RESUMO
The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.
Assuntos
Copépodes/imunologia , Ectoparasitoses/veterinária , Epitopos de Linfócito T/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Imunoglobulina M/imunologia , Animais , Proteínas de Artrópodes/imunologia , Peixes-Gato/imunologia , Ciclídeos/imunologia , Ectoparasitoses/imunologia , Peptídeos/imunologia , Proteínas Ribossômicas/imunologia , Salmo salar/imunologia , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The increasing capacity of transcriptomic analysis by high throughput sequencing has highlighted the presence of a large proportion of transcripts that do not encode proteins. In particular, long non-coding RNAs (lncRNAs) are sequences with low coding potential and conservation among species. Moreover, cumulative evidence has revealed important roles in post-transcriptional gene modulation in several taxa. In fish, the role of lncRNAs has been scarcely studied and even less so during the immune response against sea lice. In the present study we mined for lncRNAs in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynkus kisutch), which are affected by the sea louse Caligus rogercresseyi, evaluating the degree of sequence conservation between these two fish species and their putative roles during the infection process. Herein, Atlantic and Coho salmon were infected with 35 lice/fish and evaluated after 7 and 14 days post-infestation (dpi). For RNA sequencing, samples from skin and head kidney were collected. A total of 5658/4140 and 3678/2123 lncRNAs were identified in uninfected/infected Atlantic and Coho salmon transcriptomes, respectively. Species-specific transcription patterns were observed in exclusive lncRNAs according to the tissue analyzed. Furthermore, neighbor gene GO enrichment analysis of the top 100 highly regulated lncRNAs in Atlantic salmon showed that lncRNAs were localized near genes related to the immune response. On the other hand, in Coho salmon the highly regulated lncRNAs were localized near genes involved in tissue repair processes. This study revealed high regulation of lncRNAs closely localized to immune and tissue repair-related genes in Atlantic and Coho salmon, respectively, suggesting putative roles for lncRNAs in salmon against sea lice infestation.
Assuntos
Doenças dos Peixes/genética , Imunidade/genética , Infestações por Piolhos/genética , Oncorhynchus kisutch/genética , RNA Longo não Codificante/genética , Salmo salar/genética , Transcriptoma , Animais , Copépodes/imunologia , Copépodes/fisiologia , Doenças dos Peixes/imunologia , Perfilação da Expressão Gênica , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Parasita , Infestações por Piolhos/imunologia , Infestações por Piolhos/parasitologia , Oncorhynchus kisutch/imunologia , Oncorhynchus kisutch/parasitologia , Salmo salar/imunologia , Salmo salar/parasitologia , Especificidade da Espécie , Cicatrização/genéticaRESUMO
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.
Assuntos
Coinfecção/imunologia , Copépodes/imunologia , Copépodes/microbiologia , Doenças dos Peixes/imunologia , Salmo salar/imunologia , Salmo salar/microbiologia , Animais , Doenças dos Peixes/microbiologia , Vacinação/métodosRESUMO
MicroRNAs are non-coding RNA that plays a crucial role in post-transcriptional regulation and immune system regulation. On other hand, sea lice are prevalent parasites that affect salmon farming, generating different degrees of immune suppression depending on the salmon and sea louse species. Caligus rogercresseyi for example, which affects the salmon industry in Chile, decreases Th1 response, macrophage activation, TLR-mediated response and iron regulation in infected fish. In this study, we explore Atlantic salmon miRNome during infestation by C. rogercresseyi. Using small RNA sequencing, we annotated 1718 miRNAs for skin and head kidney from infected Atlantic salmon. The most abundant families identified were mir-10, mir-21, mir-30, mir-181 and let7. Significant differences were found between tissue, with 1404 annotated miRNA in head kidney and 529 in skin. Differential analysis of transcript expression indicated that at an early stage of infestation miRNA expression was higher in head kidney than in skin tissue, revealing tissue-specific expression patterns. In parallel, miRNA target prediction using 3'UTRs from highly regulated immune-related genes and iron metabolism showed that mir-140-4 and mir-181a-2-5 modulate the expression of TLR22 and Aminolevulinic acid synthase, respectively. This study contributes knowledge about the immune response of Atlantic salmon during infestation with sea lice.
