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Pathogens (Basel, Switzerland) Jul 2023The ability of () to survive in bovine milk has emerged as a serious public health concern. The first objective of this study was to evaluate the diagnostic utility of...
The ability of () to survive in bovine milk has emerged as a serious public health concern. The first objective of this study was to evaluate the diagnostic utility of IS1081-targeted real-time PCR for the detection of DNA in different fractions of bovine milk. In a model study, bovine milk samples were spiked with serially diluted BCG to investigate the detection limit of DNA in whole milk and milk fractions (cream, pellet, and pellet + cream combined) using IS1081 real-time PCR. The assay was then used to detect DNA in whole milk and milk fractions from naturally infected animals. The results showed that the IS1081 real-time PCR was more sensitive when detecting DNA in the cream layer alone and cream + pellet combined compared to whole milk or the pellet alone. While PCR-based diagnostic assays for the detection of in milk samples provide a quicker diagnostic tool for bovine tuberculosis, safe processing, and handling of -infected milk samples remain a challenge and pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to rapidly inactivate infected specimens while preserving nucleic acid for subsequent Molecular analysis. Therefore, the secondary objective of this study was to evaluate the ability of MTM to inactivate BCG in spiked milk samples as well as its ability to preserve BCG DNA for the PCR assay. The results showed that MTM can successfully inactivate BCG alone or in spiked milk samples while preserving DNA for the PCR assay. The CT values of BCG alone and spiked milk samples aliquoted in MTM and without MTM were similar at various dilutions. Taken together, our results indicate that using DNA extracted from the milk cream fraction alone or combined milk cream and pellet improved the recovery rate of DNA in bovine milk samples. MTM has the potential to provide a safe and rapid sample processing tool for inactivation in milk samples and preserve DNA for molecular diagnostics.
PubMed: 37623932
DOI: 10.3390/pathogens12080972 -
Microbial Genomics Aug 2023The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine...
The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in , , and were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature.
Topics: Humans; BCG Vaccine; Mycobacterium bovis; Phylogeny; Tuberculosis; Base Sequence
PubMed: 37526642
DOI: 10.1099/mgen.0.001077 -
One Health (Amsterdam, Netherlands) Dec 2023forms part of the complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the...
BACKGROUND
forms part of the complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in .
METHODS
This pilot study investigated the spoligotype and WGS of isolates ( = 7) from wildlife in Marloth Park (MP) and compared these with WGS data from other South African isolates. In addition, the greater resolution of WGS was used to explore the phylogenetic relatedness of isolates in neighbouring wildlife populations.
RESULTS
The phylogenetic analyses showed the closest relatives to the seven isolates from MP were isolates from wildlife in Kruger National Park (KNP), which shares a border with MP. However, WGS data indicated that the KNP and MP isolates formed two distinct clades, even though they had similar spoligotypes and identical in silico genetic regions of difference profiles.
CONCLUSIONS
isolates from MP were hypothesized to be directly linked to KNP wildlife, based on spoligotyping. However, WGS indicated more complex epidemiology. The presence of two distinct clades which were genetically distinct (SNP distance of 19-47) and suggested multiple transmission events. Therefore, WGS provided new insight into the molecular epidemiology of the isolates from MP and their relationship to isolates from KNP. This approach will facilitate greater understanding of transmission at wildlife-livestock-human interfaces and advances One Health research on tuberculosis, especially across different host species.
PubMed: 38283183
DOI: 10.1016/j.onehlt.2023.100654 -
Microorganisms Oct 2023BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely...
BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely used as a reference, belongs to the late strain clade. BCG Moreau, contrary to Pasteur, is naturally deficient in homologous recombination (HR). In this work, using a UV exposure test, we aimed to detect differences in the survival of various BCG strains after DNA damage. Transcription of core and regulatory HR genes was further analyzed using RT-qPCR, aiming to identify the molecular agent responsible for this phenotype. We show that early strains share the Moreau low survival rate after UV exposure, whereas late strains mimic the Pasteur phenotype, indicating that this increase in HR efficiency is linked to the evolutionary clade history. Additionally, RT-qPCR shows that BCG Moreau has an overall lower level of these transcripts than Pasteur, indicating a correlation between this gene expression profile and HR efficiency. Further assays should be performed to fully identify the molecular mechanism that may explain this differential phenotype between early and late BCG strains.
PubMed: 37894192
DOI: 10.3390/microorganisms11102534 -
Veterinary World Jan 2024Bovine tuberculosis (bTB) is a contagious and notifiable disease, which is prevalent in cattle populations of many countries and in several wildlife species worldwide....
BACKGROUND AND AIM
Bovine tuberculosis (bTB) is a contagious and notifiable disease, which is prevalent in cattle populations of many countries and in several wildlife species worldwide. However, the role of wildlife in the transmission and/or maintenance of bTB at the human-wild animal-animal interface and the epidemiology of zoonotic disease are poorly understood in Cameroon, where many wildlife species exist. This study aimed to estimate the prevalence and zoonotic risk factors of bTB at the cattle-wildlife-human interface in the South and East regions of Cameroon.
