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This study investigates Bacillus Calmette-Guérin (BCG) as a potential treatment for hepatocellular carcinoma (HCC), a condition often associated with unfavorable treatment outcomes. Exploiting BCGs recognized immune-boosting properties, preclinical trials are conducted using HCC mice, with a single subcutaneous dose of BCG administered post-tumor formation. Results indicate that BCG treatment effectively diminishes tumor burden and extends survival in both male and female HCC mice. Positive influences on hepatic fibrosis and metabolism are observed, leading to a reduction in lipid levels. Spatial analysis underscores BCGs tumor-specific effects, inducing the enrichment of metabolic pathways and inhibiting various cancer-related pathways. Furthermore, BCG promotes immune cell infiltration, including CD4+, CD8+ T cells, and M1 macrophages, in both v-akt murine thymoma viral oncogene homolog 1(AKT)/neutoblastoma RAS viral oncogene homolog (RAS) and β-catenin positive HCC models. Interestingly, blocking T cells, trained immunity, and Interferon-γ (IFN-γ) function reverses BCGs anti-HCC effects. In conclusion, BCG emerges as a promising treatment option for HCC, characterized by a favorable safety profile and efficacy in inhibiting fibrosis, improving metabolism, and engaging both trained immunity and T cells in therapeutic mechanisms.

Animal tuberculosis (TB) has been reported in several wildlife species in the Greater Kruger Conservation Area (GKCA), South Africa. This report describes the discovery of clinical tuberculosis, caused by Mycobacterium bovis (M. bovis), in free-ranging vervet monkeys (Chlorocebus pygerythrus). The "One Health" concept is especially relevant to TB since this is a multi-host disease with zoonotic potential and is endemic in GKCA. Vervet monkeys have become habituated to humans in tourist areas and may be a source of infection through close contact. Indirect transmission of M. bovis through environmental sources has also been suspected to present a risk of spread between host species. Clinically diseased monkeys present in two tourist areas in the GKCA, that died (n = 1) or were euthanized (n = 5), were submitted for diagnostic necropsies. The presence of pathological lesions, Ziehl-Neelsen-stained impression smears, Xpert® MTB/RIF Ultra (GXU) assay, mycobacterial culture and speciation by genomic regions of difference PCR, were used to confirm the diagnosis of M. bovis infection in these monkeys. The finding of multiple cases necessitates further investigation of TB in monkey troops living within the GKCA tourist areas to determine the source of infection and assess the risk of transmission to other animals and humans. [ABSTRACT FROM AUTHOR]

Background: The growing use of real-time PCR (qPCR) as a diagnostic method for bovine TB (bTB) requires rapid and effective DNA extraction methods, which are crucial for its success. Automated DNA extraction methods based on magnetic beads are a promising alternative to conventional silica column-based protocols (COL protocol) due to their high throughput capacity and reduced hands-on time. This study aimed to assess the performance of the MagMax CORE Nucleic Acid Purification kit and the KingFisher Flex instrument (KF protocol) as an alternative for scaling up the use of qPCR in bTB diagnosis. Methodology: Performance was evaluated with two different real-time PCR (qPCR) protocols, based on the IS6110 element and the QuantiFast and VetMAX™ (QF and VM protocols) kits, on 145 frozen tissue homogenates confirmed as either bTB-positive or negative through a composite reference standard based on microbiological culture, column-based extraction, and qPCR, as well as on negative tissue samples spiked with 106 to 103 CFU/ml of M. bovis BCG. Results: The performance of both qPCR protocols was very high on samples extracted using the KF protocol, with positive percent agreement (PPA) values of 89.04% [95% Confidence Interval (CI): 79.54–95.15%] and 93.15% [95% CI: 84.74–97.74%] for the QF and VM protocols, respectively, and negative percent agreement (NPA) values of 100% [95% CI: 95.01–100.00%]. A higher variability was identified in samples analysed with the same qPCR protocol but different extraction methods. Higher Ct values were identified for samples extracted using the KF protocol in both routine and spiked samples, likely due to using the same amount of starting material for both extraction methods, which was lower than recommended by the manufacturer for the KF protocol. Discussion: The results of this study indicate that the MagMAX CORE Nucleic Acid Purification kit coupled with a KingFisher Flex instrument is a valuable alternative for the extraction of MTBC DNA from bovine tissues. However, the increased variability and Ct values suggest that a larger amount of starting material is recommended for this methodology, warranting further studies. [ABSTRACT FROM AUTHOR]

Rationale: Mycobacterial species contain high concentrations of mycolic acids in their cell wall. Mycobacteria can pose a threat to both human health and the environment. Mass spectrometry lipidomic characterization can identify bacterial species and suggest targets for microbiological interventions. Due to the complex structures of mycolic acids and the possibility of isobaric isomers, multiple levels of separation are required for complete characterization. In this study, cyclic ion mobility (cIM) mass spectrometry (MS) was used for the analysis, separation and fragmentation of mycolic acids isomers from the bacterial species Gordonia amarae and Mycobacterium bovis. Methods: Mycolic acid isomers were interrogated from cultured G. amarae biomass and commercially available M. bovis mycolic acid extracts. These were infused into a cIM‐enabled quadrupole time‐of‐flight MS. Ions of interest were non‐simultaneously selected with the quadrupole and passed around the cyclic ion mobility device multiple times. Fragment ion analysis was then performed for the resolved isomers of the quadrupole‐selected ions. Results: Repeated passes of the cIM device successfully resolved otherwise overlapping MA isomers, allowing isomer isolation and producing an ion‐specific post‐mobility fragmentation spectrum without isomeric interference. Conclusions: Mycolic acids (MA) isomers from G. amarae and M. bovis were resolved, resulting in a high mobility resolution and low interference fragmentation analysis. These revealed varying patterns of MA isomers in the two species: G. amarae's most abundant ion of each set of MA has 1–2 conformations, while the MA + 2 m/z the most abundant ion of each set has 3–6 conformations. These were resolved after 70 passes of the cyclic device. M. bovis' most abundant ion of each keto‐MA set has 2 conformations, while the keto‐MA + 2 m/z has 1–2 conformations. These were resolved after 75 passes. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis (M. bovis) causes bovine tuberculosis (bTB). The challenges in controlling and eradicating this zoonotic disease are compounded by our incomplete understanding of the host immune response. In this study, we used high-throughput bulk RNA sequencing (RNA-seq) to characterise the response profiles of γδ T cells to antigenic stimulation using purified protein derivate from M. bovis (PPDb). γδ T cells are a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. These cells are usually classified based on the expression of a pathogen-recognition receptor, Workshop Cluster 1 (WC1), into two main subsets: WC1.1+ and WC1.2+. Previous studies have identified a preferential transcriptomic response in WC1.1+ cells during natural bTB infection, suggesting a subset-specific response to mycobacterial antigens. This follow on study tested the hypothesis that a subset specific response would also be apparent from γδ T cells from infected cattle after repeat stimulation. Peripheral blood was collected from Holstein-Friesian cattle naturally infected with M. bovis, confirmed by a single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA and stimulated with 10 μg/ml PPDb for 6 hours. After whole blood stimulation, WC1.1+ and WC1.2+ γδ T cell subsets were isolated using magnetic cell sorting (n = 5 per group). High-quality RNA was extracted from each purified lymphocyte subset (WC1.1+ and WC1.2+) to generate transcriptomes using bulk RNA sequencing, resulting in 20 RNA-seq libraries. Transcriptomic analysis revealed 111 differentially expressed genes (DEGs) common to both WC1.1+ and WC1.2+ γδ T cell compartments, including upregulation of IL1A, IL1B, IL6, IL17A, IL17F, and IFNG genes (FDR-Padj. < 0.1). Interestingly, the WC1.2+ cells showed upregulation of IL10, CCL22, and GZMA (log2FC ≥ 1.5, and FDR-Padj. < 0.1). In conclusion, while WC1.1+ and WC1.2+ γδ T cells exhibit a conserved inflammatory response to PPDb, differences in anti-inflammatory and antimicrobial gene expression between these cell subsets provide new insights into their effector functions in response to mycobacterial antigens. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis , the causative agent of bovine tuberculosis (bTB), is a zoonotic pathogen that contributes to economic losses in the cattle industry and poses a public health risk worldwide. Bacillus Calmette-Guerin, or BCG, is a live attenuated strain of M. bovis that is used for human vaccination against tuberculosis and is considered a potential vaccine candidate against bTB. However, BCG affords widely variable levels of protection against challenge and interferes with current diagnostic methods, and as such, it is not currently approved for use as a livestock or wildlife vaccine in the United States. Many efforts have been made to develop bTB vaccines that are reliable and do not interfere with diagnostic testing, but BCG continues to be the most effective option. Previous work has shown that a T helper 1 immune response is essential for protection against virulent M. bovis infection, characterized by CD4+ central and effector memory T cells. In an effort to identify an efficacious bTB intervention strategy, the study presented here used an in vitro recall response assay and concurrent evaluation of CD4+ T cell proliferation and cytokine production to characterize the surface and functional phenotypes of memory responses to BCG vaccination in cattle. Our findings enhance understanding of the bovine immune response to BCG and provide insights into the development of improved vaccines for the control of bTB. [ABSTRACT FROM AUTHOR]

