by Alexandre Brame, Innovation Manager, Nolivade, Stéphane Frouel, Aquaculture Project Manager and Maxime Hugonin, Aquaculture Product Manager, Mixscience, France

 

Alexandre Brame, Stephane Frouel and Maxime Hugonin report on a field trial run in France to evaluate the potential of a microbial solution to beneficially modulate microbiome in trout farming system and improve resistance and performance from eggs hatching to early juveniles' stage of rainbow trout Oncorhynchus mykiss.

Among the farmed aquatic species, rainbow trout is one of the most important. Some experts have projected that the world's consumption of rainbow trout will hit 950,000 metric tonnes (mt) annually in the first quarter of 2020 alone. The majority of this volume will be provided by aquaculture, with global sales volume of farmed trout expected to reach roughly 830,000 tonnes (t).

The continued relatively high cost of salmon has seen rainbow trout establish itself as a cheaper alternative fish, offering many of the same health benefits. The specialists predict a compound annual growth rate for worldwide rainbow trout market of approximately five percent over the next 10 years.

However, diseases are a primary problem in trout aquaculture and can severely impact its economic progression in many countries. The development of a fish disease is the result of the interaction between pathogens, hosts and the environment. Numerous studies have reported that water treatments may be an indirect driver in shaping the bacterial communities of the environment, skin, gills and gastrointestinal tract, commonly referred to as the 'microbiomes', of aquatic animals.

These microbes are believed to play important roles in host development, immunity, digestion and nutrition. On the contrary, deleterious bacteria may play inverted role. The diseases encountered in rainbow trout include: those caused by bacteria (Aeromonas spp., Yersinia spp., bacterial kidney disease, Flavobacterium spp., …), parasites (Gyrodactylus, Chilodonella, Trichodina, Epistylis, Trichophrya, Ichthyopthirius, Ichtyobodo, proliferative kidney disease, amoebic gill infestation, Coleps), fungi (Saprolegnia), and viruses (infectious pancreatic necrosis, viral hemorrhagic septicemia, and infectious hematopoietic necrosis).

Should a disturbance occur that causes an imbalance, or dysbiosis, in the commensal microbiome, the fish may be more vulnerable to pathogenic infection. Indeed, the gastrointestinal tract, in addition to the skin, gills and any modification of environmental water, are known to be the major routes of entry for potentially pathogenic microorganisms in fish.

The management practices designed to prevent the occurrence of diseases or the degradation of water quality are critical to a successful farming system. A strong biosecurity/prophylaxis to avoid diseases occurrence is mandatory to a successful production. Among this, the infection could be prevented by protecting hatching eggs and controlling water microflora of early stages of the fish.

The impact of novel products to preserve a stable and healthy microbiome of farmed salmonids, in particular those of microbial solutions, are receiving more attention as aquaculture practices evolve in line with restrictions on antibiotic and chemicals to treat the animal and the water.

Species of Bacillus sp. are spore-forming bacteria that are resistant to aggressive physical and chemical conditions. Various species show unusual physiological features enabling them to survive in various environmental conditions including fresh waters, marine sediments, desert sands, hot springs, arctic soils and the gastrointestinal tract of finfish and shellfish.

Application of Bacillus spp. as probiotics in feed or for bioremediation of farming water have great potential for sustainable aquaculture. Species of Bacillus spp. may play a desirable role in maintaining optimum water quality and equilibrium, which means a stress reduction, leading to an improved immuno-physiological balance, higher gut balance, better growth and enhanced survival in target aquatic animals.

In the present research, we demonstrated the potential of a microbial solution based on selected Bacillus sp. strains (Nolivade, France) directly applied in the farming water of early stages of rainbow trout.

Since it was hypothesized that supplementing farming water with microbial solution would modulate the structure of the intestinal microbiota, the primary objective of this study was to characterise the intestinal microbiota of farmed rainbow trout with or without microbial solution in the production system. Therefore, the secondary aim of this research was to evaluate the capacity of beneficial bacteria to prevent mortality or losses of zootechnical performance during a sanitary challenge (pathogenic bacteria, fungi or viruses).

Trout raceway trial

The rainbow trout trial was carried out in indoor 1300 litre (l) raceways in a private trout farm in France. Continuous water flow (with average debit of 0.6 l/s) was applied. All experimental raceways were connected to a common unit of decantation designed to mechanically filter the water. Temperature, salinity, and oxygen levels were based on ambient conditions of the river connected to the farm. Four raceways were used for control (CTRL) and four others for experimental Nolivade product (EXP).

