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Microbiology Spectrum Jun 2024Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened...
UNLABELLED
Traditionally, successful vaccines rely on specific adaptive immunity by activating lymphocytes with an attenuated pathogen, or pathogen subunit, to elicit heightened responses upon subsequent exposures. However, recent work with and other pathogens has identified a role for "trained" monocytes in protection through memory-like but non-specific immunity. Here, we used an co-culture approach to study the potential role of trained macrophages, including lung alveolar macrophages, in immune responses to the Live Vaccine Strain (LVS) of is an intracellular bacterium that replicates within mammalian macrophages and causes respiratory as well as systemic disease. We vaccinated mice with LVS and then obtained lung alveolar macrophages, or derived macrophages from bone marrow. LVS infected and replicated comparably in both types of macrophages, whether naïve or from LVS-vaccinated mice. LVS-infected macrophages were then co-cultured with either naïve splenocytes, splenocytes from mice vaccinated intradermally, or splenocytes from mice vaccinated intravenously. For the first time, we show that immune (but not naïve) splenocytes controlled bacterial replication within alveolar macrophages, similar to previous results using bone marrow-derived macrophage. However, no differences in control of intramacrophage bacterial replication were found between co-cultures with naïve macrophages or macrophages from LVS-vaccinated mice; furthermore, nitric oxide levels and interferon-gamma production in supernatants were largely comparable across all conditions. Thus, in the context of co-cultures, the data do not support development of trained macrophages in bone marrow or lungs of mice vaccinated with LVS intradermally or intravenously.
IMPORTANCE
The discovery of non-specific "trained immunity" in monocytes has generated substantial excitement. However, to date, training has been studied with relatively few microbes (e.g., Bacille Calmette-Guérin, a live attenuated intracellular bacterium used as a vaccine) and microbial substances (e.g., LPS), and it remains unclear whether training during infection is common. We previously demonstrated that vaccination of mice with Live Vaccine Strain (LVS), another live attenuated intracellular bacterium, protected against challenge with the unrelated bacterium . The present study therefore tested whether LVS vaccination engenders trained macrophages that contributed to this protection. To do so, we used a previous co-culture approach with murine bone marrow-derived macrophages to expand and study lung alveolar macrophages. We demonstrated that alveolar macrophages can be productively infected and employed to characterize interactions with LVS-immune lymphocytes. However, we find no evidence that either bone marrow-derived or alveolar macrophages are trained by LVS vaccination.
PubMed: 38940590
DOI: 10.1128/spectrum.00028-24 -
Frontiers in Cellular and Infection... 2024The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence...
BACKGROUND
The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. We designed a portable thermocycler-based real-time fluorescence loop-mediated isothermal amplification assay (cyp141-RealAmp) using six oligonucleotide primers derived from cyp141 to detect MTB. A combined number of 213 sputum samples (169 obtained from clinically diagnosed cases of pulmonary TB and 44 from a control group without tuberculosis) underwent Acid-fast bacillus (AFB) smear, culture, Xpert MTB/RIF assays, and cyp141-RealAmp assay.
RESULTS
By targeting MTB cyp141, this technique could detect as low as 10 copies/reaction within 30 min, and it was successfully rejected by other mycobacteria and other bacterial species tested. Of the 169 patients, there was no statistical difference between the detection rate of cyp141-RealAmp (92.90%, 95% CI: 89.03-96.07) and that of Xpert MTB/RIF (94.67%, 95% CI: 91.28-98.06) ( > 0.05), but both were statistically higher than that of culture (65.68%, 95% CI: 58.52-72.84) (< 0.05) and AFB (57.40%, 95% CI: 49.94-64.86) (< 0.05). Both cyp141-RealAmp and Xpert MTB/RIF had a specificity of 100%. Furthermore, a high concordance between cyp141-RealAmp and Xpert MTB/RIF was found ( = 0.89).
CONCLUSION
The cyp141-RealAmp assay was shown to be effective, responsive, and accurate in this study. This method offers a prospective strategy for the speedy and precise detection of MTB.
