Genotypic analysis of nontuberculous mycobacteria isolated from raw milk and human cases in Wisconsin.

Nontuberculous mycobacteria (NTM) compose a group of mycobacteria that do not belong to the Mycobacterium tuberculosis complex group. They are frequently isolated from environmental samples such as water, soil, and, to a lesser extent, food samples. Isolates of NTM represent a major health threat to humans worldwide, especially those who have asthma or are immunocompromised. Human disease is acquired from environmental exposures and through consumption of NTM-contaminated food. The most common clinical manifestation of NTM disease in human is lung disease, but lymphatic, skin and soft tissue, and disseminated disease are also important. The main objective of the current study was to profile the farm-level contamination of cow milk with NTM by examining milk filters and bulk tank milk samples. Five different NTM species were isolated in one dairy herd in Wisconsin, with confirmed 16S rRNA genotypes including Mycobacterium fortuitum, Mycobacterium avium ssp. hominissuis, Mycobacterium abscessus, Mycobacterium simiae, and Mycobacterium avium ssp. paratuberculosis (Mycobacterium paratuberculosis). In tank milk samples, M. fortuitum was the predominant species in 48% of the samples, whereas M. chelonae/abscessus and M. fortuitum were the only 2 species obtained from 77 and 23% of the examined filters, respectively. Surprisingly, M. avium ssp. hominissuis, M. paratuberculosis, and M. simiae were isolated from 16.7, 10.4, and 4% of the examined milk samples, respectively, but not from milk filters. Interestingly, NTM isolates from human clinical cases in Wisconsin clustered very closely with those from milk samples. These findings suggest that the problem of NTM contamination is underestimated in dairy herds and could contribute to human infections with NTM. Overall, the study validates the use of bulk tank samples rather than milk filters to assess contamination of milk with NTM. Nontuberculous mycobacteria represent one type of pathogens that extensively contaminate raw milk at the farm level. The significance of our research is in evaluating the existence of NTM at the farm level and identifying a simple approach to examine the potential milk contamination with NTM members using tank milk or milk filters from dairy operations. In addition, we attempted to examine the potential link between NTM isolates found in the farm to those circulating in humans in Wisconsin.

Drug resistance and genomic variations among Mycobacterium tuberculosis isolates from The Nile Delta, Egypt.

Tuberculosis is a global public health concern. Earlier reports suggested the emergence of high rates of drug resistant tuberculosis in Egypt. This study included 102 isolates of Mycobacterium tuberculosis collected from two reference laboratories in Cairo and Alexandria. All clinical isolates were sub-cultured on Löwenstein-Jensen medium and analyzed using both BD BACTEC MGIT 960 SIRE Kit and standard diffusion disk assays to identify the antibiotic sensitivity profile. Extracted genomic DNA was subjected to whole genome sequencing (WGS) using Illumina platform. Isolates that belong to lineage 4 represented > 80%, while lineage 3 represented only 11% of the isolates. The percentage of drug resistance for the streptomycin, isoniazid, rifampicin and ethambutol were 31.0, 17.2, 19.5 and 20.7, respectively. Nearly 47.1% of the isolates were sensitive to the four anti-tuberculous drugs, while only one isolate was resistant to all four drugs. In addition, several new and known mutations were identified by WGS. High rates of drug resistance and new mutations were identified in our isolates. Tuberculosis control measures should focus on the spread of mono (S, I, R, E)- and double (S, E)-drug resistant strains present at higher rates throughout the whole Nile Delta, Egypt.

Protective RNA nanovaccines against Mycobacterium avium subspecies hominissuis.

The induction of an effective immune response is critical for the success of mRNA-based therapeutics. Here, we developed a nanoadjuvant system compromised of Quil-A and DOTAP (dioleoyl 3 trimethylammonium propane), hence named QTAP, for the efficient delivery of mRNA vaccine constructs into cells. Electron microscopy indicated that the complexation of mRNA with QTAP forms nanoparticles with an average size of 75 nm and which have ~90% encapsulation efficiency. The incorporation of pseudouridine-modified mRNA resulted in higher transfection efficiency and protein translation with low cytotoxicity than unmodified mRNA. When QTAP-mRNA or QTAP alone transfected macrophages, pro-inflammatory pathways (e.g., NLRP3, NF-kb, and MyD88) were upregulated, an indication of macrophage activation. In C57Bl/6 mice, QTAP nanovaccines encoding Ag85B and Hsp70 transcripts (QTAP-85B+H70) were able to elicit robust IgG antibody and IFN- É£, TNF-α, IL-2, and IL-17 cytokines responses. Following aerosol challenge with a clinical isolate of M. avium ss. hominissuis (M.ah), a significant reduction of mycobacterial counts was observed in lungs and spleens of only immunized animals at both 4- and 8-weeks post-challenge. As expected, reduced levels of M. ah were associated with diminished histological lesions and robust cell-mediated immunity. Interestingly, polyfunctional T-cells expressing IFN- É£, IL-2, and TNF- α were detected at 8 but not 4 weeks post-challenge. Overall, our analysis indicated that QTAP is a highly efficient transfection agent and could improve the immunogenicity of mRNA vaccines against pulmonary M. ah, an infection of significant public health importance, especially to the elderly and to those who are immune compromised.

Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 106 CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Characterization of early immune responses elicited by live and inactivated vaccines against Johne's disease in goats.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic debilitating condition affecting ruminants causing significant economic losses to the dairy industry. Available inactivated vaccines are not effective in controlling the disease and vaccinated animals can continue to infect newly born calves. Recently, we have shown that a live-attenuated vaccine candidate (pgsN) is protective in goats and calves following challenge with virulent strains of M. paratuberculosis. To decipher the dynamics of the immune responses elicited by both live-attenuated and inactivated vaccines, we analyzed key immunological parameters of goats immunized through different routes when a marker-less pgsN vaccine was used. Within a few weeks, the inactivated vaccine triggered the formation of granulomas both at the site of inoculation and in regional lymph nodes, that increased in size over time and persisted until the end of the experiment. In contrast, granulomas induced by the pgsN vaccine were small and subsided during the study. Interestingly, in this vaccine group, histology demonstrated an initial abundance of intra-histiocytic mycobacterial bacilli at the site of inoculation, with recruitment of very minimal T lymphocytes to poorly organized granulomas. Over time, granulomas became more organized, with recruitment of greater numbers of T and B lymphocytes, which coincided with a lack of mycobacteria. For the inactivated vaccine group, mycobacterial bacilli were identified extracellularly within the center of caseating granulomas, with relatively equal proportions of B- and T-lymphocytes maintained across both early and late times. Despite the differences in granuloma-specific lymphocyte recruitment, markers for cell-mediated immunity (e.g., IFN-γ release) were robust in both injected pgsN and inactivated vaccine groups. In contrast, the intranasal live-attenuated vaccine did not elicit any reaction at site of inoculation, nor cell-mediated immune responses. Finally, 80% of animals in the inactivated vaccine group significantly reacted to purified protein derivatives from M. bovis, while reactivity was detected in only 20% of animals receiving pgsN vaccine, suggesting a higher level of cross reactivity for bovine tuberculosis when inactivated vaccine is used. Overall, these results depict the cellular recruitment strategies driving immune responses elicited by both live-attenuated and inactivated vaccines that target Johne's disease.

Systemic Neutralizing Antibodies and Local Immune Responses Are Critical for the Control of SARS-CoV-2.

Antibody measurements are primarily used to evaluate experimental and approved COVID-19 vaccines, which is unilateral considering our immune responses' complex nature. Previously, we showed that nanoparticle plasmid DNA adjuvant system, QAC, and MVA based vaccines were immunogenic against SARS-CoV-2. Here, we report on the protective efficacy of systemic humoral and mucosal cell-mediated immune responses in transgenic mice models against SARS-CoV-2 following nanoparticle immunization. Parenteral, intramuscular administration of QAC-based plasmid DNA vaccine-encoding SARS-CoV-2 S and N led to the induction of significant serum neutralizing humoral responses, which reduced viral burden in the lungs and prevented viral dissemination to the brain. In contrast, the mucosal, intranasal administration of a heterologous vaccine elicited significant mucosal cell-mediated immune responses in the lungs that limited lung viral replication. The presented results demonstrate that serum neutralizing humoral and local lung T-cell immune responses are critical for the control of SARS-CoV-2 replication.

Localized and Systemic Immune Responses against SARS-CoV-2 Following Mucosal Immunization.

The rapid transmission of SARS-CoV-2 in the USA and worldwide necessitates the development of multiple vaccines to combat the COVID-19 global pandemic. Previously, we showed that a particulate adjuvant system, quil-A-loaded chitosan (QAC) nanoparticles, can elicit robust immunity combined with plasmid vaccines when used against avian coronavirus. Here, we report on the immune responses elicited by mucosal homologous plasmid and a heterologous immunization strategy using a plasmid vaccine and a Modified Vaccinia Ankara (MVA) expressing SARS-CoV-2 spike (S) and nucleocapsid (N) antigens. Only the heterologous intranasal immunization strategy elicited neutralizing antibodies against SARS-CoV-2 in serum and bronchoalveolar lavage of mice, suggesting a protective vaccine. The same prime/boost strategy led to the induction of type 1 and type 17 T-cell responses and polyfunctional T-cells expressing multiple type 1 cytokines (e.g., IFN-γ, TNFα, IL-2) in the lungs and spleens of vaccinated mice. In contrast, the plasmid homologous vaccine strategy led to the induction of local mono and polyfunctional T-cells secreting IFN-γ. Outcomes of this study support the potential of QAC-nano vaccines to elicit significant mucosal immune responses against respiratory coronaviruses.