A Cross-sectional Study on Zoonotic and Reverse Zoonotic Transmission of Tuberculosis in Central Ethiopia


 Background: The zoonotic transmission of tuberculosis (TB) from cattle to humans has long been recognized while its reverse zoonotic transmission from humans to animals has recently been reported. The socioeconomic situations of the rural communities of Ethiopia are conducive for the zoonotic and reverse zoonotic transmission of TB between cattle and human. The objective of this study was to investigate the zoonotic and reverse zoonotic transmission TB between humans and cattle central Ethiopia.Methods: To achieve this objective a cross-sectional study was conducted on 1896 cattle slaughtered in two abattoirs and 392 TB suspicious human subjects visiting health institutions for treatment. Post mortem examination, mycobacteriological culture, and spoligotyping were used for the study. In addition, the Spoligotype International Types and VNTR (variable number of DNA tandem repeats) International Types (SITVIT2) database and the online “Run TB-Lineage” were used to identify SIT and lineages of the human and cattle isolates.Results: Gross TB lesion was detected in 4.2% (80/1896) the slaughtered cattle and predominantly (52.5%) found in the thoracic cavity. But culture positivity was only 26.3% (21/80) in suspicious lesions. Of the 21 cattle isolates, 12 (57.2%) were M. bovis while the remaining nine (42.8%) were M. tuberculosis. SIT50, SIT118 and SIT1318 were isolated from both humans and cattle. Similar to the low culture yield in cattle tissues, only 22% (86/392) the sputa from TB suspicious humans were culture positive. These 86 human isolates were classified into 81 M. tuberculosis, three M. africanum and two M. bovis by spoligotyping. The two human M. bovis isolates had the pattern of SB1443, which was not isolated from cattle in this study.Conclusion: The zoonotic and reverse zoonotic transmissions of TB were confirmed in Ethiopia by the isolation of two M. bovis from human and isolation of nine M. tuberculosis from cattle, which suggested higher role of M. tuberculosis in cattle compared to the role of M. bovis in humans.

bTB in animal populations in developing countries is due to the lack of or inadequate control measures for the socioeconomic problems associated with the presently available test and slaughter control measure [4].
The direct correlation between M. bovis infection in cattle and zoonotic TB in the human population has been well documented in industrialized countries [4]. Zoonotic TB is caused by M. bovis and transmitted from animals to humans through consumption of foods infected animals and through inhalation of cough droplets from infected animals during close physical contact with them. Zoonotic TB is also an occupational hazard for farmers, abattoir workers, butchers and veterinary professionals.
Epidemiological studies of TB in cattle and in humans in Zambia [5] and in Ethiopia [6] indicated associations between tuberculin-positive cattle and human TB. In Zambia, it was observed that households that reported a TB case within the last one year were seven times more likely to own herds containing tuberculin-positive cattle. Similarly, tuberculin positivity was three-fold higher in cattle owned by farmers with active TB than in those owned by farmers who did not have active TB in Ethiopia. These observations could suggest the transmission of mycobacteria between humans and cattle, which could be the transmission of M. bovis from cattle to humans or the transmission of M. tuberculosis from humans to cattle. A few studies in Ethiopia reported the isolation of M. bovis from sputum and ne needle aspirate of human TB cases [7][8]. According the literature review reported earlier [9], in countries where bTB is still common and pasteurization of milk is not practiced, an estimated 10-15% of human TB is caused by M. bovis and the infections of M. bovis is thought to occur through drinking raw milk [10] and through inhalation in rural areas in people who live in close contact with cattle [11].
The few studies conducted in Ethiopia are limited in their geographic coverage and representations.
Geographic regions such the East Shewa Zone of the central Ethiopia where dairy cattle and beef farming are widely practiced have not been considered. East Shewa Zone of central Ethiopia is prone to risk of transmission of mycobacteria between cattle and humans. There are many butchers in the area and cattle from the beef farms and nearby rural areas are slaughtered by butchers for selling raw meat and the area is known by the business of raw meat. In addition to beef cattle, dairy cows are kept by the farmers living in the towns and in the rural districts surrounding the towns. A signi cant percentage of raw milk are supplied to the consumers in living in the area. These common customs of raw milk and raw meat consumptions could facilitate the transmission of M. bovis from cattle to humans. On the other hand, Ethiopia stands 7 th in the 20 human TB burden countries with the incidence rate of 165 individuals per 100,000 population [12]. East Shewa Zone is one of the densely populated areas of Ethiopia with high rate of human mobility since the main road to the Port of Djibouti passes through the Zone. Therefore, there are conducive environment for the transmission of human TB, and there could be high chance of transmission of M. tuberculosis from humans to cattle because of the close physical contact between humans and their cattle. It is paramount important to investigate if there is transmission TB between cattle and humans since there are conducive epidemiological situations. The present study was conducted to investigate the zoonotic and reverse zoonotic transmission TB between humans and cattle East Shewa, central Ethiopia.

