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Volume 3, Issue 12013

ISSN: 2159-8967www.AFABjournal.com

2 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 1 - 2013

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 1 - 2013 3

Sooyoun Ahn University of Florida, USA

Walid Q. AlaliUniversity of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Debabrata BiswasUniversity of Maryland, USA

Claudia S. Dunkley University of Georgia, USA

Lawrence GoodridgeColorado State University, USA

Leluo GuanUniversity of Alberta, Canada

Joshua GurtlerERRC, USDA-ARS, USA

Yong D. HangCornell University, USA

Divya JaroniOklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael JohnsonUniversity of Arkansas, USA

Timothy KellyEast Carolina University, USA

William R. KenealyMascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

W.K. KimUniversity of Manitoba, Canada

M.B. KirkhamKansas State University, USA

Todd KostmanUniversity of Wisconsin, Oshkosh, USA

Y.M. Kwon University of Arkansas, USA

Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain

Melanie R. MormileMissouri University of Science and Tech., USA

Rama NannapaneniMississippi State University, USA

Jack A. Neal, Jr.University of Houston, USA

Benedict OkekeAuburn University at Montgomery, USA

John PattersonPurdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos RostagnoLBRU, USDA-ARS, USA

Roni ShapiraHebrew University of Jerusalem, Israel

Kalidas ShettyNorth Dakota State University, USA

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MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium

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Shiga Toxin-Producing Escherichia coli (STEC) Ecology in Cattle and Management Based Options for Reducing Fecal Shedding T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, D. J. Nisbet

39

Can Salmonella Reside in the Human Oral Cavity?S. A. Sirsat

30

Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration

R. S. Pinder, and J. A. Patterson

6

Independent Poultry Processing in Georgia: Survey of Producers’ PerspectiveE. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, S. C. Ricke

70

ARTICLES

Greenhouse Gas Emissions from Livestock and PoultryC. S. Dunkley and K. D. Dunkley

17

REVIEW

Instructions for Authors79

Introduction to Authors

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TABLE OF CONTENTS


www.afabjournal.comCopyright © 2013

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Acetogens have only been isolated in low numbers from ruminal contents, even though the majority

of acetogens isolated from ruminal contents are capable of utilizing both H2 and soluble carbohydrates

present in ruminal fluid (e.g. glucose and cellobiose). The much higher methanogenic affinity for hydro-

gen has been suggested to determine the prevalence of methanogens over acetogens in many ecosys-

tems, suggesting that other environmental factors determine the number of acetogens present in ruminal

fluid. We report the effects of carbohydrate concentration, pH and acetate concentration on the growth of

two ruminal acetogenic isolates (A10 and H3HH). The minimum amount of glucose necessary for growth

(threshold) of A10 (111 μM) and H3HH (56 μM) was greater than the glucose concentration previously de-

tected in bovine ruminal fluid (8-17 μM). However, the threshold of H3HH on cellobiose (14 μM) was much

lower than the actual concentration previously detected in ruminal fluid (110-175 μM), suggesting that this

organism could survive in the rumen using cellobiose as an energy source. Isolate A10 had a sufficiently

high threshold for cellobiose (139 μM) to suggest that, at least for certain periods, the concentration of cel-

lobiose in ruminal contents could be too low to support growth of this isolate. The growth rate of isolate

A10 was decreased by 50 % when the pH of the growth medium was lowered from 6.6 to 5.5. A similar de-

crease in growth rate was observed with isolate H3HH. Increasing the acetate concentration of the growth

medium decreased the growth of both isolates as well. Moreover, the effect of high acetate concentration

was more discernible at lower pH. The present results suggest that pH and volatile fatty acid concentration

may be key factors limiting the growth of acetogens isolated from ruminal contents.

Keywords: acetogens, rumen, hydrogen, pH, carbohydrate concentration

Correspondence: J. A. Patterson, [email protected] Tel: +1 -765-494-4826 Fax: +1-765-494-9347

Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration

R. S.Pinder1,2 and J. A. Patterson1

1Animal Science Dept, Purdue University, West Lafayette, IN 47907-10262Current address; 7855 South 600 East, Brownsburg, IN 46112

Agric. Food Anal. Bacteriol. 3: 6-16, 2013


INTRODUCTION

Hydrogen produced during microbial degrada-

tion of cellulose in ruminal contents must be elimi-

nated in order to maintain the appropriate condi-

tions for efficient conversion of cellulose to volatile

fatty acids (Zinder, 1993). Normally, methanogens

remove H2 from the rumen through methane produc-

tion from H2/CO2. However, substantial interest ex-

ists to reduce methane emissions from ruminants to

minimize this contribution to global warming. One

potential approach is to use acetogenic bacteria to

produce acetate as an alternate H2 sink. Acetogens

are a diverse group of organisms that are capable

of utilizing H2/CO2 to form acetate, a product use-

ful to the host animal. However, acetogens are typi-

cally isolated in low numbers from ruminal contents

(Morvan et al., 1994; LeVan et al., 1998) despite be-

ing capable of utilizing some of the carbohydrates

glucose, cellobiose) present in ruminal fluid (Bocca-

zzi and Patterson, 2011; Genthner et al., 1981). This

apparent contradiction suggests that other ruminal

environmental factors limit the growth of acetogens

in ruminal contents. Several possibilities exist, in-

cluding: 1) chemical factors (pH and volatile fatty

acid concentration) which interfere with the growth

of acetogens; 2) acetogens are not capable of uti-

lizing carbohydrates at the concentration present in

ruminal fluid; 3) acetogens are not capable of grow-

ing at a pace fast enough to avoid being washed out

of rumen contents; 4) acetogens cannot compete

with methanogens for H2/CO2.

The goal of the research presented herein was to

determine 1) the minimum concentration of glucose,

maltose and cellobiose needed for growth of aceto-

gens, 2 ) the growth rates of acetogens, and 3) the

influence of volatile fatty acid concentrations and pH

on growth of acetogens. We report that the growth

of isolates A10 and H3HH, two acetogenic bacteria

isolated and characterized from ruminal contents

(Boccazzi and Patterson, 2011; Pinder and Patterson,

2011, 2012) may be affected by the carbohydrate

concentrations found in ruminal fluid but that growth

of these organisms is severely influenced by the com-

bination of low pH and high acetate concentration.

MATERIALS AND METHODS

Organisms

Isolates A10 and H3HH, previously described

ruminal isolates (Boccazzi and Patterson, 2011; Pin-

der and Patterson, 2011, 2012) were used in all the

experiments described herein. These organisms

were maintained in acetogenic medium.

Growth medium

The acetogen medium of Boccazzi and Patterson

(2011) was used except where indicated otherwise.

In experiments where the initial concentration of car-

bohydrates was the treatment tested, ruminal fluid

was omitted to reduce the amount of carbohydrates

present in basal medium. In experiments where the

initial pH of the medium was one of the treatments

imposed, the composition of the medium was modi-

fied by eliminating the Na2CO3 which buffered the

medium too strongly for reliable pH adjustment be-

low 6.5. In addition, the medium was gassed with

oxygen-free 100 % N2 rather than 100 % CO2, during

preparation and dispensing. Preliminary experiments

showed that the buffering capacity of this modified

medium was sufficient for short-term (less than 12 h)

experiments with low (0.05 g/L) amounts of carbohy-

drate substrate.

Batch cultures

Glucose, maltose or cellobiose (from 0.2 μ-filter

sterilized stock solutions) were added as indicated to

tubes containing 10 mL of acetogen medium. Tubes

were inoculated with 0.1 mL of an overnight culture,

measured for the initial OD (absorbance at 660 nm)

and placed in a 37°C water bath. The contents of the

tubes were mixed and the OD of the tubes measured

and recorded at intervals as shown. For experiments

where the initial carbohydrate concentration was the

treatment regimen imposed, the growth rate (at each

carbohydrate concentration) was determined and

the threshold of carbohydrate utilization was estimat-

ed as the point where growth rate was greater than


baseline (without added carbohydrates). For experi-

ments where the pH and acetate concentration of the

media were the treatments imposed, growth rates of

the isolates was estimated by plotting the OD values

for each culture and determining the apparent maxi-

mum growth rate from the steepest portion of the

growth curve.

Continuous cultures

An anaerobic continuous culture system as de-

scribed by Boccazzi and Patterson, (2011) was used.

The growth medium consisted of acetogen medium

supplemented with 1.4 mM glucose. The medium

reservoir and growth vessels were gassed with 100

% CO2 at a flow of approximately 50 mL min-1. After

the system was reduced, the growth vessels (operat-

ing capacity; approximately 215 mL) were inoculated

with 20 mL of an overnight culture. The bacterial

culture was allowed to grow as a batch culture for

6 h after which the medium flow was initiated. The

cultures were allowed at least ten turnovers to reach

steady state conditions before sampling. Turnover

rate was determined by collecting the outflow from

the growth vessels. Sample collection was accom-

plished by diverting the outflow from the growth

vessels for 10 min into ice-cooled collecting vessels.

Aliquots of the outflow were analyzed for volatile

fatty acids (VFA), glucose and bacterial DM as de-

scribed below. Concentrations of volatile fatty acids

in the culture supernatant were determined using a

Hewlett Packard 5890 gas chromatograph (Hewlett

Packard Co., Palo Alto, CA) fitted with a glass column

packed with GP 60/80 carbopack C / 0.3% Carbowax

M / 0.1 % H3PO4 (Supelco, Inc.; Bellefonte, PA). The

injector and detector temperatures were set at 200°C

while the column temperature was set at 135°C. For-

mate concentrations in the culture supernatant were

determined using a formate dehydrogenase assay

(Schaller and Triebig, 1983). The pH of the culture

was measured immediately after sampling for vola-

tile fatty acids with an Ag combination electrode

connected to a Fisher Accumet pH meter (Fisher Sci-

entific, Pittsburg, PA). Glucose concentration in the

culture medium was assayed with a glucose oxidase

kit (Sigma Chemical Co., St. Louis, MO). The initial

glucose concentration was determined on uninocu-

lated control bottles.

After sampling, the media flow to the growth ves-

sels was adjusted to obtain the next dilution rate.

RESULTS

Effect of dilution rate on growth of iso-late A10 and H3HH

Previously, we determined the maximum growth

rate of isolate A10 to be approximately 0.47 h-1 (Pin-

der and Patterson, 2012). Both isolate A10 and isolate

H3HH attached to the surfaces of the growth vessels,

perhaps due to extracellular polysaccharide. The

net result was that apparent maximum growth rates

in continuous culture approached 1.2 h-1 (Figure 1),

This is much higher than the maximum growth rate

estimated in batch culture and is probably a result of

the wall growth continuously inculating the medium.

Similar problems have been reported for Eubacteri-

um limosum, an acetogen also isolated from ruminal

contents, which produces copious amounts of extra-

cellular polysaccharide (Loubiere et al., 1992). Nev-

ertheless, the batch culture experiments provided

sufficient information to obtain an estimate of the

maximum growth rate of isolate H3HH. The highest

growth rate (approximately 0.6 h-1) for isolate H3HH

was obtained when this organism was grown on glu-

cose (42 mM). The maximum growth rate observed

for isolate A10 was approximately 0.5 h-1 in agree-

ment with previous experiments.