Topics: Mycobacterium tuberculosis; Humans; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Molecular Diagnostic Techniques; Sputum; Tuberculosis, Pulmonary; DNA Primers; Female; Fluorescence; Adult; Male; Tuberculosis; Middle Aged
PubMed: 38938885
DOI: 10.3389/fcimb.2024.1349063 -
Microorganisms Jun 2024The Esx-1 family proteins of the Type VII secretion systems of and have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both...
The Presence of and and Other Gene Orthologs of the RD 1 Region in Non-Tuberculous Mycobacteria, Mycolicibacteria, Mycobacteroides and Mycolicibacter as Possible Impediments for the Diagnosis of (Animal) Tuberculosis.
The Esx-1 family proteins of the Type VII secretion systems of and have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both humans and animals. The presence of ESAT-6 and CFP 10 proteins, which are the most immunogenic proteins of the Esx-1 system and have been widely investigated for the immunodiagnosis of tuberculosis, in some and in , poses limitations for their use in specific diagnoses of TB. As such, to improve the specificity of the ESAT-6/CFP 10-based cell-mediated immunity (CMI) assays, other proteins encoded by genes within and outside the RD 1 region of the esx-1 locus have been evaluated as candidate antigens for CMI, as well as to investigate humoral responses in combination with ESAT-6 and or CFP 10, with varying specificity and sensitivity results. Hence, in this study, we evaluated various non-tuberculous mycobacteria (NTM), , and species genomes available on the NCBI database for the presence and composition of the RD1 region of the esx-1 locus. In addition, we also assayed by polymerase chain reaction (PCR) and sequencing of available in our culture collection for the presence and sequence diversity of and genes encoding ESAT-6 and CFP 10, respectively. Whole genome sequence (WGS) data analysis revealed the presence of RD 1 gene orthologs in 70 of the over 100 published genomes of pathogenic and non- pathogenic other than tuberculosis. Among species evaluated from our culture collection, in addition to earlier reports of the presence of and in certain , , and sp. N845T were also found to harbour orthologs of both genes. Orthologs of only were detected in , and , whereas in , and , only orthologs were detected. A phylogenetic analysis based on and sequences separated slow-growing from rapidly growing bacteria. These findings strengthen previous suggestions that and may be encoded in the majority of . The role of the Esx-1 system in both pathogenic and non-pathogenic needs further investigation, as these species may pose limitations to immunological assays for TB.
PubMed: 38930534
DOI: 10.3390/microorganisms12061151 -
Microorganisms May 2024Environmental decontamination and water disinfection practices are hallmarks of disease prevention and control in agricultural and public health settings. Informed...
Flow Cytometry Coupled with Resuscitation Assays As a High-Resolution Tool to Inform Environmental Management and Disinfection of Settings Affected by Tuberculous Mycobacteria.
Environmental decontamination and water disinfection practices are hallmarks of disease prevention and control in agricultural and public health settings. Informed fit-to-purpose biocontainment is thus dependent on methodologies accurately assessing microbial burden and viability. Also, rigorous evaluation of the efficacy of biocontrol measures implies monitoring microbial inactivation after decontamination/disinfection procedures. In this study, we used flow cytometry coupled with a resuscitation protocol to monitor the metabolic inactivation of bacteria capable of entering non-cultivable states, after the application of a chlorine-based water disinfectant. For this purpose, we used BCG as a model of slow-growing bacteria able to enter dormancy and representing a multi-host pathogen in a zoonotic disease system-animal tuberculosis-thriving both across temperate and semi-arid regions and involving environmental contamination. The biocide activity of a commercial sodium dichloroisocyanurate (NaDCC) disinfectant against BCG was evaluated through mock environmental matrix tests. Using the manufacturer-recommended dosage of NaDCC, BCG cells were apparently inactivated after 24 h upon exposure. However, we show via flow cytometry that, upon exposure to optimal growth conditions, mycobacterial cells were able to regain metabolic activity shortly after, highlighting a sublethal effect of NaDCC at the recommended commercial dosage due to reversible BCG cell damage. In contrast, increasing twice the disinfectant dosage completely inactivated BCG cells after 24 h of exposure, with full irreversible loss of metabolic activity. Methodological workflows based on conventional culture or PCR would have missed the detection of these dormant subpopulations that were in fact able to resume growth when following the recommendations of a commercial disinfectant. This study highlights the superior, high-resolution value of single-cell approaches, such as flow cytometry, to accurately assess the activity of biocides against metabolically heterogeneous and dormant pathogenic bacteria with environmental cycles, supporting data-driven prioritization of environmental management and disinfection options in contaminated vulnerable settings.
