BACKGROUND
majority of the mycobacterial species that cause human and animal tuberculosis are grouped together as members of the mycobacterium tuberculosis complex   <cit> .
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 <cit> .
although m. tuberculosis infection is the most common cause of human tuberculosis, part of other proportion of cases are due to m. bovis <cit> .
zoonotic tuberculosis is caused mainly by m. bovis that has been shown to have a very wide host range  <cit> .
the specie has been documented throughout the world with a similar impact in terms of disease occurrence  <cit> .
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  <cit> .
additionally, the kafue lechwe antelopes  found in the kafue basin have been described as feral reservoirs of btb in zambia  <cit> .
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.
<dig>  <cit> .
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  <dig>  animals,  <cit>  at a carrying density of  <dig> animals per square kilometre and approximately  <dig>  lechwe antelopes  <cit>  on a  <dig>  square kilometre wetland  <cit> .
the area is characterised by high btb with a herd level prevalence of around 50%, whereas a comparatively lower herd prevalence averaging  <dig> % has been determined in areas outside the basin  <cit> .
likewise, the corresponding kafue lechwe antelopes have been shown to have a higher btb prevalence rate  <cit> , 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  <cit>  has shown a high level of sequence homogeneity among the members .
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 <cit> .
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  and have been used in speciation of members of this complex as well as in explaining the evolution of the mtcs  <cit> .
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  <cit> .
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  analysis  <cit> .
spoligotyping results are very practical and reproducible across different laboratories internationally  <cit> .
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  <cit> .
most strains of m. bovis have one copy of is <dig> and spoligotyping is in general more discriminative when used with methods based on pcr amplification of the loci containing variable number tandem repeats   <cit> .
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
isolation and confirmation of m. bovis by mtcd-mpcr
a total of  <dig> specimens from cattle and  <dig> from lechwe antelopes were analysed to initially determine the prevalence of mtc species.
the samples were collected based on observations of gross pathological lesions upon examination.
the observations included generalized lesions involving the lungs, pleural and mediastinal lymph nodes in both lechwe and cattle.
these tuberculous lesions were observed by other workers who were investigating gross pathological distribution of tuberculous lesions in both cattle and lechwe  <cit> .
from the samples analysed,  <dig> mtc strains  were obtained with  <dig> isolates from  <dig> cattle and  <dig> from  <dig> lechwe.
following isolation m. bovis was confirmed by screening using the mtcd-mpcr analysis as previously observed  <cit> .
the mtcd-mpcr technique is very useful in the differentiation of mtc at whatever level of diagnosis as it is simple and specific  <cit> .
the presence of m. bovis in wildlife may translate into a perpetual focus of the disease  <cit>  considering that btb control in wild animals is a very difficult undertaking  <cit> .
the only workable solution would be to intensify btb testing in domestic animals so that reactors are removed.
furthermore traditional cattle herders must also be informed of the dangers of grazing their animals in areas where lechwe antelopes are present.
spoligotyping and multiple locus variable number of tandem repeats analysis
spoligotyping of the  <dig> m. bovis isolates revealed their molecular clonality .
two major spoligotype patterns  were observed accounting for  <dig> isolates , and  <dig> isolates  cattle and lechwe respectively.
two isolates not identifiable with the two major clusters was given an sb <dig> under the global spoligotype patterns diversity provided by the international data base on spoligotyping  <cit> .
this only accounted for 4% of the observed isolates.
m. bovis strains from both cattle and lechwe of the kafue basin were found to share the same spoligotype .
this spoligotype was previously reported to be the major strain circulating in cattle around the kafue basin, although by that time, no strains were determined from wild animals  <cit> .
m. bovis strains isolated outside the kafue basin were found to share a different cluster .
all the  <dig> isolates lacked spacers  <dig>   <dig>   <dig> and  <dig> to  <dig>  a characteristic feature that distinguishes m. bovis from m. tuberculosis <cit> .
