Determination of potentially mycotoxigenic fungi in coffee (Coffea arabica L.) from Nayarit.

A total of fourteen roasted coffee samples were collected from different local markets in Nayarit, Mexico. Twenty-two fungi isolates were related to the genera Aspergillus (54.54%) and Penicillium (4.5%). The strains R16 (0.33 µg/kg), 6N (1.16 µg/kg) and 11 (0.36 µg/kg) tested positive for OTA (ochratoxin A) production in PDA, the other fungi samples were not toxigenic. According to the sequence analysis of their ITS1-5.8S-ITS2 rDNA region, fungi OTA producers correspond to A. niger, A. versicolor and Byssochlamys spectabilis. These three strains were able to produce OTA when inoculated in roasted coffee in concentrations ranging from 75 to 90 µg/kg, after 21 days. Different production stages of roasted coffee (crop management, postharvest practices and storage) along with environmental conditions do not ensure mycotoxigenic fungi free products. This is the first report of OTA natural occurrence in roasted coffee from Nayarit.

Leukocyte susceptibility and immune response against Vibrio parahaemolyticus in Totoaba macdonaldi.

Vibrio parahaemolyticus is a serious pathogen that affects aquaculture. Nonetheless, to the best of our knowledge, no studies have focused on its immunological implications in Totoaba macdonaldi. Thus, the early immune response to V. parahaemolyticus in juveniles of totoaba was studied at 24 h post-infection with an in vivo study. In addition, changes in cellular innate immune parameters - phagocytosis, respiratory burst activity and viability (annexin V/propidium iodide) - were evaluated in vitro in head-kidney, spleen and thymus leukocytes at 6 and 24 h after bacterial stimulation by flow cytometry. Simultaneously, the expression levels of two immune-relevant genes (IL-1ß and IL-8) were measured by using real time PCR. During in vivo study, mRNA transcripts of IL-1ß were highly expressed in spleen, thymus and intestine and down-regulated in liver after 24 h post-infection. IL-8 gene expression was upregulated in spleen, intestine and liver compared to that of non-infected fish and down-regulated in thymus after 24 h post-infection. Generally, the results showed a significant decrease in cellular immune responses during the infection, principally in phagocytic ability and respiratory burst. The survival or viability of stimulated leukocytes was significantly reduced causing necrosis and apoptosis, indicating a robust killing response by V. parahaemolyticus. Finally the in vitro analysis showed that transcript levels of IL-1ß and IL-8 were up-regulated during stimulation with V. parahaemolyticus in head-kidney, spleen and intestine and down-regulated in thymus at any time of the experiment. Although V. parahaemolyticus has been reported to be an important pathogen for many aquatic organisms, to our knowledge this might be the first report of early-immune response in juvenile totoaba and these immune parameters may be reliable indicators and can be useful in the health control of this species.

Comparative ontogenetic development of two marine teleosts, gilthead seabream and European sea bass: New insights into nutrition and immunity.

Gilthead seabream and European sea bass are two of the most commonly farmed fish species. Larval development is critical to ensure high survival rates and thus avoid unacceptable economic losses, while nutrition and immunity are also important factors. For this reason this paper evaluates the ontogenetic development of seabream and sea bass digestive and immune systems from eggs to 73 days post-fertilisation (dpf) by assessing the expression levels of some nutrition-relevant (tryp, amya, alp and pept1) and immune-relevant (il1b, il6, il8, tnfa, cox2, casp1, tf, nccrp1, ighm and ight) genes. The results point to similar ontogenetic development trends for both species as regard nutrition and differences in some immunity related genes.

TLR21's agonists in combination with Aeromonas antigens synergistically up-regulate functional TLR21 and cytokine gene expression in yellowtail leucocytes.

The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1ß and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1ß and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail.

[Yeast irrigation enhances the nutritional content in hydroponic green maize fodder]. / La irrigación con levaduras incrementa el contenido nutricional del forraje verde hidropónico de maíz.

