Abstract
BackgroundAvian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Lactobacillus plantarum has been frequently used for expression of foreign proteins as an oral vaccine delivery system using traditional erythromycin resistance gene (erm). However, antibiotic selection markers were often used during protein expression and they pose a risk of transferring antibiotic resistance genes to the environment, and significantly restricting the application in field production. Therefore, a food-grade recombinant L. plantarum vaccine candidate would dramatically improve its application potential in the poultry industry.ResultsIn this study, we firstly replaced the erythromycin resistance gene (erm) of the pLp_1261Inv-derived expression vector with a non-antibiotic, asd-alr fusion gene, yielding a series of non-antibiotic and reliable, food grade expression vectors. In addition, we designed a dual-expression vector that displayed two foreign proteins on the surface of L. plantarum using the anchoring sequences from either a truncated poly-γ-glutamic acid synthetase A (pgsA′) from Bacillus subtilis or the L. acidophilus surface layer protein (SlpA). EGFP and mCherry were used as marker proteins to evaluate the surface displayed properties of recombinant L. plantarum strains and were inspected by western blot, flow cytometry and fluorescence microscopy. To further determine its application as oral vaccine candidate, the AMA1 and EtMIC2 genes of E. tenella were anchored on the surface of L. plantarum strain. After oral immunization in chickens, the recombinant L. plantarum strain was able to induce antigen specific humoral, mucosal, and T cell-mediated immune responses, providing efficient protection against coccidiosis challenge.ConclusionsThe novel constructed food grade recombinant L. plantarum strain with double surface displayed antigens provides a potential efficient oral vaccine candidate for coccidiosis.
Highlights
Avian coccidiosis posts a severe threat to poultry production
Enhanced green fluorescent protein (EGFP) gene was inserted into the above plasmids, resulting in two EGFP anchoring plasmid pLp-poly-γ-glutamic acid synthetase A (pgsA)′-EGFP and pLp-EGFP-S
To determine whether the expression of foreign proteins affects the growth of recombinant strains, the constructed plasmids described above were transformed into either L. plantarum NC8 or its alr deletion mutant strain L. plantarum NC8/Δalr
Summary
Avian coccidiosis posts a severe threat to poultry production. In addition to commercial attenuated vaccines, other strategies to combat coccidiosis are urgently needed. Liu et al Microb Cell Fact (2020) 19:28 parasites resistant to anti-coccidial drugs and the public pressure to limit the use of chemicals in animals [2] have prompted the development of cost-effective vaccines. Some commercial vaccines are available, such as live virulent, attenuated [3] or live-tolerant vaccines [4], some disadvantages have been noticed including the poor immunological protection, affecting weight gain and virulence reversion [5]. To overcome these problems, several novel vaccine candidates have been studied recently. Bacterial vectors originating from but not limited to Bacillus subtilis [6], Mycobacterium bovis BCG [7], Salmonella enteritidis [8] and Lactococcus lactis [9] have drawn more and more attention to be used as vectors for oral vaccines
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