Mycobacterium bovis in Burkina Faso: Epidemiologic and Genetic Links between Human and Cattle Isolates

Background In sub-Saharan Africa, bovine tuberculosis (bTB) is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations. Methodology/principal findings Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5) or humans (1) or from both (1). Moreover, these data (genetic analyses and phenetic tree) showed that the M. bovis isolates belonged to the African 1 (Af1) clonal complex (81.8%) and the putative African 5 (Af5) clonal complex (18.2%), in agreement with the results of RDAf1 deletion typing. Conclusions/Significance This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.


Introduction
Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in a broad spectrum of hosts, such as cattle, goats, sheep and wild animals to which it can be transmitted through the oral or respiratory route [1]. Humans also can acquire M. bovis generally through the aerogenous route when in close contact with infected animals, but also by consuming unpasteurized dairy products from infected animals and through the skin when handling infected carcasses [2,3,4].
In sub-Saharan Africa, bTB is a serious problem for livestock production but also a health risk for humans as most human populations live in close contact with domestic animals in which the disease is highly prevalent and imperfectly controlled [5]. Therefore, bTB has a deleterious economic burden, although this has not been quantified in Africa as yet [5,6]. In Burkina Faso, little is known about bTB epidemiology and national strategies for disease control are almost non-existent [7]. Tuberculin testing of livestock is not routinely performed and bTB screening is limited to visually checking the meat in abattoirs. Despite the high prevalence of bTB in cattle and the presence of M. bovis in 26.5% of unpasteurized milk samples, the zoonotic transmission of bTB is also poorly known [8]. In Burkina Faso, cattle breeding relies mainly on extensive transhumance and is the prerogative of few ethnic groups [7]. These populations live in close and permanent contact with their livestock and consume raw and unpasteurized dairy products that could favour M. bovis transmission [8]. Moreover, in slaughterhouses where cattle are slaughtered, butchers wear minimal protective clothing and handle infected offals with bare hands [8]. These close contacts are an important source of zoonotic transmission [7,8,9]. Although culture and species identification of the M. tuberculosis complex are not routinely performed in Burkina Faso, previous studies have suggested that M. bovis is present in 0.4% to 1.4% of isolates from patients with pulmonary tuberculosis [10,11].
The main goal of this work was thus to genetically characterize the M. bovis population in Burkina Faso in order to improve our understanding of bTB epidemiology and the circulation of M. bovis within and between the bovine and human populations in this country. We used spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis, because a combination of them is a powerful tool for the genetic characterization of M. bovis [12,13]. We also explored the presence or absence of a specific chromosomal region called RDAf1, which is a region of difference in M. bovis [14].

Human and cattle samples
The tissues showing macroscopic lesions compatible with bTB were collected from slaughtered cattle carcasses during the postmortem inspection at the slaughterhouses of Ouagadougou and Bobo Dioulasso (the two largest cities of Burkina Faso) between May and October 2011. The cattle slaughtered in these two slaughterhouses are mainly originated from neighbouring villages of Ouagadougou and Bobo Dioulasso, as well as the main areas of cattle production throughout the country and the cattle production system is mainly pastoralism. Samples were collected and transported in sterile containers at 4uC to the Mycobacteria Laboratory of the Muraz Centre (in Bobo Dioulasso) for analysis. Smear positive sputum specimens of patients with suspected pulmonary TB were collected in the framework of two studies in Burkina Faso: i) a nationwide survey on anti-tuberculosis drug resistance between 2009 and 2011 (funded by the Global Fund); ii) and a regional study in the Hauts Bassins area between 2011 and 2013 (funded by the French National Agency for Research on AIDS and Viral Hepatitis, ANRS 1224 project, ''Impact of HIV/ Mycobacterium tuberculosis co-infection on the dynamics of tuberculosis transmission in Burkina Faso'').

Isolation and identification of mycobacteria
Bovine tissue samples were processed for mycobacteria isolation following the standard procedures described by the World Organization for Animal Health [15]. Briefly, tissues with tuberculous lesions were dissected into pieces using sterile scissors and forceps, and were then crushed using sterile sea-sand, mortar and pestle. The homogenate of each sample was recovered into 50 ml sterile tube with 10 ml of sterile distilled water, and the obtained solution was homogenized on a vortex mixer for few minutes. After 5 minutes of settling, two milliliters of supernatant were decontaminated with 10 ml of NaOH at 4% according to the protocol described by Petroff's method. Patients' sputum specimens were treated according to the Petroff's method too. The obtained suspensions were inoculated in four Lowenstein-Jensen (LJ) slants, two of which were supplemented with 0.2% of sodium pyruvate. Isolates were identified as mycobacteria and as M. bovis species by Ziehl-Neelsen (ZN) staining for Acid Fast Bacilli (AFB) and a conventional biochemical method, previously described by Ledru et al. [11].
Finally, the presence of the RDAf1 deletion was determined using a multiplex PCR method with a set of three primers followed by agarose gel electrophoresis according to Müller et al. [14].