Minimum carbohydrate concentration necessary for growth

Boccazzi and Patterson (2011) described the array

of carbohydrates utilized by isolates A10 and H3HH.

The purpose of this series of experiments was to as-

sess the minimum concentration of carbohydrate

necessary for growth of isolates A10 and H3HH. The

approach used in the present experiments was to

determine the growth rate of isolates A10 and H3HH


Figure 1. Continuous culture of isolate H3HH. A continuous culture system was established as previously described The system was inoculated (approx. 10 % v/v) with an overnight culture and allowed at least ten turnovers to reach steady state conditions. An aliquot of the outflow was collected to determine bacterial mass (●) and acetate content (□). After sampling the media flow rate was adjusted and the cultures were allowed to reach steady state conditions again.


Figure 2. Influence of carbohydrate concentration on the growth rate of isolate H3HH in batch culture. Acetogen media (10 mL) containing various amounts of either glucose (♦), cellobiose (■) or maltose (●) was inoculated (0.1 mL) with an overnight culture of isolate H3HH. The OD (absorbance at 660 nm) was measured at appropriate time intervals. The growth curve of each culture was plotted and the highest growth rate at each carbohydrate concentration was deter-mined. The growth rate not attributable to the carbohydrates added (i.e., growth rate of cultures without added carbohydrates) was subtracted from the other growth rates. The vertical lines drawn on the graph represent the range of glucose and cellobiose concentrations detected in ruminal fluid. Each point is the mean of at least two (but usually three) tubes.


in acetogen medium containing concentrations of

glucose, maltose and cellobiose that ranged from

very low (10 μM, as found in the rumen) to very high

(> 1 mM, as used in substrate utilization assays). The

apparent maximum growth rates obtained at each

carbohydrate concentration were plotted as a func-

tion of the carbohydrate concentration present at

inoculation. The minimum carbohydrate concen-

tration necessary for growth was estimated as the

carbohydrate concentration below which the growth

rate of the cultures was similar to that of cultures

without added carbohydrate. Using this definition,

H3HH had a lower minimum threshold for maltose

(14 μM) and cellobiose (14 μM) than for glucose

(111 μM) (Figure 2). In contrast, isolate A10 had a

lower minimum threshold for maltose (28 μM) than

for glucose (56 μM) or cellobiose (139 μM) (Figure

3). Furthermore, the concentration of carbohydrates

needed for maximal growth rate of isolate A10 was

one magnitude less than the concentrations needed

by isolate H3HH (1 versus 10 mM).

Effect of pH and acetate concentration on growth

Factors other than growth rate, minimum con-

centration of carbohydrates needed to start growth

may also be involved in maintaining relatively low

numbers of acetogens in rumen contents. Physical

parameters (pH and VFA) have been implicated in

inhibiting growth of a number of bacterial species,

thus, the effects of pH and VFA concentration on the

growth rates of isolates A10 and H3HH were deter-

mined. Because of the growth on the wall of the

growth vessel, continuous culture data was not reli-

able, and only batch culture data is presented. Iso-

late A10 and isolate H3HH were grown in acetogen

medium without ruminal fluid, but with sodium ac-

etate (0, 50 or 100 mM) and initial pH adjusted to 6.6

or 5.5. Ruminal fluid was omitted from the medium

to reduce the basal acetate concentration from ap-

proximately 2 mM to less than 0.1 mM. Both isolate

A10 and isolate H3HH were affected by the initial

pH and acetate concentration of the medium (Table

1). At neutral pH (6.6), there was a small decrease in

growth rate with increasing acetate concentrations.

However, the growth rate of both organisms at pH

5.5 was approximately half of that obtained at pH

6.6 when no acetate was added to the medium. An

Table 1. Growth rate of isolates A10 and H3HH as affected by pH and acetate concentrations1

H3HH A10

Acetate pH 6.6 pH 5.5 pH 6.6 pH 5.5

0 mM 0.309 0.119 0.279 0.126

50 mM 0.260 0.014 0.270 0.023

100 mM 0.278 0.006 0.259 0.005


Figure 3. Influence of initial carbohydrate concentration on the growth rate of isolate A10 in batch culture. Please refer to the legend of Figure 2 for details of this experiment.


initial acetate concentration of 50 mM was sufficient

to inhibit the growth of both organisms when the pH

of the media was 5.5, and no growth occurred at pH

5.5 when initial acetate concentration was 100 mM.

DISCUSSION

The rumen evolved to provide the appropriate

conditions for microbial degradation of cellulose

and other plant structural carbohydrates (Weimer et

al., 2009). Accordingly, a number of anaerobic, car-

bohydrate-fermenting microbial species grow well

and can be isolated in high numbers from ruminal

fluid (Hungate, 1966; Oshio et al., 1987; Ricke et

al., 1996). However, the rumen is not a hospitable

environment for all microbes. The anaerobiosis, liq-

uid turnover rate, concentration of soluble carbohy-

drates and the chemical (i.e., pH and VFA concentra-

tion) characteristics of the rumen prevent the growth

of a number of bacteria that otherwise should be

able to grow on the substrates in rumen fluid. The

rumen is a highly reduced (Eh = -150 to -350 mV) an-

aerobic environment (Clarke, 1977) and oxygen in-

troduced into ruminal contents through feeding and

drinking is rapidly depleted by facultatively anaero-

bic organisms (Ellis et al., 1989). Many bacterial spe-

cies are unable to tolerate the reduced and anaero-

bic character of ruminal contents. This anaerobic

ecosystem provides the host animal with VFA, which

it can utilize as an energy source (Lin and Luchi, 1991;

Lindley et al., 1990). Although the acetogens used

in these experiments are somewhat tolerant to aero-

bic conditions (Boccazzi and Patterson, 2011), as a

group, acetogens isolated from ruminal contents are

obligate anaerobes.

The turnover rate of rumen contents also can af-

fect which organisms colonize and persist in the ru-

men. For example, fatty acid-degrading bacteria

(e.g., Anerovibrio lipoliticus) have an ample supply

of substrates in ruminal contents. However, these

organisms are unable to propagate in high numbers

in ruminal contents because their maximum growth

rate is less than the liquid turnover rate of ruminal

contents (Hungate, 1966). The result is that VFA are

available for ruminant utilization. The present results

show that maximum growth rate of isolate A10 or

H3HH would not limit establishment of high num-

bers in ruminal contents. Although we were unable

to obtain reliable data from continuous culture ex-

periments, data obtained from batch culture experi-

ments indicated that the maximum growth rate of

these organisms was approximately 0.6 h-1 for isolate

A10 and 0.5 h-1 for isolate H3HH. These generation

times are greater than the liquid turnover rate of

ruminal contents which ranges between 0.05 and 0.1

turnovers/h (Hungate, 1966; Weimer, 1992, Zinder,

1993).

The concentration of soluble carbohydrates in

ruminal fluid may also limit growth of certain bacte-

ria. As an example, Streptococcus bovis, although

routinely isolated from ruminal contents, is not found

in high numbers unless relatively high concentra-

tions of soluble carbohydrates (i.e. maltose) are

present (Russell and Baldwin, 1979). This situation

exists due to the relatively low affinity (> 1 mM) of S.

bovis for carbohydrates. In previous research (Pin-

der et al., 2012), we determined that the concentra-

tion of soluble carbohydrates in rumen fluid of cattle

consuming typical production diets ranges between

8 and 17 μM for glucose, and between 110 and 175

μM for cellobiose. The present results are important

because they indicate that the soluble carbohydrate

concentration of ruminal fluid may be sufficient for

growth by acetogens and other factors may limit

growth.

The low numbers of acetogens in ruminal con-

tents cannot be explained by the anaerobic nature

of the isolates, growth rate, or minimum carbohy-

drate concentration necessarry for growth. Thus,

other factors, such as pH or VFA concentration may

be involved in restricting colonization. The rumen

is an environment with a relatively high concentra-

tion of volatile fatty acids and frequently acidic

conditions (Hungate, 1966; Oshio et al., 1987; Rus-

sell, 1992). The concentration of total fatty acids in

ruminal fluid fluctuates considerably but is usually

within the range of 60-140 mM (Owens and Goetsch,

1988). Many factors (e.g., diet of the animal and time

in relation to feeding) affect ruminal fluid pH and


VFA concentrations. For example, high concentrate

diets cause an increase in the total concentration

of VFA in ruminal fluid as compared to diets high in

forages (Oshio et al., 1987). Moreover, ruminal VFA

concentrations fluctuate diurnally as a result of feed

ingestion by the animal. Total VFA concentrations

typically start to rise soon after feeding and peak ap-

proximately 6 h post-feeding. Similar to VFA con-

centrations, ruminal fluid pH fluctuates (generally

between pH 5 and 7) in response to diet and time

post-feeding. The pH of ruminal fluid usually is

lower in animals consuming high-concentrate diets

compared to animals consuming high forage diets.

Ruminal fluid typically decreases soon after feeding

and remains low for several h after feeding before

returning to near neutral levels.

Many bacteria (e.g., S. bovis) are capable of tol-

erating the low pH and high VFA concentrations

similar to those often detected in ruminal contents

after feeding (Russell, 1991). However, many other

bacterial species (e.g., Fibrobacter succinogenes, R.

flavefaciens, Salmonella, Veillonella, and Escherichia

coli) are sensitive to decreasing pH, especially as pH

is decreased to values of pH 6.0 and less (Dunkley

et al., 2008; Hollowell and Wolin, 1965; Kwon et al.,

1997; Ricke, 2003; Russell and Dombrowsli, 1980).

Wolin (1969) determined that the growth of E. coli is

markedly inhibited by the high volatile fatty acid con-

centrations of ruminal fluid and that this inhibition

is pH dependent. Although disagreement exists as

to the mechanism by which the presence of volatile

fatty acids in acidic conditions inhibits the growth of

certain bacteria (Russell, 1992), the fact remains that

these low pH and high VFA sensitive bacteria are un-

able to grow in ruminal contents even when specific

enrichment is attempted (Wallace et al., 1989).

The non-ruminal Clostridium thermoaceticum,

is the only acetogen that has been examined re-

garding its ability to tolerate acidic conditions and/

or high VFA concentrations. Clostridium thermo-

aceticum grows faster and produces more acetic

acid when grown at neutral pH than when grown

in acid medium (Schwartz and Keller, 1982; Wang

and Wang, 1984). Furthermore ,when exponentially-

growing cells of C. thermoaceticum were exposed

to pH’s ranging from 7 to 5, the internal pH of the

cells is maintained above 7 until the extracellular pH

dropped below 5.5 (Baronofsky et al., 1984). These

characteristics identify C. thermoaceticum as a neu-

trophile (pH optimum for growth is near 7). One

explanation of the sensitivity of neutrophiles to low

pH conditions, based on the chemiosmotic theory,

proposes that volatile fatty acids act as uncouplers

destroying the H+ gradient across the cell membrane

and are thus capable of dissipating the proton mo-

tive force of cell membranes (Freese et al., 1973;

Sheu et al., 1975; Baronofsky et al., 1984; Salmond et

al., 1984; Wallace et al., 1989). The ability of acido-

philes and acid-tolerant species to resist low pH and

high VFA concentrations is conveyed by the ability to

lower intracellular pH in the face of declining extra-

cellular pH thus maintaining a constant proton mo-

tive force across the cell membrane (Russell, 1992).

