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

• On-farm management practices that increase the risk of high Clostridium spp.
• Total Mixed Ration (TMR) were analyzed by the Most Probable Number technique.
• Clostridium isolates from TMR samples were typed using RAPD-PCR.
• Conclusions can help to Implementation of good practices in feeding sheep milking.

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 breweŕs 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.