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21 22 The objective of this study was to identify a microbial marker for pig manure contamination. 23 We quantified the persistence of four dominant bacterial groups from the pig intestinal tract 24 throughout manure handling in ten livestock operations (including aerobic digestion) using 25 molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium26 Clostridiaceae, Bacillus-Streptoccocus-Lactobacillus (BSL) and Bifidobacterium were 27 amplified and analysed by capillary electrophoresis single strand conformation polymorphism 28 (CE-SSCP). The most dominant bacterial populations were identified by cloning and 29 sequencing their 16S rRNA genes. The results showed that Bifidobacterium and, to a lesser 30 extent, members of the BSL group, were less affected by the aerobic treatment than either 31 Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found 32 in raw manure were still present in manure during land application, suggesting that they can 33 survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA 34 ITS (Internal Transcribed Sequence) of one species, Bifidobacterium thermacidophilum 35 subsp. porcinum, was sequenced and a specific pair of primers was designed for its detection 36 in the environment. Using this nested PCR assay, this potential marker was not detected in 37 samples from 30 bovine, 30 poultry, and in 28 human faeces or in 15 urban wastewater 38 effluents. As it was detected in runoff waters after spreading of pig manure, we propose this 39 marker as a suitable microbial indicator of pig manure contamination. 40 41 Author-produced version of the article published in Applied and Environmental Microbiology, 75 (15), 4967-4974 Original publication available at aem.asm.org doi:10.1128/AEM.01895-09 ha l-0 04 56 12 9, v er si on 1 12 F eb 2 01 0