Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction.

Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.

McrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle.

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme-M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.

Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure.

An acidulocomposting system for the treatment of cattle manure with little emission of ammonia gas was developed, and the structure of its microbial community was investigated by denaturing gradient gel electrophoresis (DGGE) and clone library construction. An acidulocomposting apparatus (BC20, 20 L) was operated for 79 days to treat 2 kg (wet wt) of garbage per 1 or 2 days. On day 80 of operation, the substrate was changed from garbage to cattle manure (1 kg of beef cattle manure was added to the apparatus every 2 or 3 days), and the system continued operating from days 80 to 158. The compost in the vessel was under acidic conditions at pH 5.2-5.8, and ammonia emission was below the detectable level (<5 ppm) throughout the period of cattle manure feeding. Total nitrogen and total carbon in the compost were 26-29 and 439-466 mg/g of dry weight, respectively, which are higher than those in general cattle manure compost. The main acids accumulated during operation were lactic and acetic. Sequencing analysis targeting the 16S rRNA gene revealed the stable dominance of the bacterial phylum Firmicutes, with a high proportion of the isolates belonging to the genus Bacillus. Using a culturing method with MRS agar, we isolated lactic acid bacteria (LAB) related to Pediococcus acidilactici, Weissella paramesenteroides, and Lactobacillus salivarius, indicating the existence of LAB in the system. These results indicate that acidulocomposting treatment of cattle manure is not accompanied by ammonia emission and that Bacillus and LAB may be the key components in the system.

Real-time PCR detection of the effects of protozoa on rumen bacteria in cattle.

A real-time PCR approach was used in this study to clarify the populations of major bacterial species in the rumens of faunated and unfaunated cattle. The sensitivity of this novel real-time PCR assay was evaluated by using 10(1) to 10(8) plasmid copies of target bacteria. The numbers of plasmid copies of Ruminococcus albus, Ruminococcus flavefaciens, Prevotella ruminicola, and the CUR-E cluster were higher in the unfaunated than in the faunated rumens. The CUR-E cluster belongs to the Clostridium group. In contrast, Fibrobacter succinogenes was higher in the faunated than in the unfaunated rumens. Although it is well known that an absence of protozoa brings about an increase in the bacterial population, it was clarified here that an absence of protozoa exerted differential effects on the populations of cellulolytic bacteria in cattle rumens (i.e., F. succinogenes, R. albus, and R. flavefaciens). In addition, real-time PCR analysis suggested that the CUR-E cluster was more prevalent in the unfaunated rumens.

The effect of protozoa on the composition of rumen bacteria in cattle using 16S rRNA gene clone libraries.

The effect of the presence of protozoa on the composition of rumen bacteria was investigated in cattle. Seven castrated Holstein cattle were divided into two groups: four faunated and three unfaunated, and 16S ribosomal RNA gene (rDNA) clonal libraries were constructed. A total of 312 clones were sequenced across 1,500 bp. The 151 sequences of the faunated cattle were classified into 98 operational taxonomic units (OTUs) having at least 97% similarity. The sequences derived from the faunated cattle were classified into Firmicutes (59.7%), Bacteroidetes (34.4%), Spirochaetes (2.6%), Actinobacteria (2.0%), and Proteobacteria (1.3%). Bacteroides and Prevotella (34.4%) were the major groups in the faunated cattle. The 161 sequences in the unfaunated cattle were classified into 72 OTUs. The sequences derived from the unfaunated libraries were classified into Firmicutes (65.7%), Bacteroidetes (31.1%), Proteobacteria (1.9%), and Spirochaetes (1.2%). The Clostridium botulinum group and its relatives (36.0%) were the major groups in the unfaunated cattle. An analysis by the computer program LIBSHUFF clarified that the presence of ruminal protozoa markedly affected the composition of rumen bacteria.

Culture-independent analysis of fecal microbiota in cattle.

The phylogenetic diversity of the fecal bacterial community in Holstein cattle was determined by 16S ribosomal RNA gene sequence analysis. The sequences were affiliated with the following phyla: Firmicutes (81.3%), Bacteroidetes (14.4%), Actinobacteria (2.5%), and Proteobacteria (1.4%). The Clostridium leptum subgroup was the most phylogenetically diverse group in cattle feces. In addition, a number of previously uncharacterized and unidentified bacteria were recognized in clone libraries.