# Oral administration of recombinant Bacillus subtilis spores induces protective immunity against Echinococcus granulosus infection in dogs

## TL;DR

A new oral vaccine using spores from Bacillus subtilis reduces Echinococcus infection in dogs and triggers immune responses.

## Contribution

A novel oral vaccine using recombinant Bacillus subtilis spores expressing Echinococcus proteins is shown to protect dogs against infection.

## Key findings

- The vaccine reduced Echinococcus colonization in dogs by 62.26%.
- Vaccinated dogs showed elevated immune responses with increased IFN-γ, IL-4, and IL-10 levels.
- The vaccine caused no adverse effects and reduced intestinal damage from the parasite.

## Abstract

Echinococcus granulosus sensu lato, a cestode that inhabits the small intestines of canids, causes cystic echinococcosis (CE), a globally distributed zoonosis, through its larval stage. Vaccination is a cost-effective strategy to control E. granulosus infection in dogs. However, although dogs are the definitive hosts and main sources of CE transmission, no effective oral vaccines are currently available for them.

Three E. granulosus proteins, enolase (EgENO), severin (EgSev), and cyclophilin (EgCyc), were selected as novel oral vaccine candidates. These proteins were fused to the CotB spore-coat protein and expressed on the surface of Bacillus subtilis spores. A cocktail vaccine comprising the three recombinant spores was orally administered to beagles. Two weeks after the booster immunization, each dog was challenged with 70,000 protoscoleces. At 21 days post-infection, necropsies were performed to assess the intestinal parasite burden and calculate the worm reduction rates. Fecal and serum samples were collected weekly to measure secretory IgA, IgG, and cytokine responses. Histopathological analysis of intestinal tissues was also performed.

The cocktail vaccine reduced intestinal E. granulosus colonization by 62.26% (P < 0.05) compared with B. subtilis 168 spore-only controls. Vaccinated dogs developed both mucosal and humoral immune responses against E. granulosus antigens. By day 14 post-boost immunization, serum cytokine profiling revealed that levels of IFN-γ, IL-4, and IL-10 in the vaccinated group were significantly higher than those in the control group (P < 0.05). Histopathological analysis confirmed that the vaccine caused no adverse effects and alleviated the intestinal damage induced by the parasite.

This study demonstrates that B. subtilis spores serve as a safe and effective bacterial carrier to deliver E. granulosus antigens, supporting their potential in protecting dogs against E. granulosus infection. The heterogeneity in immune responses among vaccinated dogs should be addressed in future studies to secure consistent herd-level protection.