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American Journal of Microbiological Research. 2021, 9(1), 1-8
DOI: 10.12691/ajmr-9-1-1
Open AccessReview Article

Bovine Tuberculosis: A Review of Molecular Diagnostic Methods and Impact on Public Health

Mahendra Pal1, Gemechu Berhanu2, Diba Feyisa3, Bizunesh Mideksa4 and Venkataramana Kandi5,

1Narayan Consultancy on Veterinary Public Health and Microbiology, Anand, Gujarat, India

2College of Agriculture and Veterinary Medicine, Dambi Dollo University, Ethiopia

3Hababo Guduru District Livestock and Fisheries Resource Development Office, Oromia, Ethiopia

4Department of Veterinary Laboratory Technology, Ambo University, Ethiopia

5Department of Microbiology, Prathima Institute of Medical Sciences, Karimnagar, Telangana, India

Pub. Date: January 03, 2021

Cite this paper:
Mahendra Pal, Gemechu Berhanu, Diba Feyisa, Bizunesh Mideksa and Venkataramana Kandi. Bovine Tuberculosis: A Review of Molecular Diagnostic Methods and Impact on Public Health. American Journal of Microbiological Research. 2021; 9(1):1-8. doi: 10.12691/ajmr-9-1-1


Bovine tuberculosis (BTB) is a zoonotic infectious disease of cattle, other domesticated animals, and certain wildlife populations. It has been widely distributed throughout the world, and it has been a cause for great economic loss in animal production. In developed countries, the eradication programs have reduced or eliminated TB in cattle. Many factors contribute to the persistence of the disease, such as the limitations of diagnostic tests, larger herd sizes, increase in animal movements and trade, and limited options for control, such as restrictions on whole herd depopulation. The available advanced TB diagnostic techniques can detect and differentiate the causative mycobacterial species, and help an early confirmation of the diagnosis, which is useful to design appropriate control measures in the national BTB control programs. Molecular diagnostics, such as polymerase chain reaction, spoligotyping, restriction fragment length polymorphism (RFLP), variable number tandem repeats typing (VNTR), and polymorphism GC-rich repeat sequence (PGRS) are the techniques used for concurrent detection and typing of Mycobacterium species at the strain level. The molecular epidemiology is also being used to identify the source of contamination, to determine the risk factors of BTB transmissions, to investigate the drug resistance pattern, and to track the geographic distribution and spread of clones of Mycobacteria species. The molecular diagnosis is the tool to check whether active transmission or reactivation of BTB, hence, it is better to adopt these methods for the epidemiological survey of BTB. Since BTB is a major public health problem, at least a single reference laboratory should be available for molecular based diagnosis as part of its control.

bovine tuberculosis molecular diagnostic techniques molecular epidemiology polymorphism GC-rich repeat sequence (PGRS) public health restriction fragment length polymorphism (RFLP) variable number tandem repeats typing (VNTR) zoonosis

