Molecular Analysis of Human and Bovine Tubercle Bacilli from a Local Setting in Nigeria

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2006, p Vol. 44, No /06/$ doi: /jcm Molecular Analysis of Human and Bovine Tubercle Bacilli from a Local Setting in Nigeria Simeon Cadmus, 1 Si Palmer, 2 Melissa Okker, 2 James Dale, 3 Karen Gover, 3 Noel Smith, 3 Keith Jahans, 2 R. Glyn Hewinson, 3 and Stephen V. Gordon 3 * University of Ibadan, Ibadan, Nigeria 1 ; TB Diagnosis Section, Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom 2 ; and TB Research Group, Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom 3 Received 11 February 2005/Returned for modification 14 July 2005/Accepted 1 August 2005 To establish a molecular epidemiological baseline for the strains causing tuberculosis in Nigeria, a survey of isolates from humans and cattle was carried out. Spoligotyping and variable-number tandem-repeat analysis revealed that the majority of tuberculosis disease in humans in Ibadan, southwestern Nigeria, is caused by a single, closely related group of Mycobacterium tuberculosis strains. Using deletion typing, we show that approximately 13% of the disease in humans in this sample was caused by strains of Mycobacterium africanum and Mycobacterium bovis rather than M. tuberculosis. Molecular analysis of strains of M. bovis recovered from Nigerian cattle show that they form a group of closely related strains that show similarity to strains from neighboring Cameroon. Surprisingly, the strains of M. bovis recovered from humans do not match the molecular type of the cattle strains, and possible reasons for this are discussed. This is the first molecular analysis of M. tuberculosis complex strains circulating among humans and cattle in Nigeria, the results of which have significant implications for disease control. * Corresponding author. Mailing address: TB Research Group, Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: (44) Fax: (44) s.v.gordon@vla.defra.gsi.gov.uk. Nigeria has the fourth highest burden of human tuberculosis (TB) in the world, with an incidence in 2002 of 304 cases per 100,000 and a mortality rate of 89/100,000 (19). While a directly observed treatment short-course program has been initiated, the detection of new smear-positive cases remains low, estimated at 12% in These statistics are compounded by the coinfection rate of tuberculosis patients with human immunodeficiency virus, which stood at 27% in 2002 (19). Ibadan, the capital of Oyo State, has a population of approximately 1.3 million and is a major trade route between the southern ports and rest of Nigeria. Previous studies have investigated the burden of tuberculosis in this area, with Nwachokor and Thomas reviewing data for the Ibadan area from 1966 to 1995 (15). They found that, from 1981 to 1995, there was an increase in tuberculosis in this region, with socioeconomic factors and poor health care contributing to this rise. Tuberculosis was found to predominantly affect individuals below 40 years of age, with a peak frequency in the 21- to 30-year range; deaths were highest in children under 10 years old. The situation with animal tuberculosis is less clear, as no national control strategy exists. As tuberculin testing of cattle is not routinely performed, detection of bovine tuberculosis is restricted to the observations of meat inspectors at abattoirs. The degree of zoonotic transmission of tuberculosis from animals to humans is therefore unknown. However, cultural practices exist that could facilitate transmission between cattle and humans. For example, prior to sale, cattle are fattened in close proximity to farmers homes. After being sold at markets, cattle are often slaughtered in nearby abattoirs, where the butchers wear minimal protective clothing and process offal from diseased carcasses with their bare hands. The close association between farmers and animals is exemplified by the Fulani herdsmen, who live their entire lives with their animals, offering ample opportunity for zoonotic transmission of infection. For human TB cases, bacteriological confirmation of diagnosis depends on positive Ziehl-Neelsen (ZN) stains from clinical material, with cultures not performed on a routine basis due to resource issues (9, 12). However, previous