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• The rBCG expressing rCP40 and rCP09720 proteins protect against C. pseudotuberculosis. • Mycobacterium bovis BCG/cp40 boosted with rCP40 (G5) showed 90% protection after challenge. • Besides eliciting an expected cross-immune response, rBCG induced specific humoral and cellular responses. In this study, we assessed the survival and immune response of mice vaccinated with recombinant Mycobacterium bovis BCG Pasteur that expressed the CP40 or CP09720 proteins after the mice were challenged with a C. pseudotuberculosis MIC-6 virulent strain. Six groups of mice (n = 10 mice per group) were immunised with a sterile 0.9% saline solution (G1), 106 CFU of M. bovis BCG Pasteur (G2), 106 CFU of M. bovis BCG/ cp40 (G3), 106 CFU of M. bovis BCG/ cp09720 (G4), M. bovis BCG/ cp40 boosted with rCP40 (G5), and M. bovis BCG/ cp09720 boosted with rCP09720 (G6). The highest survival rate of 90% was observed in the G5 group, followed by 80% in the G6 group and 70% in the G3 and G4 groups. Moreover, a significantly greater induction of IFN-γ and IL-10 was found in the G3 group and higher IL-17 levels were recorded in the G5 group compared to their levels in the control group (G1) (p < 0.05). A specific humoral immune response (total IgG) was found in the G5 and G6 groups on day 42 compared to the level of response in the G1 group. These results indicated that the vector vaccine elicited significantly greater survival of mice in all experimental groups after a strong virulent challenge and induced a strong immune response. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of CRX-527, a Toll-like receptor 4 agonist, as the possible adjuvant for recombinant Mycobacterium bovis Bacillus Calmette-Guerin expressing merozoite surface protein 1C (BCG-MSP-1C). Methods: The mice were immunized with BCG and BCG-MSP- 1C in the presence and absence of CRX-527. The untreated mice (injected with PBS-T80 only) were the negative control. The ability of CRX-527 to enhance IgG and its subclasses, as well as IL-4 and IFN-γ production in the serum and spleen supernatant was evaluated using ELISA. Results: Mice immunized with BCG-MSP-1C exhibited the highest production of IgGs, IL-4 and IFN-γ after third immunization. In addition, CRX-527 further promoted the production of total IgG and IgG subclasses as well as IFN-γ and IL-4 in the serum and splenocytes of immunized mice. Conclusions: CRX-527 has the potential as an adjuvant candidate for the candidate vaccines. Further study is needed to verify appropriate dosage for immunization and its efficacy. [ABSTRACT FROM AUTHOR]

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The BCG vaccines on the market have employed a Mycobacterium bovis (M. bovis) sub-strains derived from the initial strain. To date, there has been no recommendation regarding the sub-strains with the highest effectiveness when administered to humans. Because it remains the standard for Tuberculosis treatment, the quality of the BCG vaccine must be verified. The viability test is one of the parameters for BCG vaccine quality control. The culture method has become the gold standard for viability testing with various testing media. The present study aimed to evaluate the performance of Lowenstein Jensen (LJ) and Ogawa media for the viability test of Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains of M. bovis in the BCG vaccine. The number of culturable particles of each sub-strain in the BCG vaccine was estimated and statistically evaluated using the t-test. The colonies of the Pasteur 1173P2 have characteristics; tended to clump on both mediums with tiny, rough, and pale yellow/cream colors. Although the colony character of the Russian (Moscow) - 384 generally has similar feature, it did not cluster and had a smooth texture. In terms of growth rate, LJ and Ogawa media performed similarly for Pasteur 1173P2 and Russian (Moscow) - 384 sub-strains. Maximum growth is reached by the fifth week. The culturable particles of Pasteur P1173P2 sub-strains did not differ between mediums. Whereas the growth of the Russian (Moscow) - 384 sub-strains was statistically better on Ogawa media. The results of this study reveal that the performance of the media used for determining the number of culturable particles is based on the sub-strains of M. bovis present in the BCG vaccine. [ABSTRACT FROM AUTHOR]

Background and Objectives: Lipoarabinomannan is one of the components of the significant structural cell surfaces of mycobacteria and serves as an immunostimulatory factor. TNF-α and IL-12 are two examples of the anti-bacterial inflammatory cytokines that are activated and induced during infection. Materials and Methods: In this study, mannan was extracted and processed, and then Bulb/c female mice were used in three groups, one group was given BCG vaccine, the other group was given BCG vaccine with mannan adjuvant, and a non-injected group was used as a control group. Inflammatory factors interleukin-12, TNF-α, IgG and IgM were measured in mouse serum. Results: The levels of the inflammatory factors interleukin-12 and TNF-α in the serum isolated from mice receiving the BCG vaccine with mannan adjuvant showed a significant difference compared to the group that received only the BCG vaccine and the control group [IL-12] and , with P≤0.05.The examination of the level of IgG immune factors in these three groups revealed a significant difference. The group that received the BCG vaccine with mannan adjuvant showed a marked contrast compared to the group that received only the BCG vaccine and the control group, with P≤0.05. The level of IgM was higher in the group that received the BCG vaccine alone compared to the adjuvant vaccine group and the control group, with P≤0.05. Conclusion: Our results indicated that mice receiving the BCG vaccine with mannan adjuvant had significantly higher serum levels of IL-12, TNF-α, and IgG than the group receiving BCG alone.

This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in archived human sputum samples previously collected from residents who reside adjacent to the M. bovis-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for M. tuberculosis complex (MTBC) DNA but culture negative for M. tuberculosis. Amplification and Sanger sequencing of hsp65 and rpoB genes from DNA extracted from stored heat-inactivated sputum samples confirmed the presence of detectable amounts of MTBC from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and gyrB long-read amplicon deep sequencing identified M. bovis (n = 10) and M. tuberculosis (n = 7). Notably, M. bovis spoligotypes SB0130 and SB1474 were identified in 4 samples, with SB0130 previously identified in local cattle and wildlife and SB1474 exclusively in African buffaloes in the adjacent park. M. bovis DNA in sputum, from people living near the park, underscores zoonotic transmission potential in SA. Identification of spoligotypes specifically associated with wildlife only and spoligotypes found in livestock as well as wildlife, highlights the complexity of TB epidemiology at wildlife-livestock-human interfaces. These findings support the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human M. bovis infection in SA patients with positive Ultra results.