The data presented herein show that isolate A10 and

isolate H3HH, are sensitive to the pH of the culture

medium as well as the presence of volatile fatty ac-

ids in an acidic medium. These results suggest that

acetogens isolated from ruminal contents are similar

to C. thermoaceticum, E. coli and other neutrophilic

bacteria which are sensitive to low pH and high VFA

concentrations. It remains to be determined exactly

how these environmental factors (i.e., low pH and

high VFA concentrations) prevent the growth of ace-

togens isolated from ruminal contents.

Although the carbohydrate concentrations in

ruminal fluid may be marginally sufficient for growth

of isolates A10 and H3HH, the inability to grow un-

der low pH and high VFA conditions would sug-

gest that establishment of a high number of these

isolates in ruminal contents (especially with a single

inoculation) would be unlikely. However, we did not

determine if either isolate remains metabolically ac-

tive under low pH and high VFA conditions. If aceto-

gens are metabolically active under low pH and high

VFA conditions, then the possibility of using repeat-

ed (monthly, weekly or daily) inoculations to maintain

high numbers of acetogens in ruminal contents may

be possible. Another, though more difficult, pos-

sibility is to isolate or generate mutants capable of

tolerating low pH and high VFA conditions.


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ABSTRACT

In 2008 the Environmental Protection Agency (EPA) estimated that only 6.4% of U.S. greenhouse gas

(GHG) emissions originated from agriculture. Of this amount, 53.5% comes from animal agriculture. Agri-

cultural activities are the largest source of N2O emissions in the U.S. accounting for 69% of the total N2O

emissions for 2009. In animal agriculture, the greatest contributor to methane emissions is enteric fermen-

tation and manure management. Enteric fermentation is the most important source of methane in beef and

dairy production, while most of the methane from poultry and swine production originates from manure.

The main cause of agricultural nitrous oxide emissions is from the application of nitrogen fertilizers and

animal manures. Application of nitrogenous fertilizers and cropping practices are estimated to cause 78%

of total nitrous oxide emissions.

Based on the life cycle assessment of beef cattle, 86.15% of the GHGs are emitted during the production

stage, while 68.51% of emissions take place during the production of pork and 47.82% of GHG emissions

occur during the production stage of broiler chickens. The majority of the emissions from the beef cattle

production comes from enteric fermentation while manure management is the major source during swine

production and propane use during broiler poultry production.

Keywords: greenhouse gas, LCA, poultry emissions, beef emissions

Correspondence: C. S. Dunkley, [email protected]: +1 -229-386-3363 Fax: +1-229-386-3239

REVIEWGreenhouse Gas Emissions from Livestock and Poultry

C. S. Dunkley1 and K. D. Dunkley2

1Department of Poultry Science, University of Georgia, Tifton, GA 2School of Science and Math, Abraham Baldwin Agricultural College, Tifton, GA



INTRODUCTION

The primary greenhouse gases emitted by agri-

cultural activities are carbon dioxide (CO2) methane

(CH4) and nitrous oxide (N2O) (Johnson et al., 2007).

Livestock production contributes GHGs to the atmo-

sphere both directly and indirectly (IPCC, 2006). The

emissions can be classified based on the source of

the emission; 1) Mechanical, and 2) Non-mechanical.

The majority of direct CO2 emissions from animal

agriculture are usually from fossil use, for example;

the use of propane or natural gas in furnaces or in-

cinerators and the use of diesel gas to operate farm

equipment and generators results mostly in CO2

emissions (Dunkley unpublished data), this type of

emission can be described as “mechanical emis-

sions.” The use of electricity on animal production

farms results in indirect emissions since the emis-

sions do not occur on site.

For non-mechanical emissions, direct emissions

can be a by-product of digestion through enteric

fermentation (CH4 emissions). Direct emissions also

occur from the decomposition and nitrification/de-

nitrification of livestock waste (manure and urine)

where CH4 and N2O are emitted. Managed waste

that is collected and stored also emits CH4 and N2O.

Indirect emission of N2O occurs when nitrogen is lost

from the system through volatization as NH3 and Nx.

Also, indirect emissions can result from nitrogen that

is runoff or leached from manure management sys-

tems in a form other than N2O and is later converted

to N2O offsite (IPCC, 2006). Methane from enteric

fermentation and manure management are the main

sources of CH4 emissions from agricultural activities

and of all domestic livestock, dairy and beef cattle

are the largest emitters of CH4. Agricultural activities

are the largest source of N2O emissions in the US

accounting for 69% of the total N2O emissions for

2009 (EPA, 2011). The majority of the N2O emission

from animal agriculture is from manure management

which is the second largest (a far second to crop-

ping practices) N2O emitter in the agricultural sector

(IPCC, 2010). Application of nitrogenous fertilizers

and cropping practices are estimated to cause 78%

of total nitrous oxide emissions according to John-

son et al., (2007).

In 2011 the US Environmental Protection Agency

(EPA) reported that the Agricultural Sector was re-

sponsible for a total of 410.6 Tera gram CO2 equiva-

lents (Tg CO2e in 2005). Enteric fermentation and

manure management contributed a total of 200.4 Tg

CO2e which represented about 48% of the total emis-

sions from the agricultural Sector. During this period

(Figure 1.) enteric fermentation was responsible for

136.5 Tg CO2e and managed manure was respon-

sible for 63.9 Tg CO2e. In 2007, the emissions from

the Agricultural sector were 425.8 Tg CO2e a 3.7%

increase. The emissions from enteric fermentation

during this period were 141 Tg CO2e a 3.3% increase

over the 2005 period, while manure management

emissions increased to 68.8 Tg CO2e a 7.7% increase.

The GHG emissions from agriculture showed a 1.5%

reduction to 419.3 Tg CO2e in 2009 when compared

to 2007. This reduction was reflected slightly in en-

teric fermentation which was down by 0.8% to 139.8

Tg CO2e and a 2% reduction in manure management

emission to 49.5 Tg CO2e (IPCC, 2010).

EMISSIONS BASED OF MANURE MAN-AGEMENT SYSTEMS

The type of manure management system that is

used in livestock production can affect the amount

of emissions and the type of gases that are emitted.

A variety of livestock production systems operates

in the U.S. and different manure management sys-

tems are utilized depending on the type of livestock

or poultry produced (Del Grosso et al., 2008). Among

the manure management systems practiced in the

US are; pit storage, poultry with/without litter (that

is, poultry raised on a bedding material or poultry

raised in cages), dry-lot, anaerobic lagoon, pasture,

etc. (Table 1). Beef cattle can be raised using differ-

ent manure management systems and the amount of

emissions are dependent on how the manure is man-

aged. Beef cattle raised on pasture/range exhibit

relatively high N2O emissions. In this system the ma-

nure and urine from the cattle are deposited directly

on the soil reducing the likelihood of much methane


emission. When cattle are raised under conditions

where the manure is collected and spread daily and

there is no storage before it is spread onto the soil

there is low CH4 emissions and no N2O emissions.

Dairy cattle and swine reared in liquid/slurry manure

management systems have moderate to high CH4

emissions, while emissions from swine and dairy cat-

tle reared in anaerobic lagoon management systems

have variable CH4 emissions as it is mostly depen-

dent on the duration of time the manure and slurry

are stored in the lagoons. In this system, the waste

can be stored between 30 to 200 days; the longer the

storage time, the more likely the CH4 emissions will

be high. Both the liquid/slurry and anaerobic lagoon

manure systems have low N2O emissions. Poultry

reared in management systems with litter and us-

ing solid storage have relatively high N2O emissions

but low CH4 emissions. This is because the manure is

stock piled under aerobic conditions which limits the

production of CH4 (USAFGGI, 2008). Broiler, pullets,

and to an extent breeders, are reared using these

manure management systems. Commercial layers

are typically reared in high-rise cages or scrape-out/

belt systems. Here the manure is excreted onto the

floor below with no bedding to absorb moisture. The

ventilation system dries the manure as it is stored. In

some broiler breeder houses a part of the manure is

collected under the slats in the houses making it sim-

ilar to the commercial layers. In this type of manure

management system both CH4 and N2O emissions

are relatively low (IPCC, 2000).

The amount of CH4 or N2O that is emitted from

livestock also depends on environmental conditions

(Del Grosso et al., 2008). Methane is emitted under

anaerobic conditions where oxygen is not available

(Palmer and Reeve, 1993). Storage in tanks, ponds

or pits, such as those used with liquid/slurry flushing

systems encourages anaerobic conditions, therefore

more CH4 is produced (USAF 2008). Conversely, sol-

id waste storage in stacks or shallow pits promotes

Figure 1. The distribution of livestock GHG emissions by source in 2005, 2007 and 2009

63.9 68.8 67.4

136.5 141 139.8

0

20

40

60

80

100

120

140

160

2005 2007 2009

Manure Management Enteric Fermentation

Tg C

O2

equi

vale

nt


aerobic conditions which are more favorable for N2O

emissions. High temperatures and increased storage

time can also increase CH4 emissions (Del Grosso

et al., 2008). Feed characteristics also play a role in

CH4 emissions. Feed, diet, and growth rate have an

effect on the amount and quality of manure an ani-

mal produces (Monteny, 2006). Harper (2000) stated

that there was a large effect on CH4 emissions that

is contingent on the production and use of farmyard

manure. Typically, in an organic system, stock piled

manure is composted, which will increase aeration

limiting anaerobic production of CH4. Higher en-

ergy feeds result in manure with more volatile sol-

ids, which increases the substrates from which CH4

is produced (Del Grosso et al., 2008). Depending on

the species, this impact is somewhat offset because

some higher energy feeds such as that fed to poultry

are more digestible than lower quality forages fed to

ruminant animals and therefore less waste is excret-

ed. The energy content and quality of feed affects

Table 1. Description of livestock waste deposition and storage pathways

Relative Emissions

Manure Management System Description CH4 N2O

Pasture/range/paddock

Ex. beef cattle

Manure and urine from pasture and grazing ani-mals is deposited directly onto soil.

Low High

Daily Spread Manure and urine are collected and spread on fields (little or no storage prior to application).

Low Mini-mal

Solid storage

Ex. poultry

Manure and urine with or without litter are col-lected and stored long term in bulk.

Low High

Dry lot

Ex. Beef cattle

Manure and urine are deposited directly on unpaved feedlots where it is allowed to dry. It is periodically removed.

Low High

Liquid/slurry

Ex. Swine/dairy cattle

Manure and urine are collected and transported in liquid form to tanks for storage. The liquid/slurry may be stored for long periods.

Moderate to high

Low

Anaerobic Lagoon

Ex. Swine/dairy cattle

Manure and slurry are collected using a flush system and transported to lagoons for storage. It remains in lagoons for 30-200 days.

Variable Low

Pit Storage

Ex. Swine/poultry layers

Combined storage of manure and urine in pits below livestock confinements.

Moderate to high

Low

Poultry with litter

Ex. Broiler/pullet/breeders

Enclosed poultry houses utilize bedding material (ex. Wood shavings, peanut hull, rice hulls etc.). The bedding absorbs moisture and dilutes ma-nure. Litter is cleaned out typically once per year.

