E3 ubiquitin ligase CBLB regulates innate immune responses and bacterial dissemination during nontuberculous mycobacteria infection.

Nontuberculous mycobacteria (NTM) are emerging opportunistic pathogens causing pulmonary infection to fatal disseminated disease. NTM infections are steadily increasing in children and adults, and immune-compromised individuals are at a greater risk of fatal infections. The NTM disease's adverse pathology and resistance to antibiotics have further worsened the therapeutic measures. Innate immune regulators are potential targets for therapeutics to NTM, especially in a T cell-suppressed population, and many ubiquitin ligases modulate pathogenesis and innate immunity during infections, including mycobacterial infections. Here, we investigated the role of an E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (CBLB), in immunocompromised mouse models of NTM infection. We found that CBLB is essential to prevent bacterial growth and dissemination. Cblb deficiency debilitated natural killer cells, inflammatory monocytes, and macrophages in vivo. However, Cblb deficiency in macrophages did not wane its ability to inhibit bacterial growth or production of reactive oxygen species or interferon γ production by natural killer cells in vitro. CBLB restricted NTM growth and dissemination by promoting early granuloma formation in vivo. Our study shows that CBLB bolsters innate immune responses and helps prevent the dissemination of NTM during compromised T cell immunity.

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.

A DNA Prime and MVA Boost Strategy Provides a Robust Immunity against Infectious Bronchitis Virus in Chickens.

Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy.

Superinfection with SARS-CoV-2 Has Deleterious Effects on Mycobacterium bovis BCG Immunity and Promotes Dissemination of Mycobacterium tuberculosis.

An estimated one-third of the world's population is infected with Mycobacterium tuberculosis, with the majority being vaccinated with Mycobacterium bovis BCG. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat, and we must understand how SARS-CoV-2 can modulate both BCG immunity and tuberculosis pathogenesis. Interestingly, neither BCG vaccination nor tuberculosis infection resulted in differences in clinical outcomes associated with SARS-CoV-2 in transgenic mice. Surprisingly, earlier M. tuberculosis infection resulted in lower SARS-CoV-2 viral loads, mediated by the heightened immune microenvironment of the murine lungs, unlike vaccination with BCG, which had no impact. In contrast, M. tuberculosis-infected tissues had increased bacterial loads and decreased histiocytic inflammation in the lungs following SARS-CoV-2 superinfection. SARS-CoV-2 modulated BCG-induced type 17 responses while decreasing type 1 and increasing type 2 cytokines in M. tuberculosis-infected mice. These findings challenge initial findings of BCG's positive impact on SARS-CoV-2 infection and suggest potential ramifications for M. tuberculosis reactivation upon SARS-CoV-2 superinfection. IMPORTANCE Prior to SARS-CoV-2, M. tuberculosis was the leading infectious disease killer, with an estimated one-third of the world's population infected and 1.7 million deaths a year. Here, we show that SARS-CoV-2 superinfection caused increased bacterial dissemination in M. tuberculosis-infected mice along with immune and pathological changes. SARS-CoV-2 also impacted the immunity of BCG-vaccinated mice, resulting in decreased interleukin-17 (IL-17) levels, while offering no protective effect against SARS-CoV-2. These results demonstrate that SARS-CoV-2 may have a deleterious effect on the ongoing M. tuberculosis pandemic and potentially limit BCG's efficacy.

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.

Transcriptional Profiling of Early and Late Phases of Bovine Tuberculosis.