Prevalence of BTB in feedlots and dairy farms East Shewa
The average lesion prevalence was 4.2% (80/1896) in the two abattoirs. Lesion prevalence was 7.6% (48/630) in cattle slaughtered at Bishoftu abattoir while it was 2.5% (32/1266) in Adama abattoir (Table   1). However, only 26.3% of the 80 suspicious lesions were turned to be culture positive. The e distribution and frequency of lesions in different tissues of cattle was presented in Table 2. The highest proportion (52.5%) of the lesion was recorded in the lungs and thoracic lymph nodes followed by the lymph nodes of the head and neck.
Sputum culture positivity and demographic characteristics of the study subjects Of the 21 isolates, 12 (57.2%) isolates were con rmed to be M. bovis while the remaining nine (42.8%) were M. tuberculosis. The 12 M. bovis isolates were grouped into nine spoligotype patterns; three of which were clustered each consisting two isolates while the remaining six were singleton each consisting of one isolate ( Table 4). Two of the nine M. bovis spoligotype patterns were shared types (SB1265 and SB1176) while the remaining seven types were orphans. Cattle isolates of M. tuberculosis were grouped into nine spoligotype patterns in which case one isolate constituted one spoligotype pattern. Four of the nine spoligotype patterns (SIT50, SIT117, SIT118 and SIT1318) were shared types while the remaining ve were orphans (Table 4). Almost all (eight) of the nine spoligotypes of cattle isolates of M. tuberculosis were members of the Euro-American Lineage while the remaining one was a member of Indio-Oceanic Lineage.

Human isolates
A total of 86 human isolates were identi ed that were classi ed into 81 M. tuberculosis, three M. africanum and two M. bovis ( Table 5). The two isolates of M. bovis had SB1443 pattern, that has already been registered in M. bovis database. The spoligotype pattern of the human M. bovis isolates did not match with any of the patterns of the cattle M. bovis isolates. As indicated in Table 5, 75% (61/81) of the M. tuberculosis isolates were grouped under the shared spoligotypes while the remaining 25% (20/81) were grouped under orphan spoligotypes. The dominantly identi ed spoligotypes were SIT149, SIT53 and SIT118 each consisting of 18, 11 and six isolates, respectively. SIT50, SIT118 and SIT1318 which were isolated from cattle were also isolated from humans. Totally, the 81 M. tuberculosis isolates were grouped into 32 different spoligotype patterns and the majority (84.4%) these patterns were members of the Euro-American Lineage followed by the East African-Indian Lineage (12.5%) and Indio-oceanic Lineage (3.1%).
The average lesion prevalence was 4.2% (80/1896) in the two abattoirs. Lesion prevalence was 7.6% (48/630) in cattle slaughtered at Bishoftu abattoir while it was 2.5% (32/1266) in Adama abattoir (Table   1). However, only 26.3% of the 80 suspicious lesions were turned to be culture positive. The e distribution and frequency of lesions in different tissues of cattle was presented in Table 2. The highest proportion (52.5%) of the lesion was recorded in the lungs and thoracic lymph nodes followed by the lymph nodes of the head and neck.

