ORIGINAL ARTICLE Usage of a selective media (EMJH-STAFF) in primary culturing of pathogenic leptospires from bovine clinical samples A.P. Loureiro, G. Martins, P. Pinto, L. Narduche, R.C. Teixeira and W. Lilenbaum Laborat orio de Bacteriologia Veterin aria, Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense, Niter oi, Rio de Janeiro, Brazil Significance and Impact of the Study: The slow growth of leptospires and overgrowth of co-existing micro-organisms from environmental and microbiota are the major difficult to recovery Leptospira from animal clinical samples. Implementing an efficient control programme is essential to determine circulat- ing leptospires in the region and their reservoirs. This study evaluated the relationship of a selective media (EMJH-STAFF) on the recovery of pathogenic leptospires (Leptospira noguchii, Leptospira santaro- sai and Leptospira interrogans), from bovine clinical samples (urine and vaginal fluid). EMJH-STAFF seems to be an important tool in obtaining local strains for epidemiological and control purposes. Keywords cattle, culturing, isolation, Leptospira, selective media, urine, vaginal fluid. Correspondence: Walter Lilenbaum, Labo- rat orio de Bacteriologia Veterin aria, Universi- dade Federal Fluminense, Rua Hernani Mello 101, 309, Niter oi, Rio de Janeiro, 24210-030, Brazil. E-mail: mipwalt@vm.uff.br 2015/0677: received 4 April 2015, revised 2 September 2015 and accepted 3 September 2015 doi:10.1111/lam.12501 Abstract Isolation of local strains is mandatory for the success of control programs. However, clinical samples are typically contaminated by other bacteria, which impair leptospires growth. The purpose of this study was to evaluate the use of a previously reported EMJH-STAFF media in the recovery of pathogenic leptospires from bovine clinical samples, namely urine (n = 123) and vaginal fluid-VF (n = 102). EMJH-STAFF presented less contamination than EMJH (<0005), which was more evident in VF culture tubes. Nine pure leptospires cultures were obtained, six from urine (49%) and three from VF (29%). From those, seven grew on EMJH-STAFF, one on EMJH and one in both media. All the isolates were confirmed as pathogenic leptospires by lipL32- PCR, and sequencing of partial rrs showed them to belong to Leptospira noguchii, Leptospira santarosai and Leptospira interrogans species. EMJH-STAFF media was an important tool in the recovery of leptospires from bovine clinical samples. Introduction Bovine leptospirosis occurs worldwide and can result in abortions, stillbirths, premature births, weakly calves and ‘milk drop syndrome’. Thus, leptospirosis leads sig- nificantly financial losses to cattle producers. Knowledge of circulating leptospires and their reservoirs is essential to implement an effective diagnostic and successful con- trol programs (Ellis 2015). Moreover, the isolation of local strains plays an important role in increasing the sensitivity of serological diagnosis (Sarmento et al. 2012), opens new possibilities to vaccination strategies (Murray et al. 2013), and increases the understanding of local disease epidemiology (Chappel and Smythe 2012). Leptospira recovery from animal clinical samples is challenging, mainly due to the slow growth of leptospires and overgrowth of co-existing micro-organisms from environmental or normal microbiota (Saito et al. 2013). However, strictly aseptic methods of collection and sam- ples manipulation are not always possible under field con- ditions. Serial dilution technique (Lilenbaum et al. 2007), filtration (Cameron 2015), and antimicrobial selective agents (Zacarias et al. 2008; Miraglia et al. 2009; Fornazari et al. 2012; Mughini-Gras et al. 2014) have been used to prevent growth of contaminants in Leptospira cultures. Nevertheless, the reported rate of pure Leptospira cultures obtained by the usage of those methods is variable. A combination of antimicrobial selective agents, named STAFF, was developed to isolate leptospires from Letters in Applied Microbiology 61, 603--606 © 2015 The Society for Applied Microbiology 603 Letters in Applied Microbiology ISSN 0266-8254