Bovine tuberculosis, caused by Mycobacterium tuberculosis var. bovis (M. bovis), is an important enzootic disease affecting mainly cattle, worldwide. Despite the implementation of national campaigns to eliminate the disease, bovine tuberculosis remains recalcitrant to eradication in several countries. Characterizing the host response to M. bovis infection is crucial for understanding the immunopathogenesis of the disease and for developing better control strategies. To profile the host responses to M. bovis infection, we analyzed the transcriptome of whole blood cells collected from experimentally infected calves with a virulent strain of M. bovis using RNA transcriptome sequencing (RNAseq). Comparative analysis of calf transcriptomes at early (8 weeks) versus late (20 weeks) aerosol infection with M. bovis revealed a divergent and unique profile for each stage of infection. Notably, at the early time point, transcriptional upregulation was observed among several of the top-ranking canonical pathways involved in T-cell chemotaxis. At the late time point, enrichment in the cell mediated cytotoxicity (e.g., Granzyme B) was the predominant host response. These results showed significant change in bovine transcriptional profiles and identified networks of chemokine receptors and monocyte chemoattractant protein (CCL) coregulated genes that underline the host-mycobacterial interactions during progression of bovine tuberculosis in cattle. Further analysis of the transcriptomic profiles identified potential biomarker targets for early and late phases of tuberculosis in cattle. Overall, the identified profiles better characterized identified novel immunomodulatory mechanisms and provided a list of targets for further development of potential diagnostics for tuberculosis in cattle.

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.

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.

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.

A Protective Vaccine against Johne's Disease in Cattle.

Johne's disease (JD) caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is a chronic infection characterized by the development of granulomatous enteritis in wild and domesticated ruminants. It is one of the most significant livestock diseases not only in the USA but also globally, accounting for USD 200-500 million losses annually for the USA alone with potential link to cases of Crohn's disease in humans. Developing safe and protective vaccines is of a paramount importance for JD control in dairy cows. The current study evaluated the safety, immunity and protective efficacy of a novel live attenuated vaccine (LAV) candidate with and without an adjuvant in comparison to an inactivated vaccine. Results indicated that the LAV, irrespective of the adjuvant presence, induced robust T cell immune responses indicated by proinflammatory cytokine production such as IFN-γ, IFN-α, TNF-α and IL-17 as well as strong response to intradermal skin test against M. paratuberculosis antigens. Furthermore, the LAV was safe with minimal tissue pathology. Finally, calves vaccinated with adjuvanted LAV did not shed M. paratuberculosis post-challenge, a much-desired characteristic of an effective vaccine against JD. Together, this data suggests a strong potential of testing LAV in field trials to curb JD in dairy herds.

A Novel Mucosal Adjuvant System for Immunization against Avian Coronavirus Causing Infectious Bronchitis.

Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is currently a major threat to chicken health, with multiple outbreaks being reported in the United States over the past decade. Modified live virus (MLV) vaccines used in the field can persist and provide the genetic material needed for recombination and emergence of novel IBV serotypes. Inactivated and subunit vaccines overcome some of the limitations of MLV with no risk of virulence reversion and emergence of new virulent serotypes. However, these vaccines are weakly immunogenic and poorly protective. There is an urgent need to develop more effective vaccines that can elicit a robust, long-lasting immune response. In this study, we evaluate a novel adjuvant system developed from Quil-A and chitosan (QAC) for the intranasal delivery of nucleic acid immunogens to improve protective efficacy. The QAC adjuvant system forms nanocarriers (<100 nm) that efficiently encapsulate nucleic acid cargo, exhibit sustained release of payload, and can stably transfect cells. Encapsulation of plasmid DNA vaccine expressing IBV nucleocapsid (N) protein by the QAC adjuvant system (pQAC-N) enhanced immunogenicity, as evidenced by robust induction of adaptive humoral and cellular immune responses postvaccination and postchallenge. Birds immunized with pQAC-N showed reduced clinical severity and viral shedding postchallenge on par with protection observed with current commercial vaccines without the associated safety concerns. Presented results indicate that the QAC adjuvant system can offer a safer alternative to the use of live vaccines against avian and other emerging coronaviruses.IMPORTANCE According to 2017 U.S. agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67% of the industry value, with the production of >50 billion pounds of chicken meat. The economic success of the poultry industry in the United States hinges on the extensive use of vaccines to control infectious bronchitis virus (IBV) and other poultry pathogens. The majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.

