Full recombinant flagellin B from Vibrio anguillarum (rFLA) and its recombinant D1 domain (rND1) promote a pro-inflammatory state and improve vaccination against P. salmonis in Atlantic salmon (S. salar)

Flagellin is the major component of the flagellum, and a ligand for Toll-like receptor 5. As reported, recombinant
flagellin (rFLA) from Vibrio anguillarum and its D1 domain (rND1) are able to promote in vitro an upregulation of
pro-inflammatory genes in gilthead seabream (Sparus aurata) and rainbow trout (Oncorhynchus mykiss) macrophages. This study evaluated the in vitro and in vivo stimulatory/adjuvant effect for rFLA and rND1 during
P. salmonis vaccination in Atlantic salmon (Salmo salar). We demonstrated that rFLA and rND1 are molecules able
to generate an acute upregulation of pro-inflammatory cytokines (IL-1β, IL-8, IL-12β), allowing the expression of
genes associated with T-cell activation (IL-2, CD4, CD8β), and differentiation (IFNγ, IL-4/13, T-bet, Eomes,
GATA3), in a differential manner, tissue/time dependent way. Altogether, our results suggest that rFLA and rND1
are valid candidates to be used as an immuno-stimulant or adjuvants with existing vaccines in farmed salmon.

Cost-effectiveness of longitudinal surveillance for Piscirickettsia salmonis using qPCR in Atlantic salmon farms (Salmo salar) in Chile

Costs of diagnostic testing including sample collection, sampling frequency and sam- ple size are an important consideration in the evaluation of the economic feasibility of alternative surveillance strategies for detection of infectious diseases in aquatic animals. In Chile, Piscirickettsia salmonis is the primary reason for antibiotic treat- ments in farmed Atlantic salmon. In 2012, a surveillance and control programme for piscirickettsiosis was established with an overall goal of reducing antibiotic use. The present study estimated the cost-effectiveness of different sampling frequencies and sample sizes to achieve at least 95% confidence of early detection of P. salmonis at the netpen and farm levels using a validated qPCR test. We developed a stochas- tic model that incorporated variability in test accuracy, within-pen prevalence and sampling costs. Our findings indicated that the current piscirickettsiosis surveillance programme based on risk-based sampling of five moribund or dead fish from 2 to 3 netpens is cost-effective and gives a high probability of detection of P. salmonis in Atlantic salmon farms in Chile at both the netpen and farm levels. Results from this study should incentivize salmon farmers to establish cost-effective strategies for early detection of P. salmonis infection and the application of this approach to other highly infectious diseases.


Bayesian estimation of diagnostic sensitivity and specificity of a qPCR and a bacteriological culture method for Piscirickettsia salmonis in farmed Atlantic salmon (Salmo salar L.) in Chile

Early detection of piscirickettsiosis is an important purpose of government- and in- dustry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.


Atlantic salmon pre-smolt survivors of Renibacterium salmoninarum infection…

Bacterial kidney disease (BKD) is widespread in many areas of the world and can cause substantial economic losses for the salmon aquaculture industry. The objective of this study was to investigate the pathophysiological response and gene expression profiles related to the immune response at different water temperatures and to identify the best immunopathological biomarkers to define a phenotype of resistance to BKD. The abundance of msa transcripts of R. salmoninarum in the head kidney was significantly higher in infected fish at 11°C. R. salmoninarum induced significantly more severe kidney lesions, anemia and impaired renal function at 11°C. In addition, the expression pattern of the genes related to humoral and cell-mediated immune responses in infected fish at 11°C and 15°C was very similar, although R. salmoninarum induced a significantly greater downregulation of the adaptive immune response genes at the lower water temperature. These results could be due to a suppressed host response directly related to the lowest water temperature and/or associated with a delayed host response related to the lowest water temperature. Although no significant differences in survival rate were observed, fish infected at the lowest temperature showed a higher probability of death and delayed the mortality curve during the late stage of infection (35 days after infection). Thirty-three immunopathological biomarkers were identified for potential use in the search for a resistance phenotype for BKD, and eight were genes related specifically to the adaptive cell-mediated immune response.


