patients with suspected tbln or pulmonary tb who came to hospitals or health centers in study sites and provided voluntary consent were recruited into the study during 20062010 . aspirate samples and sputum samples were collected from 2,151 patients attending hospitals in gondar , woldiya , ghimbi , butajira , and negelle , ethiopia . in addition , sputum samples were collected from patients at hospitals in fiche , jinka , and filtu and at health centers at 3 suburban sites in addis ababa ( holeta , sululta , and chancho ) . samples were cultured on lwenstein - jensen medium supplemented with glycerol or pyruvate and on modified middlebrook 7h11 medium optimized for culture of m. bovis . we characterized isolates belonging to the m. tuberculosis complex by using multiplex pcr for large sequence polymorphisms ( 3,4 ) , spoligotyping ( 5 ) , and lineage - specific single - nucleotide polymorphism analysis ( 4,6 ) . isolates of selected spoligotypes were characterized by 24-loci mycobacterial interspersed repetitive unit  variable number tandem repeat ( miru - vntr ) analysis ( 7 ) . four m. tuberculosis isolates from a group of 36 with unusual spoligotype patterns were further characterized by genome sequencing ( illumina inc . , san diego , ca , usa ) . sequencing reads were mapped to the inferred most recent common ancestor of the m. tuberculosis complex ( 6 ) . a final alignment of 13,199 single - nucleotide polymorphism positions was generated and analyzed by using the neighbor - joining method with a tamura - nei evolutionary model ( www.megasoftware.net/mega_papers.php ) . characteristics of 964 cultures positive for acid - fast bacilli are summarized in table 1 . most of these isolates had an intact rd9 region , which identified them as m. tuberculosis . only 4 ( 0.4% ) of 964 isolates had undergone rd9 and rd4 deletions characteristic of m. bovis ( 3 ) . the 4 m. bovis isolates were obtained from cases of pulmonary tb , 3 of which were from patients living in pastoralist communities in southern ethiopia . the 10 nontuberculous mycobacterial isolates were identified as m. intracellulare , m. flavescens , and m. simiae ; 2 of the isolates were from patients co - infected with m. tuberculosis . tb , tuberculosis ; tbln , tb lymphadenitis in cervical lymph nodes ; ntm , nontuberculous mycobacteria ; na , not applicable . tbln samples were not collected in fiche and addis ababa . among the 954 isolates belonging to the m. tuberculosis complex , 671 ( 71% ) belonged to lineage 4 , which was the most common lineage in ethiopia . however , lineage 3 was most prevalent in the northern sites of gondar and woldiya ( 122/257 , 47% ) . two isolates with a characteristic beijing family spoligotype ( spoligotype international type [ sit ] 1 ) were identified as pseudo - beijing strains belonging to lineage 3 ( 8) . thirty - six isolates with an unusual spoligotype pattern ( missing spacers 424 ) and intact for the tbd1 region could not be assigned to known lineages . genome sequencing identified these strains as members of a new lineage ( lineage 7 ) localized between ancient lineage 1 and modern lineages 2 , 3 , and 4 of m. tuberculosis phylogeny ( figure 1 ) . this new lineage 7 was prominent among strains collected in the woldiya region ( 17/133 strains , 13% ) ( table 1 ) . genome sequence analysis of 4 strains representative of 36 related isolates identified them as members of a new phylogenetic lineage ( lineage 7 ) of m. tuberculosis , which has a phylogenetic location intermediate between ancient lineage 1 and modern lineages 2 , 3 , and 4 , and a branch point before the deletion of the tbd1 region ( 3 ) . phylogeny shown is based on 13,199 nt positions that were variable in at least 1 of the 28 m. tuberculosis complex strains represented in the tree . lineage distribution was identical between the 2 disease forms at the national level ; lineage 4 was isolated from 71% ( 442/662 ) and 70% ( 229/328 ) of pulmonary tb and tbln patients , respectively , and lineage 3 was isolated from 25% ( 153/622 ) and 24% ( 79/328 ) , respectively . the m. tuberculosis isolates encompassed 176 spoligotypes ( technical appendix table 1 ) , of which 86 patterns were new to the international genotyping database 2 ( sitvit2 ) ( www.pasteur-guadeloupe.fr:8081/sitvitdemo/ ) ( 9 ) . a total of 11% ( 101/950 ) of the isolates represented