Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups

Abstract

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.

Evaluation of Lactobacillus sobrius/L. amylovorus as a New Microbial Marker of Pig Manure

“…They are based mostly on the PCR amplification of specific genes or sequences, such as the STII toxin gene from enterotoxigenic E. coli (16), the internal transcribed spacer (ITS) sequence from Bifidobacterium thermacidophilum subsp. porcinum (26), the 16S rRNA gene of Bacteroides-Prevotella (5,27,31), and the methyl coenzyme M reductase gene from a methanogenic Archaea member (41). However, some of these methods are only qualitative, like the detection of B. thermacidophilum subsp.…”

“…Konstantinov et al (21) therefore designed a primer pair that specifically amplifies a fragment of the L. sobrius genome using real-time PCR. Finally, Jakava-Viljanen et al (13) Given the abundance of L. sobrius/amylovorus in piglet feces (19,37) and its systematic presence in raw manure (26), we tested this species as a new marker of pig fecal contamination. The aims of our study were (i) to confirm the specificity of L. sobrius/amylovorus to pig feces by analyzing five host groups (human, pig, cattle, poultry, and sheep), manure and by-products of manure treatment, runoff water, and urban wastewaters, and (ii) to estimate the suitability of this marker to identify pig fecal contamination found in surface waters.…”

“…Manure and lagoon water samples. Manure and lagoon water samples were collected from 18 piggeries located across Brittany as previously described (26). Samples were centrifuged at 16,000 ϫ g to form a pellet of approximately 250 mg (wet weight).…”

“…In the search for potential pig manure markers, we recently analyzed the dominant bacterial groups of 17 raw pig manure samples using 16S rRNA-targeted PCR and the CE-SSCP (capillary electrophoresis-single-strand conformation polymorphism) molecular typing method (26). Among the domi-nant bacterial groups (Bacteroidales, Bifidobacterium, Eubacterium-Clostridiaceae, and Bacillus-Streptococcus-Lactobacillus), we highlighted the presence of a microaerophilic species, Lactobacillus sobrius, which was isolated from piglet feces previously (19).…”

Dehydrated pork manure by-product: effect of a chitosan amendment on bacterial community and common scab incidence

“…The fact that a considerable proportion of clones in the biosolids (21 and 16% in biosolids P and PC, respectively) could not be associated with a bacterium of known taxonomic identity shows the need for further research on the microbiology of manure and manure by-products. Marti et al (2009) also reported that a high proportion of 16S rRNA gene sequences found in manure presented a low level of similarity to sequences in databases.…”