Evolutionary analysis of Mycobacterium bovis genotypes across Africa suggests co-evolution with livestock and humans.

Mycobacterium bovis is the pathogenic agent responsible for bovine tuberculosis (bTB), a zoonotic disease affecting mostly cattle, but also transmittable to humans and wildlife. Genetic studies on M. bovis allow to detect possible routes of bTB transmission and the identification of genetic reservoirs that may provide an essential framework for public health action. We used a database with 1235 M. bovis genotypes collected from different regions in Africa with 45 new Mozambican samples. Our analyses, based on phylogeographic and population genetics' approaches, allowed to identify two clear trends. First, the genetic diversity of M. bovis is geographically clustered across the continent, with the only incidences of long-distance sharing of genotypes, between South Africa and Algeria, likely due to recent European introductions. Second, there is a broad gradient of diversity from Northern to Southern Africa with a diversity focus on the proximity to the Near East, where M. bovis likely emerged with animal domestication in the last 10,000 years. Diversity indices are higher in Eastern Africa, followed successively by Northern, Central, Southern and Western Africa, roughly correlating with the regional archaeological records of introduction of animal domesticates. Given this scenario M. bovis in Africa was probably established millennia ago following a concomitant spread with cattle, sheep and goat. Such scenario could translate into long-term locally adapted lineages across Africa. This work describes a novel scenario for the spread of M. bovis in Africa using the available genetic data, opening the field to further studies using higher resolution genomic data.

Genetic diversity and potential routes of transmission of Mycobacterium bovis in Mozambique.

Bovine tuberculosis is a zoonotic disease with largely unknown impact in Africa, with risk factors such as HIV and direct contact with animals or consumption of Mycobacterium bovis infected animal products. In order to understand and quantify this risk and design intervention strategies, good epidemiological studies are needed. Such studies can include molecular typing of M. bovis isolates. The aim of this study was to apply these tools to provide novel information concerning the distribution of bovine tuberculosis in cattle in Mozambique and thereby provide relevant information to guide policy development and strategies to contain the disease in livestock, and reduce the risk associated with transmission to humans. A collection of 178 M. bovis isolates was obtained from cattle in Mozambique. Using spoligotyping and regions of difference analysis, we classified the isolates into clonal complexes, thus reporting the first characterisation of M. bovis strains in this region. Data from MIRU-VNTR typing was used to compare isolates from a number of African countries, revealing a deeply geographically structured diversity of M. bovis. Eastern Africa appears to show high diversity, suggesting deep evolution in that region. The diversity of M. bovis in Africa does not seem to be a function of recent importation of animals, but is probably maintained within each particular region by constant reinfection from reservoir animals. Understanding the transmission routes of M. bovis in Mozambique and elsewhere is essential in order to focus public health and veterinary resources to contain bovine tuberculosis.

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique.

BACKGROUND: The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. RESULTS: Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98%) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7%) cases, was the main cause of TBLN and 66.7% of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n = 19; 43.2%). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. CONCLUSIONS: M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.

Prevalence of bovine tuberculosis and risk factor assessment in cattle in rural livestock areas of Govuro District in the Southeast of Mozambique.

BACKGROUND: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is an infectious disease of cattle that also affects other domestic animals, free-ranging and farmed wildlife, and also humans. In Mozambique, scattered surveys have reported a wide variation of bTB prevalence rates in cattle from different regions. Due to direct economic repercussions on livestock and indirect consequences for human health and wildlife, knowing the prevalence rates of the disease is essential to define an effective control strategy. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional study was conducted in Govuro district to determine bTB prevalence in cattle and identify associated risk factors. A representative sample of the cattle population was defined, stratified by livestock areas (n = 14). A total of 1136 cattle from 289 farmers were tested using the single comparative intradermal tuberculin test. The overall apparent prevalence was estimated at 39.6% (95% CI 36.8-42.5) using a diagnostic threshold cut-off according to the World Organization for Animal Health. bTB reactors were found in 13 livestock areas, with prevalence rates ranging from 8.1 to 65.8%. Age was the main risk factor; animals older than 4 years were more likely to be positive reactors (OR = 3.2, 95% CI: 2.2-4.7). Landim local breed showed a lower prevalence than crossbred animals (Landim × Brahman) (OR = 0.6, 95% CI: 0.4-0.8). CONCLUSIONS/SIGNIFICANCE: The findings reveal an urgent need for intervention with effective, area-based, control measures in order to reduce bTB prevalence and prevent its spread to the human population. In addition to the high prevalence, population habits in Govuro, particularly the consumption of raw milk, clearly may potentiate the transmission to humans. Thus, further studies on human tuberculosis and the molecular characterization of the predominant strain lineages that cause bTB in cattle and humans are urgently required to evaluate the impact on human health in the region.

