Mycobacterium bovis infection at the interface between domestic and wild animals in Zambia
- Mudenda B Hang’ombe†1Email author,
- Musso Munyeme†1,
- Chie Nakajima†2,
- Yukari Fukushima2,
- Haruka Suzuki2,
- Wigganson Matandiko3,
- Akihiro Ishii2,
- Aaron S Mweene1 and
- Yasuhiko Suzuki2, 4Email author
© Hang'ombe et al.; licensee BioMed Central Ltd. 2012
Received: 19 June 2012
Accepted: 26 October 2012
Published: 14 November 2012
In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important.
A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units–Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia.
These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.
KeywordsBovine tuberculosis Cattle Mycobacterium bovis Strains Wildlife Kobus leche Kafuensis
Majority of the mycobacterial species that cause human and animal tuberculosis are grouped together as members of the Mycobacterium tuberculosis complex (MTC) [1, 2]. This Mycobacterium tuberculosis complex includes very closely related species of mycobacteria among them: M. tuberculosis, M. africanum, M. microti, M. bovis, M. caprae, M. canettii and M. pinnipedii. Although M. tuberculosis infection is the most common cause of human tuberculosis, part of other proportion of cases are due to M. bovis[3, 4].
Zoonotic tuberculosis is caused mainly by M. bovis that has been shown to have a very wide host range [4–6]. The specie has been documented throughout the world with a similar impact in terms of disease occurrence . In Zambia, BTB is not homogenously distributed, however, high prevalence rates have been recorded within and around the Kafue basin where there is extensive overlap in terms of grazing land for both wild and domestic animals [7–9]. Additionally, the Kafue lechwe antelopes (Kobus leche Kafuensis) found in the Kafue basin have been described as feral reservoirs of BTB in Zambia [10, 11]. The disease has a historical presence in the Kafue basin that predates the identification of the area as a protected ecosystem and Ramsar Site no.530 . Despite the continued reduction in annual rainfall figures under the effects of global warming, the Kafue basin still remains as one of the few lucurstrine wetland ecosystems in Zambia and Africa, supporting a surging cattle population estimated at 300,000 animals,  at a carrying density of 50 animals per square kilometre and approximately 38,000 lechwe antelopes  on a 6,000 square kilometre wetland . The area is characterised by high BTB with a herd level prevalence of around 50%, whereas a comparatively lower herd prevalence averaging 5.6% has been determined in areas outside the basin [7, 15]. Likewise, the corresponding Kafue lechwe antelopes have been shown to have a higher BTB prevalence rate [11, 16], raising questions on a possible interspecies transmission of the disease between cattle and Kafue lechwe antelopes. This is however hampered by the lack of direct evidence to conclusively ascertain this assertion.
Sequencing of the whole genome of the members of MTC [17–21] has shown a high level of sequence homogeneity among the members (99.95%). Thus a careful and detailed comparative exploration into the individual genomes of the members of this complex was employed to mine out significant differences for diagnostic capabilities. Comparative genome analysis informed us that M. bovis has a smaller genome compared with M. tuberculosis. Furthermore, M. bovis has over time lost off some genes compared to M. tuberculosis. These genomic insertion-deletions are commonly referred to as Regions of difference (RD) and have been used in speciation of members of this complex as well as in explaining the evolution of the MTCs [1, 17, 19]. Spoligotyping diagnostic technique highlights intra species differences determined by the loss of spacers at a direct repeat region in MTCs, thereby creating a fingerprint typical of a particular specie . Additionally, it is a more rapid and specific method of MTC speciation apart from being less laborious compared with biochemical, phenotypic and IS6110-restriction fragment length polymorphism (RFLP) analysis . Spoligotyping results are very practical and reproducible across different laboratories internationally [22, 23]. The technique has developed considerably and it is marked by a system of nomenclature and strain data capture and identification with a huge geographical and epidemiological relevance worldwide . Most strains of M. bovis have one copy of IS6110 and spoligotyping is in general more discriminative when used with methods based on PCR amplification of the loci containing variable number tandem repeats (VNTRs) [25, 26].
The target of this study was to molecularly characterize a population sample of M. bovis from cattle and kafue lechwe antelopes in Zambia to determine the genetic diversity and relatedness of the isolates from domestic animals and wildlife.
