Akinbowale Olajide Jenkins

Significance of the Identification in the Horn of Africa of an Exceptionally Deep Branching Mycobacterium tuberculosis Clade

Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7. Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.

Mycobacterium bovis and M. tuberculosis in Goats, Nigeria

To the Editor: Documentation of possible tuberculosis (TB) in goats in Nigeria was reported by Ojo (1) on the basis of gross lesions without culture confirmation. Livestock owners in Nigeria normally graze cattle and goats together, and this practice poses a high risk for transmission of bovine TB among these animals (1). This practice is especially a threat to goats in Nigeria because several reports have described bovine TB in cattle in Nigeria (2–5). However, reports of diagnosis of TB in goats in Nigeria are lacking. Molecular epidemiologic techniques such as deletion typing and spoligotyping have been used to characterize members of the Mycobacterium tuberculosis complex (MTC) and to provide information on transmission of mycobacterial diseases between animals and humans (6). However, because of limited resources and lack of expertise, these techniques are not commonly used in most developing nations such as Nigeria, where TB is endemic (3). Because slaughterhouses provide excellent opportunities for detecting diseases of economic and public health importance, we investigated the presence of mycobacteria in slaughtered goats with lesions suggestive of TB. The investigation was conducted at the Bodija Municipal Abattoir in Ibadan in southwestern Nigeria over a period of 6 months. Slaughtered goats were obtained from local herds and herds in northern Nigeria. A total of 10,389 male and female goats of 2 breeds (West African Dwarf and Red Sokoto) and 1–2 years of age were slaughtered; 1,387 were inspected for gross lesions of TB. Of 1,387 animals screened, 62 (4.47%) had lesions suggestive of TB in the liver, lungs, and mesenteric lymph nodes. Five (0.36%) goats were confirmed positive by culture as described (2). Deletion typing (6) with the RD9 deletion was used to distinguish M. tuberculosis from other members of the MTC. Those isolates with a deletion in this region were further investigated with primers specific for RD4. This reaction distinguishes between M. bovis, M. caprae, and other members of the MTC. Spoligotyping was performed as described (7) to type an M. tuberculosis isolate from a goat after identification of this bacterium by deletion typing. We isolated 4 strains of M. bovis and 1 strain of M. tuberculosis from the goats (Table). Spoligotyping identified the M. tuberculosis isolate as belonging to the East African Indian (EAI)–5 family in the SpolDB4 database. All M. bovis isolates were M. bovis bovis, not M. bovis caprae, according to their deletion typing profile (6). One M. bovis isolate was obtained from a male goat; the 3 remaining M. bovis isolates and the M. tuberculosis isolate were obtained from female goats. Table Results of deletion typing for Mycobacterium tuberculosis and M. bovis in goats, Nigeria* Epidemiologic inferences can be made from the results of our study. First, M. bovis, which is naturally found in cattle, was isolated from 4 slaughtered goats. Although M. bovis caprae was the M. bovis variant most frequently isolated from goats in some areas (8), in our study, only M. bovis bovis was isolated. This finding is consistent with results reported by Crawshaw et al. (9), and suggests transmission from cattle, rather than transmission from the goat reservoir. Second, because the infected goats were adult females, infection may be transmitted to their offspring. Third, M. tuberculosis was isolated from a goat. Its presence in this goat may have been caused by direct transmission from humans because this bacterium may be a natural pathogen of humans. Transmission caused by close cohabitation of goats and humans with advanced TB may occur, given the endemic nature of TB in humans in Nigeria (10). TB cases caused by EAI strains have been found in humans in southwestern Nigeria (4; S.I. Cadmus et al., unpub. data), a finding that supports zoonotic transmission of this organism from humans to goats. However, different lineages of M. tuberculosis may vary in host range, and EAI genotype strains may be adapted to human and animal hosts. Conversely, human-to-animal transmission of M. tuberculosis has been reported in Nigeria relative to infection in cattle (3,4). Thus, confirmation of TB in goats supports the possibility of risk for TB transmission between humans and animals in Nigeria. This study should be interpreted in the context of its limitations. Because the sources of the animals were unknown, we could not determine whether the organisms were imported from a neighboring country (3). In addition, we lacked information on the breed and condition of the animals. However, we have identified M. tuberculosis and TB in goats in Nigeria. Additional studies of other slaughterhouses in Nigeria are needed to confirm our results.

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria.

