Infectious Salmon Anemia Virus Shedding from Infected Atlantic Salmon (Salmo salar L.)—Application of a Droplet Digital PCR Assay for Virus Quantification in Seawater

Infectious salmon anemia virus (ISAV) infection is currently detected by fish sampling for PCR and immunohistochemistry analysis. As an alternative to sampling fish, we evaluated two different membrane filters in combination with four buffers for elution, concentration, and detection of ISAV in seawater, during a bath challenge of Atlantic salmon (Salmo salar L.) post-smolts with high and low concentrations of ISAV. Transmission of ISAV in the bath challenge was confirmed by a high mortality, clinical signs associated with ISA disease, and detection of ISAV RNA in organ tissues and seawater samples. The electronegatively charged filter, combined with lysis buffer, gave significantly higher ISAV RNA detection by droplet digital PCR from seawater (5.6 × 104 ISAV RNA copies/L; p < 0.001). Viral shedding in seawater was first detected at two days post-challenge and peaked on day 11 post-challenge, one day before mortalities started in fish challenged with high dose ISAV, demonstrating that a large viral shedding event occurs before death. These data provide important information for ISAV shedding that is relevant for the development of improved surveillance tools based on water samples, transmission models, and management of ISA.

Interactions between Rainbow Trout Eyed Eggs and Flavobacterium spp. Using a Bath Challenge Model: Preliminary Evaluation of Bacteriophages as Pathogen Control Agents

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.

Efficacy of florfenicol for control of mortality associated with Flavobacterium columnare in tilapia

The efficacy of florfenicol for control of mortality associated with Flavobacterium columnare was studied in tilapia. F. columnare T3-8/10 strain used for infection was tested for virulence by bath challenge to tilapia (body weight: BW 14 - 16 g) and antimicrobial sensitivity test. The results showed LD50 of this bacterial strain was 4.8×104 CFU/mL and it was sensitive to florfenicol. Experiment for control mortality caused by the bacterium in tilapia (BW 18 - 20 g) was designed with four treatments including negative control (uninfected fish), positive control, NT10 and NT15 (infected with LD50). Just after infection, fish in positive control, NT10 and NT15 treatments were treated with florfenicol at doses of 0, 10 and 15 mg/kg BW/day for 10 days, respectively, by feeding fish with medicated feed. Mortality of fish in positive control treatment after 14 days of infection was 54.0 ± 5.47% and statistically different in comparison with those in negative control, NT10 and NT15 treatments were 0.0, 3.0 ± 4.72 and 2.60 ± 2.51%, respectively (P < 0.05). Fish in NT10 and NT15 treatments were sampled for testing florfenicol residue in the flesh at day 1, 16, 20 and 24 after treatment. The results showed florfenicol residue levels in the flesh of sampled fish at all testing timepoints were significantly lower in comparison with the safe concentration lower than 1000 ppb regulated by the Ministry of Agriculture and Rural Development of Vietnam.