A single dose polyanhydride-based nanovaccine against paratuberculosis infection.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) causes Johne's disease in ruminants and is characterized by chronic gastroenteritis leading to heavy economic losses to the dairy industry worldwide. The currently available vaccine (inactivated bacterin in oil base) is not effective in preventing pathogen shedding and is rarely used to control Johne's disease in dairy herds. To develop a better vaccine that can prevent the spread of Johne's disease, we utilized polyanhydride nanoparticles (PAN) to encapsulate mycobacterial antigens composed of whole cell lysate (PAN-Lysate) and culture filtrate (PAN-Cf) of M. paratuberculosis. These nanoparticle-based vaccines (i.e., nanovaccines) were well tolerated in mice causing no inflammatory lesions at the site of injection. Immunological assays demonstrated a substantial increase in the levels of antigen-specific T cell responses post-vaccination in the PAN-Cf vaccinated group as indicated by high percentages of triple cytokine (IFN-γ, IL-2, TNF-α) producing CD8+ T cells. Following challenge, animals vaccinated with PAN-Cf continued to produce significant levels of double (IFN-γ, TNF-α) and single cytokine (IFN-γ) secreting CD8+ T cells compared with animals vaccinated with an inactivated vaccine. A significant reduction in bacterial load was observed in multiple organs of animals vaccinated with PAN-Cf, which is a clear indication of protection. Overall, the use of polyanhydride nanovaccines resulted in development of protective and sustained immunity against Johne's disease, an approach that could be applied to counter other intracellular pathogens.

Publisher Correction: Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic Polymorphism Associated with the Emergence of Virulent Isolates of Mycobacterium bovis in the Nile Delta.

Mycobacterium bovis is responsible for bovine tuberculosis in both animals and humans. Despite being one of the most important global zoonotic disease, data related to the ecology and pathogenicity of bovine tuberculosis is scarce, especially in developing countries. In this report, we examined the dynamics of M. bovis transmission among dairy cattle in the Nile Delta of Egypt. Animals belonging to 27 herds from 7 governorates were tested by the Single Intradermal Comparative Skin Tuberculin (SICST), as a preliminary screen for the presence of bovine tuberculosis. Positive SICST reactors were identified in 3% of the animals spread among 40% of the examined herds. Post-mortem examination of slaughtered reactors confirmed the presence of both pulmonary and/or digestive forms of tuberculosis in > 50% of the examined animals. Targeted and whole-genome analysis of M. bovis isolates indicated the emergences of a predominant spoligotype (SB0268) between 2013-2015, suggesting a recent clonal spread of this isolate within the Nile Delta. Surprisingly, 2 isolates belonged to M. bovis BCG group, which are not allowed for animal vaccination in Egypt, while the rest of isolates belonged to the virulent M. bovis clonal complex European 2 present in Latin America and several European countries. Analysis of strain virulence in the murine model of tuberculosis indicated the emergence of a more virulent strain (MBE4) with a specific genotype. More analysis is needed to understand the molecular basis for successful spread of virulent isolates of bovine tuberculosis among animals and to establish genotype/phenotype association.

Effective mosaic-based nanovaccines against avian influenza in poultry.

Avian influenza virus (AIV) is an extraordinarily diverse pathogen that causes significant morbidity in domesticated poultry populations and threatens human life with looming pandemic potential. Controlling avian influenza in susceptible populations requires highly effective, economical and broadly reactive vaccines. Several AIV vaccines have proven insufficient despite their wide use, and better technologies are needed to improve their immunogenicity and broaden effectiveness. Previously, we developed a "mosaic" H5 subtype hemagglutinin (HA) AIV vaccine and demonstrated its broad protection against diverse highly pathogenic H5N1 and seasonal H1N1 virus strains in mouse and non-human primate models. There is a significant interest in developing effective and safe vaccines against AIV that cannot contribute to the emergence of new strains of the virus once circulating in poultry. Here, we report on the development of an H5 mosaic (H5M) vaccine antigen formulated with polyanhydride nanoparticles (PAN) that provide sustained release of encapsulated antigens. H5M vaccine constructs were immunogenic whether delivered by the modified virus Ankara (MVA) strain or encapsulated within PAN. Both humoral and cellular immune responses were generated in both specific-pathogen free (SPF) and commercial chicks. Importantly, chicks vaccinated by H5M constructs were protected in terms of viral shedding from divergent challenge with a low pathogenicity avian influenza (LPAI) strain at 8 weeks post-vaccination. In addition, protective levels of humoral immunity were generated against highly pathogenic avian influenza (HPAI) of the similar H5N1 and genetically dissimilar H5N2 viruses. Overall, the developed platform technologies (MVA vector and PAN encapsulation) were safe and provided high levels of sustained protection against AIV in chickens. Such approaches could be used to design more efficacious vaccines against other important poultry infections.