Prevalence of BTB in feedlots and dairy farms East Shewa
The average lesion prevalence was 4.2% (80/1896) in the two abattoirs. Lesion prevalence was 7.6% (48/630) in cattle slaughtered at Bishoftu abattoir while it was 2.5% (32/1266) in Adama abattoir (Table  1). However, only 26.3% of the 80 suspicious lesions were turned to be culture positive. The e distribution and frequency of lesions in different tissues of cattle was presented in Table 2. The highest proportion (52.5%) of the lesion was recorded in the lungs and thoracic lymph nodes followed by the lymph nodes of the head and neck.
Sputum culture positivity and demographic characteristics of the study subjects Table 3 shows selected demographic characteristics of the human TB suspicious cases, who were used a sources sputum at the two towns. Sputum culture positivity was only 21.9% (86/392). Fifty culture positive samples were from the samples collected from Adama while 36 were from Bishoftu. Culture positivity was 18.2% (50/274) in samples collected from Adama Town while it was 30.5% (36/118) in samples collected from Bishoftu Town.

Animal isolates
Of the 21 isolates, 12 (57.2%) isolates were con rmed to be M. bovis while the remaining nine (42.8%) were M. tuberculosis. The 12 M. bovis isolates were grouped into nine spoligotype patterns; three of which were clustered each consisting two isolates while the remaining six were singleton each consisting of one isolate ( Table 4). Two of the nine M. bovis spoligotype patterns were shared types (SB1265 and SB1176) while the remaining seven types were orphans. Cattle isolates of M. tuberculosis were grouped into nine spoligotype patterns in which case one isolate constituted one spoligotype pattern. Four of the nine spoligotype patterns (SIT50, SIT117, SIT118 and SIT1318) were shared types while the remaining ve were orphans (Table 4). Almost all (eight) of the nine spoligotypes of cattle isolates of M. tuberculosis were members of the Euro-American Lineage while the remaining one was a member of Indio-Oceanic Lineage.

Human isolates
A total of 86 human isolates were identi ed that were classi ed into 81 M. tuberculosis, three M. africanum and two M. bovis ( Table 5). The two isolates of M. bovis had SB1443 pattern, that has already been registered in M. bovis database. The spoligotype pattern of the human M. bovis isolates did not match with any of the patterns of the cattle M. bovis isolates. As indicated in Table 5, 75% (61/81) of the M. tuberculosis isolates were grouped under the shared spoligotypes while the remaining 25% (20/81) were grouped under orphan spoligotypes. The dominantly identi ed spoligotypes were SIT149, SIT53 and SIT118 each consisting of 18, 11 and six isolates, respectively. SIT50, SIT118 and SIT1318 which were isolated from cattle were also isolated from humans. Totally, the 81 M. tuberculosis isolates were grouped into 32 different spoligotype patterns and the majority (84.4%) these patterns were members of the Euro-American Lineage followed by the East African-Indian Lineage (12.5%) and Indio-oceanic Lineage (3.1%). Table 3 shows selected demographic characteristics of the human TB suspicious cases, who were used a sources sputum at the two towns. Sputum culture positivity was only 21.9% (86/392). Fifty culture positive samples were from the samples collected from Adama while 36 were from Bishoftu. Culture positivity was 18.2% (50/274) in samples collected from Adama Town while it was 30.5% (36/118) in samples collected from Bishoftu Town.

