Lack of Effect of Biowish Multibio 3PS Probiotic on Bacterial Cold-Water Disease-Induced Mortality in Rainbow Trout

Bacterial Cold-Water Disease (BCWD) causes substantial mortality in salmonid hatcheries worldwide [1]. The causative agent of BCWD is Flavobacterium psychrophilum [2]. Davis [3] first identified BCWD in rainbow trout Oncorhynchus mykiss at a hatchery in West Virginia USA and labelled it peduncle disease. Since then, BCWD has also been called fin rot disease, saddleback disease, fry mortality syndrome, rainbow trout fry syndrome, rainbow trout fry mortality syndrome, bacterial disease of cold water, and cold-water disease [4]. 

Clinical signs of BCWD are numerous, ranging from tissue erosion around the caudal peduncle [3,5], whitish material along a fin margin [6], pale gill tissue, epidermal hyperplasia, lethargy, spinal abnormalities, and spiral swimming behaviors [7-12]. Reported mortality rates have ranged from 90% in rainbow trout fry [13] to 1% in juvenile rainbow trout [14]. 

Cleghorn Springs State Fish Hatchery in Rapid City, South Dakota has an endemic strain of Flavobacterium psychrophilum and regular outbreaks of BCWD [15]. BCWD-induced mortality of Shasta strain rainbow trout at the hatchery has ranged from 1% to nearly 70% [16,17]. 

Probiotics are microbial feed additives that boost the immune response of host organisms by modulating the intestinal microbiota [18,19]. Specific probiotic bacterial species and strains have been shown to reduce BCWD-associated mortality [20-22]. For example, Burbank et al. [20] identified 16 bacterial isolates from rainbow trout digestive tracts as potential probiotic candidates for BCWD control. Subsequently, Ghosh et al. [23] reported that intraperitoneally-administered Enterobacter species C6-6 could protect rainbow trout fry from Flavobacterium psychrophilum infection.  

The commercially-available probiotic BiOWiSH MultiBio 3PS contains the probiotic bacterial species Pediococcus acidilacti, Pediococcus pentasaceus, Lactobacillus plantaum, and Bacillus subtillis. Bacillus spp. and Lactobacillus spp. are the known core microbiota of rainbow trout [24-26]. Multi-Bio 3PS has successfully been used to improve immune performance in pigs and calves [27,28], Nile tilapia Oreochromis niloticus [29], and Pacific whiteleg shrimp Pennaeus vannamei [30]. 

There have been no studies examining the efficacy of a non-species-specific, commercially available probiotic such as Bio-Wish MultiBio 3PS on the control of BCWD. Thus, the objective of this study was to evaluate the effect of dietarily-administered Bio-Wish MultiBio 3PS on BCWD-induced mortality and growth of rainbow trout.

All experimentation occurred at Cleghorn Springs State Fish Hatchery, Rapid City, South Dakota, USA using degassed, and aerated spring water at a constant temperature of 11°C (total hardness as CaCO3, 360 mg L-1; alkalinity as CaCO3, 210 mg L-1; pH, 7.6; total dissolved solids, 390 mg L-1). Shasta strain rainbow trout in the study were incubated as eggs and subsequently reared similarly prior to the start of the experiment. Eight flow-through semi-square 190-liter tanks with partial (~50%) overhead covers [31] were used. Four tanks received a diet of BioVita Fry extruded feed (Bio-Oregon, Longview, Washington, USA) top-coated with BiO-WiSH MultiBio 3PS (BiO-WiSH Technologies, Cincinnati, Ohio, USA) probiotic at 0.5g per kg of feed.  BiO-WiSH MultiBio 3PS contained the probiotic bacteria Pediococcus acidilacti (≥ 1 x 10⁸ cfu/g), Pediococcus pentasaceus (≥ 1 x 10⁸ cfu/g), Lactobacillus plantaum (≥ 1 x 10⁸ cfu/g), and Bacillus subtillis (≥ 1 x 10⁷ cfu/g). Fish in the other four tanks were fed only Bio-Oregon BioVita Fry (N=4). Inflow to each tank was 7.57 l/min and water velocities were negligible (< 0.1 m/s).  

This experiment was divided into 3 trials (Table 1). Beginning on 23 February 2022, each tank received 153 g (approximately 1,860) of pre-swim-up Shasta strain rainbow trout alevins (mean ± SE; initial weight = 0.082 ± 0.00g; total length = 21.7 ± 0.2 mm; n=30). Trial 1 began at first feeding of swim-up fry on 27 February 2022, and continued for 31 days, ending on 29 March. Trial 2 began the next day and loadings were reduced to approximately 1,000 fish/tank (exactly 650 g). Trial 2 ran for 29 days, ending on 28 April. Trial 3 began the next day, and loadings were reduced to 500 fish/tank (exactly 1,110 g). Trial 3 ended on 29 June, lasting 61 days. 

Table 1: Length of time (days) and number of fish per tank for three trials with rainbow trout fed diets either with or without Bio-Wish MultiBio 3PS probiotic.

At the conclusion of each trial, the total biomass in each tank was weighed to the nearest g. In addition, 10 fish were individually weighed (0.01 g) and measured (0.1 mm). Fish were fed according to the Hatchery Constant (HC) method [32] with a planned feed conversion rate of 1.1 and a maximum growth rate of 0.075 cm/day. Vibratory feeders (Pentair Aquatic Eco-Systems, Inc., Apopka, FL, USA) connected to a timer (Sweeney Enterprises Inc., Boerne, TX, USA) delivered feed at 60-minute intervals. Mortalities were removed and recorded daily. The following formulas were used:

Data was analyzed using the SPSS (9.0) statistical program (SPSS, Chicago, Illinois, USA), with significance predetermined at P < 0.05. Individual and total tank metrics were analyzed using a one-way analysis of variance. If treatments were determined to be significantly different a post hoc means separation test was performed using the Tukey HSD test.