MATERIALS AND METHODS
We conducted a descriptive cross-sectional study from May to October 2022 in the southern region (Vallée du Ntem and Dja et Lobo) and eastern region (Haut Nyong and Lom et Djérem) of Cameroon to determine risk factors for bTB in Zebu Bororo, Goudali, Ndama, and Simmental cattle breeds. A comparative intradermal tuberculin testing (CIDT) was performed on 160 cattle randomly selected from herds using the threshold recommended by the World Organization for Animal Health. An interviewee-administered questionnaire was used to gather epidemiological data on sociodemographics, interaction between cattle and wildlife, and awareness of zoonotic tuberculosis (TB) from 90 cattle professionals. The prevalence of bTB at the herd level and associated risk factors were estimated using multiple logistic regression models.
RESULTS
Based on the comparative intradermal tuberculin test (CIDT), the estimated prevalence of bTB in 160 cattle (Zebu Bororo, Goudali, Ndama, and Simmental) in South and East Cameroon was 6.8% (4.35%-9.41%) and 1.8% (0%-3.6%) for threshold values 3 mm and 4 mm, respectively. The prevalence obtained by simple intradermal tuberculin test (IDT) was 0.6% (0%-1.2%) for a threshold value 4 mm. Univariate analysis revealed three risk factors associated with bTB with significant odds ratios (OR; p = 0.05): herd size (OR = 4.88; 95% confidence interval [CI]: 1.24-32.56); cattle aged>10 years (OR = 0.17; 95% CI: 0.05-0.53); and victims of bTB organ seizure (OR = 0.015; 95% CI: 0.002-0.067). Multivariate analysis showed that being a cattle herder and contact between wildlife and livestock due to forage was significantly associated with bTB exposure (adjusted OR = 0.02; p = 0.001).
CONCLUSION
Bovine TB is prevalent in cattle of the South and East Cameroon. Comparative IDT of cattle reared in the epidemiological and environmental context of the study areas yielded better results at a threshold of 3 mm than at a threshold of 4 mm recommended by the World Health Organization. Factors associated with exposure to/appearance of bTB were high herd size, cattle aged >10 years old, seizures of tuberculous organs, shepherding as a profession, and contact between cattle and wildlife can be due to lack of forage.
PubMed: 38406372
DOI: 10.14202/vetworld.2024.8-16 -
Cureus Dec 2023Intravesical instillation of bacillus Calmette-Guérin (BCG) is a crucial adjunctive therapy in the treatment of bladder carcinoma. Its systemic complications are rare...
Intravesical instillation of bacillus Calmette-Guérin (BCG) is a crucial adjunctive therapy in the treatment of bladder carcinoma. Its systemic complications are rare but include disseminated () infections, the diagnosis of which is a challenging task that requires keen clinical awareness. We report the case of an adult male treated with BCG who presented with fever, nonspecific constitutional symptoms, hepatic cytolysis, and cholestasis. After a detailed workup, the diagnosis was made of disseminated infection with hepatic, pulmonary, renal, and ureteral involvement. Prompt anti-tuberculosis treatment resulted in clinical and analytical improvement. This case highlights the importance of early recognition of this serious complication in patients with BCG exposure, as well as the difficulty in confirming the diagnosis for proper treatment.
PubMed: 38094875
DOI: 10.7759/cureus.50407 -
Tuberculosis (Edinburgh, Scotland) Jul 2024Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the deadliest infections in humans. Because Mycobacterium bovis Bacillus Calmette-Guérin (BCG) share...
Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the deadliest infections in humans. Because Mycobacterium bovis Bacillus Calmette-Guérin (BCG) share genetic similarities with Mycobacterium tuberculosis, it is often used as a model to elucidate the molecular mechanisms of more severe tuberculosis infection. Caveolin-1 has been implied in many physiological processes and diseases, but it's role in mycobacterial infections has barely been studied. We isolated macrophages from Wildtype or Caveolin-1 deficient mice and analyzed hallmarks of infection, such as internalization, induction of autophagy and apoptosis. For in vivo assays we intravenously injected mice with BCG and investigated tissues for bacterial load with colony-forming unit assays, bioactive lipids with mass spectrometry and changes of protein expressions by Western blotting. Our results revealed that Caveolin-1 was important for early killing of BCG infection in vivo and in vitro, controlled acid sphingomyelinase (Asm)-dependent ceramide formation, apoptosis and inflammatory cytokines upon infection with BCG. In accordance, Caveolin-1 deficient mice and macrophages showed higher bacterial burdens in the livers. The findings indicate that Caveolin-1 plays a role in infection of mice and murine macrophages with BCG, by controlling cellular apoptosis and inflammatory host response. These clues might be useful in the fight against tuberculosis.