Animal isolates
Of the 21 isolates, 12 (57.2%) isolates were con rmed to be M. bovis while the remaining nine (42.8%) were M. tuberculosis. The 12 M. bovis isolates were grouped into nine spoligotype patterns; three of which were clustered each consisting two isolates while the remaining six were singleton each consisting of one isolate ( Table 4). Two of the nine M. bovis spoligotype patterns were shared types (SB1265 and SB1176) while the remaining seven types were orphans. Cattle isolates of M. tuberculosis were grouped into nine spoligotype patterns in which case one isolate constituted one spoligotype pattern. Four of the nine spoligotype patterns (SIT50, SIT117, SIT118 and SIT1318) were shared types while the remaining ve were orphans (Table 4). Almost all (eight) of the nine spoligotypes of cattle isolates of M. tuberculosis were members of the Euro-American Lineage while the remaining one was a member of Indio-Oceanic Lineage.

Human isolates
A total of 86 human isolates were identi ed that were classi ed into 81 M. tuberculosis, three M. africanum and two M. bovis ( Table 5). The two isolates of M. bovis had SB1443 pattern, that has already been registered in M. bovis database. The spoligotype pattern of the human M. bovis isolates did not match with any of the patterns of the cattle M. bovis isolates. As indicated in Table 5, 75% (61/81) of the M. tuberculosis isolates were grouped under the shared spoligotypes while the remaining 25% (20/81) were grouped under orphan spoligotypes. The dominantly identi ed spoligotypes were SIT149, SIT53 and SIT118 each consisting of 18, 11 and six isolates, respectively. SIT50, SIT118 and SIT1318 which were isolated from cattle were also isolated from humans. Totally, the 81 M. tuberculosis isolates were grouped into 32 different spoligotype patterns and the majority (84.4%) these patterns were members of the Euro-American Lineage followed by the East African-Indian Lineage (12.5%) and Indio-oceanic Lineage (3.1%).

Discussion
In the present study, zoonotic transmission of M. bovis from cattle to humans and the reverse zoonotic transmission of M. tuberculosis from humans to cattle were investigated on 1896 cattle and 392 TB suspicious humans using mycobacterial culture and spoligotyping. TB suspicious lesions were sampled from cattle while sputum samples were the isolation mycobacteria and their identi cation using spoligotyping.
The result of the study indicated that the prevalence bTB was low in cattle slaughtered on the basis of lesion. This observation was similar with the observations made by other studies in different abattoirs in the country prior to the present study [21][22][23]. This observation can be related to the fact that almost all (about 98%) the cattle that are slaughtered in the country are indigenous zebu breed which are relatively resistant to bTB [17,[24][25][26]. Moreover, the majority of the zebu cattle that are slaughtered in Ethiopia are raised by the traditional farmers who raise their cattle under extensive cattle farming which does not favor the transmission of bTB [17]. Furthermore, the percentage of isolation of M. tuberculosis complex from lesions was low in this study similar to the results of other studies conducted earlier [18,21,27]. Such low percentage of isolation of M. tuberculosis complex in zebu cattle in Ethiopia could be associated with different factors. One of the factors could be the containment the M. tuberculosis complex in the granulomas, which are usually calci ed and could lead to the death of M. tuberculosis complex leading to the low yield of culturing of such lesions. As observed during the last two decades, most of the lesions detected in zebu cattle were localized to one anatomical site and calci ed [17,[25][26]. The other factor could be human errors that may be introduced during the transportation, storage and processing of the lesioned tissue for culturing.
The culture of positivity the sputum samples collected from TB suspicious human cases was also low. Similar to this observation, earlier study in Ethiopia reported low percentage of isolation of M. tuberculosis from the sputum of TB patients [28]. The same study indicated that 83.2% (247/297) pulmonary TB suspected patients had a negative smear results for acid fast bacilli. The chance of isolation of M. tuberculosis complex from smear negative TB cases low. Culture positivity of sputum is directly related to the availability of the bacilli in the sputum, which can also be affected on the nature of the granuloma. If the granuloma is dense and solid, the chance of excretion of the bacilli in the sputum is minimal, which in turn leads the low yield of sputum culture. In addition, if sputum proper is not collected, and if the sputum storage and processing for culture are not performed using the optimum procedures, the yield of sputum culture is low.
In the present study, M. bovis was isolated from human TB patients while on the other hand, M. tuberculosis was isolated from TB lesions of cattle. Thus, this study indicated that TB is a good example of zoonosis and reverse zoonosis. Similarly, earlier studies reported the isolation of M. bovis from human TB patients in Ethiopia although the percentage of isolation was low [29][30][31][32][33]. Besides, M. tuberculosis was isolated from TB lesions of cattle in Ethiopia and in other countries [18,29,[34][35][36][37]. Moreover, M. tuberculosis was also isolated from other species domestic animals and zoo animals [38][39][40]. Although such studies are in generally scarce in Africa, the isolation of M. tuberculosis was reported from animals in few African countries [41][42]. The possible sources of infection of animals with M. tuberculosis could be sputum, urine and feces of humans, which arise from active cases of human TB in the respiratory tract, urinary tract or gastrointestinal tract, respectively [43].
The present studies and earlier Ethiopian studies reported greater percentage of isolation of M. tuberculosis (reverse zoonosis) in cattle tissues as compared to the percentage of the isolation of M. bovis from humans. This observation could suggest that regarding B transmission in Ethiopia reverse zoonosis is more important than zoonosis, which is directly in uenced by the prevalence TB in the primary host. The prevalence of bTB in the indigenous cattle, which constitute about 98% of the Ethiopian cattle population is low, and subsequently, there is a low chance of transmission of bTB from cattle to humans. On the other hand, Ethiopia is one of the 10 countries of the world with high burden of human TB [12], which gives a high chance of transmission of M. tuberculosis from humans to cattle because of the sociocultural link of Ethiopian farmers with their cattle. But, contrary to the Ethiopian reports, studies from other African countries reported the isolation of higher percentage of M. bovis from humans as compared to the percentage of isolation of M. tuberculosis from animals [8,[44][45][46].

