Effectiveness of Sensitization Campaigns in Reducing Leprosy-Related Stigma in Rural Togo: Protocol for a Mixed Methods Cluster Randomized Controlled Trial.

BACKGROUND: In the global strategy to eliminate leprosy, there remains a need for early case detection to successfully interrupt transmissions. Poor knowledge about leprosy and leprosy-related stigma are key drivers of delayed diagnosis and treatment. Sensitization campaigns to inform and increase awareness among the general population are an integral part of many national neglected tropical disease programs. Despite their importance, the effectiveness of such campaigns has not been rigorously studied in the West African context. A multilingual rural setting with low health literacy in this region presents challenges to the potential impact of sensitization campaigns. OBJECTIVE: The primary objective of this study is to assess the causal effect of common practice community sensitization campaigns on leprosy-related knowledge and stigma at the community level and among community health volunteers. Additionally, we will test the potential of novel educational audio tools in the 15 most prominent local languages to overcome literacy and language barriers and amplify sensitization campaigns. METHODS: We will conduct a cluster randomized controlled trial using a sequential mixed methods approach in 60 rural communities across all regions of Togo, West Africa. The study features 2 intervention arms and 1 control arm, with intervention and control assignments made at the community level through randomization. Communities in intervention arm 1 will receive a sensitization campaign in line with the current Togolese national neglected tropical disease program. Communities in intervention arm 2 will receive the same sensitization campaign along with educational audio tools distributed to community households. The control arm will receive no intervention before data collection. Quantitative outcome measures on knowledge and stigma will be collected from a random sample of 1200 individuals. Knowledge will be assessed using the 9-item standardized Knowledge, Attitudes, and Practices Questionnaire. Stigma will be measured using the 7-item Social Distance Scale and the 15-item Explanatory Model Interview Catalogue Community Stigma Scale. We will estimate intention-to-treat effects at the individual level, comparing the outcomes of the intervention and control arms. In an accompanying qualitative component, we will conduct in-depth interviews with community members, community health volunteers, and health care workers in both treatment arms and the control arm to explore intervention and stigma-related experiences. RESULTS: This paper describes and discusses the protocol for a mixed methods cluster randomized controlled trial. Data collection is planned to be completed in June 2024, with ongoing data analysis. The first results are expected to be submitted for publication by the end of 2024. CONCLUSIONS: This trial will be among the first to test the causal effectiveness of community-based sensitization campaigns and audio tools to increase knowledge and reduce leprosy-related stigma. As such, the results will inform health policy makers, decision-makers, and public health practitioners designing sensitization campaigns in rural multilingual settings. TRIAL REGISTRATION: German Clinical Trials Register DRKS00029355; https://drks.de/search/en/trial/DRKS00029355. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/52106.

Feeding Saccharomyces cerevisiae fermentation postbiotic products alters immune function and the lung transcriptome of preweaning calves with an experimental viral-bacterial coinfection.

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼104 median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼1010 cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

Subcutaneous Bacillus Calmette-Guérin Administration Induces Innate Training in Monocytes in Preweaned Holstein Calves.

The bacillus Calmette-Guérin (BCG) vaccine, administered to prevent tuberculosis, is a well-studied inducer of trained immunity in human and mouse monocytes. We have previously demonstrated that aerosol BCG administration induces innate training in calves. The current study aimed to determine whether s.c. BCG administration could induce innate training, identify the cell type involved, and determine whether innate training promoted resistance to bovine respiratory syncytial virus (BRSV) infection, a major cause of bovine respiratory disease in preweaned calves. A total of 24 calves were enrolled at 1-3 d of age and blocked by age into two treatment groups (BCG, n = 12; control, n = 12). BCG was given s.c. to preweaned calves. The control calves received PBS. We observed a trained phenotype, demonstrated by enhanced cytokine production in response to in vitro stimulation with LPS (TLR-4 agonist) in PBMCs and CD14+ monocytes from the BCG group 2 wk (IL-1ß, p = 0.002) and 4 wk (IL-1ß, p = 0.005; IL-6, p = 0.013) after BCG administration, respectively. Calves were experimentally infected via aerosol inoculation with BRSV strain 375 at 5 wk after BCG administration and necropsied on day 8 postinfection. There were no differences in disease manifestation between the treatment groups. Restimulation of bronchoalveolar lavage fluid cells isolated on day 8 after BRSV infection revealed enhanced IL-1ß (p = 0.014) and IL-6 (p = 0.010) production by the BCG group compared with controls. In conclusion, results from our study show that s.c. administration of the BCG vaccine can induce trained immunity in bovine monocytes and influence cytokine production in the lung environment after BRSV infection.

Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects neonatal calves against BRSV infection.

Human respiratory syncytial virus (HRSV) is a leading cause of death in young children and there are no FDA approved vaccines. Bovine RSV (BRSV) is antigenically similar to HRSV, and the neonatal calf model is useful for evaluation of HRSV vaccines. Here, we determined the efficacy of a polyanhydride-based nanovaccine encapsulating the BRSV post-fusion F and G glycoproteins and CpG, delivered prime-boost via heterologous (intranasal/subcutaneous) or homologous (intranasal/intranasal) immunization in the calf model. We compared the performance of the nanovaccine regimens to a modified-live BRSV vaccine, and to non-vaccinated calves. Calves receiving nanovaccine via either prime-boost regimen exhibited clinical and virological protection compared to non-vaccinated calves. The heterologous nanovaccine regimen induced both virus-specific cellular immunity and mucosal IgA, and induced similar clinical, virological and pathological protection as the commercial modified-live vaccine. Principal component analysis identified BRSV-specific humoral and cellular responses as important correlates of protection. The BRSV-F/G CpG nanovaccine is a promising candidate vaccine to reduce RSV disease burden in humans and animals.

Tuberculosis poor treatment outcomes and its determinants in Kilifi County, Kenya: a retrospective cohort study from 2012 to 2019.

BACKGROUND: Tuberculosis (TB) is one of the leading causes of deaths in Africa, monitoring its treatment outcome is essential to evaluate treatment effectiveness. The study aimed to evaluate proportion of poor TB treatment outcomes (PTO) and its determinants during six-months of treatment at Kilifi County, Kenya. METHODS: We conducted a retrospective analysis of data from the TB surveillance system (TIBU) in Kilifi County, Kenya from 2012 to 2019. The outcome of interest was PTO (lost-to-follow-up (LTFU), death, transferred out, treatment failure, drug resistance) or successful treatment (cured or completed treatment). We performed time-stratified (at three months follow-up) survival regression analyses accounting for sub-county heterogeneity to determine factors associated with PTO. RESULTS: We included 14,706 TB patients, their median (IQR) age was 37 (28-50) years and 8,791 (60%) were males. A total of 13,389 (91%) were on first line anti-TB treatment (2RHZE/4RH), 4,242 (29%) were HIV infected and 192 (1.3%) had other underlying medical conditions. During 78,882 person-months of follow-up, 2,408 (16%) patients had PTO: 1,074 (7.3%) deaths, 776 (5.3%) LTFU, 415 (2.8%) transferred out, 103 (0.7%) treatment failure and 30 (0.2%) multidrug resistance. The proportion of poor outcome increased from 7.9% in 2012 peaking at 2018 (22.8%) and slightly declining to 20% in 2019 (trend test P = 0.03). Over two-thirds 1,734 (72%) poor outcomes occurred within first three months of follow-up. In the first three months of TB treatment, overweight ((aHR 0.85 (95%CI 0.73-0.98), HIV infected not on ARVS (aHR 1.72 (95% CI 1.28-2.30)) and year of starting treatment were associated with PTO. However, in the last three months of treatment, elderly age ≥50 years (aHR 1.26 (95%CI 1.02-1.55), a retreatment patient (aHR 1.57 (95%CI 1.28-1.93), HIV infected not on ARVs (aHR 2.56 (95%CI 1.39-4.72), other underlying medical conditions (aHR 2.24 (95%CI 1.41-3.54)) and year of starting treatment were positively associated with PTO while being a female (aHR 0.83 (95%CI 0.70-0.97)) was negatively associated with PTO. CONCLUSIONS: Over two-thirds of poor outcomes occur in the first three months of TB treatment, therefore greater efforts are needed during this phase. Interventions targeting HIV infected and other underlying medical conditions, the elderly and retreated patients provide an opportunity to improve TB treatment outcome.