ABSTRACT: This study determined the frequency of positive tests for bovine tuberculosis in animals and breeders/establishments in Rio Grande do Norte, Brazil. Data were provided by the Instituto de Defesa e Inspeção Agropecuária do Rio Grande do Norte (IDIARN), from its Unidade Local de Sanidade Animal e Vegetal (ULSAV’S), acquired in monthly reports issued by qualified Veterinarians under the National Program for the Control and Eradication of Brucellosis and Animal Tuberculosis (PNCEBT), from June 2012 to December 2021. For diagnosis, the comparative cervical test was used as a routine and confirmatory test. A total of 45,804 cattle were tested, 53 (0.1%) of which tested positive. Secondary data are essential in the evaluation of sanitary measures, allowing rapid generation of hypotheses about diseases and providing support for decision-making. Measures such as raising awareness among producers, sanitary control in the acquisition and sale of cows for reproduction, inspection of sanitary barriers, and conducting epidemiological surveys to understand the actual situation of this disease in the state of Rio Grande do Norte are crucial.

ABSTRACT: Bovine tuberculosis is a zoonotic disease with global distribution. This study aimed to describe its prevalence in cattle through a systematic review and meta-analysis of studies conducted all around the world. The research consisted of a systematic literature review following the precepts of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology. Cross-sectional studies that described the prevalence of the disease using the diagnostic method through an intradermal test with bovine and avian purified protein derivative (PPD) were selected. Identifying articles was performed in the PubMed, ScienceDirect, Scopus, and Web of Science databases. Of the total number of studies found (n=1,839), 60 met all criteria and were included in this review. The overall prevalence was 3.27% (2.11-5.05%) for animals and 18.09% (11.20-27.90%) for herds. Analysis of risk factors for tuberculosis in cattle was found or performed in 50 studies. The heterogeneity identified among the works included was expected, given the differences in research design, year of publication, and the number of animals sampled. It is necessary to evaluate the insertion of new ante mortem diagnostic tests into control and eradication programs, which, combined with allergic tests, may identify the largest number of animals that presented an actual positive for the disease.

Mycobacterium bovis infection, which is a prominent cause of bovine tuberculosis, has been confirmed by mycobacterial culture in African rhinoceros species in Kruger National Park (KNP), South Africa. In this population-based study of the epidemiology of M. bovis in 437 African rhinoceros (Diceros bicornis, Ceratotherium simum), we report an estimated prevalence of 15.4% (95% CI: 10.4 to 21.0%), based on results from mycobacterial culture and an antigen-specific interferon gamma release assay from animals sampled between 2016 and 2020. A significant spatial cluster of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo (Syncerus caffer) herds in the vicinity of the rhinoceros sampling location. Since African buffaloes are important maintenance hosts for M. bovis in KNP, spillover of infection from these hosts to white rhinoceros sharing the environment is suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016–2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. These study findings provide a foundation for further epidemiological investigation of M. bovis, a multihost pathogen, in a complex ecosystem that includes susceptible species that are threatened and endangered.

Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a chronic gastrointestinal disease affecting ruminants. This disease remains widespread in part due to the limitations of available diagnostics and vaccines. A representative small animal model of disease could act as a valuable tool for studying its pathogenesis and to develop new methods for paratuberculosis control, but current models are lacking. Streptomycin pre-treatment can reduce colonization resistance and has previously been shown to improve enteric infection in a Salmonella model. Here, we investigated whether streptomycin pre-treatment of mice followed by MAP gavage could act as a model of paratuberculosis which mimics the natural route of infection and disease development in ruminants. The infection outcomes of MAP were compared to M. avium subsp. hominissuis (MAH), an environmental mycobacterium, and M. bovis and M. orygis, two tuberculous mycobacteria. Streptomycin pre-treatment was shown to consistently improve bacterial infection post-oral inoculation. This model led to chronic MAP infection of the intestines and mesenteric lymph nodes (MLNs) up to 24-weeks post-gavage, however there was no evidence of inflammation or disease. These infection outcomes were found to be specific to MAP. When the model was applied to a bacterium of lesser virulence MAH, the infection was comparatively transient. Mice infected with bacteria of greater virulence, M. bovis or M. orygis, developed chronic intestinal and MLN infection with pulmonary disease similar to zoonotic TB. Our findings suggest that a streptomycin pre-treatment mouse model could be applied to future studies to improve enteric infection with MAP and to investigate other modifications underlying MAP enteritis.

This article discusses a case of atypical ulcerative cutaneous tuberculosis in a zosteriform distribution caused by Mycobacterium bovis. The patient presented with a large, persistent, indurated, erythematous plaque in the left inguinal area, along with erosions on the glans penis. Despite initial misdiagnosis and treatment for herpes simplex, the ulceration progressed. Skin biopsy and testing confirmed the presence of M. bovis. The patient responded well to antitubercular therapy and experienced complete healing of the ulcers. This case highlights the importance of considering tuberculosis as a potential diagnosis, even in atypical presentations. [Extracted from the article]

The antioxidant defense is critical for the survival of intracellular pathogens such as Mycobacterium tuberculosis complex (MTBC) species, including Mycobacterium bovis, which are often exposed to an oxidative environment caused by reactive oxygen species (ROS) in hosts. However, the signaling pathway in mycobacteria for sensing and responding to oxidative stress remains largely unclear. In this study, we characterize a TetR-type transcription regulator BCG_3893c, designated AotM, as a novel redox sensor in Mycobacterium bovis that increases mycobacterial tolerance to oxidative stress. AotM is required for the growth of M. bovis in the presence of 1 mM hydrogen peroxide. Loss of the aotM gene leads to altered transcriptional profiles with 352 genes significantly up-regulated and 25 genes significantly down-regulated. AotM recognizes a 14-bp palindrome sequence motif and negatively regulates the expression of a FAD-dependent oxidoreductase encoded by bcg_3892c. Overexpression of BCG_3892c increases intracellular ROS production and reduces the growth of M. bovis. In summary, we propose that AotM enhances the mycobacterial resistance against oxidative stress probably by inhibiting intracellular ROS production. Our findings reveal a novel underlying regulatory mechanism behind mycobacterial oxidative stress adaptation.