Gene expression associated with immune response in Atlantic salmon…

iscirickettsiosis is the most challenging disease present in the Chilean salmon industry. The aim of this study was to describe the expression of genes associated with immune response of Atlantic salmon intraperitoneally infected with LF-89 and EM-90 Piscirickettsia salmonis and vaccinated with inactivated whole-cell bacterin of P. salmonis. The fish infected with PS-LF-89 showed an anti-inflammatory response, whereas this finding was not observed in the PS-EM-90-infected fish and vaccinated fish. Fish infected with both P. salmonis isolates showed mhc1-mhc2, cd4-cd8b and igm overexpression, suggesting that P. salmonis promotes a T CD4+ and T CD8+ cell response and a humoral immune response. The vaccinated-fish exhibited mhc1, mhc2 and cd4 overexpression but a significant downregulation of cd8b and igm, suggesting that the vaccine supported the CD4+ T-cell response but did not induce an immune response mediated by CD8+ T cells or a humoral response. In conclusion, the expression pattern of genes related to the humoral and cell-mediated adaptive immune response showed up- regulation in fish infected with P. salmonis and down-regulation in vaccinated fish. The results of this study contribute to our understanding of the immune response against P. salmonis and can be used in the optimization of SRS prevention and control measures.


Gill diseases in marine salmon aquaculture with an emphasis on amoebic gill disease…

ROZAS-SERRI MA. 2019. Gill diseases in marine salmon aquaculture with an emphasis on amoebic gill disease. CAB Reviews 14(32):1-15.


Gill diseases are a growing health challenge in salmon farming worldwide, but many gaps remain in our knowledge. Gill diseases are generally complex and multifactorial disorders, often with presumable spatial and temporal distribution patterns, but are highly difficult to effectively prevent and control. The term complex gill disease (CGD) includes a wide range of clinical disease presentations on the gills of farmed salmon; usually, CGD presents from the end of summer to early winter. The pathogens involved include Neoparamoeba perurans, Tenacibaculum maritimum, Candidatus Piscichlamydia salmonis, Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii (syn. Paranucleospora theridion) and viruses, such as the Atlantic salmon paramyxovirus (ASPV) and salmon gill poxvirus (SGPV). Amoebic gill disease (AGD) is perhaps the most significant disease in terms of gill health and economic impact. AGD results in high mortality, reduced production performance and impaired fish welfare. This review summarizes and analyses CGD research, outbreaks and treatment, with a focus on AGD, as well as on knowledge gaps and avenues for future research.


Transcriptomic profiles of post-smolt Atlantic salmon challenged with Piscirickettsia salmonis…

ROZAS-SERRI MA, PEÑA A, MALDONADO L. 2018. Transcriptomic profiles of post-smolt Atlantic salmon challenged with Piscirickettsia salmonis reveal a strategy to evade the adaptive immune response and modify cell-autonomous immunity. Develepmental and Comparative Immunology 81, 348-362.


Piscirickettsiosis is the main bacterial disease affecting the Chilean salmon farming industry and is responsible for high economic losses. The development of effective strategies to control piscirickettsiosis has been limited in part by insufficient knowledge of the host response. The aim of this study was to use RNA sequencing to describe the transcriptional profiles of the responses of post-smolt Atlantic salmon infected with LF-89-like or EM-90-like Piscirickettsia salmonis. Enrichment and pathway analyses of the differentially expressed genes revealed several central signatures following infection, including positive regulation of DC-SIGN and TLR5 signalling, which converged at the NF-kB level to modulate the pro- inflammatory cytokine response, particularly in the PS-EM-90-infected fish. P. salmonis induced an IFN-inducible response (e.g., IRF-1 and GBP-1) but inhibited the humoral and cell-mediated immune responses. P. salmonis induced significant cytoskeletal reorganization but decreased lysosomal protease activity and caused the degradation of proteins associated with cellular stress. Infection with these isolates also delayed protein transport, antigen processing, vesicle trafficking and autophagy. Both P. salmonis isolates promoted cell survival and proliferation and inhibited apoptosis. Both groups of Trojan fish used similar pathways to modulate the immune response at 5 dpi, but the transcriptomic profiles in the head kidneys of the cohabitant fish infected with PS-LF-89 and PS-MS-90 were relatively different at day 35 post-infection of the Trojan fish, probably due to the different degree of pathogenicity of each isolate. Our study showed the most important biological mechanisms used by P. salmonis, regardless of the isolate, to evade the immune response, maintain the viability of host cells and increase intracellular replication and persistence at the infection site. These results improve the understanding of the mechanisms by which P. salmonis interacts with its host and may serve as a basis for the develop- ment of effective strategies for the control of piscirickettsiosis.


Comparison of gene expression in post-smolt Atlantic salmon challenged by LF-89-like…

ROZAS-SERRI M, PEÑA A, ARRIAGADA G, ENRÍQUEZ R, MALDONADO L. 2018. Comparison of gene expression in post-smolt Atlantic salmon (Salmo salar L.) challenged by LF-89-like and EM-90-like Piscirickettsia salmonis isolates reveals differences in the immune response associated with pathogenicity. Journal of Fish Diseases 41(3), 539-552.


