ABSTRACT
Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite the characterization of this pathogen in the 1800s and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host compared to other pathogenic strains of MBO. In the present study, experimental infections were performed to define attenuation. Whole genome sequencing was completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428, and 95-1315) was performed. Experimental infection studies on five calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all five animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of nine unique SNPs were missense mutations in genes with annotated functions impacting two major MBO survival and virulence pathways: (1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and (2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain's genotype-disease phenotype associations.
ABSTRACT
INTRODUCTION: Zoonoses have recently become an increasing public health problem. Zoonoses are estimated to account for 60% of all emerging infectious diseases. One particularly important zoonosis is human tuberculosis, especially tuberculosis due to Mycobacterium bovis (M. bovis), which is naturally resistant to pyrazinamide (PZA). MATERIAL AND METHODS: The patient had a pulmonary form of tuberculosis accompanied by a cough and fever. At the same time, the disease was also confirmed in 20 out of 25 cattle on the farm. The clinical specimen (sputum) was examined in accordance with the European Union (EU) laboratories' methodology. Tissue materials from cattle were verified in the National Veterinary Research Institute (NVRI), in the Bovine tuberculosis (BTB) Reference Laboratory, Pulawy, Poland and tested in accordance with the guidelines for the laboratory diagnosis of BTB. RESULTS: All M. bovis isolates represented one spoligotype, SB0120. The results of mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) evaluation showed the same genetic pattern. CONCLUSIONS: Findings from this study suggest the first confirmed interspecific transmission of Mycobacterium bovis, between a farmer and his cattle, in Poland. Present findings support the increasing concern regarding zoonotic TB that has been highlighted elsewhere.
ABSTRACT
Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance.
Subject(s)
Cattle Diseases , Mycobacterium bovis , Tuberculosis, Bovine , Cattle , Animals , Tuberculosis, Bovine/diagnosis , Immunoglobulin G , Serologic Tests/veterinary , Immunoglobulin M , Cattle Diseases/diagnosisABSTRACT
Bovine tuberculosis (bTB) control programs can be improved by combined use of tests for humoral and cell-mediated immune responses targeting multiple biomarkers of Mycobacterium bovis. To further the diagnostic benefits of this approach, we used Dual Path Platform (DPP) technology to test sera from cattle with naturally acquired bTB in the United States (US) and Spain for the presence of M. bovis antigen, IgM and/or IgG antibodies to MPB70/MPB83 fusion antigen in conjunction with tuberculin skin tests (TST) or interferon-gamma release assays (IGRA). When TST was complemented with detection of IgM and IgG antibodies, the diagnostic sensitivity increased from 85.4% to 95.1% in the US and from 64.2% to 81.5% in Spain. Likewise, adding the DPP assays enhanced IGRA diagnostic sensitivity from 82.7% to 93.8% in Spain. Detection of circulating M. bovis antigen showed added value when used in combination with the DPP antibody assays but it was limited when analyzed in the context of TST or IGRA results. Present findings support the benefits of a multi-test approach for the ante-mortem diagnosis of bTB in cattle.
Subject(s)
Cattle Diseases , Mycobacterium bovis , Tuberculosis, Bovine , Algorithms , Animals , Biomarkers , Cattle , Immunoglobulin G , Immunoglobulin M , Tuberculin Test/veterinary , Tuberculosis, Bovine/diagnosisABSTRACT
Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (â¼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.
Subject(s)
Serologic Tests , Tuberculosis, Bovine , Animals , Antibodies , Antigens, Bacterial , Cattle , Epitopes, B-Lymphocyte , Mycobacterium bovis/immunology , Polyproteins , Serologic Tests/veterinary , Tuberculosis, Bovine/diagnosisABSTRACT
Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.
Subject(s)
Antibodies, Bacterial/analysis , Immunoglobulin G/analysis , Swine Diseases , Tuberculosis, Bovine , Animals , Cattle , Immunologic Tests/veterinary , Mycobacterium bovis/immunology , Plant Extracts , Swine , Swine Diseases/diagnosis , Swine Diseases/microbiology , Tuberculosis, Bovine/diagnosisABSTRACT
Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.
Subject(s)
Immunity, Innate/genetics , Leukocytes, Mononuclear/metabolism , Mycobacterium bovis/immunology , Transcriptome/genetics , Tuberculosis, Bovine/pathology , Animals , Cattle , Gene Expression/genetics , Gene Expression Profiling/veterinary , Leukocytes, Mononuclear/cytology , Mycobacterium bovis/genetics , Oligonucleotide Array Sequence Analysis , Severity of Illness Index , Tuberculosis, Bovine/immunologyABSTRACT
Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.
Subject(s)
B-Lymphocyte Subsets/immunology , Immunologic Memory , Tuberculosis/immunology , Adaptive Immunity , Animals , Cattle , Humans , Lymphocyte Activation , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculin/administration & dosageABSTRACT
Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.
