity and mortality, but immunostimulants have been enhancing non-specific defense mechanisms conferring laying hens' protection. For this purpose, the pulp of yellow (Pouteria campechiana), white (Casimiroa edulis), and black (Diospyros digyna) sapotes were nanoencapsulated (YWB-SN) and evaluated in laying hens' peripheral blood leukocytes to test their addition to the experimental diets at a concentration of 0.5% (5g/kg of dry food) for 1 month (with two samples at days 15 and 30). The YWB-SN were safe when exposed to peripheral blood leukocytes (PBLs). The in vitro experiment showed that these nanocapsules enhanced reactive oxygen species production, and B-SN stimulated phagocytosis activity. Concerning the proinflammatory cytokine (TNF-α) transcription, this gene was upregulated after W-SN stimulation, while B-SN upregulated the IgG gene expression significantly. IgM was upregulated with any YBW-SN in PBLs after 24 h of stimulation. The in vivo study showed a notable B-SN immunostimulation in serum and an upregulation of TNF-α, IgM, and IgG mRNA transcription. Therefore, this study provides a new result of the yellow, white, and black sapote nanocapsules as a functional food for the poultry industry, highlighting the black sapote Diospyros digyna immunostimulant effect.

Development, characterization, and immunomodulation performance of spray-dried Moringa oleifera seed extract in Longfin yellowtail Seriola rivoliana.

Moringa oleifera is one of the most promising plants in aquaculture because it improves the health status, zootechnical parameters and resistance against diseases. This research evaluates the physicochemical, antioxidant values of spray-dried Moringa oleifera seed extract microencapsulates obtained at 140 and 180 °C with whey protein concentrate (WPC) and maltodextrin (MD) as wall materials in two different proportions: WPC 100% and WPC-MD (3:1). Also, immune response of peripheral blood leukocytes (PBL) of Longfin yellowtail Seriola rivoliana stimulated with spray-dried Moringa oleifera seed for 24 h was assessed. The physicochemical parameters show that the recovery yield for all the treatments was of 65% and microencapsulates demonstrated to be stable in the physicochemical tests with low solubilization times and protection against humidity. For WPC-MD (3:1)/140 °C, bioactive compound retention and antioxidant potential were higher than in other combinations. The immunological test show that any treatments was non-cytotoxic against peripheral blood leukocytes. WPC-MD (3:1)/140 °C treatment enhanced immune parameters as phagocytosis, respiratory burst, myeloperoxidase activities and nitric oxide production. Immune related genes as IL-1ß and TNF-α were up-regulated in those stimulated leukocytes with WPC-MD (3:1)/140 °C. The results suggest that this combination may be a good alternative for animal health as a medicinal and immunostimulant additive.

Cylindropuntia cholla aqueous root rich in phytosterols enhanced immune response and antimicrobial activity in tilapia Oreochromis niloticus leukocytes.

"Cacti" are rich sources of phytochemicals with antioxidant activity, and their use is mainly focused on infusions in traditional medicine in Mexico. This study characterizes the chemical compounds found in Cylindropuntia cholla root by gas chromatography coupled to mass spectrometry (GC-MS) and determines the total content of polyphenols and flavonoids, as well as their antioxidant capacity. The immunostimulatory effect of aqueous C. cholla root extract (ACcr) was evaluated at concentrations of 50, 250, 500, and 1000 µg/mL in Tilapia peripheral blood leukocytes. The results obtained by the GC-MS analysis revealed the presence of phenolic acids, flavonoid and phytosterol derivatives as ß-sitosterol and campesterol. The determination of the total polyphenol and flavonoid contents indicated that ACcr is abundant in polyphenols, showing an anti-radical capacity of scavenging free radicals, such as those of hydroxyl and superoxide, as well as an increase in lipid peroxidation inhibition capacity. Stimulation of tilapia leukocytes resulted in the increase of its phagocytic activity, respiratory burst, nitric oxide production, and superoxide dismutase activity. Finally, the results obtained for the first time allowed establishing the chemical profile of ACcr and its antimicrobial activity against three important pathogenic bacteria. The potential of this root is indicated as an additive in formulating antioxidant and immunostimulant supplements for the aquaculture and pharmaceutical industry.

