Dietary montmorillonite clay improved Penaeus vannamei survival from acute hepatopancreatic necrosis disease and modulated stomach microbiota

Acute hepatopancreatic necrosis disease (AHPND) is causing a bacterial pandemic in shrimp farming. AHPND is caused by pathogenic strains of Vibrio parahaemolyticus (VPAHPND) that produces the bacterial toxins, PirA and PirB. Shrimp exposure to AHPND-causing bacteria is hypothesized to be through the oral route, ingested into the gut where the bacteria initially colonized the stomach and then releases the binary toxins to damage the hepatopancreas. In view of changing global regulatory controls on the use of antibiotics, there is much interest in the development of functional aquafeeds. One innovative method to mitigate AHPND is to bind the bacterial toxins by adding adsorbent clay minerals into shrimp feeds. A feeding trial using triplicate groups of Penaeus vannamei post-larvae (PL30) was conducted to evaluate the efficacy of a montmorillonite (MMT) clay [Calibrin®-Z (CL)] on growth, gut health and disease resistance to AHPND. The addition of 0.25% or 0.50% CL did not significantly (P > 0.05) impact growth and feed utilization efficiency compared to control groups fed diets with 0% CL. When challenged with VPAHPND, survival of shrimp fed 0.25% or 0.50% CL were 83.3 ± 5.5% and 93.8 ± 0.1%, respectively, and were significantly higher (P < 0.05) compared to the VPAHPND-challenged positive control group (39.6 ± 10.4%) but not significantly different compared to the unchallenged negative control group (95.8 ± 2.1%). Vibrio and total cultivable bacteria counts in the hepatopancreas of shrimp fed CL-added diets were significantly lower compared to positive control. Hepatopancreas histopathology of infected shrimp fed CL-added diets showed less damage. During AHPND infection, bacterial diversity was depressed but dietary CL tended to restore stomach bacterial richness and α-diversity index. Dietary CL modulated the stomach bacterial community possibly with beneficial impact to shrimp survival. At the phylum level, Proteobacteria and Bacteroidetes were the main groups but the relative abundance of Verrucomicrobia, Acidobacteria and Firmicutes was more prevalent in the microbiota of shrimp fed CL-added diets. Bacteria of the genus Pseudoalteromonas, Tenacibaculum, and Marinimicrobium were identified as members of the healthy microbiota and Lysobacter was observed to be relatively enriched in AHPND-infected shrimp. Demequina was identified as a potential biomarker and was significantly enriched in the stomach of the CL-added groups. This is the first report on the effectiveness of dietary clay mineral in AHPND mitigation. At least 0.25% CL is suggested to be included in commercial shrimp feeds to mitigate against production and economic losses from AHPND outbreaks in shrimp farms.