Metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses

Abstract

Published studies have shown that workers in animal slaughterhouses are at a higher risk of lung cancers as compared to the general population. No specific causal agents have been identified, and exposures to several chemicals have been examined and found to be unrelated. Evidence suggests a biological aetiology as the risk is highest for workers who are exposed to live animals or to biological material containing animal faeces, urine or blood. To investigate possible biological exposures in animal slaughterhouses, we used a metagenomic approach to characterise the profile of organisms present within an aerosol sample. An assessment of aerosol exposures for individual workers was achieved by the collection of personal samples that represent the inhalable fraction of dust/bioaerosol in workplace air in both cattle and sheep slaughterhouses. Two sets of nine personal aerosol samples were pooled for the cattle processing and sheep processing areas respectively, with a total of 332,677,346 sequence reads and 250,144,492 sequence reads of 85 bp in length produced for each. Eukaryotic genome sequence was found in both sampling locations, and bovine, ovine and human sequences were common. Sequences from WU polyomavirus and human papillomavirus 120 were detected in the metagenomic dataset from the cattle processing area, and these sequences were confirmed as being present in the original personal aerosol samples. This study presents the first metagenomic description of personal aerosol exposure and this methodology could be applied to a variety of environments. Also, the detection of two candidate viruses warrants further investigation in the setting of occupational exposures in animal slaughterhouses.

Figure 1. Bioinformatics pipeline.

Approach used for the discovery of viral sequences in de novo metagenomic sequencing data from the Illumina HiSeq2000 dataset obtained from the personal aerosol samples.

Figure 2. Contig sequence diversity in personal aerosol samples.

Taxonomic assignment of BLASTN output using MEGAN for both the cattle processing area (left) and the sheep processing area (right). Kingdom level assignment is shown (A) and the proportions of contig assignments within the Eutheria branch are also shown (B). For Eutheria the proportions of Bovinae (includes cattle), Caprinae (includes sheep) and Homininae (includes human) sequences are shown, given that these were the relevant Eutheria branch organisms present within the animal slaughterhouse. BLASTN hits to all other Eutheria have been shown separately. The number of contigs assigned to each taxon is shown below the taxon name, and the percentage value is also shown (2 s.f.).

Figure 3. Confirmation of the presence of human papillomavirus 120 (A) and WU polyomavirus (B) sequences in the cattle processing area.

Agarose gel electrophoresis (2% w/v) detection of PCR amplicons showing the presence of 160 bp amplicon for WU polyomavirus and 105 bp amplicon for human papillomavirus 120. The pooled-amplified sample (B-Pool) was positive for the presence of both viruses, and results are also shown for DNA extracted from individual personal aerosol samples from each worker (original samples). Negative control samples are included (Neg), alongside a molecular marker (M; gradations in base-pairs).