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1.
Microb Pathog ; 134: 103574, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31170450

RESUMO

The present study was aimed to assess the prevalence and efficiency of techniques for the diagnosis of bovine tuberculosis (bTB) including enzyme-linked immunosorbent assay (ELISA), Gamma interferon assay (IFN-γ) and polymerase chain reaction (PCR) in comparison to skin tuberculin test and culture technique. A total of 2600 cross-breed dairy cattle in Menoufia and Daqahlia governorates were tested by the single intradermal tuberculin test where the disease prevalence was 1.8%. Serum and whole blood samples were collected from positive tuberculin reactors for ELISA and IFN-γ assay, respectively. After slaughtering of positive tuberculin reactors, the post-mortem examination was carried out and tissue samples were collected for the bacteriological examination and PCR. The percentage of visible lesions of tuberculin reactors was 78.7%, while non-visible lesions were 21.27%. Culture technique revealed that the percentage of bTB was 63.8%. The ELISA and IFN-γ assay using short-term culture filtrate (ST-CF) prepared antigen revealed higher sensitivity (72.3% and 82.9%) than the bovine purified protein derivative (PPD-B) antigen. Although prepared ST-CF antigen has great efficiency and eligibility for the diagnosis of bTB, PCR appeared to have a higher sensitivity (85.1%) than other diagnostic methods when dealing with post-mortem samples. Gamma interferon assay using ST-CF antigen is recommended for antemortem diagnosis of bTB in cattle.


Assuntos
Técnicas Bacteriológicas/métodos , Interferon gama/sangue , Mycobacterium bovis/isolamento & purificação , Tuberculose Bovina/diagnóstico , Animais , Bovinos , Técnicas de Cultura/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium bovis/imunologia , Reação em Cadeia da Polimerase/métodos , Prevalência , Sensibilidade e Especificidade , Tuberculina , Teste Tuberculínico/métodos , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia
2.
Cells ; 8(5)2019 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-31060300

RESUMO

Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis (Mtb) complex causing bovine tuberculosis (TB) and imposing a high zoonotic threat to human health. Kallikreins (KLKs) belong to a subgroup of secreted serine proteases. As their role is established in various physiological and pathological processes, it is likely that KLKs expression may mediate a host immune response against the M. bovis infection. In the current study, we report in vivo and in vitro upregulation of KLK12 in the M. bovis infection. To define the role of KLK12 in immune response regulation of murine macrophages, we produced KLK12 knockdown bone marrow derived macrophages (BMDMs) by using siRNA transfection. Interestingly, the knockdown of KLK12 resulted in a significant downregulation of autophagy and apoptosis in M. bovis infected BMDMs. Furthermore, we demonstrated that this KLK12 mediated regulation of autophagy and apoptosis involves mTOR/AMPK/TSC2 and BAX/Bcl-2/Cytochrome c/Caspase 3 pathways, respectively. Similarly, inflammatory cytokines IL-1ß, IL-6, IL-12 and TNF-α were significantly downregulated in KLK12 knockdown macrophages but the difference in IL-10 and IFN-ß expression was non-significant. Taken together, these findings suggest that upregulation of KLK12 in M. bovis infected murine macrophages plays a substantial role in the protective immune response regulation by modulating autophagy, apoptosis and pro-inflammatory pathways. To our knowledge, this is the first report on expression and the role of KLK12 in the M. bovis infection and the data may contribute to a new paradigm for diagnosis and treatment of bovine TB.


Assuntos
Apoptose , Autofagia , Imunidade Inata , Calicreínas/metabolismo , Macrófagos/patologia , Mycobacterium bovis/fisiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Bovinos , Citocinas/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Células RAW 264.7 , Transdução de Sinais , Tuberculose Bovina/patologia
3.
Prev Vet Med ; 168: 52-59, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31097123

