Simeon Cadmus

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

ABSTRACT 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.

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali, Nigeria, Cameroon, and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

African 2, a Clonal Complex of Mycobacterium bovis Epidemiologically Important in East Africa

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Causes and implications of bovine organs/offal condemnations in some abattoirs in Western Nigeria

Food animals though sources of protein and revenue to man, also serve as vehicles of disease transmission. This work reviews a three year record of slaughtered cattle in 12 abattoirs/slaughter slabs in western Nigeria to determine the economic and public health issues associated with their disease conditions. Out of 641,224 cattle slaughtered, 51,196 (7.98%) were attributable to 14 diseases/conditions including tuberculosis, pneumonia, fascioliasis, pimply gut, paramphistomosis, cysticercosis, dermatophilosis, tonsillitis, taeniasis, ascariosis, abscess, mange, mastitis and immature fetuses. Pneumonia (21.38%), fascioliasis (20.28%) and tuberculosis (7.95%) were major reasons for condemnations; least being ascariosis (0.01%). The lungs (45.66%) and liver (32.94%) accounted for most organ condemned while the heart (0.02%) was the least affected. The proportions of pneumonia, fascioliasis and immature fetuses observed were not statistically different (Mean = 3895.7; 3654.0; 3467.3); however, a significant difference existed with other conditions (MeanA= 3895.7; 3654.0; 3467.3; MeanB = 1359.7; 1057.7; 510.3). Organs/offal condemnations constituted loss of revenue and animal protein as 124,333 kilogrammes worth of meat valued in Naira at N41,613,043 (

Mycobacterium bovis and M. tuberculosis in Goats, Nigeria

332,904) was lost over the period giving an average of N13,871,014 (

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

110,968) annually. This, coupled with fetal wastage represented an economic loss; with associated public health implications.

Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control

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.

Knowledge and practices related to bovine brucellosis transmission amongst livestock workers in Yewa, south-western 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.

Risk factors associated with bovine tuberculosis in some selected herds in Nigeria

Brucellosis is a highly contagious zoonosis caused by bacteria of the genus Brucella and affecting domestic and wild mammals. In this paper, the bacteriological and serological evidence of brucellosis in Sub-Saharan Africa (SSA) and its epidemiological characteristics are discussed. The tools available for the diagnosis and treatment of human brucellosis and for the diagnosis and control of animal brucellosis and their applicability in the context of SSA are presented and gaps identified. These gaps concern mostly the need for simpler and more affordable antimicrobial treatments against human brucellosis, the development of a B. melitensis vaccine that could circumvent the drawbacks of the currently available Rev 1 vaccine, and the investigation of serological diagnostic tests for camel brucellosis and wildlife. Strategies for the implementation of animal vaccination are also discussed.