Recombinant HcGAPDH Protein Expressed on Probiotic Bacillus subtilis Spores Protects Sheep from Haemonchus contortus Infection by Inducing both Humoral and Cell-Mediated Responses

Abstract

Probiotic Bacillales are effective in controlling pathogens. Live probiotic bacteria improve the composition of the gastrointestinal microbiota, leading to a reduction in pathogen colonization. However, it remains largely unknown how probiotics regulate the host’s immunologic responses and protect the host from parasitic infection. In this study, we addressed whether Bacillales were effective against Haemonchus contortus, a parasitic nematode that infects small ruminants worldwide. Using 16S rRNA sequencing, we found that Bacillales were largely depleted in the abomasal microbiota of sheep infected with H. contortus. We constructed a recombinant Bacillus subtilis named rBSCotB-HcG that express the glyceraldehyde-3-phosphate dehydrogenase of H. contortus (HcGAPDH) on its spore surface using the Bacillus subtilis spore coat protein B (CotB) as a carrier. Mice receiving rBSCotB-HcG orally showed strong Th1-dominated immune responses. More importantly, sheep administered BSCotB-HcG per os showed increasing proliferation of the peripheral blood mononucleates, elevated anti-HcGAPDH IgG in sera, and higher anti-HcGAPDH sIgA in the intestinal mucus than the control sheep. The average weight gain of H. contortus-infected sheep treated with rBSCotB-HcG (Hc+rBSCotB-HcG) was 48.73% greater than that of unvaccinated sheep. Furthermore, these Hc+rBSCotB-HcG sheep had fewer eggs per gram of feces by 84.1% and adult worms by 71.5%. They also demonstrated greatly lessened abomasal damage by H. contortus with an abundance of probiotic species in the abomasal microbiota. Collectively, our data unequivocally demonstrate the protective roles of CotB-HcGAPDH-expressing B. subtilis spores in against H. contortus infection and showed great potential of using probiotic-based strategy in controlling parasitic nematodes of socioeconomic importance in general. IMPORTANCE Initial analyses of the abomasal microbiota of sheep using 16S rRNA sequencing suggested that probiotic bacteria played a protective role in against H. contortus infection. A recombinant Bacillus subtilis expressing a fusion protein CotB-HcGAPDH on its spore’s surface induced strong Th1 immune response in a murine model. The same probiotic recombinant, upon only one oral application, protected sheep against H. contortus infection by reducing egg shedding and decreasing adult worm loads of the parasite and increasing body weight gain of infected sheep. Both Th1 and Th2 immune responses were evident in these immunized sheep.

Isolation of Potential Probiotic Bacillus spp. from the Intestine of Nile Tilapia to Construct Recombinant Probiotic Expressing CC Chemokine and Its Effectiveness on Innate Immune Responses in Nile Tilapia

“…Such recombinant probiotics can be used as a perspective vehicle to deliver the desired characteristics and functionalities of heterologous proteins directly to the intestine. Currently, the genus Bacillus is considered an acceptable biofactory to produce heterologous proteins for various purposes of basic research and industrial interest [ 16 , 17 ]. There are many advantages to employing Bacillus spp.…”

Surface Display of Peptides Corresponding to the Heptad Repeat 2 Domain of the Feline Enteric Coronavirus Spike Protein on Bacillus subtilis Spores Elicits Protective Immune Responses Against Homologous Infection in a Feline Aminopeptidase-N-Transduced Mouse Model

“…B. subtilis is well known for its generally-recognized-as-safe certification (45). It has been widely used in animal husbandry as a feed additive due to its probiotic effects and as a vaccine vector to target the gut (35,(46)(47)(48)(49). B. subtilis spores have natural adjuvant activity, which can effectively increase T-cell reactions and promote IgA antibody production (27,50).…”

“…A positive control group received 100 μg of purified HR2P by IM injection and was boosted with the same dose at 14 days post-vaccination (dpv), whereas PBS was given by gavage to a negative control group. All six groups were immunized on days 1–3, 8–10, and 15–17 as described previously ( 35 ). Specific anti-HR2P antibodies were monitored in serum once a week after the first immunization, and the level of anti-HR2P sIgA in the intestine was detected after euthanasia in the fifth week.…”

Acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 to play roles in larval development of Haemonchus contortus

“…Rabbit anti-r Hc ACOX-1 polyclonal antibodies were prepared using a method described previously [ 64 , 65 ]. The titre of polyclonal antibodies was determined by an enzyme-linked immunosorbent assay (ELISA) as described previously [ 66 ]. Naïve serum collected prior to immunization was used as a negative control.…”