Low High

Poultry without litter

Ex. Poultry layers/broiler breeders

In high-rise cages or scrape-out/belt systems, manure is excreted onto the floor below with no bedding to absorb moisture. The ventilation system dries the manure as it is stored.

Low Low

Adapted from IPCC (2000) Chapter 4.


the amount of methane produced in enteric fermen-

tation where lower quality feed and higher quantities

of feed causes greater emissions (USAFGG, 2008).

It was reported by the EPA (2011) that an animals

feed quality and feed intake affects emission rates.

In general, lower feed quality and / or higher feed

intakes lead to higher emissions.The composition

of the waste, the type of bacteria involved, and the

conditions following excretion, all have an effect on

the production of N2O from waste management sys-

tems (EPA, 2010). In order for N2O to be emitted, the

waste must be handled aerobically where NH3 and

organic nitrogen is converted to nitrates and nitrites

(Del Grosso et al., 2008).

EMISSIONS FROM ENTERIC FERMENTA-TION AND MANAGED MANURE FROM 2005 TO 2009

Ninety-one % of emissions from enteric fermenta-

tion and managed livestock manure are in the form

of CH4 (EPA, 2011). When Monteny et al. (2001) com-

pared the distribution of methane emissions from

enteric fermentation among animal types; poultry

had the lowest amount with 0.57 lbs methane/ ani-

mal/ year when compared to dairy cattle with 185 to

271 lbs methane/ animal/ year and swine with 10.5lbs

methane/ animal/ year. In 2005, livestock emissions

from enteric fermentation and manure management

were 200.4 Tg CO2e (Table 2). Of this total, dairy

cattle and beef cattle contributed 99.3 and 30.4 Tg

CO2e respectively from enteric fermentation. Swine

contributed 1.9 Tg CO2e from enteric fermentation

while poultry contributed no emissions from enteric

fermentation. For this same period, dairy cattle were

responsible for 109.6 Tg CO2e from enteric fermen-

tation and managed livestock waste combined; beef

cattle contributed 57.4 Tg CO2e, swine contribut-

ed 22.7 Tg CO2e, while poultry contributed 4.4 Tg

CO2e. The remaining emissions (5.66 Tg CO2e) were

from other livestock animals which were not reared

in large amounts.

By 2007 (Table 3), the total amount of GHG emis-

sions from enteric fermentation and managed live-

stock waste had increased by 4.69% from emission

levels in 2005 to 209.8 Tg CO2e. This was as a result

of increases in enteric fermentation from dairy cattle

(101.6 Tg CO2e), beef cattle (32.4 Tg CO2e) swine (2.1

Tg CO2e) and horses. There were also increases in

emissions from managed livestock waste in all the

major livestock categories. Overall, during the two

year period from 2005 to 2007, dairy cattle had a

2.5% increase in emissions (112.4 Tg CO2e), beef cat-

tle had the highest percentage increase of 8.7% up

to 62.4 Tg CO2e. Swine had an increase of 6.6% up to

24.3 Tg CO2e, while poultry had a 4.5% increase (4.6

Tg CO2e) during the 2005 to 2007 period (EPA, 2011).

In 2009 (Table 4), a reduction in emissions of 1.28%

from the 2007 levels was observed even though these

emissions were not as low as in 2005. The emissions

from enteric fermentation from the major livestock

categories showed a reduction in enteric fermenta-

tion from dairy cattle (99.6 Tg CO2e), while beef cat-

tle showed an increase (33.2 Tg CO2e). Enteric fer-

mentation emissions from swine remained the same

as in 2007. For the major livestock categories overall

reductions in emissions from enteric fermentation

and managed livestock waste combined were ob-

served in all with the exception of beef cattle. Dairy

cattle had a 2% reduction down to 110.1 Tg CO2e

from the 2007 levels of 112.4 Tg CO2e. Beef cattle

had a 1.7% increase up to 63.5 Tg CO2e, swine had a

reduction of 4.9% (23.1 Tg CO2e) while poultry had a

6.5% reduction in the emissions from 2007. Of all the

major livestock categories (dairy, beef cattle, swine

and poultry) only poultry had an overall reduction

(2.2%) in emissions from 2005 to 2009 (EPA, 2011).

The emission estimates reported here were adapted

from the EPA’s 2011 report. Several modifications

to the estimates relative to the previous estimates

had an effect on the emission estimates. The modi-

fications included; the average weight assumed for

mature dairy cows from 1550 pounds used in pre-

vious inventories to 1500 pounds. There were also

slight modifications from the 2008 numbers in the

populations of calves, beef replacement and feedlot

cattle. Swine populations were also modified so that

the categories “<60 pounds” and “60- 119pounds”

changed to “<50 pounds” and “50-119 pounds”.


Table 2. Greenhouse gas emissions by livestock category and source in 2005

Enteric Fermentation Managed Livestock Waste

CH4 CH4 N2OTotal

Animal Type Tg CO2 equivalent

Dairy Cattle 99.3 2.8 7.5 109.6

Beef Cattle 30.4 21.4 5.6 57.4

Swine 1.9 19.0 1.8 22.7

Horses 3.5 0.06 0.3 3.86

Poultry 0.00 2.7 1.7 4.4

Sheep 1.0 0.1 0.4 1.5

Goats 0.30 0.00 0.0 0.3

Total 136.5 46.6 17.3 200.4



CH4 CH4 N2OTotal


Dairy Cattle 101.6 2.9 7.9 112.4

Beef Cattle 32.4 24.2 5.8 62.4

Swine 2.1 20.3 1.9 24.3

Horses 3.6 0.6 0.6 4.8

Poultry 0.00 2.8 1.8 4.6

Sheep 1.0 0.1 0.1 1.2

Goats 0.3 0.0 0.0 0.3

Total 141.0 50.7 18.1 209.8


These changes attributed to an average reduction

in emissions from dairy cattle of 11.5 Gg or 0.8% per

year and beef cattle emissions decreased an aver-

age of 0.13 Gg or less that 0.01% per year over the

entire time series relative to the previous inventory

(EPA, 2011).

Of course, in order to discuss emissions from

enteric fermentation one must consider the size

(weight) of the livestock and the number of each

type of livestock grown each year. Larger animals will

produce more methane than smaller animals and

the amount of methane emitted is increased with in-

creasing number of animals grown (Del Grosso et al.,

2011). The type of digestive system will also deter-

mine the amount of methane produced. Cattle are

ruminant animals with a four compartment stomach.

Their digestive tract is designed for microbial fer-

mentation of fibrous, high cellulose materials. One

of the by-products of microbial fermentation is meth-

ane (Stevens and Hume, 1998). Poultry and swine

are mono-gastric animals with a simple stomach and

little microbial fermentation taking place; therefore

they have less enteric methane production (Frédéric

et al., 2007). The feed quality also plays a role in the

amount of CH4 that is emitted, poorer quality high-

fiber diets will likely result in greater CH4 emissions

than higher quality diets that contains more pro-

tein (Del Grosso et al., 2011). Typically, CH4 is usu-

ally produced following the degradation of carbon

components during digestion of feed and manure

(Monteny et al., 2006). Husted (1994) stated that the

rumen was the most important site of methane pro-

duction in ruminants (breath), while in monogastric

animals such as swine and poultry, methane is usu-

ally produced in the large intestines. The manner in

which animal manure are stored whether indoors in

sub-floor pits or outdoors are also relevant sources

of CH4 production (Husted, 1994). Enteric fermen-

tation is the most important source of methane in

the dairy industry, while, the majority of CH4 emis-

sions from the pig and poultry industries originates

from manures (Monteny et al., 2006). There is also

a range in the total emissions in dairy cows that is

caused by differences in diet and housing systems.

For example; there are lower emission rates for ty-

ing stalls and higher rates for cubicle houses (Groot

Koerkamp and Uenk, 1997).



CH4 CH4 N2OTotal


Dairy Cattle 99.6 2.7 7.8 110.1

Beef Cattle 33.2 24.5 5.8 63.5

Swine 2.1 19.0 2.0 23.1

Horses 3.6 0.5 0.3 4.4

Poultry 0.0 2.7 1.6 4.3

Sheep 1.0 0.1 0.3 1.4

Goats 0.3 0.0 0.0 0.3

Total 139.8 49.5 17.9 207.2


FARM-GATE AND LIFE CYCLE ASSESS-MENT EMISSIONS

Greenhouse gas emission from the different live-

stock categories can also be evaluated based on

“Life Cycle Assessment” (LCA). This involves not

only the farm-gate emissions but also an inventory

of the material and energy inputs and the emissions

associated with each stage of production. The LCA

looks at the “cradle to grave” energy use (Guinee

et al., 2001). This assessment could include; fertil-

izer production and transportation, crop production

and transportation, feed additive manufacturing and

transportation, animal production facilities, trans-

portation to processing plants, processing, distribu-

tion to retail markets, consumer use of the product

and disposal of packaging (Guinee et al., 2001). This

can be a very complex process and researchers have

used different boundaries when approaching the

LCA for different livestock. The Environmental Work-

ing Group (2011) examined GHG emissions from

beef cattle and poultry, based on “farm-gate” emis-

sions and showed that each of the livestock category

assessed displayed differences in various areas of

production (Figure 2). Farm-gate emissions here are

based on the emissions that occur within the bounds

of the farm plus the feed production and did not in-

clude processing of the meat. The EWG reported

that the majority (7.51 kg CO2e) of GHGs was emit-

ted to produce 1 kg beef at the farm-gate was as a

result of enteric fermentation. In poultry production

the majority (1.26 kg CO2e) of GHGs emissions was

from feed production and no GHGs emissions from

enteric fermentation. To produce 1 kg of edible beef

at the farm-gate resulted in the emissions of 1.75 kg

CO2e of N2O from manure, while 0.28 kg CO2e N2O

was emitted from manure to produce 1 kg edible

chicken meat. Emissions of GHGs from energy use

at the farm-gate can also be compared for different

livestock categories. On-farm energy use to produce

1 kg of beef at the farm-gate resulted in the emission

of 0.23 kg CO2e, while to produce 1 kg chicken meat

at the farm-gate resulted in the emission of 0.26 kg

CO2e GHGs. It was also reported that 4.8 kg CO2e

was generated to produce 1 kg of edible eggs. The

majority of the emissions from the production of ed-

ible eggs occurs at the farmgate (Figure 3) and as

with chicken meat production, these emissions came

from feed production, on-farm energy use, N2O from

poultry litter and fuel combustion (EWG, 2011). The

Environmental Working Group (2011) also report-

ed LCAs from dairy production, reporting yogurt,

cheese and 2% milk LCAs. The production of whole

milk at the farm-gate resulted in 1.02 CO2e per Kg

of edible whole milk, while only 0.67 kg CO2e was

emitted per kg of edible 2% milk. Domestic cheese

production at the farm-gate resulted in the emission

of 9.09 kg per kg of edible cheese (Figure 3). For yo-

gurt production, the majority of emissions occurred

post-farm gate (1.03 kg CO2e per kg yogurt). Meth-

ane emissions from enteric fermentation were the

primary source of pre-farm-gate GHGs for cheese,

milk and yogurt production (EWG, 2011).