Results and discussion
Isolation and confirmation of M. bovisby MTCD-MPCR
Results of the MTCD-MPCR of the isolated Mycobacterial isolates from cattle and lechwe
MTCD-MPCR M. bovis +ve
Spoligotyping and Multiple locus variable number of tandem repeats analysis
Spoligotyping results by area of origin across animal species
Area of origin
No of isolates
Blue lagoon National park
Lochinvar National park
Namwala (Southern Zambia)
Lusaka (Central Zambia)
Chongwe (Central Zambia)
Kabwe (Central Zambia)
Mumbwa (Central Zambia)
Nampundwe (Central Zambia)
Characterization of M. bovis strains based on molecular tools is important in understanding the epidemiology of BTB [25, 36, 41]. These results are significant in understanding the transmission and dispersion of M. bovis strains within Zambia (Figure 2), given the high level of internal migration by the local people from the Southern part of the country to the Central and Northern regions of Zambia with their cattle. This type of internal migration may lead to the dispersion of the SB0120 strain which in this present study was found confined to the Southern regions of Zambia.
The current study has described the possible source of M. bovis in wildlife and the transmission of limited strains of M. bovis between cattle and lechwe. The identified maintenance and spread of M. bovis may become a dynamic and highly active process considering that human activities allow movement of cattle from one location to another.
Study area and sampling
Cultivation of Mycobacterial and DNA extraction
The collected samples of TB suspect were analyzed and cultured for growth as previously described . Briefly, the tissues were trimmed of fat and then a 500 mg sample was minced with sterile scissors and homogenized in a sterilized glass homogenizer. A milliliter of phosphate buffer (pH 6.8) was added and thoroughly mixed after which 1 mL of 5% sodium hydroxide was added. After incubation for 15 min at room temperature, 10 mL of phosphate buffer was added and then centrifuged at 1500 xg for 20 min. The pellet was collected and then resuspended in a final volume of 0.5 mL of phosphate buffer which was then used for inoculation onto 2% Ogawa medium. Bacterial growth was then monitored up to 8 weeks at 37°C. The resulting cultures were tentatively identified as probable Mycobacterium tuberculosis-complex by their slow growth and colony morphology. Purity and acid-fastness of the colonies were checked by Ziehl Neelsen staining. DNA was extracted from Mycobacterial colonies using DNAzol reagent (Invitrogen, Carlsbad, CA, USA) and mechanical disruption as previously described  according to the manufacturer’s instructions and dissolved in 50 μL TE buffer consisting of 10 mM Tris/HCL (pH 8.0) and 1 mM EDTA.
MTC discrimination by multiplex PCR
The Mycobacterium tuberculosis complex-discriminating multiplex PCR (MTCD-MPCR) targeting genetic regions cfp32 (a specific gene for MTC), RD9 (region of difference 9; seen only in M. tuberculosis and M. canettii) and RD12 (region of difference 12; deleted in M. bovis, M. caprae and M. canettii) was used for species differentiation according to the previously publication . The reaction mixture consisted of 7.4 μL H20, 2 μL 10 x Taq buffer, 2 μL dNTPs (2.5 mM each), 0.2 μL Taq (Takara Bio Inc, Shiga, Japan), 1 μL of DNA sample, 2.2 μL of 10 μM cfp32 primers, 0.7 μL of 5 μM RD9 primers and 0.8 μL of 5 μM RD 12 primers. The PCR was performed using the following program: denaturation for 1 min at 98°C followed by 35 cycles of 5 sec at 98°C, 20 sec at 58°C and 1 min at 68°C with final elongation at 72°C for 5 min in a thermal cycler (iCycler, Bio-Rad Laboratories Inc., Hercules, CA). The PCR products were separated by electrophoresis in a 2% agarose gel in TAE buffer, and then visualized after staining with ethidium bromide.