Bovine tuberculosis is caused by Mycobacterium bovis, a member of the M. tuberculosis complex of mycobacterial species that cause tuberculosis in humans and animals. Diagnosis of bovine tuberculosis has relied on examinations of cell-mediated immune responses to M. bovis proteins using tuberculin skin testing and/or interferon gamma release assays. Even when using these methods, disease detection during the earliest phases of infection has been difficult, allowing a window for cattle-to-cattle transmission to occur within a herd. Alternative means of diagnosis could include methods to detect M. bovis or M. bovis DNA in bodily fluids such as nasal secretions, saliva, or blood. During the first 8 weeks after experimental aerosol infection of 18 calves, M. bovis DNA was detected in nasal swabs from a small number of calves 5, 6, and 8 weeks after infection and in samples of saliva at 1, 7, and 8 weeks after infection. However, at no time could culturable M. bovis be recovered from nasal swabs or saliva. M. bovis DNA was not found in blood samples collected weekly and examined by real-time PCR. Interferon gamma release assays demonstrated successful infection of all calves, while examination of humoral responses using a commercial ELISA identified a low number of infected animals at weeks 4–8 after infection. Examination of disease severity through gross lesion scoring did not correlate with shedding in nasal secretions or saliva, and calves with positive antibody ELISA results did not have more severe disease than other calves.

Abstract Here we provide a summary of a plenary lecture delivered on Mycobacterium bovis, the bovine TB bacillus, at the M. bovis 2022 meeting held in Galway, Ireland, in June 2022. We focus on the analysis of genetic differences between M. bovis and the human pathogen Mycobacterium tuberculosis as a route to gain knowledge on what makes M. bovis function as an animal pathogen. We provide a brief historical background around M. bovis and comparative virulence experiments with M. tuberculosis, before moving to what we have learned from the studies of the M. bovis genome sequence. We discuss the need to translate knowledge on the molecular basis of virulence in M. bovis into improved control of bovine tuberculosis.

Abstract Having entered into its second century, the eradication program for bovine tuberculosis (bTB, caused by Mycobacterium bovis) in the United States of America occupies a position both enviable and daunting. Excepting four counties in Michigan comprising only 6109 km2 (0.06% of US land area) classified as Modified Accredited, as of April 2022 the entire country was considered Accredited Free of bTB by the US Department of Agriculture for cattle and bison. On the surface, the now well-described circumstances of endemic bTB in Michigan, where white-tailed deer (Odocoileus virginianus) serve as a free-ranging wildlife maintenance host, may appear to be the principal remaining barrier to national eradication. However, the situation there is unique in the U.S., and far-removed from the broader issues of bTB control in the remainder of the country. In Michigan, extensive surveillance for bTB in deer over the last quarter century, and regulatory measures to maximize the harvest of publicly-owned wildlife, have been implemented and sustained. Prevalence of bTB in deer has remained at a low level, although not sufficiently low to eliminate cattle herd infections. Public attitudes towards bTB, cattle and deer, and their relative importance, have been more influential in the management of the disease than any limitations of biological science. However, profound changes in the demographics and social attitudes of Michigan’s human population are underway, changes which are likely to force a critical reevaluation of the bTB control strategies thus far considered integral. In the rest of the U.S. where bTB is not self-sustaining in wildlife, changes in the scale of cattle production, coupled with both technical and non-technical issues have created their own substantial challenges. It is against this diverse backdrop that the evolution of whole genome sequencing of M. bovis has revolutionized understanding of the history and ecology of bTB in Michigan, resolved previously undiscernible epidemiological puzzles, provided insights into zoonotic transmission, and unified eradication efforts across species and agencies. We describe the current status of bTB eradication in the U.S., how circumstances and management have changed, what has been learned, and what remains more elusive than ever.

Abstract Bovine tuberculosis (bTB) was first diagnosed in cattle in South Africa in 1880 and proclaimed a controlled disease in 1911. Testing of cattle for bTB is voluntary and only outbreaks of disease are reported to the National Department of Agriculture so the prevalence of the disease in cattle is largely unknown. There is a Bovine Tuberculosis Scheme which is aimed at the control of bTB in cattle but the same measures of test and slaughter, and the quarantining of the property apply to wildlife as well. bTB was first diagnosed in wildlife in a greater kudu in the Eastern Cape in 1928 and has to date been found in 24 mammalian wildlife species. The African buffalo has become a maintenance host of the disease, which is considered endemic in the Kruger National Park, the Hluhluwe-iMfolozi Park and the Madikwe Game Park. Control of bTB at the wildlife-livestock interface is difficult because of spill-over and spill-back between species. Only buffalo are required by law to be tested before translocation, but bTB has been introduced to the Madikwe Game Park probably by the translocation of other infected wildlife species. There is no national control strategy for the control of bTB in wildlife. Indirect tests have been developed to test for bTB in eight species, 6 of which can be considered endangered. More research needs to be done to develop an effective and efficient vaccine to combat the transmission of bTB within and between species. New policies need to be developed that are effective, affordable and encompassing to control the spread of bTB in South Africa.

Bovine tuberculosis is endemic in Nigeria with control measures as provided by the laws of the country being minimally enforced mostly at the abattoirs only. This study focused on bovine tuberculosis in Adamawa and Gombe States. Tuberculosis lesions were observed in 183 of 13,688 slaughtered cattle in the regions between June and December 2020. Analysis of tissue samples resulted in 17 Mycobacterium bovis isolates, predominantly from Gombe State. Spoligotyping identified four spoligotypes, including SB0944, SB1025, SB1104, and one novel pattern. MIRU-VNTR analysis further differentiated these spoligotypes into eight profiles. All isolates belonged to the Af1 clonal complex. The study emphasises the need for broader coverage and more isolates to comprehensively understand the molecular epidemiology of bovine tuberculosis in Nigeria. To enhance research and surveillance, a cost-effective approach is proposed, utilising a discriminatory VNTR panel comprising five or nine loci. The five-locus panel consists of ETR-C, QUB26, QUB11b, MIRU04, and QUB323. Alternatively, the nine-locus panel includes ETR-A, ETR-B, QUB11a, and MIRU26. Implementing this approach would provide valuable insights into the genetic diversity of M. bovis strains in Nigeria. These findings are crucial for developing effective control measures and minimising the impact of bovine tuberculosis on both animal and human health.

