70th SWCS International Annual Conference July 26-29, 2015 Greensboro, NC

1. Agricultural Water as a Potential Source of
Bacterial Contamination to Fresh Produce: A
Rainfall Simulation Study
1
Fawzy Hashem, 1
Brett Smith, 1
Tamador Khairi, 1
Arthur Allen,
2
Ray Bryant, and Patricia Millner2
1
University of Maryland Eastern Shore (UMES) and 2
USDA ARS‐
70th
SWCS International Annual Conference
Greensborro, NC
July 26-29, 2015

2. Tomatoes
Celery
Peppers
Cucumber
Melons
Spinach
Lettuce
Cabbage
Kale
Fresh Produce Implicated in Contamination
Outbreaks
 Leafy greens have accounted for 34% of all
contaminated food outbreaks since 1996
 Lettuce linked to more food illness
outbreaks than any other leafy green product
http://www.biodiversityexplo
rer.org/plants/amaranthacea
e/beta_vulgaris.htm
http://www.greenhousetomatoesoutsi
de.com/gghtosp.htm

3. Listeria monocytogenes
E. coli O157:H7: account for 20%
of contamination outbreaks where
the agent was known
Salmonella : account for 30% of
contamination outbreaks
Staphylococcus
Foodborne viruses
Major Microorganisms Involved in Fresh
Produce Contamination

4.  Suspected Field Production Factors (3E’s)
 Environment
 Water: Irrigation, Flooding, Runoff,
 Soil Amendments: Manure and compost
 Vectors: Insects & wildlife
 Dust Particles and Bioaerosols: proximity to
grazed pastures, ranges, feedlots or dairies
 Employees: Hands, gloves, coughs, sanitation
 Equipment: Tractors, implements, harvesting,
containers
Potential Causes of Fresh Produce Contamination
at the Field Level

5. Manure Application to Agricultural Fields
Manure Broadcasting
Manure subsurface
Injection

6. Presence of bacterial and parasitic human pathogens in animal manures
Type of Manure
Pathogen Bovine Avian Porcine Ovine
Equin
e Caprine
Bacteria
Campylobacter spp. + + + + +
Clostridium perfringens + + +
Escherichia coli (shiga-
toxigenic) + +
Escherichia coli O157:H7 + + + + +
Listeria monocytogenes + + + +
Salmonella spp. + + + + + +
Yersinia enterocolitica + +
Parasites
Cryptosporidium spp. + + + +
Giardia + + +
Harris, L. et al. 2013. A Framework for Developing Research Protocols for Evaluation of Microbial Hazards and Controls During Production That
Pertain to the Application of Untreated Soil Amendments of Animal Origin on Land Used to Grow Produce
That May Be Consumed Raw. J Food Prot. 76: 1062-84.

7. Goal and Objectives
Main Goal: To minimize fresh produce contamination
with bacterial pathogens at the farm level.
Objectives: To study avenues leading to fresh produce
Contamination at the farm level:
– Soil amendments: Manure
– Irrigation and runoff water
– Bioaerosol/dust particles generated from farm
operations

8. Survival of enteric pathogens in fields amended
with animal manure
Manure Pathogen Survival Study (MPSS)
• Compare the survival of attenuated (stx-negative) E. coli
O157:H7 to non-pathogenic E. coli in animal manures on
three different sites in the Mid-Atlantic region
• Determine if the 270-day period (FDA proposed rule)
from manure application to harvest of fresh produce is
sufficient to prevent transfer of enteric pathogens to
produce crops

9. Manure Pathogen Survival Study (MPSS)
• Manure Types: Poultry Liter, dairy manure, dairy
liquid, horse manure
• Bacteria:
– Attenuated O157 and non-pathogenic E. coli
strains, which had been isolated from produce-
growing environments (Suslow, UC-Davis)
– Inoculum was prepared in sterile manure extract
• Spread manure (solid or liquid) to conventional or
organic soils, and then spray manure with non-
pathogenic/ O157- E. coli at either low or high
populations
• Use both direct plating and mini-MPN methods to
recover and enumerate E. coli populations

10. Southeast Agricultural
Research & Extension Center
(SEAREC), Manheim, PA
Soil type: Hagerstown silt
loam (SL)
University of Maryland
Eastern Shore, Princess
Anne, MD
Soil type: Othello silt
loam
USDA ARS, Beltsville
Area Research Center,
Beltsville MD
Soil type: Keyport and
Matawan
Organically- and
conventionally-managed
plots
Study sites

11. Non-pathogenic E. coli populations inoculated into manure-
amended soils at low populations (UMES): Summer-Fall 2012
LogCFU(MPN)/gdryweight
2 rain events
between
days 7 and
14 – 0.87 in.
6 rain events
between
days 28 and
57 – 1.97 in
Days
0

12. Attenuated E. coli O157:H7 populations inoculated into
manure-amended soils at low populations (UMES, MD):
Summer-Fall 2012
2 rain events
between days
7 and 14 –
0.87 in.
6 rain events
between days
28 and 57 – 1.97
in
LogCFU(MPN)/gdryweight
Days
E. Coli O157:H7 Low Inoculum

13. Purpose:
To Examine the Microbial Quality of Runoff
Water Collected After Passage Through Manure
Amended Soil
To assess the transport of Salmonella and E. coli
from manure amended soils through runoff water
following rainfall simulation events
Rainfall Simulation Study

14. Rationale and Context
Rainfall stimulated runoff water can be an avenue for
contamination of water sources and fresh produce by
pathogenic microorganisms
Animal manure applied to cropland can harbor
pathogens
When runoff water develops, it carries pathogens from
the original site of application to waterways and crop
fields where pathogens enter the food chain
Human sickness may occur as a result of drinking/use
of contaminated water or consumption of contaminated
fresh produce
Microbial Quality of Runoff Water Collected After
Passage Through Manure Amended Soil

15. Rainfall Simulation Study
Four Rainfall simulation events were conduced over
three years
Rainfall simulation events (40 min @, 7 cm hˉ¹)
• Soil: Silt loam Othello series soil
– Boxes (100x20 x7.5 cm): packed (1.2 g cmˉ³ bulk density) set
at 3% slope,
– 9, 5-mm base drainage holes, 5-cm lip and a collection gutter
on one end, leading to collection vessel
Treatments: Poultry litter, broiler manure, dairy manure,
and NH4SO4 (control) applied to achieve 150 kg PAN ha-1
;
poultry litter ± incorporated after application
5 replicates

16. Rainfall simulation study (cont.)
Soil boxes amended with manure Rainfall simulator
Soil boxes and runoff water collection bottles

17. Schematic of packed soil runoff box. Box with 5 cm lip on the forward end is packed with soil to 5cm
deep. Runoff that spills over the 5 cm lip is collected in an attached gutter that is shielded against
rainfall; 5 mm diameter holes (9) allow water that infiltrates the soil to drain from the boxes and prevent
ponding. A nipple attached near the forward edge of the bottom of the gutter allows runoff water to drain
into funnels and collection bottles positioned below the nipple. (Kibet et al., 2014)

18. Bacterial Analysis
• Runoff water (150 mL) collected from each box was
assayed for the presence of total coliforms, E. coli,
Salmonella, and E. coli O157:H7
• IDEXX Quanti-trays TM /2000 were used to detect the
presence of generic E. coli and total coliforms
100 ml water samples were added to IDEXX Quanti-
trays; trays were incubated at 35 C for 18-24 hrs⁰
 Most Probable Number (MPN/100 mL) for each
sample was reported

19. Materials and Methods (cont.)
• Samples (2,50 µL) were spiral-plated onto Xylose-
Lysine- Tergitol 4 (XLT4) and MacConkey Sorbitol
Agar (CTSMAC) agar to detect the presence or
absence of Salmonella and E. coli O157:H7,
respectively.
• BAX-PCR was used to confirm the identity of
presumptive colonies of Salmonella and E. coli
O157:H7.

20. Materials and Methods(cont)Materials and Methods(cont)
• Most-Probable Number (MPN) approach, IDEXX
Quanti-Tray TM2000 system incubated at 44.5 C forᵒ
18 hrs, was used to determine the presence of generic
E. coli and fecal coliforms.
Preparation of water samples in lab
Darkyellow wells are
positive for
fecal coliforms.
Florescent wells are positive
forgeneric E. coli.

21. Initial concentrations (Log10
CFU/ml) of Salmonella and E.
coli in soil and manure treatments

22. A) Concentrations
(Log10
CFU/ml) of
E. coli in runoff
water (both
rainfall
simulations) from
packed soil boxes
amended with
various
treatments;
B) Concentrations
(Log10
CFU/ml) of
Salmonella in
runoff water
(both rainfall
simulations) from
packed soil boxes
amended with
various
treatments
Means with similar
letters are not
significantly
different

23. A) Concentrations
(Log10
CFU/ml) of
E. coli in runoff
water (both rainfall
simulations);
B) Concentrations
(Log10
CFU/ml) of
Salmonella in runoff
water (both rainfall
simulations)
Means with similar letters are not
significantly different
Results of
Runoff Study 1

24. Runoff Water
Rainfall Simulations
 E. coli and Salmonella were detected in runoff water from
dairy slurry and liquid swine manure amended soils
 E. coli was detected in runoff from poultry compost
amended soil during both rainfall simulations
 Salmonella and E. coli 0157:H7 was never detected in any
runoff water during rainfall simulation 2
 There was a significant effect on total bacteria
concentrations found in runoff water and the type of
treatment applied to soil (p<0.001 for E. coli and p<0.01
for Salmonella, respectively)
Results

25. Conclusion
•Runoff water from all treatments showed the presence of
total coliforms and generic E. coil, but their concentrations
varied among treatments.
•Salmonella and E. coli O157: H7 were not detected in any
treatments.
•Concentrations of generic E. coli in broadcast poultry
litter and dairy manure were significant in rainfall event 2.

26. Pathogen movement by runoff water is highly
capable of occurring, resulting in contamination of
waterways or entrance into the food chain
Pathogen loads carried by surface water are higher
immediately after manure is applied to land
Pathogens can be retained in soil
Manure type influences the number of pathogens
that are available for movement by runoff water
Conclusions

27. Acknowledgements
USDA-ARS Collaborators: Millner, Sharma &
Bryant
Funding Agencies:
USDA-NIFA
US-FDA
USDA-ARS
UMES Ag Exp. Station and Mr. Earle Canter