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

Molecular characterisation of Mycobacterium bovis isolated from African buffaloes ( Syncerus caffer ) in Hluhluwe-iMfolozi Park in KwaZulu-Natal, South Africa

Bovine tuberculosis (BTB), a chronic disease of mammals caused by Mycobacterium bovis, is a threat to South African wildlife. It has been reported that African buffaloes (Syncerus caffer) are reservoir hosts of BTB in South African wildlife populations. This study reports on the molecular identification and typing of 31 M. bovis isolates collected between 1993 and 2008, mainly from buffaloes but also from two lions and a bush pig, in the Hluhluwe-iMfolozi Park (HiP) in KwaZulu-Natal. To study the dynamics of BTB in the buffalo populations, 28 M. bovis isolates from the HiP and epidemiologically related parks were characterised using regions of difference deletion analysis for species identification and spoligotyping, variable number of tandem repeats (VNTR), polymorphic G-C-rich sequences and IS6110 restriction fragment length polymorphism (RFLP) genotyping methods. At least three distinct M. bovis genotypes were found amongst HiP samples. The combination of VNTR typing (using a 16-loci panel) and IS6110 RFLP revealed the presence of three additional genetic profiles in individual buffaloes, demonstrating that the highest level of discrimination was achieved by these typing methods. One of the observed spoligotypes (SB0130) was dominant and represented 75% of isolates from buffaloes. A novel M. bovis spoligotype (SB1474), which is reported for the first time in this study, was observed in 14.3% of isolates from buffaloes. Based on the observed genetic relationships, the findings suggest independent introductions from at least three unrelated sources. These findings improve the knowledge regarding the diversity of circulating M. bovis strains in the HiP.

Mycobacterium bovis infection in livestock workers in Ibadan, Nigeria: evidence of occupational exposure.

SETTING Bovine tuberculosis (TB) is endemic in the cattle population in Nigeria. Livestock workers are at risk of Mycobacterium bovis infection and unaware of their health status. OBJECTIVE To determine the occurrence of pulmonary M. bovis infection among livestock workers. DESIGN A cross-sectional study of livestock traders was conducted for TB through screening of sputum samples using a simple random sampling method coupled with oral interview on the assumption of sub-clinical pulmonary TB infection. Specimens were cultured, and the isolates analysed using molecular typing techniques. RESULTS Overall, 10% (7/70) of the livestock traders had a positive culture indicative of M. tuberculosis complex, which were differentiated into M. bovis (n = 2) and M. tuberculosis (n = 5) using deletion typing. Further spoligotyping analyses of the M. bovis and two available M. tuberculosis isolates classified the strains as SB1432 and SB09444 and LAM_10 CAM and T1 using respectively www.mbovis.org and spotclust databases. Prolonged cough and >3 years in the livestock trade were risk factors for infection. CONCLUSION We confirm that there is undetected pulmonary M. bovis infection among livestock traders in Nigeria. Further studies on the role of occupationally exposed workers in the transmission of M. bovis infection to the larger community are required.

Mycobacterium bovis, but also M. africanum present in raw milk of pastoral cattle in north-central Nigeria

Using deletion typing technique, five mycobacteria isolated from unpasteurised milk samples from cows in north-central Nigeria were characterized as Mycobacterium bovis (n = 4) and M. africanum (n = 1). This report emphasizes that transmission between the animal and human reservoir is a serious threat in Nigeria.

Mycobacterium tuberculosis and Mycobacterium africanum in stools from children attending an immunization clinic in Ibadan, Nigeria.

BACKGROUND Tuberculosis is a major cause of childhood morbidity and mortality in Nigeria. Diagnosis of childhood tuberculosis is a global challenge making early treatment a mirage. In this study we investigated the stools of children for the presence of mycobacteria. METHODS Stool samples from children aged 3 days to 3 years who presented for postnatal immunization at a large university-based clinic in Nigeria, were subjected to Ziehl-Neelsen staining. Samples with acid-fast bacilli were further processed using mycobacterial culture, spoligotyping, and deletion typing. RESULTS One hundred and ninety-two stool samples from different children were collected and processed. Thirty (15.6%) had acid-fast bacilli. Of these, eight had Mycobacterium tuberculosis and one had Mycobacterium africanum. CONCLUSIONS Approximately 5% (9/192) of apparently well children had evidence of potentially serious tuberculosis infection. The usefulness of stool specimens for diagnosing pediatric tuberculosis warrants further investigation.