Abstract Background Tuberculosis (TB) has been an important public health concern in Bangladesh. The most common cause of human TB is Mycobacterium tuberculosis, while bovine TB is caused by Mycobacterium bovis. Objective The objective of this study was to determine the frequency of TB in individuals with occupational exposure to cattle and to detect Mycobacterium bovis among cattle in slaughterhouses in Bangladesh. Methods Between August 2014 and September 2015, an observational study was conducted in two government chest disease hospitals, one cattle market, and two slaughterhouses. [Correction added on 27 June 2023, after first online publication: In the preceding sentence, the year “2014” has been added after the word “August”.] Sputum samples were collected from individuals who met the criteria for suspected TB and had been exposed to cattle. Tissue samples were collected from cattle that had low body condition score(s). Both humans and cattle samples were screened for acid‐fast bacilli (AFB) by Ziehl–Neelsen (Z‐N) staining and cultured for Mycobacterium tuberculosis complex (MTC). Region of difference (RD) 9‐based polymerase chain reaction (PCR) was also performed to identify Mycobacterium spp. We also conducted Spoligotyping to identify the specific strain of Mycobacterium spp. Results Sputum was collected from a total of 412 humans. The median age of human participants was 35 (IQR: 25–50) years. Twenty‐five (6%) human sputum specimens were positive for AFB, and 44 (11%) were positive for MTC by subsequent culture. All (N = 44) culture‐positive isolates were confirmed as Mycobacterium tuberculosis by RD9 PCR. Besides, 10% of cattle workers were infected with Mycobacterium tuberculosis in the cattle market. Of all TB (caused by Mycobacterium tuberculosis) infected individuals, 6.8% of individuals were resistant to one or two anti‐TB drugs. The majority of the sampled cattle (67%) were indigenous breeds. No Mycobacterium bovis was detected in cattle. Conclusions We did not detect any TB cases caused by Mycobacterium bovis in humans during the study. However, we detected TB cases caused by Mycobacterium tuberculosis in all humans, including cattle market workers.

ABSTRACTWe analyzed the pan-genome and gene content modulation of the most diverse genome data set of the Mycobacterium tuberculosis complex (MTBC) gathered to date. The closed pan-genome of the MTBC was characterized by reduced accessory and strain-specific genomes, compatible with its clonal nature. However, significantly fewer gene families were shared between MTBC genomes as their phylogenetic distance increased. This effect was only observed in inter-species comparisons, not within-species, which suggests that species-specific ecological characteristics are associated with changes in gene content. Gene loss, resulting from genomic deletions and pseudogenization, was found to drive the variation in gene content. This gene erosion differed among MTBC species and lineages, even within M. tuberculosis, where L2 showed more gene loss than L4. We also show that phylogenetic proximity is not always a good proxy for gene content relatedness in the MTBC, as the gene repertoire of Mycobacterium africanum L6 deviated from its expected phylogenetic niche conservatism. Gene disruptions of virulence factors, represented by pseudogene annotations, are mostly not conserved, being poor predictors of MTBC ecotypes. Each MTBC ecotype carries its own accessory genome, likely influenced by distinct selective pressures such as host and geography. It is important to investigate how gene loss confer new adaptive traits to MTBC strains; the detected heterogeneous gene loss poses a significant challenge in elucidating genetic factors responsible for the diverse phenotypes observed in the MTBC. By detailing specific gene losses, our study serves as a resource for researchers studying the MTBC phenotypes and their immune evasion strategies.IMPORTANCEIn this study, we analyzed the gene content of different ecotypes of the Mycobacterium tuberculosis complex (MTBC), the pathogens of tuberculosis. We found that changes in their gene content are associated with their ecological features, such as host preference. Gene loss was identified as the primary driver of these changes, which can vary even among different strains of the same ecotype. Our study also revealed that the gene content relatedness of these bacteria does not always mirror their evolutionary relationships. In addition, some genes of virulence can be variably lost among strains of the same MTBC ecotype, likely helping them to evade the immune system. Overall, our study highlights the importance of understanding how gene loss can lead to new adaptations in these bacteria and how different selective pressures may influence their genetic makeup.

Background: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. Methods: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. Results: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. Conclusions: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a zoonotic disease that can spread from animals to humans as well as from human to human. Little research has been conducted on bovine tuberculosis prevalence and molecular characterization in the western part of Ethiopia. To investigate this, a cross-sectional study was conducted on slaughtered cattle at the Nekemte municipal abattoir between January 2020 and June 2021. A detailed postmortem examination, culture, acid-fast staining technique, molecular characterization using RD4 deletion, and spoligotyping were all carried out. Based on a detailed postmortem examination, the overall prevalence of bovine tuberculosis was 7.8% (80 of 1020). Mycobacterium isolation confirmed only 12.5% (10/80) of the suspected tuberculosis tissue lesions. With acid-fast bacilli staining, all Mycobacterium spp. isolates (n = 10) were positive. However, only 9/10 isolates were confirmed to be M. bovis with RD4 molecular deletion typing. Spoligotyping revealed that 55.6% (5/9) of the isolate patterns had previously been reported, but 44.4% (4/9) of the isolates were new. In the current investigation, it was discovered that 80% (4/5) of the M. bovis strains circulating in the cattle population of study regions were SB2233 (2/5) and SB0134 (2/5), whereas 20% (1/5) of the strains corresponded to SB1176, which is compatible with previously documented M. bovis spoligotypes. These findings suggested that M. bovis was the main cause of bovine tuberculosis in the study area and posed a risk of disease transmission from cattle to humans due to low levels of public health awareness. As such, improved awareness among citizens and the development of control policies are warranted. [ABSTRACT FROM AUTHOR]

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB. [ABSTRACT FROM AUTHOR]

