# The Protective Effects and Immunological Responses Induced by a Carboxymethyl Cellulose Microcapsule-Coated Inactivated Vaccine Against Largemouth Bass Ranavirus (LMBRaV) in Largemouth Bass (Micropterus salmoides)

## TL;DR

A new oral vaccine using carboxymethyl cellulose microcapsules effectively protects largemouth bass against ranavirus by boosting immune responses and reducing viral load.

## Contribution

The novel use of CMC microcapsules to deliver an inactivated vaccine orally, enhancing immune response and survival in largemouth bass.

## Key findings

- CMC microcapsules successfully release vaccine antigens in the intestinal tract of largemouth bass.
- The micro-CMC@LIV group showed significantly higher neutralizing antibody titers and immune gene expression compared to uncoated vaccines.
- The micro-CMC@LIV group had an 82.14% relative percent survival and lower viral loads in key organs after LMBRaV challenge.

## Abstract

Background: Epizootics of largemouth bass ranavirus (LMBRaV) in largemouth bass (Micropterus salmoides) populations are associated with elevated mortality and significant financial losses. Given the lack of effective and safe medication to treat this disease, oral vaccination, which directly targets the intestinal mucosal immune system, is crucial for disease resistance. Methods: This study utilized carboxymethyl cellulose (CMC) to coat LMBRaV inactivated vaccine (LIV) (micro-CMC@LIV). The morphology and characteristics of the CMC microcapsules were determined. In vitro simulated gastric and intestinal conditions were used to validate that the microcapsules could tolerate gastric conditions and subsequently release their contents in the intestinal tract. This was confirmed using CMC-coated coumarin 6 (C6) fluorescence microcapsules. Results: After the oral administration of micro-CMC@LIV, the detection of LMBRaV major capsid protein confirmed effective antigen release and absorption in the midgut and hindgut. Neutralizing antibody titers were significantly higher (1:81.71) in the micro-CMC@LIV group compared to the uncoated vaccine group (1:21.69). The expression of genes linked to the innate and adaptive immune systems was upregulated post-micro-CMC@LIV treatment. Following the LMBRaV challenge, the micro-CMC@LIV group exhibited a relative percent survival (RPS) of 82.14%, significantly higher than the uncoated vaccine group (61.61%). Droplet digital PCR analysis revealed significantly lower viral loads in the liver, spleen, and head kidney of the micro-CMC@LIV group compared to the control group and the uncoated vaccine group. Conclusions: These results collectively suggest that the CMC-coated LIV can be effectively delivered to the intestinal tract and induce robust antibody and immune responses, providing a reliable method for preventing and controlling LMBRaV disease in the largemouth bass industry.