88,753 research outputs found
Flow cytometric detection of gamma interferon can effectively discriminate Mycobacterium bovis BCG-vaccinated cattle from M. bovis-infected cattle
No full textMycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-gamma) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-gamma-producing lymphocytes by flow cytometric analysis of intracellular IFN-gamma expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-gamma-expressing CD4+ T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4+ T lymphocytes expressing IFN-gamma, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle
Global Gene Expression and Systems Biology Analysis of Bovine Monocyte-Derived Macrophages in Response to In Vitro Challenge with Mycobacterium bovis
No full textpeer-reviewedBackground
Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array.
Results
Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis.
Conclusions
The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.Science Foundation Ireland (www.sfi.ie) Investigator grants (Nos: SFI/01/F.1/B028 and SFI/08/IN.1/B2038); Department of Agriculture, Fisheries and Food (www.agriculture.ie) Research Stimulus Grant (No: RSF 06 405); European Union Framework 7 (http://cordis.europa.eu/fp7) Project Grant (No: KBBE-211602-MACROSYS); Irish Research Council for Science, Engineering and Technology (IRCSET) funded Bioinformatics and
Systems Biology PhD Programme (http://bioinfo-casl.ucd.ie/PhD)
Social group size affects Mycobacterium bovis infection in European badgers (Meles meles)
No full text1. In most social animals, the prevalence of directly transmitted pathogens increases in larger groups and at higher population densities. Such patterns are predicted by models of Mycobacterium bovis infection in European badgers (Meles meles). 2. We investigated the relationship between badger abundance and M. bovis prevalence, using data on 2696 adult badgers in 10 populations sampled at the start of the Randomized Badger Culling Trial. 3. M. bovis prevalence was consistently higher at low badger densities and in small social groups. M. bovis prevalence was also higher among badgers whose genetic profiles suggested that they had immigrated into their assigned social groups. 4. The association between high M. bovis prevalence and small badger group size appeared not to have been caused by previous small-scale culling in study areas, which had been suspended, on average, 5 years before the start of the current study. 5. The observed pattern of prevalence might occur through badgers in smaller groups interacting more frequently with members of neighbouring groups; detailed behavioural data are needed to test this hypothesis. Likewise, longitudinal data are needed to determine whether the size of infected groups might be suppressed by disease-related mortality. 6. Although M. bovis prevalence was lower at high population densities, the absolute number of infected badgers was higher. However, this does not necessarily mean that the risk of M. bovis transmission to cattle is highest at high badger densities, since transmission risk depends on badger behaviour as well as on badger density
Molecular detection of Mycobacterium bovis in the environment
No full textAn investigation was carried out to determine the presence and persistence of
Mycobacterium bovis in the environment. Soil samples were taken in April 2000 from a
farm in Ireland which had undergone a bovine tuberculosis outbreak some four months
prior. Total community DNA was extracted from these samples and PCR carried out
targeted to two genes specific for the Mycobacterium tuberculosis complex; mpb64 and
mpb70. These genes were detected in soil samples taken from entrances to two badger
sets and in soil from two sites where the infected cattle grazed. Further analysis of DNA
using oligonucleotide primers specific for the 16S rRNA genes of slow-growing
mycobacteria was carried out. This revealed the presence of 16S rRNA genes relating to
Mycobacterium bovis. RT-PCR was also carried out using these primers on total
community RNA. Sequences relating to M. bovis were found, indicating that the DNA
and RNA came from viable, intact cells in the soil, and that M. bovis persists in soil for
up to four months after contamination of the soil. Sampling was repeated in November
2002 following a second TB outbreak in January 2001. DNA sequences for mpb64 and
mpb7O were only found in the samples from the badger setts, as were 16S rRNA
sequences.
The survival of the vaccine strain M. bovis BCG was also determined, using soil
microcosms experiments in defined environmental conditions. M. bovis BCG was found
to survive for longest at temperatures of 37°C and at soil wetting levels of 30%.
Culturable cells could not be detected after 60 days, however DNA and RNA relating to
M. bovis BCG could be detected up to 18 months after initial inoculation. This suggests
the cells persisted in a viable non-culturable state. Experiments to determine the rate of
persistence of DNA in soil were carried out. DNA was found to persist for no longer than
10 days in soil.
