Epidemiology and Molecular Characterization of Mycobacterium Bovis in Humans and Cattle and Assessment of its Zoonotic Importance in Central Ethiopia
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Date
2021-11
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Addis Ababa University
Abstract
Mycobacterium bovis (M. bovis) is a member of the Mycobacterium tuberculosis complex (MTBC) and causes tuberculosis in humans (zoonotic tuberculosis-zTB) and animals, mainly in cattle (bovine tuberculosis-bTB). There were limited studies on zTB in Ethiopia but also a reliable estimate of bTB prevalence in cattle in central Ethiopia is missing. In addition no whole
genome sequencing (WGS) based M .bovis studies had been performed in Ethiopia before this study. Also, there has been a limited effort in search for an alternative diagnostic method to culture to detect M. bovis in clinical specimens. Therefore, due to these research gaps, this study, which combined bTB and zTB, was conducted from 2018 to 2021 in central Ethiopia (Addis Ababa, Sebeta, Holeta, Sululta, Sendafa and Bishoftu) with the objective of generating epidemiological and molecular data to update our understanding on bTB/zTB. The bTB study in cattle involved a cross sectional one-stage cluster sampling survey of dairy farms in central Ethiopia using tuberculin skin testing and the collection of additional data by questionnaire to estimate the prevalence of bTB and identify potential risk factors contributing to bTB
transmission. For the zTB part, surveillance of TB in humans was carried out among individuals working in bTB infected dairy farms, patients presented at selected health centers, and exposure to risk factors was assessed using questionnaire. From consenting TB suspected individuals, demographic and clinical information was collected by questionnaire. Sputum and Fine Needle
Aspirates (FNA) samples were collected from suspected cases. In addition, isolation of M. bovis was done from raw milk collected from tuberculin skin test positive cows and from cattle tissue lesions. The genetic diversity of M. bovis isolates was examined using spoligotyping and whole genome sequencing (WGS) analysis. Furthermore, in this study the performance of a TaqMan real time PCR (RT-PCR) assay as an alternative diagnostic method to culture was evaluated. Two hundred ninety-nine (n=299) dairy herds in the six study areas were randomly selected, from which 5,675 cattle were tested. The overall prevalence of bTB after standardisation for herd-size in the population was 54.4% (95% CI 48.7-60%) at the herd level, and it was 24.5% (95% CI 23.3-25.8) at the individual animal level. A Generalized Linear Mixed Model (GLMM) was used to explore risk factors association with bTB status. We found that herd size, animal age, bTB history at farm, and breed were significant risk factors. With regard to zTB, among 110 DFWs in 73 bTB infected dairy farms, 41 had at least one of the symptoms that are typical for
TB. Three DFWs had swollen nodes at their neck, a symptom typical for TB lymphadenitis. In assessment of risk factors: raw milk consumption was practiced by more than two thirds of theDFWs with symptoms of TB (68.2%) and over half of DFWs with symptoms did not think TBcould be transmitted via raw milk consumption. Overall in the surveillance of zTB (active and passive), a total of 167 specimens (sputum=131; FNA=36) were collected from 161 TB suspected individuals for the isolation of M. bovis. Of these processed specimens, three samples with M. bovis were detected in total (1.8%, n=167). And of these three, one M. bovis isolate was sequenced and the genotype was spoligotype SB1476 which was previously reported from cattlein Ethiopia suggesting possible zoonotic transmission. With regard to isolation and characterization of M. bovis from cattle, out of 827 cattle (abattoirs and dairy farms), 76 of them(9.2%) had tuberculous lesion. From these tuberculous lesions, 62 isolates (n=137 samples) from42 animals were confirmed to be M. bovis. Similarly out of 975 milking cows which were tuberculin skin test positive (37.8%, n=2582), 490 composite raw milk samples were collectedand of these 11 (2.2%) yield M. bovis isolates showing evidence that raw milk is not safe and can be a source of infection for human TB due to M. bovis. The genetic diversity of 74 M. bovisisolates (one being a human isolate) was assessed and ten different spoligotypes were recorded. In cattle spoligotype SB1176 was the most prevalent type (n=31, 44.3%) followed by SB0133(n=11, 15.7%). Our WGS analysis with a total of 55 M. bovis isolates sequenced (one being ahuman isolate) showed three clonal complexes clearly segregating in the phylogeny: African 2(Af2; n=47), European 3 (Eu3; n=7) and Unknown8 (n=1). In addition, the present study reported for the first time clonal complex European 3 (Eu3) from Ethiopia. With regard toTaqMan assay performance evaluation - the assay performance on 440 clinical samples was variable for different specimens and overall performed well for sputum samples for all targets.
In conclusion, this study recorded high prevalence of bTB in dairy cattle in central Ethiopia. M.bovis prevalence in humans in central Ethiopia was low; however, further investigation is neededin all regions at national level given bTB is endemic in cattle in Ethiopia. Knowledge gap onbTB and M. bovis isolation from milk, showed that there is a clear potential for zoonotic transmission and needs further investigation. The TaqMan RT-PCR assay is a promising methodology for the diagnosis of zTB and bTB, but with further validation works needed usingdifferent targets and specimens.
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Keywords
Mycobacterium bovis,humans ,cattle,Zoonotic tuberculosis-zTB