Subject(s)
Antibodies, Bacterial/immunology , Antigens, Bacterial/immunology , Host-Pathogen Interactions/immunology , Mycobacterium bovis/immunology , Tuberculosis, Bovine/immunology , Tuberculosis, Bovine/microbiology , Animals , Antibodies, Bacterial/blood , Cattle , Immunoassay , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunologyABSTRACT
Mycobacterium bovis is the cause of tuberculosis in most mammalian species, most notably cattle and other members of the family Bovidae; however, many species of the family Cervidae are also susceptible. In North America, tuberculosis has been identified in both captive and free-ranging cervids. Captive cervids are tested for tuberculosis following many of the same guidelines applied to cattle, including intradermal tuberculin testing using M. bovis purified protein derivative (PPD). Both captive and free-ranging deer and elk have been implicated as the source of infection for many cattle herds. Vaccination with the human vaccine M. bovis BCG has been considered as one possible tool to aid in eradication of tuberculosis from cattle and both captive and free-ranging cervids. Studies in cattle have demonstrated that BCG vaccination can induce false positive intradermal tuberculin test reactions in some cattle. Similar findings have been reported for red deer. We orally vaccinated white-tailed deer with BCG and showed that vaccination can induce false positive skin test reactions in some deer and that the rate of false positive reactions is greater with a higher vaccine dose.
Subject(s)
BCG Vaccine/immunology , Deer , Mycobacterium bovis/immunology , Tuberculin Test/veterinary , Tuberculosis/veterinary , Vaccination/veterinary , Animals , Tuberculosis/prevention & controlABSTRACT
Tuberculosis (TB) remains a leading cause of death from infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine TB and zoonotic TB infection. γδ T cells are known to participate in the immune control of mycobacterial infections. Data in human and nonhuman primates suggest that mycobacterial infection regulates memory/effector phenotype and adaptive immune functions of γδ T cells. To date, the impact of M. bovis infection on bovine γδ T cells and their effector and memory differentiation remains unknown. In this study, we show that circulating γδ T cells from M. bovis-infected cattle can be differentiated based on the expression of CD27, which is indicative of their capacity to respond to virulent M. bovis infection: CD27+ γδ T cells proliferated in response to M. bovis Ag and, thus, may comprise the adaptive γδ T cell compartment in cattle. We further show that bovine M. bovis-specific γδ T cells express surface markers characteristic of central memory T cells (CD45R-CD27+CD62Lhi) and that M. bovis-specific CD4 and γδ T cells both upregulate the expression of the tissue-homing receptors CXCR3 and CCR5 during infection. Our studies contribute significantly to our understanding of γδ T cell differentiation during TB infection and provide important insights into the link between phenotypic and functional subsets in the bovine. Accurate characterization of γδ T cell effector and memory-like responses induced during mycobacterial infection will contribute to improved strategies for harnessing the γδ T cell response in protection against TB for humans and animals.
Subject(s)
Leukocyte Common Antigens/metabolism , Mycobacterium bovis/immunology , T-Lymphocyte Subsets/immunology , Tuberculosis, Bovine/immunology , Tuberculosis/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolism , Animals , Cattle , Cell Differentiation , Cell Proliferation , Cells, Cultured , Humans , Immunologic Memory , Lymphocyte Activation , Receptors, Antigen, T-Cell, gamma-delta/genetics , Receptors, CCR5/metabolism , Receptors, CXCR3/metabolismABSTRACT
Tuberculosis (TB) in humans is a global public health concern and the discovery of animal cases of Mycobacterium tuberculosis (Mtb) infection and disease, especially in multi-host settings, also has significant implications for public health, veterinary disease control, and conservation endeavors. This paper describes a fatal case of Mtb disease in a free-ranging African elephant (Loxodonta africana) in a high human TB burden region. Necropsy revealed extensive granulomatous pneumonia, from which Mtb was isolated and identified as a member of LAM3/F11 lineage; a common lineage found in humans in South Africa. These findings are contextualized within a framework of emerging Mtb disease in wildlife globally and highlights the importance of the One Health paradigm in addressing this anthroponotic threat to wildlife and the zoonotic implications.
ABSTRACT
Using multi-antigen print immunoassay and DPP® VetTB Assay approved in the United States for testing captive cervids and elephants, we analyzed antibody recognition of MPB83 and CFP10/ESAT-6 antigens in Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis and in white-tailed deer (Odocoileus virginianus), fallow deer (Dama dama), elk (Cervus elaphus), and cattle (Bos taurus) infected with Mycobacterium bovis. Serum IgG reactivity to MPB83 was found in the vast majority of tuberculous cattle and cervid species among which white-tailed deer and elk also showed significant CFP10/ESAT-6 recognition rates with added serodiagnostic value. In contrast, the infected elephants developed antibody responses mainly to CFP10/ESAT-6 with MPB83 reactivity being relatively low. The findings demonstrate distinct patterns of predominant antigen recognition by different animal hosts in tuberculosis.