Comparison of the capillary and agarose electrophoresis based multiple locus VNTR (variable number of tandem repeats) analysis (MLVA) on Mycobacterium bovis isolates

Electrophoretic techniques that can be used for genotyping of bacterial pathogens ranges from manual, low-cost, agarose gels to high-throughput capillary electrophoresis sequencing machines. These two methods are currently employed in the electrophoresis of PCR products used in multiple locus VNTR (variable number of tandem repeats) analysis (MLVA), i.e. the agarose electrophoresis (AE) and the capillary electrophoresis (CE). Some authors have suggested that clusters generated by AE are less reliable than those generated by CE and that the latter is a more sensitive technique than the former when typing Mycobacterium tuberculosis complex (MTC) isolates. Because such a claim could have significant consequences for investigators in this field, a comparison was made on 19 Belgian Mycobacterium bovis strains which had previously been genotyped using CE VNTR analysis. The VNTR profiles of the CE VNTR analysis were compared with those obtained by AE VNTR analysis at 14 VNTR loci. Our results indicated that there were no differences in copy numbers at all loci tested when the copy numbers obtained by the AE VNTR analysis were compared with those obtained by CE VNTR analysis. The use of AE VNTR analysis in mycobacterial genotyping does not alter the sensitivity of the MLVA technique compared with the CE VNTR analysis. The AE VNTR can therefore be regarded as a viable alternative in moderately equipped laboratories that cannot afford the expensive equipment required for CE VNTR analysis and data obtained by AE VNTR analysis can be shared between laboratories which use the CE VNTR method.

Field application of immunoassays for the detection of Mycobacterium bovis infection in the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is considered the most important maintenance host of bovine tuberculosis (BTB) in wildlife in Southern Africa. The diagnosis of Mycobacterium bovis infection in this species mostly relies on the single intradermal comparative tuberculin test (SICTT). As an alternative, the BOVIGAM® 1G, an interferon-gamma (IFN-γ) release assay, is frequently used. The test performance of cell-mediated immunity (CMI-) and humoral immunity (HI-) based assays for the detection of M. bovis infections in buffaloes was compared to identify the test or test combination that provided the highest sensitivity in the study. Buffaloes were sampled during the annual BTB SICTT testing in the Hluhluwe-iMfolozi-Park (KwaZulu-Natal, South Africa) during June 2013. A total of 35 animals were subjected to the SICTT, 13 of these tested positive and one showed an inconclusive reaction. CMI-based assays (BOVIGAM® 1G (B1G) and BOVIGAM® 2G (B2G)) as well as a serological assay (IDEXX TB ELISA) were used to further investigate and compare immune responsiveness. Thirteen SICTT positive buffaloes and one inconclusive reactor were slaughtered and a post-mortem (PM) examination was conducted to confirm BTB. Lesions characteristic of BTB were found in 8/14 animals (57.1%). Test results of individual assays were compared with serial and parallel test interpretation and the sensitivity was calculated as a percentage of test positives out of the number of SICTT positive animals with granulomatous lesions (relative sensitivity). The B1G assay showed the highest individual sensitivity (100%; 8/8) followed by the B2G assay (75%; 6/8) and the IDEXX TB ELISA (37.5%; 3/8). Therefore, using in parallel interpretation, any combination with the B1G showed a sensitivity of 100% (8/8), whereas combinations with the B2G showed a 75% sensitivity (6/8). Out of the 21 SICTT negative animals, 7 animals showed responsiveness in the B2G or IDEXX TB ELISA. In conclusion, this study has shown that the BOVIGAM® IFN-γ assay had the highest test performance.

The first report of Escherichia fergusonii isolated from non-human primates, in Africa

The aim of this study was to determine the resistance phenotypes of selected enteric bacteria isolated from non-human primates at a wildlife-human interface. Bacterial isolates from faecal samples of non-human primates at two wildlife rehabilitation centres in South Africa were screened for the presence of Escherichia coli. The biochemical characterisation of E. coli and E. coli-like bacteria revealed both adonitol positive and sorbitol negative strains – a unique characteristic of Escherichia fergusonii and Escherichia coli K99. Further tests were carried out to identify the isolates, namely growth on Simmons citrate agar supplemented with 2% adonitol and biochemical tests based on their ability to ferment cellobiose and d-arabitol. Antimicrobial sensitivity was determined with microbroth dilution tests employing microtitre plates with 21 different antimicrobial drugs. Molecular characterisation was done with a duplex polymerase chain reaction (PCR) assay that targeted the yliE and EFER_1569 genes. E. fergusonii strains were confirmed by the presence of a 233 bp segment of the yliE gene and a 432 bp segment of the EFER_1569 gene. Twenty-three E. coli-like bacteria were confirmed as E. fergusonii based on the confirmatory tests and they were in 100% agreement. Approximately 87% of them were resistant to polymyxins B and E (colistin) as well as the carbapenem group with occasional resistance to amikacin. This is the first reported isolation and identification of E. fergusonii strains in non-human primates. The findings point to E. fergusonii as a possible emerging pathogen of zoonotic importance.