LptD-antigen system on gold nanoparticles: an innovative strategy in the nanovaccine development.

Nanovaccine development is a growing research field in which the development of new carriers and bioconjugation approaches is a priority. In this sense, this report describes for the first time, the development of a novel conjugate that consists of gold nanoparticles (AuNPs) obtained by a one-step synthesis using an immunogenic peptide of the Lipopolysaccharide-assembly protein LptD fromVibrio parahaemolyticusbacteria as a reducing and capping agent. The resultingLptD@AuNPscompounds were fully characterized and the results showed the high capacity of the peptide to form complexes and reduce gold ions. The reaction yield estimated was higher than 83% and the chemical integrity of the peptide on the NP surface revealed a tyrosine amino acid bonding on the AuNP surface. Furthermore, theLptD@AuNPsystem showed high colloidal stability in a wide pH range (3-11 pH values), where the hydrodynamic diameter and Zeta potential behavior were strongly influenced by the functional groups of the antigenic peptide. The cytotoxicity assays showed that the obtained system is safe for mouse leukocytes, while immunized mice withLptD@AuNPsproduced specific IgG antibodies. These encouraging results revealed the efficacy of some antigenic peptides as reducers and capping agents, in addition, opening the path to determine immunogenicity and immunoprotective efficacy of theLptD@AuNPsystem against the disease induced byVibrio parahaemolyticus.

ß-Glucan bioactivities from Cystobasidium benthicum in Totoaba macdonaldi thymus cells.

The overuse of antibiotics in aquaculture has led to serious concerns on microbial resistance and chemical residues. Novel sources of immunostimulants could help to solve this problem by stimulating the immune system to fight against pathogens. Therefore, this study aims to explore the immunostimulant potential of Cystobacidium benthicum-ß-glucans (Cb-ßG) using thymus cells from Totoaba (Totoaba macdonaldi), a recently farmed fish species in Mexico. The Cb-ßG was characterized and tested for its own antioxidant capacity. Then, a Cb-ßG safety experiment was carried out in thymus cells by evaluating the effects on immune parameters and immune-related genes. Cb-ßG had a molecular weight of 2.32 kDa, comprised of ß-1,3-1,6-glucan (53.4%), and showed strong antioxidant capacity compared to that of the positive antioxidant control. Cb-ßG had no toxic effects of thymus cells and enhanced phagocytic, respiratory burst, myeloperoxidase and superoxide activities. Additionally, immune-related genes implicated in recognition and effector functions of yeast glucans were up-regulated (Toll like receptor 2, C-type lectin family 17 member A, colony-stimulating factor 1 receptor 2, macrophage mannose receptor 1, and Interleukin-1ß). In conclusion, the glucan -characterized physically-chemically from the yeast C. benthicum (Cb-ßG)- was safe, had strong antioxidant capacity to scavenge free radicals, and stimulated immune parameters and immune-related gene expressions on thymus cells from Totoaba macdonaldi.

Composition, antioxidant capacity, intestinal, and immunobiological effects of oregano (Lippia palmeri Watts) in goats: preliminary in vitro and in vivo studies.

