Mycobacterium Bovis Infection Research Articles

LCN2 regulates the gut microbiota and metabolic profile in mice infected with Mycobacterium bovis.

Our study addresses the critical knowledge gap regarding the specific influence of lipocalin 2 (LCN2) in the context of Mycobacterium bovis infection, particularly focusing on its role in the gut environment. Utilizing LCN2 knockout (Lcn2-/-) mice, we meticulously assessed changes in the gut microbiota and metabolic components following M. bovis infection. Our findings reveal alterations in the gut microbial community, emphasizing the potentially crucial role of LCN2 in maintaining stability. Furthermore, we observed significant shifts in specific microbial communities, including the enrichment of Akkermansia muciniphila, known for its positive impact on intestinal health and immune regulation. The implications of our study extend beyond understanding the dynamics of the gut microbiome, offering insights into the potential therapeutic strategies for gut-related health conditions and microbial dysbiosis.

The safety of overdose and repeat administrations of BCG Danish strain 1331 vaccine in calves and pregnant heifers

Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, is a zoonotic disease in cattle that represents a significant ongoing challenge to cattle farming productivity and the livelihoods of livestock farmers in the UK. Vaccination of cattle with BCG could directly target the ability of M. bovis to proliferate within vaccinates, restricting bTB pathogenesis and onward disease transmission, and represent a step change in the tools available to help control bTB in farmed cattle. A Marketing Authorisation (MA) is required before a cattle BCG vaccine could be sold and supplied as a veterinary medicine within the UK and this requires comprehensive data supporting vaccine quality, efficacy and, most importantly, its safety. We carried out two independent Good Laboratory Practice (GLP) studies in which the safety of BCG vaccination in cattle was stringently tested through overdose and repeat vaccine administrations in young calves and pregnant heifers. Mild and generally short-lived reactions to vaccinations were observed in some animals, most commonly increases in body temperature and swelling at vaccine injection sites, but these did not have a negative impact on the overall health status of vaccinates. BCG was not shed in the saliva, faeces, milk or urine from vaccinated animals and its dissemination was limited to injection site tissues and associated lymph nodes. Overall, young calves and pregnant heifers vaccinated with BCG remained in good general health, and the vaccinated pregnant heifers had normal pregnancies and gave birth to healthy calves. Obtaining a Marketing Authorisation for a cattle BCG vaccine is a critical milestone in the progress towards the eventual use of BCG vaccination in cattle as an additional bTB control tool within the UK; these pivotal GLP vaccine safety studies generated the detailed and essential target animal safety data needed to support this.

Mycobacterium bovis Infection through Inhalation in Children in the Same Family

Mycobacterium bovis Infection through Inhalation in Children in the Same Family

Selenium nanoparticles enhance mucosal immunity against Mycobacterium bovis infection

Selenium nanoparticles (SeNPs) enhance the immune response as adjuvants, increasing the efficacy of viral vaccines, including those for COVID-19. However, the efficiency of mucosal SeNPs in boosting vaccine-induced protective immunity against tuberculosis remains unclear. Therefore, this study aims to investigate whether the combination of SeNPs with the AH antigen (Ag85A-HspX) can boost respiratory mucosal immunity and thereby enhance the protective effects against tuberculosis. We synthesized SeNPs and assessed their impact on the immune response and protection against Mycobacterium bovis (M. bovis) as a mucosal adjuvant in mice, administered intranasally at a dose of 20 µg. SeNPs outperformed polyinosinic-polycytidylic acid (Poly IC) in stimulating the maturation of bone marrow-derived dendritic cells (BMDCs), which enhanced antigen presentation. SeNPs significantly activated and proliferated tissue-resident memory T cells (TRMs) and effector CD4+ T cells in the lungs. The vaccines elicited specific antibody responses in the respiratory tract and stimulated systemic Th1 and Th17 immune responses. Immunization with AH and SeNPs led to higher levels of mucosal secretory IgA in bronchoalveolar lavage fluid (BALF) and secretory IL-17 in splenocytes. Moreover, SeNPs immunized mice showed reduced M. bovis infection loads and inflammatory lesions in the lungs post-challenge. Notably, immunization with AH and SeNPs significantly reduced bacterial load in the lungs, achieving the lowest levels compared to all other tested groups. This study calls for pre-clinical investigation of AHB-SeNPs as an anti-bovine tuberculosis vaccine and for exploring its human vaccine potential, which is anticipated to aid in the development of innovative vaccines or adjuvants.

