For many people, cows are a clean, docile animal viewed at state or local fairs or seen wandering around pastures on a drive through the countryside. However, managing a herd of dairy animals is no small task. Farmers desire healthy animals that consistently give milk every day. For cows housed primarily in barns, the quality of the bedding used in the stall is important. Not only should bedding be comfortable but also clean, a task made difficult by cows that have no concern about where they are when they are eliminating waste. This creates the possibility a cow could be lying on top of manure for several hours a day and possibly infecting their udders with bacteria like Escherichia coli strain O157:H7, causing mastitis and negatively affecting milk production and cow health. Reusable sand bedding is an option increasingly used for dairy cows, with studies indicating a lower risk of mastitis in herds with sand bedding versus organic bedding (e.g., sawdust). Research by Westphal et al. discovered why sand bedding is able to suppress growth of pathogenic bacteria.
Because sand bedding is recycled, the researchers decided to take samples of bedding from a free-stall barn at four different points in the bedding lifecycle: Fresh sand added to the stall, used sand from the stall, sand that had been washed after use and washed sand that had been sitting in a pile waiting to be recycled back into the barn. Samples also were taken during three different seasons, as prevalence of O157:H7 peaks in summer compared to autumn or winter. To test the ability of sand bedding to suppress growth, two 150g samples were either heated to 80°C for 30 minutes or left unheated. Because the researchers hypothesized that the ability of sand bedding to prevent pathogenic bacterial growth was based on similarity to soil (i.e., the microbial communities present are able to control plant pathogens), they wanted to test if heating the sand bedding would destroy the ability of the sand to suppress O157:H7 growth.
The sand bedding with or without heat treatment was inoculated with E. coli O157:H7 expressing green fluorescent protein (GFP), and samples taken one day, one week and two weeks postinoculation. The results supported the researcher’s hypothesis: there were fewer GFP-expressing O157:H7 bacteria in the untreated bedding samples compared to heat-treated samples regardless of season the sample was taken. The greatest suppressive effect was seen in recycled bedding, not a surprising result because any microbial community present would stabilize when sand bedding is left to sit outside before being reused. However, Westphal et al. noted that both heated and unheated samples demonstrated declining growth of E. coli O157:H7 over time. The researchers speculated that there were suppression factors that were not heat-sensitive. Unfortunately, there was no true negative control for sand bedding because the fresh bedding consisted of new sand mixed with recycled sand at a 1:1 ratio. I would be interested to learn how fresh sand that had not been near any cows fared in this experiment.
To learn more about which microbes might be involved in keeping growth of E. coli O157:H7 low in sand bedding, DNA was extracted from 0.25g of sand and used for PCR amplification of 16S rRNA genes using a fluorescently labeled primer. A portion of the PCR product was then digested with the restriction enzyme MspI and the size of resulting fragments was analyzed. These terminal restriction fragments (TRFs) were subjected to interval plotting and linear regression to determine if there were fragments found in abundance in the growth-suppressive bedding, comparing heated versus nonheated samples. There were different results from the two analysis methods, so the researchers selected the ones determined to be abundant in both analyses. Westphal et al. also screened for microbial TRFs present in untreated sand bedding in the samples that showed the greatest suppressive effect in the first day after inoculation, generally the washed or recycled sand or both. Overall, 11 TRFs were identified across all samples analyzed. These fragments were then cloned, the inserts sequenced and the sequence compared against sequence databases using BLAST analysis. The TRFs under 100bp in size were found in the Cytophaga-Flexibacter-Bacteroidetes group, the closest matches being uncultured bacteria from compost or manure, appropriate considering the origin of these samples. TRFs of 140–300bp were mainly associated with two bacterial groups, the gammaproteobacteria and the firmicutes, while the largest TRFs (480–510bp) were associated with the gamma- and betaproteobacteria. These TRFs had closest matches to uncultured bacteria from cattle feedlots, wastewater or compost. Two TRFs had no BLAST results.
The data in this experiment do seem to support the hypothesis that bacteria present as a community in bedding sand for dairy cattle can suppress the growth of pathogenic E. coli O157:H7. The microbial community constituents based on sequence comparison of the isolated TRFs are consistent with bacterial sequences found around cattle even if a single bacterium species could not be identified. Research like this is important for everyone who enjoys drinking milk and consuming dairy products. Knowing that a bedding choice can not only improve the health of animals but prevent a pathogenic organism from entering the human food supply allows farmers to make informed choices on managing their milking operations.
Westphal, A., Williams, M.L., Baysal-Gurel, F., LeJeune, J.T.and McSpadden Gardener, B.B. (2011). General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding. Applied and Environmental Microbiology, 77 (6), 2113-21 PMID: 21257815