A number of different GHG emission values from

LCA have been published for different livestock cat-

egories (Table 4). Based on these publications the

emissions from beef production at the farm-gate

ranged from 14.8 to 20 kg CO2e/kg of product at

the farm-gate with an average of 16.25 kg CO2e/kg

of product at the farm-gate. The figures for swine

ranged from 3.4 to 6.4 kg CO2e/kg of product at

the farm-gate with an average of 4.82 kg CO2e/kg

of product at the farm-gate, while the emissions

for poultry ranged from 2.33 to 4.6 kg CO2e/kg of

product at the farm-gate with an average of 3.09 kg

CO2e/kg of product at the farm-gate. According to

reports by EWG (2011), beef cattle LCA emissions in

kg CO2e/kg of consumed food was 27 kg. They also

reported that the LCA for pork was 12.1 kg CO2e/kg

of consumed food, while chicken had an LCA of 6.9

kg CO2e/kg of consumed food (Figure 3).

The LCA emissions that were calculated by the

EWG included the production emissions. This in-

cluded the emissions before the product left the

farm plus all avoidable and unavoidable waste. Cal-

culations were also done to include post-production

emissions which included processing, transport, re-

tail, cooking and waste disposal (EWG, 2011). Of the

27 kg CO2e emitted to produce 1 kg of beef (con-

sumed) only 3.73 kg CO2e was post farm-gate emis-


Figure 2. Sources of beef and poultry production emissions

Figure 3. LCA production and post-production emissions of beef and dairy cattle, swine and poultry

7.51

4.67

1.75

0.590.23

0

1.26

0.280

0.26

0

1

2

3

4

5

6

7

8

Enteric Fermentation CH4

Feed Production Manure N20 Manure CH4 On-farm Energy Use

kg CO2e/kg

edible meat

at farm-gate

Beef Cattle Poultry

Farmgate Emissions per kg consumed meat

23.27

11.778.29

3.3 3.631.17 0.92

3.73

1.7 3.81

3.61.17

1.03 0.98

0

5

10

15

20

25

30

Beef Cattle Cheese Swine Poultry Eggs Yogurt Milk (2%)

kg C

O2e

/kg

cons

umed

mea

t

Production Emissions Post-production Emissions

LCA Emissions for Beef Cattle, Dairy, Swine and Poultry


sions (Figure 3). A total of 3.81 kg CO2e was emitted

post farm-gate to produce 1 kg of consumed pork,

while 3.3 kg CO2e from the total 6.9 kg emitted to

produce 1 kg chicken (consumed) was post farm-

gate.

From the LCA emissions it is clear that the major-

ity (86%) of the emissions from beef cattle produc-

tion occur during the production stage while only

14% of the LCA emissions occur post-production

(Figure 4). This is similar to swine where the major-

ity (69%) of emissions was also observed during the

production stage. A different scenario was observed

for the poultry LCA where 48% of the emissions were

observed during the production stage.

CONCLUSIONS

Of the major livestock animals reared, emissions

from poultry production systems generate the low-

est levels of emissions to produce one kg CO2e/kg

meat at farm-gate while dairy cattle produce the

lowest levels of emissions to produce one kg CO2e/

kg product at farm-gate. Dairy cattle emit the high-

est levels of GHG per animal followed by beef cattle

and swine. The majority of the emissions from beef

production come from enteric fermentation and

feed production with the cow to calf and the steer

calf stages generating more than 65% of the total

GHG emissions from this livestock category. In all the

stages of beef production, high levels of CH4 from

enteric fermentation are generated. For dairy cattle,

the majority of emissions are from enteric fermen-

tation, similar to beef cattle production. Methane

emission from manure storage and feed production

Figure 4. Percent production and post-production emissions for beef and dairy cattle, swine and poultry LCA

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Beef Cattle Cheese Swine Poultry Eggs Yogart Milk (2%)

Production Emissions Post-production Emissions


in dairy cattle production also contributes to high

levels of GHGs. Swine production emits GHGs pri-

marily from manure management and fuel combus-

tion. Only a small amount of CH4 is emitted during

digestion when compared to ruminants. At least one

third of GHG emitted from swine production is from

post farm-gate activities. The largest contributor

to GHG emissions from poultry production is feed

production. The highest emissions from poultry on-

farm activities are from fuel combustion from energy

use and manure management. In broiler production

post farm-gate emission makes up more than half

of all the emission, while post farm-gate emissions

from egg farm operations accounts for less than one

quarter of the total emissions.

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6.4 DERFA, 2008

3.4-4.2 Pelletier, 2010

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4.62 EWG, 2011

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4.6 DERFA, 2008

2.36 Pelletier, 2008

3.1 Wiltshire, 2006

2.33 EWG, 2011


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global_change/AFGGInventory1990_2005.htm.




ABSTRACT

The oral cavity is a dynamic environment with several niches for attachment of a variety of flora. The

dominant flora in the mouth are comprised of anaerobic Gram-positive bacteria. Salmonella is a Gram-

negative bacterium which, according to the literature, is found very rarely in the parotid gland of the mouth

of humans. One of the most important characteristics of bacteria in the oral cavity is their ability to attach

to the mucosal cells. Salmonella has displayed ability to attach to the epithelial cells of the intestine and

have a variety of fimbiral lectins, type I fimbriae, and flagella which aid attachment to a variety of cells. This

review details the ability of Salmonella to cause disease and the potential of Salmonella as a pathogen in

the oral cavity of humans.

Keywords: Salmonella, dental, oral cavity

INTRODUCTION

Salmonella is a facultative intracellular Gram-neg-

ative pathogen transmitted by the ingestion of con-

taminated food and water. Depending on the spe-

cies, Salmonella may cause either typhoid fever or

enteritis. S. Typhi causes typhoid fever which is a se-

rious systemic infection. S. Typhimurium causes gas-

troenteritis which is localized infection in the intes-

Correspondence:Sujata A. Sirsat, [email protected]: +1 -713-743-2624 Fax: +1-713-743-3696

tines of humans (Takaya et al., 2005). The incidence

of illness due to salmonellosis has risen in the past

three decades. Foods such as eggs, poultry, peanut

butter, raw sprouts and most commonly implicated

in foodborne Salmonella outbreaks (Maki, 2009).

One of the largest outbreak of Salmonella Ty-

phimurium occurred in Chicago in 1984 when

200,000 people acquired the pathogen via pasteur-

ized milk contaminated with non-pasteurized milk

(Tafazoli et al., 2003). Salmonella outbreaks in foods

can be prevented by proper food handling practices

and thorough cooking of poultry and eggs. Improv-

REVIEWCan Salmonella Reside in the Human Oral Cavity?

Sujata A. Sirsat1

1 Conrad N. Hilton College of Hotel and Restaurant Management, University of Houston, Houston, TX 77204-3028



ing standards of quality testing and animal rearing

practices will also be advantageous (Tafazoli et al.,

2003). However, contaminated ready-to-eat (RTE)

foods pose a challenge since these are not pro-

cessed before consumption (Koo et al., 2012). More

recently, Salmonella outbreaks have occurred in

ground beef, mangoes, cantaloupe, and pet food

(CDC, 2012a; CDC, 2012b; CDC, 2012c; CDC, 2013).

The evidence of Salmonella in the oral cavity

dates back to 430 BC (Sulonen et al., 2007). There

have been a few cases reported of Salmonella infec-

tion in the parotid gland of the mouth (Sulavik et al.,

1997; Suárez and Rüssmann, 1998; Stern et al., 2001;

Sulakvelidze et al., 2001; Stone 2002; Sturny et al.,

2003; Strindelius et al., 2004; Stern et al., 2005; Stern

et al., 2006). The parotid gland is the salivary gland in

humans that facilitates the early digestion of starch

and swallowing (Thesleff et al., 1988).These cases are

most often caused by Salmonella due to secondary

infections (Stern et al., 2001). As will be discussed in

the following sections, Salmonella has a tremendous

ability of attach to epithelial cells of the intestine of

the host. The focus of this review is to discuss the

potential ability of Salmonella to survive and attach

to the oral mucosa and propose possible methods

to study this interaction.

GENETICS OF SALMONELLA PATHO-GENESIS

It is believed that Salmonella virulence has

evolved as a result of horizontal gene transfer (John-

ston et al., 1996). This concept is supported by the

fact that large numbers of virulence genes are clus-

tered within its chromosome. The five Salmonella

Pathogenicity Islands (SPI) identified in Salmonella

are located in various regions on the chromosomes

and contain sets of virulence genes (Foster and

Spector, 1995). Invasion is believed to be controlled

by genetic and environmental regulators. Envi-

ronmental signals such as high pH (Cheung et al.,

1999), low osmolarity (Darwin and Miller, 1999) and

low oxygen (Cirillo et al., 1998) are believed to in-

crease invasion of Salmonella in the host. This has

been proven by testing the effects of these factors

on Salmonella strains containing lacZ fusions in the

invasion genes and performing ß–galactosidase as-

says and improved invasion of Salmonella in tissue

culture cell lines (Cheung et al., 1999; Davidson and

Harrison, 2002). In addition, (Sirsat et al., 2011) dem-

onstrated that sublethal heat stress may increase the

attachment ability of Salmonella in Caco-2 cells. SPI-

1 encodes a type III secretion system (TTSS) which is

essential for the process of cell invasion. The protein

forms a secretory “needle complex” which spans the

inner and outer bacterial membrane (Jones, 2005).

Control of invasion genes leads to the formation of

the type III secretion apparatus at the point of infec-

tion (Bowe et al., 1998). This secretion system is used

by microorganisms to translocate virulence-associat-

ed effector proteins into the cytoplasm of the host

cells resulting in a cross-talk which leads to down

stream responses such as membrane ruffling on the

surface of the host cell followed by the formation

of phagosomes which may internalize the bacteria

(Lucas and Lee, 2000; Liu et al.,2008). SPI-1 specifi-

cally codes for transcriptional regulators such as hilA

(Davidson and Harrison, 2002), hilC (Chenoweth et

al., 2007), and hilD (Chatfield et al., 1992). The hilA

gene has been shown to be required for the expres-

sion of three other invasion genes: invF, sspC, and

orgA (Davidson and Harrison, 2002). The gene invF is

a transcriptional regulator (Enoch, 2007), sspC codes

for an invasion protein (Libby et al., 1994), and orgA

product is a component of an export machinery

system (Leverentz et al., 2001). PhoPQ is a regula-

tory two-component system, which is not contained

within SPI-1 but is crucial for invasion phenotype of

Salmonella (Liljemark and Gibbons, 1972; Lichten-

steiger and Vimr, 2003). This two-component system

responds to extracellular cation levels (Lillard, 1980).

In conditions of low cation concentration, sensor

kinase PhoQ phospohorylates the regulator PhoP

which activates pag transcription. Induction and ex-

pression of pag genes are required for the survival of

the bacteria in the macrophage (Lewis et al., 2009).

Other pathogenicity islands, SPI-2 and SPI-5, encode

genes which are responsible for pathogenesis of Sal-

monella after invasion of the host (Shumway, 1990).


Salmonella attachment to epithelial cells is de-

pendent on lectin-like adhesions which recognize

specific glycoconjugate receptors on the host cells.

Salmonella has several types of fimbrial lectins in-

cluding SEF14. SEF17, and SEF21 (Teplitski et al.,

2003). Other fimbriae such as plasmid-encoded

fimbriae (PEF) and long polar fimbriae (LPF) of Sal-

monella are also believed to be involved in the ad-

hesion of the bacteria to villus of the small intestine

and the Peyers patches (Terai et al., 2005; Mihaljevic

et al., 2007). Studies done by (Teplitski et al., 2003)

have shown that flagella also play an important role

in the attachment of Salmonella to epithelial cells of

the host by functioning to move to appropriate sites.

