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Abortion in buffalo-breeding industry is one of the key roots of economic losses in the livestock sector. Brucella spp., Leptospira spp. and Campylobacter fetus are main pathogens because of the potential impact on veterinary and human health. This study assessed the relative performance of ELISA and multiplex PCR (mPCR) assay using different primer sets for the detection of venereal pathogens. Variety of samples (blood, urine, preputial washings and semen) were collected from a total of 160 buffalo breeding bulls of Semen Production Unit, Qadirabad and private dairy farms in Punjab Province of Pakistan. Serum and total genomic DNA was extracted for molecular detection by ELISA and PCR respectively. This study comprised 9 sets of primers to optimize multiplex with respect to melting temperature and crosslinking among primers. ELISA assay for Brucella was 2.5%, whereas for Leptospira and Campylobacter fetus it was 1.88% for each. With respect to development of triplex and duplex PCR, results of our assay were completely consistent with monoplex PCR using a combination of newly designed and reported primers. All ELISA suspected samples found negative for Brucella, Leptospira and C. fetus when tested with both monoplex and triplex PCR. This multiplex PCR assay provides a valuable complementary tool in routine, simultaneous and robust detection of these three genital diseases and revealing epidemiological facts about abortion measures in buffalo. [ABSTRACT FROM AUTHOR]