Genetic diversity and population structure analyses
Several diversity indices, including the genotypic diversity (Gd = the number of different genotypes divided by the total number of samples using the combination of MIRU-VNTR and Spoligotyping data), the allelic diversity per locus and the mean genetic diversity (H s ) were calculated. The population structure was explored by estimating the F st (index of genetic differentiation between samples) value (0 = no differentiation and 1 = fixation of alternative alleles). The allelic diversity, the H s and the F st were calculated using F-STAT, version 2.9.3 with the 26 MIRU-VNTR loci [23].

Author Summary
Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis in livestock and wild animals. Humans can acquire this germ by aerogenous route when in close contact with infected animals, or by consuming unpasteurized dairy products from infected animals and also through the skin when handling infected carcasses. For the present study in Burkina Faso, M. bovis strains were collected from slaughtered animals during routine veterinarian inspection at the slaughterhouses of Bobo Dioulasso and Ouagadougou and also from patients with suspected pulmonary tuberculosis. The isolates were genetically characterized using three techniques: spoligotyping, MIRU-VNTR and RDAf1 deletion analysis. Our results highlight two aspects of M. bovis epidemiology that are crucial for disease control: i) M. bovis circulates between Burkina Faso and its neighbouring countries and ii) M. bovis is transmitted mainly between cattle, but also between cattle and humans, and potentially between humans. This study stresses the need to develop an efficient strategy to control M. bovis transmission, but also the difficulty to implement control measures because of the complex epidemiology of bovine tuberculosis in Burkina Faso.

Phenetic tree and statistical analyses
Genetic relationships among isolates were built with the UPGMA (unweighted pair group method with arithmetic average) and NJ (Neighbour Joining) clustering methods using the MIRU-VNTR and spoligotyping data. The Phylip and Populations packages [24] were used for tree elaboration based on the Nei's distance, and Treedyn for tree visualization and annotation [25].

Ethical considerations
The recruitment of human patients and the collection of bovine samples were done according to protocols approved by the Ethics

M. bovis strains in human and cattle samples
Among the sputum samples collected between 2009 and 2013, the presence of M. bovis was biochemically confirmed in 5/269 (1.85%) samples from the nationwide survey and in 2/307 (0.65%) samples from the regional study respectively. Patients originated from cities that were quite distant one from each other ( Table 1)
The RDAf1 deletion was detected in 27/33 isolates (81.8%). For these samples, as expected, the spoligotyping signatures revealed the absence of spacer 30.

Phenetic analysis
We obtained comparable trees with UPGMA and NJ methods (data not shown). Only the UPGMA is presented here in order to facilitate the confrontation of tree with the spoligotyping data ( Fig. 1). From the tree, we could distinguish two groups (Fig. 1). Group I had six strains (18.2%) that were characterized by the absence of spacers 4 and 5, the presence of spacer 30 in the spoligotype signatures and of the RDAf1 region. This group of     strains appeared similar to the one described by Müller et al. [26], provisionally called African 5 (Af5) clonal complex. Even if these strains revealed specific spoligotype signatures as described above, no genomic deletion allowed to characterize this group as a well defined clonal complex [14,26,27,28]. In this study, this group of strains is thus called ''putative Af5 clonal complex''. Group II included 27 strains that were assigned to the African 1 (Af1) clonal complex based on two criteria: (i) the absence of spacer 30 in the spoligotype; and (ii) the presence of the specific RDAf1 deletion in the genome (Fig. 1) [14].

Genetic diversity and population structure of M. bovis in Burkina Faso
The mean genetic diversity (H s ) and the genotypic diversity (Gd) were respectively 0.187 and 0.79. As expected, the genetic differentiation between Af1 and the putative Af5 strains was high and significant [group II (n = 27) versus group I (n = 5); F st = 0.35; p,0.05]. In addition to the RDAf1 deletion and spoligotype signatures, three MIRU-VNTR loci (QUB-3232, QUB-26 and MIRU 26) allowed assigning the isolates to the Af1 or the putative Af5 clonal complex because the number of repetitions (n) is $5 for putative Af5 and n#1 for Af1 with QUB-3232 locus, n$5 for putative Af5 and n#4 for Af1 with locus QUB-26, n = 7 or 1 for putative Af5, and 1,n,7 for Af1 with MIRU 26. Nevertheless, these differences should be confirmed on a larger sample with isolates from different regions.