Impact of Hypoxia-Inducible Factor-1α on Host Immune Metabolism and Tissue Damage During Mycobacterium bovis Infection

Abstract Hypoxia-inducible factor-1α (HIF-1α) is a pivotal regulator of metabolic and inflammatory responses. This study investigated the role of HIF-1α in Mycobacterium bovis infection and its effects on host immune metabolism and tissue damage. We evaluated the expression of immunometabolism markers and matrix metalloproteinases (MMPs) in cells infected with M. bovis, and following HIF-1α inhibition in vitro. To understand the implications of HIF-1α inhibition on disease progression, mice at different infection stages were treated with the HIF-1α inhibitor, YC-1. Our results revealed an upregulation of HIF-1α in macrophages after M. bovis infection, facilitating enhanced M1 macrophage polarization. Blockade of HIF-1α moderated these responses but escalated MMP activity, hindering bacterial control. Consistent with our in vitro results, early-stage treatment of mice with YC-1 aggravated pathological alterations and tissue damage, while late-stage HIF-1α inhibition proved beneficial in managing the disease. Our findings underscored the nuanced role of HIF-1α across different phases of M. bovis infection.

Tuberculosis in wild animals in India.

India is renowned for its complex megadiverse ecosystems and abundant biodiversity. Bovine tuberculosis (bTB) often remains synonymous with Mycobacterium bovis infection in cattle. The domain of tuberculosis (TB) among wild animals, induced by members of the Mycobacterium tuberculosis complex organisms (MTBC), is often underexplored and underreported in India. Within this context, instances of wild animal tuberculosis (wTB) have manifested across both captive and free-roaming animals. The sources contributing to wTB in animals can be human, animal, or environmental factors, thus illuminating the complex transmission pathways. The diagnosis of wTB continues to pose a formidable challenge, a consequence of the expansive taxonomic diversity in both the host and the pathogen. Complications inherent in acquiring samples from wildlife, the absence of standardized diagnostic protocols, limited insights into infection prevalence, and resource constraints compound diagnosis. Amidst these, adopting the comprehensive One Health paradigm surfaces as an imperative, accentuating the interconnectedness bridging human, animal, and environmental health. Recognizing key stakeholders and fostering intersectoral collaboration to provide enhanced diagnostic techniques driven by skilled personnel and advanced infrastructure play pivotal roles in a comprehensive strategy. Additionally, leveraging vaccination efforts contributes to effective control. A national wTB surveillance program is a cornerstone, ensuring an integrated and holistic approach to disease management. Through this review, we delve into the current landscape of wTB in India, unveiling its multifaceted challenges, and further explore the multifarious strategies that the One Health approach proffers in this dynamic endeavor.

Mycobacterium bovis vaccination and subsequent experimental infection outcomes are associated with changes in vitamin D status in dairy calves.