Conclusion
Zoonotic transmission TB was observed by the isolation of two M. bovis from human subjects while the reverse zoonotic transmission was con rmed by isolation of nine M. tuberculosis isolates from cattle tissues. This observation could suggest that the prevalence of M. tuberculosis infection in cattle is higher than the prevalence of M. bovis infection in humans.

Study design
This study was a cross-sectional study in which the zoonotic transmission of M. bovis from cattle to humans and the reverse zoonotic transmission of M. tuberculosis from humans to cattle were investigated in the East Shewa Zone of central Ethiopia.

Study setting
The eld sample collection part of the study was conducted from August 2015 to August 2016 at Adama and Bishoftu towns East Shewa Zone of Oromia Regional State, central Ethiopia (Figure 1). Bishoftu and Adama towns are located at 47km and 95km southeast of Addis Ababa, respectively. These two towns are known by beef production and selling of beef meat. Cattle from the beef farms and rural areas surrounding the two towns are slaughtered in the abattoirs located at these towns and raw meat consumption is very common in the area. In addition to beef cattle, dairy cows are kept by the farmers living in these towns and in the rural districts surrounding the two towns. Thus, a signi cant percentage of raw milk are supplied to the consumers in living in these towns and the surrounding districts. Unfortunately, as BTB is prevalent in dairy farms in the country and these dairy farms are not expected to be free from the disease.

Sample size
The determination was based on the availability of fund and logistics to support the research activities of the project. Since the main objective of this research was to evaluate the transmission of M. tuberculosis between cattle and humans, it was tried to collect the maximum number of samples by examining large number of cattle and also collecting large number sputum samples. No statistical calculation was used to estimate the sample size in both human and cattle studies. Thus, the available fund and logistics could cover the examination of 1,896 cattle at the two (1266 at Adama and 630 at Bishoftu) abattoirs. In addition, 392 (274 from Adama and 118 from Bishoftu) sputum samples were collected from the two towns.

