Comparative study on cultural and molecular methods for detection of Brucella melitensis in sheep and goat

Title: Comparative study on cultural and molecular methods for detection of Brucella melitensis in sheep and goat

Authors: KB Patel, SI Patel, BK Patel, HC Chauhan, AG Bhagat and BS Chandel

Source: Ruminant Science (2017)-6(2):309-314.

Cite this reference as: Patel KB, Patel SI, Patel BK, Chauhan HC, Bhagat AG and Chandel BS (2017). Comparative study on cultural and molecular methods for detection of Brucella melitensis in sheep and goat. Ruminant Science 6(2):309-314.


Brucellosis due to Brucella melitensis is widespread in India and is considered to be the major cause of abortion in small ruminants incurring severe economic loss. In the present study, a total 170 clinical samples of vaginal swab, placental cotyledons, aborted foetal organs and stomach contents, hygroma and orchitis fluid, whole blood and milk were collected but unable to cultural isolation of the Brucella organism. Among the three different genus specific primer pairs used for amplification by PCR, Only B4/B5 and JPF/JPR primer pair detected three samples positive for Brucella DNA from 170 clinical samples.  Proper selection of primer pair is essential, as it was affected by the presence of genomic DNA and finally PCR efficiency. Out of combination of three species specific primers pairs (omp31, ORF A0503 and B. abortus+ IS711) used, only omp31 primer pair produced desired amplicon of 720bp in three clinical samples. This indicated that these animals were infected with Brucella melitensis. Comparatively PCR was found more suitable method for detection Brucella in clinical samples as compared to cultural methods because 3 samples were found positive by PCR which were negative in cultural isolation.


Al-Gardia MA, Khairani-Bejo S, Zunita Z and Omar AR (2011). Detection of Brucella melitenisis in blood samples collected from goats. Journal of Animal and Veterinary Advances 10(11):1437-1444.

Al-Tememy HAH, Al-jubort KH and Abdul-majeed BA (2013). Pathological and molecular diagnosis of Brucella melitensis in the fetal and placental tissues of aborted ewes in Al-Najaf city. Kufa Journal for Veterinary Medical Sciences 4(1):28-40.

Amin AS, Hamdy ME and Ibrahim AK (2001). Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Veterinary Microbiology 83(1):37-44.

Avsever ML, Erdenlig S, Eskiizmirliler SN, Tunaligil S, Ozden M, Baklan EA, Saytekin AM, Saglam G and Aydemir S (2013). Identification, biotyping, and antibiotic susceptibilities of Brucella spp. isolated from goat fetuses. Bornova Veteriner Bilimleri Dergisi 35(49): 9-13.

Bailey GG, Krahn JB, Drasar BS and Stoker NG (1992). Detection of Brucella melitensis and Brucella abortus by DNA amplification. Journal of Tropical Medicine and Hygiene 95(4): 271-275.

Bamaiyi PH, Noor SA and Mohamed AZ (2012b). Seroprevalence and economic impact of eradicating zoonotic brucellosis in Malaysia: A case study of Melaka state of Malaysia. Veterinary World 5(7):398-404.

Bhanu RV, Gunaseelan L, Subramanian A and Yale G (2013). A study on bovine brucellosis in an organized dairy farm. Veterinary World 6(9):681-685.

Bricker BJ and Halling SM (1994). Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella melitensis, Brucella ovis and Brucella suis bv 1 by PCR. Journal of Clinical Microbiology 32(11):2660-2666.

Chauhan HC, Patel KB, Patel SI, Patel BK, Chandel BS, Bhagat AG,  Patel MV, Patel SS, Patel AC, Shrimali MD and Shome Rajeshwari (2017). Serological survey of brucellosis in camel of Gujarat. International Journal of Current Microbiology and Applied Sciences 6(4):1815-1821.

Dadawala AI, Chandel BS, Chauhan HC, Kumar Pankaj, Bhagat AG and Shah NM (2012). Detection of brucella antibody by using different serological tests in cattle from north Gujarat. Ruminant Science 1(2):145-148.

Dubey Praveshika, Patel KB, Patel BK, Chauhan HC, Chandel BS, Patel SS, Shrimali MD, Kala JK, Patel MG, Patel AC, Rajgor Manish, Patel MA and Modi AN (2017). Molecular detection of brucella organism from milk and milk products. International Journal of  Current Microbiology and Applied Sciences 6(4):1087-1091.