RESUMO

The IFN-γ (interferon gamma) assay is used in Ireland as an ancillary diagnostic test to the single intradermal comparative tuberculin test (SICTT) to maximise the detection of Mycobacterium bovis infected animals (bTB) in cattle herds. Understanding the relationships between herd and animal risk factors and IFN-γ test results is critical to enable the development and evaluation of policy measures on how best to use the test. In this study, we set out to characterise Irish herds with IFN-γ test positive animals in terms of herd size, number of SICTT reactors and number of IFN-γ positive tests, and to evaluate the IFN-γ test in terms of the test cut-off values. The results showed that larger herds with more SICTT reactors were likely to have more IFN-γ positives in the herd, and herds with an IFN-γ test positive animal that was also positive for bTB lesions at post-mortem had higher numbers of IFN-γ positive animals in the herd. Raising the cut-off values for the IFN-γ test only marginally decreased the combined sensitivity of the IFN-γ and the SICTT for diagnosis of bTB lesioned animals. The analysis has provided valuable information on the performance of the IFN-γ test as it is used under current bTB infection levels in Ireland.


Assuntos
Interferon gama/sangue , Mycobacterium bovis , Tuberculose Bovina/diagnóstico , Animais , Bovinos , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/veterinária , Irlanda , Sensibilidade e Especificidade , Teste Tuberculínico/veterinária , Tuberculose Bovina/sangue , Tuberculose Bovina/imunologia
4.
Vet Microbiol ; 230: 1-6, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827373

RESUMO

Bovine tuberculosis (BTB) is a disease of economic and zoonotic importance caused mainly by Mycobacterium bovis. In addition to the tuberculin skin test, an interferon-gamma (IFN-γ) release assay (IGRA) blood test has been incorporated in the BTB control programs of numerous countries as an ancillary test to the skin test. A potential disadvantage of the IGRA assay is that it relies solely on the measurement of a single readout (i.e. IFN-γ) for the detection of BTB. In this study we have assessed the practical use of CXCL10 as an additional biomarker for the diagnosis of BTB in the setting of the current testing approach alongside IGRA. To do so, we have assessed both IFN-γ and CXCL10 readouts in blood cultures from a variety of different BTB cattle groups stimulated with standard tuberculin reagents and also with more specific defined antigens (ESAT-6, CFP-10 and Rv3615c). When using a tuberculin based whole blood assay, CXCL10 alone could not substitute for IFN-γ as the analyte measured in the test without reducing the sensitivity of detecting BTB animals. However, when used as an additional test readout, CXCL10 identified BTB animals that failed to induce IFN-γ responses. When tested in non-infected animals, the use of the dual biomarker system had the potential to lower overall test specificity, however this could be overcome by raising the cut-off values for CXCL10 test positivity. Taken together, the results demonstrate that in particular settings, measurement of CXCL10 has the potential to complement the current use of IFN-γ in blood assays to maximise the detection of BTB.


Assuntos
Quimiocina CXCL10/sangue , Interferon gama/sangue , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Biomarcadores/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática/veterinária , Testes de Liberação de Interferon-gama/veterinária , Mycobacterium bovis/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/veterinária , Teste Tuberculínico/veterinária , Reino Unido
5.
PLoS One ; 14(2): e0212751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794653

RESUMO

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine for use against tuberculosis (TB); however, it is known to reduce childhood mortality from infections other than TB. The unspecific protection induced by BCG vaccination has been associated with the induction of memory-like traits of the innate immune system identified as 'trained' immunity. In humans and mouse models, in vitro and in vivo BCG training leads to enhanced production of monocyte-derived proinflammatory cytokines in response to secondary unrelated bacterial and fungal pathogens. While BCG has been studied extensively for its ability to induce innate training in humans and mouse models, BCG's nonspecific protective effects have not been defined in agricultural species. Here, we show that in vitro BCG training induces a functional change in bovine monocytes, characterized by increased transcription of proinflammatory cytokines upon restimulation with the toll-like receptor agonists. Importantly, in vivo, aerosol BCG vaccination in young calves also induced a 'trained' phenotype in circulating peripheral blood mononuclear cells (PBMCs), that lead to a significantly enhanced TLR-induced proinflammatory cytokine response and changes in cellular metabolism compared to PBMCs from unvaccinated control calves. Similar to the long-term training effects of BCG reported in humans, our results suggest that in young calves, the effects of BCG induced innate training can last for at least 3 months in circulating immune populations. Interestingly, however, aerosol BCG vaccination did not 'train' the innate immune response at the mucosal level, as alveolar macrophages from aerosol BCG vaccinated calves did not mount an enhanced inflammatory response to secondary stimulation, compared to cells isolated from control calves. Together, our results suggest that, like mice and humans, the innate immune system of calves can be 'trained'; and that BCG vaccination could be used as an immunomodulatory strategy to reduce disease burden in juvenile food animals before the adaptive immune system has fully matured.


