The goal of this study is to determine if survival and detection of Listeria in the drains of poultry further-processing plant is affected by the presence of bacterivorous protozoa. The project has been split into two phases: the first of which is to sample floor drains to identify drains colonized with Listeria and drains that are consistently free of the organism; and the second phase is to analyze the fauna found in the drains by DNA analysis to determine what species of protozoa are present in the drains.
The first phase has to be done in real time; that is, samples from drains are immediately processed and cultured for Listeria. The second phase, the DNA analysis, is more efficiently performed in batches and can be done with frozen samples.
Status (As of November 2011)
The original design for this project was to amplify ribosomal genes found in floor-drain samples, clone them and then perform sequence analysis to identify a subset of the clones. A reasonable number of clones by that protocol would be in the range of two hundred, plus or minus. The plan was changed when a new technology called “massively parallel sequencing (MPS)” became available. With MPS a great number of fragments of DNA can be sequenced without cloning. Investigators applied this technology to the floor-drain samples collected from poultry further-processing plants and were rewarded with 45,098 individual sequences of eukaryotic ribosomal genes.
The identifications of the sequences were largely predictable with a few surprises. The primers that were used to perform the sequences were designed to not amplify ribosomal genes from chicken and when analysis was done by DGGE there did not appear to be any chicken ribosomal genes in the product. However, by MPS 19,133 (42%) of the sequences were from chicken. Most of the rest of the sequences were from assorted fungi (17 different genera).
The results from protozoa were again unexpected. Only one genus of free-living bacterivorous protozoa was recovered. This was a poorly described amoeba, Rhogostoma (40 total sequences, approximately 0.1% of the total sequences). Investigators also found that 117 sequences were identified as members of the genus Eimeriidae. Eimeriidae are pathogenic protozoa that live in the intestinal lining of their hosts. A striking feature of the Eimeriidae is their fastidious requirement for a specific host. Although investigators were not able to identify the Eimeriidae sequences to species level, they may have come from chickens, which might indicate intestinal content contamination of the drains in the further-processing plant. If this is the case, it raises questions as to whether the other species found in the floor-drains are resident or transient contaminants from the raw poultry meat.
The data can only be expressed as proportion or percent of the total analyzed. Investigators do not currently have the technology to determine the absolute number individuals of each species in the samples. With certain assumptions relative abundance can be calculated by relating the number of sequences of one species compared to all the others in the data set. In this way, it can be estimated that there were approximately 500-fold fewer Rhogostoma cells than there were cells from chickens (it needs to be remembered that the red blood cells of birds contain nuclei with DNA, unlike mammals). However, the samples did not have any noticeable coloration from chicken blood.
Analysis of the capabilities of Rhogostoma to ingest and kill Listeria is the next obvious step in this project. The only pure cultures that can be found are a pair of isolates in the Culture Collection of Algae and Protozoa, located in the United Kingdom. These two cultures were originally isolated from marine sources and may not be representative of the species found. In future studies, investigators will specifically isolate the protozoa from the floor drains and attempt to maintain them in their laboratories. Investigators are also working on reefing the PCR primers and conditions used for MPS to exclude chicken and fungal contaminants of the protozoa samples.