molecular studies have previously shown that clonality implies active transmission of disease  <cit> .
thus our results in the kafue basin where there was high clonality between isolates from lechwe and cattle, suggest an active transmission of m. bovis between the two animal species.
the spoligotyping results showed that sb <dig>  clustered outside the kafue basin suggesting that none of the lechwe antelopes harboured m. bovis with such a spoligo pattern.
mlva analysis identified  <dig> distinct clusters, which are corresponding to either sb <dig> or sb <dig> .
within the cluster possessing the spoligo pattern sb <dig>  there were  <dig> groups and  <dig> strain distinguishable one another by  <dig> loci, miru <dig> and qub <dig>  the cluster possessing the spoligotype sb <dig> was differentiated in  <dig> groups.
nineteen strains,  <dig> from lechwe and  <dig> from cattle, consisted of a major group and other  <dig> groups or singletons were discriminated from the major group by a single mlva locus difference each.
the spoligotype sb <dig> seems to be a descendant pattern of sb <dig> lacking spacer  <dig> and also clustered in sb <dig> group with  <dig> locus  difference.
the high isolation frequency of spoligotype sb <dig> in cattle and lechwe conforms to the finding of previous works from zambia  <cit>  and may demonstrate dominance of this spoligotype despite the small sample size.
the sb <dig> spoligotype has a considerable degree of geographical dispersion in zambia and other african countries such as algeria  <cit>  and south africa  <cit> .
this spoligotype is also common in continental europe  <cit> .
such a strain could have been introduced through livestock development schemes of trying to improve african beef local breeds.
this finding has been highlighted by different workers  <cit> .
spoligotype sb <dig> has been reported in this work for the first time in zambia.
this spoligotype has been documented in south africa  <cit> , which has a close livestock trade link with zambia.
to our knowledge, this is the first study conducting molecular characterization of m. bovis strains from cattle and lechwe in zambia.
however, routine studies have already previously revealed the presence of btb in these animals  <cit> .
in this study, results have shown genetic relatedness between m. bovis in cattle and lechwe antelopes of the kafue basin.
it is important to note that the area around lochnivar national park was used for ranching purposes by the early settlers from south africa.
this area was only gazetted as an animal sanctuary in  <dig>  it is the only place where kafue lechwe antelopes antelopes are confined.
the animals are semi-aquatic living in large groups near water and as such are confined to the area shown in figure  <dig> .
characterization of m. bovis strains based on molecular tools is important in understanding the epidemiology of btb  <cit> .
these results are significant in understanding the transmission and dispersion of m. bovis strains within zambia , 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 sb <dig> strain which in this present study was found confined to the southern regions of zambia.
CONCLUSIONS
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.

 majority of the mycobacterial species that cause human and animal tuberculosis are grouped together as members of the mycobacterium tuberculosis complex. 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. zoonotic tuberculosis is caused mainly by m. bovis that has been shown to have a very wide host range. 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. additionally, the kafue lechwe antelopes found in the kafue basin have been described as feral reservoirs of btb in zambia. 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.. 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 animals, at a carrying density of animals per square kilometre and approximately lechwe antelopes on a 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 % has been determined in areas outside the basin. likewise, the corresponding kafue lechwe antelopes have been shown to have a higher btb prevalence rate, 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 has shown a high level of sequence homogeneity among the members. 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 and have been used in speciation of members of this complex as well as in explaining the evolution of the mtcs. 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 analysis. spoligotyping results are very practical and reproducible across different laboratories internationally. 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 is and spoligotyping is in general more discriminative when used with methods based on pcr amplification of the loci containing variable number tandem repeats. 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.  