The inhibitory effect of nisin on Mycobacterium avium ssp. paratuberculosis and its effect on mycobacterial cell wall.

Infection with Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) is a widespread problem in the United States and worldwide, and it constitutes a significant health problem for dairy animals with a potential effect on human health. Mycobacterium paratuberculosis is easily transmitted through consumption of contaminated milk; therefore, finding safe methods to reduce the mycobacterial load in milk and other dairy products is important to the dairy industry. The main objective of the current study was to investigate the effect of natural products, such as bacteriocins designated as "generally regarded as safe" (GRAS), on the survival of M. paratuberculosis in milk. Commercially synthesized bacteriocin (nisin) was used to examine its effect on the survival of laboratory and field isolates of M. paratuberculosis and in contaminated milk. Surprisingly, nisin had a higher minimum inhibitory concentration (MIC) against the laboratory strain (M. paratuberculosis K10), at 500 U/mL, than against field isolates (e.g., M. paratuberculosis 4B and JTC 1281), at 15 U/mL. In milk, growth of M. paratuberculosis was inhibited after treatment with levels of nisin that are permissible in human food at 4°C and 37°C. Using both fluorescent and scanning electron microscopy, we were able to identify defects in the bacterial cell walls of treated cultures. Our analysis indicated that nisin reduced membrane integrity by forming pores in the mycobacterial cell wall, thereby decreasing survival of M. paratuberculosis. Thus, nisin treatment of milk could be implemented as a control measure to reduce M. paratuberculosis secreted in milk from infected herds. Nisin could also be used to reduce M. paratuberculosis in colostrum given to calves from infected animals, improving biosecurity control in dairy herds affected by Johne's disease.

Genomic analysis of the emergence of drug-resistant strains of Mycobacterium tuberculosis in the Middle East.

Tuberculosis (TB) represents a significant challenge to public health authorities, especially with the emergence of drug-resistant (DR) and multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis. We sought to examine the genomic variations among recently isolated strains of M. tuberculosis in two closely related countries with different population demography in the Middle East. Clinical isolates of M. tuberculosis from both Egypt and Saudi Arabia were subjected to phenotypic and genotypic analysis on gene and genome-wide levels. Isolates with MDR phenotypes were highly prevalent in Egypt (up to 35%) despite its relatively stable population structure (sympatric pattern). MDR-TB isolates were not identified in the isolates from Saudi Arabia despite its active guest worker program (allopatric pattern). However, tuberculosis isolates from Saudi Arabia, where lineage 4 was more prevalent (>65%), showed more diversity than isolates from Egypt, where lineage 3 was the most prevalent (>75%). Phylogenetic and molecular dating analyses indicated that lineages from Egypt were recently diverged (~78 years), whereas those from Saudi Arabia were diverged by over 200 years. Interestingly, DR isolates did not appear to cluster together or spread more widely than drug-sensitive isolates, suggesting poor treatment as the main cause for emergence of drug resistance rather than more virulence or more capacity to persist.

Biomarkers for Early Stages of Johne's Disease Infection and Immunization in Goats.