Spoligotyping of M. bovisisolates using micro-spoligoarrays
Spoligotyping was performed according to the procedure by Kamerbeek and co-workers with slight modifications . Forty-three spacer-sequence probes were covalently bound to the membrane (Pall Co., NY, USA). The primers used were DRa (GGTTTTGGGTCTGACGAC) and DRb (CCGAGAGGGGACGGAAAC). A hot start PCR was done by mixing 1 μL each of the 10 μM primer, 7.3 μL H2O, 3 μL of 5 x colorless Go Taq buffer (Promega™, Fitchburg, WI), 1.5 μL of PCR DIG Labeling Mix (Roche), 0.2 μL of Go Taq DNA Polymerase (5 units/μL; Promega) and 1 μL of DNA sample in 15 μL total reaction mix per tube. The PCR reaction was initiated by denaturation at a temperature of 98°C for 1 min, followed by 40 cycles of 98°C for 5 sec, 55°C for 10 sec and 72°C for 30 sec with a final elongation step at 72°C for 1 min in a thermal cycler. The 500 times diluted PCR product in hybridization buffer was heat denatured at 95°C for 5 minutes and immediately cooled on ice to leave the DNA single stranded. Hybridization was performed by placing the nitrocellulose membranes (Pall Co., NY, USA) for incubation at 60°C for 1 hour. After which, the membrane was washed in TBST-E (0.1% Tween-20 and 1mM EDTA-2Na in TBS) at 60°C for 1 min and then10 min, finally 1 min. The membrane was then dried at room temperature. DIG on the nitrocellulose membranes were reacted with a 1000 times diluted Ant-Digoxigenin-POD [poly], Fab fragments (Roche), with TBSTE-E at room temperature for 30 min. Then the membranes were sequentially washed in TBST-E at room temperature for 1 min, 10 min and 1 min. Then, POD on the membranes was detected by TMB solution (TMB Peroxidase Substrate Kit, Vector Labs Inc™, Burlingame, CA) according to the manufacturer’s protocol.
Multiple locus variable number of tandem repeats (MLVA) assay of M. bovisisolates
The isolates were further genotyped by PCR amplification using primers targeting 26 VNTR loci . Two different PCR reaction mixtures were conducted according to the loci. Locus MIRU10, 16, 24, 26, 27, 39, 40, ETR-B, F, VNTR-424, 3690 were done under betaine 1.0 M, whilst locus MIRU2, 4, 20, 23, 31, ETR-A, C, QUB-11a, 11b, 26, 3232, 3336, VNTR-1955, 2401, 4156 were performed under GCII buffer (Takara). The PCR reaction mixture under 1.0 M betaine buffer was conducted in a mixture consisted of 6.3 μL H2O, 0.4 μL of dNTP (10 mM each), 3.0 μL of 5x colorless Go Taq buffer, 3.0 μL of betaine (5 M), 0.6 μL of Primer 1 and 0.6 μL of Primer 2, 0.1 μL of Go Taq (5 units/ μL) DNA Polymerase, and 1 μL of DNA sample. An initial denaturation step of 95°C for 5 min was followed by 32 cycles at 95°C for 15 sec, 58°C for 20 sec and 72°C for 1 min with a final elongation step at 72°C for 1 min in a thermalcycler. The PCR reaction mixture under the GCII buffer consisted of 4.8 μL H2O, 0.4 μL of dNTP (10 mM each), 7.5 μL of 2 x GCII buffer, 0.6 μL of primers, 0.1 μL of Go Taq (5 units/ μL) DNA Polymerase (Promega), and 1 μL of DNA sample. An initial denaturation step at 95°C for 5 min was followed by 32 cycles of 95°C for 15 sec, 50°C for 20 sec and 72°C for 45 sec with a final elongation step at 72°C for 1 min in a thermalcycler. All the samples were electrophoresed on a 2% agarose gel to identify the repeat numbers.
HBM, MM, CN, WM and ASM are veterinarians.
Polymerase chain reaction
Mycobacterium tuberculosis complex-discriminating multiplex PCR
Mycobacterial interspersed repetitive units–variable-number tandem repeats
Regions of difference.
The authors are grateful to Mr L. Moonga and Mr E. Mulenga from the School of Veterinary Medicine, University of Zambia for technical assistance. This work was supported in part by the Directorate of Research and Graduate Studies of the University of Zambia to HBM and MM, in part by J-GRID; the Japan Initiative for Global Research Network on Infectious Diseases to YS, in part by the Global Center of Excellence (COE) Program, “Establishment of International Collaboration Centers for Zoonosis Control” from MEXT to YS, in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) to YS and CN, and in part by a grant from U.S.-Japan Cooperative Medical Science Programs to YS.
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