Cat-to-Human Transmission of Mycobacterium bovis, United Kingdom.

Human infection with Mycobacterium bovis is reported infrequently in the United Kingdom. Most cases involve previous consumption of unpasteurized milk. We report a rare occurrence of 2 incidents of cat-to-human transmission of M. bovis during a cluster of infection in cats.

Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal.

Investigation of the cause of geographic disparities in IDEXX ELISA sensitivity in serum samples from Mycobacterium bovis-infected cattle.

Accurately identifying Mycobacterium bovis-infected cattle is critical for bovine tuberculosis prevention and control. One method for identifying infected cattle is an ELISA developed by IDEXX laboratories, which detects antibodies to two M. bovis proteins, MPB70 and MPB83. The assay's sensitivity varies by geographic region, with sensitivities of 77%, 45%, and 9% in bovine serum samples from the United Kingdom (n = 126), the United States (n = 146), and Mexico (n = 128), respectively. We hypothesized that geographically-biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may explain these differing sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries. For each gene, a single, common sequence was identified in most genomes of the M. bovis strains collected in all three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA-positive genomes and three of the five ELISA-negative genomes contained the most common sequence of all four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain the geographic disparities in IDEXX ELISA sensitivity.

Ageing is associated with molecular signatures of inflammation and type 2 diabetes in rat pancreatic islets.

AIMS/HYPOTHESIS: Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas. METHODS: Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods. RESULTS: Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1). CONCLUSIONS/INTERPRETATION: Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.

Why doesn't bovine tuberculosis transmit between humans?

Tuberculosis caused by Mycobacterium tuberculosis is an important bacterial pathogen of man. This human-adapted pathogen was ancestral to a lineage of animal-adapted strains which cause similar disease in many different mammals but are unable to transmit between humans. How did the animal-adapted strains lose the ability to transmit between humans?

Overview and phylogeny of Mycobacterium tuberculosis complex organisms: implications for diagnostics and legislation of bovine tuberculosis.

Members of the Mycobacterium tuberculosis complex (MTBC) cause a serious disease with similar pathology, tuberculosis; in this review, bovine tuberculosis will be considered as disease caused by any member of the MTBC in bovids. Bovine tuberculosis is responsible for significant economic loss due to costly eradication programs and trade limitations and poses a threat to both endangered and protected species as well as to public health. We here give an overview on all members of the MTBC, focusing on their isolation from different animal hosts. We also review the recent advances made in elucidating the evolutionary and phylogenetic relationships of members of the MTBC. Because the nomenclature of the MTBC is controversial, its members have been considered species, subspecies or ecotypes, this review discusses the possible implications for diagnostics and the legal consequences of naming of new species.

Mycobacterium bovis infection in domestic pigs in Great Britain.

Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), infects a wide range of wild and domestic mammals. Despite a control programme spanning decades, M. bovis infection levels in cattle in Great Britain (GB) have continued to rise over recent years. As the incidence of infection in cattle and wildlife may be linked to that in swine, data relating to infection of pigs identified at slaughter were examined in this study. Between 2007 and 2011, almost all M. bovis-infected pigs originated from farms in the South-West and West-Midland regions of England. The data suggest that pigs raised outdoors or on holdings with poor biosecurity may be more vulnerable to infection with M. bovis. In the majority of cases, the same strains of M. bovis were found in pigs and cattle, despite that fact that direct contact between these species was rarely observed. Genotyping and geographical mapping data indicated that some strains found in pigs may correlate better with those present in badgers, rather than cattle. In consequence, it is proposed that pigs may represent a useful sentinel for M. bovis infection in wildlife in GB. Given the potential implications of this infection for the pig industry, and for the on-going effort to control bovine TB, the importance of understanding the epidemiology and pathogenesis of M. bovis infection, as well as monitoring its prevalence, in pigs should not be underestimated.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group.

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

Surveillance of bovine tuberculosis and risk estimation of a future reservoir formation in wildlife in Switzerland and Liechtenstein.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4-7.8) but none of the 269 red deer (0%; 0-1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.

Molecular genotyping of Mycobacterium bovis isolated from cattle tissues in the North West Region of Cameroon.