INTRODUCTION- Tuberculosis (TB) is a chronic granulomatous infection principally caused by Mycobacterium TB and less frequently by ingestion of Mycobacterium bovis infected unpasteurized cow’s milk or by other atypical mycobacteria. It presents a great social and economic problem and is one of the major factors responsible for the high morbidity and mortality in India. The incidence of tuberculous cervical lymphadenopathy now accounts for two third of the extra pulmonary tuberculous lymphadenopathy. Most of these are supposed to be tuberculous in origin because of greater incidence of pulmonary tuberculosis in our country. At the same time there are other causes of lymph-adenopathy which are usually misdiagnosed as tuberculosis. It has been a common problem for both to clinicians as well as pathologist from to diagnose tuberculosis. Methos and materials- The present work is carried out in 100 clinically suspected cases of tuberculous cervical lymphadenitis attending E.N.T., Surgery, Paediatrics and Medicine Department of central India institute as an outdoor/indoor patient during the period of one year. Patients with enlarged cervical lymph nodes with a history suggestive of tuberculosis were included after taking an informed consent. Results- Study was done on 100 clinically suspected cases of tuberculous cervical lymphadenitis, Tuberculosis was diagnosed in 57% cases by FNAC, smear and culture together, the maximum incidence of tuberculosis was observed in second and third decades, Females were more affected (64%) than males with the ratio of 1:2.3. By FNAC 42% accuracy was obtained, 30% cases were AFB smear positive in our study this rate of incidence is nearer to other authors. In our culture study, 57 cases were diagnosed as tuberculous and 4 cases as non-tuberculous cervical lymphadenitis. Culture positive was higher in granulomatous necrotic lesions. Sensitivity, specificity and predictive values of culture study were significantly higher than FNAC and smear. These methods of investigation need considerable experience and confidence of a pathologist who perform the procedure for a better result. When culture was taken as Gold Standard, cytology was found to be more sensitive than smear. Conclusions – from this study we concluded that Both FNAC and smear are quick, simple, less traumatic and cost-effective methods and used as a routine investigating procedure in OPD of urban and semi-urban hospitals. Simplicity of these techniques (FNAC & Smear) combined with early availability of results and good diagnostic accuracy warrants their clinical application. Missed cytological diagnosis and isolation of non-tuberculous mycobacteria justify culture studies on all suspected tuberculous lymphadenitis cases. [ABSTRACT FROM AUTHOR]

Type-1 and type-3 interferons (IFNs) are important for control of viral replication; however, less is known about the role of Type-2 IFN (IFNγ) in anti-viral immunity. We previously observed that lung infection with Mycobacterium bovis BCG achieved though intravenous (iv) administration provides strong protection against SARS-CoV-2 in mice yet drives low levels of type-1 IFNs but robust IFNγ. Here we examine the role of ongoing IFNγ responses to pre-established bacterial infection on SARS-CoV-2 disease outcomes in two murine models. We report that IFNγ is required for iv BCG induced reduction in pulmonary viral loads, an outcome dependent on IFNγ receptor expression by non-hematopoietic cells. Importantly, we show that BCG infection prompts pulmonary epithelial cells to upregulate IFN-stimulated genes with reported anti-viral activity in an IFNγ-dependent manner, suggesting a possible mechanism for the observed protection. Finally, we confirm the anti-viral properties of IFNγ by demonstrating that the recombinant cytokine itself provides strong protection against SARS-CoV-2 challenge when administered intranasally. Together, our data show that a pre-established IFNγ response within the lung is protective against SARS-CoV-2 infection, suggesting that concurrent or recent infections that drive IFNγ may limit the pathogenesis of SARS-CoV-2 and supporting possible prophylactic uses of IFNγ in COVID-19 management. The role of interferon-γ (IFNγ) in anti-viral immunity has been unclear. Here the authors show that bacterial-induced or intranasally administered IFNγ effectively restricts SARS-CoV-2 infection in mice through effects on non-hematopoietic cells. [ABSTRACT FROM AUTHOR]

Even in the face of the COVID-19 pandemic, Tuberculosis (TB) continues to be a major public health problem and the 2nd biggest infectious cause of death worldwide. There is, therefore, an urgent need to develop effective TB diagnostic methods, which are cheap, portable, sensitive and specific. Raman spectroscopy is a potential spectroscopic technique for this purpose, however, so far, research efforts have focused primarily on the characterisation of Mycobacterium tuberculosis and other Mycobacteria, neglecting bacteria within the microbiome and thus, failing to consider the bigger picture. It is paramount to characterise relevant Mycobacteriales and develop suitable analytical tools to discriminate them from each other. Herein, through the combined use of Raman spectroscopy and the self-optimising Kohonen index network and further multivariate tools, we have successfully undertaken the spectral analysis of Mycobacterium bovis BCG, Corynebacterium glutamicum and Rhodoccocus erythropolis. This has led to development of a useful tool set, which can readily discern spectral differences between these three closely related bacteria as well as generate a unique spectral barcode for each species. Further optimisation and refinement of the developed method will enable its application to other bacteria inhabiting the microbiome and ultimately lead to advanced diagnostic technologies, which can save many lives. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is an intracellular bacterium that causes a highly contagious and potentially lethal tuberculosis (TB) in humans. It can maintain a dormant TB infection within the host. DosR (dormancy survival regulator) (Rv3133c) has been recognized as one of the key transcriptional proteins regulating bacterial dormancy and participating in various metabolic processes. In this study, we extensively investigate the still not well-comprehended role and mechanism of DosR in Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) through a combined omics analysis. Our study finds that deleting DosR significantly affects the transcriptional levels of 104 genes and 179 proteins. Targeted metabolomics data for amino acids indicate that DosR knockout significantly upregulates L-Aspartic acid and serine synthesis, while downregulating seven other amino acids, including L-histidine and lysine. This suggests that DosR regulates amino acid synthesis and metabolism. Taken together, these findings provide molecular and metabolic bases for DosR effects, suggesting that DosR may be a novel regulatory target. [ABSTRACT FROM AUTHOR]

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique. [ABSTRACT FROM AUTHOR]

New efficient mycobacterial inhibitors based on 5-substituted 2-thiouridine derivatives have been described. A series of new 5-alkynyl-substituted 2-thiouridines have been synthesized in good yields by the palladium-catalyzed Sonogashira cross-coupling of 5-iodo-2-thiopyrimidine base with terminal alkynes in DMF at room temperature. The presence of a sulfur atm at C2 of the pyrimidine ring has been shown not to affect the yield of the target compounds. The synthesized 2-thiouridine derivatives were evaluated for their antimyco-bacterial activity against Mycobacterium bovis and Mycobacterium tuberculosis at concentrations of 0.1 to 100 μg/mL using microplate Alamar Blue assay (MABA). The compounds showed high antimycobacterial activity against both tested strains. The MIC50 values for 2-thionucleosides 14–16 (0.28–0.75 μg/mL) were much superior to those of the reference drugs rifampicin, D-cycloserine, and isoniazid, which makes these compounds promising for further more detailed study. [ABSTRACT FROM AUTHOR]

