Current vaccination strategy against Piscirickettsia salmonis in Chile based only on the EM-90 genogroup shows incomplete cross-protection for the LF-89 genogroup.

Piscirickettsia salmonis, the primary bacterial disease in Chilean salmon farming, necessitates a constant refinement of control strategies. This study hypothesized that the current vaccination strategy for SRS control in the Chilean Atlantic salmon aquaculture industry, which has been in place since 2017 (ALPHA JECT® 5.1 plus LiVac®), solely relies on vaccines formulated with the EM-90 genogroup of P. salmonis (PS-EM-90), triggering a partial cross-immunity response in fish infected with the LF-89 genogroup (PS-LF-89). Relative Percent Survival (RPS) and cell-mediated immune (CMI) response were evaluated in Atlantic salmon post-smolts vaccinated with the standard vaccination strategy but challenged with both PS-EM-90 and PS-LF-89, in addition to other vaccination strategies considering primo vaccination and booster with other commercial vaccines and the possible enhancing effects of the combination with a natural immunomodulator (PAQ-Xtract®) administered orally. The intraperitoneal (I.P.) challenge was performed after 2395°-days (DD) after the start of the immunostimulant delivery, 1905 DD after the primo vaccination, and 1455 DD after the booster vaccination. Unvaccinated fish showed 73.6 and 41.7 % mortality when challenged with PS-EM-90 and PS-LF-89, respectively. Fish infected with PS-LF-89 died significantly faster (21 days post-infection, dpi) than fish challenged with PS-EM-90 (28 dpi) (p = 0.0043) and had a higher probability of death (0.4626) than fish challenged with PS-EM-90. RPS had a significant positive correlation with the PS-EM-90 load of the P. salmonis genogroup (r = 0.540, p < 0.01) but not with the PS-LF-89 load (r = 0.155, p > 0.05). This demonstrated that the immunization strategies were more effective in lowering PS-EM-90 loads, resulting in higher survival rates in fish challenged with PS-EM-90. The current industry vaccination strategy recorded a 100 % RPS when fish were challenged with PS-EM-90, but the RPS dropped significantly to 77 % when fish were challenged with PS-LF-89, meaning that the strategy did not show complete cross-protection. But after adding PAQ-Xtract®, the RPS improved from 77 % to 92 % in fish that were vaccinated with the standard method but then challenged with PS-LF-89. The most effective vaccination strategy was based on LiVac® as primo vaccination and ALPHA JECT® 5.1 plus LiVac® as booster vaccination, with or without PAQ-Xtract®, in both PS-EM-90 (100 %) and PS-LF-89 (96 %) challenged fish. The serum concentration of anti-P. salmonis IgM did not show a correlation with the protection of immunization strategies expressed in survival. Low serum IL-12 and high serum IFNγ concentrations showed a correlation with higher bacterial loads and lower survival. Aggregate analysis showed a significant correlation between higher numbers of CD8+ cells in the head-kidney, higher fish survival, and a lower bacterial load. The immunization strategies were safe for fish and induced only mild microscopic lesions in the gut. Taken together, our results help to better understand the biological interaction between P. salmonis and post-smolt vaccinated Atlantic salmon to deepen the knowledge on vaccine-induced protection, CMI immune response, and cross-immunity applied to improve the current immunization strategy for SRS control in the Chilean salmon industry.

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 sample 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 treatments 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 stochastic 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 industry-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.

Gene expression associated with immune response in Atlantic salmon head-kidney vaccinated with inactivated whole-cell bacterin of Piscirickettsia salmonis and pathogenic isolates.

Piscirickettsiosis 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 upregulation 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.

Co-Infection by LF-89-Like and EM-90-Like Genogroups of Piscirickettsia Salmonis in Farmed Atlantic Salmon in Chile: Implications for Surveillance and Control of Piscirickettsiosis.

Piscirickettsiosis (SRS), caused by Piscirickettsia salmonis, is the main infectious disease that affects farmed Atlantic salmon in Chile. Currently, the official surveillance and control plan for SRS in Chile is based only on the detection of P. salmonis, but neither of its genogroups (LF-89-like and EM-90-like) are included. Surveillance at the genogroup level is essential not only for defining and evaluating the vaccination strategy against SRS, but it is also of utmost importance for early diagnosis, clinical prognosis in the field, treatment, and control of the disease. The objectives of this study were to characterize the spatio-temporal distribution of P. salmonis genogroups using genogroup-specific real-time probe-based polymerase chain reaction (qPCR) to discriminate between LF-89-like and EM-90-like within and between seawater farms, individual fish, and tissues/organs during early infection in Atlantic salmon under field conditions. The spatio-temporal distribution of LF-89-like and EM-90-like was shown to be highly variable within and between seawater farms. P. salmonis infection was also proven to be caused by both genogroups at farm, fish, and tissue levels. Our study demonstrated for the first time a complex co-infection by P. salmonis LF-89-like and EM-90-like in Atlantic salmon. Liver nodules (moderate and severe) were strongly associated with EM-90-like infection, but this phenotype was not detected by infection with LF-89-like or co-infection of both genogroups. The detection rate of P. salmonis LF-89-like increased significantly between 2017 and 2021 and was the most prevalent genogroup in Chilean salmon aquaculture during this period. Lastly, a novel strategy to identify P. salmonis genogroups based on novel genogroup-specific qPCR for LF-89-like and EM-90-like genogroups is suggested.

First Report of Aeromonas veronii as an Emerging Bacterial Pathogen of Farmed Nile Tilapia (Oreochromis niloticus) in Brazil.