Investigations into the role of vitamin D (vitD) in the immune response of cattle are limited. The objectives of this study were therefore to investigate the association between circulating vitD concentration, TB vaccination and Mycobacterium bovis (M. bovis) infection outcomes in 24 dairy calves (<8 weeks old) which were housed throughout and fed a bodyweight-based allowance. The study design incorporated 2 phases: vaccination (experimental wk 0–52) and experimental infection phase (wk 52–65). Vaccinated calves (n = 12) received a subcutaneous injection of a live-attenuated TB strain at wk 0, whereas unvaccinated ones (n = 12) were injected with saline. All animals were infected with 7,600 cfu of M. bovis 52 weeks post-vaccination, and lung and lymph nodes tissues were assessed for pathology following euthanasia after wk 65. Blood samples were taken throughout wk 0–65. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were measured using a commercial ELISA. A mixed-effects linear regression model revealed significantly higher 25(OH)D concentrations in the vaccinates post-infection (wk 65) compared with the non-vaccinates. Linear regression analysis between 25(OH)D concentration and the level of M. bovis-driven pathology revealed a negative linear relationship i.e., higher concentrations were associated with lower pathology scores, irrespective of vaccination status. No correlation was detected between interferon-γ cytokine production and vitD concentration. Overall, the results support a significant role for vitD in the development of effective immunity of cattle against M. bovis. Gaining insight into the interaction between TB vaccination, M. bovis infection and vitD could potentially guide the optimization of vaccination protocols and future TB control strategies.

Negative Regulation of Autophagy during Macrophage Infection by Mycobacterium bovis BCG via Protein Kinase C Activation.

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.

Intra- and interspecies infectious neighbourhoods as determinant parameters for Mycobacterium bovis infection among badgers in southwestern France

The epidemiological system for Mycobacterium bovis in France involves cattle and, in some areas, wildlife species (mainly badgers and wild boar). This multi-host aspect complicates the control and eradication prospects for bovine tuberculosis in endemic areas, despite the surveillance and control measures implemented for decades in this officially tuberculosis-free European country. To improve control measures, and to manage spillback transmission from badgers to cattle, it is necessary to clarify the transmission mechanisms of M. bovis in these epidemiological systems. We modelled a badger population from a southwestern endemic area by a Dirichlet tessellation based on a sett census conducted by local hunters and trappers between 2013 and 2015. We then used a logistic regression model to test the association between the infection status of setts and computed variables depicting three types of transmission (intraspecific, interspecific and landscape-associated). The apparent prevalence of infected setts was of 40.5%. Two variables were significantly associated with the probability for a sett to be infected: the proportion of neighbouring setts that were infected (OR: 3.19 [2.04–5.17]95%) and the presence of nearby pastures belonging to an infected farm (OR: 2.33 [1.13–4.89]95%]. While badger culling measures have been implemented according to the national TB control plan in the study area since 2012 (in the vicinity of infected farms and their pastures), our results clearly highlight the need to reinforce measures aimed at reducing both intraspecific and interspecific infection pressure. For this purpose, the promising prospect of badger vaccination could be considered, along with biosecurity measures.

Temporal dynamics of the early immune response following Mycobacterium bovis infection of cattle

Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.

Assessing the dynamics of Mycobacterium bovis infection in three French badger populations

Assessing the dynamics of Mycobacterium bovis infection in three French badger populations

Exploring the genetic factors behind the discrepancy in resistance to bovine tuberculosis between African zebu cattle and European taurine cattle

Caused by the pathogenic agent Mycobacterium bovis, bovine tuberculosis (bTB) is a major concern in cattle breeding due to both its zoonotic potential and economic impact. Greater resistance to this disease has been reported in certain African zebu breeds compared to European taurine breeds. However the genetic basis for the lower susceptibility to bTB infection observed in zebu cattle remains poorly explored. This study was conducted on whole genome sequencing data of three bTB infection-resistant African zebu breeds and two bTB infection-susceptible taurine breeds to decipher the genetic background. A set of four selection signature statistics based on linkage disequilibrium, site frequency spectrum, and population differentiation were used on SNPs whereas between population variance based VST and t-test were used on CNVs. As a complement, genes from previous literature reported as candidate genes for bTB resistance were also inspected to identify genetic variations. Interestingly, the resulting nine candidate genes had deleterious missense variants (SHC3, IFNGR1, TLR2, TLR6, IL1A, LRRK2, EP300 and IRAK4) or a CNV difference (CD48) segregating between the groups. The genes found in the study play a role in immune pathways activated during Mycobacterium infection, contributing to the proliferation of immune cells and the granuloma formation, ultimately modulating the outcome of the infectious event. In particular, a deleterious variant in the LRRK2 gene, whose deficiency has been linked to improved prognosis upon tuberculosis infection, was found in the bTB infection-resistant zebu breeds. Therefore, these genes constitute credible candidates in explaining the discrepancy in Mycobacterium bovis infection susceptibility among different breed.