The objective of this study was to evaluate the effect of irrigation with yeasts (Debaryomyces hansenii var. Fabry, Yarowia lipolytica YIBCS002, Yarowia lipolytica var. BCS and Candida pseudointermedia) on the final nutritional content of hydroponic green maize fodder (Zea Zea mays L.), applied at different fodder growth stages (1. seed-seedling stage, 2. seedling-plant 20cm, 3. during all the culture). Irrespective of the fodder growth stages at which they were applied, all yeasts tested enhanced the content of raw protein, lipids, ash, moisture and energy. The percentage of electrolytes (Na, K, Cl, sulphates, Ca and Mg) showed different responses depending on the kind of yeast applied; D. hansenii exhibited the highest increment in all electrolytes, except for phosphorous. We conclude that the addition of yeasts belonging to the genera Debaryomyces, Candida and Yarowia to the irrigation solution of hydroponic systems enhances the nutrient content of green fodder. This kind of irrigation can be applied to generate high commercial value cultures in limited spaces.

La irrigación con levaduras incrementa el contenido nutricional del forraje verde hidropónico de maíz / Yeast irrigation enhances the nutritional content in hydroponic green maize fodder

El objetivo del presente estudio fue evaluar el efecto de la irrigación con las levaduras Debaryomyces hansenii var. Fabry, Yarowia lipolytica YIBCS002, Yarowia lipolytica var. BCS y Candida pseudointermedia sobre el contenido nutricional final del forraje verde hidropónico de maíz (Zea mays L. ), al ser efectuada en diferentes etapas de crecimiento de aquel (fase semilla-plántula o fase plántula-planta 20 cm), o bien durante todo el cultivo. Todas las levaduras incrementaron el contenido de proteína cruda, lípidos, cenizas, humedad y energía bruta, independientemente de la etapa de crecimiento del forraje en las que fueron aplicadas. El porcentaje de electrólitos (Na, K, Cl, sulfatos, Ca y Mg) varió en función de la levadura aplicada; D. hansenii incrementó todos los electrólitos, excepto el P. Se concluye que la adición de levaduras del género Debaryomyces, Candida y Yarowia en la solución de riego de sistemas hidropónicos mejora el contenido de nutrientes del forraje verde. Esta práctica puede contribuir a la generación de cultivos de valor comercial en espacios limitados

The objective of this study was to evaluate the effect of irrigation with yeasts (Debaryomyces hansenii var. Fabry, Yarowia lipolytica YIBCS002, Yarowia lipolytica var. BCS and Candida pseudointermedia) on the final nutritional content of hydroponic green maize fodder (Zea Zea mays L.), applied at different fodder growth stages (1. seed-seedling stage, 2. seedling-plant 20 cm, 3. during all the culture). Irrespective of the fodder growth stages at which they were applied, all yeasts tested enhanced the content of raw protein, lipids, ash, moisture and energy. The percentage of electrolytes (Na, K, Cl, sulphates, Ca and Mg) showed different responses depending on the kind of yeast applied; D. hansenii exhibited the highest increment in all electrolytes, except for phosphorous. We conclude that the addition of yeasts belonging to the genera Debaryomyces, Candida and Yarowia to the irrigation solution of hydroponic systems enhances the nutrient content of green fodder. This kind of irrigation can be applied to generate high commercial value cultures in limited spaces.

Covalently Cross-Linked Arabinoxylans Films for Debaryomyces hansenii Entrapment.

In the present study, wheat water extractable arabinoxylans (WEAX) were isolated and characterized, and their capability to form covalently cross-linked films in presence of Debaryomyces hansenii was evaluated. WEAX presented an arabinose to xylose ratio of 0.60, a ferulic acid and diferulic acid content of 2.1 and 0.04 µg∙mg(-1) WEAX, respectively and a Fourier Transform Infra-Red (FT-IR) spectrum typical of WEAX. The intrinsic viscosity and viscosimetric molecular weight values for WEAX were 3.6 dL∙g(-1) and 440 kDa, respectively. The gelation of WEAX (1% w/v) with and without D. hansenii (1 × 10(7) CFU∙cm(-2)) was rheologically investigated by small amplitude oscillatory shear. The entrapment of D. hansenii decreased gel elasticity from 1.4 to 0.3 Pa, probably by affecting the physical interactions between WEAX chains. Covalently cross-linked WEAX films containing D. hansenii were prepared by casting. Scanning electron microscopy images show that WEAX films containing D. hansenii were porous and consisted of granular-like and fibre microstructures. Average tensile strength, elongation at break and Young's modulus values dropped when D. hansenii was present in the film. Covalently cross-lined WEAX containing D. hansenii could be a suitable as a functional entrapping film.

