Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids

Background: Tumour necrosis factor-alpha (TNF-\u3b1) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-\u3b1 has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-\u3b1 detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-\u3b1 antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-\u3b1 of salmonid fish. TNF-\u3b1 epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-\u3b1, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-\u3b1 positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-\u3b1 function

The expression of TRPV channels, prostaglandin E2 and pro-inflammatory cytokines during behavioural fever in fish

A fever, or increased body temperature, is a symptom of inflammation, which is a complex defence reaction of the organism to pathogenic infections. After pathogens enter the body, immune cells secrete a number of agents, the functions of which stimulate the body to develop a functional immune and fever response. In mammals it is known that \{PGE2\} is the principal mediator of fever. The extent to which \{PGE2\} and other pro-inflammatory cytokines such as TNF-α, IL-6, or IL-1β could be involved in the induction of behavioural fever in fish remains to be clarified. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including \{TRPV1\} and TRPV2, which are activated by heat. Here we show that members of the \{TRP\} family, \{TRPV1\} and TRPV4, may participate in the coordination of temperature sensing during the behavioural fever. To examine the behavioral fever mechanism in Salmo salar an infection with IPNV, infectious pancreatic necrosis virus, was carried out by an immersion challenge with 10 ×105 PFU/mL-1 of IPNV. Behavioural fever impacted upon the expression levels of both \{TRPV1\} and \{TRPV4\} mRNAs after the viral challenge and revealed a juxtaposed regulation of \{TRPV\} channels. Our results suggest that an increase in the mRNA abundance of \{TRPV1\} is tightly correlated with a significant elevation in the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and PGE2) in the Pre-Optic Area (POA) and cytokine release in plasma. Together, these data indicate that the reduction of \{TRPV4\} expression during behavioural fever may contribute to the onset of behavioural fever influencing movement toward higher water temperatures. Our data also suggest an effect of \{TRPV\} channels in the regulation of behavioural fever through activation of \{EP3\} receptors in the central nervous system by \{PGE2\} induced by plasma-borne cytokines. These results highlight for first time in mobile ectotherms the key role of pro-inflammatory cytokines and \{TRPV\} channels in behavioural fever that likely involves a complex integration of prostaglandin induction, cytokine recognition and temperature sensing

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profil

Heritability of immunity traits and resistance of Atlantic salmon against the sea louse Caligus rogercresseyi

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon

Induction of foxp3 during the Crosstalk between Antigen Presenting Like-Cells MHCII+CD83+ and Splenocytes CD4+IgM− in Rainbow Trout

In fish, the spleen is one of the major immune organs in the animal, and the splenocytes could play a key role in the activation and modulation of the immune response, both innate and adaptive. However, the crosstalk between different types of immune cells in the spleen has been poorly understood. In this work, an in vitro strategy is carried out to obtain and characterize mononuclear splenocytes from rainbow trout, using biomarkers associated with lymphocytes (CD4 and IgM) and antigen-presenting cells (CD83 and MHC II). Using these splenocytes, co-cultures of 24 and 48 h are used to determine the gene expression of master transcriptional factors that coordinate the polarization of T cells (t-bet, gata3, and foxp3). The results show a proportional upregulation of foxp3 (compared to t-bet and gata3) in co-cultures (at 24 h) of IFNγ-induced splenocytes with and without stimulation of Piscirickettsia salmonis proteins. In addition, foxp3 upregulation was established in co-cultures with IFNγ-induced cells and in cells only stimulated previously with P. salmonis proteins at 48 h of co-culture. These results show a potential communication between antigen-presenting-like cells and lymphocyte in the spleen, which could be induced towards a Treg phenotype

Yeast as a protein source during smoltification of Atlantic salmon (Salmo salar L.), enhances performance and modulates health