Background: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2-5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathophysiology of early infection with M. paratuberculosis is still not well understood and represents a key hurdle toward the development of better diagnostics. Methods: The present study employed a large-scale RNA-Sequencing technology to better understand early stages of M. paratuberculosis infection and immunization. Specifically, gene expression profiles of peripheral blood mononuclear cells (PBMCs) from infected or vaccinated goats were compared to controls. Results: When compared to the naïve control goats, we identified a large number of transcripts (N = 226, 1018, 1714) that were differentially expressed in the M. paratuberculosis-infected goats, goats vaccinated with live attenuated or inactivated vaccines. There were also 1133 differentially expressed (DE) transcripts between vaccinated goats and infected ones. Bioinformatics evaluation of the DE genes indicated the regulation of a large number of genes with immunity and inflammatory functions including IL-18BP, IFN-γ, IL-17A, NOS2, LIPG, and IL-22. Interestingly, a large number of goat genes (N = 667) were regulated whether live or inactivated vaccine were used. Some of the regulated genes (e.g., IL-17A, IFN-γ) continued its unique transcriptional profile up to 12 months post-challenge. Conclusion: Overall, transcriptome analysis of infected and/or immunized goats identified potential targets for developing early diagnostics for Johne's disease and a potential approach to differentiate infected from vaccinated animals. A similar approach could be used to analyze later stages of Johne's disease or other chronic infections.

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer.

When advising farmers on how to control Johne's disease in an infected herd, one of the main recommendations is to avoid feeding waste milk to calves and instead feed calf milk replacer (CMR). This advice is based on the assumption that CMR is free of viable Mycobacterium avium ssp. paratuberculosis (MAP) cells, an assumption that has not previously been challenged. We tested commercial CMR products (n = 83) obtained from dairy farms around the United States by the peptide-mediated magnetic separation (PMS)-phage assay, PMS followed by liquid culture (PMS-culture), and direct IS900 quantitative PCR (qPCR). Conventional microbiological analyses for total mesophilic bacterial counts, coliforms, Salmonella, coagulase-negative staphylococci, streptococci, nonhemolytic Corynebacterium spp., and Bacillus spp. were also performed to assess the overall microbiological quality of the CMR. Twenty-six (31.3%) of the 83 CMR samples showed evidence of the presence of MAP. Seventeen (20.5%) tested positive for viable MAP by the PMS-phage assay, with plaque counts ranging from 6 to 1,212 pfu/50 mL of reconstituted CMR (average 248.5 pfu/50 mL). Twelve (14.5%) CMR samples tested positive for viable MAP by PMS-culture; isolates from all 12 of these samples were subsequently confirmed by whole-genome sequencing to be different cattle strains of MAP. Seven (8.4%) CMR samples tested positive for MAP DNA by IS900 qPCR. Four CMR samples tested positive by both PMS-based tests and 5 CMR samples tested positive by IS900 qPCR plus one or other of the PMS-based tests, but only one CMR sample tested positive by all 3 MAP detection tests applied. All conventional microbiology results were within current standards for whole milk powders. A significant association existed between higher total bacterial counts and presence of viable MAP indicated by either of the PMS-based assays. This represents the first published report of the isolation of viable MAP from CMR. Our findings raise concerns about the potential ability of MAP to survive manufacture of dried milk-based products.

Superior Protection from Live-Attenuated Vaccines Directed against Johne's Disease.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the etiological agent of Johne's disease in ruminants. Johne's disease is an important enteric infection causing large economic losses associated with infected herds. In an attempt to fight this infection, we created two novel live-attenuated vaccine candidates with mutations in sigH and lipN (pgsH and pgsN, respectively). Earlier reports in mice suggested these vaccines are promising candidates to fight Johne's disease in ruminants. In this study, we tested the performances of the two constructs as vaccine candidates using the goat model of Johne's disease. Both vaccines appeared to provide significant immunity to goats against challenge from wild-type M. paratuberculosis The pgsH and pgsN constructs showed a significant reduction in histopathological lesions and tissue colonization compared to nonvaccinated goats and those vaccinated with an inactivated vaccine. Unlike the inactivated vaccine, the pgsN construct was able to eliminate fecal shedding from challenged animals, a feature that is highly desirable to control Johne's disease in infected herds. Furthermore, strong initial cell-mediated immune responses were elicited in goats vaccinated with pgsN that were not demonstrated in other vaccine groups. Overall, the results indicate the potential use of live-attenuated vaccines to control intracellular pathogens, including M. paratuberculosis, and warrant further testing in cattle, the main target for Johne's disease control programs.