The development of vaccines and effective diagnostic methods for bovine tuberculosis requires an understanding of the immune response against its causative agent, Mycobacterium bovis. Although this disease is primarily investigated and diagnosed through the assessment of cell-mediated immunity, the role of B cells and antibodies in bovine tuberculosis has been relatively undervalued and understudied. Current evidence indicates that circulating M. bovis-specific antibodies are not effective in controlling the disease. However, local humoral immune responses may contribute to either defence or pathology. Recent studies in animal models and cattle vaccine trials suggest a potential beneficial role of B cells in tuberculosis control. This review discusses the role of B cells and antibodies in bovine tuberculosis and explores antibody-based diagnostics for the disease, including traditional techniques, such as different ELISA, new platforms based on multiple antigens and point-of-care technologies. The high specificity and sensitivity values achieved by numerous antibody-based tests support their use as complementary tests for the diagnosis of bovine tuberculosis, especially for identifying infected animals that may be missed by the official tests. [ABSTRACT FROM AUTHOR]

This report by the European Food Safety Authority and the European Centre for Disease Prevention and Control presents the results of zoonoses monitoring and surveillance activities carried out in 2023 in 27 Member States (MSs), the United Kingdom (Northern Ireland) and 10 non‐MSs. Key statistics on zoonoses and zoonotic agents in humans, food, animals and feed are provided and interpreted historically. In 2023, the first and second most reported zoonoses in humans were campylobacteriosis and salmonellosis, respectively. For both agents, an increase in the absolute number of cases was observed in comparison with 2022. Fifteen MSs and the United Kingdom (Northern Ireland) reached all the established targets in poultry populations with regard to the reduction in Salmonella prevalence for the relevant serovars. Salmonella samples from carcases of various animal species, and samples for Campylobacter quantification from broiler carcases, were more frequently positive when performed by the competent authorities than when own‐checks were conducted. Shiga toxin‐producing Escherichia coli (STEC) was the third most reported zoonotic agent in humans, followed by Yersinia enterocolitica and Listeria monocytogenes. L. monocytogenes and West Nile virus infections were the most severe zoonotic diseases, with the highest percentage of hospitalisations among cases and the highest case fatality rates. Twenty‐seven MSs and the United Kingdom (Northern Ireland) reported a slight decrease in food‐borne outbreaks in 2023 overall in comparison with 2022, although the overall number of reported human cases and hospitalisations increased. Salmonella Enteritidis remained the most frequently reported causative agent for reported cases and food‐borne outbreaks. Salmonella in 'eggs and egg products' was the agent/food pair of most concern. In 2023 this combination caused the largest number of outbreaks and cases among all agent/food combination and ranked second in number of hospitalisations. Salmonella was also the causative agent associated with the majority of multi‐country outbreaks reported in the EU in 2023. This report also provides updates on brucellosis, echinococcosis, Q fever, rabies, toxoplasmosis, trichinellosis, tuberculosis due to Mycobacterium bovis or M. caprae, and tularaemia. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis), remains the leading cause of death from a single infectious agent globally. Intracellular survival is crucial for their virulence; yet, the underlying mechanisms are not fully understood. This study aimed to demonstrate the significance of a previously unannotated bovine gene ENSBTAG00000011305 in M. bovis intracellular survival. This gene was termed NMRAL2_Bovine due to its inclusion of the NmrA domain which has a relation to nitric oxide (NO) production. We used CRISPR/Cas9 to knock out NMRAL2_Bovine in bovine lung epithelial cells and observed a significant decrease in M. bovis-induced cell death and the intracellular bacterial count, alongside increased NO levels. A transcriptome analysis revealed the upregulation of pathways linked to NO, IL-6, and TNF-α production, which was confirmed by the increased expression of iNOS, IL-6, and TNF-α. Correspondingly, Western blotting indicated that key signaling pathways, including NF-κB and MAPK, were activated. In conclusion, our findings determined that NMRAL2_Bovine functions as a negative regulator of the inflammatory response induced by M. bovis infection at the cellular level and, thereby, provide a novel insight into TB pathogenesis and a potential target for developing novel host-directed therapies against TB. [ABSTRACT FROM AUTHOR]

Mycobacterial infections, caused by various species within the Mycobacterium genus, remain one of the main challenges to global health across the world. Understanding the complex interplay between the host and mycobacterial pathogens is essential for developing effective diagnostic and therapeutic strategies. Host long noncoding RNAs (lncRNAs) have emerged as key regulators in cellular response to bacterial infections within host cells. This review provides an overview of the intricate relationship between mycobacterial infections and host lncRNAs in the context of Mycobacterium tuberculosis and non-tuberculous mycobacterium (NTM) infections. Accumulation of evidence indicates that host lncRNAs play a critical role in regulating cellular response to mycobacterial infection within host cells, such as macrophages, the primary host cells for mycobacterial intracellular survival. The expression of specific host lncRNAs has been implicated in the pathogenesis of mycobacterial infections, providing potential targets for the development of novel host-directed therapies and biomarkers for TB diagnosis. In summary, this review aims to highlight the current state of knowledge regarding the involvement of host lncRNAs in mycobacterial infections. It also emphasizes their potential application as novel diagnostic biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