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.

Dietary administration of ß-1,3/1,6-glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression.

It is widely known that ß-glucans and probiotic bacteria are good immunostimulants for fish. In the present work we have evaluated the dietary effect of ß-1,3/1,6-glucan (isolated from Laminarina digitata) and Pdp 11 (Shewanella putrefaciens, probiotic isolated from gilthead seabream skin), single or combined, on growth, humoural (seric level of total IgM antibodies and peroxidase and antiprotease activities) and cellular innate immune response (peroxidase and phagocytic activities of head-kidney leucocytes), as well as the expression of immune-related genes in gilthead seabream (Sparus aurata). Four treatment groups were established: control (non-supplemented diet), Pdp 11 (10(9) cfu g(-1)), ß-1,3/1,6-glucan (0.1%) and ß-1,3/1,6-glucan + Pdp 11 (0.1% and 10(9) cfu g(-1), respectively). Fish were sampled after 1, 2 and 4 weeks of feeding. Interestingly, all supplemented diets produced increments in the seabream growth rates, mainly the Pdp 11-suplemented diet. Overall, Pdp 11 dietary administration resulted in decreased serum IgM levels and peroxidase activity. However, the seric antiprotease activity was increased in fish fed with both supplements together. Furthermore, ß-1,3/1,6-glucan and combined diet increased phagocytic activity after 2 or 4 weeks. At gene level, IL-1ß and INFγ transcripts were always up-regulated in HK but only the interleukin reached significance after 4 weeks in the group fed with ß-glucan. On the contrary, IgM gene expression tended to be down-regulated being significant after 1 week in seabream specimens fed with ß-glucan or ß-glucan plus Pdp 11. These results suggest that ß-1,3/1,6-glucan and Pdp 11 modulate the immune response and stimulates growth of the gilthead seabream, one of the species with the highest rate of production in Mediterranean aquaculture.

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 dietary ß-1,3/1,6-glucan on the antioxidant and digestive enzyme activities of Pacific red snapper (Lutjanus peru) after exposure to lipopolysaccharides.

The effect of ß-1,3/1,6-glucan, derived from yeast, on growth, antioxidant, and digestive enzyme performance of Pacific red snapper Lutjanus peru before and after exposure to lipopolysaccharides (LPS) was investigated. The ß-1,3/1,6-glucan was added to the basal diet at two concentrations (0.1 and 0.2 %). The treatment lasted 6 weeks, with sampling at regular intervals (0, 2, 4, and 6 weeks). At the end of this period, the remaining fish from either control or ß-glucan-fed fish were injected intraperitoneally with LPS (3 mg kg(-1)) or with sterile physiological saline solution (SS) and then sampled at 0, 24, and 72 h. The results showed a significant increase (P < 0.05) in growth performance after 6 weeks of feeding with ß-glucan. Superoxide dismutase (SOD) activity in liver was significantly higher in diets containing 0.1 % ß-glucan in weeks 4 and 6, compared to the control group. ß-Glucan supplementation at 0.1 and 0.2 % significantly increased aminopeptidase, trypsin, and chymotrypsin activity. At 72 h after injection of LPS, we observed a significant increase in catalase activity in liver from fish fed diets supplemented with 0.1 and 0.2 % ß-glucan; SOD activity increased in fish fed with 0.1 % ß-glucan in relation to those injected with SS. Feed supplemented with ß-1,3/1,6-glucan increased growth, antioxidant activity, and digestive enzyme activity in Pacific red snapper.

Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) during commercial production.

The resident microbiota of three oyster species (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) was characterised using a high-throughput sequencing approach (pyrosequencing) that was based on the V3-V5 regions of the 16S rRNA gene. We analysed the changes in the bacterial community beginning with the postlarvae produced in a hatchery, which were later planted at two grow-out cultivation sites until they reached the adult stage. DNA samples from the oysters were amplified, and 31 008 sequences belonging to 13 phyla (including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) and 243 genera were generated. Considering all life stages, Proteobacteria was the most abundant phylum, but it showed variations at the genus level between the postlarvae and the adult oysters. Bacteroidetes was the second most common phylum, but it was found in higher abundance in the postlarvae than in adults. The relative abundance showed that the microbiota that was associated with the postlarvae and adults differed substantially, and higher diversity and richness were evident in the postlarvae in comparison with adults of the same species. The site of rearing influenced the bacterial community composition of C. corteziensis and C. sikamea adults. The bacterial groups that were found in these oysters were complex and metabolically versatile, making it difficult to understand the host-bacteria symbiotic relationships; therefore, the physiological and ecological significances of the resident microbiota remain uncertain.

