Search Results (238)

Bovine viral diarrhea virus (BVDV) is a globally significant pathogen affecting both domestic livestock and wildlife, including white-tailed deer (WTD; Odocoileus virginianus). While experimental infections have demonstrated WTD susceptibility to BVDV, natural infections and associated reproductive outcomes remain scarcely documented. Here, we report the first confirmed case of naturally occurring BVDV-1 infection associated with fetal mummification in farmed WTD in Florida. A two-year-old doe experienced a stillbirth involving two mummified fetuses, which were submitted for necropsy and laboratory diagnostics. Gross findings included diarrhea and underdeveloped eyes in the fetuses, along with small white nodules indicative of amnion nodosum. While not harmful, this condition suggests underlying fetal compromise or intrauterine stress. Virus isolation using Vero E6 and bovine turbinate cell lines, along with a reverse transcription PCR (RT-PCR) assay specifically developed in this study, confirmed the presence of BVDV-1 (Pestivirus bovis) RNA in both maternal and fetal samples, suggesting vertical transmission. Sanger sequencing of RT-PCR amplicons further verified the virus species as BVDV-1. Differential diagnostics for other pathogens, including bluetongue virus, epizootic hemorrhagic disease virus, Mycobacterium spp., and Toxoplasma gondii, were negative. These findings underscore the importance of using biosecurity measures and including BVDV in the differential diagnosis of abortions to reduce the risk of BVDV transmission and potential outbreaks on deer farms, particularly those close to cattle operations. The molecular tools developed in this study provide a robust framework for improved detection and monitoring of BVDV in both wildlife and livestock populations. Full article

Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI. Full article

Background: Zoonotic infection with Mycobacterium bovis continues to occur, particularly in regions lacking bovine tuberculosis surveillance and where the consumption of unpasteurized dairy products, including artisanal cheeses, is common. We describe the clinical and microbiological characteristics, diagnostic procedures, and treatment outcomes of individuals with HIV with M. bovis infection. Methods: We conducted a retrospective study analyzing sociodemographic, clinical, microbiological, and computed tomography (CT) data, as well as treatment outcomes, in 12 patients with HIV with confirmed M. bovis infection. These findings were compared with those of 14 individuals with HIV diagnosed with Mycobacterium tuberculosis infection during the same period. Results: Consumption of unpasteurized dairy products was significantly associated with M. bovis. Patients with M. bovis infection had higher CD4+ T-cell counts compared to those with M. tuberculosis infection (p = 0.01, r = 0.45). All M. bovis cases presented with extrapulmonary disease. CT imaging in M. bovis infection more frequently demonstrated retroperitoneal lymphadenopathy, hepatosplenomegaly, and splenic abscesses compared to M. tuberculosis infection. Microbiological identification was exclusively from extrapulmonary sites in all M. bovis cases. Surgical interventions, including abscess drainage or splenectomy, were significantly more common among M. bovis patients. Conclusions: M. bovis infection in individuals with HIV is characterized by consistent extrapulmonary, often abdominal, involvement. Surgical procedures are frequently required for both diagnosis and management. Targeted efforts to identify M. bovis are warranted, particularly in high-burden regions where unpasteurized dairy consumption remains prevalent. Full article

Extracellular vesicles (EVs) can be distributed in various bodily fluids, such as serum and urine, and play an essential role in immune regulation, substance transport, and other aspects. Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which places a tremendous burden on public health prevention and control within society. Researchers are committed to developing various diagnoses and treatment plans to eliminate TB effectively. The results of some studies conducted to date demonstrate that the serum EVs of TB patients, which carry components related to Mtb, can be used as relevant markers for TB detection and improve diagnostic efficiency. However, no relevant reports exist on the particular physiological functions such EVs perform, thus warranting further exploration. In this study, we collected serum EVs from both healthy individuals and TB patients. After identifying the morphology, concentration, and expression of classic markers (CD63, CD81, and CD9) of EVs, we explored their physiological functions at the cellular level and their physiological functions and effects on BCG colonization in the lungs at the mouse level. It was found that EVs were abundant in TB patients and healthy individuals, and the number of CD63 and CD9 markers co-expressed on the surface of serum EVs in healthy individuals was greater than that in TB patients. Serum EVs in patients with TB can stimulate cells to secrete more immune cytokines, such as TNF-α and IL-6, compared with those in healthy individuals; induce an increase in the M1/M2 ratio of macrophages in the peripheral blood mononuclear cells of mice; and inhibit the colonization of Mycobacterium bovis bacillus Calmette Guérin (BCG) in the lungs of mice. In addition, they can inhibit the occurrence of inflammatory responses in the lung tissue of mice. The above results suggest that serum EVs in TB patients may exert their physiological function by regulating immune responses. This finding also indicates that exploring serum EVs in TB patients with regard to their physiological functions shows excellent potential. Full article