There was no significant difference in mortality between treatments in each of the three trials (Table 2). Trial 1 had the highest mortality rate at 7.43 ± 0.89% in the probiotic treatment and 9.44 ± 1.47% in the control. Mortality was greatly reduced to less than 0.34% for both treatments during trial 2 and less than 0.08% in trial 3. Ending tank weights and feed conversion ratio were not significantly different between the treatments in trials 1 and 3. However, in trial 2, ending tank weight was significantly higher in the control tanks and feed conversion ratio was significantly lower compared to the probiotic treatment tanks No significant differences were found for individual fish lengths, weights, SGR, or K for any trial of the experiment (Table 3). 

Table 2: Total tank initial weight (kg), ending weight (kg), FCR, and % mortality for three consecutive trials with rainbow trout Oncorhynchus mykiss receiving a diet either with or without BioWish MutliBio 3PS as a probiotic. 

Columns with a superscript letter are significantly different from each other (p ≤ 0.05). 

Table 3: Individual fish starting length (mm), end length (mm), start weight (g), end weight (g), SGR, and K for three consecutive trials with rainbow trout Oncorhynchus mykiss receiving a diet either with or without BioWish MutliBio 3PS as a probiotic.

The BiO-WiSH MultiBio 3PS probiotic used in this study did not significantly impact survival or growth of rainbow trout. While this is the first study to use MultiBio 3PS on cold water fish, it has been shown to be effective on other species. For example, MultiBio 3PS has been shown to increase disease resistance to Streptococcus iniae in Nile tilapia [29] and improve immune performance in pigs and calves [27,28] Contrarily, MultiBio 3PS did not increase immune gene expression in channel catfish Ictalurus punctatus [33]. It also did not improve gut or intestinal bacterial profiles on Pacific White shrimp [34,35]. 

Probiotics used in rainbow trout culture have been shown to improve water quality [36], stimulate an improved immune response [37-39], reduce mortality [40], and stimulate respiratory burst activity [41]. Furthermore, after compiling the first meta-analysis looking at probiotic effects on rainbow trout, Rahimi et al. [42] found that probiotic use enhanced most of the components of the immune systems of rainbow trout in most studies.  The benefits of probiotics have also been studied for disease resistance in other fish and crustaceans. Pirarat et al. [43] demonstrated that after feeding tilapia Oreochromis niloticus with Lactobacillus rhamnosus ATCC 53103 for 2 weeks, stimulation of cellular immunity was detected as demonstrated by an increase in phagocytic activity. Similar observations have been described by Balcazar et al. [44], who demonstrated a positive effect on humoral immune response following probiotic administration in brown trout Salmo trutta. Studies have shown that the use of Bacillus sp. in cropping systems has improved the water quality, survival, growth rate and health status of juvenile giant prawn Penaeus monodon and reduced the number of pathogenic vibrios [45].  

The overall mortalities in this study were consistent with the 10% BCWD-induced mortality typically observed at Cleghorn Springs Hatchery [16]. Other studies have observed rainbow trout mortality rates as low as 1% [14] and as high as 90% [13] due to BCWD. 

Most of the mortality in this study occurred in the first trial when the trout only had innate immunity. Martin et al. [17] suggested that diets leading to faster growth and more rapid development of the secondarily developed adaptive immunity may impact fish responses to future BCWD outbreaks. Prior to the development of adaptive immunity, young fish are more susceptible to opportunistic environmental pathogens such as F. psychrophilum [46-49]. 

The BioOregon starter diet used in this trial contains beta-glucans, nucleotides and a high level of vitamins designed to stimulate the immune system. It is possible that the positive effects of the probiotic might be suppressed with such a nutritionally complete diet with other immune-stimulating ingredients [50-52]. 

Burbank et al. [20] suggested that bacterial strains specific to rainbow trout GI tracts should be used as potential probiotic candidates. Bacillus spp. and Lactobacillus spp. are found naturally at high levels in the core microbiota of rainbow trout [24-26]. These bacterial species closely correspond to two of the four listed bacterial species in MultiBio 3PS; Lactobacillus plantaum, and Bacillus subtillis. As long as the concentrations of probiotic were sufficient for rainbow trout diets, and the water temperature was not too low to activate the bacteria, the species present in MultiBio 3PS would have been expected to stimulate an immune response in the fish. 

The probiotic was prepared and applied to the feed according to manufacturer’s specifications at 0.5g per kg of feed. It is unknown if the probiotic dissolved off the feed or diluted into the water before ingestion, although the fish were feeding readily at the water’s surface. The concentration of bacterial cells used should have been sufficient since the 10^7-8 cfu/g^-1 of each probiotic species contained in the MultiBio 3PS was the same concentration of bacterial cells used in most other trials utilizing similar strains with rainbow trout [20,53-55]. 

In conclusion, while MultiBio probiotics have a record of success in warmwater fish, shrimp, pigs, and cows, its effect was negligible in this study. Future tests should examine the use of different concentrations, different temperatures, different delivery methods, and different species, strains, and sizes of fish.

The authors would like to thank all their co-workers at Cleghorn Springs State Fish Hatchery for assisting with carrying out this experiment. Authors would also like to thank Alexis Gerber and Breah Rosner for assisting with formatting of this manuscript.

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