(1) Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with limited treatment options and poor prognosis. Bacillus Calmette–Guérin (BCG), a live attenuated strain of Mycobacterium bovis, has been used as an immunotherapeutic agent in bladder cancer and has shown non-specific beneficial effects. This report presents a unique case of GBM regression following BCG therapy for bladder cancer, suggesting the potential systemic immunomodulatory effects of BCG on GBM. (2) Case Presentation: A 67-year-old male with a history of bladder cancer treated with BCG presented with neurological symptoms. Imaging revealed two GBM lesions, and surgery was performed to remove one. Subsequently, the patient experienced complete tumor regression after initial stability. (3) Conclusions: This case highlights the potential of BCG or other immunotherapies in GBM treatment and underscores the need for further research. Understanding the immunomodulatory effects of BCG on GBM could lead to innovative therapies for this devastating disease; although, overcoming the immune evasion mechanisms in the brain is a significant challenge. Further investigation is warranted to explore this promising avenue of research. [ABSTRACT FROM AUTHOR]

Purpose: The correlation between the incidence of type 1 diabetes mellitus (T1DM) and tuberculosis or bacillus Calmette-Guérin (BCG) vaccination rate in individuals aged <15 years was investigated using worldwide data. Methods: The incidence of T1DM, rate of BCG vaccination, and incidence of tuberculosis were obtained from the Diabetes Atlas 9th edition of the International Diabetes Federation and the Global Health Observatory data repository of the World Health Organization. Gross domestic product (GDP) per capita and population data by country were obtained from the World Bank and United Nations, respectively. Results: GDP per capita negatively correlated with the incidence of tuberculosis and positively correlated with the incidence of T1DM (coefficient=-0.630 and 0.596, respectively; all P<0.001). The incidence of T1DM and tuberculosis was significantly associated with the Organisation for Economic Cooperation and Development (OECD) status (P<0.001). After adjusting for GDP per capita, regional grouping, and OECD status, the incidence of T1DM negatively correlated with that of tuberculosis (R²=0.729, P=0.009). However, there was no association between the BCG vaccination rate and incidence of T1DM (P=0.890). Conclusion: There was a negative correlation between the incidence of tuberculosis and T1DM in children and adolescents aged <15 years at the country level. [ABSTRACT FROM AUTHOR]

Objectives: To examine the utility and efficacy of a multifaceted protocol for the administration of intravesical bacillus Calmette–Guerin (BCG) for non‐muscle‐invasive bladder cancer (NMIBC). Subjects and methods: A multicenter retrospective review was conducted among 83 patients undergoing Fremantle protocol intravesical BCG for NMIBC within 4 major hospitals in Western Australia between January 2016 and December 2018. The Fremantle protocol consists of weekly BCG instillations for 6 weeks during the induction phase, followed by monthly BCG instillations for 10 months during the maintenance phase with integrated clearance‐to‐proceed algorithms for urine MSU checks, flexible cystoscopies performed at 3 monthly intervals during maintenance BCG, and repeat GA cystoscopies with four quadrant bladder biopsies routinely obtained following the completion of induction and maintenance treatment. Results: For patients undergoing Fremantle protocol BCG, 98.8% (82/83) and 75.9% (63/83) of patients completed their induction and maintenance courses of BCG, respectively. Induction BCG was delivered over a median duration of 35 days (range 34–84 days), and maintenance BCG was delivered over a median duration of 266 days (range 1–682 days). The tumor recurrence rate was 10.8% (9/83) at the time of post‐induction biopsies, 2.4% (2/83) during maintenance treatment, 0% (0/60) at the time of post‐maintenance biopsies, and 8.8% (5/57) after a median further follow‐up of 16 months (range 0–51 months). Conclusion: The Fremantle protocol appears to be a safe and effective BCG regimen with several advantages over other BCG protocols, including high completion rates, low recurrence rates, and being highly pragmatic. [ABSTRACT FROM AUTHOR]

The bacillus Calmette–Guérin (BCG) is an attenuated bacterium derived from virulent Mycobacterium bovis. It is the only licensed vaccine used for preventing severe forms of tuberculosis in children. Besides its specific effects against tuberculosis, BCG administration is also associated with beneficial non-specific effects (NSEs) following heterologous stimuli in humans and mice. The NSEs from BCG could be related to both adaptive and innate immune responses. The latter is also known as trained immunity (TI), a recently described biological feature of innate cells that enables functional improvement based on metabolic and epigenetic reprogramming. Currently, the mechanisms related to BCG-mediated TI are the focus of intense research, but many gaps are still in need of elucidation. This review discusses the present understanding of TI induced by BCG, exploring signaling pathways that are crucial to a trained phenotype in hematopoietic stem cells and monocytes/macrophages lineage. It focuses on BCG-mediated TI mechanisms, including the metabolic-epigenetic axis and the inflammasome pathway in these cells against intracellular pathogens. Moreover, this study explores the TI in different immune cell types, its ability to protect against various intracellular infections, and the integration of trained innate memory with adaptive memory to shape next-generation vaccines. [ABSTRACT FROM AUTHOR]

According to the World Health Organization (WHO), around 1 million children worldwide are diagnosed with tuberculosis each year. The Bacillus Calmette–Guérin (BCG) vaccine has been used around the world for over 100 years. The complications of the BCG vaccination can occur in about 0,06% of children and include local or systemic adverse reactions. Due to the close analogy between the vaccine strain and other species of the Mycobacterium tuberculosis complex (MTBC), molecular methods are recommended for differential diagnosis of Vaccine adverse events (VAE) after BCG. The ability to quickly and specifically identify BCG is important in view of different treatment regimens. The aim of the study was to assess the usefulness of genetic testing for Mycobacterium bovis BCG in the paraffin-embedded specimens' methods. We describe two cases of VAE in immune-compromised children presenting with osteoarticular changes that had been clinically suspected of tuberculosis and led to molecular identification through GeneXpert, GenoType MTBC, and Spoligotyping. Results: Mycobacterium bovis BCG was detected in osteoarticular changes embedded in paraffin block of two patients. Conclusion: Genetic tests using paraffin-embedded materials allow for quick identification and differential diagnosis of patients with Tuberculosis and VAE after BCG. This is an important issue, especially in cases where the tissue has only been submitted for histopathological examination without microbiological diagnostics for tuberculosis. [ABSTRACT FROM AUTHOR]