European badgers (Meles meles) play an important role in the epidemiology of bovine tuberculosis (caused by Mycobacterium bovis) in England, but little is known about the prevalence of atypical mycobacteriosis. Badgers are also known to be infected by other infectious agents, and the relationship between mycobacteriosis and concomitant infections needs further investigation. Overall, 88 badger carcasses from the Midlands of England collected between July 2016-August 2017 were selected for histopathological examination based on the degree of autolysis (mild), mycobacterial culture results and a balanced sex ratio. Mycobacteria were cultured from 44 badgers, of which 31 were mycobacteria belonging to the M. tuberculosis complex (MTBC) (based on IS6110 PCR and Hsp64 and/or rRNA PCR and sequencing) and 13 were non-tuberculous atypical mycobacteria (NTM). Mycobacteria were not cultured from the remaining 44 animals. Histologically, the most common findings were silica-laden macrophages (85%), granulomas (53%), sarcocystosis (47%), nephritis (31%), portal/periportal hepatitis (26%), ulcerative dermatitis (18%). Culturable mycobacteriosis was associated with higher prevalence of granulomas (p < 0.001) and lower prevalence of hepatitis (p = 0.003). NTM (M. nonchromogenicum, M. avium complex, M. hassiacum, M. malmoense, M. vaccae.) infections were associated with granulomatous pneumonia, and M. malmoense was associated with pyogranulomatous and ulcerative dermatitis. In conclusion, this study describes, for the first time, histological lesions associated with NTM in badgers, the histomorphology of which was similar to those caused by MTBC. In addition, the negative relationship between mycobacteriosis and periportal hepatitis may indicate a complex relationship between mycobacteriosis and other diseases, as previously observed with tuberculosis. [ABSTRACT FROM AUTHOR]

The franciscana (Pontoporia blainvillei) is a small cetacean endemic to the southwestern Atlantic Ocean in a vulnerable conservation status. Their habitat is restricted to shallow waters and estuaries. In 2020, fresh female carcass of franciscana (juvenile/subadult) was found dead alone (individual stranding) on a public access beach in the Rio Negro Estuary in "Balneario El Cóndor", Argentinian Patagonia. Grossly, a widespread granulomatous lesion compatible with tuberculosis were observed. A sample of mesenteric lymph nodes with granulomatous lesions (~ 50 g) was collected for bacteriological culture and molecular identification. Bacterial growth was observed in Stonebrink media and Ziehl Neelsen staining revealed acid-fast bacilli. Identification of a Mycobacterium bovis strain with the SB0288 spoligotype was obtained. The spoligotype detected here has already been reported in cattle and humans. Presumably, fluids and feces of infected livestock and wild animals, or their carcasses, may contaminate the water. Thus, this report demonstrates the potential risk of zoonotic tuberculosis transmission through wastewater. Contaminated wastewater is eventually a threat to animal species living in the area, and potentially becomes a zoonotic risk. [ABSTRACT FROM AUTHOR]

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results: The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions: In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment. [ABSTRACT FROM AUTHOR]

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process. [ABSTRACT FROM AUTHOR]

Background: Zoonotic human tuberculosis (TB) caused by Mycobacterium bovis (M. bovis) is as vital as Mycobacterium tuberculosis, however with scarce available information. We aimed to use whole-genome sequencing (WGS) technology to take a deep insight into the circulating genotypes of human M. bovis and the genomic characteristics underlying virulence and drug resistance. Methods: The study included smear positive Ziehl-Neelsen samples from patients with suspected tuberculosis. Samples were cultured on Lowenstein-Jensen media and suspected colonies of M. bovis were selected to undergo DNA extraction and WGS. Data was analysed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), and online bioinformatics tools. A phylogenetic tree was constructed for our sequenced strains, in addition to a set of 59 previously sequenced M. bovis genomes from different hosts and countries. Results: Out of total 112 mycobacterial positive cultures, five M. bovis were isolated and underwent WGS. All sequenced strains belonged to Mycobacterium tuberculosis var bovis, spoligotype BOV_1; BOV_11. Resistance gene mutations were determined in 100% of strains to pyrazinamide (pncA and rpsA), isoniazid (KatG and ahpC), ethambutol (embB, embC, embR and ubiA), streptomycin (rpsl) and fluoroquinolones (gyrA and gyrB). Rifampin (rpoB and rpoC) and delamanid (fbiC) resistance genes were found in 80% of strains. The major represented virulence classes were the secretion system, cell surface components and regulation system. The phylogenetic analysis revealed close genetic relatedness of three sequenced M. bovis strains to previous reported cow strains from Egypt and human strains from France, as well as relatedness of one M. bovis strain to four human Algerian strains. One sequenced strain was related to one cow strain from Egypt and a human strain from South Africa. Conclusions: All sequenced M. bovis isolates showed the same spoligotype, but diverse phylogeny. Resistance gene mutations were detected for anti-TB drugs including pyrazinamide, isoniazid, streptomycin, ethambutol, fluoroquinolones, cycloserine, rifampin and delamanid. The virulence profile comprised genes assigned mainly to secretion system, cell surface components and regulation system. Phylogenetic analysis revealed genetic relatedness between our isolates and previously sequenced bovine strains from Egypt as well as human strains from other nearby countries in the region. [ABSTRACT FROM AUTHOR]

Abstract Background Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. Results The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. Conclusions In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Abstract Background Zoonotic human tuberculosis (TB) caused by Mycobacterium bovis (M. bovis) is as vital as Mycobacterium tuberculosis, however with scarce available information. We aimed to use whole-genome sequencing (WGS) technology to take a deep insight into the circulating genotypes of human M. bovis and the genomic characteristics underlying virulence and drug resistance. Methods The study included smear positive Ziehl-Neelsen samples from patients with suspected tuberculosis. Samples were cultured on Lowenstein-Jensen media and suspected colonies of M. bovis were selected to undergo DNA extraction and WGS. Data was analysed using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), and online bioinformatics tools. A phylogenetic tree was constructed for our sequenced strains, in addition to a set of 59 previously sequenced M. bovis genomes from different hosts and countries. Results Out of total 112 mycobacterial positive cultures, five M. bovis were isolated and underwent WGS. All sequenced strains belonged to Mycobacterium tuberculosis var bovis, spoligotype BOV_1; BOV_11. Resistance gene mutations were determined in 100% of strains to pyrazinamide (pncA and rpsA), isoniazid (KatG and ahpC), ethambutol (embB, embC, embR and ubiA), streptomycin (rpsl) and fluoroquinolones (gyrA and gyrB). Rifampin (rpoB and rpoC) and delamanid (fbiC) resistance genes were found in 80% of strains. The major represented virulence classes were the secretion system, cell surface components and regulation system. The phylogenetic analysis revealed close genetic relatedness of three sequenced M. bovis strains to previous reported cow strains from Egypt and human strains from France, as well as relatedness of one M. bovis strain to four human Algerian strains. One sequenced strain was related to one cow strain from Egypt and a human strain from South Africa. Conclusions All sequenced M. bovis isolates showed the same spoligotype, but diverse phylogeny. Resistance gene mutations were detected for anti-TB drugs including pyrazinamide, isoniazid, streptomycin, ethambutol, fluoroquinolones, cycloserine, rifampin and delamanid. The virulence profile comprised genes assigned mainly to secretion system, cell surface components and regulation system. Phylogenetic analysis revealed genetic relatedness between our isolates and previously sequenced bovine strains from Egypt as well as human strains from other nearby countries in the region.

