Genetic diversity and geographical distribution of strains of mycobacterium tuberculosis complex in Ethiopia: Review

Tuberculosis (TB) is a bacterial disease caused by Mycobacterium tuberculosis complex (MTBC), which is a leading cause of death worldwide. The major species of MTBC are M. africanum, M.canettii, M.microti, M.bovis, M.tuberculosis [1]. The members of M.tuberculosis complex are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA sequences but differ widely in terms of their host tropisms, phenotypes, and pathogenicity.


Tuberculosis (TB) is a bacterial disease caused by
Ethiopia is one of the 22 high burden countries with high annual TB incidence of 247 cases/100,000 population in 2012 [2]. M. tuberculosis is the most common cause of human TB, but an unknown proportion of cases are due to M. bovis. Human TB caused by M. bovis (bovine tuberculosis; BTB) is clinically indistinguishable from TB caused by M. tuberculosis and can only be differentiated by laboratory methods [3]. Specifi c data on zoonotic BTB transmission is very scarce in the developing world because the diagnosis of TB most often relays on sputum microscopy only. However, with recent advancement in molecular methods for characterization of M. tuberculosis complex including spoligotyping [4], deletion typing [5], MIRU-VNTR methods [6] identifi cation of members of M. tuberculosis complex at species strain level has been possible.
One of the members of M. tuberculosis complex, M. bovis has been considered as the classical causative agent of BTB in cattle; however, the organism has also been isolated from different species of livestock, wildlife and also from human which become a serious public health problem [1,7]. Molecular strain typing (genotyping) has contributed signifi cantly to the understanding of TB epidemiology and has helped to improve TB control by providing information

Abstract
In Ethiopia, tuberculosis (TB) is one of the major infectious diseases with wide spread geographic distribution and endemic nature, which has been well documented both in human and livestock of the country. TB in livestock has an important economic and public health signifi cance although the actual prevalence of animal tuberculosis at the national level is yet unknown. Identifi cation of the etiology of tuberculosis in human and different species of livestock has a paramount signifi cance in order to understand the transmission pattern, the pathogenesis, and control of the diseases. So far, detection of animal TB in Ethiopia has been carried out most commonly on the basis of tuberculin skin testing, abattoir meat inspection and very rarely on bacteriological techniques. These diagnostic methods can not differentiate the specifi c species of Mycobacterium tuberculosis complex or other species /strains of Mycobacterium, hence utilization of advanced molecular techniques to characterize and genotype the causative agents is important. Genotyping of Mycobaterium species allows understanding the genetic diversity, transmission dynamics and evolutionary/phylogenic relationships of the isolates in different hosts of animal and human. The aim of this paper is, therefore, to review molecular genetic diversity and distribution of mycobacterial infection due to M.tuberculosis complex (mainly M.bovis, M.tuberculosis) and non-tuberculous mycobacterium in Ethiopia in addition to highlighting the zoonotic signifi cance of mycobacterial species originated from livestock and to show the feasible prevention and control options of tuberculosis in cattle and other animals. on transmission dynamics, determining the importance of reactivation versus exogenous re-infection, investigating/ confi rming outbreaks, confi rmation of laboratory crosscontamination and to identify the clonal spread of successful clones, including multi-drug-resistant ones [8,9]. Furthermore, molecular typing has revealed that the MTBC has a diverse population structure with manifold lineages that show large differences in their geographical occurrence. Several typing techniques have been used to characterize isolates of MTBC including IS6110 restriction fragment length polymorphism (RFLP), spoligotyping and MIRU-VNTR methods as the most frequently used [4,5]. Genotyping of MTBC is used to identify and distinguish MTBC into distinct species, strains lineages and/or sublineages that are quite useful for TB tracking and control and examining host-pathogen relationships [8].
In Ethiopia, a number of researches have been carried out

General description of genus mycobacterium
Mycobacterium is a pathogenic bacterium that belongs to the class Actinomycetes, order Actinomycetales and family Mycobac-  Figure 1 [5,8].
Mycobacteria are resistant to decolorization due to its thick cell wall and high lipid content [12]. This property is termed acid fastness, so that Mycobacterium is commonly referred to as acid-fast bacilli. In contrast, these microorganisms are not readily stained with the Gram stain method and are considered weakly Gram-positive. The rods tend to stain irregularly and often have a beaded appearance. They are slow growers, i.e. they require more than 7 days forming colonies when sub-cultured on Lowenstein-Jensen media. M. tuberculosis forms rough colonies on Lowenstein-Jensen solid media. Colonies formed by M. bovis are smooth with irregular edges on egg based media [13]. The genus Mycobacterium is most closely related to the genera Rhodococcus and Nocardia and all three genera have a similar cell wall type and are acid fast but comparatively Mycobacterium have characteristics of slow growth rate [14].