This study investigated Lippia palmeri Watt (oregano) phytochemical compounds, their antioxidant capacity, and immunological effects on goat peripheral blood leukocytes (PBL), and on the presence of intermediate polar compounds in goat feces fed dietary oregano. The polar and nonpolar fractions of L. palmeri W. were characterized and phytochemical contents and antioxidant capacity were determined. Twelve healthy Anglo-Nubian goats were used for the in vivo trials, which were randomly assigned to control fed with basal diet, or oregano group fed with basal diet + 2.6% (DM basis) dried oregano leaves. Goat peripheral blood leukocytes (PBL) were isolated for the in vitro study, and PBL were stimulated with oregano extracts at 100 and 150 µg/mL after 24 h. For the in vivo trial, dietary oregano (2.6% on DM basis) was evaluated in the goats for 90 days. Relatively high abundance of carvacrol and thymol phytochemical compounds was found in oregano. The highest antioxidant capacity of oregano extracts was detected at 100 and 150 µg/mL. Nitric oxide production, phagocytosis, and superoxide dismutase activities increased (p < 0.05) in stimulated PBL with oregano extracts, whereas the pro-inflammatory (TNF-α and IL-1ß) transcription and antioxidant (CAT and GPX-4) genes downregulated. In the in vivo experiment, dietary oregano enabled the detection of nine compounds found in goat feces, from which caproic (C6) was in a high relative quantity compared with the control group. Oregano has phytochemical compounds with strong antioxidant capacity that protect cells against oxidative stress damage and could modulate immune response and feces composition in goats.

Immunostimulatory and antioxidant effects of supplemental feeding with macroalga Sargassum spp. on goat kids.

Algae are a rich source of bioactive compounds and health properties that have been narrowly explored in goat production systems. The aim of this study was to determine the effect of feeding diets supplemented with Sargassum spp. on antioxidant status and immune parameters in goat kids. The diets were as follows: control (basal diet without alga), Sargassum spp. 2.5% (Ss2.5), and Sargassum spp. 5% (S5) fed over a 70-day period. A total of 11 body tissues, intestinal mucus, and blood serum were sampled at necropsy. Protein content, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), lysozyme, and anti-protease activities were determined, as well as immunoglobulin A (IgA) and immunoglobulin G (IgG). The results indicated that Sargassum spp. supplementation increased protein content in six tissues. Antioxidant activities (SOD and CAT) and immune-related (lysozyme, MPO, and anti-protease) activities were statistically higher (P < 0.05) in Sargassum spp. groups compared with control in several tissues, intestinal mucus, and serum. Imunoglobulin A levels in intestinal mucus were higher (P < 0.05) in Sargassum spp.-supplemented groups than the control group. In conclusion, diet supplementation of Sargassum spp. improves the antioxidant status and enhances the immune parameters in goats. Sargassum spp. dietary supplementation is proposed as strategy to strengthen antioxidant status and stimulate the immune system, which helps in the control of opportunistic pathogens in goats.

Oral administration of Debaryomyces hansenii CBS8339-ß-glucan induces trained immunity in newborn goats.

Beta-glucans from yeast can induce trained immunity in in vitro and in vivo models. Intraperitoneal doses of ß-glucans in mammals have shown to induce trained immunity, but the training effects of orally administering ß-glucans are unknown. Newborn goats are susceptible to infections in the neonatal stage, so the induction of trained immunity could improve animal survival. This study aimed to describe the in vitro effects of immunological training by ß-glucan from Debaryomyces hansenii (ß-Dh) on caprine monocytes, as well as its in vivo effects using oral doses on newborn goats upon challenge with lipopolysaccharide (LPS). Hence in vitro, goat monocytes trained with ß-Dh up-regulated the gene expression of macrophage surface markers (CD11b and F4/80) whereas enhanced cell survival and high phagocytic ability was found upon LPS challenge. In the in vivo experiment, newborn goats stimulated with two doses (day -7 and - 4) of ß-Dh (50 mg/kg) and challenged (day 0) with LPS showed an increase in respiratory burst activity, IL-1ß, IL-6, and TNFα production in plasma, and transcription of the macrophage surface markers. This study has demonstrated for the first time that trained immunity was induced with oral doses of ß-glucan upon LPS challenge in mammals using newborn goats.

Methylmercury, cadmium and arsenic(III)-induced toxicity, oxidative stress and apoptosis in Pacific red snapper leukocytes.

