Deployment of a Reservoir-Targeted Vaccine Against Borrelia burgdorferi Reduces the Prevalence of Babesia microti Coinfection in Ixodes scapularis Ticks.

In the Northeast and upper Midwest of the United States, Babesia microti and Borrelia burgdorferi use Ixodes scapularis ticks as vector and Peromyscus leucopus mice as major reservoir host. We previously established, in a 5-year field trial, that a reservoir-targeted outer surface protein A vaccine reduces the prevalence of B. burgdorferi-infected ticks. We accessed ticks and mouse blood samples collected during the trial, extracted total DNA, and amplified the B. microti 18S rRNA gene. Vaccine deployment reduced the prevalence of ticks coinfected with B. microti and that of mice infected with B. microti. Breaking the enzootic cycle of B. burgdorferi may reduce the incidence of babesiosis.

Field evaluation of a novel oral reservoir-targeted vaccine against Borrelia burgdorferi utilizing an inactivated whole-cell bacterial antigen expression vehicle.

Blacklegged ticks (Ixodes scapularis) are the principal vector for Borrelia burgdorferi, among other infectious agents, in the northeastern, mid-Atlantic, and upper midwestern USA. White-footed mice (Peromyscus leucopus) are the primary and most competent reservoir host of B. burgdorferi in the Northeast. Live reservoir-targeted vaccines (RTVs) to limit enzootic transmission of B. burgdorferi were previously developed and successfully evaluated in laboratory and controlled field trials. A novel, inactivated RTV was developed to minimize regulatory and market challenges facing previous RTVs based on live bacterial or viral vehicles. Thirty-two residential properties in Redding, Connecticut, participated in a field trial of an orally delivered, inactivated RTV efficacy study (2015-2016). During the two-year vaccination period, a significant decrease in the percentage of B. burgdorferi-infected I. scapularis larvae parasitizing P. leucopus was observed, as was a significant reduction in the percentage of infected P. leucopus on RTV-treated properties when compared to control properties. This novel inactivated RTV was effective in reducing numbers of B. burgdorferi-infected I. scapularis and B. burgdorferi-infected P. leucopus on properties where it was distributed.

Effect of orally administered B. subtilis-cNK-2 on growth performance, immunity, gut health, and gut microbiome in chickens infected with Eimeria acervulina and its potential as an alternative to antibiotics.

This study investigated the best oral delivery strategy (gavage or feed) for the B. subtilis expressing the chicken anti-microbial peptide cNK-2 (B. subtilis-cNK-2) in comparison to monensin, in chickens challenged with Eimeria acervulina (E. acervulina). A total of 120 broiler chickens were randomly allocated into 5 treatment groups in a completely randomized design: 1) uninfected chickens fed with basal diet (NC), 2) E. acervulina-infected chickens fed a basal diet (PC), 3) E. acervulina-infected chickens fed a basal diet supplemented with 90 mg monensin/kg feed (MO), 4) E. acervulina-infected chickens fed a basal diet and orally gavaged with B. subtilis-cNK-2 at 1 × 1010 cfu/d (CNK-O), and 5) E. acervulina-infected chickens fed a basal diet mixed with B. subtilis-cNK-2 at 1 × 1010 cfu/kg feed (CNK-F). The challenge consisted of 5,000 sporulated E. acervulina oocysts through oral gavage on d 15. Body weights were measured on d 7, 14, 21, and 23. Duodenal tissue and digesta samples were collected at 6 d postinfection (dpi) to assess the gut integrity, oxidative stress, mucosal immunity, and the gut microbiome. Fecal samples were collected from 6 to 8 dpi to enumerate the oocyst shedding. Chickens in the CNK-O group showed improved (P < 0.05) growth performance, gut integrity, and mucosal immunity compared to PC, comparable to chickens in the MO group. Chickens in the MO, CNK-F, and CNK-O treatment groups all showed lower (P < 0.05) oocyst shedding compared to PC chickens. Moreover, distinct cytokine profile, oxidative stress measures, tight junction proteins, and shifts in the gut microbiome with associated functional changes were observed in all challenge groups. In conclusion, we showed that the oral administration of B. subtilis-cNK-2 improved growth performance, enhanced local protective immunity, and reduced fecal oocyst shedding in broiler chickens infected with E. acervulina, demonstrating potential use of B. subtilis-cNK-2 as an alternative to antibiotics to protect chickens against coccidiosis.