Retracted: Pathological, Histological, and Molecular Based Investigations Confirm Novel Mycobacterium bovis Infection in Boselaphus tragocamelus.

[This retracts the article DOI: 10.1155/2022/7601463.].

Antemortem detection of Mycobacterium bovis in nasal swabs from African rhinoceros

Mycobacterium bovis (M. bovis) infection has been identified in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros populations in Kruger National Park, South Africa. However, it is unknown whether M. bovis infected rhinoceros, like humans and cattle, can shed mycobacteria in respiratory secretions. Limited studies have suggested that rhinoceros with subclinical M. bovis infection may present minimal risk for transmission. However, recent advances that have improved detection of Mycobacterium tuberculosis complex (MTBC) members in paucibacillary samples warranted further investigation of rhinoceros secretions. In this pilot study, nasal swab samples from 75 rhinoceros with defined infection status based on M. bovis antigen-specific interferon gamma release assay (IGRA) results were analysed by GeneXpert MTB/RIF Ultra, BACTEC MGIT and TiKa–MGIT culture. Following culture, speciation was done using targeted PCRs followed by Sanger sequencing for mycobacterial species identification, and a region of difference (RD) 4 PCR. Using these techniques, MTBC was detected in secretions from 14/64 IGRA positive rhinoceros, with viable M. bovis having been isolated in 11 cases, but not in any IGRA negative rhinoceros (n = 11). This finding suggests the possibility that MTBC/M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment.

Host factor RBMX2 promotes epithelial cell apoptosis by downregulating APAF-1's Retention Intron after Mycobacterium bovis infection.

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.

Label-Free Citrate-Stabilized Silver Nanoparticles-Based, Highly Sensitive, Cost-Effective, and Rapid Visual Method for the Differential Detection of Mycobacterium tuberculosis and Mycobacterium bovis.

Tuberculosis poses a global health challenge, and it demands improved diagnostics and therapies. Distinguishing between Mycobacterium tuberculosis (M. tb) and Mycobacterium bovis (M. bovis) infections holds critical "One Health" significance due to the zoonotic nature of these infections and inherent resistance of M. bovis to pyrazinamide, a key part of the directly observed treatment, short-course (DOTS) regimen. Furthermore, most of the currently used molecular detection methods fail to distinguish between the two species. To address this, our study presents an innovative molecular-biosensing strategy. We developed a label-free citrate-stabilized silver nanoparticle aggregation assay that offers sensitive, cost-effective, and swift detection. For molecular detection, genomic markers unique to M. tb and M. bovis were targeted using species-specific primers. In addition to amplifying species-specific regions, these primers also aid the detection of characteristic deletions in each of the mycobacterial species. Post polymerase chain reaction (PCR), we compared two highly sensitive visual detection methods with respect to the traditional agarose gel electrophoresis. The paramagnetic bead-based bridging flocculation assay successfully discriminates M. tb from M. bovis with a sensitivity of ∼40 bacilli. The second strategy exploits citrate-stabilized silver nanoparticles, which aggregate in the absence of amplified dsDNA on the addition of sodium chloride (NaCl). This technique enables the precise, sensitive, and differential detection of as few as ∼4 bacilli. Our study hence advances tuberculosis detection, overcoming the challenges of M. tb and M. bovis differentiation and offering a quicker alternative to time-consuming methods.