Assuntos
Imunidade Inata , Imunidade nas Mucosas , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/farmacologia , Tuberculose Bovina/imunologia , Vacinação , Aerossóis , Animais , Bovinos , Citocinas/imunologia , Feminino , Humanos , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Receptores Toll-Like/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/prevenção & controle
6.
Int J Mol Sci ; 20(4)2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30791397

RESUMO

Cyclic GMP-AMP synthase (cGAS) is an important cytosolic DNA sensor that plays a crucial role in triggering STING-dependent signal and inducing type I interferons (IFNs). cGAS is important for intracellular bacterial recognition and innate immune responses. However, the regulating effect of the cGAS pathway for bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis (M. bovis) infection is still unknown. We hypothesized that the maturation and activation of BMDCs were modulated by the cGAS/STING/TBK1/IRF3 signaling pathway. In this study, we found that M. bovis promoted phenotypic maturation and functional activation of BMDCs via the cGAS signaling pathway, with the type I IFN and its receptor (IFNAR) contributing. Additionally, we showed that the type I IFN pathway promoted CD4⁺ T cells' proliferation with BMDC during M. bovis infection. Meanwhile, the related cytokines increased the expression involved in this signaling pathway. These data highlight the mechanism of the cGAS and type I IFN pathway in regulating the maturation and activation of BMDCs, emphasizing the important role of this signaling pathway and BMDCs against M. bovis. This study provides new insight into the interaction between cGAS and dendritic cells (DCs), which could be considered in the development of new drugs and vaccines against tuberculosis.


Assuntos
Células Dendríticas/imunologia , Fator Regulador 3 de Interferon/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium bovis , Nucleotidiltransferases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tuberculose Bovina/imunologia , Tuberculose Bovina/metabolismo , Animais , Bovinos , Diferenciação Celular , Células Dendríticas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Interferon Tipo I/metabolismo , Camundongos , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Tuberculose Bovina/microbiologia
7.
Int J Mol Sci ; 20(1)2018 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-30577452

RESUMO

It is widely accepted that different strains of Mycobacterium tuberculosis have variable degrees of pathogenicity and induce different immune responses in infected hosts. Similarly, different strains of Mycobacterium bovis have been identified but there is a lack of information regarding the degree of pathogenicity of these strains and their ability to provoke host immune responses. Therefore, in the current study, we used a mouse model to evaluate various factors involved in the severity of disease progression and the induction of immune responses by two strains of M. bovis isolated from cattle. Mice were infected with both strains of M. bovis at different colony-forming unit (CFU) via inhalation. Gross and histological findings revealed more severe lesions in the lung and spleen of mice infected with M. bovis N strain than those infected with M. bovis C68004 strain. In addition, high levels of interferon-γ (IFN-γ), interleukin-17 (IL-17), and IL-22 production were observed in the serum samples of mice infected with M. bovis N strain. Comparative genomic analysis showed the existence of 750 single nucleotide polymorphisms and 145 small insertions/deletions between the two strains. After matching with the Virulence Factors Database, mutations were found in 29 genes, which relate to 17 virulence factors. Moreover, we found an increased number of virulent factors in M. bovis N strain as compared to M. bovis C68004 strain. Taken together, our data reveal that variation in the level of pathogenicity is due to the mutation in the virulence factors of M. bovis N strain. Therefore, a better understanding of the mechanisms of mutation in the virulence factors will ultimately contribute to the development of new strategies for the control of M. bovis infection.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Mycobacterium bovis , Tuberculose Bovina/genética , Tuberculose Bovina/microbiologia , Animais , Biópsia , Bovinos , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/genética , Pulmão/patologia , Camundongos , Tipagem de Sequências Multilocus , Mutação , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Polimorfismo de Nucleotídeo Único , Baço/patologia , Tuberculose Bovina/imunologia , Virulência/genética , Fatores de Virulência
8.
Vet Immunol Immunopathol ; 203: 52-56, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30243373