Piscirickettsiosis is the main bacterial disease affecting the Chilean salmon farming industry and is responsible for high economic losses. The aim of this study was to describe and comparatively quantify the immune response of post-smolt Atlantic salmon infected by cohabitation with fish bearing LF-89-like and EM-90-like Piscir- ickettsia salmonis. The expression of 17 genes related to the immune response was studied in head kidney from cohabitant fish by RT-qPCR. Our results at the transcriptomic level suggest that P. salmonis is able to manipulate the kinetics of cytokine production in a way that might constitute a virulence mechanism that pro- motes intracellular bacterial replication in cells of Atlantic salmon. This strategy involves the creation of an ideal environment for the microorganism based on induction of the inflammatory and IFN-mediated response, modulation of Th1 polar- ization, reduced antigen processing and presentation, modulation of the evasion of the immune response mediated by CD8+ T cells and promotion of the CD4+ T-cell response during the late stage of infection as a mechanism to escape host defences. This response was significantly exacerbated in fish infected by PS-EM-90 compared with fish infected by PS-LF-89, a finding that is probably associated with the higher pathogenicity of PS-EM-90.


Comparative pathogenesis of piscirickettsiosis in Atlantic salmon (Salmo salar L.)…

ROZAS-SERRI M, ILDEFONSO R, PEÑA A, ENRÍQUEZ R, BARRIENTOS S, MALDONADO L. 2017. Comparative pathogenesis of piscirickettsiosis in Atlantic salmon (Salmo salar L.) post-smolt experimentally challenged with LF-89-like and EM-90-like Piscirickettsia salmonis isolates. Journal of Fish Diseases 40(10), 1451-1472.


Piscirickettsiosis (SRS) is the most prevalent bacterial disease in Chilean salmon aquaculture and is responsible for high economic losses. The aim of this study was to comparatively characterize the pathogenesis of SRS in post-smolt Atlantic salmon during the early and late stages of infection with Piscirickettsia salmonis LF-89-like (PS-LF-89) and EM-90-like (PS-EM-90) using a cohabitation challenge. The patho- genesis of cohabitant fish infected with the two isolates was relatively different due to cohabitant fish infected with PS-EM-90 showing higher cumulative mortality and shorter time until death compared with PS-LF-89 fish. PS-LF-89 caused an SRS infection characterized by kidney and liver lesions, whereas PS-EM-90 caused sys- temic and haemorrhagic disease characterized by kidney, liver, heart, brain, skeletal muscle and intestine lesions. Decreased serum concentration of total proteins and albumin as well as increased serum ALT, AST and creatinine levels in fish infected with both isolates confirmed that changes in liver and kidney function occurred dur- ing infection. Tissue damage, expressed as an SRS histoscore, showed a strong posi- tive correlation with the bacterial load expressed as abundance of P. salmonis 16S rRNA transcripts in the livers and kidneys of fish affected with either isolate, but the correlation was significantly higher in fish infected with PS-EM-90. The results contribute to improving the understanding of the bacteria–host interaction.


Piscirickettsiosis and Piscirickettsia salmonis in fish: a review

ROZAS MA, ENRÍQUEZ VR. 2014. Piscirickettsiosis and Piscirickettsia salmonis in fish: a review. Journal of Fish Diseases 37:163-188.


The bacterium Piscirickettsia salmonis is the aetio- logical agent of piscirickettsiosis a severe disease that has caused major economic losses in the aquaculture industry since its appearance in 1989. Recent reports of P. salmonis or P. salmonis-like organisms in new fish hosts and geographical regions have increased interest in the bacterium. Because this gram-negative bacterium is still poorly understood, many relevant aspects of its life cycle, virulence and pathogenesis must be investigated before prophylactic procedures can be properly designed. The development of effective control strategies for the disease has been limited due to a lack of knowledge about the biology, intracellular growth, transmission and virulence of the organism. Piscirickettsiosis has been difficult to control; the failure of antibiotic treatment is common, and currently used vaccines show vari- able long-term efficacy. This review summarizes the biology and characteristics of the bacterium, including its virulence; the infective strategy of P. salmonis for survival and evasion of the host immune response; the host immune response to invasion by this pathogen; and newly described features of the pathology, pathogenesis, epidemiol- ogy and transmission. Current approaches to the prevention of and treatment for piscirickettsiosis are discussed.