1. Biological Soil Amendments of Animal Origin: Pathogen Persistence
2/12/19
Keith R. Schneider, Ph.D.
Food Science and Human Nutrition
Gainesville, FL 32611

2. Biological Soil Amendments of Animal Origin
Currently we’ve completed three concurrent studies
Two studies looked at Salmonella content in chicken litter
The first in conjunction with UC Davis, Univ of Ariz, Univ of Delaware and UF (M. Danyluk)
The second is an independent, UF-only study looking at extrinsic factors affecting survival
A third study looked at presence of pathogen E. coli in bovine manure (also with UCD, UA and UD)

3. Biological Soil Amendments of Animal Origin (BSAAO)
Expect further guidance
Raw manure
To be further studied...
*90/120 NOP standard
Treated
Physical/chemical process (validated)
Composting (prescribed conditions)
No time interval, BUT:
Must meet microbial standard

4. Poultry Litter Survey
Poultry houses in the United States produce 10.2 million tons of litter each year. This high nitrogen containing by-product is frequently applied to agricultural lands as an alternative to synthetic fertilizers
Pictures from Danyluk Lab

5. Poultry Litter Survey 1
Poultry litter was collected from 13 broiler and breeder farms in Florida (2), Georgia (2), Alabama (7), and Texas (2).
Work from Danyluk Lab

6. Poultry Litter Survey
Work from Danyluk Lab

7. Summary of Study 1
490 poultry litter samples were collected from 13 farms.
Salmonella was detected in 34 (6.9%) of the total samples collected
Six of the 13 farms from which litter was sampled tested positive for Salmonella
More farms visited in the fall season (50%, 6/12) tested positive for Salmonella than farms sampled in the Spring (9%, 1/11)
The prevalence of Salmonella in spring samples (4.8%) was lower than in the fall season (8.9%)

8. Chicken Litter Survey – Study 2
Untreated chicken litter sampled from 18 farms in North Florida
Each location sampled three separate times (n=54)
Samples represent partial cleanout (removal of caked litter) after a flock has been removed
Goal of this study was to look into factors that affect Salmonella survival
Work from Schneider Lab

9. Chicken Litter Survey – Parameters Tested
Microbial
Chemical
Physical
Total aerobic plate count (APC) pH
Moisture Content
Salmonella MPN
Nitrogen
Total Ash
Ammonia Nitrogen
Total Solids
Phosphorus (P2O5)
Organic Matter
Potassium (K2O)
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10. Chicken Litter Survey – Microbiology Results
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11. Chicken Litter Survey – Microbiology Results
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12. Chicken Litter Survey – Chemical and Physical Results
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13. Chicken Litter Survey – Chemical and Physical Results
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14. Chicken Litter Survey – Microbiology Results
Salmonella Concentration in Chicken Litter Samples (n = 54)
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15. Chicken Litter Survey – Salmonella Serotyping
Subset of representative Salmonella isolates sent for serotyping (n=47)
12 unique Salmonella serotypes were identified
Most common serotypes:
Typhimurium - 13
Kentucky - 8
Enteritidis - 7
Mbandaka - 7

16. Chicken Litter Survey – Antimicrobial Testing
The only resistances identified were to tetracycline (14 isolates), sulfisoxazole (11 isolates), and streptomycin (7 isolates)
18/47 (38%) isolates were resistant to at least 1 antimicrobial agent
14/47 (30%) isolates were resistant to 2 antimicrobials
11 were resistant to sulfisoxazole and tetracycline
3 were resistant to streptomycin and tetracycline
4/47 (9%) isolates were resistant to 1 antimicrobial
These isolates were all resistant to streptomycin
None were resistance to more than 2 antimicrobials
7/47 (15%) isolates showed intermediate sensitivity to 1 antimicrobial agent
1/47 (2%) isolate showed intermediate sensitivity to 1 antimicrobial agent AND resistance to 1 antimicrobial agent
23/47 (49%) isolates were sensitive to all antimicrobial agents tested

17. Chicken Litter Survey – Summary Study 2
Salmonella was recovered from 18/18 (100%) farms and 33/54 (61%) of the litter samples collected.
Significant (p<0.02) seasonal variation was found among APC counts, with Spring samples having the highest mean APC (9.2 log CFU/g).
The average APC from all litter samples was 8.8 log CFU/g (Table 1).
None of the microbial, chemical, or physical parameters measured were significant predictors for Salmonella MPN/g in chicken litter.
Work from Schneider Lab

18. Bovine Manure Survey

19. STEC - Bovine Manure Pathogen Survey
539 total samples
13 farms
3 piles per farm
7 samples per pile
Two visits per farm (fall/winter, spring/summer) (one exception)
Florida farms (n=12) Texas farm (n=1)
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20. Summary of stx1 and/or stx2 positive samples
Farm Visited
Storage Locations†
Samples
Sampled 25 77
539
Positive 18 29
49^
% Positive
     72%*
37% 9%
* 12 of 13 farms were stx1 and/or stx2 positive (92%)
^ 23 samples were found positive initially, and 26 samples were identified after screening rfbE positive samples for stx1/stx2
† Storage locations (screened solids, barn, lagoon, pile)
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Table 1. Manure Pathogen Survey summary stx1 and/or stx2 positives
Farm Visits
Storage Locations†
Samples (7 per location)
Sampled 25 77
539
Positive 18 29
49^
% Positive
72%*
37% 9%
Table 1. Manure Pathogen Survey summary stx1 and/or stx2 positives
Farm Visits
Storage Locations†
Samples (7 per location)
Sampled 25 77
539
Positive 18 29
49^
% Positive
72%*
37% 9%