Introduction: Mycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs. Materials and methods: In this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification. Results: The majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA. Discussion: Identification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa. [ABSTRACT FROM AUTHOR]

Background: Control of Tuberculosis (TB) infection is mainly the result of productive teamwork between T-cell populations and antigen presenting cells (APCs). However, APCs activation at the site of initiating cellular immune response during BCG early infection is not completely understood. Methods: In this study, we injected C57BL/6 mice in intravenous (i.v) or subcutaneous (s.c) route, then splenic or inguinal lymph node (LN) DCs and MΦs were sorted, and mycobacteria uptake, cytokine production, antigen presentation activity, and cell phenotype were investigated and compared, respectively. Results: Ag85A-specific T-cell immune response began at 6 days post BCG infection, when BCG was delivered in s.c route, Th17 immune response could be induced in inguinal LN. BCG could induce high level of activation phenotype in inguinal LN MΦs, while the MHC II presentation of mycobacteria-derived peptides by DCs was more efficient than MΦs. Conclusions: The results showed that BCG immunized route can decide the main tissue of T-cell immune response. Compared with s.c injected route, APCs undergo more rapid cell activation in spleen post BCG i.v infection. [ABSTRACT FROM AUTHOR]

Bacillus Calmette-Guérin (BCG) Danish strain 1331 (CattleBCG) is currently the lead vaccine candidate for the control of bovine tuberculosis (TB) in cattle in GB, where prior vaccination has shown to result in a significant reduction in bovine TB pathology induced by infection with Mycobacterium bovis (M. bovis). A critical knowledge gap in our understanding of CattleBCG is the duration of immunity post vaccination at the minimum intended vaccine dose. To this end, we performed an experiment where calves were vaccinated with a targeted dose of 106 CFU and, after a period of 52 weeks, experimentally infected with M. bovis. Post mortem examination performed 13 weeks after infection revealed a statistically significant reduction in the severity of TB pathology in the CattleBCG vaccinated group compared with the unvaccinated control group. Additionally, this study allowed us to further assess the diagnostic performance of a defined antigen DIVA reagent (DST-F) developed to detect infected amongst vaccinated animals. Our results demonstrate that when used in a skin test format, DST-F showed high specificity (100 %) in BCG-vaccinated animals when tested prior to infection, whilst detecting all infected animals when re-tested after infection. Furthermore, we also present results supporting the use of the DST-F reagent in an interferon-gamma release assay. In conclusion, the results of this study demonstrate a 52-week duration of immunity following administration of a minimum dose of CattleBCG. This evidence will be a fundamental component in our efforts to apply for UK marketing authorisation to enable vaccination of cattle as a significant additional control measure in the ongoing fight against bovine TB in GB. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis onco-BCG bacilli used in immunotherapy of bladder cancer are candidates for training of immune cells towards microbial pathogens. Increasing antibiotic resistance of gastric pathogen Helicobacter pylori (Hp) prompts the search for new anti-Hp and immunomodulatory formulations. Colonization of gastric mucosa by Hp through mucin 5 AC (MUC5AC) ligands could potentially be a therapeutic target. The aim of this study was to examine the ability of onco-BCG mycobacteria to reduce Hp adhesion to gastric epithelial cells using Cavia porcellus model. Animals were inoculated per os with 0.85% NaCl, Hp alone, onco-BCG alone or with onco-BCG and Hp. After 7/28 days Mucin5AC and Hp binding to gastric epithelium were assessed in gastric tissue specimens by staining with anti-Mucin5AC and anti-Hp antibodies, respectively, both fluorescently labeled. Primary gastric epithelial cells were treated ex vivo with live Hp or Hp surface antigens (glycine extract or lipopolysaccharide) alone or with onco-BCG. In such cells MUC5AC and Hp binding were determined as above. Mycobacteria reduced the amount of MUC5AC animals infected with Hp and in gastric epithelial cells pulsed in vitro with Hp components. Decrease of MUC5AC driven in cell cultures in vitro and in gastric tissue exposed ex vivo to mycobacteria was related to diminished adhesion of H. pylori bacilli. Vaccine mycobacteria by diminishing the amount of MUC5AC in gastric epithelial cells may reduce Hp adhesion. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), attributed to the Mycobacterium tuberculosis complex, is one of the most serious zoonotic diseases worldwide. Nevertheless, the host mechanisms preferentially leveraged by Mycobacterium remain unclear. After infection, both Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (MB) bacteria exhibit intimate interactions with host alveolar macrophages; however, the specific mechanisms underlying these macrophage responses remain ambiguous. In our study, we performed a comparative proteomic analysis of bovine alveolar macrophages (BAMs) infected with MTB or MB to elucidate the differential responses of BAMs to each pathogen at the protein level. Our findings revealed heightened TB infection susceptibility of BAMs that had been previously infected with MTB or MB. Moreover, we observed that both types of mycobacteria triggered significant changes in BAM energy metabolism. A variety of proteins and signalling pathways associated with autophagy and inflammation-related progression were highly activated in BAMs following MB infection. Additionally, proteins linked to energy metabolism were highly expressed in BAMs following MTB infection. In summary, we propose that BAMs may resist MTB and MB infections via different mechanisms. Our findings provide critical insights into TB pathogenesis, unveiling potential biomarkers to facilitate more effective TB treatment strategies. Additionally, our data lend support to the hypothesis that MTB may be transmitted via crossspecies infection. [ABSTRACT FROM AUTHOR]