Methylmercury (MeHg), cadmium (Cd) and arsenic (As(III)) are among the most toxic metals in aquatic systems that have been associated with multiple animal and human health problems. This study investigated cytotoxic, oxidative stress, and apoptosis effects on fish leukocytes following their exposure to metals. A preliminary study indicated that leukocytes exposed to MeHg at a concentration of 0.01 mM, Cd at 0.05 mM, and As(III) at 2 mM showed a time-dependent cell viability reduction (around 40%), so they were selected for further experiments. To evaluate the effect of MeHg, Cd and As(III) on Pacific red snapper Lutjanus peru, we measured cytotoxicity, reactive oxygen species, antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)), nitric oxide production, apoptosis-related and immune-related genes on head-kidney and spleen leukocytes following exposure to MeHg (0.01 mM), Cd (0.05 mM) and As(III) (2 mM) for 30 min and 2 h. Reactive oxygen species (ROS) generation highly increased in time-dependent doses in head-kidney leukocytes compared with the control group. Regarding antioxidant activity, SOD increased significantly in leukocytes exposed to any heavy metals after two h. Expressly, CAT activity decreased in those leukocytes exposed to Cd and As(III). Apoptotic function genes (Casp-2, Casp-3, and Casp-7) strongly up-regulated after heavy metal exposure, but Cd was more toxic. Finally, granzyme A and perforin 1 strongly up-regulated in leukocytes exposed to MeHg and As(III) compared with the control group. Our data showed that MeHg, Cd, and As(III) might have been cytotoxic and induced oxidative stress and apoptosis with possible biological consequences in fish.

Biosynthesis of ß-d-glucan­gold nanoparticles, cytotoxicity and oxidative stress in mouse splenocytes.

This study reports biosynthesis of gold-nanoparticles (AuNPs) by using ß-d-glucans isolated from the yeast Yarrowia lypolitica D1. ß-d-glucans serve as reducing and stabilizing mediators that induce the formation of AuNPs on the outer surface of the own ß-d-glucan. The systems were physicochemically characterized by ultraviolet visible (UV-Vis) spectroscopy, high-resolution transmission electron microscopy (HR-TEM), scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDS), and dynamic light scattering (DLS) analyses. The results revealed the generation of AuNPs with quasi-spherical shape or large one dimension (1D) gold-nanostructures (AuNSs) depending on the HAuCl4 concentration. A cytotoxic study was assessed in mouse splenocytes. Contrary to that expected, important cytotoxicity was found in all ß-d-gluc+AuNPs systems by an oxidative stress increase. This study discusses the cytotoxic mechanism, suggesting that the resulting ß-d-gluc+AuNPs systems may not be candidates for the formulation of immunostimulants or nanocarriers for biomedical applications.

Antioxidant, intestinal immune status and anti-inflammatory potential of Chenopodium ambrosioides L. in fish: In vitro and in vivo studies.

Chenopodium ambrosioides L. has been used for centuries as traditional medicine in many clinical situations. The objectives of this study were first to assess the nutraceutical potential of C. ambrosioides L. extract through analyses of its chemical composition and antioxidant properties, followed by assessing toxicity and antioxidative activities on fish splenocytes. The second one was to perform an in vivo study using dietary C. ambrosioides L. extract (0.0, 0.5, 1.0 and 2.0%; w/w) for 15 and 30 days (2-week and 4-week treatments) to assess associated-intestine health status by short-chain fatty production, antioxidant enzyme activities and anti-inflammatory effects on Pacific red snapper (Lutjanus peru). Non-polar and polar fractions were detected by gas chromatography/mass spectrometry (GC-MS) in C. ambrosioides, of which the most abundant compounds were carvacrol, phytol, squalene, vitamin E and sucrose. The extract of C. ambrosioides L. enhanced a considerable antiradical and reducing power; fish splenocytes responded positively with higher (88%) cell viability than control. The production of nitric oxide and superoxide anion, as well as superoxide dismutase and catalase activities, were also enhanced in splenocytes treated with C. ambrosioides L. The in vivo study results showed that acetate was the major short-chain fatty acid found in fish receiving C. ambrosioides L. after week four. Pro-inflammatory cytokine gene expression in intestine was modulated in fish fed with C. ambrosioides L. at any time of the experimental trial. In addition, the histological findings suggested that its extract did not cause inflammatory damage in intestine. Overall, the results suggest that C. ambrosioides L. is safe for immune cells and promoting intestinal health status of fish through antioxidant and anti-inflammatory effects, making it an interesting additive in functional diets.