single spoligotypes , and 62% ( 591/950 ) were included in 10 major spoligotype clusters ( table 2 ) . * sit , spoligotype international type ; sitvit2 , sit international genotyping database 2 ; go , gondar ; wo , woldiya ; gi , ghimbi ; fi , fiche ; aa , addis ababa ; bu , butajira ; nfj , negelle , filtu , and jinka ; ptb , pulmonary tb ; tbln , tb lymphadenitis in cervical lymph nodes . there was no difference in cluster distribution between pulmonary tb and lymph node tb isolates ; 10% and 11% , respectively , were single types , and 64% and 60% , respectively , were included in dominant clusters . two large clusters representative of lineage 4 ( sit 149 ) and lineage 3 ( sit 25 ) were further characterized by miru - vntr typing ( technical appendix table 2 ) and network analysis ( figure 2 ) . in each case , variable number tandem repeat ( miru - vntr ) networks of major spoligotype clusters , ethiopia , 20062010 . two large spoligotype clusters from lineage 4 ( a ) ( 90 isolates of sopligotype 149 ) and lineage 3 ( b ) ( 73 isolates of spoligotype 25 ) were further characterized by 24-loci miru - vntr typing ( technical appendix table 2 ) . minimum - spanning trees were calculated for each cluster by using miru - vntrplus ( www.miru-vntrplus.org ) . genotypes l and e1 include > 15 isolates and remaining genotypes include < 15 isolates . genotypes indicated by a black circle were isolated from patients with pulmonary tuberculosis ( tb ) ; those in light gray were isolated from patients with tb lymphadenitis in cervical lymph nodes ( tbln ) ; and those in dashed circles were isolated from patients with pulmonary tb and those with tbln . all 4 m. bovis isolates from humans showed typical bovine spoligotype profiles lacking spacers 3 , 9 , 16 , and 3943 . in addition , they lacked spacers 47 and had deletions of rdaf2 , which are features that define strains of the african 2 clonal complex of m. bovis reported from tb - infected cattle in ethiopia ( 10 ) . the frequency of m. bovis in persons in this study ( 0.4% ) is similar to that found in other studies of human tb in africa ( 11 ) and south and central america ( 12 ) , but much lower than that observed among selected populations in tanzania ( 16% ) ( 13 ) , ethiopia ( 17% ) ( 14 ) , and mexico ( 28% ) ( 15 ) . these findings indicate that the overall contribution of m. bovis to human tb is minor but greater in specific areas . in ethiopia , monitoring of zoonotic transmission is needed in urban areas with high rates of bovine tb associated with intensive farming of imported dairy cattle ( r. firdessa et al . data ) and among pastoralist populations from which human m. bovis cases were identified in this study . zoonotic transmission of m. bovis can be excluded as the predominant cause of the high national incidence of tbln in ethiopia . mapping of disease networks by spoligotyping and miru - vntr analysis showed an integrated distribution of the 2 disease forms , which suggested that cases of tbln arise from within the pulmonary tb transmission network , rather than from an external zoonotic source . we identified a novel phylogenetic lineage of m. tuberculosis ( lineage 7 ) in multiple sites and at a high frequency in woldiya in the northeastern highlands of ethiopia . screening of the sitvit2 database ( 9 ) and the us centers for disease control and prevention national tuberculosis genotyping surveillance network database ( l.s . comm . ) identified 23 ( 0.03% ) of 90,000 isolates as members of lineage 7 ; all were isolated from patients whose country of origin ( when known ) was in the horn of africa . lineage 7 is of considerable evolutionary interest because it represents a phylogenetic branch intermediate between the ancient and modern lineages of m. tuberculosis ( 3,4,6 ) . strain frequencies of respective spoligotype international types ( sits ) of mycobacterium tuberculosis isolated in this study , results of large and small sequence polymorphism typing , and typing results of 24-loci mycobacterial interspersed repetitive unit variable number tandem repeats for isolates of spoligotypes sit 25 and sit 149 , ethiopia , 20062010 .
molecular typing of 964 specimens from patients in ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for m. bovis . 
 we report a novel phylogenetic lineage of m. tuberculosis strongly associated with the horn of africa .