Abstract Bovine tuberculosis is one of the most important common infectious diseases between humans and livestock. Cow’s milk can be investigated as one of the transmission reservoirs of the disease. Our study was conducted to investigate the presence of Mycobacterium bovis (M. bovis) DNA in cow’s milk from different regions of Lorestan province using Touch-down PCR (TD-PCR) method. So, 100 milk samples from industrial and traditional cattle farms were collected and evaluated according to the animal breed, the average age of the animal and the region where the animal was kept. Seven (26.9%) out of the 26 cow’s milk samples contaminated with Mycobacterium spp., were diagnosed as M. bovis positive. The cattle with an average age of more than 5 years were most infected with Mycobacterium. Also, the crossbred cattle with an average age of 3 to 5 years, which were kept and raised in tropical areas, showed the highest rate of M. bovis infection. Based on our knowledge, this is the first study regarding the presence of Mycobacterium in cow’s milk in Iran.

Abstract In the United Kingdom, the management of bovine tuberculosis (bTB) challenges the coexistence of people and wildlife. Control of this cattle disease is hindered by transmission of its causative agent, Mycobacterium bovis, between cattle and badgers Meles meles. Badger culling has formed an element of bTB control policy for decades, but current government policy envisions expanding badger vaccination. Farming leaders are sceptical, citing concerns that badger vaccination would be impractical and potentially ineffective. We report on a 4‐year badger vaccination initiative in an 11 km2 area which, atypically, was initiated by local farmers, delivered by scientists and conservationists, and co‐funded by all three. Participating landholders cited controversies around culling and a desire to support neighbours as their primary reasons for adopting vaccination. The number of badgers vaccinated per km2 (5.6 km−2 in 2019) exceeded the number culled on nearby land (2.9 km−2 in 2019), and the estimated proportion vaccinated (74%, 95% confidence interval [CI] 40%–137%) exceeded the 30% threshold predicted by models to be necessary to control M. bovis. Farmers were content with how vaccination was delivered, and felt that it built trust with wildlife professionals. The percentage of badgers testing positive for M. bovis declined from 16.0% (95% CI 4.5%–36.1%) at the start of vaccination to 0% (95% CI 0%–9.7%) in the final year. With neither replication nor unvaccinated controls, this small‐scale case study does not demonstrate a causal link between badger vaccination and bTB epidemiology, but it does suggest that larger‐scale evaluation of badger vaccination would be warranted. Farmers reported that their enthusiasm for badger vaccination had increased after participating for 4 years. They considered vaccination to have been effective, and good value for money, and wished to continue with it. Synthesis and applications: Although small‐scale, this case study suggests that badger vaccination can be a technically effective and socially acceptable component of bTB control. A wider rollout of badger vaccination is more likely if it is led by the farming community, rather than by conservationists or government, and is combined with scientific monitoring. Read the free Plain Language Summary for this article on the Journal blog.

Bovine tuberculosis is one of the most important common infectious diseases between humans and livestock. Cow's milk can be investigated as one of the transmission reservoirs of the disease. Our study was conducted to investigate the presence of Mycobacterium bovis (M. bovis) DNA in cow's milk from different regions of Lorestan province using Touch-down PCR (TD-PCR) method. So, 100 milk samples from industrial and traditional cattle farms were collected and evaluated according to the animal breed, the average age of the animal and the region where the animal was kept. Seven (26.9%) out of the 26 cow's milk samples contaminated with Mycobacterium spp., were diagnosed as M. bovis positive. The cattle with an average age of more than 5 years were most infected with Mycobacterium. Also, the crossbred cattle with an average age of 3 to 5 years, which were kept and raised in tropical areas, showed the highest rate of M. bovis infection. Based on our knowledge, this is the first study regarding the presence of Mycobacterium in cow's milk in Iran. [ABSTRACT FROM AUTHOR]

Fungal endophytes, symbiotic microorganisms residing within plants, are renowned for producing bioactive secondary metabolites with diverse beneficial properties. We investigated the antimicrobial potential of fungal endophytes isolated from Solanum mauritianum, an invasive weed, against clinically significant bacterial pathogens. Selected fungal endophytes (Penicillium chrysogenum, Fusarium sp., and Paracamarosporium leucadendri) were isolated from the plant's leaves and fruits. Their crude extracts were tested against various referenced strains, such as Mycobacterium species (M. smegmatis ATCC 607 and M. bovis ATCC 27290), Staphylococcus aureus ATCC 6571, Bacillus subtilis ATCC 11774, Klebsiella species (K. pneumoniae ATCC 10031 and K. oxytoca ATCC 8724), Escherichia coli ATCC 10536, and Pseudomonas aeruginosa ATCC 10145, using the Kirby-Bauer disk diffusion method. Resazurin Microtiter Assay was used for the determination of the minimum inhibitory concentration. The chemical nature of the secondary metabolites in the crude extracts produced by fungal endophytes was evaluated using high-resolution liquid chromatography–mass spectrometry (LC-MS) using water and acetonitrile gradient. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS) was employed for untargeted metabolomics. LC-QTOF-MS/MS identified 63 bioactive compounds across the three endophytes. P. chrysogenum had the highest activity against S. aureus and M. smegmatis (1.15 mg/mL and 0.02 mg/mL, respectively), while P. leucadendri demonstrated moderate activity against M. smegmatis (2.91 mg/mL) and E. coli (1.16 mg/mL). Fusarium sp. exhibited the broadest spectrum of antibacterial activity, with MIC values ranging from 0.03 mg/mL (B. subtilis) to 10 mg/mL (M. smegmatis). P. leucadendri produced 29 metabolites, Fusarium sp. had 23 identified metabolites, and a total of 11 metabolites were identified from P. chrysogenum. The fruits of the plant, accounting for 60%, appeared to be the most abundant in the endophyte diversity when compared to the stems and leaves. This study highlights the potential of fungal endophytes from S. mauritianum as a source of novel bioactive compounds, particularly against multidrug-resistant pathogens, contributing to the ongoing efforts to combat antimicrobial resistance. [ABSTRACT FROM AUTHOR]