Mycobacterium bovis is the causative pathogen of bovine tuberculosis (bTB), a disease that affects cattle and other mammals, including humans. Currently, there is no efficient vaccine against bTB, underscoring the need for novel immunization strategies. The M72 fusion protein, composed of three polypeptides derived from Mycobacterium tuberculosis and M. bovis, has demonstrated protective efficacy against M. tuberculosis in clinical trials when combined with the AS01E adjuvant. Given the established efficacy of nanocapsule formulations as vaccine delivery systems, this study evaluated a novel immunization strategy combining BCG with either full-length M72 or a truncated M72 fused to a streptococcal albumin-binding domain (ABDsM72). Both antigens were encapsulated in chitosan/alginate nanocapsules and assessed in a murine M. bovis challenge model. Priming with BCG followed by an M72 boost significantly improved splenic protection compared to BCG alone, but it did not enhance pulmonary protection. Notably, boosting with ABDsM72 further increased the proportion of CD4+KLRG1-CXCR3+ T cells in the lungs of M. bovis-challenged mice, a key correlate of protective immunity. These findings demonstrate that chitosan/alginate-encapsulated antigens enhance BCG-induced immunity, supporting their potential as next-generation vaccine candidates for bTB control. Full article

Mycobacterium tuberculosis (Mtb) is a major global health threat, exacerbated by the emergence of antibiotic-resistant strains. This study investigated fluoroquinolone resistance protein A (MfpA), which enhances mycobacterial survival by targeting host mitochondria and regulating apoptosis. Wild-type (WT) and knockout (KO) Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strains, a common model for Mtb, were utilized to examine host cell responses. Compared to WT strains, KO strains showed reduced colony-forming units (CFUs), elevated TNF-α and IL-6 levels, and increased apoptosis. MfpA was found to localize to mitochondria, increasing ROS production and disrupting mitochondrial membrane potential. Transcriptomic analysis revealed that MfpA modulated the NF-κB signaling pathway, regulating the expression of gadd45β. These results suggest that MfpA drives both antibiotic resistance and virulence by suppressing apoptosis via the mitochondrial and NF-κB pathways, promoting mycobacterial persistence. Studies using BCG provide valuable insight into Mtb’s survival mechanisms, highlighting MfpA’s dual role in resistance and pathogenesis. Full article

Background: The prevalence of pulmonary nontuberculous mycobacteria (NTM) is increasing in Europe and North America. Most pulmonary NTM cases are caused by Mycobacterium avium complex (MAC). The treatment of pulmonary MAC is suboptimal with failure rates ranging from 30% to 40% and there is a need to develop new vaccines. Methods: We tested the ability of two whole-cell vaccines, DAR-901 (heat-killed M. obuense) and BCG (live-attenuated M. bovis), to induce MAC cross-reactive immunity by first immunizing BALB/c mice and then performing IFN-γ ELISPOT assays after overnight stimulation of splenocytes with live MAC. To study the ability of these vaccines to protect against MAC infection, BALB/c mice were vaccinated with DAR-901 (intradermal) or BCG (subcutaneous or intranasal) and challenged with aerosolized MAC 4 weeks later. A group of mice vaccinated with BCG were also treated with clarithromycin via gavage. Lung colony-forming units (CFU) in immunized mice and unvaccinated controls were quantified 4 weeks after infection. Histopathology was used to quantify lung inflammation and flow cytometry was used to study lung immunity in BCG-vaccinated and unvaccinated mice following MAC infection. To increase the safety profile of mucosal BCG vaccination, we studied BCG with a “kill switch” (tetR BCG) in scnn1b-transgenic mice (i.e., mice prone to cystic fibrosis-type lung diseases). Results: Our results showed that (i) DAR-901 induced cross-reactive immunity to MAC to a similar level as BCG, (ii) DAR-901 and BCG protected against aerosol MAC challenge, (iii) mucosal BCG vaccination, compared to systemic BCG and DAR-901 vaccinations, provided the best protection against MAC challenge, (iv) BCG vaccination did not interfere with anti-MAC activities of clarithromycin, (v) BCG-vaccinated mice had increased inflammation and increased frequencies of activated CD4 and CD8 T cells following MAC infection, and (vi) doxycycline treatment of tetR BCG-vaccinated mice decreased lung BCG CFU without affecting MAC immunity. Conclusions: Both DAR-901 and BCG vaccinations induce MAC cross-reactive immunity and protect against aerosolized MAC challenges. Mucosal BCG vaccination provides the best protection and TetR BCG could enhance the safety of mucosal BCG vaccination. Full article

