1. P. Millner1, K. White1, L. Graham2, N. (Tong) Liu3, J. Coffie2, R. Stonebraker2, D. Clark2, F. Hashem2, M. Sharma1
1)USDA-ARS, Beltsville, MD, 2)University of Maryland Eastern Shore, Princess Anne, MD, 3)University of Maryland, College Park, MD
Animal manure composts (biological soil amendments, BSAs) are
used to enhance quality and fertility of soils. When produced by
validated processes, composts are approved for use by USDA-
National Organic Program rules and currently proposed FDA
Produce Safety rules. Likewise, heat-treated poultry litter pellets
(htPLP) produced by a validated process are approved BSAs.
Nutrient-rich, microbiologically-unbuffered htPLP top-dressed on
compost-amended soil poses a potential risk of rapid colonization
and survival by pathogenic bacteria inadvertently introduced to
BSAs and then subsequently transferred to crops.
Effect of Top-Dressing Compost and Heat-Treated Poultry Litter Pellets on Survival of E. coli
in Dairy Manure Compost-Amended Soils
MATERIALS AND METHODS
INTRODUCTION
SIGNIFICANCE
RESULTS
 The attO157 populations declined from ~2.0 to ≤0.02
LogMPN/gdw in CDM-amended plots by day 3 in the fall and
did not recover after top-dressing with CDM, htPLP, or
inorganic fertilizer.
 Populations of gEc were greater and more persistent in all
BSA treatments compared to attO157 populations (p<0.05).
 Rainfall during 1-29dpi sustained gEc populations to 2.5-
3.8LogMPN/gdw; after 30 dpi and several days <0⁰C, with
ample rainfall (i.e, soil moisture), gEc was detectable at by
MPN in all 16 plots at 119 dpi and in 1 of 16 plots at180 dpi.
 After top-dressing, rainfall, and below freezing
temperatures, spinach was frosted and was not harvestable;
gEc populations ranged from 0.95(htPLP only)-1.5(CDM only)
logMPN/gdw soil on 119dpi.
 Inoculum: A 5-strain cocktail (1 L) of five rifampicin-resistant E.
coli : 3 generic E. coli (gEc) and 2 attenuated strains of E. coli
O157:H7 (attO157), growth conditioned in sterile poultry litter
extract, were sprayed (~4.92LogCFU/mL) onto 2m2 plots amended
or not with dairy manure solids or htPLP prior to tillage
 Plots were seeded to spinach 1 day post-inoculation (dpi) and top-
dressed with either CDM, htPLP, or fertilizer according to nitrogen
requirements 35dpi. Soil samples (n=160) were collected on 0-119
and 180 dpi.
 Bacterial Enumeration: Samples (30g) each suspended in 120mL
buffered peptone water were spiral or spread plated onto Sorbitol
MacConkey-rifampicin agar (SMACR); plates were incubated at
42oC. Mini-MPN was used to determine populations when plate
counts were below the detection limit for gEc and attO157. All
MPN-enriched wells were streaked (2µL) onto ChromagarO157 for
confirmation of gEc (blue) and attO157 (mauve) colonies.
 Bacterial presence: For populations undetectable by MPN, sample
enrichments (30g/120mL, at 42oC,24h ) were streaked onto
SMACR to determine presence /absence. Suspect attO157 were
confirmed by latex agglutination.
 Poultry litter pellets top-dressed on soil with or
without prior incorporation of dairy manure solids
inoculated with gEc and attO157 did not increase
persistence of gEc or attO157 substantially compared to
soil amended only with CDM or that top-dressed with
CDM.
 Populations of attO157 decline more rapidly than
those of gEc in soil amended at agronomic rates with
mature (120-day) CDM even with adequate soil
moisture and subsequent top-dressing with CDM, htPLP,
or inorganic fertilizer.
Figure 1A-D. Survival of gEc and attO157 in soil with BSAs (as measured by log
CFU/gdw (gram dry weight): CT, compost tilled; CTTD, compost tilled and compost
top-dressed; CTPPTD, compost tilled and poultry pellets top-dressed; PPTD, poultry
pellets top-dressed; NOTE: B - modified x-axis scale shows early stage population
dynamics /declines; C-Total Daily Rainfall, mm; D- Mean daily ground temperature-⁰C.
, htPLP, or fertilizer.
A randomized complete block (n=4) field experiment conducted in
Beltsville, MD examined survival of non-pathogenic E. coli (gEc) and
attenuated E. coli O157:H7 (attO157) in soil amended with and
without composted dairy manure solids (CDM), and top-dressed
with or without either CDM, htPLP, or inorganic fertilizer.
PURPOSE
-20
-15
-10
-5
0
5
10
15
20
25
30
0 10 20 30 40 50 60 70 80 90 100 110
Days Post-Inoculation
BARC - Maximum and Minumum Temperatures - ⁰C (10/15/14 - 2/11/15)
Maximum Minimum
0
5
10
15
20
25
30
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116
Precipitation(mm)
Rainfall Daily Total
ACKNOWLEDGEMENTS
This research was funded by USDA-ARS-Environmental
Microbial and Food Safety Laboratory project “Microbial
Ecology and Safety of Fresh Produce” and a Specific
Cooperative Agreement between USDA-ARS and the
University of Maryland Eastern Shore, in collaboration
with the U.S. Food and Drug Administration Produce
Safety Staff.
0
1
2
3
4
5
6
0 10 20 30 40 50 60 70 80 90 100 110
Log10CFUorMPN/gdws
generic E. coli in Soil CT
CTTD
CTPPTD
PPTD
A
B
D
C
-1
0
1
2
3
4
5
6
0 1 3 7 9 14 29 48 62 119
Log10CFUorMPN/gdw
Days Post-Inoculation
E. coli O157:H7 in Soil CT
CTTD
CTPPTD
PPTD