Comparative proteome analysis revealed the differences in response to both Mycobacterium tuberculosis and Mycobacterium bovis infection of bovine alveolar macrophages.

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

Spatial association of Mycobacterium bovis infection in cattle and badgers at the pasture interface in an endemic area in France.

Despite control and surveillance programmes, Mycobacterium bovis, the main aetiologic agent of bovine tuberculosis (bTB), is still detected on cattle farms and in wildlife populations in France, especially in badgers in the French Côte-d'Or département. The aim of our study was to find out if infected badgers were trapped significantly closer to pastures of infected farms than non-infected badgers and, if so, to determine the most efficient distance around those pastures for badger trapping, particularly for surveillance purposes. We studied two subareas (southern and northern), chosen based on natural barriers to badger movements and according to the presence of pastures belonging to infected farms (POIFs) and infected or non-infected badgers. In each subarea, we computed the shortest distances D0 and D between badgers trapped a given year n between 2015 and 2019 (n=59 infected and n=1535 non-infected badgers for D0; n=53 infected and n=1476 non-infected badgers for D) and POIFs designated as infected between the year n - 4 and n+1 (respectively n=373 and n=388 POIFs). D0 was calculated without considering spoligotypes, while D was calculated considering the possible epidemiological link between infected badgers and POIFs by using bTB spoligotype information. Then, we computed the observed mean and median of the D0 and D distances and used a bootstrap analysis to test if infected badgers were found significantly closer to POIFs than non-infected badgers. We observed that infection of badgers was not independent of distance from POIF in both subareas but distances (D0 or D) were different between the northern and southern subarea. In the northern subarea, which displays a mosaic landscape (mean and median D distances were respectively 612m and 303m for infected badgers), infected badgers indeed were trapped closer to POIFs, considering D0 and D. In the southern subarea, predominantly forested, infected badgers were significantly closer to POIFs than non-infected badgers when considering D0 but not for D (mean and median D distances were respectively 7148m and 4831m for infected badgers). These results will help to determine the most efficient distance from POIFs to trap badgers to determine their infection status in countryside landscapes. They also highlight the need to better understand the epidemiological systems at play in more forested landscapes where badgers may behave differently or other susceptible sympatric wild species might play a more important role in the circulation of M. bovis, both phenomena contributing to badger infection at greater distances from POIFs.

"Spotting" Mycobacterium bovis infection in leopards (Panthera pardus) - novel application of diagnostic tools.

Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (TB) which poses a threat to many of South Africa's most iconic wildlife species, including leopards (Panthera pardus). Due to limited tests for wildlife, the development of accurate ante-mortem tests for TB diagnosis in African big cat populations is urgently required. The aim of this study was to evaluate currently available immunological assays for their ability to detect M. bovis infection in leopards. Leopard whole blood (n=19) was stimulated using the QuantiFERON Gold Plus In-Tube System (QFT) to evaluate cytokine gene expression and protein production, along with serological assays. The GeneXpert® MTB/RIF Ultra (GXU®) qPCR assay, mycobacterial culture, and speciation by genomic regions of difference PCR, was used to confirm M. bovis infection in leopards. Mycobacterium bovis infection was confirmed in six leopards and individuals that were tuberculin skin test (TST) negative were used for comparison. The GXU® assay was positive using all available tissue homogenates (n=5) from M. bovis culture positive animals. Mycobacterium bovis culture-confirmed leopards had greater antigen-specific responses, in the QFT interferon gamma release assay, CXCL9 and CXCL10 gene expression assays, compared to TST-negative individuals. One M. bovis culture-confirmed leopard had detectable antibodies using the DPP® Vet TB assay. Preliminary results demonstrated that immunoassays and TST may be potential tools to identify M. bovis-infected leopards. The GXU® assay provided rapid direct detection of infected leopards. Further studies should aim to improve TB diagnosis in wild felids, which will facilitate disease surveillance and screening.

Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) 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 M. bovis 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 M. bovis 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.