Morphology and biochemical composition
Mycobacterium is a fastidious, slow growing, lipid-rich, hydrophobic and acid fast bacterial rod shape which resists decolorization with acid alcohol. It has no outer membrane; rather it has a cell wall made up of different macromolecules, namely peptidoglycans, arabinogalactan, mycoclic acid and lipopolysaccharide or lipoarabinomannan (LAM) which is anchored plasma membrane ( Figure 2). Mycoclic acid is the major component of the cell wall envelope, greater than 50% by weight, a signifi cant number which are responsible for their resistance to humoral defense mechanism, disinfectants, acids and alkalis. The staining characteristic of M. tuberculosis is due to the mycoclic acid which resists decolorization by acid alcohol Figure 2.

Genotyping of mycobacterium tuberculosis Complex
The genomics of Mycobacterium tuberculosis complex have proved considerably more diffi cult to elucidate than those of many other bacteria. This is due to the diffi culties presented by extremely slow growth, the unique cell wall composition of the organism, the need for protection of laboratory personnel and a paucity of cloning vehicles [16]. For many years, it was thought that human tuberculosis evolved from the bovine disease by adaptation of an animal pathogen to the human host. This hypothesis is based on the property of M. tuberculosis to be almost exclusively a human pathogen, whereas M. bovis has a much broader host range. In addition, RFLP has poor discriminatory power for isolates with low numbers of insertion sites such as M. bovis [17]. Spoligotyping is a rapid, polymerase chain reaction (PCR)-based method for genotyping strains of the MTBC.
It is easier to perform and requires smaller amounts of DNA than RFLP analysis but its discriminatory capacity is inferior to RFLP. Spoligotyping is useful in discriminating strains with low IS6110 copy numbers such as M. bovis and it can be performed on nonviable organisms. The clinical usefulness of spoligotyping is determined by its rapidity, both in detecting causative bacteria and in providing epidemiologic information on strain identities [4]. and exchange between laboratories [8,9]. humans, and many other animals [1,18].

Several prevalence studies have been performed recently
showed that BTB is endemic in cattle; however, prevalence varies depending on the geographical areas, breeds and husbandry practices. Abattoir and dairy farm studies from central Ethiopia have reported prevalence between 3.5 and 13.5% and locally in peri-urban Addis Ababa up to 50% [19][20][21]. In contrast, lower prevalence of 0.9% was reported in traditionally kept zebu cattle [22]. Other livestock than cattle have also been investigated. Based on gross pathology, prevalence of 5-10% was reported in camels slaughtered at Dire Dawa abattoir in eastern Ethiopia and in Addis Ababa abattoir .The observed variability of BTB disease frequency in Ethiopia might well be infl uenced by different livestock production systems (rural/pastoral/peri-urban) and different geographic and climatic contexts. Transmission of BTB seems to be higher in intensive peri-urban settings when compared to extensive rural and pastoral areas [23,24].

Genetic diversity and geographical distribution
In Ethiopia a number of molecular epidemiological studies on BTB in livestock were carried out in different regions of the country [19,20,24,25]. According to Berg, et al. [20] a total of seven different spoligotypes were identifi ed, of which SB0133, SB1476, and SB1176 were more prevalent. SB0133  predominates in Jinka but it also represented in AddisAbaba, Gimbi and Woldia. SB1476 is the most common pattern in both Gimbi and Gonder but it was found in Jinka and Butajira. SB1176 is the most prevalent among the samples from Addis Ababa but can also be seen Butajira, Gonder and Woldiya. The remaining four spoligotypes patterns, SB0134, SB1488, SB1489 and SB1477, are all highly related to SB0133, and isolates with these patterns were mostly collected from Addis Ababa or Jinka abattoirs. The largest diversity of M. bovis strains was found in Addis Ababa abattoir with fi ve different spoligotypes , likely refl ecting the wide geographical area (Figure 4) from which cattle were sourced [20].

