Characterization of a Naturally Occurring Vibrio parahaemolyticus Infection and Investigation of a Dietary Humic Substance on Growth and Disease Resistance, and Immune Response to V. parahaemolyticus in Pacific White Shrimp (Litopenaeus vannamei) Cultured in Low Salinity Water

Abstract

Vibrio parahaemolyticus is a Gram-negative bacterium commonly found in estuarine, marine, and coastal environments. It is known to induce disease throughout the global shrimp aquaculture industry. This thesis includes two separate studies, including the characterization of a V. parahaemolyticus isolate (ARS-2-2024-GL) recovered from a naturally occurring outbreak which caused mass mortality in Pacific white shrimp (PWS; Litopenaeus vannamei) reared in a low salinity (4 ppt) recirculating system (RAS; 363 L system) and a 6-week trial which investigated the effect of a humic Reed-Sedge Peat substance, Menefeed 150© (hereafter, MFG 150), on growth of PWS followed by a bacterial challenge of V. parahaemolyticus to evaluate immunostimulant properties conferred by this unique product. ARS-2-2024-GL was originally isolated from the intestine of a PWS stocked in a RAS comprised of 16 tanks containing 10 PWS each, with an average of 2.63 g each. Observed clinical signs associated with the episode included pale and watery hepatopancreas, erratic swimming, lethargy and mortality. The moribund and deceased PWS were necropsied, and microbial samples were aseptically collected and cultured on Difco™ marine agar for the general isolation of bacterial samples and selective CHROMagar™ Vibrio for the isolation and detection of Vibrio spp. Biochemical and phenotypic profiles of the isolate were developed using the API 20 E© strip system, Biolog GEN III ID Microplate identification system, and Fatty Acid Methyl Ester Analysis (FAME). 16S rRNA sequencing confirmed the isolate identification as V. parahaemolyticus (99.6% Vibrio parahaemolyticus). Preliminary bacterial challenges were also carried out in PWS using ARS-2-2024-GL to determine the virulence of the isolate and identify an appropriate challenge method and dosage. Furthermore, PWS were subjected to both immersion and reverse gavage to determine the most effective route of delivery. The findings from the challenges suggest the potential of ARS-2-2024-GL to cause mortality at 1.30 x 107 CFU per shrimp. A 20% re-isolation of daily mortalities was sampled from the intestines, hepatopancreases, and gills of the deceased individuals during both challenges, which yielded 100% recovery of V. parahaemolyticus from the diseased shrimp, satisfying Koch’s postulate that a microorganism must be re-isolated from the inoculated host and identified as being identical to the original causative agent. For the growth trial, practical diets were formulated (36% protein; 6% lipid) to contain varying levels of MFG 150 and produced at the Aquatic Animal Nutrition Laboratory at the School of Fisheries, Aquaculture, and Aquatic Sciences, Auburn University (Auburn, Alabama). The PWS were randomly assigned a treatment (0, 1, 2, and 4 g/kg MFG 150) and stocked at 15 shrimp (average initial weight of 1.16 g) per tank, with seven tanks per treatment, in a static aquarium system in which daily water exchanges were administered. Feed was offered to shrimp four times a day. At the end of the six-week trial, shrimp were counted and group weighed. Three shrimp per tank were used to obtain intestines and hepatopancreas samples. Gene expression was analyzed to determine immune-related gene interactions impacted by dietary treatment. Three to six additional shrimp per treatment were sampled for proximate analysis. Following the growth trial, remaining shrimp were redistributed within their respective treatments into quadruplicate and stocked at 15 shrimp per tank to carry out a bacterial challenge via reverse gavage injection of V. parahaemolyticus at 1.23 x 108 CFU per shrimp. The treatment group provided with the basal diet was injected with 100 µL each of naïve marine broth as the negative control. The positive control group was also fed the basal diet injected with V. parahaemolyticus at 1.37 x 108 CFU per shrimp. There was significantly higher weight gain (%) (p < 0.05) for shrimp fed 1 g/kg MFG 150 than those fed 4 g/kg MFG 150. All treatments offered MFG 150 experienced significant protection from a known virulent strain of V. parahaemolyticus compared to the negative control (p = 0.0005, p = 0.00009, and p = 0.000007, respectively). This could be due to upregulation of immune-related genes. However, the only gene that was measured in this study to have a significant difference between treatments was tumor necrosis factor alpha (tnf-α) (p = 0.017). Dietary supplementation of MFG 150 resulted in several benefits, such as an increase in weight gain percentage at 1 g/kg MFG150 and a significantly higher survival rate when challenged with V. parahaemolyticus for L. vannamei reared in low salinity conditions, and the substance could potentially be a beneficial disease management tool to shrimp producers that struggle to manage this recurring bacterial disease.