Comparison of multiplex and ordinary PCR for diagnosis of paratuberculosis and tuberculosis in blood samples (buffy coat) of cattle and buffaloes

Document Type : Short paper


1 MVSc Student in Veterinary Microbiology, Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India

2 Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India

3 Directorate of Livestock Farm, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India


Background: Paratuberculosis and tuberculosis (TB) caused by Mycobacterium avium paratuberculosis (MAP) and Mycobacterium tuberculosis complex (MTC), respectively are economically important, chronic debilitating diseases affecting the dairy herds and are also potential zoonotic threats. Aims: Differential diagnosis of paratuberculosis and TB in blood samples of cattle and buffaloes. Methods: In this study, an in-house developed multiplex polymerase chain reaction (PCR) targeting MAP, Mycobacterium bovis and Mycobacterium smegmatis was used in blood samples (buffy coat) parallel with IS900 PCR and esxB PCR for diagnosis of paratuberculosis and TB, respectively; in a total of 202 cattle and buffaloes. Results: Out of 202 animals, 12 (5.9%) and 17 (8.4%) animals were positive for MAP by multiplex PCR and IS900 PCR, respectively; from which only 8 (4%) animals were positive by both tests; whereas 4 and 9 animals were exclusively positive by multiplex PCR and IS900 PCR, respectively. None of the animals were found to be positive for M. bovis and M. smegmatis by the multiplex PCR. However, the esxB PCR detected 13 (6.4%) animals positive for TB. In fact, 3 (1.5%) animals were found to be co-infected by both paratuberculosis and TB. Conclusion: The in-house multiplex PCR detected MAP in buffy coat and there was a fair degree of agreement between the multiplex PCR and IS900 PCR in detection of MAP DNA though the latter detected more number of animals to be positive for MAP. Besides, esxB PCR showed a high diagnostic potential and can be used for diagnosis of TB from blood.


Main Subjects

Bakshi, CS; Shah, DH; Verma, R; Singh, RK and Malik, M (2005). Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR. Vet. Microbiol., 109: 211-216.
Barrington, GM; Gay, JM; Eriks, IS; Davis, WC; Evermann, JF; Emerson, C; O’Rourke, JL; Hamilton, MJ and Bradway, DS (2003). Temporal patterns of diagnostic results in serial samples from cattle with advanced paratuberculosis infections. J. Vet. Diagn. Invest., 15: 195-200.
Bhide, M; Chakurkar, E; Tkacikova, L; Barbudhe, S; Novak, M and Mikula, I (2006). IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep. Vet. Microbiol., 112: 33-41.
Bolaños, CAD; de Paula, CL; Guerra, ST; Franco, MMJ and Ribeiro, MG (2017). Diagnosis of mycobacteria in bovine milk: an overview. Rev. Inst. Med. Trop. São Paulo. 59: e40.
Brahma, D; Narang, D; Chandra, M; Gupta, K; Singh, A and Kaur, G (2017). Diagnosis of Mycobacterial infections (Tuberculosis and Paratuberculosis) in tissue samples using molecular (inhouse multiplex PCR, PCR and TaqMan real-time PCR), histopathology and immuno-histochemical techniques. Trop. Biomed., 34: 1-17.
Carvalho, IA; Vinicius, CEB; Souza, IM; Zanardo, LG; Filho, JDR; Gomes, MJP and Moreira, MAS (2012). Diagnosis of Paratuberculosis in cattle: microbiological culture, serology and PCR. Braz. J. Microbiol., 43: 581-585.
Dikshit, M; Sharma, RJ; Adsool, AD and Chaphalkar, SR (2012). ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis in making diagnostic tool for TB. J. Biotechnol. Lett., 3: 28-30.
Franco, MM; Paes, AC; Ribeiro, MG; Pantoja, JC; Santos, AC; Miyata, M; Leite, CQ; Motta, RG and Listoni, FJ (2013). Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil. BMC Vet. Res., 9: 85.
Gao, A; Odumeru, J; Raymond, M; Hendrick, S; Duffield, T and Mutharia, L (2009). Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis. Can. J. Vet. Res., 73: 58-64.
Gümüşsoy, KS; İça, T; Abay, S; Aydin, F and Hizlisoy, H (2015). Serological and molecular diagnosis of paratuberculosis in dairy cattle. Turk. J. Vet. Anim. Sci., 39: 147-153.
Gwozdz, JM; Thompson, KG; Manktelow, BW; Murray, A and West, DM (2000). Vaccination against para-tuberculosis of lambs already infected experimentally with Mycobacterium avium subspecies paratuberculosis. Aust. Vet. J., 78: 560-566.
Hermon-Taylor, J (2009). Mycobacterium avium subspecies paratuberculosis, Crohn’s disease and the Doomsday scenario. Gut Pathog., 1: 15. doi: 10.1186/1757-4749-1-15.
Juste, RA; Garrido, JM; Geijo, MV; Elguezabal, N; Aduriz, G; Atxaerandio, R and Sevilla, I (2005). Comparison of blood PCR and ELISA for detection of Mycobacterium avium subsp. paratuberculosis infection in sheep and cattle. J. Vet. Diagn. Invest., 17: 354-359.
Khol, JL; Wassertheurer, M; Sodoma, E; Revilla-Fernández, S; Damoser, J; Österreicher, E; Dünser, M; Kleb, U and Baumgartner, W (2013). Long-term detection of Mycobacterium avium subspecies para-tuberculosis in individual and bulk tank milk from a dairy herd with a low prevalence of Johne’s disease. J. Dairy Sci., 96: 3517-3524.
Naser, SA; Ghobrial, G; Romero, C and Valentine, JF (2004). Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet. 364: 1039-1044.
OIE (2009). Chap. 2.4.6: Bovine tuberculosis. Terrestrial Manual. World Organization for Animal Health, Paris, France.
OIE (2019b). Accessed on 04.02.2019.