Topics: Animals; Caveolin 1; Apoptosis; Mycobacterium bovis; Mice, Knockout; Mice, Inbred C57BL; Macrophages; Tuberculosis; Sphingomyelin Phosphodiesterase; Autophagy; Host-Pathogen Interactions; Disease Models, Animal; Bacterial Load; Cytokines; Ceramides; Liver; Cells, Cultured; Mice; Inflammation Mediators; Time Factors
PubMed: 38547568
DOI: 10.1016/j.tube.2024.102493 -
IScience Feb 2024Monocyte exhaustion with sustained pathogenic inflammation and immune-suppression, a hallmark of sepsis resulting from systemic infections, presents a challenge with...
Monocyte exhaustion with sustained pathogenic inflammation and immune-suppression, a hallmark of sepsis resulting from systemic infections, presents a challenge with limited therapeutic solutions. This study identified Methoxy-Mycolic Acid (M-MA), a branched mycolic acid derived from Bacillus Calmette-Guérin (BCG), as a potent agent in alleviating monocyte exhaustion and restoring immune homeostasis. Co-treatment of monocytes with M-MA effectively blocked the expansion of Ly6C/CD38/PD-L1 monocytes induced by LPS challenges and restored the expression of immune-enhancing CD86. M-MA treatment restored mitochondrial functions of exhausted monocytes and alleviated their suppressive activities on co-cultured T cells. Independent of TREM2, M-MA blocks Src-STAT1-mediated inflammatory polarization and reduces the production of immune suppressors TAX1BP1 and PLAC8. Whole genome methylation analyses revealed M-MA's ability to erase the methylation memory of exhausted monocytes, particularly restoring Plac8 methylation. Together, our data suggest M-MA as an effective agent in restoring monocyte homeostasis with a therapeutic potential for treating sepsis.
PubMed: 38323001
DOI: 10.1016/j.isci.2024.108978 -
Frontiers in Veterinary Science 2023Bovine tuberculosis (bTB), caused by () infection in cattle, is an economically devastating chronic disease for livestock worldwide. Efficient disease control measures...
Bovine tuberculosis (bTB), caused by () infection in cattle, is an economically devastating chronic disease for livestock worldwide. Efficient disease control measures rely on early and accurate diagnosis using the tuberculin skin test (TST) and interferon-gamma release assays (IGRAs), followed by culling of positive animals. Compromised performance of TST and IGRA, due to BCG vaccination or co-infections with non-tuberculous mycobacteria (NTM), urges improved diagnostics. Lateral flow assays (LFAs) utilizing luminescent upconverting reporter particles (UCP) for quantitative measurement of host biomarkers present an accurate but less equipment- and labor-demanding diagnostic test platform. UCP-LFAs have proven applications for human infectious diseases. Here, we report the development of UCP-LFAs for the detection of six bovine proteins (IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10), which have been described by ELISA as potential biomarkers to discriminate infected from naïve and BCG-vaccinated cattle. We show that, in line with the ELISA data, the combined PPDb-induced levels of IFN-γ, IL-2, IL-6, CCL4, and CXCL9 determined by UCP-LFAs can discriminate challenged animals from naïve (AUC range: 0.87-1.00) and BCG-vaccinated animals (AUC range: 0.97-1.00) in this cohort. These initial findings can be used to develop a robust and user-friendly multi-biomarker test (MBT) for bTB diagnosis.
PubMed: 37655261
DOI: 10.3389/fvets.2023.1193332 -
MSystems Feb 2024Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A...
Tuberculosis remains the most pervasive infectious disease and the recent emergence of drug-resistant strains emphasizes the need for more efficient drug treatments. A key feature of pathogenesis, conserved between the human pathogen and the model pathogen is the metabolic switch to lipid catabolism and altered expression of virulence genes at different stages of infection. This study aims to identify genes involved in sustaining viable intracellular infection. We applied transposon sequencing (Tn-Seq) to , an unbiased genome-wide strategy combining saturation insertional mutagenesis and high-throughput sequencing. This approach allowed us to identify the localization and relative abundance of insertions in pools of transposon mutants. Gene essentiality and fitness cost of mutations were quantitatively compared between growth and different stages of infection in two evolutionary distinct phagocytes, the amoeba and the murine BV2 microglial cells. In the genome, 57% of TA sites were disrupted and 568 genes (10.2%) were essential, which is comparable to previous Tn-Seq studies on and . Major pathways involved in the survival of during infection of are related to DNA damage repair, lipid and vitamin metabolism, the type VII secretion system (T7SS) ESX-1, and the Mce1 lipid transport system. These pathways, except Mce1 and some glycolytic enzymes, were similarly affected in BV2 cells. These differences suggest subtly distinct nutrient availability or requirement in different host cells despite the known predominant use of lipids in both amoeba and microglial cells.IMPORTANCEThe emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system / to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for to identify conservation of virulence pathways between hosts.
Topics: Animals; Mice; Humans; Virulence; Microglia; Mycobacterium marinum; Amoeba; Dictyostelium; Tuberculosis; Mycobacterium tuberculosis; Lipids
PubMed: 38270456
DOI: 10.1128/msystems.01326-23