PubMed: 38930449
DOI: 10.3390/microorganisms12061068 -
Medicina (Kaunas, Lithuania) May 2024: Bacillus Calmette-Guérin (BCG) vaccine administration has been suggested to prevent glucose metabolism abnormalities and fatty liver in genetically obese mice;...
: Bacillus Calmette-Guérin (BCG) vaccine administration has been suggested to prevent glucose metabolism abnormalities and fatty liver in genetically obese mice; however, it is not clear whether the beneficial effects of BCG are also observed in the progression of glucose intolerance induced by a high-fat diet (HFD). Therefore, the effects of BCG vaccination on changes in glucose tolerance and insulin response were investigated in HFD-fed C57BL/6 mice. : We used the BCG Tokyo 172 strain to determine effects on abnormalities in glucose metabolism. For vaccination, five-week-old male mice were injected intraperitoneally with BCG and maintained on a HFD for three weeks. The mice were regularly subjected to intraperitoneal glucose tolerance and insulin tolerance tests (IGTTs and ITTs). These tests were also performed in mice transplanted with bone marrow cells from BCG-vaccinated donor mice. : Significant effects of BCG vaccination on blood glucose levels in the IGTTs and ITTs were observed from week 12 of the experiment. BCG vaccination significantly improved changes in fasting glucose and insulin levels, insulin resistance indexes, and glucagon-to-insulin ratios in conjunction with the HFD at the end of the experiment. Significant inhibitory effects in the IGTTs and ITTs on glucose intolerance were also observed with transplantation with bone marrow cells derived from BCG-vaccinated donor mice. : BCG vaccination significantly delayed glucose intolerance progression, suggesting a beneficial effect of BCG on the pathogenesis of type 2 diabetes. It has also been suggested that the effects of BCG vaccination may be at least partially due to an immune memory (trained immunity) for hematopoietic stem and progenitor cells of the bone marrow.
Topics: Animals; Mice, Inbred C57BL; Diet, High-Fat; Glucose Intolerance; BCG Vaccine; Mice; Male; Blood Glucose; Insulin Resistance; Disease Progression; Glucose Tolerance Test; Insulin; Disease Models, Animal; Vaccination
PubMed: 38929483
DOI: 10.3390/medicina60060866 -
Cells Jun 2024causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival...
causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.
Topics: Mycobacterium tuberculosis; Cytokines; Macrophages; Mycobacterium bovis; Mice; Bacterial Proteins; Animals; Humans; NF-kappa B; Microbial Viability; Bacterial Adhesion
PubMed: 38920649
DOI: 10.3390/cells13121019 -
International Journal of... Apr 2024Bacille Calmette-Guérin (BCG) is a live-attenuated vaccine routinely administered to newborns to prevent severe forms of tuberculosis (TB) in TB-endemic countries.... (Review)
Review
Infantile Disseminated Bacille Calmette-Guérin Disease with Hemophagocytosis and Mimicking Juvenile Myelomonocytic Leukemia: A Case Report with Concise Literature Review.
Bacille Calmette-Guérin (BCG) is a live-attenuated vaccine routinely administered to newborns to prevent severe forms of tuberculosis (TB) in TB-endemic countries. Disseminated BCG vaccine disease is a classic feature of children with human immunodeficiency virus (HIV) or primary immunodeficiency disorders (PIDs) and is associated with high mortality. We report a case of a 6-month-old infant with disseminated BCG disease and hemophagocytic lymphohistiocytosis mimicking juvenile myelomonocytic leukemia with no demonstrable features of HIV or PID even after extensive laboratory work-up and succumbed to progressive disease. Disseminated BCG disease is a rare and potentially fatal complication of BCG vaccine, and prompt immunological evaluation complemented by initiation of 4-drug antitubercular therapy and definitive treatment with antiretroviral therapy or hematopoietic stem cell transplant is warranted.
