Evaluation of Probiotics Solutions in Shrimp Aquaculture and Their Effectiveness Against Acute Hepatopancreatic Necrosis Disease Caused By Vibrio parahaemolyticus Strain A3

Abstract

As the demand for farmed shrimp continues to grow worldwide, the use of probiotics to address the sustainability of aquaculture fisheries has gained much attention. Emerging diseases in shrimp aquaculture, such as acute hepatopancreatic necrosis disease (AHPND), have devastating economic impacts in countries that largely depend on this activity. The relevance of this research lies on the fact that it explores the potential of using probiotics to mitigate the negative effects of AHPND in shrimp aquaculture. The scope of these studies includes survival of probiotic microbes in typical aquaculture water conditions, the effectiveness of probiotics in vitro and in vivo against the pathogenic strain of Vibrio parahaemolyticus that causes AHPND, and the effects of probiotics on the bacterial community composition in aquaculture water and gastrointestinal tract of shrimp after an induced AHPND infection. The microorganisms chosen as probiotics for this research include a lactic acid bacterium, a yeast and a photosynthetic bacterium. Informal feedback from shrimp farmers in Thailand and Vietnam revealed positive results against AHPND when using a commercially available probiotic containing multiple species of microorganisms from these probiotic groups. This research was divided into four studies. The first study (Chapter 2) evaluated the growth of the three different probiotic microbes in two different salinity conditions commonly found in intensive shrimp production systems to determine whether they could be further considered as potential candidates. The hypothesis was that the NaCl concentrations of the media may not have an effect on acid production, growth and cell morphology of the microorganisms being evaluated due to their metabolic mechanisms of adaptation to differences in osmotic pressure. The probiotic microbes were cultured in nutrient media enriched with 1 and 2% NaCl. Microbial survival, acidity and cell morphology between treatments were compared using enumeration by serial dilutions and plating, pH measurements and scanning electron microscopy imaging, respectively. The results showed that salinity levels up to 2% NaCl did not affect the growth of lactic acid bacteria and yeast. Photosynthetic bacteria grown in media with 1% NaCl showed a 24-hour delay in comparison to the control and a prolonged lag phase that lasted 48 hours when the media contained 2% NaCl. Therefore, the hypothesis was partially supported. Based on these results, all three probiotic microbes demonstrated to be suitable for application in aquaculture ponds with up to 2% salinity. The second study (chapter 3) aimed at determining the inhibitory effects of eight different formulations of probiotic solutions against the pathogenic strain of V. parahaemolyticus in vitro. The hypothesis of this study was that probiotic solutions containing whole microbial cultures of multiple microbial types including lactic acid bacteria may have a greater inactivation of the pathogen. The probiotic formulations consisted of individual cultures, combinations of the three probiotic microbes, and a commercially available probiotic formulation. The inhibitory effects were evaluated following a disk diffusion test on solid media by comparing diameters of zones of inhibition, and a challenge test in liquid media by comparing pathogen survival after exposure to probiotic solutions. Findings revealed inhibition zones with greater diameters in disks treated with whole microbial cultures (min: 7.83 mm, max: 11.33 mm) versus disks treated with only supernatants (min: 7.00 mm, max: 8.50 mm). Results from the challenge in liquid media tests showed greater inactivation of the pathogen after 48 h (6.56±0.07 to 5.43±0.03 log10 reduction) when treated with lactic acid bacteria alone and in combination with other microbial types. From these results, the hypothesis was supported and it was concluded that probiotic solutions including a lactic acid bacterium, the combination of lactic acid bacterium and photosynthetic bacterium and the combination of lactic acid bacterium, yeast and photosynthetic bacterium may be used to effectively inhibit AHPND in shrimp aquaculture. The third study (chapter 4) explored the effects of probiotic solutions on live shrimp (Litopenaeus vannamei) pretreated with probiotics for 7 days prior to challenging them with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that higher shrimp survival and weight gains would be observed when shrimps are exposed to probiotics solutions with multiple microbial types in the water and feed. Water quality parameters (dissolved oxygen, temperature, acidity, salinity and total ammonia nitrogen), difference in shrimp weight increase and shrimp survival were compared between probiotic treatments and controls. Treatments included: (1) a lactic acid bacterium alone (Pro.Sol1), (2) a lactic acid bacterium and a photosynthetic bacterium (Pro.Sol2), (3) the combination of a lactic acid bacterium, a yeast, and a photosynthetic bacterium (Pro.Sol3), and (4) a commercial probiotic (Com.Pro) and the results showed shrimp survival of 11.7, 26.7, 36.7 and 73.3%, respectively. Also, treatments Pro.Sol3 and Com.Pro resulted in higher weight gains (19.7 and 31.2%, respectively) versus the negative control (11.2%). Moreover, onset of the disease was delayed in all treatments as follows: 12 h with Pro.Sol1, 20 h with Pro.Sol2, 22 h with Pro.Sol3, and 26 h with Com.Pro. From these results, the hypothesis was supported and it was concluded that probiotics have the potential to effectively mitigate the effects of AHPND in the shrimp aquaculture. Finally, the fourth study (chapter 5) evaluated the effects of probiotics on the bacterial diversity of the gastrointestinal tract of shrimp as well as variation of bacterial and fungal diversity in the water before and after challenging shrimp with the pathogenic V. parahaemolyticus strain causing AHPND. The hypothesis of this study was that probiotic solutions with multiple microbial types may be able to maintain the microbial composition of the shrimp GI tract and aquaculture water preventing an increase in relative abundance of the family Vibrionaceae. Next generation sequencing was conducted using an Illumina MiSeq™ and primers specific for bacterial V4 hypervariable region of the 16S rRNA gene. The results obtained from the GI tract of shrimp revealed that the relative abundance of the family Vibrionaceae significantly increased in treatments with high mortalities, whereas treatments with higher survivals showed no significant difference in relative abundance of Vibrionaceae family members (P>0.05) in comparison to the negative control. The Shannon diversity index values (abundance and evenness) of the bacterial communities revealed that the treatment with the highest survival had the highest Shannon index value (4.69±0.133) whereas the treatment with lowest survival had the lowest Shannon index value (0.17±0.004). The results obtained from water samples did not show a higher abundance of the family Vibrionaceae, and diversity was maintained after infection (Shannon index 4.64±0.58). Regarding fungal diversity in water samples, Shannon index values revealed no significant changes before (3.627±0.37) and after infection (3.664±0.18) except for Pro.Sol3 (2.859±0.56) and Com.Pro (1.795±0.50), which included yeast in their formulation. Thus, the hypothesis of this study was partially supported since the results revealed that while all probiotics maintained the diversity of microbial composition in the water, only those probiotic solutions with various microbial types in the formulation maintained the diversity of the microbial composition in the GI tract of shrimp providing protection against AHPND.