Diversity studies were carried out on the farm samples and on Warwick soil, to determine
the diversity of the mycobacterial population. 16S rRNA analysis was carried out and
showed the presence ofsequences relating to M. bovis, Al. hiberniae, M. avium, Al. fallax,
and M. farcinogenes
Whole genome sequencing reveals local transmission patterns of mycobacterium bovis in sympatric cattle and badger populations
No full textWhole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled ‘reservoir’ host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers
Genome-wide transcriptional profiling of peripheral blood leukocytes from cattle infected with Mycobacterium bovis reveals suppression of host immune genes
No full textBackground
Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), a pathological infection with significant economic impact. Recent studies have highlighted the role of functional genomics to better understand the molecular mechanisms governing the host immune response to M. bovis infection. Furthermore, these studies may enable the identification of novel transcriptional markers of BTB that can augment current diagnostic tests and surveillance programmes. In the present study, we have analysed the transcriptome of peripheral blood leukocytes (PBL) from eight M. bovis-infected and eight control non-infected age-matched and sex-matched Holstein-Friesian cattle using the Affymetrix® GeneChip® Bovine Genome Array with 24,072 gene probe sets representing more than 23,000 gene transcripts.
Results
Control and infected animals had similar mean white blood cell counts. However, the mean number of lymphocytes was significantly increased in the infected group relative to the control group (P = 0.001), while the mean number of monocytes was significantly decreased in the BTB group (P = 0.002). Hierarchical clustering analysis using gene expression data from all 5,388 detectable mRNA transcripts unambiguously partitioned the animals according to their disease status. In total, 2,960 gene transcripts were differentially expressed (DE) between the infected and control animal groups (adjusted P-value threshold ≤ 0.05); with the number of gene transcripts showing decreased relative expression (1,563) exceeding those displaying increased relative expression (1,397). Systems analysis using the Ingenuity® Systems Pathway Analysis (IPA) Knowledge Base revealed an over-representation of DE genes involved in the immune response functional category. More specifically, 64.5% of genes in the affects immune response subcategory displayed decreased relative expression levels in the infected animals compared to the control group.
Conclusions
This study demonstrates that genome-wide transcriptional profiling of PBL can distinguish active M. bovis-infected animals from control non-infected animals. Furthermore, the results obtained support previous investigations demonstrating that mycobacterial infection is associated with host transcriptional suppression. These data support the use of transcriptomic technologies to enable the identification of robust, reliable transcriptional markers of active M. bovis infection.This work was supported by Investigator Grants from Science Foundation Ireland (Nos: SFI/01/F.1/B028 and SFI/08/IN.1/B2038), a Research Stimulus Grant from the Department of Agriculture, Fisheries and Food (No: RSF 06 405) and a European Union Framework 7 Project Grant (No: KBBE-211602-MACROSYS). KEK is supported by the Irish Research Council for Science, Engineering and Technology (IRCSET) funded Bioinformatics and Systems Biology PhD Programme http://bioinfo-casl.ucd.ie/PhD
Aspectos imunopatológicos da interação do mycobacterium bovis em hospederios bovinos naturalmente infectados e coinfectados com vírus da leucose enzoótica bovina (BLV)
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2013.A pesquisa racional de novos candidatos a antígenos vacinais ou imunodiagnóstico destinados ao controle da tuberculose bovina (bTB) requer o conhecimento básico da imunopatogênese da doença. No entanto, ainda há escassez de informações sobre os aspectos imunopatológicos da interação Mycobacterium bovis-hospedeiro durante a infecção natural de bovinos. Na análise de uma coorte de 349 bovinos positivos no teste tuberculínico (PPD), em 247 bovinos foi confirmada a infectação natural pelo M. bovis. Dos animais positivos para M. bovis, 92% (228/247) não apresentavam sinais sugestivos da doença, entretanto, apresentavam lesões de bTB graves e disseminadas. Além disso, a disseminação das lesões de bTB estava correlacionada positivamente com a gravidade da patologia da doença e carga bacteriana viável no tecido. Adicionalmente, o encapsulamento do granuloma foi correlacionado negativamente com o crescimento do M. bovis nos tecidos, bem como com a gravidade da patologia, sugerindo que o encapsulamento é um mecanismo dinâmico e pode ser eficaz para controlar a proliferação/disseminação micobacteriana intragranuloma durante a infecção natural. Além disso, a avaliação detalhada da resposta granulomatosa nos pulmões demonstrou que o número de células gigantes multinucleadas tipo Langhans correlaciona negativamente com a contagem micobacteriana. Em contraste, a população de neutrófilos na resposta granulomatosa tuberculosa foi associado com o aumento na proliferação do M. bovis intragranuloma. Os achados aqui apresentados sugerem que o encapsulamento e as células gigantes multinucleadas estão associados ao controle da infecção pelo M. bovis, enquanto que os neutrófilos podem servir como um biomarcador celular de proliferação bacteriana durante a infecção natural. Como prova de conceito de que a dinâmica celular incitada durante a infecção por M. bovis influencia o controle do crescimento micobacteriano, analisamos parâmetros imunopatológicos durante a co-infecção natural com o vírus da leucose enzoótica bovina (BLV), um patógeno que modula a resposta imune adaptativa. Bovinos coinfectados com M. bovis e BLV