ABSTRACT Mycobacterium bovis and Mycobacterium tuberculosis are the most relevant among pathogenic mycobacteria, both belonging to the M. tuberculosis complex (MTC). Samples of blood, liver, spleen, kidneys, lungs and caseous tubercles were collected from a free‐ranging juvenile black capuchin monkey (Sapajus nigritus) showing non‐specific signs of illness. Macroscopic findings included emaciation, a caseous lesion in a tooth and gingiva, disseminated nodules in both lungs and left kidney parenchyma and caseous nodules on the pleura and mesentery. The lesions suggested MTC infection, a diagnosis subsequently supported in the lung by bacilloscopy, immunochromatography and PCR. A multiplex PCR further validated the presence of M. bovis genes. Cases of tuberculosis in platyrrhine primates have only been reported in animals maintained in captivity. We describe for the first time the pathological and molecular findings of M. bovis infection in a free‐ranging platyrrhine monkey within an area of intense human–wildlife interaction, which has important implications from a One Health perspective. [ABSTRACT FROM AUTHOR]

Epidemiologic research on zoonotic tuberculosis historically used Mycobacterium bovis as a surrogate measure; however, increased reports of human tuberculosis caused by other animal-associated Mycobacterium tuberculosis complex members like Mycobacterium orygis necessitates their inclusion. We performed a retrospective cohort study including persons infected with any animal-lineage M tuberculosis complex species in Alberta, Canada, from January 1995 to July 2021, identifying 42 patients (20  M bovis , 21  M orygis , 1 M caprae). Demographic, epidemiologic, and clinical characteristics were compared against persons with culture-confirmed M tuberculosis infection. The proportion of culture-positive infections caused by M orygis increased continuously from 2016 to 2020. Significantly more females at a higher median age were impacted by M orygis , with all patients originating from South Asia. Mycobacterium bovis caused significantly more extrapulmonary disease and disproportionately impacted young females, particularly those pregnant or postpartum. All infections were acquired abroad. These findings can aid in developing targeted public health interventions. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are involved in the breakdown of lung extracellular matrix and the consequent release of Mycobacterium tuberculosis into the airways. Recent studies indicate that kallikrein-related peptidase 12 (KLK12) regulate MMP-1 and MMP-9, suggesting that targeting the KLK12 gene could be a promising tuberculosis (TB) treatment. To maximise therapeutic potential, this strategy of silencing KLK12 needs to be delivered to the pathogenic cell population while preserving the immunoprotective and tissue homeostatic functions of other lung macrophages. Our research found that KLK12 is highly expressed in M2 macrophages, leading us to design mannose-based bovine serum albumin nanoparticles (MBNPs) for delivering siRNA to silence KLK12 in these cells. The results of in vitro experiments showed that MBNPs could accurately enter M2 macrophages and sustainably release KLK12-siRNA with the help of mannose and mannose receptor targeting. The results of the in vivo experiments showed that MBNPs could reach the lungs within 1 h after intraperitoneal injection and peaked at 6 h. MBNPs increased collagen fibre content in the lungs by decreasing the levels of KLK12/MMPs thereby limiting the progression of TB. Importantly, MBNPs provided greater alleviation of pulmonary TB symptoms and reduced bacterial load in both TB and drug-resistant TB models. These findings provide an alternative and effective option for the treatment of TB, especially when drug resistance occurs. RNA interference using small interfering RNA (siRNA) can target various genes and has potential for treating diseases such as tuberculosis (TB). However, siRNAs are unstable in the blood and within cells. This study presents bovine serum albumin nanoparticles encapsulating KLK12-siRNA (BNPs) synthesized via desolvation. A mannose layer was added (MBNPs) to target mannose receptors on M2 macrophages, facilitating endocytosis. The low pH-responsive MBNPs enhance lysosomal escape for siRNA delivery, downregulating the KLK12 pathway. Tests confirmed that MBNPs effectively inhibited Mycobacterium bovis proliferation, reduced granulomas, and decreased inflammation in a mouse model. This research aims to reduce antibiotic use, shorten treatment duration, and provide a novel TB treatment option. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. Results: Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. Conclusions: Human lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level. [ABSTRACT FROM AUTHOR]

In recent years, there has been an increase in the number of reported cases of Mycobacterium microti infection in various animals, which can interfere with the ante-mortem diagnosis of animal tuberculosis caused by Mycobacterium bovis. In this study, whole genome sequencing (WGS) was used to search for protein-coding genes to distinguish M. microti from M. bovis. In addition, the population structure of the available M. microti genomic WGS datasets is described, including three novel Belgian isolates from infections in alpacas. Candidate genes were identified by examining the presence of the regions of difference and by a pan-genome analysis of the available WGS data. A total of 80 genes showed presence-absence variation between the two species, including genes encoding Proline-Glutamate (PE), Proline-Proline-Glutamate (PPE), and Polymorphic GC-Rich Sequence (PE-PGRS) proteins involved in virulence and host interaction. Filtering based on predicted subcellular localization, sequence homology and predicted antigenicity resulted in 28 proteins out of 80 that were predicted to be potential antigens. As synthetic peptides are less costly and variable than recombinant proteins, an in silico approach was performed to identify linear and discontinuous B-cell epitopes in the selected proteins. From the 28 proteins, 157 B-cell epitope-based peptides were identified that discriminated between M. bovis and M. microti species. Although confirmation by in vitro testing is still required, these candidate synthetic peptides containing B-cell epitopes could potentially be used in serological tests to differentiate cases of M. bovis from M. microti infection, thus reducing misdiagnosis in animal tuberculosis surveillance. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (BTB) still represents a significant public health concern and economic issue for livestock breeders in Egypt. This research investigates the incidence of bovine tuberculosis in cattle slaughtered at the central abattoir in Aswan, Egypt, in 2023. A total of 720 cattle were checked antemortem and postmortem at the abattoir. The suspected lesions were analyzed with acid-fast staining, microscopy, histology, ELISA, and RT-PCR. Based on gross tubercle lesions, the overall occurrence of bovine tuberculosis in cattle slaughtered at Aswan abattoirs was 3.2% (23/720), with thoracic lymph nodes and lungs exhibiting the highest frequency of tubercle lesions compared to other organs. According to employed diagnostic tests, the prevalence of tuberculosis was estimated as 68.6, 61, 56.5, and 78.3% for microscopy, histopathology, ELISA, and RT-PCR, respectively. These findings highlight the incidence of bovine tuberculosis and the dissemination of tuberculous lesions among cattle slaughtered at Aswan abattoirs. Furthermore, such prevalence emphasizes the urgent need for a practical disease control approach and an organized surveillance system in the cattle population. One Health strategy is strongly advised to mitigate the zoonotic transmission to people and economic losses in the cattle industry. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