Participants of the study
The study subjects were comprised of cattle slaughtered at these two abattoirs and human TB patients who visited the heath care centers at the two towns during the study period. Post mortem examination for BTB was conducted on cattle, which were originated from the beef farms in located either at the two towns or in the surrounding districts. Similarly, sputum samples of individuals who were living in these towns and or the surrounding districts plus who had connections with either dairy or beef cattle were used for bacteriological and molecular analysis.

Variables
The primary outcome was the isolation the same species of M. tuberculosis complex from both cattle and humans, which could suggest the transmission of TB between cattle and humans. Other secondary outcomes were percentage of cattle with TB lesion in their tissues and the percentage of suspicious TB lesion that were positive by culture and spoligotyping. With regard to the human study, the secondary outcome was the percentage of TB suspicious patients who were positive by culture and spoligotyping. The diagnostic criteria for cattle study were gross BTB lesion, and con rmation by culture and spoligotyping. While the diagnostic criteria for human study was clinical examination by the responsible health professionals in the health care centers, and then further con rmation of culturing of sputum and spoligotyping the isolates. The nal con rmation of the transmission of M. tuberculosis complex species between cattle and humans was made by spoligotyping.

Sample collection
As the primary objective the study was to con rm the existence of the transmission of M. tuberculosis species between humans and animals, focus was made on the search of BTB lesions in tissues of cattle and then collection of suspicious lesions. Data on the body condition and origin of each study animal were collected. However, data on the age of the animal not collected. In addition, the tissues from which suspicious lesions detected were recorded. The BTB suspicious tissue samples were collected into universal bottles with normal saline and transported to the TB laboratory Aklilu Lemma Institute of Pathobiology of the Addis Ababa University in cold chain. In total, 1896 cattle were examined at the two abattoirs and 80 BTB suspicious lesions were collected for culturing. The number of the animal recruited was dependent on the availability of logistics and convenience of the slaughterhouses.
Sample collection in humans was conducted as part of the routine diagnostic procedures of TB and sputum samples submitted to the hospitals and health centers by 392 TB suspicious cases for diagnosis were shared. Thus, 274 sputum samples were collected from Adama Town while 118 sputum samples were collected from Bishoftu Town. The sputa samples were transported in in cold chain to the TB laboratory of the Aklilu Lemma Institute of Pathobiology of the Addis Ababa University for culturing.

Post-mortem examination and tissue sampling
Body condition scoring was made using the method developed earlier based on the different prominent anatomical structures such as the visibility of the ribs, the gluteal muscles, tuber coxae, the head of the tail, transverse processes and others [13][14] Postmortem examination was performed following the protocol set previously by another researcher [15]. Each of the seven lobes of the lungs were thoroughly inspected and palpated for any suspicious gross TB lesions. Similarly, mandibular, retropharyngeal, cranial and caudal mediastinal, left and right bronchial, hepatic, and mesenteric lymph nodes were sliced into 2mm size sections and then inspected for the presence of visible lesions according to the protocol described earlier [16][17]. The tissues showing macroscopic lesions compatible with bTB were collected into universal bottle in 0.9% saline solution and then transported to the laboratory in cold chain. A total of 80 suspicious lesions were collected from the total 1896 cattle investigated, and processed for culturing as described below.
Culturing of sputum and tissues samples Regular visits were made to the hospital and health centers in the study area for collection of sputum samples that were submitted to the routine diagnosis of TB. In total, 392 sputum samples were collected and transported to the TB laboratory in cold chain for culturing. The samples were stored at -20 o C freezer until being thawed and processed for culturing. Culturing of sputum samples was done on Lowenstein-Jensen (LJ) media following the procedure used earlier [6]. Similarly, suspicious tissue samples were processed for culturing on LJ media following the procedure used by other researchers [1]. The colonies were harvested and re-suspended in sterile distilled water and heat-killed at 80 o C for 50 minutes so that mycobacterial DNA is released and used for subsequent spoligotyping.