Olsen, I; Barletta, RG and Thoen, CO (2010). Mycobacterium. In: Gyles, CL; Prescott, JF; Songer, JG and Thoen, CO (Eds.), Pathogenesis of bacterial infections in animals. (4th Edn.), Ames, Iowa, Wiley-Blackwell. PP: 113-132.

Pinxteren, LAHV; Ravn, P; Agger, EM; Pollock, J and Andersen, P (2000). Diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP-10. Clin. Diagn. Lab. Immunol., 7: 155-160.
Quinn, PJ; Markey, BK; Leonard, FC; Fitzpatrick, ES; Fanning, S and Hartigan, PJ (2011). Mycobacterium species. In: Quinn, PJ; Markey, BK; Leonard, FC; Fitzpatrick, ES; Fanning, S and Hartigan, PJ (Eds.),
Veterinary microbiology and microbial disease
. (2nd Edn.), Chapter 23, Oxford, Wiley-Blackwell. PP: 250-262.
Rogerson, BJ; Jung, YJ; LaCourse, R; Ryan, L; Enright, N and North, RJ (2006). Expression levels of Mycobacterium tuberculosis antigen encoding genes versus production levels of antigen specific T cells during stationary level lung infection in mice. Immunology. 118: 195-201.
Shah, DH; Verma, R; Bakshi, CS and Singh, RK (2002). A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. FEMS Microbiol. Lett., 214: 39-43.
Singh, SV; Singh, PK; Gupta, S; Chaubey, KK; Singh, B; Kumar, A; Singh, AV and Kumar, N (2013). Comparison of microscopy and blood-PCR for the diagnosis of clinical Johne’s disease in domestic ruminants. Iran. J. Vet. Res., 14: 345-349.
Singh, PK; Singh, SV; Kumar, H; Sohal, JS and Singh, AV (2010). Diagnostic application of IS900 PCR using blood as a source sample for the detection of Mycobacterium avium subspecies paratuberculosis in early and subclinical cases of caprine paratuberculosis. Vet. Med. Int., Article ID 748621, 8 pages.
Singh, SV; Singh, AV; Singh, R; Sharma, S; Shukla, N; Mishra, S; Singh, PK; Sohal, JS; Kumar, H; Patil, PK; Misra, P and Sandhu, KS (2008). Sero-prevalence of bovine Johne’s disease in buffaloes and cattle population of north India using indigenous ELISA kit based on native Mycobacterium avium subsp. paratuberculosis ‘Bison type’ genotype of goat origin. Comp. Immunol. Microbiol. Infect. Dis., 31: 419-433.
Siqueira, FM; Lopes, CE; Snell, GG and Gomes, MJP (2016). Identification of Mycobacterium smegmatis in bovine Mastitis. Acta Sci. Vet., (Suppl. 1), 44: 166.
Stott, AW; Jones, GM; Humphry, RW and Gunn, GJ (2005). Financial incentive to control paratuberculosis (Johne’s disease) on dairy farms in the United Kingdom. Vet. Rec., 156: 825-831.
Vary, PH; Anderson, PR; Green, E; Taylor, JH and McFadden, JJ (1990). Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. J. Clin. Microbiol., 28: 933-937.
Youssef, DGS; Sallam, FA; Darwish, SF and Amin, AS (2014). Evaluation of conventional and real-time PCR assays for molecular diagnosis of Johne’s disease in dairy cattle. Int. J. Curr. Microbiol. App. Sci., 3: 969-981.
Zali, MHS; Farajnia, F; Yahyapour, H; Moslemzadeh, T and Hashempour, A (2014). Detection of Mycobacterium bovis in cattle suspected to tuberculosis by PCR method in Urmia-Iran. BEPLS., 3: 152-157.