Discussion
We present here the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The 6.8% prevalence of bTB in cattle recorded in the present study on the basis of observable lesions in carcasses is lower than in previously published works. A 19% prevalence was reported for the Bobo Dioulasso slaughterhouse, fifteen years ago [8].
Nevertheless, our finding is in agreement with results in other Sahelian countries [6,29,30,31]. Despite this still high prevalence of bTB in cattle in our study, the prevalence of M. bovis in human TB was low (0.6-1.85%), as that reported in other studies in this country [7,8,10,11]. However, prevalences could have been underestimated because only patients with pulmonary TB were included in the present and previous studies. Classically, M. bovis is mainly responsible for extra-pulmonary TB [8]. In any case, the presence of glycerol in LJ medium may have negative impact on the yield of M. bovis growth. The use of LJ medium with 0.5% of sodium pyruvate and without glycerol could allow the optimization of this mycobacterial species isolation in Burkina Faso.
In our study, the combination of spoligotyping and RDAf1 deletion analysis showed that all M. bovis strains belonged either to the Af1 clonal complex, also found in other countries of West-Central Africa, such as Mali, Cameroon, Nigeria, Chad and Niger [14,32], or to the putative Af5 clonal complex, also previously described in Mali [26]. In Burkina Faso, the putative Af5 strains are geographically located in the Western Region (Bobo Dioulasso and Solenzo), an area bordering Mali. The presence of common or related genotypes between Burkina Faso and Mali can be explained by the transhumance activities between these countries and by the transit of Malian livestock on their way to the South, for instance to Ghana and Nigeria. Spoligotype signatures belonging to the putative Af5 clonal complex have also been reported in Europe [14,33]. The putative Af5 clonal complex could have been introduced in these African regions from Europe, possibly via North Africa [26,33,34]. In the Af1 clonal complex, the SB0944 spoligotype signature is defined as the most recent common ancestor (progenitor) and is the most frequent pattern within this group (40% in Chad, 46.1% in Nigeria and 62.7% in Cameroon). It was also the most abundant (52%) in our study [14]. The spread of the Af1 clonal complex over this large area of West-Central Africa could be explained by the long distance transhumance for livestock production in the Sahel, mainly practised by the Fulani pastoralists [26]. This points out the difficulty to develop an efficient strategy to stop bTB transmission.
As expected, MIRU-VNTR typing revealed more polymorphisms than spoligotyping with a high genotypic diversity, but a low genetic diversity because the majority of the analysed loci presented a low mean allelic diversity. Despite a significant genetic differentiation, the low number of isolates analyzed in this study does not allow any assumption about the chronological emergence of these two groups of strains. Nevertheless, to explain the predominance of the Af1 clonal complex in many countries, Müller and al. have suggested that Af1 might have a selective advantage compared to the putative Af5 [14].
When we compared the M. bovis population of Burkina Faso with the populations from Mali, Chad, Nigeria and Cameroon described in Müller et al. [14], we observed a significant genetic differentiation (data not shown). Studies conducted in Chad and Nigeria showed inter-country variation in terms of discriminatory power of MIRU-VNTR loci. These studies and the present one had eight markers in common and only ETR A and ETR B had very high discriminatory power, while MIRU 2 had a very low or no discriminatory power [12,35]. On the basis of the polymorphic loci, each country presents a specific pool of genotypes, although they also share several genotypes with the neighbouring countries. As suggested by Müller et al., these different genetic patterns could be explained by specific evolutionary processes (such as genetic drift and/or selection pressure) depending on the ecosystem set after the emergence and spread of Af1 and putative Af5 progenitors in each country [14].
From an epidemiological point of view, despite the absence of reliable and accurate conventional data the finding that specific spoligotype signatures/MIRU-VNTR patterns were shared only by bovine isolates or by bovine and human isolates suggests a recent transmission within the cattle population and between cattle and humans. Moreover, two patients were infected by Af1 strains with the same genotype (spoligotype plus MIRU-VNTR results). The sputum samples from these two patients were processed at the same medical centre (TB Diagnosis and Treatment Centre of Dafra, Bobo Dioulasso) and at the Mycobacteria Laboratory of the Muraz Centre in 2011, but not at the same time, thus excluding intra-laboratory contamination. Nevertheless, the epidemiological link could not be definitively established for this cluster. Different routes of contamination could be responsible for the infection: i) contamination by a common animal or food-borne source; ii) human to human transmission. Indeed inter-human transmission cannot be excluded since intra-familial and community based transmissions of human TB cases due to Af1 strains of M. bovis have been already suspected [36,37].
In conclusion, our study shows that two groups of M. bovis circulate in Burkina Faso; a major group belonging to the Af1 clonal complex and a minor group belonging to the putative Af5 clonal complex. Furthermore, the comparison with data from other African regions indicates an inter-country transmission associated with a country-specific evolution. Finally, the clusters suggest current transmission that occurs mainly within cattle populations, less frequently between cattle and humans and possibly between humans. This study points out the difficulty to develop an efficient national control strategy of bTB in Burkina Faso.