Medium-term storage of calf beddings affects bacterial community and effectiveness to inactivate zoonotic bacteria

Abstract

Land-spreading of animal faecal wastes -such as animal beddings- can introduce zoonotic enteropathogens into the food system environment. The study evaluated the effectiveness of animal beddings naturally contaminated by calf manure to reduce E. coli O157:H7 or Salmonella enterica. The two pathogens were introduced separately as a four strains-cocktail and at high (>6.5 Log10 g-1) concentration into bedding materials, and their inactivation over a 10 weeks-period was monitored by using a Most Probable Number (MPN) enumeration method. Inactivation of E. coli O157:H7 was more effective in the bedding inoculated immediately after collection from calf pens than in the beddings inoculated after a 2 months-pre-storage period: E. coli O157:H7 levels were reduced by 6.6 Log10 g-1 in unstored bedding (0.5 Log10 g-1 recovered; 95%CI: 0.0-1.2), and by 4.9 Log10 g-1 in pre-stored bedding (2.2 Log10 g-1 recovered; 95%CI: 1.5-2.8) with a significant (p<0.05) difference between unstored and pre-stored. S. enterica was inactivated less effectively as counts were reduced by one order of magnitude, with no significant difference in inactivation between unstored and pre-stored beddings. Low levels of naturally occurring E. coli O157 and Salmonella spp. were detected in the non-inoculated beddings, as well as in the straw prior to use in the animal facility. To better understand the possible biological processes involved, the bacterial community present in the beddings was characterised by short-read 16S rRNA sequencing. Pre-storage of the bedding affected the composition but not the diversity of the bacterial community. Analyses of the key bacterial phyla suggested that the presence of a diverse and stable bacterial community might facilitate inactivation of the introduced pathogens, and a possible role of bacterial orders associated with lignocellulolytic resources. Overall, the study contributed to the understanding of the fate of zoonotic bacteria introduced in animal beddings during storage and identified bedding storage practices pre-and post-use in animal facilities that could be important to prevent the risk of zoonosis dissemination to the environment or to the dairy herds.

Fig 1

Inactivation of E. coli O157:H7 and S. enterica in unstored (filled diamond) and pre-stored (hollow circle) straw+manure bedding (a, c) and straw (b, d). Each MPN counts were obtained from triplicate bags. Bars are 95% confidence intervals for the geometric mean counts. * indicates significant difference at p = 0.05 between unstored and pre-stored geometric mean counts within an incubation time.

Fig 2

Bray-Curtis principal coordinate analysis (PCoA) illustrating the changes in bacterial community beta-diversity in the four beddings types over incubation time. Each symbol represents a sample, with distance between samples calculated using Bray-Curtis similarity measures. Dashed ellipses indicate 95% confidence intervals of the ordinations for named clusters.

Fig 3

Change of relative abundance of major bacterial phyla across bedding types (a: straw; b: straw + manure) with and without pre-storage treatment and over incubation time. Only the phyla with abundance above 1.0% were plotted. Bars are standard error of mean (n = 6).