Orally delivered Bacillus subtilis expressing chicken NK-2 peptide stabilizes gut microbiota and enhances intestinal health and local immunity in coccidiosis-infected broiler chickens.

We recently reported a stable Bacillus subtilis-carrying chicken NK-lysin peptide (B. subtilis-cNK-2) as an effective oral delivery system of an antimicrobial peptide to the gut with therapeutic effect against Eimeria parasites in broiler chickens. To further investigate the effects of a higher dose of an oral B. subtilis-cNK-2 treatment on coccidiosis, intestinal health, and gut microbiota composition, 100 (14-day-old) broiler chickens were allocated into 4 treatment groups in a randomized design: 1) uninfected control (CON), 2) infected control without B. subtilis (NC), 3) B. subtilis with empty vector (EV), and 4) B. subtilis with cNK-2 (NK). All chickens, except the CON group, were infected with 5,000 sporulated Eimeria acervulina (E. acervulina) oocysts on d 15. Chickens given B. subtilis (EV and NK) were orally gavaged (1 × 1012 cfu/mL) daily from d 14 to 18. Growth performances were measured on d 6, 9, and 13 postinfection (dpi). Spleen and duodenal samples were collected on 6 dpi to assess the gut microbiota, and gene expressions of gut integrity and local inflammation makers. Fecal samples were collected from 6 to 9 dpi to enumerate oocyst shedding. Blood samples were collected on 13 dpi to measure the serum 3-1E antibody levels. Chickens in the NK group showed significantly improved (P < 0.05) growth performance, gut integrity, reduced fecal oocyst shedding and mucosal immunity compared to NC. Interestingly, there was a distinct shift in the gut microbiota profile in the NK group compared to that of NC and EV chickens. Upon challenge with E. acervulina, the percentage of Firmicutes was reduced and that of Cyanobacteria increased. In NK chickens, however, the ratio between Firmicutes and Cyanobacteria was not affected and was similar to that of CON chickens. Taken together, NK treatment restored dysbiosis incurred by E. acervulina infection and showed the general protective effects of orally delivered B. subtilis-cNK-2 on coccidiosis infection. This includes reduction of fecal oocyst shedding, enhancement of local protective immunity, and maintenance of gut microbiota homeostasis in broiler chickens.

Administration of an Orally Delivered Substrate Targeting a Mammalian Zoonotic Pathogen Reservoir Population: Novel Application and Biomarker Analysis.

Reservoir-targeted vaccines (RTVs) have the potential to be effective at breaking the transmission cycle of many tick-borne pathogens including, but not limited to, Borrelia burgdorferi, B. miyamotoi, B. mayonii, Babesia microti, and Anaplasma phagocytophilum. To determine what proportion of a wild reservoir species we could effectively target, we distributed an experimental non-RTV Rhodamine B (RhB)-coated pellet formulation devoid of nutrient supplementation using bait boxes with ad libitum access, in battery-operated time-release bait stations, and by hand broadcast. Regardless of distribution method, a total of 208 of 242 (86%) white-footed mouse (Peromyscus leucopus) captures were positive for RhB by either pelage staining or by detecting fluorescent expression in vibrissae under a microscope. In bait box locations, 91% of captured mice were RhB-positive, 89% in hand broadcast locations, and 80% in time-release station locations. Based on results, we are confident that the bait formulation was readily accepted regardless of distribution technique, reached a substantial proportion of the reservoir population, and provides an effective vehicle to deliver a range of RTVs to targeted, wild, pathogen reservoir populations.