The next section discusses host immune reaction to

combat virulent Salmonella.

IMMUNE REACTION TO COMBAT SAL-MONELLA

Salmonella enters the host via contaminated food

or water (Threlfall, 2006). The pathogen exhibits in-

creased acid-tolerance response when exposed to

the low pH of the stomach (Leistner and Gorris, 1995).

Following this, Salmonella enters the small intestine

and moves towards and adheres to the intestinal ep-

ithelium due to expression of several fimbriae genes

(Leistner, 2000). Cytoskeletal rearrangements occurs

once the bacteria adhere to the epithelial surface

causing host membrane ruffling which leads to en-

closing adherent bacteria in vesicles (Li et al., 2002).

Salmonella invasion into epithelial cells leads to

recruitment of neutrophils into the intestinal lumen.

One of the significant factors that lead to neutrophil

recruitment is the secretion of IL-8 by the epithelial

cells (Takaya et al., 2005). Following this, Salmonella

enters the macrophages leading to the activation of

its virulence mechanisms which further leads to its

survival and replication in the host. In some cases

Salmonella may enter the blood and cause second-

ary infections colonizing in various organs of the

body (Lewis et al., 2009).

Salmonella also has the potential to activate the

adaptive immune system. Activation of cytokines

leads to downstream activation of both the T helper

(Th) and T cytotoxic (Tc) cells (Thomson et al., 1977;

Testerman et al., 2002). Tc cells either kill the intra-

cellular bacteria or release live bacteria when they

lyse. Th cells activate B cells which may produce an-

tibodies which could aid in opsonizing the bacteria

(Thomson et al.,1977; Testerman et al., 2002). Mice

studies have shown that the depletion of Th cells

had a more pronounced effect than the depletion

of Tc cells. Transfer of Th cells to naïve recipients

induced more effective vaccination compared to

transfer of Tc cells. The following section discusses

the microbial ecology of the mouth (Mittrucker and

Kaufmann, 2000).

ORAL MICROBIAL ECOLOGY

Bacteria have been found in the mouths of infants

shortly after birth. Organisms such as Escherichia

coli are transient microorganisms. Others such as S.

salivarius and lactobacilli are more characteristic of

adult flora and are found within a few days (Takaya

et al., 2002). Studies have shown that maternal sa-

liva may act as a source of some Gram-negative

anaerobes in the oral microflora of infants even be-

fore tooth eruption (Könönen et al., 2007). Oral flora

rapidly changes once teeth erupt in the mouth since

teeth provide an additional unique surface for bacte-

rial colonization. Saliva is another common environ-

mental influence in the mouth since it washes away

non-adherent bacteria and has inherent buffering

properties (Russell and Melville, 1978). Additionally,

other factors such as diet, oral hygiene, and general

health also influence the nature of oral flora (Tatusov

et al., 2003).

The mouth contains several different types of

bacteria. This could be because of the availability

of several different niches available for bacterial at-

tachment. These surfaces are the teeth, mucosal epi-

thelial cells, and the bacterial layers that constitute

the plaque on the teeth (Whittaker et al.,1996). This

topic has been extensively reviewed by (Tafazoli et

al., 2003; Takaya et al., 2004). The majority of bacteria

belong to streptococci genus which is a Gram-posi-


tive filamentous organism. In particular, Streptococ-

cus mutans are especially important as they cause

dental caries (Gibbons and Houte, 1975; Gibbons,

1984). S. mitis and S. sanguis are other streptococ-

ci that are present in lower numbers (Liljemark and

Gibbons, 1972). Other Gram-positive rods such as

those belonging to the Actinomyces species are also

present. These are involved in some periodontal dis-

eases. Gram-negative organisms such as Neisseria

and Veillonella may be also present on the oral cav-

ity. Most of these bacteria are anaerobic and include

members of Bacteroides, Fusobacterium, and Vibrio

(Gibbons, 1984).

The indigenous oral flora exerts competition to-

ward bacteria found elsewhere in the human body

and hence it is rare to isolate Salmonella, E. coli or

Staphylococcus aureus from the mouth. This is be-

cause all the attachment sites are taken up by the

indigenous flora and their numbers are a lot higher

as compared to the potential pathogenic bacteria

(Tavazoie et al., 1999).

There are several forces that regulate the coloni-

zation of bacteria in the mouth. The most important

factor however is bacterial adherence to surfaces

in the mouth. Attachment properties are especially

crucial for microorganisms since there is a risk of be-

ing washed out of the oral cavity entirely (Tafazoli et

al., 2003). The mouth has proven to be a particularly

interesting model for studying adhesion between

host and pathogens since it has several different mi-

crocosms which can be easily sampled. Additionally,

there is increased interest to study these interactions

because microorganisms cause periodontal diseas-

es. It has been demonstrated that dietary sucrose

is required for accumulation of microorganisms on

teeth surfaces (Taitt et al., 2004).

SALMONELLA IN THE ORAL CAVITY

Evidence of Salmonella in the oral cavity has been

seen as far back as 430 BC when (Sulonen et al.,

2007) set out to determine the probable cause of

the “Plague of Athens” using skeleton dental pulp

from the mass burial site. The researchers performed

polymerase chain reaction (PCR) and screened six

different pathogens using specific DNA primers.

They concluded that the pathogen in the dental

pulp of the skeletons was Salmonella or a pathogen

very closely related to Salmonella.

The parotid gland is the salivary gland that is of-

ten affected due to inflammation (Takaya et al., 2004;

Takaya et al., 2005). Parotitis usually manifests in in-

fants, elderly persons, and the immunocompromised

(Takaya et al., 2005). The most common pathogen

that is associated with bacterial parotitis is Staphylo-

coccus aureus (Takaya et al., 2005). Other organisms

such as Haemophilus influenzae, Klebsiella pneu-

monia, and Salmonella spp. are not as frequently

isolated (Takaya et al., 2004). Nine cases of parotid

abscess due to Salmonella has been reported in the

literature to date (Sulavik et al., 1997; Suárez and

Rüssmann, 1998; Stern et al., 2001; Sulakvelidze et

al., 2001; Stone 2002; Sturny et al., 2003; Strindelius

et al., 2004; Stern et al., 2005; Stern et al., 2006).

As described in the previous sections, Salmonella

causes these types of infections after it enters the

gastrointestinal tract and is disseminated into the

bloodstream. The secondary areas of colonization

are usually diseased tissue and trauma sites (Stern et

al., 2001). (Stern et al., 2001) presented a case study

of a 50-year old patient who had parotid abscess.

This individual may have been infected orally or it

could have been a result of a secondary infection

due to Salmonella. However, the patient did not

show symptoms of gastroenteritis and hence may

have been a carrier of the pathogen eventually re-

sulting in Salmonella spreading to the bloodstream

and the manifestation of parotid abscess.

Saliva contains the enzymes lysozyme and lac-

toferrin (Tafazoli et al., 2003) and Gram-negative

pathogens such as Salmonella and E. coli have been

shown to be sensitive toward lactoferrin as it acts

as an antimicrobial (Takaya et al., 2002). In addition,

these antimicrobial enzymes interact with each other

in a synergistic or additive manner (Tenovuo, 1998).

Lysozyme acts on Gram-negative bacteria by hydro-

lyzing 1,4-beta-linkages in the bacterial cell walls

(Proctor et al., 1988). Lactoferrin interacts with the

lipolysacharide of the Gram negative cell membrane


(Appelmelk et al., 1994). This may explain why such

few cases of Salmonella persistence are evident in

the oral cavity. Independent studies have found that

specific Salmonella mutations may lead to resistance

against lysozyme (Sanderson et al., 1974).

POTENTIAL STUDIES

As discussed in previous sections, the key to bac-

terial presence and survival in the mouth is attach-

ment to various sites in the oral cavity. This is espe-

cially challenging in the mouth due to the constant

washing of non-adherent bacteria by saliva. Also,

saliva contains lysozyme which has been shown to

be effective against Gram-negative organisms such

as Salmonella and E. coli (Takaya et al., 2002). One of

the initial studies to examine Salmonella in the oral

cavity would be to test the effect of saliva on the vi-

ability of Salmonella. This would give investigators

an indication if the concentration of lysozyme in sa-

liva is inhibitory to Salmonella. This can be done by

collecting saliva from humans and adding cultures of

Salmonella grown overnight and testing viability by

the plate count method.

Tenor et al. (2004) prepared epithelial cell suspen-

sions to test the adherence of bacteria to the cells of

oral cavity and similar studies could be performed

for Salmonella to test its capability of adherence to

these types of cells. A flow-chart of the method is

Figure 1. Flow chart of an experimental method to test the ability of bacterial attachment to hu-man epithelial cells from the oral cavity

Collect human epithelial cells by scraping oral cavity with wooden

stick

Wash cells to remove unattached bacteria and diluted to 105 cells/ml

Mix cell suspensions with bacteria

and incubate at 35°C for 30 min

Wash cells to removed unattached bacteria

Stain bacteria and enumerate under the microscope


demonstrated in Figure 1. The method can be de-

scribed briefly as follows. The researchers collected

human epithelial cells by scraping oral mucosal cells

with a wooden stick and twirled the applicator in

saline to dislodge the cells. The cells were subse-

quently washed free of unattached bacteria and ad-

justed with saline to 105 cells/mL. Following this, the

authors tested the ability of various bacteria to ad-

here to the epithelial cells. This was done by mixing

cell suspensions of the bacteria with cell suspensions

of the epithelial cells and incubating the mixture at

35°C for 30 min. The epithelial cells were washed to

remove non-adherent bacteria. The bacteria were

stained in order to observe and enumerate the cells

under the microscope. A more quantitative method

would involve conducting viable bacteria studies by

recovering cells as petri plate counts and assessing

the number of Salmonella that adhere to the human

epithelial cells. This study would offer the means for

assessing the capability of Salmonella to compete

with the normal flora of the human oral cavity. Also,

the results may indicate whether Salmonella has the

necessary mechanisms to attach to surfaces of the

mouth.

Tenor et al. (2004) performed electron microscopy

studies after mixing bacterial cultures and epithelial

cells to observe the adherence property of bacteria.

Similar studies could be performed with Salmonella

and human epithelial cells. The electron microscopy

analyses would be particularly useful in demonstrat-

ing visually, the ability of Salmonella to adhere to

these cells in the presence of other bacterial com-

petition.

CONCLUSIONS

The ability to adhere to a variety of surfaces avail-

able in the oral cavity of the mouth is of utmost

important to the flora that primarily resides in the

mouth. Salmonella has been observed in some cas-

es of parotid gland infection; however, this type of

infection is often secondary in nature and extremely

rare. Lysozyme and lactoferrin, key enzymes in the

saliva may act as antimicrobial agents against Sal-

monella; however, some strains may be resistant to

these enzymes. Additional studies are needed to

understand the implications of Salmonella in the

oral cavity and complications that may results down-

stream. The proposed experiments may be able to

identify whether Salmonella possesses gene(s) that

encodes for the necessary fimbrial lectins to attach

to surfaces of the oral cavity.