Assuntos
Copépodes/imunologia , Doenças dos Peixes/imunologia , Rim Cefálico/fisiologia , MicroRNAs/genética , Doenças Parasitárias em Animais/imunologia , Salmo salar/imunologia , Pele/patologia , Animais , Chile , Biologia Computacional , Ectoparasitoses , Rim Cefálico/parasitologia , Imunidade/genética , Imunomodulação , Ferro/metabolismo , Especificidade de Órgãos , Salmo salar/parasitologia , Análise de Sequência de RNA , Pele/parasitologia , TranscriptomaRESUMO
Sea lice infestations are a particular concern in the salmonid aquaculture industry due to damaging effects on fish growth, disease/infection susceptibility, and survival. Despite the impacts of sea lice parasitism, few studies have determined corresponding physiological thresholds, or the quantity of sea lice that can trigger measurable effects in the host immune response. The present study evaluated the mRNA expressions of immune-related genes in Salmo salar (Atlantic salmon) under infestation challenges with contrasting loads of the sea louse Caligus rogercresseyi. Specifically, two groups of S. salar were infected with either 35 (i.e. low parasitic load) or 100 (i.e. high parasitic load) copepodids per fish. At 14 days post-infestation, the mRNA levels of immune-related genes (e.g. related to oxidative stress, pro- and inflammatory responses, and the adaptive TH1/TH2 pathways) were assessed through RT-qPCR. Significant differences were found in relation to parasitic load, suggesting density-dependent effects that activated the S. salar immune system. Higher parasitic load promoted strong inflammatory and oxidative stress responses that were correlated with the TH1 immune response. This study highlights the molecular signatures for distinct parasitic loads, providing new perspectives towards fully understanding parasite-host interactions.
Assuntos
Copépodes/imunologia , Ectoparasitoses/veterinária , Doenças dos Peixes/imunologia , Interações Hospedeiro-Parasita/imunologia , Salmo salar , Animais , Chile , Ectoparasitoses/imunologia , Ectoparasitoses/parasitologia , Doenças dos Peixes/parasitologia , Densidade Demográfica , Células Th1/metabolismoRESUMO
The Toll and IMD signaling pathways represent one of the first lines of innate immune defense in invertebrates like Drosophila. However, for crustaceans like Caligus rogercresseyi, there is very little genomic information and, consequently, understanding of immune mechanisms. Massive sequencing data obtained for three developmental stages of C. rogercresseyi were used to evaluate in silico the expression patterns and presence of SNPs variants in genes involved in the Toll and IMD pathways. Through RNA-seq analysis, which used 20 contigs corresponding to relevant genes of the Toll and IMD pathways, an overexpression of genes linked to the Toll pathway, such as toll3 and Dorsal, were observed in the copepod stage. For the chalimus and adult stages, overexpression of genes in both pathways, such as Akirin and Tollip and IAP and Toll9, respectively, were observed. On the other hand, PCA statistical analysis inferred that in the chalimus and adult stages, the immune response mechanism was more developed, as evidenced by a relation between these two stages and the genes of both pathways. Moreover, 136 SNPs were identified for 20 contigs in genes of the Toll and IMD pathways. This study provides transcriptomic information about the immune response mechanisms of Caligus, thus providing a foundation for the development of new control strategies through blocking the innate immune response.
Assuntos
Proteínas de Artrópodes/genética , Copépodes/genética , Copépodes/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais , Receptores Toll-Like/genética , Transcriptoma , Animais , Proteínas de Artrópodes/metabolismo , Chile , Simulação por Computador , Copépodes/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de RNA , Receptores Toll-Like/metabolismoRESUMO
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.
Assuntos
Antígenos/imunologia , Copépodes/imunologia , Doenças dos Peixes/prevenção & controle , Salmo salar/parasitologia , Vacinas , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos/química , Antígenos/genética , Aquicultura , Sequência de Bases , Chile , Clonagem Molecular , Copépodes/química , Copépodes/genética , DNA Complementar/química , Feminino , Doenças dos Peixes/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Noruega , Filogenia , Conformação Proteica , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , TilápiaRESUMO
The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.
Assuntos
Anticorpos/imunologia , Doenças dos Peixes/imunologia , Imuno-Histoquímica/veterinária , Peroxirredoxinas/imunologia , Salmonidae/imunologia , Animais , Células Cultivadas , Copépodes/imunologia , Ectoparasitoses/imunologia , Ectoparasitoses/veterinária , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Rim Cefálico/imunologia , Macrófagos/imunologia , Vibrio/imunologia , Vibrioses/imunologia , Vibrioses/veterináriaRESUMO
Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.