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[1]  Hlokwe, T.M., Van Helden, P. and Michel, A. (2013). Evaluation of the discriminatory power of variable number of tandem repeat typing of Mycobacterium bovis isolates from Southern Africa. Tran Em Dis., 60: 111-120.
[2]  Pal, M. and Boru, B.G. (2012). Zoonotic significance of Mycobacterium bovis infection. Journal of Natural History 8: 86-89.
[3]  Pal, M., Zenebe, N. and Rahman, M.T. (2014). Growing significance of Mycobacterium bovis in human health. Microb H., 3: 29-34.
[4]  Terefe, D. (2014). Gross pathological lesions of BTB and efficiency of meat inspection procedure to detect infected cattle in Adama municipal abattoir. JVMAH. 2: 48-53.
[5]  Cosivi, O., Grange, J.M., Dabron, C.J., Raviglione, M.C., Fujikura, T., Cousins, D., Robinson, R. A., Huchzermeyer, H.F., de Kantor I. and Meslin F.X. (1998). Zoonotic TB due to Mycobacterium bovis in developing countries. Emerging Infectious Diseases 4: 1-59-70.
[6]  Pal, M. (2007). Zoonoses. Second Edition. Satyam Publishers, Jaipur, India.
[7]  Le Roex, N., Van Helden, P.D., Koets, A.P. and Hoal, E.G. (2013). Bovine TB in livestock and wildlife: Physiol Genom., 45: 631-637.
[8]  Rodriguez-Campos, S., Smith, N.H., Boniotti, M.B. and Aranaz, A. (2014). Overview and phylogeny of Mycobacterium tuberculosis complex organisms: Implications for diagnostics and legislation of bovine tuberculosis. Res Vet Sci. 97: 5-19.
[9]  Awah-Ndukum, J., Kudi, A.C., Bradley, G., Titanji, V.K. and Fon-Tebug, S. (2013). Prevalence of bovine tuberculosis in cattle in the highlands of Cameroon based on the detection of lesions in slaughtered cattle and tuberculin skin tests of live cattle. Vet Med., 57: 59-76.
[10]  Radostits, O.M., Gay, C.C., Hinchelift, K.W. and Constabel, P.D. (2007). Veterinary Medicine. A text book of the disease of cattle, sheep, pig, goat and horses.10thedn. Elsevier, London. Pp: 1007-1040.
[11]  Ereqat, S., Nasereddin, A.H., Levine, K. and Al-Jawabreh, A. (2013). First-time detection of Mycobacterium bovisin livestock tissues and milk in the West Bank, Palestinian territories. PLoS Neglected Trop Dis., 7: 1371.
[12]  Skuce, R.A., Allen, A.R. and McDowell, S.W.J. (2011). Bovine tuberculosis (TB): A review of cattle-to-cattle transmission, risk factors and susceptibility. Bacteriology Branch Veterinary Sciences Division Agrifood and Biosciences Institute, 1: 115-167.
[13]  Ewnetu, L., Melaku, A. and Birhanu, A. (2012). BTB Prevalence in Slaughtered Cattle at Akaki Municipal Abattoir Based on Meat Inspection Methods. Glob Vet.,5: 541-545.
[14]  Mamo, G., Abebe, F., Worku, Y., Hussein, N., Legesse, M. and Ameni, G. (2013). Bovine tuberculosis and its associated risk factors in pastoral and agro-pastoral cattle herds of Afar Region, Northeast Ethiopia. Journal of Veterinary Medicine and Animal Health, 6: 171-179.
[15]  Duarte, E.L., Domingos, M., Amado, A. and Botelho, A. (2009). Spoligotype diversity of M. bovisa nd Mycobacterium capraeanimal isolates. Vet Microbiol.,130: 415-421.
[16]  Malama, S., Muma, J.B. and Mbulo, G. (2013). Isolation of M.ycobacterium bovis from human sputum in Zambia: Public health and diagnostic significance. J Infect Dis Ther. 1:3.
[17]  Batool, B.T., Tareen, A., Ahmed, S.S., Ejaz, H., Kakar, M.A., Awan, M.A. and Shahwani, M.N. (2017). Prevalence of zoonotic tuberculosis and brucellosis in animals of Quetta and Pish in Districts, Balochistan. Pakistan J. Zool., 49: 387-389.
