RESUMO
Salmonid rickettsial septicaemia (SRS) is a contagious disease caused by Piscirickettsia salmonis, an intracellular bacterium. SRS causes an estimated economic loss of $700 million USD to the Chilean industry annually. Vaccination and antibiotic therapy are the primary prophylactic and control measures used against SRS. Unfortunately, commercially available SRS vaccines have not been shown to have a significant effect on reducing mortality. Most vaccines contain whole inactivated bacteria which results in decreased efficacy due to the limited ability of the vaccine to evoke a cellular mediated immune response that can eliminate the pathogen or infected cells. In addition, SRS vaccine efficacy has been evaluated primarily with Salmo salar (Atlantic salmon). Vaccine studies using Oncorhynchus mykiss (rainbow trout) are scarce, despite SRS being the leading cause of infectious death for this species. In this study, we evaluate an injectable vaccine based on P. salmonis proteoliposome; describing the vaccine security profile, capacity to induce specific anti-P. salmonis IgM and gene expression of immune markers related to T CD8 cell-mediated immunity. Efficacy was determined by experimental challenge with P. salmonis intraperitoneally. Our findings indicate that a P. salmonis proteoliposome-based vaccine is able to protect O. mykiss against challenge with a P. salmonis Chilean isolate and causes a specific antibody response. The transcriptional profile suggests that the vaccine is capable of inducing cellular immunity. This study provides new insights into O. mykiss protection and the immune response induced by a P. salmonis proteoliposome-based vaccine.
Assuntos
Vacinas Bacterianas/administração & dosagem , Doenças dos Peixes/prevenção & controle , Oncorhynchus mykiss , Infecções por Piscirickettsiaceae/veterinária , Proteolipídeos/uso terapêutico , Sepse/veterinária , Vacinação/veterinária , Animais , Chile , Doenças dos Peixes/microbiologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/prevenção & controle , Sepse/microbiologia , Sepse/prevenção & controleRESUMO
An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1ß and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only the innate immune responses, but did not protect Salmo salar against P. salmonis. These results suggest that the knowledge of the formulation of vaccines based on P. salmonis proteins is useful as an effective therapy, this demonstrates the importance of the different research tools to improve the study of the different immune responses, resistance to diseases in the Atlantic salmon. We suggest that this vaccine can help prevent widespread infection by P. salmonis, in addition to being able to be used as a booster after a primary vaccine to maintain high levels of circulating protective antibodies, greatly helping to reduce the economic losses caused by the pathogen.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Peixes , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae , Salmo salar , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/prevenção & controle , Infecções por Piscirickettsiaceae/veterinária , Salmo salar/imunologia , Salmo salar/microbiologiaRESUMO
Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.
Assuntos
Anticorpos Antibacterianos/imunologia , Doenças dos Peixes/imunologia , Lisossomos/imunologia , Macrófagos/imunologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/imunologia , Salmão/imunologia , Animais , Doenças dos Peixes/microbiologia , Lisossomos/microbiologia , Macrófagos/microbiologia , Infecções por Piscirickettsiaceae/veterinária , Salmão/microbiologiaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as key regulators in diverse biological processes across taxa. However, despite the importance of these transcripts, little is known about their role during the immune response in salmonids. Because of this, we use deep sequencing technologies to explore the microRNA-based transcriptomic response of the Atlantic salmon (Salmo salar) to the intracellular bacteria Piscirickettsia salmonis, one of the main threats to salmon aquaculture in Chile. Hence, 594 different miRNAs were identified from head kidney and spleen transcriptomic data. Among them, miRNA families mir-181, mir-143 and mir-21 were the most abundant in control groups, while after infection with P. salmonis, mir-21, mir-181 and mir-30 were the most predominant families. Furthermore, transcriptional analysis revealed 84 and 25 differentially expressed miRNAs in head kidney and spleen respectively, with an overlapping response of 10 miRNAs between the analyzed tissues. Target prediction, coupled with GO enrichment analysis, revealed that the possible targets of the most regulated miRNAs were genes involved in the immune response, such as cortisol metabolism, chemokine-mediated signaling pathway and neutrophil chemotaxis genes. Among these, predicted putative target genes such as C-C motif chemokine 19-like, stromal cell-derived factor 1-like, myxovirus resistance protein 2 and hepcidin-1 were identified. Overall, our results suggest that miRNA expression in co-modulation with transcription activity of target genes is related to putative roles of non-coding RNAs in the immune response of Atlantic salmon against intracellular bacterial pathogens.