During 2022, AIDS claimed a life every minute and about 9.2 million HIV-infected people were not on treatment. In addition, a person living with HIV is estimated to be 20–30 times more susceptible to developing active tuberculosis. Every year, 130,000 infants are newly infected, with vertical transmission being the main cause of pediatric HIV infection. Thus, the development of an effective, safe, and accessible vaccine for neonates and/or adults is an urgent need to prevent or control HIV infection or progression to AIDS. An effective HIV vaccine should induce long-lasting mucosal immunity, broadly neutralizing antibodies, innate immunity, and robust stimulation of CD4+ and CD8+ T-cell responses. Recombinant BCG is a promising live-attenuated bacterial vaccine vector because of its capacity to stimulate T-cell immunity. As a slow-growing microorganism, it provides prolonged low-level antigenic exposure upon infecting macrophages and APCs, potentially stimulating both effector and memory T-cell responses. BCG is considered safe and is currently administered to 80% of infants in countries where it is part of the national immunization program. Additionally, BCG offers several benefits as a live vaccine vehicle since it is cost-effective, easy to mass-produce, and heat stable. It is also well-suited for newborns, as maternal antibodies do not interfere with its efficacy. Furthermore, BCG has a strong safety profile, having been administered to over three billion people as a TB vaccine. In this review, we provide an extensive summary of the literature relating to immunogenicity studies in animal models performed since 2011. Moreover, we provide a comprehensive analysis of the key factors influencing the design of recombinant BCG as a live vaccine vehicle: (i) expression vectors; (ii) selection of HIV immunogen; (iii) promoters to regulate gene expression; (iv) BCG strain and BCG codon optimization; (v) genetic plasmid stability; (vi) influence of preexisting immunity, route, and dose immunization; and (vii) safety profile. Full article

Background: Bovine tuberculosis (bTB) is an infectious disease which causes significant damage to the farming industry and remains a disease of global significance. Although control strategies have focused on a test and cull approach primarily based around specific cell-mediated immune responses, serological assays are increasingly being used as a supplementary test alongside skin testing and interferon-gamma release (IGRA) assays. The UK is moving towards the use of the Bacillus Calmette–Guérin (BCG) vaccination of cattle as an additional targeted control tool against bTB. However, there are concerns over its potential impact on the outcomes of bTB diagnostic tests and other non-TB assays, such as serological tests for Mycobacterium avium subsp. paratuberculosis (MAP). Methods: We investigated the performance of commercially available serology tests designed to detect bTB and MAP using serum samples from BCG-vaccinated animals which were subsequently infected with Mycobacterium bovis (M. bovis). Results: BCG vaccination per se did not significantly impact the specificity of serological diagnostic tests for bTB or Johne’s disease. However, increased numbers of false-positive responses in bTB serology tests were seen in BCG-vaccinated animals 3 weeks following a tuberculin skin test, where up to 23% and 54% of animals gave a positive result in IDEXX and Enferplex tests, respectively. Furthermore, M. bovis infection gave rise to false-positive test results for Johne’s disease, irrespective of the animals’ prior BCG vaccination status. Conclusions: Caution should be taken when assessing results from serology tests for bTB if tuberculin skin testing has occurred shortly before collection of blood from BCG-vaccinated cattle. Furthermore, these results highlight the potential for misdiagnosis of MAP infection when using serology tests in bTB-infected cattle. Full article