[18]  Palomino, J.C. and Leao, S.C. (2007). Tuberculosis: From Basic Science to Patient Care. Amedeo Challenge, Belgium. Pp: 687.
[19]  Lambert, P. and Sandoe, J. (2011). Medical Microbiology and Infection, 5th edn. Edited by Elliott, T. and Casey, A. Blackwell Publishing Ltd. Aston University, Birmingham, UK. Pp: 1-11.
[20]  Bilal, S., Iqbal, M., Murphy, P. and Power, J. (2010). Human bovine tuberculosis – remains in the differential. J Med Mic.,59: 1379-1382.
[21]  Higgins, J., Camp, P., Farrell, D. Bravo, D., Pate, M. and Robbe-Austerman, S. (2011). Identification of Mycobacterium species gene sequencing Vet Res.,7: 77.
[22]  Marie-France, H., Maria Laura, B. and Claude, S. (2009). Classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach. Vet Res., 40: 50.
[23]  Simons, S., Van Ingen, J., Hsueh, P. and Van Soolingen, D. (2011): Non tuberculous Mycobacteria in Respiratory Tract Infections, Eastern Asia. Emer Infec Dis. Pp: 343-349.
[24]  Liebana, E., Johnson, L., Gough, J., Durr, P., Jahans, K. and Downs, S.H. (2008). Pathology of naturally occurring bovine tuberculosis in England and Wales. Vet J. 176 (3): 354.
[25]  Cassidy, J.P. (2006). The pathogenesis and pathology of BTB with insights from studies of tuberculosis in humans and laboratory animal models. Vet Microbiol.,112: 151-161.
[26]  Gannon, B.W., Hayes, C.M. and Roe, J.M. (2007). Survival rate of airborne Mycobacterium bovis. Res in Vet Sci. 82: 169-172.
[27]  Olsen, I., Barletta, R.G., Thoen, C.O. (2010). Pathogenesis of Bacterial Infections in Animals. 4thedn. Ames, IA: Wiley-Blackwell Publishing. Pp: 113-32.
[28]  Forrelad, M.A., Klepp, L.I., Gioffre, A., Sabio-Garcia, J., Morbidini, H.R. and Bigi, F. (2013). Virulence factor of MTBC.Virul. 4(1): 3-66.
[29]  Thoen, C.O. and Barletta, R.G. (2014). Pathogenesis of tuberculosis caused by Mycobacterium bovis. In: Thoen, C.O., Steele, J.H., Kaneene, J.B. (eds). Zoonotic Tuberculosis: M. bovisand Other Pathogenic Mycobacteria. Ames, IA: Wiley-Blackwell. Pp: 51-62.
[30]  OIE. (2009). Bovine Tuberculosis. Office of International des Epizootics, World organization for animal health manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees) 6thedn. Paris. Pp: 1-16.
[31]  Tschopp, R., Hattendorf. J., Roth, F. and Shaw A. (2012). Cost estimate of bovine tuberculosis to Ethiopia. Curr Top Microbiol Immunol. 3: 245-261.
[32]  Constable, P.D., Hichcliff, K.W., Done, S.H. and Grunberg, W. (2017). Systemic and multiorgan disease in veterinary medicine 11thedn.Elseveir Health Science W.B. Saunders. Pp: 2002-2214.
[33]  Domingo, M., Vidal, E. and Marca, A. (2014). Pathology of BTB.Res Vet Sci.,97: 20-29.
[34]  Water, W., Maggioli, M.F. and Palmer.M.V. (2014). Relevance of bovine tuberculosis research to the understanding of human disease: historical perspective, approaches and immunologic mechanisms. Vet Immunol and Immunopathol. 159:113-132.
[35]  Perez-Lago, L., Navarro, Y. and Garcia-de-Viedma, D. (2013) Current knowledge and pending challenges in zoonosis caused by Mycobacterium bovis: A review. Research in Veterinary Science, 97: S94-S100
[36]  Palmer, M.V., Thacker, T.C., Gort, C.A. and Corner, L.A. (2012). Mycobacterium bovis: A Model pathogen at the interface of livestock, wildlife, and humans. Vet Med Internat.1:17.