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ABSTRACT

Cattle can be naturally colonized with foodborne pathogenic bacteria such as Shiga Toxin-producing E.

coli (STEC) in their gastrointestinal tract. While these foodborne pathogens are a threat to food safety, they

also cause human illnesses via cross contamination of other foods and the water supply, as well as via direct

animal contact. In order to further curtail these human illnesses and ensure a safe and wholesome food

supply, research into preharvest pathogen reduction controls and interventions has grown in recent years.

This review addresses the ecology of STEC in cattle and potential controls and interventions that have been

proposed or implemented to reduce STEC in cattle. We focus in this review on the use of management

practices and the effects of diet and water management. Implementation of preharvest strategies will not

eliminate the need for good sanitation procedures in the processing plant and during food preparation

and consumer handling. Instead, live-animal management interventions must be implemented as part of

a multiple-hurdle approach that complements the in-plant interventions, so that the reduction in pathogen

entry to the food supply can be maximized.

Keywords: E. coli O157:H7, EHEC, cattle, management

INTRODUCTION

One of the largest food safety (and economic) im-

pacts on the cattle industry has been the emergence

Correspondence: Todd Callaway, [email protected]: +1-979-260-9374 Fax: +1-979-260-9332.

of Shiga Toxin-producing Escherichia coli (STEC)

bacteria, which are part of the natural reservoir in ru-

minant animals such as cattle (Karmali et al., 2010).

STEC-caused illnesses cost the American economy

more than $1 billion each year in direct and indirect

costs from more than 175,000 human illnesses (Scal-

lan et al., 2011; Scharff, 2010). Furthermore, since

the emergence of the “poster child” of STEC, E. coli

REVIEWShiga Toxin-Producing Escherichia coli (STEC) Ecology in Cattle

and Management Based Options for Reducing Fecal Shedding

T. R. Callaway1, T. S. Edrington1, G. H. Loneragan2, M. A. Carr3, D. J. Nisbet1

1Food and Feed Safety Research Unit, USDA/ARS, 2881 F&B Rd., College Station, TX 778452Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409

3Research and Technical Services, National Cattlemen’s Beef Association, Centennial, CO 80112

Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies neither approval of the product, nor exclusion of others that

may be suitable.



O157:H7, more than $2 billion dollars have been

spent by the cattle industry to combat STEC in pro-

cessing plants (Kay, 2003).

While post-harvest pathogen-reduction strate-

gies have been largely successful at reducing direct

foodborne illness, these processing interventions

have not been perfect (Arthur et al., 2007; Barkocy-

Gallagher et al., 2003), in large part because avenues

of human exposure include indirect routes (LeJeune

and Kersting, 2010; Nastasijevic, 2011). In order to

further curtail human illnesses and ensure a safe and

wholesome food supply, research into preharvest

pathogen reduction controls and interventions has

grown in recent years (Callaway et al., 2004; LeJeune

and Wetzel, 2007; Oliver et al., 2008; Sargeant et al.,

2007).

The impact of using pathogen reduction strate-

gies focused on the environmental contamination

and exposure routes at the live animal stage are like-

ly to have large impacts on resulting human illnesses

(Rotariu et al., 2012; Smith et al., 2012). Reduction

of STEC can also yield public health improvements

in rural communities (LeJeune and Kersting, 2010)

and amongst attendees of agricultural fairs, ro-

deos and open farms (Keen et al., 2006; Lanier et

al., 2011). Thus, the logic underlying focusing on

reducing foodborne pathogenic bacteria in live

cattle is straightforward: 1) reducing the amount of

pathogens entering processing plants will reduce

the burden on the plants and render the in-plant

interventions more effective; 2) reducing horizontal

pathogen spread from infected animals (especially

in “supershedders”) in transport and lairage; 3) will

reduce the pathogenic bacterial burden in the envi-

ronment and wastewater streams; and 4) will reduce

the direct risk to those in direct contact with animals

via petting zoos, open farms, rodeos and to animal

workers. This review addresses the microbial ecol-

ogy of STEC colonization of cattle and controls and

interventions that have been proposed or imple-

mented to reduce STEC in live cattle in the areas

of: 1) Management practices and transport, and 2)

Cattle water and feed management.

EHEC, STEC, VTEC AND NON-O157:H7’S: A PRIMER

Although the relatively recent (1982) emergence

of E. coli O157:H7 into public view makes it seem

that this organism is a new arrival in the food chain,

data indicates that this organism is far more ancient,

having arisen between 400 and 70,000 years ago

(Law, 2000; Riley et al., 1983; Wick et al., 2005; Zhou

et al., 2010). Although a variety of acronyms have

been applied to the “hamburger bug”, they belong

to a single group that acquired toxin genes from Shi-

gella via a gene transfer event (Kaper et al., 2004;

Karmali et al., 2010; Wick et al., 2005). Research-

ers refer to these pathogens often interchangeably

as Enterohemorrhagic E. coli (EHEC), Shiga toxin-

producing E. coli (STEC), or Verotoxin-producing E.

coli (VTEC). While E. coli O157:H7 was the first of

the STEC’s to be recognized as a major food safety

threat, recently the other “non O157:H7 STEC” have

been increasingly implicated in human illness out-

breaks (Bettelheim, 2007; Fremaux et al., 2007). Be-

cause of this linkage, the “gang of six” non-O157 se-

rogroups (O26, O45, O103, O111, O121, and O145)

have joined O157:H7 as being classified as adulter-

ants in beef (USDA/FSIS, 2012). Since this declara-

tion, focus has shifted on understanding the general-

ized STEC ecology, rather than simply focusing on E.

coli O157:H7 (Gill and Gill, 2010).

For years, researchers (including the present au-

thors) assumed that in general, all of the non-O157

STEC would behave similarly to O157:H7 in a physi-

ological and ecological sense. However, recent

research has found that in addition to the genetic

divergence seen in O157:H7 lineages (Zhang et al.,

2007), there appear to be significant physiological

differences between and within non-O157 STEC

which may play a role in the ecological niche occu-

pied in the ruminant gastrointestinal tract by these

non-O157 serotypes (Bergholz and Whittam, 2007;

Free et al., 2012; Fremaux et al., 2007). While these

and other physiological differences need to be inves-

tigated further, and their roles in the gastrointestinal

microbial population must be determined, it appears

that the O157:H7 serotype is well adapted to survive


in cattle (García et al., 2010; O’Reilly et al., 2010) and

that other STEC serotypes can also live in the gastro-

intestinal tract of cattle (Arthur et al., 2002; Chase-

Topping et al., 2012; Joris et al., 2011; Monaghan et

al., 2011; Polifroni et al., 2012; Thomas et al., 2012),

and be transferred into ground beef (Bosilevac and

Koohmaraie, 2011; Fratamico et al., 2011).

While we understand much of how E. coli O157:H7

behaves in the gastrointestinal tract and farm envi-

ronment, we know very little about the ecology of

other STEC in those environments (Monaghan et al.,

2011; Polifroni et al., 2012). Thus this review focuses

on pre-harvest pathogen interventions based upon

E. coli O157:H7 data. We hypothesize that non-

O157:H7 STEC will largely behave in a broadly simi-

lar fashion to E. coli O157:H7 in the gastrointestinal

and farm environment; however this is an educated

assumption and that imposed limitation must be un-

derstood. Thus readers must be aware that most of

the research referred to in this review is based upon

E. coli O157:H7 specifically, and may or may not ap-

ply to all STEC.

ECOLOGY OF STEC AND GASTROIN-TESTINAL COLONIZATION

Because E. coli O157:H7 (and to some degree

non-O157 STEC) co-evolved along with its host it is

uniquely well-fitted to survive in the gastrointestinal

tract of cattle as a commensal type organism (Law,

2000; Wick et al., 2005). While E. coli O157:H7 can

live in the rumen of cattle (Rasmussen et al., 1999),

the site of primary colonization is the terminal rec-

tum (Naylor et al., 2003; Smith et al., 2009a). This

organism produces a potent cytotoxin (Shiga toxin)

that does not seriously impact its preferred host (cat-

tle) because they lack toxin receptors (Pruimboom-

Brees et al., 2000), but this same toxin causes serious

illness in humans colonized by E. coli O157:H7 (Kar-

mali et al., 2010; O’Brien et al., 1992). Unfortunately,

this means that the natural commensal-type relation-

ship between STEC (including O157 and non-O157)

and cattle ensures that this organism can be passed

on to meat products and consumers of beef (Fe-

rens and Hovde, 2011). This transmission most fre-

quently occurs during summer months, and is linked

to a summer increase in the prevalence of E. coli

O157:H7 in cattle (Edrington et al., 2006a; Lal et al.,

2012; Naumova et al., 2007; Ogden et al., 2004; Wells

et al., 2009), not just an increase in consumption or

a change of cooking habits by consumers (Money et

al., 2010; Williams et al., 2010a). It has been sug-

gested that neuroendocrine factors may play a role

in E. coli O157:H7 (Edrington et al., 2006a; Green et

al., 2004), as may signaling between host and intes-

tinal microbial populations or within STEC popula-

tions via quorum-sensing (Edrington et al., 2009b;

Sperandio, 2010; Sperandio et al., 2001). Further

possible interactions within the microbial ecosystem

of the rumen are demonstrated in the preferential

consumption of E. coli O157:H7 by ruminal protozoa

(Epidinium), and increased populations in the pres-

ence of Dasytricha (Stanford et al., 2010).

Because of the nature of STEC survival in the

ruminant gut, it is no surprise that it persists in fe-

cal deposits (Dargatz et al., 1997; Jiang et al., 2002;

Maule, 2000; Yang et al., 2010) and in soils (Bolton

et al., 2011; Semenov et al., 2009; Van Overbeek et

al., 2010). This allows E. coli O157:H7 to cycle within

pens and farms in a fecal-oral route (Russell and Jar-

vis, 2001), recirculating within groups or individual

animals (Arthur et al., 2010). The presence of super-

shedding cattle (Chase-Topping et al., 2008) in the

population can further enhance this horizontal trans-

mission within a herd or a pen of cattle (Arthur et

al., 2010; Arthur et al., 2009; Cobbold, 2007; LeJeune

and Kauffman, 2006). However, the host/dietary/mi-

crobial factors underlying the “supershedder” status

of cattle remains unknown, as do factors that allow

simple gut colonization by E. coli O157:H7. Thus it

is apparent that the farm/pen/facility environment

plays an important role in STEC colonization and re-

circulation, as well as via direct and indirect transmis-

sion to human farm workers/visitors and consumers

(Ihekweazu et al., 2012; LeJeune and Kersting, 2010;

Smith et al., 2012; Stacey et al., 2007; Strachan et al.,

2006).


MANAGEMENT PRACTICES AND TRANSPORTATION

Good management of cattle is critical for efficient

animal production, but to date no typical “manage-

ment” procedures have been shown to affect colo-

nization or shedding of foodborne pathogens (Ellis-

Iversen et al., 2008; Ellis-Iversen and Van Winden,

2008; LeJeune and Wetzel, 2007), some practices

may reduce horizontal transmission and recirculation

of STEC within a herd of cattle (Ellis-Iversen and Wat-

son, 2008). However, the use of management tools

like the squeeze chute (crush) to process cattle has

been shown to increase the odds of hide contami-

nation with E. coli O157(Mather et al., 2007). Yet, in

spite of this lack of evidence regarding impacts on

food safety, good management practices are criti-

cal to ensuring animal health and welfare (Morrow-

Tesch, 2001).