Topics: Humans; Leukemia, Myelomonocytic, Juvenile; Infant; Lymphohistiocytosis, Hemophagocytic; BCG Vaccine; Tuberculosis; Diagnosis, Differential; Fatal Outcome; Male; Mycobacterium bovis; Antitubercular Agents
PubMed: 38916394
DOI: 10.4103/ijmy.ijmy_48_24 -
Microbiology Spectrum Jun 2024A human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and...
UNLABELLED
A human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate JKD8049 in naïve and bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days ( = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this model and highlights both the promise and the problems with trying to induce protective immunity against .
IMPORTANCE
In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of JKD8049 (our proposed CHIM strain). We also demonstrate that bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.
PubMed: 38916323
DOI: 10.1128/spectrum.00555-24 -
Frontiers in Veterinary Science 2024Wildlife represents an increasingly important source of pathogens of medical and veterinary importance. Surveillance in wildlife offers an insight on current...
INTRODUCTION
Wildlife represents an increasingly important source of pathogens of medical and veterinary importance. Surveillance in wildlife offers an insight on current epidemiological status of selected pathogens and help to prevent spillovers to humans and livestock.
MATERIAL AND METHODS
Our study included 312 wild ruminants belonging to five species: Roe deer ( = 134), red deer ( = 113), Alpine chamois ( = 53), European mouflon ( = 10) and Alpine ibex ( = 2). Seven pathogens that may have profound effect on human/livestock health and economic viability of the farms were tested using serological methods.
RESULTS
Antibodies against spp., subsp. (MAP) and were detected in 34.62% (108/312), 0.96% (3/312), 2.24% (7/312), 0, 0.96% (3/312), 0, 0.64% (2/312) of animals tested, respectively. Because of low prevalences, risk factors were assessed only for . Sex (female>male) and species (roe deer>red deer, roe deer>Alpine chamois) were significantly associated with the positive outcome, while age was not.
DISCUSSION
Adult males had the lowest prevalence which offers future research opportunities. The lower seroprevalence of most investigated pathogens suggests game meat, if properly cooked, as being relatively safe for human consumption. This is the first study investigating the seroprevalence and associated risk factors of selected pathogens in wild ruminants in Slovenia.
PubMed: 38915887
DOI: 10.3389/fvets.2024.1415304 -
Scientific Reports Jun 2024Bovine tuberculosis (bTB) is endemic and has a substantial impact on the livestock sector in Ethiopia and other low and middle-income countries (LMICs). With a national...
Bovine tuberculosis (bTB) is endemic and has a substantial impact on the livestock sector in Ethiopia and other low and middle-income countries (LMICs). With a national emphasis on dairy farm intensification to boost milk production and spur economic growth, the incidence of bTB is anticipated to rise. However, Ethiopia, like other LMICs, lacks a comprehensive national bTB control strategy due to the economic and social infeasibility of traditional test-and-cull (TC) approaches. To inform the development of such a strategy, we evaluated the effectiveness and feasibility of TC and test-and-segregation (TSg) strategies for bTB control on Ethiopian dairy farms. A TC approach was used at Farm A [N = 62; comparative cervical test (CCT) > 4 mm, starting prevalence 11.3%] while TSg was implemented at Farm B (N = 45; CCT > 4 mm, prevalence 22.2%), with testing intervals of 2-4 months. Both strategies achieved a reduction in bTB prevalence to 0%, requiring seven rounds of TC over 18 months at Farm A, and five rounds of TSg over 12 months at Farm B's negative herd. The results show that adopting more sensitive thresholds [CCT > 0 mm or single cervical test (SCT) > 2 mm] during later rounds was pivotal in identifying and managing previously undetected infections, emphasizing the critical need for optimized diagnostic thresholds. Cost analysis revealed that TC was approximately twice as expensive as TSg, primarily due to testing, labor, and cow losses in TC, versus construction of new facilities and additional labor for TSg. This underscores the economic and logistical challenges of bTB management in resource-limited settings. Taken together, our study highlights an urgent need for the exploration of alternative approaches including TSg and or vaccination to mitigate within herd transmission and enable implementation of bTB control in regions where TC is not feasible.
Topics: Cattle; Animals; Tuberculosis, Bovine; Ethiopia; Dairying; Prevalence; Feasibility Studies; Farms; Female; Mycobacterium bovis
PubMed: 38906922
DOI: 10.1038/s41598-024-64884-x