Sheep and goat tuberculosis in Ethiopia
TB in goats and sheep is caused by members of M. tuberculosis complex predominantly by M. bovis and M. caprae and few caused by M. tuberculosis. The overall animal prevalence of TB in small ruminants was 0.5% (95% CI: 0.2%-0.7%) at ≥4mm and 3.8% (95% CI: 3%-4.7%) at cutoff ≥2mm [26]. Caprine tuberculosis (TB) caused mainly by M. bovis and M. caprae poses a risk to goat health and production in developing world .Goats may become infected with M. bovis when sharing pastures with infected cattle, at watering points, market places and shared night shelters Figure 5.

Genetic diversity and geographical distribution
Mycobacteriological culture and molecular characterization of isolates from goats resulted in isolation of M. tuberculosis strain SIT149 [26]; SIT53 [27] and non tuberculous mycobacteria as causative agents of tuberculosis and tuberculosis like diseases in goats, respectively. The isolation of Mycobacterium tuberculosis in goats suggests a potential transmission of the causative agent from human. The SIT149 strain of M. tuberculosis is a dominant strain in Ethiopia and it was a common isolate in human pulmonary TB patients from the same Afar Pastoral Region. The isolation of the SIT53 strain of M. tuberculosis from goats suggests transmission from humans [27].

Camel tuberculosis in ethiopia
Tuberculosis caused by M. bovis is the most common form of tuberculosis in domestic camels. The prevalence of camel TB was 10.04% (91/906) on the basis of pathology [24] . Tuberculosis as a zoonosis from camel to human also plays an important role among nomadic people where milk and milk products are consumed raw [28]. The occurrences of TB lesions in camels were relatively higher in the younger and older camels than other age groups. Older animals are affected by TB which could be due to the fact that older animals have weaker immune system. The higher frequency of lesion in younger camels could be due to the less developed immunity [29] Young camels can also be easily infected with higher doses of mycobacteria via colostrums from infected camel in a similar way, as it occurs in cattle.

Genetic diversity and geographical distribution
Molecular epidemiological studies on Camel tuberculosis indicated that one of the strains which caused a generalized disseminated TB in camel was SB0133 [24], whereas the other strain was SB1953 which has been recently reported to the database. In Ethiopia, a number of studies reported new strains with specifi c spoligotype pattern in cattle. On the other hand, the isolation of SB0133 M. bovis strains in the present study from camel of pastoral area of Ethiopia is in line with the isolation of this strain from cattle of southern Ethiopia [20]. The ma jority of camel TB lesions were caused by NTM [24]. On the other hand, Gumi [25] have characterized M. tuberculosis strain SIT149 using spoligotype from disseminated generalized TB cases of camel and NTM as a causative agents of camel TB in south east pastoral camels of Ethiopia [25].

Zo onotic tuberculosis in Ethiopia
In developing countries, like Ethiopia, TB is widely distributed. The proportion of which BTB contributes to the total of tuberculosis cases in humans. It depends on the prevalence of the disease in animals, socioeconomic conditions, consumer habits, practiced food hygiene and medical prophylaxis measures. The primary bacterium that causes TB in humans is M. tuberculosis. In countries where BTB in cattle is still highly prevalent, pasteurization is not widely practiced  M. tuberculosis still responsible for most cases of death due to infectious diseases after HIV. The incidence of pulmonary tuberculosis caused by M. bovis is higher in farm workers than in urban inhabitants [32]. In rural areas of Ethiopia most people drink raw milk and do have extremely close attachment with cattle (such as sharing shelter) that intensifi es the transmission and spread of BTB [10]

Prevention and control
The basic strategies required for control and elimination of bovine tuberculosis are well known and well defi ned.
However, because of fi nancial constraints, scarcity of trained professionals, lack of political will, as well as the underestimation of the importance of zoonotic tuberculosis in both the animal and public health sectors by national governments and donor agencies, control measures are not applied or are applied inadequately in most developing countries [3]. Cattle should not be treated at all and as such farm animals with tuberculosis must be slaughtered (culled) [33]. This is because the risk of shedding the organisms, hazards to humans and potential for drug resistance make treatment controversial. As this disease is primarily transmitted from cattle to humans in milk, Also, the role of wild fauna in the epidemiology of tuberculosis in livestock and humans need not be ignored, as they have been reported to serve as a reservoir of the pathogen. Animal husbandry practices should be improved upon to reduce contact between domestic livestock and wild ruminants especially during grazing. Vaccination is practiced in human medicine, but it is not widely used as a preventive measure in animals [35][36][37].