research studies have used cultures to determine the relative contributions of Mycobacterium tuberculosis and Mycobacterium bovis to tuberculosis in Nigeria. In the Lagos region, Idigbe and colleagues (7) found that of 102 sputum samples found to be positive for M. tuberculosis complex organisms, 4 were M. bovis; hence, in their setting, tuberculosis due to M. bovis was a relatively minor component. These studies relied on bacteriological methods (niacin production, nitrate reduction, etc.) to differentiate M. tuberculosis from M. bovis. However, molecular typing of M. tuberculosis complex isolates provides a rapid means for discriminating members of the M. tuberculosis complex, a process which can often be difficult when using classical bacteriological methods. The identification of M. bovis is critical for determining the impact of zoonotic transmission of infection to humans, as it gives impetus to the adoption of public health measures such as the pasteurization of milk, cooking of meat, and control of tuberculosis in domestic animals. Our study was conducted to survey strains of the M. tuberculosis complex in humans in Ibadan and to determine the burden of tuberculosis caused by strains other than M. tuberculosis in the human population. At the same time, we surveyed strains recovered from cattle in the same region to determine whether there was any evidence of zoonotic 29

2 30 CADMUS ET AL. J. CLIN. MICROBIOL. transmission. In carrying out this work, two TB-referral hospitals, a private resident s cattle herd, and a major abattoir in the city of Ibadan were visited over 2 years to allow a strain collection to be built. Our study showed that there was an endemic M. tuberculosis strain circulating in the human population in this area and that infection due to M. bovis was present in the human population studied. The implications of our results to tuberculosis control in the area are discussed. MATERIALS AND METHODS Human samples. Human samples were collected from two major TB-referral hospitals in Ibadan, Nigeria: Jericho Chest Hospital and University College Hospital. ZN staining was used to screen the samples to identify sputum-positive TB patients; culture was preformed on the ZN-positive samples with the remainder of the sample using two slopes each of Lowenstein-Jensen medium with pyruvate and/or glycerol. Similarly, fine-needle aspirates from patients with cervical lymphadenitis were collected by a clinician for routine ZN screening, with any remaining volume from these aspirates then cultured as described above. Farm and abattoir samples. As no national bovine TB-control policy is in place, we targeted a local herd and abattoir to sample local M. bovis strains. The cattle that were tuberculin tested under this project were from a private herd of about 170 head, situated approximately 20 km from the Jericho Chest Hospital and University College Hospital. The abattoir used is the biggest in the city of Ibadan and has recorded a high number of cases of bovine TB over the years. It is situated approximately 2 km from University College Hospital and approximately 5 km from Jericho Chest Hospital. Samples were collected from diseased cattle showing gross TB lesions during routine postmortem inspections. Affected organs included the lungs, livers, hearts, spleens, muscles, kidneys, and associated lymph nodes. Processing of samples. The processing of lesions and sputum samples was based on the Becton Dickinson digestion and decontamination procedure. The same procedure was carried out for processing both the sputum and cattle samples (for the cattle samples, grinding with pestle and mortar was first done with the addition of sterile distilled water before the procedure). Using a sterile, 50-ml centrifuge tube with a screw cap, equal amounts of specimen and activated NALC (N-acetyl-L-cysteine)-NaOH of about 5 ml each were added. The centrifuge tube was capped and mixed on a vortex-type mixer until the specimen was liquefied. The mixture was allowed to stand at room temperature for 15 min with occasional gentle shaking. Prepared phosphate buffer was added to the 15-ml mark on the centrifuge tube and mixed, followed by centrifugation for 15 to 20 min at 3,000 g. The supernatant was carefully decanted, and 