RESUMO

Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis (M. bovis), is a major economic disease of livestock worldwide. Vaccination is considered as a potentially sustainable adjunct to the current control strategy. Cattle vaccination with the live attenuated M. bovis bacillus Calmette-Guerin (BCG) confers variable protection; the reasons for this variability are not understood. Indoleamine 2, 3-dioxygenase (IDO), through the catalysis of tryptophan, is thought to have an immunoregulatory role in the immune response to Mycobacterium tuberculosis (M. tuberculosis). In this work, we used immunohistochemistry and digital image analysis to evaluate the presence of IDO in granulomas at different stages of development in cattle that had been BCG-vaccinated or not and then challenged with M. bovis. Our results show that the expression of IDO in granulomas from non-vaccinated M. bovis challenged animals is higher than in granulomas from BCG-vaccinated M. bovis challenged animals. Thus, it is possible that vaccination with BCG prevents the induction of what are thought to be host immunosuppressive pathways by M. bovis, which contribute to pathology during the disease.


Assuntos
Vacina BCG/imunologia , Granuloma/veterinária , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Mycobacterium bovis/imunologia , Tuberculose Bovina/enzimologia , Animais , Vacina BCG/farmacologia , Bovinos , Granuloma/enzimologia , Granuloma/imunologia , Granuloma/metabolismo , Linfonodos/enzimologia , Linfonodos/metabolismo , Tuberculose Bovina/imunologia , Tuberculose Bovina/metabolismo
9.
PLoS One ; 13(7): e0201253, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30063728

RESUMO

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)-an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor-has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants.


Assuntos
Adjuvantes Imunológicos/farmacologia , Memória Imunológica/efeitos dos fármacos , Lectinas Tipo C/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Receptor 3 Toll-Like/imunologia , Vacinas contra a Tuberculose/farmacologia , Adjuvantes Imunológicos/química , Animais , Bovinos , Masculino , Paratuberculose/imunologia , Paratuberculose/patologia , Paratuberculose/prevenção & controle , Linfócitos T/patologia , Vacinas contra a Tuberculose/química , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/patologia , Tuberculose Bovina/prevenção & controle
10.
Vet Res ; 49(1): 69, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-30021619

RESUMO

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host-pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.


Assuntos
Fator de Indução de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Bovinos/fisiologia , Endodesoxirribonucleases/metabolismo , Macrófagos/virologia , Tuberculose Bovina/imunologia , Animais , Caspases/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Mycobacterium bovis/fisiologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores
11.
Tuberculosis (Edinb) ; 111: 143-146, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30029900

RESUMO

ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes -esxA and esxB - remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings.


Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Mutação , Mycobacterium bovis/genética , Polimorfismo Genético , Tuberculose Bovina/microbiologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Sequência de Bases , Bovinos , Sequência Conservada , Genótipo , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Fenótipo , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Virulência/genética
12.
Elife ; 72018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29866255

RESUMO

Vaccination of cattle against bovine Tuberculosis (bTB) has been a long-term policy objective for countries where disease continues to persist despite costly test-and-slaughter programs. The potential use of vaccination within the European Union has been linked to a need for field evaluation of any prospective vaccine and the impact of vaccination on the rate of transmission of bTB. We calculate that estimation of the direct protection of BCG could be achieved with 100 herds, but over 500 herds would be necessary to demonstrate an economic benefit for farmers whose costs are dominated by testing and associated herd restrictions. However, the low and variable attack rate in GB herds means field trials are unlikely to be able to discern any impact of vaccination on transmission. In contrast, experimental natural transmission studies could provide robust evaluation of both the efficacy and mode of action of vaccination using as few as 200 animals.