C-type lectin 17A and macrophage-expressed receptor genes are magnified by fungal ß-glucan after Vibrio parahaemolyticus infection in Totoaba macdonaldi cells.

C-type lectins are a principal carbohydrate recognition mechanism as glucans on cell surfaces. This study identified and investigated molecular characterization and immune roles of a novel c-type lectin 17A from Totoaba macdonaldi (TmCLEC17A), which were described in head-kidney leukocytes after immunostimulation with fungal ß-glucan 197A and Vibrio parahaemolyticus infection. This nucleotide sequence from totoaba was acquired using NGS and bioinformatics tools. Its full-length cDNA sequence consisted of 1128 bp (including the stop codon) and an open reading frame (ORF) of 771 bp encoding a 256 amino acid protein, 5´-UTR of 48 bp and 3´-UTR of 309 bp. The TmCLEC17A protein revealed a C-terminal-C-type lectin (CTL, also named carbohydrate-recognition domain, CRD), a N-terminal trans-membrane domain and a coiled coil motif, showing the highest similarity (80%) and identity (96%) with Larimichthys crocea. Fungal ß-glucan 197A plus V. parahaemolyticus enhanced transcriptions of CLEC17A and TLR2 significantly besides the macrophage receptors, such as macrophage mannose receptor 1 and macrophage colony-stimulating factor 1 receptor 2. In addition, natural resistance-associated macrophage protein 2 was significantly up-regulated in leukocytes challenged with live V. parahaemolyticus. Overall, these results indicated that CLEC17A might be implicated in T. macdonaldi innate immunity as a pattern recognition receptor; fungal ß-glucan 197A could stimulate cellular immune mechanisms in head-kidney leukocytes; and it could be used as potential immunostimulant in fish aquaculture.

Molecular characterization and expression analyses of toll like receptor-5 induced by Vibrio parahaemolyticus antigens in Pacific red snapper.

Toll-like receptor 5 (TLR5) is a member of TLRs family responsible for the bacterial flagellin recognition in vertebrates. Herein, the TLR5M gene structure of Pacific red snapper (Lutjanus peru) was characterized. The full-length cDNA of LpTLR5M comprises an open reading frame (ORF) of 2715 bp, encoding a polypeptide of 904 amino acids including 9 LRRs (residues 119-562) and one LRR-CT domain (residues 593-646) at the extracellular region, and a TIR domain (residues 710-904) in the cytoplasmic region. The amino acid sequence in L. peru TLR5 showed high identity (66-69%) with TLR5 from Paralichthys olivaceus and Scophthalmus maximus. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of LpTLR5M mRNA in all the examined tissues, with higher levels in intestine, liver, and head-kidney. Furthermore, expression of LpTLR5M and five cytokine genes was also investigated 24 h and one week post-stimulation in fish intraperitoneally injected with ToxA, live V. parahaemolyticus (Vp) or V. parahaemolyticus Lysate antigens. TLR5M was significantly induced in fish infected with Vp. The pro-inflammatory cytokines IL-6, IL8 and IL-12 were significantly up-regulated in head-kidney in fish stimulated with Vp, while in intestine upregulation was observed following ToxA or Lysate injection. In contrast, IL-17 mRNA was significantly up-regulated in the intestine from fish infected with live Vp at 24 h post-injection. The results indicate that Lysate and Vp antigens can induce an immune response via TLR5M and that cytokines have an important role in the defense mechanisms against V. parahaemolyticus.