Yeast produced from lignocellulosic biomass has the potential to serve as a high-quality protein source with health benefits, especially during critical stages of the Atlantic salmon life cycle, such as during seawater transfer (SWT). In this study, we evaluated the effect of adding 25% Candida utilis yeast to salmon feed on growth performance and overall health by using morphometry, immunohistochemistry, cytokine enzyme-linked immunosorbent assays (ELISA) and gene expression analysis during and after SWT. There were four dietary treatments: 1) control diet in freshwater (FW) and seawater (SW) (Control); 2) control diet in FW and a yeast-based diet in SW (Control/Yeast); 3) yeast-based diet in FW and SW (Yeast); 4) yeast-based diet in FW and a control diet in SW (Yeast/Control). Our results showed that fish fed the yeast diet throughout the FW and SW period achieved higher feed intake and higher growth rate than fish fed the control diet. Morphometric and immunochemical analyses of the distal intestine (DI) revealed decreased length and number of CD3 labeled cells in the simple folds of fish fed control diet, while no changes were observed in fish fed the yeast diet. Furthermore, yeast significantly decreased the secretion of IFNγ, TNFα, IL-1β, IL-8, and modulated the gene expression of aquaporin 8 (aqp8ab), superoxide dismutase (sod1) and major histocompatibility complex 1 (mhc1) in DI, suggesting reduced inflammatory processes in yeast fed fish. These findings indicate that Candida utilis yeast is a promising alternative protein source with functional properties in diets for smolting Atlantic salmon before and after SWT.publishedVersio

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress

Background Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. Methods We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. Results The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. Conclusions Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri

Commercial Vaccines Do Not Confer Protection against Two Genogroups of <i>Piscirickettsia salmonis</i>, LF-89 and EM-90, in Atlantic Salmon

In Atlantic salmon, vaccines have failed to control and prevent Piscirickettsiosis, for reasons that remain elusive. In this study, we report the efficacy of two commercial vaccines developed with the Piscirickettsia salmonis isolates AL100005 and AL 20542 against another two genogroups which are considered highly and ubiquitously prevalent in Chile: LF-89 and EM-90. Two cohabitation trials were performed to mimic field conditions and vaccine performance: (1) post-smolt fish were challenged with a single infection of LF-89, (2) adults were coinfected with EM-90, and a low level coinfection of sea lice. In the first trial, the vaccine delayed smolt mortalities by two days; however, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 60.3%, vaccinated: 56.7%; p = 0.28). In the second trial, mortality started three days later for vaccinated fish than unvaccinated fish. However, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 64.6%, vaccinated: 60.2%, p = 0.58). Thus, we found no evidence that the evaluated vaccines confer effective protection against the genogroups LF-89 and EM-90 of P. salmonis with estimated relative survival proportions (RPSs) of −9% and −12%, respectively. More studies are necessary to evaluate whether pathogen heterogeneity is a key determinant of the lack of vaccine efficacy against P. salmonis

Norway spruce extracts (NSEs) as bioactive compounds in novel feeds: Effect on intestinal immune-related biomarkers, morphometry and microbiota in Atlantic salmon pre-smolts

Compounds from woody biomass are promising candidates as feed additives to improve fish health and welfare of Atlantic salmon (Salmo salar) during the productive cycleng. Thus, conidendrin- and isorhapontin-based extracts (NSEs) from Norway spruce (Picea abies) were evaluated at different levels in a four-week trial. A commercial-like diet (D1) was supplemented with 0.2% of a commercial β-glucan (D2) or with NSEs at 0.02%, 0.1% and 0.2% (D3-D8). No significant differences were found in growth, intestinal morphometry and microbiota at the genus level. Immune-related biomarkers in distal intestine from D5 and D6 indicated an activation of a pro-inflammatory response by the up-regulation of il1b, as in D2. Interestingly, isorhapontin-based extract at higher inclusion level (D8), showed a down-regulation of both pro- and anti-inflammatory- genes, suggesting maintenance of homeostasis. These results showed the importance of source and inclusion levels of bioactive compounds on the regulatory balance associated with fish immune response