Background/Objectives: In vitro, vancomycin (VAN) and tetrahydrolipstatin (THL) together have been shown to synergistically inhibit Mycobacterium tuberculosis (Mtb), the world’s most infectious killer. The poor oral bioavailability of VAN and THL and predominant tropism of Mtb infection to the lungs and alveolar macrophages make pulmonary administration highly attractive. This study aimed to develop and assess the efficacy of dry powders for inhalation of VAN microparticles embedded with THL. Methods: The dry powders produced by spray-drying, with or without hydrogenated castor oil (HCO), were characterized for their physicochemical properties among others by HPLC-DAD. The fast-screening impactor was used to determine powder aerodynamic properties, and VAN and THL releases were established from the paddle over disk method. Biological activities were assessed in a new M. bovis-infected macrophage model and in Mtb-infected mice. Results and Discussion: The addition of 25% HCO enables co-deposition (fine particle dose) at the desired weight ratio and co-releasing of VAN and THL in aqueous media. Microparticles with 0% to 50% HCO drastically reduced cytoplasmic Mycobacterium bovis survival (99.9% to 62.5%, respectively), with higher efficacy at low HCO concentration. Consequently, VAN/THL with or without 25% HCO was evaluated in Mtb-infected mice. Although no decrease in Mtb lung burden was observed after two weeks of administration, the endotracheal administration of VAN 500 mg/kg and THL 50 mg/kg with 25% HCO administrated three times during five days concomitantly with daily oral rifampicin (10 mg/kg) demonstrated 2-fold bacterial burden reduction compared to the group treated with RIF alone. Conclusions: HCO was crucial for obtaining a fine particle dose at the synergistic weight ratio (VAN/THL 10:1) and for releasing both drugs in aqueous media. With oral administration of the first-line rifampicin, the dry powder VAN/THL/25% HCO was able to exert a potential anti-tubercular effect in vivo in Mtb-infected mice after five days. Full article

Although the global incidence of tuberculosis has declined in recent years, tuberculosis remains a major global public health challenge. The Mycobacterium tuberculosis complex (MTBC) including M. tuberculosis, M. bovis, M. microti, etc., is the deadliest Mycobacterium spp. that needs more attention. Research on M. microti is significant as it is a zoonotic pathogen that can spread between animals and humans. By exploring the function of a transglutaminase in M. microti (MmTG), which is widely distributed in Mycobacterium and other species, a potential cytotoxic effector has been characterized. MmTG inhibits cell proliferation by inducing the phosphorylation of RIPK1 (receptor-interacting serine/threonine-protein kinase 1) and the Cys159 of MmTG is the highly conserved residue related to its cytotoxicity. Understanding MmTG and its homologs can provide more insights into the pathogenic mechanisms of mycobacteria and contribute to the development of more effective therapeutic strategies against mycobacterial infections. Full article

Mycobacterium is one of the most dangerous pathogens of both animals and humans. Bovine tuberculosis (BTB) is a disease caused by mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC), which spreads mainly among domestic cattle but also to mammals other than cattle. The transmission of MTBC between different species requires research and epidemiological investigations to control its spread. When multiple species are a reservoir of infection, it poses a significant public health and veterinary concern. In this study, the diagnosis of alpaca, cattle, horses, dogs, a sheep and a cat from one farm suspected of bovine tuberculosis was performed. The animals (except for one horse, the dogs and the cat) were euthanised after the intradermal tuberculin tests. Mycobacterial isolation from animal tissue samples was performed. The obtained Mycobacterium strains were genotyped using spoligotyping and mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) methods. The isolates from a horse, two cows, a sheep and an alpaca were classified as Mycobacterium (M.) bovis. The single M. bovis spoligotype SB0666 pattern was isolated, and the MIRU-VNTR results presented the same 222632237401435 patterns. The molecular investigation uncovered information on the relationship of Mycobacterium bovis. Full article

Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines induced by the mycobacterial components make CFA the powerful immunostimulating adjuvant. In these studies, the adjuvant effects are investigated in a select population of cells, and the changes, if any, with the metabolic alterations in the systemic compartment are unclear. We investigated whether the effects of IFA and CFA can be influenced by the metabolic shifts in mice immunized with saline, IFA, or CFA using Mycobacterium tuberculosis var. bovis Bacillus Calmette–Guérin (BCG) as a positive control. After seven days of immunization, we analyzed the serum metabolite profiles using liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to identify metabolic features between the groups. The data revealed that, in the scores space, the CFA and BCG groups were more closely aligned compared to the saline group, while the IFA group displayed an intermediate profile. Furthermore, comparisons between the CFA and BCG groups showed more significant perturbations in lipid and amino acid metabolism, particularly involving glycerophospholipids, cysteine, and aromatic amino acids. In contrast, comparisons between the BCG and IFA groups indicated a more pronounced disruption in central energy metabolism pathways, such as the citric acid cycle and pyruvate metabolism. Together, the data suggest that the serum metabolite profiles in response to IFA and CFA might play a role in modulating the immune responses. Full article

Background: The increasing prevalence of drug-resistant tuberculosis (TB) underscores the urgent need for novel antimicrobial agents. Methods: This study integrates cultivation optimization, nuclear magnetic resonance (NMR) fingerprinting, and principal component analysis (PCA) to explore microbial secondary metabolites as potential anti-TB agents. Results: Using the combined approach, 11 bioactive compounds were isolated and identified, all exhibiting anti-Mycobacterium bovis BCG activity. Notable findings include borrelidin, a potent threonyl-tRNA synthetase inhibitor with broad biological activities, and L-O-Lac-L-Val-D-O-Hiv-D-Val, a peptide isolated for the first time from a plant endophyte, demonstrating broad-spectrum antimicrobial activity. Additionally, elaiophylin and polycyclic tetramate macrolactams (PTMs) displayed significant bactericidal effects, with elaiophylin achieving complete BCG inhibition at 72 h and PTMs marking their first reported anti-TB activity. The study also identified bafilomycins as potent scaffolds for anti-TB drug development, showcasing rapid bactericidal activity at low MIC values. Conclusions: These findings emphasize the value of microbial metabolites as a reservoir of bioactive compounds and provide new avenues for developing next-generation anti-TB therapies. Full article

The domestic dog (Canis lupus familiaris) is a competent host for Mycobacterium (M.) bovis infection but no ante mortem diagnostic tests have been fully validated for this species. The aim of this study was to compare the performance of ante mortem diagnostic tests across samples collected from dogs considered to be at a high or low risk of sub-clinical M. bovis infection. We previously tested a total of 164 dogs at a high risk of infection and here test 42 dogs at a low risk of infection and 77 presumed uninfected dogs with a combination of cell-based and/or serological diagnostic assays previously described for use in non-canid species. The interferon-gamma release assay (IGRA) using peripheral blood mononuclear cells (PBMCs) identified the highest number of test-positive animals (85, 52%), with a suggested specificity of 97.3%, whilst a whole-blood IGRA was found to be unreliable. The production of antigen-specific tumour necrosis factor-alpha (TNF-α) by PBMC in response to a cocktail of ESAT-6 and CFP-10 peptides correlated very strongly with the overall IGRA results, suggesting future diagnostic potential. All three serological assays employed in this study (Idexx M. bovis Ab ELISA, [Idexx Laboratories, Westbrook, ME, USA], DPP VetTB lateral flow assay [Chembio, Medford, NY, USA], and comparative PPD ELISA [in-house]) identified seropositive dogs but, overall, the test-positive rate for the serological assays was only one third that of the cellular-based assays. Circulating serum cytokine concentrations of interferon gamma and TNF-α were not statistically different between the high- and low-risk groups of dogs. While many dogs in the high-risk group had serum biochemical abnormalities, these did not correlate with the findings from the diagnostic TB tests. This study demonstrates, for the first time, the utility of two cellular and three serological assays for detecting sub-clinical M. bovis infections of dogs. Whilst the data suggest a high test specificity for all assays evaluated, further work is needed to validate the sensitivity and specificity of individual or combinations of tests using sufficient numbers of dogs of a known infection status. Full article