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In the United Kingdom, the management of bovine tuberculosis (bTB) challenges the coexistence of people and wildlife. Control of this cattle disease is hindered by transmission of its causative agent, Mycobacterium bovis, between cattle and badgers Meles meles.Badger culling has formed an element of bTB control policy for decades, but current government policy envisions expanding badger vaccination. Farming leaders are sceptical, citing concerns that badger vaccination would be impractical and potentially ineffective.We report on a 4‐year badger vaccination initiative in an 11 km2 area which, atypically, was initiated by local farmers, delivered by scientists and conservationists, and co‐funded by all three. Participating landholders cited controversies around culling and a desire to support neighbours as their primary reasons for adopting vaccination.The number of badgers vaccinated per km2 (5.6 km−2 in 2019) exceeded the number culled on nearby land (2.9 km−2 in 2019), and the estimated proportion vaccinated (74%, 95% confidence interval [CI] 40%–137%) exceeded the 30% threshold predicted by models to be necessary to control M. bovis. Farmers were content with how vaccination was delivered, and felt that it built trust with wildlife professionals.The percentage of badgers testing positive for M. bovis declined from 16.0% (95% CI 4.5%–36.1%) at the start of vaccination to 0% (95% CI 0%–9.7%) in the final year. With neither replication nor unvaccinated controls, this small‐scale case study does not demonstrate a causal link between badger vaccination and bTB epidemiology, but it does suggest that larger‐scale evaluation of badger vaccination would be warranted.Farmers reported that their enthusiasm for badger vaccination had increased after participating for 4 years. They considered vaccination to have been effective, and good value for money, and wished to continue with it.Synthesis and applications: Although small‐scale, this case study suggests that badger vaccination can be a technically effective and socially acceptable component of bTB control. A wider rollout of badger vaccination is more likely if it is led by the farming community, rather than by conservationists or government, and is combined with scientific monitoring. Read the free Plain Language Summary for this article on the Journal blog. Read the free Plain Language Summary for this article on the Journal blog [ABSTRACT FROM AUTHOR]

Cutaneous tuberculosis is an infection caused by Mycobacteria tuberculosis, the rare Mycobacterium bovis and the bacillus Calmette-Guérin vaccine. This disease has many clinical types with diverse clinical manifestations, mainly includes lupus vulgaris, tuberculosis verrucosa cutis, orificial tuberculosis and scrofuloderma that are difficult to identify. We report a case of cutaneous tuberculosis in a female who presented with disseminated papular and nodular lesions on her face and hands. The results of skin biopsy, PCR, and IGRA test contributed to the diagnosis. All lesions were resolved leaving only superficial scars after 5 months treatment. [ABSTRACT FROM AUTHOR]

The diagnosis of drug‐resistant tuberculosis (TB) by molecular testing of Mycobacterium tuberculosis drug resistance genes is becoming increasingly common clinically. However, M. bovis, as an uncommon pathogen of human TB, may interfere with the test results. A comprehensive understanding of phylogenetically informative mutations in the drug resistance genes of M. bovis is required to distinguish true resistance‐conferring mutations. We analyzed 53 drug resistance genes in 165 M. bovis isolated from humans using whole‐genome sequencing data and found that 98.2% (162/165) of isolates have pyrazinamide intrinsic genotypic resistance, owing to the H57D mutation in the pncA gene. 12.1% (20/165) of M. bovis isolates were resistant to drugs other than pyrazinamide. Furthermore, we discovered 18 phylogenetically informative mutations that differed between M. bovis and the major lineages 1–4 of M. tuberculosis. Additionally, we reported false‐positive ethambutol resistance caused by M. bovis infection due to the phylogenetically informative mutation embB E378A. This study is crucial for gaining insights into the genetic characterization and drug resistance of M. bovis prevalent in humans, as well as contributing to the development of more accurate molecular diagnostic methods and detection tools for drug resistance. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis variant bovis (M. bovis) is a highly pathogenic environmental microorganism that causes bovine tuberculosis (bTB), a significant zoonotic disease. Currently, "test and culling" is the primary measure for controlling bTB, but it has been proven to be inadequate in animals due to their high susceptibility to the pathogen. Selective breeding for increased host resistance to bTB to reduce its prevalence is feasible. In this study, we found a vital host-dependent factor, RBMX2, that can potentially promote M. bovis infection. By knocking RBMX2 out, we investigated its function during M. bovis infection. Through transcriptome sequencing and alternative splicing transcriptome sequencing, we concluded that after M. bovis infection, embryo bovine lung (EBL) cells were significantly enriched in RNA splicing associated with apoptosis compared with wild-type EBL cells. Through protein/molecular docking, molecular dynamics simulations, and real-time quantitative PCR, we demonstrated that RBMX2 promotes the apoptosis of epithelial cells by upregulating and binding to apoptotic peptidase activating factor 1 (APAF-1), resulting in the alternative splicing of APAF-1 as a retention intron. To our knowledge, this is the first report of M. bovis affecting host epithelial cell apoptosis by hijacking RBMX2 to promote the intron splicing of downstream APAF-1. These findings may represent a significant contribution to the development of novel TB prevention and control strategies. [ABSTRACT FROM AUTHOR]

Background: In addition to conventional treatment and modifications in physical activity and diet, alternative strategies have been investigated to manage, prevent, or delay diabetes in humans. In this regard, one strategy has relied on the immunomodulatory properties of mycobacteria, whereby Bacillus Calmette-Guerin, an attenuated live strain of Mycobacterium bovis, has been shown to improve glycemic control in patients with diabetes and to alleviate hyperglycemia in selected murine models of diabetes. A novel heat-killed (HK) whole-cell preparation of Mycobacterium aurum (M. aurum) is currently under development as a potential food supplement; nevertheless, its potential bioactivity remains largely unknown. Thus, the present study investigated the potential prophylactic anti-diabetic effects of HK M. aurum in streptozotocin (STZ)-induced diabetic mice. Methods: Mice were divided into three groups: the STZ-induced diabetic group was injected with a single intraperitoneal high dose of STZ, the HK M. aurum-treated diabetic group was prophylactically treated with three doses of HK M. aurum 6 weeks before STZ injection, and the control non-diabetic group was given three intradermal injections of borate-buffered saline and an intraperitoneal injection of citrate buffer. Liver lactate dehydrogenase (LDH), uncoupling protein 2 (UCP2), and glucose transporter 2 (GLUT2) and skeletal muscle LDH, UCP3, and GLUT4 protein expression levels in different mouse groups were determined by Western blot. Results: Our results indicated that HK M. aurum did not cause any significant changes in glycemic levels of normal non-diabetic mice. Prophylactic administration of three doses of HK M. aurum to diabetic mice resulted in a significant reduction in their blood glucose levels when compared to those in control diabetic mice. Prophylactic treatment of diabetic mice with HK M. aurum significantly restored their disturbed protein expression levels of liver UCP2 and LDH as well as of skeletal muscle UCP3. On the other hand, prophylactic treatment of diabetic mice with HK M. aurum had no significant effect on their liver GLUT2 and skeletal muscle GLUT4 and LDH protein expression levels. Conclusions: Our findings provide the first evidence that HK M. aurum possesses a hyperglycemia-lowering capacity and might support its future use as a food supplement for the amelioration of diabetes. [ABSTRACT FROM AUTHOR]