Tuberculosis infection among youths in overcrowded university hostels in Kenya: a cross-sectional study.

BACKGROUND: Tuberculosis (TB) remains a top global health problem and its transmission rate among contacts is higher when they are cohabiting with a person who is sputum smear-positive. Our study aimed to describe the prevalence of TB among student contacts in the university and determine factors associated with TB transmission. METHODS: We performed a cross-sectional study with an active contact case finding approach among students receiving treatment at Kilifi County Hospital from January 2016 to December 2017. The study was conducted in a public university in Kilifi County, a rural area within the resource-limited context of Kenya. The study population included students attending the university and identified as sharing accommodation or off-campus hostels, or a close social contact to an index case. The index case was defined as a fellow university student diagnosed with TB at the Kilifi County Hospital during the study period. Contacts were traced and tested for TB using GeneXpert. RESULTS: Among the 57 eligible index students identified, 51 (89%) agreed to participate. A total of 156 student contacts were recruited, screened and provided a sputum sample. The prevalence of TB (GeneXpert test positive/clinical diagnosis) among all contacts was 8.3% (95% CI 4.5-14%). Among the 8.3% testing positive 3.2% (95% CI 1.0-7.3%) were positive for GeneXpert only. Sharing a bed with an index case was the only factor significantly associated with TB infection. No other demographic or clinical factor was associated with TB infection. CONCLUSION: Our study identified a high level of TB transmission among university students who had contact with the index cases. The study justifies further research to explore the genetic sequence and magnitude of TB transmission among students in overcrowded university in resource limited contexts.

Feeding Saccharomyces cerevisiae fermentation products lessens the severity of a viral-bacterial coinfection in preweaned calves.

We have previously reported that supplementation with Saccharomyces cerevisiae fermentation products (SCFP) ameliorates clinical signs and lung pathology following experimental bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. The objectives of this study were to determine the effect of SCFP supplementation on the metabolic and endocrine responses, and disease outcome of a viral-bacterial coinfection in preweaned calves. Twenty-seven, 1- to 2-d-old Holstein-Angus cross calves were enrolled in the study; one SCFP calf was removed from the trial during the pre-challenge phase due to complications from nephritis. Calves were assigned to two treatment groups: control or SCFP-treated, base milk replacer with 1 g/d SCFP (Smartcare, soluble formula) and calf starter top dressed with 5 g/d SCFP (NutriTek, insoluble formula). Calves were infected with BRSV on day 21, followed 6 d later by intratracheal inoculation with Pasteurella multocida (PM). Calves were euthanized on day 10 post-viral infection. Calves receiving SCFP had reduced thoracic ultrasonography scores on day 7 post-viral infection (P = 0.03) and a tendency toward reduced scores on day 10 post-viral infection (P = 0.09). Calves receiving SCFP also had less severe lung pathology scores at necropsy (P = 0.06). No differences between treatments were observed in lung viral loads (P = 0.48) or bacterial lung recovery (P = 0.34); however, there was a distinction in the lung location for PM recovery, with PM isolated more frequently from the cranial lobes in SCFP-treated calves, but more frequently from the caudal lobes of control calves. Calves treated with SCFP tended (P = 0.07) to have higher serum IL-6 concentrations following the coinfection. Calves treated with SCFP had lower concentrations of serum nonesterified fatty acids and beta-hydroxybutyric acid compared with controls following experimental challenge (P = 0.03 and P = 0.08, respectively), suggesting metabolic changes favoring growth and development. There were no differences between groups in gene expression of insulin receptor, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor, or haptoglobin in the liver. Results from this study suggest that supplementing with SCFP may moderate the impact of a respiratory viral-bacterial coinfection on preweaned calves through metabolic and immune modifications.

Applications of Nanovaccines for Disease Prevention in Cattle.

