[Cu(NN1)2]ClO4, a Copper (I) Complex as an Antimicrobial Agent for the Treatment of Piscirickettsiosis in Atlantic Salmon.

Piscirickettsia salmonis is the pathogen that most affects the salmon industry in Chile. Large quantities of antibiotics have been used to control it. In search of alternatives, we have developed [Cu(NN1)2]ClO4 where NN1 = 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one. The antibacterial capacity of [Cu(NN1)2]ClO4 was determined. Subsequently, the effect of the administration of [Cu(NN1)2]ClO4 on the growth of S. salar, modulation of the immune system and the intestinal microbiota was studied. Finally, the ability to protect against a challenge with P. salmonis was evaluated. The results obtained showed that the compound has an MIC between 15 and 33.9 µg/mL in four isolates. On the other hand, the compound did not affect the growth of the fish; however, an increase in the transcript levels of IFN-γ, IL-12, IL-1ß, CD4, lysozyme and perforin was observed in fish treated with 40 µg/g of fish. Furthermore, modulation of the intestinal microbiota was observed, increasing the genera of beneficial bacteria such as Lactobacillus and Bacillus as well as potential pathogens such as Vibrio and Piscirickettsia. Finally, the treatment increased survival in fish challenged with P. salmonis by more than 60%. These results demonstrate that the compound is capable of protecting fish against P. salmonis, probably by modulating the immune system and the composition of the intestinal microbiota.

Optimization of florfenicol dose against Piscirickettsia salmonis in Salmo salar through PK/PD studies.

Salmonid Rickettsial Septicemia (SRS) is the disease of greatest economic importance in the Chilean salmon farming industry, causing high mortality in fish during the final stage of their productive cycle at sea. Since current, commercially available vaccines have not demonstrated the expected efficacy levels, antimicrobials, most commonly florfenicol, are still the main resource for the treatment and control of this pathogen. The aim of this study was to determine the most appropriate single dose of florfenicol, administered through medicated feed, for the treatment of Piscirickettsia salmonis (P. salmonis), using pharmacokinetic/pharmacodynamic (PK/PD) models. Previously, Minimum Inhibitory Concentrations (MICs) of florfenicol were determined for 87 P. salmonis isolates in order to define the epidemiological cut-off point (COWT). The most commonly observed MIC was 0.125 µg mL-1 (83.7%). The COWT value was 0.25 µg mL-1 with a standard deviation of 0.47 log2 µg mL-1 and 0.36 log2 µg mL-1, for Normalized resistance interpretation (NRI) method and ECOFFinder method, respectively. A MIC of 1 µg mL-1 was considered the pharmacodynamic value (PD) to define PK/PD indices. Three doses of florfenicol were evaluated in fish farmed under controlled conditions. For each dose, 150 fish were used and blood plasma samples were collected at different time points (0-48 hours). PK parameters were obtained from curves representing plasma concentrations as a function of time. The results of Monte Carlo simulation indicate that at a dose of 20 mg/Kg l.w. of florfenicol, administered orally as medicated feed, there is 100% probability (PTA) of achieving the desired efficacy (AUC0-24h/MIC>125). According to these results, we suggest that at the indicated dose, the PK/PD cut-off point for florfenicol versus P. salmonis could be 2 µg mL-1 (PTA = 99%). In order to assess the indicated dose in Atlantic salmon, fish were inoculated with P. salmonis LF-89 strain and then treated with the optimized dose of florfenicol, 20 mg/Kg bw for 15 days.

Assessing concentration of antibiotics in tissue during oral treatments against piscirickettsiosis.

The use of antimicrobials in aquaculture is increasingly being scrutinized. In Chile, piscirickettsiosis accounts for approximately 90% of the total volume of antibiotics used in marine aquaculture. Treatment failures are frequently reported, but there is limited information on why this occurs. Fish producers have started assessing the level of antibiotics in fish tissues during and immediately after in-feed treatments to determine if they are adequately medicating their fish. In this study, we evaluated the probability of finding antibiotic concentrations in muscle tissue above the minimum inhibitory concentration for 90% of the P. salmonis isolates (MIC90) recently tested in Chile, for two antibiotics commonly used in aquaculture. We found that the proportion of fish with antibiotic concentrations above the MIC90 varied, depending on the product used, species, day of sample collection, and size category of fish within a cage. The proportion of fish above the MIC90 was lower in fish treated with florfenicol than in fish treated with oxytetracycline. Using a mixed-effects logistic model, we modeled the probability of antibiotic concentrations above MIC90 when fish were treated with florfenicol. Our model suggested lower probabilities of having concentrations above MIC90 in Atlantic salmon than in rainbow trout when samples were collected 14 days after the treatment started compared to 7 days, and in the smaller fish within a cage. We discuss these findings and hypothesize about potential issues with treating large populations of fish with in-feed antimicrobials.

Retrospective analysis of antibiotic treatments against piscirickettsiosis in farmed Atlantic salmon Salmo salar in Chile.

Piscirickettsiosis is the most prevalent salt-water infectious disease in farmed salmonids in Chile. Antimicrobials are used to treat this disease; however, there is growing concern about the poor response to therapeutants on some fish farms. The objective of this study was to assess whether factors such as type of antibiotic used, average fish weight, temperature at the beginning of the treatment, and mortality at the time of treatment administration affect the probability of treatment failure against piscirickettsiosis. Pen-level treatment and production information for the first treatment event from 2014 pens on 118 farms was used in a logistic mixed model to assess treatment failure. We defined a failed treatment as when the average mortality 3 wk after the treatment was above 0.1%. Farm and company were included in the model as random effects. We found that the antibiotic product, mortality level before the treatment, and fish weight at the start of the treatment all had a significant effect on treatment outcome. Our results suggest that antibiotic treatment success is higher if the treatment is administered when mortality associated with piscirickettsiosis is relatively low. We discuss the effect of weight on treatment success and its potential relationships with husbandry practices and drug pharmacokinetics.

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.