B-cell activating CpG ODN 1668 enhance the immune response of Pacific red snapper (Lutjanus peru) exposed to Vibrio parahaemolitycus.

B-class CpG ODN 1668 is known to possess clear immunostimulatory properties. In this study, we investigated the potential ability of CpG ODN 1668 to enhance the immune response of Pacific red snapper exposed to Vibrio parahaemolyticus. Four different treatments were evaluated in Pacific red snapper: (1) stimulatory CpG ODN 1668, (2) stimulatory CpG ODN 1668 and V. parahaemolyticus, (3) exposure only to V. parahaemolyticus and (4) PBS. Samples were taken at 24, 72, 168 and 240 h of stimulation/infection. The results show that intraperitoneal injection of CpG-ODN 1668 enhanced the anti-protease, superoxide dismutase and catalase activities in serum. CpG ODN 1668 upregulated TLR9 and IgM gene expression in head-kidney, intestine and skin, with higher expression in head-kidney. A higher correlation was observed between TLR9 and IgM in head-kidney and intestine. Finally, no histopathological damages were observed in fish stimulated with CpG ODN 1668. In contrast, melanomacrophages-like structures were present in higher numbers in infected fish. Taken together, these results indicate that CpG ODN 1668 activates innate immune response and upregulate the TLR9 and IgM-mediated immune response. These results may be exploited for the control of Vibriosis in farmed Pacific red snapper.

Molecular cloning and comparative responses of Toll-like receptor 22 following ligands stimulation and parasitic infection in yellowtail (Seriola lalandi).

TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. In this study, we cloned the full-length cDNA sequence of yellowtail (Seriola lalandi) TLR22 (SlTLR22). The complete cDNA sequence of SlTLR22 was 4208 bp and encodes a polypeptide of 961 amino acids. Analysis of the deduced amino acid sequence indicated that SlTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 91-633) and one C-terminal LRR domain (LRR-CT, residues 693-744) in the extracellular region, and a TIR domain (residues 800-943) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced SlTLR22 has the highest sequence identity to turbot TLR22 (76%). Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SlTLR22 mRNA in all examined tissues with higher levels in the head kidney, intestine, skin and spleen. Further, SlTLR22 expression was significantly up-regulated following TLR ligands injection with lipopolysaccharide (LPS), CpG ODN2006 and polyinosinic: polycytidylic acid (poly I:C) in spleen and liver. Amyloodinium ocellatum infection also induced a high expression of SlTLR22 in spleen, intestine, muscle, skin and gill, with maximum increases ranging from 1000 to 100 fold upon different ligands and organs. Finally, histological examination in gill tissue confirmed infection by the parasite and histopathological lesion was observed also in spleen and skin. These findings suggest a possible role of SlTLR22 in the immune responses to the infections of a broad range of pathogens that include DNA and RNA viruses and parasites.

Dietary administration of microalgae alone or supplemented with Lactobacillus sakei affects immune response and intestinal morphology of Pacific red snapper (Lutjanus peru).

The aim of this study was to evaluate the effect of dietary microalgae Navicula sp single or in combination with Lactobacillus sakei on growth performance, humoral immune parameters and intestinal morphology in Pacific red snapper, Lutjanus peru. The experimental fish were grouped into four treatment diets which were a control diet (commercial diet, Control), silage microalgae Navicula sp plus L. sakei (10(6) CFU g(-1), Navicula + L. sakei), lyophilized microalgae (Navicula) and L. sakei (10(6) CFU g(-1), L. sakei). The blood and intestine samples were collected on week 4 and 8. The weight gain showed an additive effect of Navicula + L. sakei at 8 weeks of treatment compared with fish fed control diet. Overall, physiological parameters such as total protein and hemoglobin were increased in fish fed with Navicula and L. sakei diets at 4 and 8 weeks of feeding assay, respectively. There was a significant improvement in immune parameters, principally in myeloperoxidase, lysozyme, total antiproteases activities and IgM in fish fed with Navicula + L. sakei and L. sakei diets at 4 or 8 weeks of treatments. Serum antioxidant capabilities revealed significant increase in phosphatase alkaline, esterase, protease, superoxide dismutase and catalase in groups which received diet supplemented with Navicula + L. sakei and L. sakei diets. Finally, light microscopy observations revealed no effect of experimental diets on microvilli height. Curiously, the presence of vacuoles inside the enterocytes was significant higher in the intestine of L. sakei group after four or six weeks of feeding. Elevated intraepithelial leucocyte levels and melanomacrophages centers were observed in fish fed Navicula or control diets at any time of the experiment. To conclude, the results of the present study demonstrate that the fish that were fed with Navicula + L. sakei or L. sakei diets yielded significantly better immune status and antioxidant capabilities.