Mycophage endolysins are highly diverse and modular enzymes composed of domains involved in peptidoglycan binding and degradation. Mostly, they are characterized by a three-module design: an N-terminal peptidase domain, a central catalytic domain and a C-terminal peptidoglycan binding domain. Previously, the affinity of cell wall binding domains (CBDs) to the mycobacterial peptidoglycan layer was shown for some of these endolysins. In this study, an in depth screening was performed on twelve mycophage endolysins. The discovered CBDs were characterized for their binding affinity to Mycobacterium (M.) bovis bacille Calmette-Guérin (BCG), a largely unexplored target and an attenuated strain of M. bovis , responsible for bovine tuberculosis. Using homology-based annotation, only four endolysins showed the presence of a known peptidoglycan binding domain, the previously characterized pfam 01471 domain. However, analysis of the secondary structure aided by AlphaFold predictions revealed the presence of a C-terminal domain in the other endolysins. These were hypothesized as new, uncharacterized CBDs. Fusion proteins composed of these domains linked to GFP were constructed and positively assayed for their affinity to M. bovis BCG in a peptidoglycan binding assay. Moreover, two CBDs were able to fluorescently label M. bovis BCG in milk samples, highlighting the potential to further explore their possibility to function as CBD-based diagnostics. • Previously characterized pfam 01471 domain was annotated in mycophage endolysins. • Mycophage endolysins contain new, uncharacterized cell-wall binding domains. • Cell-wall binding domains fused to GFP fluorescently labelled M. bovis BCG in milk. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by SARS-CoV-2 virus, has been one of the top public health threats across the world over the past three years. Mycobacterium bovis BCG is currently the only licensed vaccine for tuberculosis, one of the deadliest infectious diseases in the world, that is caused by Mycobacterium tuberculosis. In the past decades, recombinant M.bovis BCG has been studied as a novel vaccine vector for other infectious diseases in humans besides tuberculosis, such as viral infections. In the current study, we generated a recombinant M. bovis BCG strain AspikeRBD that expresses a fusion protein consisting of M. tb Ag85A protein and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using synthetic biology technique. Our results show that the recombinant M. bovis BCG strain successfully expressed this fusion protein. Interestingly, the recombinant M. bovis BCG strain AspikeRBD significantly induced SARS-CoV-2 spike-specific T cell activation and IgG production in mice when compared to the parental M.bovis BCG strain, and was more potent than the recombinant M.bovis BCG strain expressing SARS-CoV-2 spike RBD alone. As expected, the recombinant M. bovis BCG strain AspikeRBD activated an increased number of M. tb Ag85A-specific IFNγ-releasing T cells and enhanced IgG production in mice when compared to the parental M.bovis BCG strain or the BCG strain expressing SARS-CoV-2 spike RBD alone. Taken together, our results indicate a potential application of the recombinant M. bovis BCG strain AspikeRBD as a novel dual vaccine against SARS-CoV-2 and M. tb in humans. 2,3 [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in gd T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. gd T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific gd T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-g ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. gd T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and gd-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ gd T cell compartment (absolute log2 FC = 1.5 and FDR Padj. = 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, gd T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is a licensed vaccine against tuberculosis. It requires attenuated live bacteria to be effective, possibly because actively secreted proteins play a critical role in inducing anti-tuberculosis immunity. BCG also functions as an effective adjuvant. Moreover, the effects of BCG components as adjuvants are not important as those of attenuated live BCG, which is used in cancer immunotherapy. However, the BCG secreted proteins have not been paid attention in anticancer immunity. To understand mycobacterial secreted proteins' function, we investigate immune responses to BCG culture filtrate proteins (CFP). Here, CFP strongly induce both antigenspecific CD4+ T cells and specific CD8+ T cells, which may be functional cytotoxic T lymphocytes (CTLs). In this study, we clearly demonstrate that CFP acts as an adjuvant for CTL induction against specific co-administered proteins and propose CFP as a new protein adjuvant. The CTL response shows potent anticancer effects in mice. These findings could provide insight into the contribution of mycobacterial secreted proteins in both anticancer and antimycobacterial immunity. [ABSTRACT FROM AUTHOR]

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Healthcare workers (HCWs) may have different response to Bacillus Calmette-Guérin (BCG) vaccination due to previous occupational exposure to Mycobacterium particles. We report subgroup analysis of the BATTLE trial, comparing BCG effects in HCWs vs non-HCWs. This was a secondary analysis of a trial. The BATTLE trial was a double-blind placebo-controlled phase III clinical trial that investigated BCG revaccinating adults who were recently infected with SARS-CoV-2 virus. BCG and placebo recipients were sub-grouped based on regular occupational contact with patients into HCWs (48 BCG and 50 placebo) and non-HCWs (124 BCG and 134 placebo). Weekly COVID-19 symptom progression and injection site reactions were compared between subgroups on weeks one, two, three, and six follow-ups. HCWs were more likely to complain of itching on the injection site early after injection (OR = 2.5, p = 0.049). They developed peeling and crusting on the site of injection faster than non-HCWs (during the second week, p = 0.033 and 0.040, OR = 3.3 and 2.7, respectively). HCWs were also more likely to maintain their papule or develop a late onset pustule during later weeks (weeks four and six, p = 0.024 and 0.006, OR = 2.2 and 8.6, respectively). In terms of COVID-19 symptom progression, recovery from anosmia was more likely in the non-HCWs who received BCG (week six, p Holm's corrected = 0.002, OR = 3.3). HCWs' local reaction to BCG injection was slightly more rapid and more intense, possibly due to their occupational exposure. BCG may also ameliorate COVID-19 induced inflammation and anosmia in non-HCWs but not HCWs. Therefore, HCWs might be less likely to benefit from BCG vaccination. ClinicalTrials.gov register number NCT04369794. [ABSTRACT FROM AUTHOR]

Background Bovine Tuberculosis is a respiratory disease caused by the pathogen Mycobacterium bovis (M. bovis) that infects cattle. Though rare, this disease can also affect humans, as well as domestic and wild animals, making it a serious concern. Therefore, searching for alternative and new vaccines with high efficiency and safety is the main goal in bovine tuberculosis prophylaxis. New vaccines, known as vector vaccines, have the potential to become safe and effective alternatives to the traditional BCG vaccine. In this study, two major immunodominant proteins of M. bovis Esat-6 and TB10.4 were utilized to create a vector vaccine for bovine tuberculosis. Methods The Esat-6 and TB10.4 genes were amplified by PCR. The amplified and purified PCR products were sequenced by the Sanger method. Assembly and multiple alignments of amplicon nucleotides were carried out in the MEGA 11 software. Result Two genes of the local strain 0078-M. bovis-8/RIBSP were sequenced. The nucleotide sequences were deposited in the GenBank database. Comparative analysis of the nucleotide sequences of the ESAT-6 and TB10.4 genes established 100% identity of the compared strains of Mycobacterium. Conclusion Through the use of phylogenetic analysis, it has been confirmed that the amplified genes are related to the mycobacteria genus. This discovery allows the development of a vector vaccine against bovine tuberculosis utilising these genes. [ABSTRACT FROM AUTHOR]