Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction W. Lilenbaum a, * , R. Varges a , P. Ristow b , A. Cortez c , S.O. Souza c , L.J. Richtzenhain c , S.A. Vasconcellos c a Veterinary Bacteriology Laboratory, Department of Microbiology and Parasitology, Universidade Federal Fluminense, 24210-130 Niteroi, RJ, Brazil b Laboratory of Pathology and Molecular Biology, Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Salvador, 40296-710 Salvador, Brazil c Department of Preventive Veterinary Medicine and Animal Health, Faculty of Veterinary Medicine and Zootechny, University of São Paulo, 05508-900, SP, Brazil article info Article history: Accepted 24 December 2008 Keywords: Leptospira Leptospirosis Sheep Goat PCR Carriers Control abstract Few studies were conducted on the diagnosis and control of small ruminants’ leptospirosis. Thirteen goat herds and seven sheep flocks located in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity by MAT (Microscopic Agglutination Test), 19 and 40 seropositive goats and sheep, respectively, were selected, and urine samples were col- lected for bacteriology and PCR. For both species of animals, the most prevalent reactions were due to serogroups Sejroe and Shermani. Although leptospires were observed by darkfield microscopy in eight samples, pure isolates were obtained by bacteriological culture from only two samples. However, twelve urine samples (six goats and six sheep) were positive by PCR. Based on these findings, we consider that the combined use of MAT as a screening test followed by urine PCR for the direct detection of Leptospira spp. DNA was adequate for the identification of carrier animals among goats and sheep. These are valu- able tools for the control of leptospirosis in small ruminants. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction In spite of its occurrence and economic importance, leading to reproductive problems and overall impaired productivity, few studies have been conducted on the diagnosis and control of small ruminants’ leptospirosis. The efficacy of control programs for lep- tospirosis in sheep and goats, as well as in cattle, relies mainly on the correct identification of Leptospira spp. carriers (Faine et al., 2000; Magajevski et al., 2005; Ruiz et al., 2005). Sheep and goats are able to develop chronic renal infection (Gerritsen et al., 1994) and maintain persistent leptospiruria, perpetuating infec- tion in the herd (Cousins et al., 1989). Goats and sheep are considered less susceptible to leptospirosis than other domestic farm animal species, e.g. cattle (Leon-Vizcaino et al., 1987; Ciceroni et al., 2000). They may develop the chronic form of the disease; which is characterized by impaired fertility, neonatal deaths, abortions and decreased milk production, causing substantial economic losses (Ellis, 1994). Besides, small ruminants are able to develop chronic renal infection and maintain persistent leptospiruria, disseminating bacteria to other animal species as well as to humans. A recent study in Brazil showed that 11.1% of goats were seroreactive to Leptospira with a predominance of serovar Hardjo (Lilenbaum et al., 2007a). Leptospires of the Grippotyphosa serogroup were isolated from dairy goats (Lilen- baum et al., 2007b), whereas Leptospira noguchii was recently iso- lated from sheep (Silva et al., 2007). The standard serological diagnosis of leptospirosis is the micro- scopic agglutination test (MAT), which is recommended for the diagnosis on a herd-screening basis. It relies on the seroreaction with live bacteria representative of local serovares. Nevertheless, the correlation of serology with the presence of bacteria in the kid- neys or in extra-renal locations is not evident and the direct detec- tion of the organism is necessary to reliably identify carriers, as part of an efficacious control program (Faine et al., 2000; Magajev- ski et al., 2005). Efforts to identify carriers are directed towards the detection of the agent or its DNA in urine and other tissues. Available tech- niques include dark field direct microscopic examination (DFM), bacterial isolation, and the detection of leptospiral DNA by PCR (polymerase chain reaction) (Lucchesi et al., 2004). Since lepto- spiral cultivation is laborious, time-consuming, and contamina- tion of cultures may occur, other approaches are welcome to identify carriers of leptospires. PCR has been used to detect Leptospira spp. in clinical specimens as bovine urine (Magajevski et al., 2005; Bomfim and koury, 2006) and vaginal fluids/semen of goats and sheep (Lilenbaum et al., 2008), with encouraging re- sults. The purpose of the present study was to study the preva- lence of Leptospira infection in sheep and goats with reduced fertility, as well as to evaluate the use of urine PCR as a potential 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.12.014 * Corresponding author. Tel.: +55 21 2629 2435; fax: +55 21 2629 2432. E-mail address: mipwalt@vm.uff.br (W. Lilenbaum). Research in Veterinary Science 87 (2009) 16–19 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc