1. University of Maryland College Park School of Public Health
Animal Feed Components and The Impacts of Land-applying Manure on Soil Bacterial Biodiversity and Antibiotic Resistance
Amy R. Sapkota, PhD, MPH
University of Maryland College Park School of Public Health

2. Background
Animal-based food products constitute a large proportion of the U.S. diet
U.S. per capita consumption of total meats is 90.5 kg/year (199 lbs/year) (USDA, 2005)
Quality of food products is directly related to animal feeding practices
Farms and feeding practices have undergone changes
Small family farms  Concentrated animal feeding operations
Corn or grass diet  Complex mixtures
U.S. feed industry has grown to be the largest feed producer in the world
>120 million tons of feed
$25 billion industry
Total meats includes: beef, pork, veal, lamb, poultry, fish, shellfish
Source: USDA,

3. What is in Animal Feed and Does it Affect Human Health?
Previously, no comprehensive, peer-reviewed source existed regarding specific feed ingredients and their potential impacts on human health
Existing sources focused on one feed contaminant and one health effect

4. Methods We reviewed the literature and FDA, USDA, and CDC databases:
To summarize existing data on current feed production practices and feed ingredients
To describe biological, chemical and other etiologic agents that have been detected in feed
To evaluate evidence whether current feeding practices may lead to adverse human health effects
To identify data gaps that prevent comprehensive assessments of human health risks associated with feed
Compiled a review article targeted to public health researchers

5. What is in animal feed?
Thousands of feed ingredients are used to make animal feed
Source: Sapkota et al EHP 115;5:

6. Etiologic Agents Detected in Feed and Potential Human Health Effects
Source: Sapkota et al EHP 115;5:

7. From Animal Feed Ingredients to Human Health

8. We need increased federal funding and surveillance from “farm to fork”
Take-home Messages
No comprehensive nationwide animal feed surveillance system to monitor:
The amounts and types of specific feed ingredients
The levels of contaminants in feeds and food products
Human health effects data are not appropriately linked to the minimal amount of surveillance data that do exist
Difficult to pinpoint the extent to which human health risks are associated with animal feed
….including rendered animals, animal waste, antibiotics, metals and fats…
This was published in EHP, and now for the first time this data can be accessed in the peer-reviewed literature
We need increased federal funding and surveillance from “farm to fork”

9. The Impacts of Land-applying Manure on Soil Bacterial Biodiversity and Antibiotic Resistance

10. Background
500 million to 1 billion tons of animal manure is land-applied each year
Manure contains nutrients and contaminants, including antibiotic residues and resistant bacteria
When swine manure is land-applied, resistance genes can be transferred between swine-associated bacteria and soil bacteria, perpetuating the spread of resistance (Agerso 2005; Jensen 2002)
We hypothesized that antibiotic selective pressures resulting from the land-application of swine manure could modify soil bacterial biodiversity as well
Sources: Agerso et al Appl Env Microbiol 71: ; Jensen et al Env Int 28:

11. Study Objectives
To use traditional culture techniques and metagenomic methods to explore antibiotic resistance and bacterial diversity in agricultural soils amended with swine manure compared to control soils

12. Methods Spectinomycin Spectinomycin = Aminocyclitol antibiotic
Sapkota et al. [In preparation].

13. Methods: Sample Analysis
Culture techniques:
Culturable bacteria (susceptible and resistant) were isolated from each sample and identified by PCR and sequencing
Isolates were tested for the aad(A) resistance gene and the resulting genes were sequenced
Metagenomic methods:
Total DNA was extracted from each sample
16S rRNA genes in each sample were amplified, purified, and labeled using an in vitro transcription method
RNA was hybridized to a 16S rRNA-based taxonomic microarray
Microarray results were analyzed by principal components analysis
The aad(A) resistance gene was quantified using real-time PCR, cloned and sequenced

14. Bacterial Biodiversity in Culturable Isolates
Sapkota et al. [In preparation].

15. Bacterial Biodiversity in Metagenomic DNA Samples
Metagenomic microarray approach revealed more soil biodiversity, in general, and more differences between manure-amended and control soils.
Sapkota et al. [In preparation].

16. Spectinomycin Resistance in Culturable Isolates
The aad(A) resistance gene was only detected in isolates from manure samples and soils impacted by manure.

17. aad(A) Resistance Gene in Metagenomic DNA Samples
Similar to results observed in culturable isolates
Higher copy numbers of the aad(A) resistance gene were in soils impacted by manure
Currently, we are analyzing all aad(A) resistance gene sequences from culturable bacteria and metagenomic samples to understand diversity in the gene
Samples
Standards NC
Is the gene similar in all samples or does it exhibit genetic diversity?

18. Conclusions
Microarray results indicate that some differences exist in bacterial biodiversity between soils amended with swine manure and control soils
Spectinomycin-resistant bacteria were isolated only from manure samples and soils recovered from pig grazing fields
The aad(A) resistance gene was only detected in manure samples and soils impacted by manure
The application of swine manure to soils impacts both antibiotic resistance and bacterial biodiversity in soil
This could be attributed to a variety of factors, including antibiotic selective pressures and the addition higher levels of nutrients to soil bacterial communities.

19. Acknowledgements
Funding: Center for a Livable Future, Johns Hopkins Bloomberg School of Public Health
École Centrale de Lyon Colleagues: Timothy Vogel, Pascal Simonet, Elizabeth Navarro, Maude David, Jean-Michel Monier, Audra Nemir, Benoit Remenant, Saliou Fall, Sandrine Demaneche
JHU Colleagues: Kellogg Schwab, Ellen Silbergeld, Robert Lawrence, Shawn McKenzie, Polly Walker, Lisa Lefferts