[37]  Thoen C, Lobue P, and de Kantor I. (2006). The importance of Mycobacterium bovis as a zoonosis. Vet Microbiol. 112(2-4): 339-45.
[38]  Ashford DA, Whitney E, Raghunathan P and Cosivi O. (2001). Epidemiology of selected mycobacteria that infect humans and other animals. Rev Sci Technol, Office Int des Epizoot., 20: 105-112.
[39]  Abubakar, U.B., Ameh, J.I., Abdulkadir, I.A., Silasu, I., Okaiyeto, S.O. and Kudi, A.C. (2011). Bovine tuberculosis in Nigeria: Vet. Res.,4: 24-27.
[40]  Kemal J, Sibhat B, Abraham A, et al. (2019). Bovine tuberculosis in eastern Ethiopia: prevalence, risk factors and its public health importance. BMC Infect Dis.19 (1):39.
[41]  Awada, L., Tizzani, P., Erlacher-Vindel, E., Forcella, S. and Caseres, P. (2018). Bovine Tuberculosis worldwide picture. In: Bovine Tuberculosis. Chambers, M., Gordon, S., Olea-Popelka, F. and Barrow, P. (eds). CBA International: 461-591.
[42]  Carslake, D., Grant, L.E. and Green, J. (2011). Endemic cattle diseases: Comparative epidemiology and governance. Philos Trans Royal Soc Biol Sci., 366: 1975-1986.
[43]  Alemu, J., Mamo, G., Ameni, G. and Pal, M. (2016). Molecular epidemiology of bovine tuberculosis in cattle and its public health implications in Gambella region, Ethiopia. Molecular Microbiology Res 6: 1-16.
[44]  Pal, M., Zenebe, N., Amare, T. and Woldemarium, T. (2017). An abattoir-based study on bovine tuberculosis in Debre Zeit, Ethiopia. World’s Veterinary Journal 7: 101-107.
[45]  Bailey, S.S., Crawshaw, T.R., Smith, N.H. and Palgrave, M.V. (2013). M.ycobacterium bovis infection in domestic pigs in Great Britain. J Vet., 198(2): 391-397.
[46]  Girmay, G., Pal, M., Deneke, D., Weldesilasse, G. and Eqar, Y. (2012). Prevalence and public health importance of bovine tuberculosis in and around Mekelle town, Ethiopia. International J Livestock Res. 2 (2): 180-188.
[47]  Amit, K.V., Ruchi, T., Sandip, C., Neha, M., Kuldeep, D. and Shoorgh, B. (2014). Insight into bovine tuberculosis (BTB), various approaches for its diagnosis, control and its public health concerns. Asian J Anim and Vet Advan., 9 (6): 324-344.
[48]  Nwanta, J.A., Joseph, I. and Ezema, W. (2010). Zoonotic tuberculosis: A review of epidemiology, clinical presentation. J Publ Health and Epidemiol 2 (6):118-124.
[49]  Medeiros dos Santos, L., Marrasi, C.D. and Lilenbuam, W. (2010). Potential application of new diagnostic methods for controlling BTB in Brazil. Braz J Microbiol. 41: 531-541.
[50]  Rowe MT and Donaghy J.(2008) Mycobacterium bovis: the importance of milk and dairy products as a cause of human tuberculosis in the UK. A review of taxonomy and culture methods, with particular reference to artisanal cheeses International Journal of Dairy Technology. 61 (4):317-326.
[51]  Varello, K., Pezzolato, M., Mascarino, D., Ingravalle, F. and Caranelli, M. (2008). Comparison of histological techniques for the diagnosis of bovine tuberculosis in the framework of eradication programs. J Vet Diagn Invest. 20: 164-169.
[52]  Bezos, J., Casal, C., Romero, B., Schroeder, B. and Raeber, A. (2014). Current ante-mortem techniques for the diagnosis of bovine tuberculosis. Res in Vet sci. 97: 44-52.
[53]  Good, M., Bakker, D., Duignan, A., and Collins D.M. (2018). The history of in vivo Tuberculin Testing in bovines: Tuberculosis, a “One Health” Issue. Frontiers in Vet Sci., 5(10): 3389.