Iron bioavailability in larvae yellow snapper (Lutjanus argentiventris): cloning and expression analysis of ferritin-H.

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, ferritin H subunit was cloned from the larvae of yellow snapper, Lutjanus argentiventris, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the LaFeH was 1231 bp in length encoding 177 amino acids with a predicted molecular mass (MW) about 20.82 kDa and theoretical isoelectric point (pI) of 5.79. Amino acid alignment revealed that LaFeH shared high similarity with other known ferritins. It shared high degree identity to the ferritin H subunits of Lates calcarifer (99%), Takifugu rubripes (97%) and Dicentrarchus labrax (97%), and low identity to that of human (82%) and mouse (84%). By real-time PCR assays, the mRNA transcripts of LaFeH was found to be higher expressed in head-kidney, eye, heart and brain. Moreover, mRNA expression levels of LaFeH was measured by real-time PCR in larvae exposed with graded levels of iron (6.8 µg/ml and 13.6 µg/ml (Fe2x and Fe4x, respectively) and an iron chelation assay. Results showed that the expression of the LaFeH mRNA increased gradually with Fe2x in water. The LaFeH gene expression declined with increasing iron exposure levels at Fe4x. Finally, we can observe a high expression of LaFeH gene in larvae exposed to iron chelation therapy at 2 h; however this increase was gradually decreasing over time. In summary, the LaFeH gene expression for larvae yellow snapper showed a dose-depend increase following the iron treatment. These data indicated that iron bioavailability regulates LaFeH at transcriptional level in larvae yellow snapper. Further studies are necessary to ascertain their role in the immune response in teleost fish.

Effects of marine silages enriched with Lactobacillus sakei 5-4 on haemato-immunological and growth response in Pacific red snapper (Lutjanus peru) exposed to Aeromonas veronii.

Combined effects of marine silages enriched with Lactobacillus sakei 5-4 were evaluated on growth performance, immune activity and disease resistance of Pacific red snapper (Lutjanus peru) against Aeromonas veronii infection. The experimental fish were divided into three groups which were fed with each one of the following diets: silage-probiotic-free diet (control, C group), Pacific creole-fish silage diet supplemented with live L. sakei (10(6) CFU g(-1)) (FSLact group) and Humboldt squid silage diet supplemented with live L. sakei (10(6) CFU g(-1)) (SSLact group) for 6 weeks. After 6 weeks, fish were immunocompromised with pathogenic A. veronii and spleen and liver samples were processed for histopathological studies. Generally, the results showed enhanced growth performance in fish fed the diet containing SSLact at 6 and 7 weeks compared with fish fed control diet. Addition of SSLact had an increase in plasmatic protein at week 6 and post-challenge. Hemoglobin concentration increased after challenge in fish fed with SSLact compared to control group. At week 6 and post-challenge the results indicated that, the fish groups which received diet supplemented with SSLact revealed significant increase in humoral immune parameters. Histologically, fish fed C diets showed marked fatty degeneration and great activation of melanomacrophage centers compare with SSLact and FSLact groups. These results support the idea that the marine silages with squid as protein source enriched or combined with L. sakei 5-4 increases the body weight and stimulates the physiological and humoral immune parameters in Pacific red snapper infected with A. veronii.