Subject(s)
Antibodies, Bacterial/blood , Cattle/immunology , Deer/immunology , Elephants/immunology , Tuberculosis/veterinary , Animals , Antigens, Bacterial/blood , Antigens, Bacterial/immunology , Bacterial Proteins/blood , Bacterial Proteins/immunology , Cattle/microbiology , Deer/microbiology , Elephants/microbiology , Immunoassay/methods , Immunoglobulin G/blood , Mycobacterium bovis/immunology , Mycobacterium tuberculosis/immunology , Serologic Tests/methods , Serologic Tests/veterinary , Tuberculosis/diagnosis , Tuberculosis/epidemiology , Tuberculosis/immunology , Tuberculosis, Bovine/diagnosis , Tuberculosis, Bovine/epidemiology , Tuberculosis, Bovine/immunology , United States/epidemiologyABSTRACT
Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.
Subject(s)
Intraepithelial Lymphocytes/immunology , Tuberculosis, Bovine/immunology , Animals , Bacterial Toxins/metabolism , Cattle , Coculture Techniques/veterinary , Cytokines/metabolism , In Situ Hybridization/veterinary , Lung/immunology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Male , Mycobacterium bovis , Real-Time Polymerase Chain Reaction/veterinary , Sequence Analysis, RNA/veterinary , TranscriptomeABSTRACT
Bovine tuberculosis (TB), caused by Mycobacterium bovis, remains an important zoonotic disease posing a serious threat to livestock and wildlife. The current TB tests relying on cell-mediated and humoral immune responses in cattle have performance limitations. To identify new serodiagnostic markers of bovine TB, we screened a panel of 101 recombinant proteins, including 10 polyepitope fusions, by a multiantigen print immunoassay (MAPIA) with well-characterized serum samples serially collected from cattle with experimental or naturally acquired M. bovis infection. A novel set of 12 seroreactive antigens was established. Evaluation of selected proteins in the dual-path platform (DPP) assay showed that the highest diagnostic accuracy (â¼95%) was achieved with a cocktail of five best-performing antigens, thus demonstrating the potential for development of an improved and more practical serodiagnostic test for bovine TB.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Mycobacterium bovis/immunology , Serologic Tests/methods , Tuberculosis, Bovine/immunology , Animals , Cattle , Immunoassay/methodsABSTRACT
Between 1940 and 2004, more than 335 emerging infectious disease events were reported in the scientific literature. The majority (60%) of these events involved zoonoses, most of which (72%) were of wildlife origin or had an epidemiologically important wildlife host. Because this trend of increasing emerging diseases likely will continue, understanding the pathogenesis, transmission, and diagnosis of these diseases in the relevant wildlife host is paramount. Achieving this goal often requires using wild animals as research subjects, which are vastly different from the traditional livestock or laboratory animals used by most universities and institutions. Using wildlife in infectious disease research presents many challenges but also provides opportunities to answer questions impossible to address by using traditional models. Cervid species, especially white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and red deer (Cervus elaphus), are hosts or sentinels for several important pathogens, some of which are zoonotic. The long history of infectious disease research using white-tailed deer, conducted at ever-increasing levels of sophisticated biosecurity, demonstrates that this type of research can be conducted safely and that valuable insights can be gained. The greatest challenges to using wildlife in infectious disease research include animal source, facility design, nutrition, animal handling, and enrichment and other practices that both facilitate animal care and enhance animal wellbeing. The study of Mycobacterium bovis infection in white-tailed deer at the USDA's National Animal Disease Center serves to illustrate one approach to address these challenges.
Subject(s)
Animal Husbandry , Deer , Mycobacterium bovis/physiology , Tuberculosis/veterinary , Animals , Animals, Wild , Biomedical Research , Containment of Biohazards/veterinary , Deer/classification , Female , MaleABSTRACT
Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge.
Subject(s)
BCG Vaccine , Feces/microbiology , Tuberculosis, Bovine/prevention & control , Volatile Organic Compounds/isolation & purification , Animals , Animals, Domestic , Animals, Wild , Cattle , Humans , Mycobacterium bovis/isolation & purification , Mycobacterium bovis/pathogenicity , Tuberculosis, Bovine/microbiologyABSTRACT
BACKGROUND: Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. RESULTS: Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. CONCLUSIONS: Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.
Subject(s)
Antibodies, Bacterial/blood , Enzyme-Linked Immunosorbent Assay/veterinary , Tuberculosis, Bovine/diagnosis , Animals , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Female , Immunoglobulin G/immunology , Male , Mycobacterium bovis/immunology , Time Factors , Tuberculin Test/veterinaryABSTRACT
Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.
Subject(s)
Blood Chemical Analysis/methods , Lipopolysaccharides/blood , Tuberculosis, Bovine/blood , Animals , Cattle , ImmunoassayABSTRACT
The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.