[54]  OIE. (2006). Manual of diagnostic tests and vaccines for terrestrial animals (online). Paris: Bovine tuberculosis. Available at: Accessed 7 Oct 2007.
[55]  Whelan AO, Clifford D, Upadhyay B, et al.(2010). Development of a skin test for bovine tuberculosis for differentiating infected from vaccinated animals. J Clin Microbiol. 48(9):3176-3181.
[56]  Asiimwe, J. (2008). Molecular characterization of Mycobacterium bovis isolates from selected slaughters houses in Kampala. A thesis for Bachelor of Science degree, Uganda.
[57]  Gabriela, E.V., Susana, F.V. and Clara, I.E. (2017). Virulence Factors and Pathogenicity of Mycobacterium Research and Development, Wellman Ribon, Intech Open, Available from: development/virulence-factors-and-pathogenicity-of- mycobacterium.
[58]  Minion, J., Leung, E., Talbot, E., Dheda, K. and Pai, M. (2011). Diagnosing tuberculosis with urine lipoarabinomannan: systematic review and meta-analysis. Eur Respir J 38: 1398-1405.
[59]  Whipple, D.L., Palmer, M.V., Slaughter, R.E. and Jones, S.L. (2001). Comparison of purified protein derivatives and effect of skin testing on results of a commercial gamma interferon assay for diagnosis of tuberculosis in cattle. J Vet Diagn Invest. 13: 117-122.
[60]  McLernon, J., Costello, E., Flynn, O., Madigan, G. and Ryan, F. (2010). “Evaluation of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and spoligotyping for genotyping of Mycobacterium bovis isolates and a comparison with restriction fragment length polymorphism typing,” J Clin Microbiol. 48(12): 4541-4545.
[61]  Guta S, Casal J, Napp S, et al.(2014) Epidemiological investigation of bovine tuberculosis herd breakdowns in Spain 2009/2011. PLoS One. 9 (8): e104383. Published 2014 Aug 15.
[62]  Brosch, R., Gordon, S.V., Marmiesse, M., Brodin, P., Buchrieser, C., and Cole, S.T. (2012). A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proceeding of the National Academy of Science, USA.99: Pp: 3684-3689.
[63]  Bifani, P., Kurepina, N. and Mathema, B. (2009). Genotyping of Mycobacterium tuberculosis clinical isolates using IS6110-based restriction fragment length polymorphism analysis. Methods Mol Biol. 551: 173-188.
[64]  Van, S.D. (2008). Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements. J Intern Med. 249:1-26.
[65]  Ogbaini-Emovon, E. (2009). Current trends in the laboratory diagnosis of tuberculosis. Benin J Postgrad Med. 11:79-90.
[66]  Sharma, R. and Gupta, V. (2010). Spoligotyping for the Detection of Mycobacterium tuberculosis complex bacteria. Asian J Biochem. 6: 29-37.
[67]  Rozo, A.J. and Ribon, W. (2010). Molecular tools for Mycobacterium tuberculosis genotyping. Rev Salud Publica. 12: 510-521.
[68]  O’Brien, R., Flynn, O., Costello, E., and Rogers, M. (2008). Identification of a novel DNA probe for strain typing Mycobacterium bovis by restriction fragment length polymorphism analysis. J Clin Microbiol.38:1723-1730.
[69]  Araujo, C.P., Osorio, A.A., Jorge, K. and Ramos, C.A. (2014). Direct detection of MTBC in bovine and bubaline tissues through nested-PCR. Brazilian J Micro.2: 633-640.
[70]  Ramos, D.F., Tavares, L., Da Silva, P.E. and Dellagostin, O.A. (2014). Molecular typing of Mycobacterium bovis isolates: A review. Brazilian J Micr. 2:365-372.
[71]  Yang ZH, Ijaz K, Bates JH, Eisenach KD, Cave MD.(2000). Spoligotyping and polymorphic GC-rich repetitive sequence fingerprinting of mycobacterium tuberculosis strains having few copies of IS6110. J Clin Microbiol. 38(10): 3572-6.