apresentaram maior carga micobacteriana intragranuloma e uma doença mais grave. Na análise histomorfológica das lesões granulomatosas, observamos uma diminuição significativa na população de linfócitos, seguido por um aumento no número de neutrófilos e menor deposição de tecido conjuntivo. Estes dados sugerem uma alteração no fluxo de células para o ambiente intragranuloma devido a defeitos linfocitários. Consistente com essa hipótese, observamos uma menor resposta de hipersensibilidade do tipo tardia (DTH) em animais com linfocitose persistente (LP-BLV+). Estas evidências indicam que a infecção pelo BLV está associada à perda da homeostase da resposta imune celular do hospedeiro e possivelmente ao aumento da suscetibilidade do hospedeiro a bTB. Em conjunto, nossos dados integram a resposta granulomatosa do hospedeiro com a carga infecciosa micobacteriana e revelam novos elementos para o entendimento da imunopatogênese da bTB que poderiam ser empregados na implementação de estratégias racionais de controle da tuberculose bovina.<br.Abstract : Rational discovery of novel immunodiagnostic and vaccine candidateantigens to control bovine tuberculosis (bTB) requires knowledge ofdisease immunopathogenesis. However, there remains a paucity ofinformation on the Mycobacterium bovis-host immune interactionsduring the natural infection. Analysis of 247 naturally tuberculinic testpositive (PPD) M. bovis-infected cattle revealed that 92% of theseanimals were found to display no clinical signs, but presenteddisseminated bTB-lesions positively correlated with both pathologyseverity score and viable tissue bacterial loads. Additionally, granulomaencapsulation negatively correlated with M. bovis growth as well aspathology severity, suggesting that encapsulation is an effectivemechanism to control bacterial proliferation during natural infection.Moreover, multinucleated giant cell numbers were found to negativelycorrelate with bacterial counts in lung granulomas. In contrast,neutrophil numbers in the granuloma were associated with increased M.bovis proliferation. To better understand the role of the granulomatousresponse for the control of mycobacterial growth during infection by M.bovis, immunopathological parameters during co-infection with naturalbovine leukemia virus (BLV), a pathogen that modulates the adaptiveimmune response, were analyzed. Cattle co-infected with M. bovis andBLV had higher mycobacterial intragranuloma load and more severedisease. In the histomorphological analysis of granulomatous lesions,we observed a significant decrease in the lymphocyte populationfollowed by an increase in neutrophils and a reduction in connectivetissue deposition. These data suggest an alteration in the cell flow for theintragranuloma environment due to defective lymphocytes. Consistentwith this hypothesis, we observed a lower delayed-type hypersensitivity(DTH) response in animals with persistent lymphocytosis (LP-BLV+).This evidence indicate that BLV infection is associated with a loss ofhomeostasis of the host cellular immune response and possiblyincreased susceptibility of the host to bTB. Together, our data integratethe host granulomatous response with the mycobacterial infectious loadand reveal new elements for understanding the immunopathogenesis ofbTB which can be used in the implementation of rational strategies tocontrol bovine tuberculosis
The prevalence, distribution and severity of detectable pathological lesions in badgers naturally infected with Mycobacterium bovis
No full textThe Randomized Badger Culling Trial (RBCT) began in 1998 to determine the impact of badger culling in controlling bovine tuberculosis in cattle. A total of 1166 badgers (14% of total)proactively culled during the RBCT were found to be tuberculous, offering a unique opportunity to study the pathology caused by Mycobacterium bovis in a large sample of badgers. Of these, 39% of adults (y6% of all adults culled) had visible lesions (detectable at necropsy) of bovine tuberculosis ; cubs had a lower prevalence of infection (9%) but a higher percentage of tuberculous cubs (55.5%) had visible lesions. Only ~1% of adult badgers had extensive, severe pathology. Tuberculous badgers with recorded bite wounds (~5%) had a higher prevalence of visible lesions and a different distribution of lesions, suggesting transmission via bite wounds. However, the predominance of lesions in the respiratory tract indicates that most transmission occurs by the respiratory route
Molecular epidemiology and diagnosis of "Mycobacterium bovis" infections in African cattle
No full textMycobacterium bovis is the major causative agent of bovine tuberculosis (BTB) and
part of the Mycobacterium tuberculosis complex (MTBC). BTB can have an impact
on the national and international economy, affects the ecosystem via transmission to
wildlife and is of public health concern due to its zoonotic potential. Although still
present in some industrialized countries, BTB today mostly affects developing
countries lacking the resources to apply expensive test and slaughter schemes. In
Africa, the disease is present virtually on the whole continent; however, little accurate
information on its distribution and prevalence is available. Evaluations of antemortem
tests for the diagnosis of BTB in Africa are scarce but a prerequisite to
identify appropriate tools for future disease control programs. Spoligotyping and
variable number of tandem repeat (VNTR) typing of M. bovis strains isolated from
cattle in the UK has revealed the predominance of a single clonal complex of strains
with subtypes of the complex being geographically localized to specific regions within
the country. Spoligotype patterns of strains isolated from cattle from several countries
throughout the world have been reported in a number of recent studies and the
construction of databases (www.Mbovis.org, SPOLDB4) has facilitated their
comparison and helped elucidate the distribution of specific strain families.