2 ml of phosphate buffer of ph 6.8 was added to resuspend the sediment. The suspension was inoculated onto Lowenstein-Jensen slopes with pyruvate and/or glycerol and incubated at 37 C for between 8 and 12 weeks. Isolates were harvested for molecular typing analysis by scrapping the growth from a slope into 200 microliters of sterile distilled water and heating at 80 C for 1 h. Spoligotyping. Spoligotyping was performed as previously described by Kamerbeek et al. (8) with minor modifications. The direct repeat (DR) region was amplified by PCR with oligonucleotide primers derived from the DR sequence. Twenty-five microliters of the following reaction mixture was used for the PCR: 12.5 l of HotStarTaq Master Mix (QIAGEN; this solution provides a final concentration of 1.5 mm MgCl 2 and 200 M each deoxynucleoside triphosphate.), 2 l of each primer (20 pmol each), 5 l of the suspension of heat-killed cells (approximately 10 to 50 ng), and 3.5 l of distilled water. The mixture was heated for 15 min at 96 C and subjected to 30 cycles of 1 min at 96 C, 1 min at 55 C, and 30 s at 72 C. The amplified product was hybridized to a set of 43 immobilized oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus. After hybridization, the membrane was washed twice for 10 min in 2 SSPE (1 SSPE is 0.18 M NaCl, 10 mm NaH 2 PO 4, and 1 mm EDTA [ph 7.7]) 0.5% sodium dodecyl sulfate at 60 C and then incubated in 1:4,000-diluted streptavidin-peroxidase conjugate (Boehringer) for 45 to 60 min at 42 C. The membrane was washed twice for 10 min in 2 SSPE 0.5% sodium dodecyl sulfate at 42 C and rinsed with 2 SSPE for 5 min at room temperature. Hybridizing DNA was detected by the enhanced chemiluminescence method (Amersham) and by exposure to X-ray film (Hyperfilm ECL; Amersham) as specified by the manufacturer. Patterns were numbered and prefixed with NH if from human isolates and N if isolated from cattle. Deletion analysis. The use of deletion analysis for the typing of M. tuberculosis complex strains has been previously described (2, 16). In our analysis, we used primers directed against the TbD1, RD4, RD7, RD9, and RD10 loci to generate a deletion profile that would allow speciation of the isolate. The HotStarTaq Master Mix system from QIAGEN was used for PCRs, with primers described previously by Brosch and colleagues (2, 16). The reaction mixture was 25 microliters of HotStarTaq Master Mix, 2 microliters of each primer (at 10 pmol/ l), and distilled water to a final volume of 50 microliters. The PCR cycle was performed on a Perkin-Elmer GeneAmp machine using an initial hot start of 95 C for 15 min, followed by 38 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min; a final extension step of 72 C for 15 min completed the cycle. Products were visualized by electrophoresis through 1% agarose gels. VNTR analysis. The variable-number tandem-repeat (VNTR) method uses six targets (A through F) originally described by Frothingham and Meeker- O Connell (6). These loci vary in the length of internal repeat units, giving alleles that vary in size. Strains can then be called on the basis on the number of repeats at each allele, e.g., would have seven copies of allele A, 5 of allele B, etc. Primers used were as described previously by Frothingham and Meeker- O Connell (6) for the M. tuberculosis exact tandem repeat A to F loci. Primer product sizes were predicted for each locus, and primers were fluorescently labeled to allow analysis of all six loci in a single lane of a polyacrylamide gel. Labels were as follows: FAM for loci A and E, VIC for B and C, and NED for D and F. PCRs were set up using the QIAGEN HotStarTaq Master Mix with the following mix: 10 l of Master Mix, 0.5 l of each primer (at 10 pmol/ml), 7 l of water, and 2 l of the suspension from the heat-killed cells. The thermal cycle used for amplification was an initial denaturation at 94 C for 15 min, followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 68 C for 1 min, and extension at 72 C for 2 min; a final extension at 72 C for 10 min was followed by a holding step at 4 C. PCR products were pooled for each strain, such that the six alleles for each strain could be run in a single lane, and products were then electrophoresed through polyacrylamide gels. Tracked gel images