Small Ruminant Research 139 (2016) 39–45 Contents lists available at ScienceDirect Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres Importance of on-farm management practices on lactate-fermenting Clostridium spp. spore contamination of total mixed ration of Manchega ewe feeding. Determination of risk factors and characterization of Clostridium population C. Arias a , B. Oliete a , S. Sese ˜ na b , L. Jiménez a , Ll. Palop b , M.D. Pérez-Guzmán a , R. Arias a,∗ a Centro Regional de Selección y Reproducción Animal (CERSYRA), Instituto Regional de Investigación y Desarrollo Agroalimentario y Forestal de Castilla-La Mancha, Consejería de Agricultura, Medio Ambiente y Desarrollo Rural de la Junta de Comunidades de Castilla-La Mancha, 13300 Valdepe˜ nas, Spain b Departamento de Química Analítica y Tecnología de Alimentos, Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Avda, Carlos III s/n, 45071, Toledo, Spain a r t i c l e i n f o Article history: Received 6 June 2015 Received in revised form 27 April 2016 Accepted 4 May 2016 Available online 5 May 2016 Keywords: Total mixed ration Butyric bacteria Farm-management RAPD-PCR 16S-ARDRA a b s t r a c t This work studies the on-farm management practices that increase the risk of developing high lactate- fermenting Clostridium spp. spore counts in Total Mixed Ration (TMR) of Manchega sheep. Moreover the ecology of the Clostridium population was studied in relation to those management practices. A total of 136 TMR samples belonging of 23 Manchega sheep flocks were analyzed for lactate-fermenting Clostridium spores by the Most Probable Number technique (MPN). Information about the feeding characteristics was also collected from the 23 flocks. A logistic regression analysis indicated silages and wet brewer´ ıs grains used for feeding as the on-farm management risk factors that lead to an increase of Clostridium spp. spore counts. A total of 155 Clostridium isolates from TMR samples were typed using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The 56 different genotypes obtained were subsequently identified by restriction analysis of 16S-rRNA gene (16S-ARDRA), so three populations were observed: 92.90%, 5.81% and 1.29% corresponding to Clostridium (C.) sporogenes, Clostridium beijerinkii and Clostridium butyricum respectively. The risks factors of high Clostridium spp. spore counts also increased Clostridium population diversity and favoured the presence of C. butyricum. The results confirmed that these risk factors should be taken into account in developing strategies in the control of Clostridium spp. spores contamination in TMR. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Spain is one of the largest producers of dairy sheep in European Union. Sheep milk production in Spain has increased to 457.6 thou- sand tonnes in 2014 (MAGRAMA, 2014). Manchega sheep is one of the most important native dairy sheep in Spain (529.505 sheep) and its principal production area is Castilla-La Mancha region, with an estimated milk production of 73 million liters of milk in 2015 (ESROM, 2015). The sheep milk production is influenced by many factors: genetic, season, management, health, feeding, etc. The quality of feeding is very important in current’s dairy sheep production systems. Feeding is one of the highest costs in these herds and directly affects the quality of milk. In commercial farms, Total Mixed Ration (TMR) in milking sheep is used. In its prepara- ∗ Corresponding author. E-mail address: rarias@jccm.es (R. Arias). tion it is common to use silage and other wet products. Previous studies in cattle (Colombari et al., 2005; Vissers et al., 2006; Julien et al., 2008) and in dairy sheep (Arias et al., 2013) have shown that these raw materials are sources of lactate-fermenting Clostridium spp. spores contamination in milk and these microorganisms are responsible of the late-blowing defect in ripened cheeses (Garde et al., 2011). Studies in cattle demonstrate that it is necessary to use good management practices in feeding to prevent the butyric spores con- tamination increase (Gaggiotti et al., 2007; Vissers et al., 2007a; Borreani and Tabacco, 2008). In ewe milk, there are few studies (Salmerón et al., 2002; Scintu et al., 2004), and they have not estab- lished the management practices affecting Clostridium spp. spore contamination in TMR. For this reason, the aims of this study are to determine the management practices in dairy sheep production systems considered as risk factors to obtain high Clostridium spp. spore counts (>5 10 3 spores g −1 ) in TMR and to determine how these practices affect Clostridium populations. http://dx.doi.org/10.1016/j.smallrumres.2016.05.003 0921-4488/© 2016 Elsevier B.V. All rights reserved.