Brazil is one of the world's leading producers of Nile tilapia, Oreochromis niloticus. However, the industry faces a major challenge in terms of infectious diseases, as at least five new pathogens have been formally described in the last five years. Aeromonas species are Gram-negative anaerobic bacteria that are often described as fish pathogens causing Motile Aeromonas Septicemia (MAS). In late December 2022, an epidemic outbreak was reported in farmed Nile tilapia in the state of São Paulo, Brazil, characterized by clinical signs and gross pathology suggestive of MAS. The objective of this study was to isolate, identify, and characterize in vitro and in vivo the causative agent of this epidemic outbreak. The bacterial isolates were identified as Aeromonas veronii based on the homology of 16S rRNA (99.9%), gyrB (98.9%), and the rpoB gene (99.1%). A. veronii showed susceptibility only to florfenicol, while it was resistant to the other three antimicrobials tested, oxytetracycline, enrofloxacin, and amoxicillin. The lowest florfenicol concentration capable of inhibiting bacterial growth was ≤0.5 µg/mL. The phenotypic resistance of the A. veronii isolate observed for quinolones and tetracycline was genetically confirmed by the presence of the qnrS2 (colE plasmid) and tetA antibiotic-resistant genes, respectively. A. veronii isolate was highly pathogenic in juvenile Nile tilapia tested in vivo, showing a mortality rate ranging from 3 to 100% in the lowest (1.2 × 104) and highest (1.2 × 108) bacterial dose groups, respectively. To our knowledge, this study would constitute the first report of highly pathogenic and multidrug-resistant A. veronii associated with outbreaks and high mortality rates in tilapia farmed in commercial net cages in Brazil.

Why Does Piscirickettsia salmonis Break the Immunological Paradigm in Farmed Salmon? Biological Context to Understand the Relative Control of Piscirickettsiosis.

Piscirickettsiosis (SRS) has been the most important infectious disease in Chilean salmon farming since the 1980s. It was one of the first to be described, and to date, it continues to be the main infectious cause of mortality. How can we better understand the epidemiological situation of SRS? The catch-all answer is that the Chilean salmon farming industry must fight year after year against a multifactorial disease, and apparently only the environment in Chile seems to favor the presence and persistence of Piscirickettsia salmonis. This is a fastidious, facultative intracellular bacterium that replicates in the host's own immune cells and antigen-presenting cells and evades the adaptive cell-mediated immune response, which is why the existing vaccines are not effective in controlling it. Therefore, the Chilean salmon farming industry uses a lot of antibiotics-to control SRS-because otherwise, fish health and welfare would be significantly impaired, and a significantly higher volume of biomass would be lost per year. How can the ever-present risk of negative consequences of antibiotic use in salmon farming be balanced with the productive and economic viability of an animal production industry, as well as with the care of the aquatic environment and public health and with the sustainability of the industry? The answer that is easy, but no less true, is that we must know the enemy and how it interacts with its host. Much knowledge has been generated using this line of inquiry, however it remains insufficient. Considering the state-of-the-art summarized in this review, it can be stated that, from the point of view of fish immunology and vaccinology, we are quite far from reaching an effective and long-term solution for the control of SRS. For this reason, the aim of this critical review is to comprehensively discuss the current knowledge on the interaction between the bacteria and the host to promote the generation of more and better measures for the prevention and control of SRS.

Reference Intervals for Blood Biomarkers in Farmed Atlantic Salmon, Coho Salmon and Rainbow Trout in Chile: Promoting a Preventive Approach in Aquamedicine.

The mission of veterinary clinical pathology is to support the diagnostic process by using tests to measure different blood biomarkers to support decision making about farmed fish health and welfare. The objective of this study is to provide reference intervals (RIs) for 44 key hematological, blood biochemistry, blood gasometry and hormones biomarkers for the three most economically important farmed salmonid species in Chile (Atlantic salmon, coho salmon and rainbow trout) during the freshwater (presmolt and smolt age range) and seawater stages (post-smolt and adult age range). Our results confirmed that the concentration or activity of most blood biomarkers depend on the salmonid species, age range and/or the interaction between them, and they are often biologically related to each other. Erythogram and leukogram profiles revealed a similar distribution in rainbow trout and coho salmon, but those in Atlantic salmon were significantly different. While the activity of the most clinically important plasma enzymes demonstrated a similar profile in Atlantic salmon and rainbow trout, coho salmon demonstrated a significantly different distribution. Plasma electrolyte and mineral profiles showed significant differences between salmonid species, especially for rainbow trout, while Atlantic salmon and coho salmon demonstrated a high degree of similarity. Furthermore, electrolytes, minerals and blood gasometry biomarkers were significantly different between age ranges, suggesting a considerably different distribution between freshwater and seawater-farmed fish. The RIs of clinically healthy fish described in this study take into account the high biological variation of farmed fish in Chile, as the 3.059 individuals came from 78 different fish farms, both freshwater and seawater, and blood samples were collected using the same pre-analytical protocol. Likewise, our study provides the Chilean salmon farming industry with standardized protocols that can be used routinely and provides valuable information to improve the preventive vision of aquamedicine through the application of blood biomarkers to support and optimize health, welfare and husbandry management in the salmon farming industry.

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-1ß, IL-8, IL-12ß), allowing the expression of genes associated with T-cell activation (IL-2, CD4, CD8ß), 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.

Atlantic Salmon Pre-smolt Survivors of Renibacterium salmoninarum Infection Show Inhibited Cell-Mediated Adaptive Immune Response and a Higher Risk of Death During the Late Stage of Infection at Lower Water Temperatures.

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 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.

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.

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-κB 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 development of effective strategies for the control of piscirickettsiosis.