were then analyzed using GeneScan and Genotyper to call the alleles at each locus. In the typing nomenclature, the presence of an asterisk over the number of repeats at the D locus denotes the presence of a truncated repeat; the F locus contains two discrete repeats, hence 3.1 means three repeats of the first, followed by a single copy of the second. RESULTS AND DISCUSSION Tuberculosis strains from humans. Human strains were collected both from sputum samples (55 in total) and fine-needle cervical aspirates (5 in total). The latter samples were collected to determine whether there was any evidence of M. bovis in these extrapulmonary presentations, but difficulties in culture and collection meant that only a few samples were available for molecular analysis. The spoligotype and VNTR patterns of 60 strains cultured from humans are shown in Table 1. In total, we identified 51 strains of M. tuberculosis, 6 of Mycobacterium africanum type I, and 3 of M. bovis. Among the 51 strains of M. tuberculosis, a total of 18 different spoligotype patterns were identified; however, one pattern was most common, present in 35 out of the 51 isolates, and was designated NH1. The spoligotype pattern of this dominant strain is characteristic of the Cameroon family of M. tuberculosis strains (deletion of spacers 23 to 25 [13]). All but four isolates of this common strain share the same VNTR pattern ( *-3-3.1). Hence, the majority of tuberculosis in humans in this Nigerian area is caused by a dominant group of strains that form part of the Cameroon family of M. tuberculosis. The dominance of the Cameroon family of M. tuberculosis in Cameroon and Nigeria may represent the clonal expansion of strains within each region and supports the observation of a stable association of specific clones with geographically localized human populations (4, 19). A distinct group of six isolates from humans (strains 71, 75, 80, 100, 118, and 135) had identical VNTR profiles and a spoligotype pattern associated with strains of M. africanum (absence of spacers 8, 9, and 39). Deletion analysis of these isolates showed that the regions corresponding to TbD1,

3 VOL. 44, 2006 ANALYSIS OF NIGERIAN M. TUBERCULOSIS COMPLEX STRAINS 31 TABLE 1. Spoligotypes and VNTR profiles of human isolates Strain a Spoligotype (age Sexb [yr]) no. c Spoligotype patternd VNTR 59* F (2) N7 5554* * F (25) NH3 RF 71* F (9) NH5 4243* * M (3) NH6 3233* * M (9) NH8 4243* M (24) NH * M (26) NH M (29) NH1 4243* F (30) NH1 4243* M (55) NH1 4243* F (24) N6 5554* M (50) NH15 RF 96 M (34) NH * M (34) NH1 4243* F (27) NH1 4243* M (24) NH * M (25) NH1 4243* F (29) NH1 2243* M (32) NH1 3243* M (21) NH1 4243* F (22) NH * M (45) N4 4344* M (44) NH1 4243* M (26) NH1 4243* F (45) NH1 4243* M (27) NH1 4243* F (27) NH1 4243* M (18) NH M (26) NH1 4243* F (48) NH * M (26) NH1 4243* M (22) NH1 4243* F (30) NH * F (31) NH F (27) NH1 4243* M (42) NH * F (20) NH1 4243* F (27) NH * M (29) NH1 4243* M (70) NH1 2243* M (21) N2 3233* F (22) NH1 4243* F (30) NH * M (26) NH1 4243* F (48) NH1 4243* F (25) NH1 4243* M (45) NH1 4243* M (44) NH1 4243* M (26) NH1 4243* F (45) NH * M (27) NH * F (27) NH1 4243* M (20) NH * M (18) NH * M (26) NH1 4243* F (32) NH1 3243* F (33) NH1 4243* M (42) NH1 4243* M (45) NH1 4243* F (35) NH1 4243*33.1 a Strains marked with an asterisk were cultured from fine-needle aspirates. b F, female; M, male. c Spoligotypes N6 and N7 were given designations SB1027 and SB1029, respectively, at d, spacer present. Profiles are formed by the presence or absence of 43 spacers. RD10, RD4, and RD7 were present, but the RD9 region was deleted (Table 2). Hence, they all appear to be typical subtype I, West African M. africanum strains (11). Two of the M. africanum isolates were from cervical aspirates of children suffering from tuberculous lymphadenitis; whether this observation is significant is unclear. In a PCR-based analysis of 35 strains recovered from fine-needle aspirates of patients in Ethiopia, 29 were positive for M. tuberculosis, while 6 were positive for M. bovis (10). However, in this latter study, it would not have been possible to differentiate between M. tuberculosis and M. africanum, so it is possible that some of the M. tuberculosis strains may have been M. africanum. Therefore, whether M.