Assuntos
Controle de Doenças Transmissíveis/métodos , Mycobacterium bovis/imunologia , Tuberculose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Bovinos , Tuberculose Bovina/imunologia , Tuberculose Bovina/transmissão
13.
Vet Immunol Immunopathol ; 196: 35-47, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29695323

RESUMO

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON®-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Interferon gama/farmacologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium bovis/imunologia , Paratuberculose/imunologia , Teste Tuberculínico/veterinária , Tuberculose Bovina/imunologia , Animais , Formação de Anticorpos/imunologia , Bovinos , Testes de Liberação de Interferon-gama/veterinária , Masculino , Paratuberculose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Tuberculose Bovina/diagnóstico
14.
Microb Genom ; 4(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29557774

RESUMO

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection.


Assuntos
Imunidade Inata , Macrófagos Alveolares , Mycobacterium bovis , Mycobacterium tuberculosis , Transcriptoma , Tuberculose Bovina , Tuberculose Pulmonar , Animais , Bovinos , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Proteômica , Tuberculose Bovina/genética , Tuberculose Bovina/imunologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia
15.
Vet Microbiol ; 214: 89-92, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29408038

RESUMO

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.


Assuntos
Anticorpos Antibacterianos/sangue , Bovinos/imunologia , Cervos/imunologia , Elefantes/imunologia , Tuberculose/veterinária , Animais , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/sangue , Proteínas de Bactérias/imunologia , Bovinos/microbiologia , Cervos/microbiologia , Elefantes/microbiologia , Imunoensaio/métodos , Imunoglobulina G/sangue , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/imunologia , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/imunologia , Estados Unidos/epidemiologia
16.
Sci Rep ; 8(1): 894, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343690

RESUMO

The Mycobacterium tuberculosis complex (MTBC) is the collective term given to the group of bacteria that cause tuberculosis (TB) in mammals. It has been reported that M. tuberculosis H37Rv, a standard reference MTBC strain, is attenuated in cattle compared to Mycobacterium bovis. However, as M. tuberculosis H37Rv was isolated in the early 1930s, and genetic variants are known to exist, we sought to revisit this question of attenuation of M. tuberculosis for cattle by performing a bovine experimental infection with a recent M. tuberculosis isolate. Here we report infection of cattle using M. bovis AF2122/97, M. tuberculosis H37Rv, and M. tuberculosis BTB1558, the latter isolated in 2008 during a TB surveillance project in Ethiopian cattle. We show that both M. tuberculosis strains caused reduced gross pathology and histopathology in cattle compared to M. bovis. Using M. tuberculosis H37Rv and M. bovis AF2122/97 as the extremes in terms of infection outcome, we used RNA-Seq analysis to explore differences in the peripheral response to infection as a route to identify biomarkers of progressive disease in contrast to a more quiescent, latent infection. Our work shows the attenuation of M. tuberculosis strains for cattle, and emphasizes the potential of the bovine model as a 'One Health' approach to inform human TB biomarker development and post-exposure vaccine development.


Assuntos
Bacillus/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Bovina/imunologia , Tuberculose/imunologia , Animais , Biomarcadores/metabolismo , Bovinos , Feminino , Humanos , Tuberculose/metabolismo , Tuberculose/microbiologia , Tuberculose Bovina/metabolismo , Tuberculose Bovina/microbiologia
17.
Microb Pathog ; 115: 343-352, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29197526

RESUMO

The immune signalling genes during the challenge of bovine macrophages with bacterial products derived from tuberculosis causing bacteria in cattle were investigated in the present study. An in-vitro cell culture model of bovine monocyte-derived macrophages were challenged to Mycobacterium bovis. Macrophages from healthy and already infected animals can both be fully activated during M. bovis infection. Analysis of mRNA abundance in peripheral blood mononuclear cells from M. bovis infected and non-infected cattle were performed as a controls. Cells of treatment were challenged after six days for six hours incubation at 37 °C, with 5% CO2, to total RNA was extracted then cDNA labelling, hybridization and scanning for microarray methods have been developed for microarray based immune related gene expression analysis. The differential expressions twenty genes (IL1, CCL3, CXCR4, TNF, TLR2, IL12, CSF3, CCR5, CCR3, MAPT, NFKB1, CCL4, IL6, IL2, IL23A, CCL20, IL8, CXCL8, TRIP10, CXCL2 and IL1B) implicated in M. bovis response were examined Agilent Bovine_GXP_8 × 60 K microarray platform. Cells of treatment were challenged after six days for six hours incubation then pathways analysis of Toll like receptor and Chemokine signalling pathway study of responsible genes in bovine tuberculosis. The PBMC from M. bovis infected cattle exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel genes expression program due to M. bovis exposure. It will guide future studies, regarding the complex macrophage specific signalling pathways stimulated upon phagocytosis of M. bovis and role of signalling pathways in creating the host immune response to cattle tuberculosis.