In an attempt to gain insights into the population structure of M. bovis in Africa, we
have isolated strains of M. bovis from cattle carcasses with gross visible lesions at
abattoirs in Dakar (Senegal), Bamako (Mali), Sarh (Chad), Morogoro (Tanzania),
Algiers (Algeria) and Blida (Algeria). These mycobacteria were subjected to
spoligotyping and VNTR typing. A specific region of difference, which we have
named RDAf1, was previously found to be absent in strains from Chad and presence
of this deletion was assessed in our strain collection by PCR. In collaboration with
others, additional strains of M. bovis from other African countries were subjected to molecular typing.
At the abattoir of Sarh in Chad, 954 cattle were subjected to single intra-dermal
comparative cervical tuberculin (SICCT) testing and two recently developed
fluorescence polarization assays (FPA) prior to slaughter. Animal carcasses
underwent standard meat inspection. Gross visible lesions were extracted, analyzed
by microscopy and cultured. Cultured acid-fast bacilli (AFB) were further
characterized by molecular techniques. The different diagnostic tests were evaluated
using a sub-population of animals with either a PCR confirmed MTBC infection or no visible lesions. In addition, a Bayesian model for the evaluation of multiple diagnostic
tests in the absence of a gold standard method was developed.
In collaboration with others, we have identified a clonal complex of strains of M. bovis
present at high frequency in cattle in population samples from Chad, Cameroon,
Nigeria and Mali. This closely related group of bacteria is defined by the RDAf1
chromosomal deletion and can be identified by the absence of spacer 30 in its
spoligotype pattern. We have named this group of strains the Bovis African1 (Af1)
clonal complex. Strains of the Af1 clonal complex were not detected in population
samples from other regions in Africa or other parts of the world, suggesting that the
Af1 clonal complex is geographically localized to sub-Saharan West-Central Africa.
VNTR typing allowed to distinguish sub-populations of the Af1 clonal complex, which
were geographically localized to different countries. This was an unexpected result
suggesting that the movement of strains between countries is not common in this
area. In Mali, in addition to Af1, a second clonal group of M. bovis has been detected
and matching VNTR patterns for some of its strains and strains from France could
indicate a French origin. In Tanzania, also two clonal complexes of M. bovis were
detected by spoligotyping with one clade showing a link to strains, previously
identified in Uganda and Ethiopia and the second clade showing a link to strains
previously isolated in South Africa. M. bovis strains isolated from Algerian cattle were
closely related to strains from continental Europe and especially France. In
conclusion, our work has revealed important insights into the population structure of
M. bovis in Africa and suggests the presence of distinct clonal complexes of strains,
geographically localized to specific areas of the continent.
Our Bayesian model estimated the true BTB prevalence amongst the slaughterhouse
cattle population in Sarh, Chad to be at 8%. The Bayesian and the gold standard test
evaluation methods indicated that the ideal cut-off for positive SICCT test
interpretation should be lowered from > 4 mm (OIE standard cut-off) to > 2 mm, in
the Chadian setting. This result is of practical relevance and likely to apply to other
countries in sub-Saharan Africa. Using this cut-off, sensitivity and specificity of
SICCT was estimated at around 65% and 90%, respectively. Both FPA tests showed
a sensitivity of less than 50% but specificities of at least 90%. Our results suggested
that a substantial amount of lesions detected at the abattoir have been caused by other organisms than M. bovis
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