4 32 CADMUS ET AL. J. CLIN. MICROBIOL. Strain TABLE 2. Deletion analysis of human isolates Deletion typing for the presence of a : TbD1 RD9 RD10 RD7 RD4 59 ND ND ND ND ND ND ND ND ND ND RF ND ND ND ND ND ND 113 ND 115 ND 116 ND 118 ND 119 ND 120 ND 121 ND 122 ND 123 ND 124 ND 125 ND 126 ND 128 ND 129 ND 131 ND 134 ND 135 ND 137 ND 138 ND 139 ND 140 ND 141 ND 142 ND 143 ND 144 ND 145 ND 146 ND 147 ND 148 ND 150 ND 151 ND 152 ND 153 ND 154 ND a ND, not determined;, region present;, region deleted; RF, repeat fail. africanum is a significant cause of lymphadenitis remains to be determined. Three of the human isolates, 59, 92, and 107, were found to be M. bovis, both on the basis of a characteristic spoligotype (absence of spacers 3, 9, 16, and 39 through 43), and deletion analysis. (The TbD1 locus was present, but RD9, RD7, and RD4 were all deleted in strains 92 and 107.) The spoligotype pattern of isolate 107 was the same as that of two of the M. bovis isolates from cattle (N4), but the VNTR profile of the human isolate ( *-3-3.1) was different from those of the cattle isolates. Isolate 92 had a unique spoligotype (termed N6) but the same VNTR ( *-3-3.1) as isolate 59. This latter isolate, which was from a child with cervical lymphadenopathy, had a very similar spoligotype to N6, the difference being that it lacked spacer 36, suggesting a common evolutionary descent. Strains from cattle. The spoligotypes and VNTR patterns of 17 M. tuberculosis complex strains isolated from infected cattle are shown in Table 3. Six different spoligotypes were found; two were M. tuberculosis-like in that they contained spacers 3, 16, and 40 through 43, while the remaining four were typical M. bovis patterns. The four M. bovis spoligotype patterns were compared with the M. bovis spoligotype database ( to determine whether they had been previously described. Two patterns (SB0944 and SB0952) had been previously described by Njanpop-Lafourcade and colleagues (14) in a survey of M. bovis isolates from Cameroon, a country neighboring Nigeria. The remaining two patterns did not appear in the database and were therefore designated N4 and N5. All the M. bovis spoligotype patterns recovered from cattle in this study can be generated from the SB0944 spoligotype pattern through a single-step deletion of spacers; e.g., SB0952 was generated by the deletion of spacers 13 through 17, N4 by the deletion of spacers 31 and 32, and N5 by loss of spacers 5 and 6. If, as has been suggested, spoligotype patterns evolve by the deletion of spacer units only (17), then pattern SB0944 may represent the spoligotype pattern of the ancestral strain of this group of closely related strains. Strains with spoligotype SB0944 have also been described as dominant in Cameroon (14). Hence, the Nigerian and Cameroon strains seem to share a common evolutionary origin. This may be due to cattle trading links between the countries, as cattle are imported and exported between Nigeria and neighboring African countries. The nine strains with the SB0944 pattern showed minor polymorphisms in VNTR profiles. Four strains, sharing VNTR profile *-3-3.1, were isolated from the same farm, suggesting a common source of infection. Four strains from abattoir samples all showed VNTR profile * While the shared molecular type suggests an epidemiological connection, the lack of records for the slaughtered animals made retrospective tracing difficult. An epidemiological connection can also be argued for strains 21, 34, and 37, which share spoligotype SB0952 and VNTR profile *-3-3.1, and strains 33 and 45 (spoligotype N4, VNTR *-3-3.1). There was no apparent association between cattle breed and M. bovis molecular type, although our sample size was in all probability too small to have revealed a link. Two strains isolated from cattle, 20 and 17, had spoligotype patterns that putatively identified them as M. tuberculosis and M. africanum, respectively. Deletion analysis (Table 4) showed that strain 20 was deleted at the TbD1 locus but was positive for the presence of loci RD9, RD10, RD4, and RD7. This combination of markers places it as a modern M. tuberculosis strain, as defined by Brosch and colleagues (2). VNTR analysis was inconclusive, since repeat testing of one or more of the alleles gave variable repeat numbers; therefore, we could not give the sample a definitive VNTR score and scored it as a

