Abstract
The surface of beef cattle feedlot pens is commonly conceptualized as being packed uncomposted manure. Despite the important role that the feedlot pen may play in the transmission of veterinary and zoonotic pathogens, the bacterial ecology of feedlot surface material is not well understood. Our present study characterized the bacterial communities of the beef cattle feedlot pen surface material using 3647 full-length 16S rDNA sequences, and we compared the community composition of feedlot pens to the fecal source material. The feedlot surface composite was represented by members of the phylum Actinobacteria (42%), followed by Firmicutes (24%), Bacteroidetes (24%), and Proteobacteria (9%). The feedlot pen surface material bacterial communities were clearly distinct from those of the feces from animals in the same pen. Comparisons with previously published results of feces from the animals in the same pen reveal that, of 139 genera identified, only 25 were present in both habitats. These results indicate that, microbiologically, the feedlot pen surface material is separate and distinct from the fecal source material, suggesting that bacteria that originate in cattle feces face different selection pressures and survival challenges during their tenure in the feedlot pen, as compared to their residence in the gastrointestinal tract.
Introduction
Since much of the mass of the feedlot pen surface comes from fecal deposits, one could expect the bacterial communities of FSM to be similar to that of feces. Our hypothesis was that once the feces leaves the animal, the fecal community structure does not remain intact. Although the FSM is commonly conceptualized as being packed uncomposted manure (Ferguson et al., 2005), we propose that, microbiologically, these two substrates are distinct. While we are beginning to understand the microbial communities associated with cattle feces, relatively little is known about the bacteria comprising the microbial landscape of FSM. Additionally, relationships between bacterial members of the feces and FSM habitat communities are unknown. To address this question, we characterized the bacterial diversity of FSM samples, and compared the FSM community structure to previously characterized fecal community data (Durso et al., 2010) collected at the same time from animals housed in the same pen.
Materials and Methods
Replicate FSM surface samples were collected from the top 5 cm of a 10 cm2 area of unconsolidated FSM (air temperature 26°C, soil temperature 19°C) at three locations along the feed bunk. Samples were placed on ice, homogenized, and placed at −80°C within 1 hour of collection. Total DNAs from thawed, homogenized FSM samples were isolated, amplified, cloned, screened for quality and chimeras, and aligned as previously described (Durso et al., 2010). DOTUR (Schloss and Handelsman, 2005) was used to assign operational taxonomic units (OTUs). Approximate species and class level assignments were generated based on OTU cutoffs of 3% and 15% sequence similarity levels, respectively.
Results and Discussion
We examined 3647 full-length 16S rRNA gene clones from FSM samples (GenBank accession nos. FJ671600-FJ675664). Rarefaction curves were used to determine the extent of sequence coverage and suggest a thorough level of coverage of the predominant bacterial classes within FSM (Schloss and Handlesman, 2006). Two species richness estimators were used to predict the total number of OTUs in FSM samples: Chao I (709; 95% confidence interval 594–883) and Abundance Coverage Estimator (ACE) (689; 95% confidence interval 601–812). These numbers are significantly higher than reports from cattle feces which identified 142, or 478 bacterial species (Dowd et al., 2008; Durso et al., 2010) but below the estimated 2000 found in Minnesota soil (Schloss and Handelsman, 2006).
Bacterial community structure of FSM
Actinobacteria were the most abundant phylum in FSM, representing 42% (n = 1516) of clones, followed by Firmicutes (24%, n = 897), Bacteroidetes (24%, n = 891), and Proteobacteria (9%, n = 316). Phyla represented by 1% or less of clones included Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Gemmatimonadetes, Planctomycetes, Tenericutes, Thermomicrobia, and TM7. 370 total OTUs were identified from FSM, only 54 of which were found in all libraries examined. The 10 most frequently observed FSM genera inferred by RDP classification of our 16S rDNA sequences are listed in Figure 1.
Ten most frequently isolated genera from the beef cattle feces and feedlot. Taxonomic names assigned using Ribosomal Database Project “classifier” tool with 95% confidence threshold and 90% sequence similarity cutoff. Genera are listed in order of decreasing occurrence.
Comparison of FSM and fecal communities
We found that FSM communities are distinct from those of the fecal source material. Only 25 (18%) of 139 total genera from feces and FSM were present in both habitats, 21 (15%) were found only in feces, and 93 (67%) were found exclusively in FSM. Actinobacteria and Proteobacteria were more frequently identified in FSM, and Firmicutes and Bacteroidetes were more frequently identified in feces. The Actinobacteria are a group of high G+C Gram-positive bacteria that commonly produce secondary metabolites, including antibiotics (Bull and Stach, 2007). It may be possible that among this group are individuals that naturally produce substances inhibitory to the growth or survival of bacterial pathogens in FSM. Bacteroides was the only bacterial genus that was frequently identified from both habitats (Fig. 1). Bacteroides is a Gram-negative intestinal anaerobe, and it is likely that the clones sequenced from the secondary habitat are a result of high numbers of Bacteroides deposited in the feedlot pen via feces. Thirty-eight percent of fecal clones and 23% from FSM showed low homology with previously characterized organisms, suggesting that a large component of both habitats is comprised of uncharacterized or poorly understood bacteria. When overall richness was calculated for cattle feces and FSM using the same number of clones (3647 from FSM and 3647 randomly chosen from cattle), the bacterial communities were distinct in terms of richness as estimated by Chao, and ACE indices (Chaofeces 478, ChaoFSM 709, ACEfeces 416, and ACEFSM 689), but had similar diversity indices as measured by Shannon and Simpson (Shannonfeces 4.21, ShannonFSM 4.26, Simpsonfeces 0.0330, and SimpsonFSM 0.0349).
Conclusions
In conclusion, this is the first study to examine bacterial communities of the preharvest feedlot environment, and the largest study to date of FSM microbiota using full-length 16S rDNA sequencing. Our results demonstrate that bacterial communities of FSM are separate and distinct from those of the cattle fecal source material. While feces may be the source of much of the mass of the FSM, the bacterial profile of the feces and that of the feedlot pen differ greatly, suggesting that zoonotic pathogens originating in cattle feces face different selection pressures and survival challenges during their tenure in the feedlot pen, as compared to their residence in the bovine gastrointestinal tract.
Footnotes
Acknowledgments
We thank Ron Mlejnek, Steve Simcox, Bob Lee, and Sandy Fryda-Bradley for technical assistance; Melissa Durso and Tammy Sorensen for help with 
; Joan Rosch for secretarial assistance; Randy Bradley, Phil Anderson, and William Dailey for IT support; and the MARC cattle crew for their assistance with sample collection. This research was supported by the U.S. Department of Agriculture, Agricultural Research Service, National Program 108.
Disclaimer
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Disclosure Statement
No competing financial interests exist.