21. Confirmed E. coli O157 Presumptive Positive Samples by qPCR targeting the rfbE gene
Farm Visited
Storage Locations†
Samples
Sampled 25 77
539
Positive
 23* 43 97
% Positive
 92%
 56%
  18%
* All 13 farms were positive for rfbE
† Storage locations (screened solids, barn, lagoon, pile)
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22. Prevalence of stx-positive and rfbE-positive samples based on manure storage type.
Pile Sample Type*
Screened Solids (n=84)
Barn (n=112)
Lagoon (n=126)
Pile (n=217)
Surface
(n=81)
Internal (n=199)
stx + 7 8% 14
13%
11% 6% 5 13 7%
rfbE + 10 9% 34
27% 46
21% 16
20% 37
19%
*There were instances of barn storage being classified as a internal sample if the manure was piled high enough to take an internal sample from the pile. There was a significant difference in rfbE-presence and storage type (P<0.05). No significant difference was observed between stx-presence and storage type (P>0.05).
The eaeA gene codes for Escherichia coli strains
The rfbE codes for O157:H7
Table 3. Prevalence of stx-positive and rfbE-positive samples based on manure storage type.
Positive sample
Manure Storage Type
Pile Sample Type*
Screened Solids (n=84)
Barn (n=112)
Lagoon (n=126)
Pile (n=217)
Toe
(n=81)
Gut (n=199)
stx + 7 8% 14
13%
11% 6% 5 13 7%
rfbE + 10 9% 34
27% 46
21% 16
20% 37
19%
*There were instances of barn storage being classified as a gut sample if the manure was piled high enough to take an internal sample from the pile. There was a significant difference in rfbE presence and storage type (P<0.05). No significant difference between stx-presence and storage type was noted (P>0.05).
Table 3. Prevalence of stx-positive and rfbE-positive samples based on manure storage type.
Positive sample
Manure Storage Type
Pile Sample Type*
Screened Solids (n=84)
Barn (n=112)
Lagoon (n=126)
Pile (n=217)
Toe
(n=81)
Gut (n=199)
stx + 7 8% 14
13%
11% 6% 5 13 7%
rfbE + 10 9% 34
27% 46
21% 16
20% 37
19%
*There were instances of barn storage being classified as a gut sample if the manure was piled high enough to take an internal sample from the pile. There was a significant difference in rfbE presence and storage type (P<0.05). No significant difference between stx-presence and storage type was noted (P>0.05).

23. Estimated concentration (MPN/g) of STEC in stx1/2 positive manure samples.
Work from Schneider Lab

24. STEC Survival in Soil
Main factors for survival in soils
-Microbial community
-Nutrient availability
-Soil structure
-Temperature
Problem: FDA’s harvest interval for soil amendments is currently reserved
Altered microbial communities increases pathogen survival
Florida soil – high sand, low clay content
Future research: how is survival influenced by soil particle size and organic matter?
Autoclaved soil
Natural soil
Unfortunately for consumers, STEC is a hot topic.. Shiga toxin producing E. coli. Remember the recent romaine lettuce nationwide outbreak from Yuma AZ? Same bug. Thought to be from irrigation water contaminated from feedlot in close proximity.
STEC survives for weeks, to months in soils, and even for up to a year in some studies. Problematic because it only takes a few cells to make someone sick.
- Our previous research involved a large prevalence survey in manure across FL, other Universities (Delaware, UC Davis, Arizona) doing the same thing. About 20% of samples had E. coli O157.
- Now doing lab studies to figure out which natural microbes can reduce survival times, also how organic matter influences survival. So far, we have seen when we autoclave soil we get rid of certain microbes, which increases survival substantially. Now the goal is to figure out what specific microbes in the natural soil that lowers survival times.
In the survival graph, you see the concentration of bacteria on the y axis, and survival (in days) on the x axis. You can see that STEC survives much longer in the autoclaved soil. This is at 30C
Future research focused on organic matter levels and soil particle size. Complicated questions, soil is the most diverse ecosystem on Earth.

25. STEC Survival in Soil Autoclaved soil Natural soil
Unfortunately for consumers, STEC is a hot topic.. Shiga toxin producing E. coli. Remember the recent romaine lettuce nationwide outbreak from Yuma AZ? Same bug. Thought to be from irrigation water contaminated from feedlot in close proximity.
STEC survives for weeks, to months in soils, and even for up to a year in some studies. Problematic because it only takes a few cells to make someone sick.
- Our previous research involved a large prevalence survey in manure across FL, other Universities (Delaware, UC Davis, Arizona) doing the same thing. About 20% of samples had E. coli O157.
- Now doing lab studies to figure out which natural microbes can reduce survival times, also how organic matter influences survival. So far, we have seen when we autoclave soil we get rid of certain microbes, which increases survival substantially. Now the goal is to figure out what specific microbes in the natural soil that lowers survival times.
In the survival graph, you see the concentration of bacteria on the y axis, and survival (in days) on the x axis. You can see that STEC survives much longer in the autoclaved soil. This is at 30C
Future research focused on organic matter levels and soil particle size. Complicated questions, soil is the most diverse ecosystem on Earth.

26. Summary
We’re finding pathogenic microorganisms routinely in raw manure
While present, the numbers are typically low.
While FDA hasn’t determined a waiting period yet, the current USDA standard for organic production (90/120 days) still seems like a good recommendation.
Even thought the numbers are low, nothing substitutes for proper composting and handling of any raw manure and litter.

27. Questions