Innate Immunity Provides Biomarkers of Health for Teleosts Exposed to Nanoparticles

In recent years, the unique properties of nanoparticles have fostered novel applications in various fields such as biology, pharmaceuticals, agriculture, and others. Unfortunately, their rapid integration into daily life has also led to environmental concerns due to uncontrolled release of nanoparticles into the aquatic environment. Despite increasing awareness of nanoparticle bioaccumulation in the aquatic environment, much remains to be learned about their impact on aquatic organisms and how to best monitor these effects. Herein, we provide the first review of innate immunity as an emerging tool to assess the health of fish following nanoparticle exposure. Fish are widely used as sentinels for aquatic ecosystem pollution and innate immune parameters offer sensitive and reliable tools that can be harnessed for evaluation of contamination events. The most frequent biomarkers highlighted in literature to date include, but are not limited to, parameters associated with leukocyte dynamics, oxidative stress, and cytokine production. Taken together, innate immunity offers finite and sensitive biomarkers for assessment of the impact of nanoparticles on fish health

I am uncertain” vs “it is uncertain”. How linguistic markers of the uncertainty source affect uncertainty communication

Two psychological sources of uncertainty bear implications for judgment and decision-making: external uncertainty is seen as stemming from properties of the world, whereas internal uncertainty is seen as stemming from lack of knowledge. The apparent source of uncertainty can be conveyed through linguistic markers, such as the pronoun of probability phrases (e.g., I am uncertain vs. It is uncertain). Here, we investigated whether and when speakers use different pronoun subjects as such linguistic markers (Exp. 1 and 2) and what hearers infer from them (Exp. 3 and 4). Speakers more often described higher probabilities and knowable outcomes with internal probability phrases. In dialogue, speakers mirrored the source of their conversational partner. Markers of the source had a main effect or interacted with the probability conveyed and speaker expertise to shape the judgments and decisions of hearers. For example, experts voicing an internal probability phrase were judged as more knowledgeable than experts using an external probability phrase whereas the result was the opposite for lay speakers. We discuss how these findings inform our understanding of subjective uncertainty and uncertainty communication theories

Coinfection takes its toll: Sea lice override the protective effects of vaccination against a bacterial pathogen in Atlantic salmon

Vaccination is considered crucial for disease prevention and fish health in the global salmon farming industry. Nevertheless, some aspects, such as the efficacy of vaccines, can be largely circumvented during natural coinfections. Sea lice are ectoparasitic copepods that can occur with a high prevalence in the field, are frequently found in co-infection with other pathogens, and are highly detrimental to fish health. The aim of this case-control study was to evaluate the interaction between the detrimental effects of coinfection and the protective effects of vaccination in fish. We used the interaction between the sea louse Caligus rogercresseyi, the bacterial pathogen Piscirickettsia salmonis, and their host, the Atlantic salmon Salmo salar, as a study model. Our results showed that coinfection decreased the accumulated survival (AS) and specific growth rate (SGR) of vaccinated fish (AS = 5.2 ± 0.6%; SGR = −0.05 ± 0.39%) compared to a single infection of P. salmonis (AS = 42.7 ± 1.3%; SGR = 0.21 ± 0.22%). Concomitantly, the bacterial load and clinical signs of disease were significantly increased in coinfected fish. Coinfection may explain the reduced efficacy of vaccines in sea cages and highlights the need to test fish vaccines in more diverse conditions rather than with a single infection.CONICYT-Chile though project FONDECYT N°1140772Cooperative Research Programme Fellowships of OECD (PCI 2015-CONICYT) awarded to J.A.G and P.C.C.F. was supported by PONTIFICIA UNIVERSIDAD CATÓLICA DE VALPARAÍSO (Proyecto VRIEA-PUCV Postdoctorado) and CONICYT-Chile as a Postdoctoral fellowship (FONDECYT N°3170744)D.T. was supported by CONICYT-Chile as a Postdoctoral fellowship (FONDECYT N° 74170029