Spoligotyping of mycobacterial isolates
Spoligotyping was performed on 107 M. tuberculosis complex isolates at the Aklilu Lemma Institute of Pathobiology, Addis Ababa University, following the standard operating procedure that was used by Berg et al. [18] and primarily developed by Kamerbeek et al. [19]. The DNA released by heat-killing of the colonies was used as a template to amplify the direct repeat (DR) region of M. tuberculosis complex by polymerase chain reaction (PCR) using oligonucleotide primers derived from the DR sequence, RDa (5'GGTTTTGGGTTTGAACGAC3') and RDb (5'CCGAGAGGGGACG GAAAC3') primers [19]. The total volume of the reaction the PCR reaction mixture was 25 μl and constituted of 12. 10 minutes in 2 x SSPE-0.5% sodium dodecyl sulfate at 42 o C and rinsed with 2 x SSPE for 5 minutes at room temperature. Hybridizing DNA was detected by the enhanced chemiluminescence method (Amersham, Biosciences, Amersham, UK) and by exposure to X-ray lm (Hyper lm ECL, Amersham). A mixture of 10 ml of ECL reagent 1 and 10 ml of ECL reagent 2 was prepared, and then added onto the membrane, and the membrane was rinsed in the solution for 5min at room temperature. Then, the membrane was attached onto a lm in the dark room and placed in the cassette and incubated for 15 minutes a room temperature. Thereafter the lm was removed and placed in a developer solution for 2 minutes. The lm was removed from the developer and rinsed with tap water for 15 seconds and then placed in a xer solution for 1 minute. Thereafter, the lm was dried and used for interpretation of the result. The presence of the spacer was identi ed as a black square while absence of the spacer was identi ed as a white square on the lm. Thereafter, the black squares were converted to 1 while the white squares were converted to 0 and then transferred to the spoligotype international types-VNTR international types (SITVIT) database for the identi cation of the spoligotype international types (SIT) and the lineages of the isolates.

Data analysis
Proportion was used to show the number of cattle with TB lesions. Tissue lesion and sputum culture positivity were presented using proportions. Comparisons of proportions was made by using chi-square (c 2 ) test. Statistical signi cance was considered at P<0.05. In molecular aspect of the study, the identi cation of the SIT numbers and lineages of isolates was done using SITVIT1Database and Run TB-Lineage. The results of spoligotyping were converted into octal and binary formats, and then entered into query box so that the names of the strains are retrieved from the database if the spoligotype pattern of the strain in question ts the pattern that has already been registered in the SPolDB4 database and at http://www.pasteur-guadeloupe.fr:8081/SITVITDemo/ (SITVIT1Database) [20]. If the pattern of the strain in question has not been registered in the database prior to this study, the strain was considered as an orphan. The lineages were also generated by entering binary and octal formats into the query box of SITVIT1Database and Run TB-Lineage. Declarations and nancial reports on annual basis. In addition, the nal scienti c and nancial reports were submitted to the AAU.

Competing Interests
The authors declare that there are no competing interests Availability of data and materials All data generated and /or analyzed during the current study are not publicly available but they are kept at the Data Management Unit of the Aklilu Lemma Institute of Pathobiology, Addis Ababa University. They can be available by request through the corresponding author.
Ethical approval and consent to participate The study was approved by the Institutional Review Board (IRB) of the Oromia Health Bureau (Reference No. IRB/BEFO/HBTFH/1-8/3647). Verbal consent was included in the proposal and was approved by the IRB. The IRB approved verbal consent since the project was PhD student's research project and did not involve international collaboration and material transfer. Thus, informed consent was obtained verbally from the patients for using sputum samples for this research. Furthermore, permission was obtained from the owners of animals for post mortem examination and sampling