Bioreduction of Sheep Carcasses Effectively Contains and Reduces Pathogen Levels under Operational and Simulated Breakdown Conditions

Options for the storage and disposal of animal carcasses are extremely limited in the EU after the introduction of the EU Animal By-products Regulations (ABPR; EC/1774/2002), leading to animosity within the livestock sector and the call for alternative methods to be validated. Novel storage technologies such as bioreduction may be approved under the ABPR provided that they can be shown to prevent pathogen proliferation. We studied the survival of <i>Enterococcus faecalis</i>, <i>Salmonella</i> spp., <i>E. coli</i> O157 and porcine parvovirus in bioreduction vessels containing sheep carcasses for approximately 4 months. The vessels were operated under two different scenarios: (A) where the water within was aerated and heated to 40 °C, and (B) with no aeration or heating, to simulate vessel failure. Microbial analysis verified that pathogens were contained within the bioreduction vessel and indeed reduced in numbers with time under both scenarios. This study shows that bioreduction can provide an effective and safe on-farm storage system for livestock carcasses prior to ultimate disposal. The findings support a review of the current regulatory framework so that bioreduction is considered for approval for industry use within the EU

Intracellular enzyme (glycosyl hydrolase) activities associated to bacterial enzyme extracts isolated from two wild Iberian lynx fecal samples and rumen content from four rumen-fistulated and non-lactating Holstein cows.

<p>(A) Average potential hydrolysis rates (n = 2, ± standard deviation in three technical replicates) in protein extracts from two wild lynxes (Eva and Granadilla) captured in the same area and under the same protocols. (B) Average potential hydrolysis rates (n = 4, ± standard deviation in three technical replicates) in protein extracts from rumen content from four rumen-fistulated and non-lactating Holstein cows. (C) Comparative average glycosidase activity for lynx gut and cow rumen protein extracts; the fold difference is specifically shown based on data provided in panels A and B. In all cases, enzyme activity was quantified using a BioTek Synergy HT spectrophotometer by measuring release of <i>p</i>-nitrophenol (<i>p</i>NP) using a protein amount of 6.34 µg (for Eva), 7.74 µg (for Granadilla), 15.83 µg (for SRF) and 15.42 µg (for LAB), and [substrate] of 1 mg ml⁻¹ (from a 10 mg ml⁻¹ stock solution) in 20 mM glycine buffer, pH 9.0, <i>T</i> = 30 °C, in a final volume of 50 µl. The different substrates used as specifically shown. Note: activity against <i>p</i>NP derivatives of GalNAc or other mucus-associated sugars could not be determined because they are not commercially available.</p

Model representing the potential sugar uptake system feeding the central lynx metabolism through the hydrolysis of carbohydrate-containing substrates derived directly from animal tissues or indirectly from plant/sugar biomass most likely derived from prey nutrition sources.

<p>Model representing the potential sugar uptake system feeding the central lynx metabolism through the hydrolysis of carbohydrate-containing substrates derived directly from animal tissues or indirectly from plant/sugar biomass most likely derived from prey nutrition sources.</p

Heat map and clustering of predicted genes encoding GHs in the lynx, termite, bovine rumen, panda, wallaby and canine gastrointestinal metagenomes.

<p>The heat map colors represent the relative percentages of GH families within each sample. Hierarchical clustering based on relative percentage of predicted genes encoding GHs is specifically shown.</p

Comparison of lynx with other animal-associated (mammals and arthropoda) gut metagenomes.

<p>Clustering of gut metagenomes available within IMG/M based on functional composition of KEGGs.</p

Overview of the relative abundance of phylogenetic prokaryotic groups recovered from the lynx distal gut based on SSU rRNA tag sequences extracted from the pyrosequences.

<p>All reads belonging to phylum Proteobacteria identified related to Betaproteobacteria, which is only cited in the Figure.</p

Temperature (A) and pH (B) optima and stability (C and D) of the purified GHF43 R_09-02 protein.

