Effect of a spore-based Mannheimia haemolytica vaccine on immune responses and respiratory microbiota in sheep

Mannheimia haemolytica is an opportunistic pathogen associated with respiratory disease in ruminants. Currently available vaccines do not provide complete protection against infection from M. haemolytica and therefore novel methods are needed for its mitigation. The aim of this study was to develop a mucosal vaccine against M. haemolytica, using Bacillus subtilis spores as an adjuvant and evaluate the effect of vaccination on the immune response and respiratory microbiota in sheep. A chimeric protein (MhCP) containing a tandem repeat of neutralizing epitopes from M. haemolytica leukotoxin A (NLKT) and outer membrane protein PlpE was expressed to produce antigen for adsorption to B. subtilis spores. Spore-bound antigen (Spore-MhCP) was prepared and administered to sheep via two mucosal routes (intranasal and intragastric), whereas the unbound antigen MhCP was delivered via intranasal and intramuscular routes. Additionally, unbound spores were evaluated intranasally, and the intranasal administration of saline was used as the control treatment. When compared to control, intranasal immunization with Spore-MhCP generated the strongest secretory IgA-specific response against both PlpE and NLKT in all samples (nasal swab, bronchoalveolar lavage, and feces), and exhibited a tendency to enhance immune responses more rapidly than intranasally delivered unbound MhCP. From nasopharyngeal samples, 16S rRNA gene sequencing revealed that 27 genera were altered in abundance in the intranasal Spore-MhCP group, compared to the control group across time. Among them, a notable decrease in Mannheimia was observed in the intranasal Spore-MhCP group (d14-35; P < 0.01), possibly resulting from vaccine-induced antibodies. Because proliferation of M. haemolytica in the upper respiratory tract is a prerequisite to lung infection, this spore-bound vaccine may offer protection against proliferation and subsequent lung infection.