An epidemiological study was carried out to determine the Mycobacterium bovis strains causing bovine tuberculosis (TB) in cattle in North West Cameroon. Suspected TB lesions from slaughtered cattle were cultured on Lowenstein-Jensen and Middlebrook 7 H9 media to isolate mycobacteria agents for molecular genotyping using deletion analysis and spoligotyping. PCR-based genomic deletion typing showed that 54 of 103 tubercle bacilli isolated from cattle tissue were M. bovis strains and the African 1 clonal complex was widespread in affected cattle. Spoligotyping analysis revealed a closely related group of five M. bovis strains. SB0953, the dominant spoligotype pattern, and four new patterns identified as SB2161, SB2162, SB2663 and SB2664 according to the www.Mbovis.org international spoligotype database were identified. These spoligotypes were similar to other M. bovis strains recovered from bordering regions and other parts of Africa. The findings provided useful facts on the zoonotic risks of bovine TB and overwhelming evidence of the significance of M. bovis infection to human TB in the North West Region of Cameroon. The study revealed that bovine TB was widespread in cattle destined for human consumption and also has important implications for the control of TB in animals and humans in Cameroon.

The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex.

BACKGROUND: M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the "modern" M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle. CONCLUSIONS/SIGNIFICANCE: The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.

European 2--a clonal complex of Mycobacterium bovis dominant in the Iberian Peninsula.

Mycobacterium bovis isolates from the Iberian Peninsula are dominated by strains with spoligotype patterns deleted for spacer 21. Whole-genome sequencing of three Spanish strains with spacer 21 missing in their spoligotype pattern revealed a series of SNPs and subsequent screening of a selection of these SNPs identified one in gene guaA that is specific to these strains. This group of strains from the Iberian Peninsula missing spoligotype spacer 21 represents a new clonal complex of M. bovis, defined by the SNP profile with a distinct spoligotype signature. We have named this clonal complex European 2 (Eu2) and found that it was present at low frequency in both France and Italy and absent from the British Isles.

The global distribution and phylogeography of Mycobacterium bovis clonal complexes.

The consequences of the clonality of the Mycobacterium tuberculosis complex are described and, in particular, the identification of clonal complexes. Clonal complexes are groups of strains all descended from a single cell that was the most recent common ancestor (MRCA) of the clonal complex and all bearing characteristics derived from the MRCA. Three clonal complexes of Mycobacterium bovis have been identified and called African 1, African 2 and European 1. Members of each clonal complex have a distinct spoligotype signature and are identified by a unique deletion present in each member of the clonal complex. The African 1 and African 2 clonal complexes are geographically localised to Central-West Africa and East Africa, respectively and have not been found in cattle outside of these regions. However, the European 1 clonal complex is globally distributed and has been identified in the British Isles, former British colonies, The Americas as well as Kazakhstan and Korea. It is suggested that modern cattle breeds, such as Herefords, bred in the UK in the 18th Century, would provide a good vehicle for the global distribution of this closely related group of strains. The phylogeography of M. bovis and M. tuberculosis are compared and in particular the diversity of M. tuberculosis in Africa, compared with the localised dominance of M. bovis clonal complexes, is highlighted. Finally, the practical use of M. bovis clonal complexes is reviewed, however, a more significant use of M. bovis clonal complexes is to generate testable hypotheses to understand the pathogenicity and spread of this important veterinary pathogen. This first look at the phylogeography of M. bovis clonal complexes has shown surprising geographical localisation of molecular types but also hints strongly that much of the worldwide distribution of bovine pathogen resulted from human trade in cattle within the last 200 years.

Naming spoligotype patterns for the RD9-deleted lineage of the Mycobacterium tuberculosis complex; www.Mbovis.org.

This www.Mbovis.org website and associated databases were initiated in response to the need of the Mycobacterium bovis molecular typing community for standardisation of nomenclature for spoligotype patterns. The purpose of the website is to provide standardised names for spoligotype patterns from strains of the RD9-deleted, lineage of the Mycobacterium tuberculosis complex. Each unique spoligotype pattern is named by 'SB' followed by a four integer number (e.g. SB0120) and the database currently records over 1400 patterns submitted by the research community over the last 9 years. The database holds information and authoritative names for spoligotype patterns from both clades of Mycobacterium africanum, M. bovis (antelope), Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium caprae and M. bovis. The history of the database is described, as well as the method for submitting new patterns to the database and the functionality of the website.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets.

