Probiotic
Enterococcus faecalis
surface-delivering key domain of EtMIC3 proteins: immunoprotective efficacies against
Eimeria tenella
infection in chickens
Authors: Pan Xinghui , Kong Rui , Liu Qiuju , Jia Zhipeng , Bai Bingrong , Chen Hang , Zhi Wenjing , Wang Biao , Ma Chunli , Ma Dexing 
Published: October 2023

Enterococcus faecalis
MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the
Eimeria tenella
microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant
E. faecalis
were recorded. The immunized chickens were challenged with 4.0 × 10
4
E. tenella
-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of
E. faecalis
, respectively. Oral immunizations with recombinant
E. faecalis
, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (
P
< 0.01). The groups immunized with four recombinant
E. faecalis
showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic
E. faecalis
delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of
Eimeria
infection.

IMPORTANCE

Avian coccidiosis caused by
Eimeria
brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time,
Eimeria
infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot.
Eimeria
parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens.
Eimeria tenella
microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the
Eimeria
invasion.
Enterococcus faecalis
is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by
E. faecalis,
respectively. Oral immunizations were performed to investigate immune protective effects against
Eimeria
infection.