isolation and confirmation of m. bovis by mtcd-mpcr a total of specimens from cattle and from lechwe antelopes were analysed to initially determine the prevalence of mtc species. the samples were collected based on observations of gross pathological lesions upon examination. the observations included generalized lesions involving the lungs, pleural and mediastinal lymph nodes in both lechwe and cattle. these tuberculous lesions were observed by other workers who were investigating gross pathological distribution of tuberculous lesions in both cattle and lechwe. from the samples analysed, mtc strains were obtained with isolates from cattle and from lechwe. following isolation m. bovis was confirmed by screening using the mtcd-mpcr analysis as previously observed. the mtcd-mpcr technique is very useful in the differentiation of mtc at whatever level of diagnosis as it is simple and specific. the presence of m. bovis in wildlife may translate into a perpetual focus of the disease considering that btb control in wild animals is a very difficult undertaking. the only workable solution would be to intensify btb testing in domestic animals so that reactors are removed. furthermore traditional cattle herders must also be informed of the dangers of grazing their animals in areas where lechwe antelopes are present. spoligotyping and multiple locus variable number of tandem repeats analysis spoligotyping of the m. bovis isolates revealed their molecular clonality. two major spoligotype patterns were observed accounting for isolates, and isolates cattle and lechwe respectively. two isolates not identifiable with the two major clusters was given an sb under the global spoligotype patterns diversity provided by the international data base on spoligotyping. this only accounted for 4% of the observed isolates. m. bovis strains from both cattle and lechwe of the kafue basin were found to share the same spoligotype. this spoligotype was previously reported to be the major strain circulating in cattle around the kafue basin, although by that time, no strains were determined from wild animals. m. bovis strains isolated outside the kafue basin were found to share a different cluster. all the isolates lacked spacers and to a characteristic feature that distinguishes m. bovis from m. tuberculosis. molecular studies have previously shown that clonality implies active transmission of disease. thus our results in the kafue basin where there was high clonality between isolates from lechwe and cattle, suggest an active transmission of m. bovis between the two animal species. the spoligotyping results showed that sb clustered outside the kafue basin suggesting that none of the lechwe antelopes harboured m. bovis with such a spoligo pattern. mlva analysis identified distinct clusters, which are corresponding to either sb or sb. within the cluster possessing the spoligo pattern sb there were groups and strain distinguishable one another by loci, miru and qub the cluster possessing the spoligotype sb was differentiated in groups. nineteen strains, from lechwe and from cattle, consisted of a major group and other groups or singletons were discriminated from the major group by a single mlva locus difference each. the spoligotype sb seems to be a descendant pattern of sb lacking spacer and also clustered in sb group with locus difference. the high isolation frequency of spoligotype sb in cattle and lechwe conforms to the finding of previous works from zambia and may demonstrate dominance of this spoligotype despite the small sample size. the sb spoligotype has a considerable degree of geographical dispersion in zambia and other african countries such as algeria and south africa. this spoligotype is also common in continental europe. such a strain could have been introduced through livestock development schemes of trying to improve african beef local breeds. this finding has been highlighted by different workers. spoligotype sb has been reported in this work for the first time in zambia. this spoligotype has been documented in south africa, which has a close livestock trade link with zambia. to our knowledge, this is the first study conducting molecular characterization of m. bovis strains from cattle and lechwe in zambia. however, routine studies have already previously revealed the presence of btb in these animals. in this study, results have shown genetic relatedness between m. bovis in cattle and lechwe antelopes of the kafue basin. it is important to note that the area around lochnivar national park was used for ranching purposes by the early settlers from south africa. this area was only gazetted as an animal sanctuary in it is the only place where kafue lechwe antelopes antelopes are confined. the animals are semi-aquatic living in large groups near water and as such are confined to the area shown in figure . characterization of m. bovis strains based on molecular tools is important in understanding the epidemiology of btb. these results are significant in understanding the transmission and dispersion of m. bovis strains within zambia, 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 sb 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. 
a pcr identified and 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 sb where isolates outside the kafue basin clustered into different nodes of sb and sb 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. 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.
 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 and 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 sb where isolates outside the kafue basin clustered into different nodes of sb and sb 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. bovine tuberculosiscattlemycobacterium bovisstrainswildlifekobus leche kafuensis 