Protein nanoparticles Made of recombinant Viral antigens : a Promising Biomaterial for Oral Delivery of Fish Prophylactics

In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as "nanopellets" (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated

Caracterización molecular del reflejo inflamatorio y modulación de la respuesta inmune frente a infecciones en peces

El mantenimiento de la homeostasis del sistema inmune innato es fundamental, ya que éste debe mantener una respuesta equilibrada frente a los patógenos. En mamíferos, uno de los mecanismos por el cual se mantiene la homeostasis es el reflejo inflamatorio, un circuito neural que modula la respuesta inflamatoria previniendo la posibilidad de una sobreactivación. El primer objetivo de esta tesis fue establecer si el reflejo inflamatorio descrito en mamíferos también estaba presente en otros vertebrados, y para ello nos centramos en su estudio en teleósteos, más específicamente en trucha (Oncorhynchus mykiss). Una pieza clave del sistema del reflejo inflamatorio es el receptor nicotínico de acetilcolina alfa 7 (α7nAChR). La secuencia del receptor α7nACh de trucha está altamente conservada en comparación con otras especies y el análisis filogenético lo agrupa con otras especies de peces. En estudios in vivo, la estimulación intraperitoneal con lipopolisacárido (LPS) y un análogo del ARN de doble cadena (poly (I:C)), aumentan la expresión del receptor α7nACh en diferentes tejidos y, además, modulan los niveles de acetilcolina en plasma y en bazo. Adicionalmente, el receptor se expresa en macrófagos aislados de bazo y de riñón anterior, en ambos casos siendo capaz de unir α-bungarotoxina, un antagonista específico del receptor α7nACh. Nuestros resultados muestran que existe una modulación de la nicotina (un agonista del receptor) en la expresión génica de moléculas inflamatorias en macrófagos frente a una estimulación con poly (I:C) y no con LPS, a diferencia de estudios realizados en mamíferos. Esta modulación se realiza a través del receptor α7nACh, ya que la adición de un antagonista específico, produce una disminución del efecto de la nicotina. En conclusión, los resultados presentados otorgan indicios de la existencia de un sistema de reflejo inflamatorio en peces, con similitudes y diferencias con el sistema descrito en mamíferos. La mayoría de los teleósteos responden a los patógenos con un potente sistema inmune innato, lo que convierte la modulación de éste, en una buena herramienta para mejorar la capacidad de respuesta de los peces ante una infección. El sistema inmune de los teleósteos puede ser estimulado a través de patrones moleculares asociados a patógenos (PAMPs), y a su vez, por citoquinas, como potenciadores no específicos. La segunda hipótesis de esta tesis es que nanopartículas proteicas, conocidas como cuerpos de inclusión (CI), pueden ser utilizados como plataformas de entrega de inmunoestimulantes. En este contexto, fueron desarrollados CI con (citoquinas) y sin (proteínas fluorescentes) una función inmunológica en peces. Los CI son internalizados in vitro por hepatocitos del pez cebra (Danio rerio) y por macrófagos de trucha, modulando la expresión génica de genes relacionados con la respuesta inflamatoria. Los resultados de la biodistribución in vivo en trucha mostraron que los CI son acumulados en bazo y en riñón anterior, y son endocitados por células inmunes relevantes como, los macrófagos. Los diferentes CI producidos y administrados intraperitonealmente, protegen al pez cebra frente a una infección bacteriana letal (Pseudomonas aeruginosa), siendo mayor la protección otorgada por los CI formados por citoquinas. Adicionalmente, los CI mantienen su morfología y función frente a condiciones extremas de pH y temperatura, así como durante la liofilización. Al ser administrados oralmente en truchas, los CI llegan a los dos primeros tramos del intestino y son absorbidos por células presentes en la mucosa intestinal, las cuales, por su localización en la mucosa podrían corresponderse con linfocitos intraepiteliales (IELs). Nuestros resultados sugieren que los CI pueden ser una alternativa a los métodos actuales como plataforma de entrega de inmunoestimulantes y proteínas por vía oral sin necesidad de encapsulación adicional, siendo éste uno de los principales objetivos de la industria de cultivo de peces.The inflammatory reflex is a neural circuit that modulates the innate immune system response preventing an overactivation. The first hypothesis of this thesis was to establish whether the inflammatory reflex described in mammals was also present in other vertebrates, and we focused on its study in Oncorhynchus mykiss. We cloned and characterised one of the main components of the inflammatory reflex: the alpha 7 nicotinic acetylcholine receptor (α7nAChR). The in vivo stimulation with LPS and poly (I:C) increased the receptor gene expression in different tissues and, also, modulated acetylcholine levels in plasma and spleen. The receptor was expressed in trout macrophages and was able to bind bungarotoxin, a specific receptor antagonist. Our results showed that nicotine modulates the gene expression of inflammatory genes in macrophages through α7nAChR after the stimulation with poly (I:C). In conclusion, the results provide evidences of evolutionary conservation of the inflammatory reflex in vertebrates. Teleost respond to an infections with the activation of a potent innate immune system aiming to eliminate pathogens. Thus, the modulation of the innate system would be an excellent target to improve the responsiveness of the fish to an infection. We have been used protein nanoparticles, known as inclusion bodies (IBs) as immunostimulants delivery platform. The IBs were efficiently internalized in vitro by both zebrafish hepatocytes (Danio rerio) and trout macrophages, and when IBs were administered in vivo they accumulated in the spleen and the head kidney of trout. Importantly, the IBs administered by intraperitoneal injection protect zebrafish against a lethal bacterial infection (Pseudomonas aeruginosa). The orally administered IBs were uptaken by cells of the intestinal mucosa. Our results suggest that the IBs could be an attractive alternative to stimulation of the innate immune system and can be administered orally overcoming the harsh conditions of the gastrointestinal tract