Assuntos
Rim Cefálico/fisiologia , MicroRNAs/genética , Neutrófilos/fisiologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/genética , Salmo salar/genética , Baço/fisiologia , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Quimiotaxia/genética , Chile , Rim Cefálico/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala , Hidrocortisona/metabolismo , Imunidade Inata/genética , Infecções por Piscirickettsiaceae/imunologia , Salmo salar/imunologia , Baço/microbiologia , TranscriptomaRESUMO
INTRODUCTION: Piscirickettsia salmonis (P. salmonis) is the aetiological bacterium of the contagious disease piscirickettsiosis or salmonid rickettsial septicaemia (SRS) and causes significant economic losses to aquaculture production in Chile. Current strategies to control infection are i) indiscriminate antibiotic use and ii) vaccination with predominantly P. salmonis bacterin vaccines that do not provide acceptable levels of protection against piscirickettsiosis. Areas covered: This review covers the basic biology of P. salmonis, clinical piscirickettsiosis and disease control, the development of current P. salmonis vaccines, innate and adaptive immunity and a 5-year plan to develop new piscirickettsiosis vaccines. Expert commentary: Fundamental knowledge is lacking on the complexities of P. salmonis-host interactions, relating to bacterial virulence and host innate and adaptive immune responses, which needs to be addressed. The development of new P. salmonis vaccines needs the application of comprehensive 'omics' technologies to identify candidate vaccine antigens capable of stimulating long-lasting protective immune responses.
Assuntos
Vacinas Bacterianas/administração & dosagem , Doenças dos Peixes/prevenção & controle , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/veterinária , Sepse/veterinária , Animais , Aquicultura/métodos , Chile , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/prevenção & controle , Salmão , Sepse/microbiologia , Sepse/prevenção & controle , TrutaRESUMO
Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called "salmon rickettsial syndrome". Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected-cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.
Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Macrófagos/imunologia , Oncorhynchus mykiss/imunologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/imunologia , Animais , Linhagem Celular , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Hepcidinas/imunologia , Interleucina-10/imunologia , Interleucina-12/imunologiaRESUMO
Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples.
Assuntos
Vacinas Bacterianas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Luminescência , Piscirickettsia/imunologia , Animais , Reprodutibilidade dos TestesRESUMO
Rickettsial organisms are well-known fish pathogens in both natural and culture environments. This study reports an outbreak of disease in red tilapia larvae caused by piscirickettsia-like organisms (PLOs), which lasted from June until October 2009. Severe mortality was recorded almost exclusively in larvae and postlarvae aged 1-22 days old. Although clinical or gross findings were not evident in diseased fish, histopathology revealed severe necrosis of the epidermis and gill epithelium, with concomitant changes in the underlying skeletal muscle as being the most relevant microscopic lesions. Although PLOs were visible with the routine hematoxylin eosin technique, they were better observed with Giemsa and toluidine blue stains. Transmission electron microscopy revealed that the bacterium was located within the cytoplasm and phagolysosoma-like structures of epithelial cells from the gills and the skin. The bacteria measured 0.9 ± 0.2 µm × 2.1 ± 0.6 µm and had a double cell membrane (the outer one having undulating projections), with variable electron-dense and electron-lucent areas. Ultrastructurally, abundant myelin figures surrounded the microorganisms within host cell cytoplasm. Results indicated that Piscirickettsia-like organisms can cause massive epithelial cell damage associated with concomitant alteration of the electrolyte balance.
Assuntos
Surtos de Doenças/veterinária , Doenças dos Peixes/microbiologia , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Tilápia , Animais , Aquicultura , Colômbia/epidemiologia , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/patologia , Brânquias/microbiologia , Brânquias/patologia , Brânquias/ultraestrutura , Histocitoquímica/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Piscirickettsia/ultraestrutura , Infecções por Piscirickettsiaceae/epidemiologia , Infecções por Piscirickettsiaceae/patologiaRESUMO
Effective oral immunization systems may be very helpful to the salmon industry, particularly during the seawater growth stages in which vaccination through injection is not possible. During the seawater growing stage, fish become more susceptible to several types of disease, due to the natural decay of vaccine-induced immune responses. In this study, we demonstrate the immune response and efficacy of a new salmonid rickettsial septicaemia (SRS) oral vaccine, developed using MicroMatrix™ Technology. The vaccine, which is administered together with daily feed ration, induces a specific immune response at local and systemic levels. Anti-Piscirickettsia salmonis specific antibodies were detected as soon as 300 degree-days after vaccination. Furthermore, oral vaccination was able to protect fish against a lethal pathogen challenge when administered either as a primary vaccination or as a booster for an injected vaccine. Results show that oral vaccination is an efficacious treatment for the prevention of SRS outbreaks throughout the salmon culture period.
Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Piscirickettsiaceae/veterinária , Salmo salar/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Doenças dos Peixes/imunologia , Imunidade nas Mucosas , Imunização Secundária , Imunoglobulina M/sangue , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/prevenção & controle , Aumento de PesoRESUMO
The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsia salmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified and expressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infected salmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies have allowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identified it as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading frame encoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed in vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterization was attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential of ChaPs was determined with serum from naturally infected fish.