Habtamu TT, Rathore R, Dhama K and Karthik K (2013). Isolation and molecular detection of Brucella melitensis from disease outbreak in sheep and B. abortus from cattle farm by IS711 and omp2a gene based PCR. International Journal of Current Research 5(7):1920-1925.

Kala MS (2009). Serological, cultural and molecular methods for detection of brucella infection of bovines in North Gujarat. MVSc thesis submitted to SDAU, Sardarkrushinagar, Gujarat.

Kotadiya AJ (2012). Serological, cultural and molecular detection of brucella infection of sheep in Gujarat. MVSc thesis submitted to SDAU, Sardarkrushinagar, Gujarat.

Lavaroni O, Aguirre N, Vanzini V, Lugaresi C and Torioni de echaide S (2004). Assessment of polymerase chain reaction (PCR) to diagnose brucellosis in a brucella infected herd. Revista Argentina de Microbiología 36(3):101-106.

Leal-Klevezas DS, Lopez-Merino A, Martinez-Soriano JP, Martinnez-Soriano JP (1995). Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. Journal of Clinical Microbiology 33(12):3087-3090.

Leary SO, Sheahan M and Sweeney T (2006). Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Research in Veterinary Science 81(2):170-176.

Leyla G, Kadri G and Umran O (2003). Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Veterinary Microbiology 93(1):53-61.

Navarro E, Escribano J, Fernandez J and Solera J (2002).Comparison of three different PCR methods for detection of Brucella spp in human blood samples. FEMS Immunology and Medical Microbiology 34(2):147-151.

Panchasara HH, Patel JS and Patel PR (2015). Seroprevalence of brucella infection in organized farms and field conditions in Gujarat. Ruminant Science 4(1):37-41.

Patel BK, Patel KB, Patel SI, Modi AN, Chauhan HC, Shrimali MD, Dodia VD and Chandel BS (2017a). Diagnosis of brucellosis using molecular techniques from various clinical samples. International Journal of Agriculture, Environment and Biotechnology 10(2):267-270.

Patel KB, Chauhan HC, Patel BK, Patel SS, Shrimali MD, Kala JK, Modi AN, Rajgor Manish, Patel MA, Patel AC, Patel MG and Chandel BS (2017b). Clinico-seroepidemiology and molecular characterization of brucellosis in animals. International Journal of Current Microbiology and Applied Sciences 6(4):222-231.

Poester FP, Nielsen K, Samartino LE and Yu WL (2010). Diagnosis of Brucellosis. Open Veterinary Science Journal 4:46-60.

 Romero C, Pardo M, Grillo MJ, Diaz R,  Blasco JM and Lopez-Goni I (1995). Evalution of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. Journal of Clinical Microbiology 33(12):3198-3200.

Sayour AE and Azzam RA (2014). Epidemiological characterization and pathogenicity of prevalent brucella strains in Egypt. Annals of Veterinary and Animal Science 77-85.

Shrimali MD, Shah NM, Chandel BS, Chauhan HC, Patel SS, Patel KB, Patel BK, Bhagat AG,  Patel SI, Dadawala AI, Shah JD, Rajgor Manish, Pandya RP,  Patel AC, Patel MA, Kala JK and Patel MG (2017). Isolation, identification and molecular characterization of Brucella abortus from bovines. Journal of Pure and Applied Microbiology 11(2):933-939.

Stella M, Constantine A, Efimia S, Eudoxia D and Athina K (2007). Evaluation of different PCR assays for early detection of acute and relapsing brucellosis in humans in comparison with conventional methods. Journal of Clinical Microbiology 45(4):1211-1218.

Sutariya MB, Roy A, Kumar P and Patel PK (2014). Seroprevalence of brucellosis in sheep and goats of Gujarat. Indian Journal of Small Ruminants 20(1):128-130.

Tsolis RM, Seshadri R, Santos RL, Sangari FJ, Lobo JM, Jong MF, Ren Q, Myers G, Brinkac LM, Nelson W, Robert TD, Angiuoli S, Khouri H, Dimitrov G,  Robinson JR, Stephanie M, Walker RL, Elzer PE, Hassan KA and Paulsen IT (2009). Genome Degradation in Brucella ovis corresponds with narrowing of its host range and tissue tropism. PLoS ONE 4(5):5519.

Vizcaino N, Cloeckaert A, Zygmunt MS and Dubray G (1996). Cloning, nucleotide sequence, and expression of omp31 gene coding for an immunogenic major outer membrane protein. Infection and Immunity 64(9):3744-3751.