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

Immunomodulation Evidence of Nanostructured Recombinant Proteins in Salmonid Cells

Recent studies have demonstrated that immune-related recombinant proteins can enhance immune function, increasing host survival against infectious diseases in salmonids. This research evaluated inclusion bodies (IBs) of antimicrobial peptides (CAMPIB and HAMPIB) and a cytokine (IL1βIB and TNFαIB) as potential immunostimulants in farmed salmonids. For this purpose, we produced five IBs (including iRFPIB as a control), and we evaluated their ability to modulate immune marker gene expression of three IBs in the RTS11 cell line by RT–qPCR. Additionally, we characterized the scale-up of IBs production by comparing two different scale systems. The results showed that CAMPIB can increase the upregulation of tnfα, il1β, il8, and il10, HAMPIB significantly increases the upregulation of tnfα, inos, and il10, and IL1βIB significantly upregulated the expression of tnfα, il1β, and cox2. A comparison of IL1βIB production showed that the yield was greater in shake flasks than in bioreactors (39 ± 1.15 mg/L and 14.5 ± 4.08 mg/L), and larger nanoparticles were produced in shake flasks (540 ± 129 nm and 427 ± 134 nm, p < 0.0001, respectively). However, compared with its shake flask counterpart, the IL1βIB produced in a bioreactor has an increased immunomodulatory ability. Further studies are needed to understand the immune response pathways activated by IBs and the optimal production conditions in bioreactors, such as a defined medium, fed-batch production, and mechanical bacterial lysis, to increase yield.info:eu-repo/semantics/publishedVersio

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

Immunization of zebrafish with VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs.

<p>Survival curves after i.p. injection of VP1GFP (ClpA^-) and iRFP-H6 (BL21(DE3)) IBs at 150 μg/fish and challenge with <i>P</i>. <i>aeruginosa</i> PAO1 (4.9x10⁷ cfu/animal) (n = 15). Untreated zebrafish that had been infected with PAO1 at LD₅₀ were used as a mortality control. Significant differences were analyzed using the Log-rank test, **, <i>p<</i>0.01.</p