<p>The parameters were determined using <i>p</i>NPβX as the substrate. (<b>A</b>) For the optimum temperature determination, the pH was adjusted to 6.0 (sodium acetate 20 mM). (<b>B</b>) The optimum pH was determined in the range of pH 4.0–9.0 at 34°C. The buffers (100 mM) used were as follows: acetate (pH 4.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0) and Tris-HCl (pH 8.0–9.0). In both cases, the <i>k</i>_cat value was determined using an [E] ranging from 0 to 12 nM and a substrate concentration of 70 mM. Activity at 100% refers to 230.3±13.7 s⁻¹ at pH 6.0 and 34°C. (<b>C</b>) The time lost normalised quantification of the R_09-02 activity levels (with <i>p</i>NPβX) at 34°C and pH 6.0 (sodium acetate 20 mM) is shown. Protein (1.5 μg) was incubated, and the activity was determined as described in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#s2" target="_blank">Methods</a></b>. (<b>D</b>) The effect of chemical reagents and metal ions on the hydrolase activity (<i>p</i>NPβX). The concentrations of the various chemicals ranged from 2 mM (black) and 5 mM (light grey) to 10 mM (dark grey), and the relative activities were defined using the activity ratio without the added chemicals. The optimal pH (6.0) and temperature (34°C) were used in the assays. All of the measurements were analysed in triplicate, and error bars are indicated. The error bars represent the standard deviation of three replicates from a single protein preparation.</p

Contribution of GHF43 proteins to plant polymer hydrolysis in the rumen.

<p>(<b>A</b>) The relative abundance and distribution of glycosyl hydrolase families in the metagenome from the bovine rumen microbiome. The data include the pyrosequencing data of 4 metagenomic samples, including fibre-adherent and pooled liquid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#pone.0038134-Brulc1" target="_blank">[10]</a>. (<b>B</b>) New pathways of pentose and hexose digestion by the R_09-02-like enzymes in bovine rumen. The scheme indicates that R_09-02 may contribute to the digestion of arabinoxylans (a common activity associated with GHF43 enzymes: grey arrows) and gluco- and galactooligosaccharides (black arrows) derived from amylose/starch and galactans.</p

Structural models of R-03_04 (A), R-03_05 (B) and R-09_02 (C).

<p>The putative catalytic residues (general acid, base and transition state stabilisers) are depicted in red. The left panel shows the overall folding of the protein. The right panel is a close-up view of the catalytic site, indicating the catalytic residues and other highly conserved residues among the GHF43 enzymes that may establish polar (yellow) or hydrophobic (violet) contacts with the substrate at the −1 subsite. The middle panel shows the solvent-accessible surface close to the catalytic site. As a reference, the location of a xylobiose molecule (green) is given according to the structural superimposition with the β-xylosidase from <i>Geobacillus stearothermophilus</i> (PDB code 2EXJ).</p

Phylogenetic and modular characteristics of the GHF43 proteins identified in the present study.

<p>(<b>A</b>) The scheme of modular arrangements in the biochemically characterised GHF43 enzymes. The catalytic module is represented with a green box. The single representative of type D (Uniprot code P45796) is predicted to contain domains in the C-terminal extension a CBM6 and a CBM36 module (dark and light blue ovals, respectively) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#pone.0038134-JamalTalabani1" target="_blank">[42]</a>. In one case of the type B enzymes (Uniprot code Q45071), a CBM6 domain is predicted as a Pfam hit in the C-terminal domain. (<b>B</b>) Phylogenetic tree of the catalytic domains of the biochemically characterised GHF43 enzymes. The GHF43 catalytic modules were selected according to the predictions as Pfam hits, before Clustal alignment. The modular type (according to the scheme in [A]) and the Uniprot or NCBI (underlined) accession code of the original protein are indicated in each case. The GHF43 enzymes analysed in this study (R_03-04, R_03-05, R_09-02) are included and highlighted with a box. Those enzymes include xylosidases (Xyl), arabinosidases (Ara), bifunctional xylosidases/arabinosidases with similar activities for both substrate types (Xyl-Ara) or with certain preference for one or another (Xyl > Ara or Ara > Xyl), galactosidase (Gal) and the multifunctional R_09-02; enzymes with more than one catalytic domain were not included. The letters in brackets indicate the type of GH. The numbers on the branches indicate bootstrap values greater than 50%. Phylogenetic analysis of protein sequences was conducted with <i>MEGA</i> 4.0 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#pone.0038134-Tamura1" target="_blank">[43]</a> using the Neighbor-Joining treeing method and Poisson correction. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#pone.0038134.s015" target="_blank">Table S7</a></b> contains a list of bibliographic records that provided experimental support for enzymes described in the Figure.</p