Vaccines are one of the most important tools available to prevent and reduce the incidence of infectious diseases in cattle. Despite their availability and widespread use to combat many important pathogens impacting cattle, several of these products demonstrate variable efficacy and safety in the field, require multiple doses, or are unstable under field conditions. Recently, nanoparticle-based vaccine platforms (nanovaccines) have emerged as promising alternatives to more traditional vaccine platforms. In particular, polymer-based nanovaccines provide sustained release of antigen payloads, stabilize such payloads, and induce enhanced antibod- and cell-mediated immune responses, both systemically and locally. To improve vaccine administrative strategies and efficacy, they can be formulated to contain multiple antigenic payloads and have the ability to protect fragile proteins from degradation. Nanovaccines are also stable at room temperature, minimizing the need for cold chain storage. Nanoparticle platforms can be synthesized for targeted delivery through intranasal, aerosol, or oral administration to induce desired mucosal immunity. In recent years, several nanovaccine platforms have emerged, based on biodegradable and biocompatible polymers, liposomes, and virus-like particles. While most nanovaccine candidates have not yet advanced beyond testing in rodent models, a growing number have shown promise for use against cattle infectious diseases. This review will highlight recent advancements in polymeric nanovaccine development and the mechanisms by which nanovaccines may interact with the bovine immune system. We will also discuss the positive implications of nanovaccines use for combating several important viral and bacterial disease syndromes and consider important future directions for nanovaccine development in beef and dairy cattle.

Mycoplasma bovis delay in apoptosis of macrophages is accompanied by increased expression of anti-apoptotic genes, reduced cytochrome C translocation and inhibition of DNA fragmentation.

Bacterial pathogens have evolved to manipulate host cell death and survival pathways for their intracellular persistence. Understanding the ability of a bacterium to induce or inhibit cell death is essential for elucidating the disease pathogenesis and suggesting potential therapeutic options to manage the infection. In recent years, apoptosis inhibition by different bacteria has been suggested as a mechanism of survival by allowing the pathogen to replicate and disseminate in the host. Mycoplasma bovis has evolved mechanisms to invade and modulate apoptosis of bovine peripheral blood mononuclear cells (PBMC), red blood cells (RBCs), primary macrophages and monocytes. To date, these mechanisms are poorly understood. Using apoptosis assays such as Annexin V binding, caspases activity, reactive oxygen species production, DNA fragmentation and differential gene expression we set out to determine how M. bovis modulates macrophage survival. Using the BoMac cell line, we report a significant reduction in STS-induced apoptosis through caspase dependent manner. Besides activating the NF-kß pathway and inhibiting caspases 3, 6 and 9, M. bovis strain Mb1 also inhibits production of reactive oxygen species and DNA fragmentation of the host cell. We also report a significant up-regulation of the anti-apoptotic genes Bcl-2 and Bcl-XL upon infection. Our results indicate that M. bovis strain Mb1 inhibits the intrinsic pathway of apoptosis and up-regulate survival genes in BoMac cells.

Th-17 cell mediated immune responses to Mycoplasma bovis proteins formulated with Montanide ISA61 VG and curdlan are not sufficient for protection against an experimental challenge with Mycoplasma bovis.

The current avenues for prevention and/or control of Mycoplasma bovis infection in cattle involve antibiotic treatment of affected animals, herd management practices including separation and or culling infected animals, and the use of commercial vaccines, which offer limited protection. Some bacterin vaccines may cause negative reactions; therefore a different approach is needed, such as the use of recombinant vaccines based on protective antigens formulated with effective adjuvants. The role of Th-17 immune responses in protection against bacterial infections has been investigated for several pathogens. In this study, our goal was to identify M. bovis antigens that may elicit Th-17 protective responses. We tested a vaccine containing M. bovis proteins formulated with Montanide ISA61™ VG and curdlan. After vaccination, the animals were challenged using a BHV-1/M. bovis co-infection model. We detected IL-17 and other cytokines in supernatants of PBMCs incubated with the recall antigens. In addition, we detected antibody and PBMC proliferative responses to the antigens. Despite observing slight decreases in the proportion of the lung lesions and in weight loss in the vaccinated group, we concluded that Th-17 responses to the antigens used here were not protective.