Goats are natural hosts of Mycobacterium (M.) bovis, and affected herds can be the cause of significant economic losses. Similarites in disease course and lesions of M. bovis infections in goats and M. tuberculosis in humans make goats good models for human tuberculosis. The aim of this investigation was to characterize M. bovis challenge models in goats. For this, goats were endobronchially inoculated with three doses of M. bovis or culture medium. Clinical signs, shedding, and immune responses were monitored until 146 days post inoculation (dpi). At necropsy, lesions were examined by computed tomography, histology, and bacteriological culture. Infected goats did not develop clinical signs. M. bovis was cultured from feces, but never from nasal swabs. IGRAs were positive from 28 dpi onwards, antibodies at 140 dpi, and SICCT at 146 dpi. The increase in CD25+, IFN-γ+, and IFN-γ-releasing T-cell subpopulations was time-related, but not dose-dependent. All infected goats developed paucibacillary granulomas in the lungs and regional lymph nodes. M. bovis was regularly cultured. Dose-dependent effects included the size of pulmonary lesions, caverns, intestinal lesions, and early generalization in the high-dose group. In summary, reproducible challenge models with dose-dependent differences in lesions were established, which may serve for testing vaccines for veterinary or medical use. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, poses significant zoonotic and economic challenges globally. The current prevention and treatment options are limited and increasingly complicated by the emergence of multidrug‐resistant strains. This study employs reverse vaccinology and immunoinformatics to design a multi‐epitope subunit vaccine targeting the MPB83, ArfA, DnaK, GrpE, and LpqH proteins of M. bovis. The T‐cell and B‐cell epitopes of the candidate vaccine were predicted and evaluated for antigenicity, allergenicity, and toxicity. The promising epitopes were then assembled into three vaccine constructs (bTBV1, bTBV2, and bTBV3) using appropriate adjuvants, pan HLA DR‐binding epitope (PADRE), and linkers. The constructs were analyzed for physicochemical properties, 3D structure, cytokines induction and stability, followed by molecular docking with bovine CD molecules and toll‐like receptor, TLR‐9. Among the candidates, bTBV3 was chosen as one of the most promising vaccine candidates due to its high aliphatic index (67.60), lowest instability score (27.26), and a strong binding affinity. Molecular dynamics simulations and the results of interactions between the vaccine–receptor complexes (eigenvalue 2.318704e‐06) show that the vaccine construct bTBV3 is stable. In silico immune simulation findings, such as elevated IgM levels and increased Th cell populations, suggest that the designed multi‐epitope vaccine candidate bTBV3 elicits robust humoral and cellular immune responses, confirming the vaccine's potential efficacy. Additionally, codon optimization (CAI: 0.997 and GC: 54.687%) and in silico cloning facilitated efficient expression in E. coli. This study highlights the potential of bioinformatics‐driven approaches in developing effective subunit vaccines against bTB, providing a foundation for experimental validation and future applications in combating this pervasive zoonotic disease, bovine tuberculosis. [ABSTRACT FROM AUTHOR]

Cutaneous tuberculosis is a rare manifestation of extrapulmonary tuberculosis caused by Mycobacterium tuberculosis in most cases and rarely by Mycobacterium bovis. Diagnosis may be challenging due to a wide range of clinical findings and similarities to other chronic dermatoses, leading to delayed treatment. We present a case of scrofuloderma in a 4‐year‐old girl that arose from a contiguous spread from the anterior mediastinum with associated pulmonary involvement. [ABSTRACT FROM AUTHOR]

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis. Author summary: Tuberculosis, caused by Mycobacterium tuberculosis, is still one of the world's deadliest infectious diseases. However, the origins and rise of M. tuberculosis as a successful pathogen are not well understood. Here, we report the isolation and characterisation of a marine sponge-derived mycobacterium (M. spongiae) from the Great Barrier Reef that has striking genotypic similarity to M. tuberculosis, with 80% average nucleotide identity. We further show by proteomic and lipidomic analyses that M. spongiae shares virulence factors and unique cell wall lipids with the tubercle bacillus. In spite of these conserved genotypic and phenotypic features, M. spongiae was not virulent in a mouse model of infection, leading us to investigate its potential as a vaccine strain or genetic donor for enhancing the current BCG vaccine for tuberculosis. Our findings contribute to understanding the evolutionary origins of M. tuberculosis and provide further insights into its pathogenesis. [ABSTRACT FROM AUTHOR]

We report here on the characterisation in mice of a noninvasive bacille Calmette-Guérin (BCG) skin challenge model for assessing tuberculosis (TB) vaccine efficacy. Controlled human infection models (CHIMs) are valuable tools for assessing the relevant biological activity of vaccine candidates, with the potential to accelerate TB vaccine development into the clinic. TB infection poses significant constraints on the design of a CHIM using the causative agent Mycobacterium tuberculosis (Mtb). A safer alternative is a challenge model using the attenuated vaccine agent Mycobacterium bovis BCG as a surrogate for Mtb, and intradermal (skin) challenge as an alternative to pulmonary infection. We have developed a unique noninvasive imaging system based on fluorescent reporters (FluorBCG) to quantitatively measure bacterial load over time, thereby determining a relevant biological vaccine effect. We assessed the utility of this model to measure the effectiveness of 2 TB vaccines: the currently licenced BCG and a novel subunit vaccine candidate. To assess the efficacy of the skin challenge model, a nonlinear mixed effect model was built describing the decline of fluorescence over time. The model-based analysis identified that BCG vaccination reduced the fluorescence readout of both fluorophores compared to unvaccinated mice (p < 0.001). However, vaccination with the novel subunit candidate did not alter the fluorescence decline compared to unvaccinated mice (p > 0.05). BCG-vaccinated mice that showed the reduced fluorescent readout also had a reduced bacterial burden in the lungs when challenged with Mtb. This supports the fluorescence activity in the skin as a reflection of vaccine induced functional pulmonary immune responses. This novel noninvasive approach allows for repeated measurements from the challenge site, providing a dynamic readout of vaccine induced responses over time. This BCG skin challenge model represents an important contribution to the ongoing development of controlled challenge models for TB. We need better models to assess the efficacy of early tuberculosis vaccine candidates and prioritise some for larger scale trials. This study introduces a novel non-invasive skin challenge model using a fluorescent BCG reporter strain that enables measuring of vaccine-induced immune responses. [ABSTRACT FROM AUTHOR]

New insights in the urinary microbiome have led to a better understanding being built of the shifts in bacterial representations from health to disease; these hold promise as markers for diagnosis and therapeutic responses. Although several efforts have been made to identify a "core urinary microbiome", different fingerprints have been identified in men and women that shift with age. The main bacterial groups overall include Firmicutes, Actinobacteria, Fusobacteria, and Bacteroidetes. Although patients with bladder cancer have a microbiome that is similar to that of healthy individuals, differences have been observed at the species level with Fusobacterium nucleatum and Ralstonia, and at the genus level with Cutibacterium. Different bacterial representations may influence extracellular matrix composition, affecting tumor metastatic spreading and tumorigenic metalloproteinase expression. Furthermore, gene expression affecting targets of immune therapy, such as PD-L1, has been associated with changes in bacterial representations and therapeutic response to BCG. This comprehensive review aims to examine the influence of the urinary microbiome in bladder cancer. [ABSTRACT FROM AUTHOR]