Dietary administration of microalgae Navicula sp. affects immune status and gene expression of gilthead seabream (Sparus aurata).

Effects of silage microalgae enriched with a probiotic and lyophilized microalgae were evaluated on main immune parameters and different gene expression of gilthead seabream (Sparus aurata L.). A total of 60 seabream were grouped into 3 treatment diets which were a control diet (commercial diet) without microalgae (C), commercial diet supplemented with silage microalgae Navicula sp. plus Lactobacillus sakei 5-4 (10(6) CFU g(-1)) (SM), and commercial diet supplemented with lyophilized microalgae (LM) for 4 weeks. Generally, the results showed a significant increase in the immune parameters, principally in leucocyte peroxidase, phagocytosis and complement activities in fish fed with SM diet compared to control group. About the gene expression in head-kidney, transcript levels (Interleukin-8, Interleukin-1ß and ß-defensin) were upregulated in fish fed with SM after 4 weeks of treatments. However, the gene expression was upregulated in intestine from fish fed with LM with significant difference in transferrin and cyclooxygenase 2 gene at 2 weeks, and in occludin, transferrin, interleukin-8 and interleukin-1ß at 4 weeks. Finally, about the digestive enzymes, LM diet caused an upregulated of α-amylase and alkaline phosphatase genes at 2 weeks; however SM diet caused an upregulated trypsin gene at 4 weeks. SM diet a higher enhancing effect on gilthead seabream immune parameters than that observed when using LM. Furthermore, dietary administration of microalgae Navicula sp. provokes upregulation of several genes in the gut that correlates with slight inflammation. Further studies are needed to know if this diatom could be useful for administering as diet supplement for farmed 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.

Cytosolic manganese superoxide dismutase genes from the white shrimp Litopenaeus vannamei are differentially expressed in response to lipopolysaccharides, white spot virus and during ontogeny.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme usually located in mitochondria. There are only a few examples of cytosolic MnSOD (cMnSOD). In the shrimp Litopenaeus vannamei, we have previously characterized three cMnSOD cDNAs and their differential tissue-specific expression. To obtain insights about their genomic organization, we characterized the three corresponding cMnSOD genes, named them cMnsod1, cMnsod2, and cMnsod3 and studied their specific expression during ontogeny, response to lipopolysaccharides (LPS) and white spot virus infection (WSSV) in hemocytes from shrimp. The first two genes contain five introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions, while the third one is intron-less. We analyzed 995 nucleotides upstream cMnsod2, but no classical promoter sequences were found. The deduced products of the three cMnSOD genes differ in two amino acids and there are four silent changes. cMnsod3 expression is modulated by WSSV and cMnsod2 by LPS. cMnsod2 is expressed from eggs to post larval stage during ontogeny. This is the first report of crustacean cMnSOD multigenes that are differently induced during the defense response and ontogeny.

Immune response of gilthead seabream (Sparus aurata) following experimental infection with Aeromonas hydrophila.

The Gram-negative bacteria Aeromonas hydrophila is a heterogeneous organism that causes the disease known as motile aeromonad septicaemia, which is responsible for serious economic loss in seabream culture due to bacterial infections. However, the immune mechanisms involved in this disease in fish are still poorly understood. For the purpose of this study, gilthead seabream (Sparus aurata L.) specimens received a double intraperitoneal injection of bacterial inoculums: a primary infection with 1 × 10(7) cell ml(-1) A. hydrophila, followed by a secondary infection with 1 × 10(8) cell ml(-1) fourteen days later. Changes in cellular innate immune parameters - phagocytosis, respiratory burst activity and peroxidase leucocyte content - were evaluated 24 and 48 h after each injection. Simultaneously, the expression levels of nine immune-relevant genes (TLR, NCCRP-1, HEP, TCR, IgM, MHC-IIα, IL-1ß, C3 and CSF-1R) were measured in the head-kidney, spleen, intestine and liver, by using q-PCR. Generally, the results showed a significant decrease in cellular immune responses during the primary infection and a significant enhanced during the second infection, principally in respiratory burst and peroxidase activity, thus indicating a recovery of the immune system against this bacterial pathogen. Finally, transcript levels of immune genes were down-regulated during the first infection, except for the IL-1ß gene. In contrast, mRNA expression levels during the re-infection were significantly up-regulated. The results seem to suggest a relatively fast elimination of the bacteria and recovery of fish during the secondary infection.