[72]  Rekha, V.B., Gunaseelan, L., Pawar, G., Nassiri, R. and Bharathy, S. (2015). Molecular detection of M. tuberculosis from bovine milk samples. J Adv Vet. Anim Res .2: 80-83.
[73]  Figueiredo, E.E., Júnior, C.A., Furlanetto, L.V., Silva, F.G., Duarte, R.S., Silva, J.T., Lilenbaum, W. and Paschoalin, V.M. (2012). Molecular techniques for identification of species of the Mycobacterium tuberculosis complex: The use of multiplex PCR and an adapted HPLC method for identification of Mycobacterium bovis and diagnosis of bovine tuberculosis. Understanding tuberculosis: Global Experiences and Innovative Approaches to the Diagnosis, Cuiba, Brasil, Pp. 411-432.
[74]  Muller, B., Durr, S., Alonso, S., Hattendorf, J., Laisse, C. and Parsons, S. (2013). Zoonotic Mycobacterium bovis-induced tuberculosis in humans. Emerg Infect Dis., 19(6): 899-908.
[75]  Smith, N.H., Gordon, S.V., de la Rua-Domenech, R., Clifton-Hadley, R.S. and Hewinson, R.G. (2006). Bottle necks and broom sticks: the molecular evolution of Mycobacterium bovis. Nat Rev Microbiol. 4(9):670-681.
[76]  Huang, Z.Y., de Boer, W.F. and Prins, H. (2013). Dilution effect in bovine tuberculosis: risk factors for regional disease occurrence in Africa. Proc R Soc B.,280: 1-7.
[77]  Van Ingen, J., de Zwaan, R., Dekhuijzen, R., Boeree, M. and Van Soolingen, D. (2009). Region of difference 1 in nontuberculous mycobacterium species adds a Phylogenetic and taxonomical character. J Bacteriol. 191(18):5865-5867.
[78]  Zeweld, S.W. (2014). Cultural and molecular detection of zoonotic tuberculosis and its public health impacts in selected districts of Tigray region, Ethiopia. Sokoto. J Vet Sci. 12:1-12.
[79]  Iwnetu, R., Woldeamanuel, Y., Asfaw, M. and Gebrekirstos, C. (2009). Comparative performance of diagnostic tests for mycobacterial lymphadenitis in a high-burden country.J Infect Dis. 41: 462-468.
[80]  Duarte, E.L., Domingos, M., Amado, A., Cunha, M.V. and Botelho, A. (2010). MIRUVNTR adds discriminatory value to groups of Mycobacterium bovis and Mycobacterium caprae strains defined by Spoligotyping. Vet Microbiol.,143: 299-306.
[81]  Parriers, P.M., Andrade, G.I., Nascimento, T. and Gomes H.M. (2012). Spoligotyping and VNTR analysis of M.ycobacterium bovis isolates from cattle in Brazil. Mem Inst Oswaldocruz. 107:64-73.
[82]  Supply, P., Alexi, C., Lesjean, S. and Rusch-Gerdes, S. (2006). Proposal for standardization of optimized Mycobacterial interspersed repetitive unit-variable number tandem repeat typing of Mycobacterium tuberculosis. J ClinMicrobiol.12:498-510.
[83]  Skuce, R.A. Bryne, A.W., Lahuerta-Marin, A. and Allen, A. (2018). M. bovis typing and surveillance in BTB.CBA International. Pp: 58-79.
[84]  Gormley, E. and Corner, L.A. (2013). Control strategies for wildlife tuberculosis in Ireland. TransboundEmerg Dis., 60(1): 128-135.
[85]  Good, M. and Duignan, A. (2011). Perspective on the history of bovine tuberculosis and the role of tuberculin in bovine tuberculosis eradication. Vet Med Internat., 1-11.
[86]  Palmer MV, and Waters WR. (2011). Bovine tuberculosis and the establishment of an eradication program in the United States: role of veterinarians. Vet Med Int. 81: 6345.
[87]  Arnot LF, and Michel A. (2020). Challenges for controlling bovine tuberculosis in South Africa. Onderstepoort J Vet Res. 87(1): e1-e8. Published 2020 Feb 27.