Agricultural Water as a Potential Source of Bacterial Contamination to Fresh Produce: A

Rainfall Simulation Study

1Fawzy Hashem, 1Brett Smith, 1Tamador Khairi, 1Arthur Allen, 2Ray Bryant, and Patricia Millner2

1University of Maryland Eastern Shore (UMES) and 2USDA‐ARS

70th SWCS International Annual ConferenceGreensborro, NCJuly 26-29, 2015

TomatoesCeleryPeppersCucumberMelons

SpinachLettuceCabbageKale

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.biodiversityexplore

r.org/plants/amaranthaceae/

beta_vulgaris.htm

http://www.greenhousetomatoesoutsi

de.com/gghtosp.htm

Listeria monocytogenesE. coli O157:H7: account for 20%

of contamination outbreaks where the agent was known

Salmonella : account for 30% of contamination outbreaks

StaphylococcusFoodborne viruses

Major Microorganisms Involved in Fresh Produce Contamination

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

Manure Application to Agricultural Fields

Manure Broadcasting

Manure subsurface Injection

Presence of bacterial and parasitic human pathogens in animal manures

Type of Manure

Pathogen Bovine Avian Porcine OvineEquin

e CaprineBacteria 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.

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

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

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

Southeast Agricultural Research & Extension Center

(SEAREC), Manheim, PA Soil type: Hagerstown silt

loam (SL)

University of Maryland Eastern Shore, Princess

Anne, MDSoil type: Othello silt

loam

USDA ARS, Beltsville Area Research Center,

Beltsville MDSoil type: Keyport and

Matawan Organically- and

conventionally-managed plots

Study sites

Non-pathogenic E. coli populations inoculated into manure-amended soils at low populations (UMES): Summer-Fall 2012

Lo

g C

FU

(M

PN

)/g

dry

wei

gh

t

2 rain events between

days 7 and 14 – 0.87 in.

6 rain events between

days 28 and 57 – 1.97 in

Days

0

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

Lo

g C

FU

(M

PN

)/g

dry

wei

gh

t

Days

E. Coli O157:H7 Low Inoculum

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

Rationale and ContextRainfall 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

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 application5 replicates

Rainfall simulation study (cont.)

Soil boxes amended with manure Rainfall simulator

Soil boxes and runoff water collection bottles

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)

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

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.

Materials and Methods(cont)Materials and Methods(cont)

• Most-Probable Number (MPN) approach, IDEXX Quanti-Tray TM2000 system incubated at 44.5 ᵒC for 18 hrs, was used to determine the presence of generic E. coli and fecal coliforms.

Preparation of water samples in labDark yellow wells are positive for fecal coliforms.

Florescent wells are positive for generic E. coli.

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

treatments

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

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

Runoff WaterRainfall 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

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.

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 landPathogens can be retained in soil

Manure type influences the number of pathogens that are available for movement by runoff water

Conclusions

Acknowledgements

USDA-ARS Collaborators: Millner, Sharma & Bryant

Funding Agencies:

USDA-NIFA

US-FDA

USDA-ARS

UMES Ag Exp. Station and Mr. Earle Canter