Effects of polyamines on cellular innate immune response and the expression of immune-relevant genes in gilthead seabream leucocytes.

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes (IgM, MHCIα, MHCIIα, C3, IL-1ß, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them (C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.

Oral delivery of live yeast Debaryomyces hansenii modulates the main innate immune parameters and the expression of immune-relevant genes in the gilthead seabream (Sparus aurata L.).

Microorganisms isolated from fish can be used as prophylactic tools for aquaculture in the form of probiotic preparations. The purpose of this study was to evaluate the effects of dietary administration of the live yeast Debaryomyces hansenii CBS 8339 on the gilthead seabream (Sparus aurata L.) innate immune responses. Seabream were fed control or D. hansenii-supplemented diets (10(6) colony forming units, CFU g(-1)) for 4 weeks. Humoral (seric alternative complement and peroxidase activities), and cellular (peroxidase, phagocytic, respiratory burst and cytotoxic activities) innate immune parameters and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) were measured from serum, head-kidney leucocytes and liver, respectively, after 2 and 4 weeks of feeding. Expression levels of immune-associated genes, Hep, IgM, TCR-beta, NCCRP-1, MHC-II alpha, CSF-1R, C3, TNF-alpha and IL-1 beta, were also evaluated by real-time PCR in head-kidney, liver and intestine. Humoral immune parameters were not significantly affected by the dietary supplementation of yeast at any time of the experiment. On the other hand, D. hansenii administration significantly enhanced leucocyte peroxidase and respiratory burst activity at week 4. Phagocytic and cytotoxic activities had significantly increased by week 2 of feeding yeast but unchanged by week 4. A significant increase in liver SOD activity was observed at week 2 of feeding with the supplemented diet; however CAT activity was not affected by the dietary yeast supplement at any time of the experiment. Finally, the yeast supplemented diet down-regulated the expression of most seabream genes, except C3, in liver and intestine and up-regulated all of them in the head-kidney. These results strongly support the idea that live yeast Debaryomyces hansenii strain CBS 8339 can stimulate the innate immune parameters in seabream, especially at cellular level.

Effects of dietary arginine on hematological parameters and innate immune function of channel catfish.

The effects of elevated dietary arginine on the hematology and immune function of juvenile channel catfish Ictalurus punctatus were evaluated by means of in vivo and in vitro experiments. Healthy juvenile channel catfish (average weight, 34.8 g) were fed casein-gelatin-based diets containing 28% crude protein and supplemented with crystalline L-arginine (ARG) at 0.5, 1, 2, or 4% of diet. An intact-protein diet containing 1.3% arginine also was included to investigate the effects of amino acid form (crystalline-free amino acids versus intact protein). Each purified diet was fed to apparent satiation to triplicate groups of fish for 6 weeks. At the end of the experimental feeding period, the fish were injected intraperitoneally with two doses (3 d apart) of 2 mg lipopolysaccharide/kg body weight. Six days after the initial injection, the fish were anesthetized and tissue samples were obtained to evaluate hematological and humoral and cellular immune parameters, including phagocytic activity of peritoneal macrophages, hemoglobin, hematocrit, mean corpuscular volume (MCV), blood cell counts, plasma protein, and hepatic superoxide dismutase activity. High dietary levels (4% ARG) resulted in significantly higher levels of hemoglobin, hematocrit, and circulating erythrocytes. Dietary ARG did not significantly affect MCV and the number of circulating leukocytes, lymphocytes, eosinophils, and monocytes. In vitro, a moderate level (2 mM) of ARG in the culture media was found to be ideal in significantly enhancing phagocytosis. This study demonstrates that some aspects of the immune system of channel catfish are sensitive to changes in dietary ARG.