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter-enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

Mutations in the regulatory network underlie the recent clonal expansion of a dominant subclone of the Mycobacterium tuberculosis Beijing genotype.

The Beijing genotype family is an epidemiologically important sub-group of Mycobacterium tuberculosis. It has been suggested that the high frequency of the Beijing isolates in some areas could be explained by selective advantages. Some evidence suggests that the emerging and most frequently isolated "Typical Beijing" lineage has the ability to circumvent BCG-induced immunity. To investigate the phylogeny of the Beijing genotype of M. tuberculosis, the genome of six Beijing strains from three different countries was sequenced with next-generation sequencing. The phylogeny of these strains was established using single nucleotide polymorphisms (SNPs). The three Typical Beijing strains clustered very tightly in the Beijing phylogeny suggesting that Typical Beijing strains represent a monophyletic lineage and resulted from recent diversification. Typing of 150 M. tuberculosis strains with a subset of the SNPs and comparison of the IS6110 restriction-fragment length polymorphism (RFLP) patterns of these strains to a database of 1522 Beijing RFLP patterns revealed that about 80% of all Beijing strains belong to the Typical Beijing subclone, which indicates clonal expansion. To identify the genomic changes that are characteristic for all Typical Beijing strains and to reconstruct their most recent common ancestor, the presence of SNPs was assayed in other Beijing strains. We identified 51 SNPs that define the minimal set of polymorphisms for all Typical Beijing strains. Nonsynonymous polymorphisms in genes coding for the regulatory network were over-represented in this set of mutations. We suggest that alterations in the response to environmental signals may have enabled Typical Beijing strains to develop the emerging phenotype.

African 2, a clonal complex of Mycobacterium bovis epidemiologically important in East Africa.

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Poor maternal nutrition leads to alterations in oxidative stress, antioxidant defense capacity, and markers of fibrosis in rat islets: potential underlying mechanisms for development of the diabetic phenotype in later life.

Low birth weight is associated with glucose intolerance, insulin resistance, and type 2 diabetes (T2D) in later life. Good evidence indicates that the environment plays an important role in this relationship. However, the mechanisms underlying these relationships are defined poorly. Islets are particularly susceptible to oxidative stress, and this condition combined with fibrosis is thought to be instrumental in T2D pathogenesis. Here we use our maternal low-protein (LP) rat model to determine the effect of early diet on oxidative stress and fibrosis in pancreatic islets of male offspring at 3 and 15 mo of age. Islet xanthine oxidase (XO) expression was increased in 15-mo LP offspring, which suggests increased oxidative-stress. Manganese superoxide-dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), and heme oxygenase-1 (HO-1) (antioxidant enzymes) were reduced significantly in LP offspring, which indicated impairment of oxidative defense. Expression of fibrosis markers collagen I and collagen III also increased in 15-mo LP offspring. Angiotensin II receptor type I (AT(II)R(1)), induced by hyperglycemia and oxidative-stress, was significantly up-regulated in 15-mo LP offspring. Lipid peroxidation was also increased in 15-mo LP animals. We conclude that maternal protein restriction causes age-associated increased oxidative stress, impairment of oxidative defense, and fibrosis. These findings provide mechanisms by which suboptimal early nutrition can lead to T2D development later in life.

The tempo and mode of molecular evolution of Mycobacterium tuberculosis at patient-to-patient scale.

A total of six polymorphisms were identified by comparing the genomes of the first and the last isolate of a well-characterized transmission chain of Mycobacterium tuberculosis involving five patients over a 12 and a half year period. The six polymorphisms consisted of four single nucleotide changes (SNPs), a tandem repeat polymorphism (TRP) and a previously identified IS6110 transposition event. These polymorphic sites were surveyed in each of the isolates from the five patients in the transmission chain. Surprisingly, five of the six polymorphisms accumulated in a single patient in the transmission chain; this patient had been non-compliant to tuberculosis treatment. This first insight into the tempo and mode of molecular evolution in M. tuberculosis at the patient-to-patient level suggests that the molecular evolution of the pathogen in vivo is characterized by periods of relative genomic stability followed by bursts of mutation. Whatever the mechanism for the accumulation of mutations, this observation may have profound consequences for the application of vaccines and therapeutic drugs, the management and treatment of disease outbreaks of M. tuberculosis, the most important bacterial pathogen of humans.