Prevalence of bovine tuberculosis and risk factor assessment in cattle in rural livestock areas of Govuro District in the Southeast of Mozambique

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is an infectious disease of cattle that also affects other domestic animals, free-ranging and farmed wildlife, and also humans. In Mozambique, scattered surveys have reported a wide variation of bTB prevalence rates in cattle from different regions. Due to direct economic repercussions on livestock and indirect consequences for human health and wildlife, knowing the prevalence rates of the disease is essential to define an effective control strategy. Methodology/principal findings: A cross-sectional study was conducted in Govuro district to determine bTB prevalence in cattle and identify associated risk factors. A representative sample of the cattle population was defined, stratified by livestock areas (n = 14). A total of 1136 cattle from 289 farmers were tested using the single comparative intradermal tuberculin test. The overall apparent prevalence was estimated at 39.6% (95% CI 36.8-42.5) using a diagnostic threshold cut-off according to the World Organization for Animal Health. bTB reactors were found in 13 livestock areas, with prevalence rates ranging from 8.1 to 65.8%. Age was the main risk factor; animals older than 4 years were more likely to be positive reactors (OR = 3.2, 95% CI: 2.2-4.7). Landim local breed showed a lower prevalence than crossbred animals (Landim × Brahman) (OR = 0.6, 95% CI: 0.4-0.8). Conclusions/significance: The findings reveal an urgent need for intervention with effective, area-based, control measures in order to reduce bTB prevalence and prevent its spread to the human population. In addition to the high prevalence, population habits in Govuro, particularly the consumption of raw milk, clearly may potentiate the transmission to humans. Thus, further studies on human tuberculosis and the molecular characterization of the predominant strain lineages that cause bTB in cattle and humans are urgently required to evaluate the impact on human health in the region

Mycobacterium tuberculosis Beijing genotype is associated with HIV infection in Mozambique.

The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit - variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where a distinct group of four isolates had deletion of RD150, a signature of the "sublineage 7" recently emerging in South Africa. The same group was very similar to South African "sublineage 7" by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from South Africa, in association with HIV infection.

Characterization of tuberculous lesions in naturally infected African buffalo (Syncerus caffer).

Tuberculosis pathology was studied on 19 African buffalo (Syncerus caffer) from a herd in the Hluhluwe-iMfolozi Park in South Africa. The animals tested positive with the comparative intradermal tuberculin test and were euthanized during a test-and-cull operation to decrease prevalence of bovine tuberculosis (bTB) in the park. The lymph nodes and lungs were examined grossly for presence of tuberculous lesions, which were scored on a 0-5 scale for macroscopic changes. The gross lesions were examined histologically and classified into grade I, II, III, or IV according to a grading system used for bTB lesions in domestic cattle. Macroscopic lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (in 16 animals) and mediastinal (in 11 animals). All four grades of microscopic lesions were observed, grade II lesions were the most frequent. Mycobacterium bovis was detected by PCR in 8 out of 19 animals, and acid-fast bacilli were seen in 7 out of 19 animals, together both techniques identified mycobacteria in 5 out of 19 animals. Lesions were paucibacillary, as acid-fast bacilli were only rarely observed. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is by inhalation.

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.

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.

Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique.

BACKGROUND: Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique. RESULTS: A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2.The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages. CONCLUSIONS: The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.

African 1, an epidemiologically important clonal complex of Mycobacterium bovis dominant in Mali, Nigeria, Cameroon, and Chad.

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.