5 VOL. 44, 2006 ANALYSIS OF NIGERIAN M. TUBERCULOSIS COMPLEX STRAINS 33 TABLE 3. Spoligotypes and VNTR profiles of bovine isolates Sample a Cattle breed b c Spoligotype pattern Spoligotype designation d VNTR pattern 4* N dama SB *33.1 7* WF SB * * WF SB * * WF SB * WF NH27 RF 20 WF NH1 RF 21 WF SB * RB N4 5544* WF SB * WF SB * RB SB * WF SB * WF N5 5554* RB N4 5544* RB SB * WF SB * WF SB *33.1 a Strains marked with an asterisk were isolated from cattle kept in a local herd; all the rest are from abattoir samples. b WF, While Futani; RB, Red Bororo. c, spacer present. Profiles are formed by the presence or absence of 43 spacers. d Spoligotypes N4 and N5 were given the designations SB1025 and SB1026, respectively, at repeat fail (RF). The identification of M. tuberculosis in cattle is intriguing; while human-to-cattle transmission of M. tuberculosis has been reported (1), it is generally held that disease in cattle due to M. tuberculosis is less severe than that caused by M. bovis (5). However, the possibility that the isolation of M. tuberculosis in this sample was a result of crosscontamination cannot be ruled out. Similarly, the isolation of an M. africanum strain was interesting, as while it is virulent for cattle, it has been only rarely isolated from this host (3, 18); however, again cross-contamination cannot be ruled out. The three M. bovis strains isolated from humans did not match those from cattle in either spoligotype or VNTR type, although they all had spoligotype patterns similar to the dominant pattern found in cattle. This surprising result may reflect the small number of strains analyzed from cattle; further sampling may reveal a greater diversity than that shown here. Strain TABLE 4. Deletion analysis of bovine isolates Deletion typing for the presence of a : TbD1 RD9 RD10 RD4 RD ND ND ND ND ND RF a ND, not determined;, region present;, region deleted; RF, repeat fail. However, the isolation rate of M. bovis from our sputum samples (3 M. bovis strains cultured from 55 sputum samples) was similar to that of Idigbe and colleagues, who isolated 4 M. bovis strains from 102 sputum samples (7). CONCLUSIONS This study is the first to detail molecular types of M. tuberculosis complex strains from cattle and humans in Nigeria and establishes a baseline for future investigations. We highlight the need for molecular characterization of clinical isolates to ensure that correct estimates are made of the true burden of infection due to M. bovis and M. africanum in developing countries; approximately 13% of the disease in humans in this area was caused by strains of the RD9-deleted lineage (M. africanum type I and M. bovis) rather than M. tuberculosis. A more expansive analysis of the impact of M. bovis infection on human health also seems warranted in light of our finding that M. bovis infection was evident in the human population. Finally, the finding of a number of M. africanum isolates from patients with extrapulmonary tuberculosis will require further sampling to determine whether this is a local phenomenon. Hence, the application of molecular typing to these local isolates has opened up a number of areas for future work that could have a significant impact on national disease control. ACKNOWLEDGMENTS We thank the following people for making this work possible: Martin Vordermeier (VLA) for organizing the visit to VLA Weybridge and his hospitality; David Alonge (University of Ibadan) and Jaiyeola Thomas and Abideen Olayiwola Oluwasola (University College Hospital, Ibadan) for support and guidance; Felix Obaseki of the TB Laboratory, University College Hospital, Ibadan; and the TB Laboratory staff of Jericho Chest Hospital, Ibadan. We also express our sincere thanks to Markus Hilty (Swiss Tropical Institute) for advice and discussion. Work at the VLA was funded by the Department for Environment, Food, and Rural Affairs.

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