Before hatching, 50,000 trout eggs were deposited on special shelves dispatched in each raceway. Initially, the eggs were immersed for one day in a solution containing the microbial solution (2g of product diluted into in 10l of water), and then it was applied under its original powder form, directly in water from day one post hatching to day 60. This microbial solution was applied at a dose equivalent to 1.10e5 CFU/l of raceway's water.

At day 60, for microbiota analysis, 12 young juveniles were sampled per group, rinsed with sterile water and then crushed to extract DNA in order to demonstrate changes in the structure of the microbiota of rainbow trout.

The samples were stored in freezer -80º C until processing. For total DNA extraction, the commercial QIAamp® DNA Stool Mini kit (QIAGEN) was used according to the manufacturer's instructions. Then, the V4 region of the 16S ribosome subunit gene was amplified with primers containing overlapping region with Illumina platform primers.

After verification of the amplicons quality, samples were sequenced using the Illumina MiSeq (paired-end library) platform with the 250-cycle V2 kit. The bioinformatics analysis was performed through MOTHUR v.1.36.1 software. Taxonomic classification was obtained using SILVA Database.

Finally, cumulative mortality and growth performance were measured. Veterinary diagnosis was done all along the trial to identify diseases which occurred at the farm during the experimental period and to observe how they affect fish.

Impact of the microbial solution on gut microbiota, disease resistance and performance of trout

Comprehensive taxonomic baselines of microbial taxa can bring a good overview of the healthy status of rainbow trout. It is assumed that bacterial genera commonly found in the intestine of fish, including Vibrio, Aeromonas, Clostridium, Flavobacterium, Edwarsiella, Pseudomonas, Photobacterium, Renibacterium, Serratia, Yersinia, are strains that are capable of causing disease.

Many of these microbes often comprise minor components of the intestinal microbiome and only emerge as opportunists when the fish is stressed or when its immune system is compromised. Much of the literature often refers to imbalances or perturbations in the fish intestinal microflora and the potential adverse effects that can arise as a result.

In the present study, after 61 days of treatment, gut biodiversity and abundance of trout gut was clearly modulated by the microbial solution and differences appeared between CTRL and EXP groups.

Bacteria of the genus Aeromonas, Clostridium and Serratia were significantly or at least numerically more present in control group indicating that the healthy status of trout of control group was potentially altered and fish were more susceptible to be challenged by external stress occurring during the growth period.

One major sanitary event appeared between day 23 and day 30 of the experiment  when an important occurrence of Saprolegnia spp associated to high presence of Aeromonas were diagnosed in the gut of the control group. These pathogens most often become pathogenic to fish only when fish are stressed, injured, in a poor nutritional state, temperature shocked, or otherwise debilitated.

A number of predisposing factors are involved in the development of fungal infection in fish—the factors affect both the fish and fungus, and a combination of factors rather than any single condition ultimately leads to infection. It has long since been believed that the fungi responsible for Saprolegniasis are secondary pathogens, and lesions are commonly seen after handling and after traumatic damage to the skin that could be caused by overcrowded conditions or in conjunction with pollution or bacterial or viral infections. Skin and gill lesions are by far the most frequently observed but there have been reports of infection of internal organs.

The utilisation of the microbial solution on the eggs and directly in the water during the early stages of the trout development had a protective effect against infestation of juveniles by Saprolegnia and Aeromonas since the fish were less injured and survival was significantly better at the end of the trial. Thus, at the end of the nursery period, the average mortality was of 14.3 percent for experimental raceways whereas it reached 21.1 percent for control raceways, meaning a relative improvement of 33 percent.

This protection of fish by the microbial solution had positive effects on final growth performance since final weight of experimental group was slightly higher than control. Average final individual weight of control group was 1.58g versus 1.65g for the experimental group, meaning a relative improvement of 4.2 percent.

Conclusion

Should the fish be challenged by fungi associated to opportunistic bacteria, a variety of external disinfectants may be used. They include use of malachite green, copper sulfate, potassium permanganate, and formalin. All these products are controversial since they may have an adverse effect on environment.

This microbial product developed by Nolivade seems to be a new and promising route as a preventive solution in trout farming systems. This study has shown effectiveness to reduce the susceptibility of the fish during the early stages of its life, when it faces environmental changes and in case of high pathogenic pressure.

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