Mycoplasma bovis-Induced Inhibition of Bovine Peripheral Blood Mononuclear Cell Proliferation Is Ameliorated after Blocking the Immune-Inhibitory Programmed Death 1 Receptor.

Mycoplasma bovis-induced immune suppression is a major obstacle faced by the host for controlling infections. M. bovis impairment of antigen-specific T-cell responses is achieved through inhibiting the proliferation of peripheral blood mononuclear cells (PBMCs). This impairment may contribute to the persistence of M. bovis infection in various sites, including lungs, and its systemic spread to various organs such as joints, with the underlying mechanisms remaining elusive. Here, we elucidated the role of the immune-inhibitory receptor programmed death 1 (PD-1) and its ligand (PD-L1) in M. bovis infection. Flow cytometry (FCM) analyses revealed an upregulation of PD-L1 expression on tracheal and lung epithelial cell lines after M. bovis infection. In addition, we found increased PD-L1 expression on purified lung lavage macrophages following M. bovis infection by FCM and determined its localization by immunofluorescence analysis comparing infected and control lung tissue sections. Moreover, M. bovis infection increased the expression of the PD-1 receptor on total PBMCs and in gated CD4+ and CD8+ T-cell subpopulations. We demonstrated that M. bovis infection induced a significant decrease in CD4+ PD-1INT and CD8+ PD-1INT subsets with intermediate PD-1 expression, which functioned as progenitor pools giving rise to CD4+ PD-1HIGH and CD8+ PD-1HIGH subsets with high PD-1 expression levels. We blocked PD-1 receptors on PBMCs using anti-PD-1 antibody at the beginning of infection, leading to a significant restoration of the proliferation of PBMCs. Taken together, our data indicate a significant involvement of the PD-1/PD-L1 inhibitory pathway during M. bovis infection and its associated immune exhaustion, culminating in impaired host immune responses.

Induction of a balanced IgG1/IgG2 immune response to an experimental challenge with Mycoplasma bovis antigens following a vaccine composed of Emulsigen™, IDR peptide1002, and poly I:C.

Prevention and or control of Mycoplasma bovis infections in cattle have relied on the treatment of animals with antibiotics; herd management including separation and or culling infected animals; and the use of vaccines with limited protection. Due to the negative reactions and incomplete protection observed after vaccination with some bacterin-based vaccines, there is a need to put more efforts in the development of recombinant-based vaccines. However, the arsenal of antigens that may be suitable for a fully protective vaccine is rather limited at this point. We have tested a vaccine formulation containing M. bovis proteins formulated with adjuvants that have been shown to aid in the protection against other pathogens. After vaccinations, the animals were challenged using a BHV-1/M. bovis co-infection model. While the PBMC proliferation and cytokine responses to the antigens in the vaccine were negligible, humoral responses reveal that eight antigens elicit a balanced IgG1/IgG2 response although this was not enough to confer protection against M. bovis.

Effect of Mycoplasma bovis on bovine neutrophils.

Mycoplasma bovis (M. bovis) is a small bacterium that lacks a cell wall. M. bovis infection results in chronic pneumonia and polyarthritis syndrome (CPPS), otitis media, conjunctivitis, and meningitis in feedlot cattle and mastitis in dairy cattle. Numerous studies of peripheral mononuclear cells (PBMC) indicate that M. bovis evades host immunity through targeted effects on immune cell activity, including inhibition of effector function and simultaneous aberrant activation of immune cell activity that has no effect on protection against the bacterium. Few studies have addressed the interaction between M. bovis and neutrophils, one of the most important cell subsets of innate immunity. We hypothesized that M. bovis modifies specific neutrophil activities to support its persistence and systemic dissemination. In this study, we demonstrate that M. bovis enhances neutrophil apoptosis, stimulates production of pro-inflammatory cytokines, IL-12 and TNF-α, inhibits production of nitric oxide (NO) but augments elastase release. We also show that IL-17 an inflammatory cytokine produced by Th-17 cells does not enhance the capacity of neutrophils to destroy M. bovis. These findings present novel mechanisms of mycoplasma evasion of host innate immunity and provide potential opportunities for immuno-therapeutic interventions.