Assuntos
Regulação da Expressão Gênica/imunologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Animais , Bovinos , Células Cultivadas , Macrófagos/microbiologia , Fagocitose/genética , RNA Mensageiro/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Tuberculose Bovina/microbiologia
18.
Transbound Emerg Dis ; 65(1): 96-104, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28168855

RESUMO

Bovine tuberculosis (bTB) is highly prevalent in intensive dairy farms of the urban "milk-sheds" in Ethiopia, and vaccination could be a cost-effective disease control strategy. In the present study, the efficacy of Bacillus Calmette-Guerin (BCG) to protect against bTB was assessed in Holstein-Friesian calves in a natural transmission setting. Twenty-three 2-week-old calves were subcutaneously vaccinated with BCG Danish SSI strain 1331, and matched 26 calves were injected with placebo. Six weeks later, calves were introduced into a herd of M. bovis-infected animals (reactors) and kept in contact with them for 1 year. In vitro and in vivo immunological tests were performed to assess immune responses post-vaccination and during exposure. Successful vaccine uptake was confirmed by tuberculin skin test and IFN-γ responses in vaccinated calves. The kinetics of IFN-γ responses to early secretory antigen target 6 and culture filtrate protein 10 (ESAT6 and CFP10, respectively) and tuberculin skin test responses post-exposure suggested that the animals were infected early after being placed in contact with the infected herd as immunological signs of infection were measurable between 2 and 4 months post-initial exposure. Protection was determined by comparing gross and microscopic pathology and bacteriological burden between vaccinated and control calves. BCG vaccination reduced the proportions of tissues with visible pathology in vaccinates compared to control calves by 49% (p < .001) with 56%, 43%, 72%, and 38% reductions in the proportion of lesioned tisues in head, thoracic, abdominal lymph nodes, and lungs, respectively (p-values .029-.0001). In addition, the lesions were less severe grossly and microscopically in vaccinated calves than in non-vaccinated calves (p < .05). The reduction in the overall incidence rates of bTB was 23%, 28%, and 33% on the basis of the absence of gross pathology, M. bovis culture positivity, and histopathology, respectively, in vaccinated animals. In conclusion, BCG vaccination reduced the frequency and severity of the pathology of bTB significantly, which is likely to reduce onwards transmission of the disease.


Assuntos
Vacina BCG/administração & dosagem , Mycobacterium bovis/imunologia , Tuberculose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Animais Recém-Nascidos/imunologia , Anticorpos Antibacterianos/sangue , Bovinos , Etiópia/epidemiologia , Interferon gama , Pulmão/patologia , Linfonodos/patologia , Teste Tuberculínico , Tuberculose Bovina/imunologia , Tuberculose Bovina/transmissão
19.
Front Immunol ; 9: 3159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30804949

RESUMO

Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.


Assuntos
Colectinas/imunologia , Proteínas do Sistema Complemento/imunologia , Mycobacterium bovis/imunologia , Soroglobulinas/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Animais , Biomarcadores , Bovinos , Colectinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Soroglobulinas/metabolismo , Células THP-1 , Tuberculose Bovina/metabolismo
20.
PLoS One ; 12(11): e0188448, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29155877

RESUMO

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.


Assuntos
Anticorpos Antibacterianos/biossíntese , Vacina BCG/administração & dosagem , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Interferon gama/biossíntese , Mycobacterium bovis/efeitos dos fármacos , Tuberculose Bovina/prevenção & controle , Animais , Bovinos , Formaldeído , Temperatura Alta , Esquemas de Imunização , Imunogenicidade da Vacina , Injeções Subcutâneas , Interferon gama/metabolismo , Masculino , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Vacinas Atenuadas , Vacinas Vivas não Atenuadas
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