Background Tuberculosis (TB), predominantly caused by Mycobacterium tuberculosis (MTB) infection, remains a prominent global health challenge. Macrophages are the frontline defense against MTB , relying on autophagy for intracellular bacterial clearance. However, MTB can combat and evade autophagy, and it influences macrophage polarization, facilitating immune evasion and promoting infection. We previously found that heparin-binding hemagglutinin (HBHA) inhibits autophagy in A549 cells; however, its role in macrophage autophagy and polarization remains unclear. Methods Bacterial cultures, cell cultures, Western blotting, immunofluorescence, macrophage infection assays, siRNA knockdown, and enzyme-linked immunosorbent assay were used to investigate HBHA's impact on macrophages and its relevance in Mycobacterium infection. Results HBHA inhibited macrophage autophagy. Expression of recombinant HBHA in Mycobacterium smegmatis (rMS-HBHA) inhibited autophagy, promoting bacterial survival within macrophages. Conversely, HBHA knockout in the Mycobacterium bovis bacillus Calmette-Guérin (BCG) mutant (BCG-ΔHBHA) activated autophagy and reduced bacterial survival. Mechanistic investigations revealed that HBHA may inhibit macrophage autophagy through the Toll-like receptor 4–dependent PI3K-AKT-mTOR signaling pathway. Furthermore, HBHA induced macrophage M2 polarization. Conclusions Mycobacterium may exploit HBHA to suppress the antimicrobial immune response in macrophages, facilitating intracellular survival and immune evasion through autophagy inhibition and M2 polarization induction. Our findings may help identify novel therapeutic targets and develop more effective treatments against MTB infection. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB) is a chronic inflammatory disease primarily caused by Mycobacterium bovis. The infection affects domestic animals and wildlife, posing a zoonotic risk to humans. To understand the dynamics of transmission and genetic diversity in Italy's M. bovis population, we conducted whole-genome sequencing (WGS) analysis on two prevalent genotypes, belonging to Spoligotype SB0120, identified in different geographical and temporal contexts. By comparing these genomes with international M. bovis isolates, we identified a distinct clade within the lineage La1.2, encompassing the Italian SB0120 isolates, indicating a genomic segregation of Italian M. bovis from other European isolates. Within Italy, a significant level of genetic variability emerged across regions, while isolates within epidemiologically linked outbreaks exhibited minimal genetic diversity. Additionally, isolates derived from cattle and wild boars within a tuberculosis hotspot in Central Italy and from cattle and black pigs in Sicily formed unified clonal clusters. This indicates the presence of persistent strains circulating in the examined regions. The genetic diversity within herds was limited, as specific clones endured over time within certain herds. This research enhances our comprehension of the epidemiology and transmission patterns of bTB in Italy, thereby aiding the development of precise control strategies and disease management. Using WGS and implementing standardized protocols and databases will be pivotal in combating bTB and promoting One-Health approaches to address this noteworthy public health concern. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18–50 years, who were both M tuberculosis -naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov , NCT02709278 , and is complete. Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2–11] vs 4% [1–13], p=0·62; respiratory 7% [1–19] vs 4% [1–9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5–17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [−1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported—a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020. [ABSTRACT FROM AUTHOR]

Background Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. Methods A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) method. Results Spoligotyping identified 34 types and classified them into 14 clusters. East African–Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter–Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. Conclusions This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Bovine tuberculosis is caused by Mycobacterium bovis, which causes tuberculosis in humans and animals. Diagnosis of bovine tuberculosis has relied on examination of immune responses to M. bovis; however, 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 rather than the animal's response to infection. DNA-from M. bovis was detected in nasal swabs and saliva from a small number of experimentally infected calves during the first 8 weeks after experimental infection. Although DNA from M. bovis could be detected, no culturable M. bovis was recovered from nasal swabs or saliva. Moreover, M. bovis DNA was not found in blood samples collected weekly. Successful infection of all calves was demonstrated using an interferon gamma release assay. Identification of infected animals through the detection of M. bovis will require the use of alternative samples or alternative assays. 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 FROM AUTHOR]

In a multi-host system, understanding host-species contribution to transmission is key to appropriately targeting control and preventive measures. Outbreak reconstruction methods aiming to identify who-infected-whom by combining epidemiological and genetic data could contribute to achieving this goal. However, the majority of these methods remain untested on realistic simulated multi-host data. Mycobacterium bovis is a slowly evolving multi-host pathogen and previous studies on outbreaks involving both cattle and wildlife have identified observation biases. Indeed, contrary to cattle, sampling wildlife is difficult. The aim of our study was to evaluate and compare the performances of three existing outbreak reconstruction methods (seqTrack, outbreaker2 and TransPhylo) on M. bovis multi-host data simulated with and without biases. Extending an existing transmission model, we simulated 30 bTB outbreaks involving cattle, badgers and wild boars and defined six sampling schemes mimicking observation biases. We estimated general and specific to multi-host systems epidemiological indicators. We tested four alternative transmission scenarios changing the mutation rate or the composition of the epidemiological system. The reconstruction of who-infected-whom was sensitive to the mutation rate and seqTrack reconstructed prolific super-spreaders. TransPhylo and outbreaker2 poorly estimated the contribution of each host-species and could not reconstruct the presence of a dead-end epidemiological host. However, the host-species of cattle (but not badger) index cases was correctly reconstructed by seqTrack and outbreaker2. These two specific indicators improved when considering an observation bias. We found an overall poor performance for the three methods on simulated biased and unbiased bTB data. This seemed partly attributable to the low evolutionary rate characteristic of M. bovis leading to insufficient genetic information, but also to the complexity of the simulated multi-host system. This study highlights the importance of an integrated approach and the need to develop new outbreak reconstruction methods adapted to complex epidemiological systems and tested on realistic multi-host data.

Animal tuberculosis (TB) has been reported in several wildlife species in the Greater Kruger Conservation Area (GKCA), South Africa. This report describes the discovery of clinical tuberculosis, caused by Mycobacterium bovis (M. bovis), in free-ranging vervet monkeys (Chlorocebus pygerythrus). The “One Health” concept is especially relevant to TB since this is a multi-host disease with zoonotic potential and is endemic in GKCA. Vervet monkeys have become habituated to humans in tourist areas and may be a source of infection through close contact. Indirect transmission of M. bovis through environmental sources has also been suspected to present a risk of spread between host species. Clinically diseased monkeys present in two tourist areas in the GKCA, that died (n = 1) or were euthanized (n = 5), were submitted for diagnostic necropsies. The presence of pathological lesions, Ziehl-Neelsen-stained impression smears, Xpert® MTB/RIF Ultra (GXU) assay, mycobacterial culture and speciation by genomic regions of difference PCR, were used to confirm the diagnosis of M. bovis infection in these monkeys. The finding of multiple cases necessitates further investigation of TB in monkey troops living within the GKCA tourist areas to determine the source of infection and assess the risk of transmission to other animals and humans.

Almost every aspect of cancer can be influenced by microbiota including tumor onset, progression, and response to therapy. The increasing evidence of the role of microbiota in human health and disease has reinvigorated the interest in designing microbial products that can affect cancer outcomes. Researchers have made numerous attempts to develop safe, engineered biotherapeutic products for cancer treatment using synthetic biology tools. Despite the progress, only Bacillus Calmette-Guérin is approved for human use. Here, we highlight the recent advances and current challenges in using live bacteria as cancer therapeutics.