Bedding and pen surfaces

E. coli O157:H7 can live for a long period of time

in manure, soil and other organic materials (Jiang

et al., 2002; Maule, 2000; Winfield and Groisman,

2003) and can be transmitted successively through

their environment (Semenov et al., 2010; Semenov

et al., 2009). Cattle, especially dairy cows, are bed-

ded on materials that are largely chosen on animal

health and welfare grounds. Unfortunately, bedding

material can harbor bacteria that are responsible for

mastitis, as well as foodborne pathogenic bacteria

that can be spread between cattle (Davis et al., 2005;

Oliver et al., 2005; Richards et al., 2006; Wetzel and

LeJeune, 2006). Researchers have shown that urine

increases growth of E. coli O157:H7 on bedding, po-

tentially by providing substrate for growth (Davis et

al., 2005). Modeling research has shown that an in-

crease in bedding cleaning frequency would increase

the death rate of E. coli O157:H7 (Vosough Ahmadi

et al., 2007). Further studies have demonstrated that

the use of very dry bedding reduced E. coli O157:H7

prevalence on farms (Ellis-Iversen et al., 2008; Ellis-

Iversen and Van Winden, 2008). Researchers have

shown that sand bedding reduced transmission of E.

coli O157:H7 between dairy cows, resulting in lower

populations of E. coli O157:H7 in cattle bedded on

sand compared to sawdust (LeJeune and Kauffman,

2005; Westphal et al., 2011). It is suspected that this

difference was due to desiccation or reduced nutri-

ent availability.

Feedlot surfaces were thought to contain ma-

nure-like bacterial populations, but recent molecular

studies have indicated that the bacterial communi-

ties of feedlot surfaces are complex, yet utterly dis-

tinct from fecal bacterial populations (Durso et al.,

2011). This suggests that traits that favor survival

in the gastrointestinal tract (anaerobic, warm, dark)

do not favor survival on the feedlot surface (aerobic,

cooler, sunlit). Surfaces such as pond ash do not im-

pact survival of E. coli O157 (Berry et al., 2010), how-

ever studies and anecdotal evidence indicates that a

greater number of cattle shed E. coli O157:H7 when

housed in muddy pen conditions than cattle from

pens in normal condition and that the condition of

the pen floor may influence the prevalence of cat-

tle shedding the organism and the ability of E. coli

O157:H7 to survive dry conditions (Berry and Miller,

2005; Smith et al., 2001; Smith et al., 2009b). Studies

have recently demonstrated that sunlight can reduce

E. coli O157:H7 populations on pen surfaces (Berry

and Wells, 2012) and in water systems (Jenkins et

al., 2011). Overall, bedding or pen cleaning will not

eliminate E. coli O157:H7 from any farm or feedlot

environment, but it may slow spread within a herd or

between penmates.

Manure Management

E. coli O157:H7 and other STEC survive in manure

and can persist for a lengthy period of time (up to

21 months) (Bolton et al., 2011; Fremaux et al., 2007;

Hutchison et al., 2005; Kudva et al., 1998; Varel et

al., 2008). Although there are differences amongst

STEC strains in their ability to persist in manure,

these appear to be related to the oxidative capacity

of each strain (Franz et al., 2011). The presence of

a native bacterial population in manure reduces E.

coli O157:H7 survival in soils (Van Overbeek et al.,

2010). The amendation of manure in soil can result in


STEC uptake directly by plants, including food crops

(Franz and Van Bruggen, 2008; Jiang et al., 2002; Se-

menov et al., 2010; Semenov et al., 2009). Rainfall

events can also wash STEC from cattle feces (stored

or in fields) into drinking or irrigation water supplies

(Anonymous, 2000; Cook et al., 2011; Ferguson et al.,

2007; Oliveira et al., 2012; Pachepsky et al., 2011). As

the mean temperature of manure rises during stor-

age the survival of E. coli O157:H7 is reduced (Se-

menov et al., 2007), indicating that composting can

enhance manure safety, thus reducing human illness-

es (Graham and Nachman, 2010; Kelley et al., 1999).

There have been few studies that have isolated

STEC consistently from cattle waste lagoons (Purdy

et al., 2010). This is potentially due to the oxidized

nature of the lagoon, or the presence of a native mi-

crobial population. In waste water lagoons, there are

protozoa that preferentially consume E. coli O157:H7

(Ravva et al., 2010), possibly explaining at least some

of the difference between E. coli O157:H7 survival in

manure with that of limited survival in dairy lagoons

(Ravva et al., 2006). Research has demonstrated that

the addition of chemical oxidants to wastewater

lagoons can reduce pathogen populations (Luster-

Teasley et al., 2011).

Biosecurity

Farm biosecurity is critical for animal health and

welfare, especially in regard to animal diseases, but

to date there has been little direct impact demon-

strated on foodborne pathogenic bacteria such as

E. coli O157:H7 (Ellis-Iversen and Van Winden, 2008).

Research has shown that other animal species, ro-

dents, insects and birds and boars can carry STEC at

least transiently (Branham et al., 2005; Cernicchiaro

et al., 2012; French et al., 2010; Rice et al., 2003; Sán-

chez et al., 2010; Wetzel and LeJeune, 2006). Mix-

ing of sheep with cattle has been shown to increase

the risk of cattle shedding STEC (Stacey et al., 2007),

and a positive correlation between cattle and sheep

density was found, at least in the UK (Strachan et al.,

2001). Other diverse factors such as the presence

of dogs, pigs, or wild geese on the farm have been

linked to an increased risk of E. coli O157:H7 shed-

ding (Gunn et al., 2007; Synge et al., 2003). Ruminant

animals other than cattle do carry E. coli O157:H7

(French et al., 2010; Hussein et al., 2000; Sargeant

et al., 1999), and this includes sheep and deer that

often share the same pasture, feed bunks and wa-

ter supplies (Bolton et al., 2012; Branham et al.,

2005). Other researchers have found that flies and

other insects on farms can carry STEC from one loca-

tion to another (Ahmad et al., 2007; Hancock et al.,

1998; Keen et al., 2006; Talley et al., 2009). Further-

more, wild migratory birds such as starlings (Carlson

et al., 2011a; Carlson et al., 2011b; Cernicchiaro et

al., 2012; Wallace et al., 1997; Wetzel and LeJeune,

2006), cowbirds and egrets (Callaway, unpublished

data) can carry STEC (and other foodborne patho-

gen) between pens, and even between farms long

distances apart. While these effects are probably

minimal in their direct impact on food safety within a

farm, they represent vectors for pathogens to move

between “clean” groups of cattle or farms.

Cattle grouping

Many farms are closed to entry by animals from

other farms to prevent animal disease transmission.

Closed herds prevent spread of E. coli O157:H7 (and

other pathogens) from one farm to another (Ellis-

Iversen et al., 2008; Ellis-Iversen and Van Winden,

2008; Ellis-Iversen and Watson, 2008). However

some studies have shown that a closed farm does

not impact E. coli O157:H7 incidence on farms (Cob-

baut et al., 2009). The results of this study suggest

that E. coli O157:H7 should be considered common

to groups of feedlot cattle housed together in pens

(Smith et al., 2001), thus keeping groups together

throughout their time on a farm, or in a feedlot, with-

out introducing new members to groups appears to

reduce horizontal transmission between animals.

A further benefit of grouping cattle involves the

use of age as a segregating factor. Young cattle (es-

pecially heifers) shed more E. coli O157:H7 than do

older cattle (Cobbaut et al., 2009; Cray and Moon,

1995; Smith et al., 2001). While it is not possible

to segregate calves from cows in the beef indus-

try, there is potential benefit to keeping same-age


groups of calves together as they are transported

and enter backgrounding or feedlot operations to

prevent horizontal transmission between groups.

Off-site rearing of dairy heifers may be an important

solution to reducing foodborne pathogens, as has

been shown in regard to Salmonella (Hegde et al.,

2005), and the risk of transmission back to the farm

by heifers returning from an off-site facility was found

to be low (Edrington et al., 2008).

Animal density may also play a role in the horizon-

tal spread of E. coli O157:H7 and other foodborne

pathogens (Vidovic and Korber, 2006) as well as the

vertical spread to humans (Friesema et al., 2011b).

Densely packed animals have a great chance of con-

tamination with fecal spread. However increased an-

imal density reduces the physical footprint and may

allow for more efficient and effective waste handling.

It has been shown that higher animal density can be

linked to an increased risk of carriage of some STEC,

including O157:H7 (Frank et al., 2008; Vidovic and

Korber, 2006). Other European studies have also

found an effect of animal density on human STEC

illnesses (Friesema et al., 2011a; Haus-Cheymol et

al., 2006), yet Canadian researchers found a variable

impact (Pearl et al., 2009). Further studies found

that increased stocking density increased shedding

of STEC, independent of group size (Stacey et al.,

2007; Strachan et al., 2006).

The issue of “supershedders” complicates re-

search into effects of animal density and pathogen

shedding (Arthur et al., 2009; Cernicchiaro et al.,

2010; Chen et al., 2012; LeJeune and Kauffman, 2006;

Stanford et al., 2005). If supershedders do exist long

term, rather than simply being a transient phase of

infection, then there are interactive effects of animal

density and pathogen density in the animal that must

be accounted for (Matthews et al., 2006; Matthews et

al., 2009). The role of super-shedding animals (even

if a transient phenomenon) cannot be discounted

in the contamination of hides during transport and

lairage, especially in dense conditions (Arthur et al.,

2010; Arthur et al., 2009).

Transportation and lairage

Handling and transport to processing plants or

feedlots or other farms causes stress (see below) and

may spread E. coli O157:H7 due to physical contact

or fecal contamination, and trailers used may spread

pathogens between lots or loads of cattle (Mather

et al., 2007). Studies have indicated that transport

did not affect STEC populations in cattle, however

in these studies E. coli O157:H7 populations were

very low initially (Barham et al., 2002; Minihan et

al., 2003; Reicks et al., 2007; Schuehle Pfeiffer et al.,

2009). However, other studies have found that trans-

port caused an increase in fecal shedding of E. coli

O157:H7 (Bach et al., 2004). Researchers found that

transporting cattle more than 100 miles doubled the

risk of having positive hides at slaughter compared to

cattle shipped a short distance, though the question

of time in close-confinement versus being in transit

was not examined (Dewell et al., 2008). In another

study, longer transport times were correlated with

increased levels of fecal shedding of E. coli O157:H7

(Bach et al., 2004). It was also demonstrated that a

combination of transport and lairage did not lead to

an increase in the number or prevalence of E. coli

O157:H7 from cattle (Fegan et al., 2009).

The presence of a high shedding animal in a

trailer has been shown to increase the odds of other

animals within the load being hide-positive for E.

coli O157:H7 (Arthur et al., 2010; Arthur et al., 2009;

Fox et al., 2008). However, it should be noted that

both low- and high-shedding cattle can be respon-

sible for the spread within and between truckloads

(Dodd et al., 2010). Cattle trailers can be important

fomites of E. coli O157:H7 to uninfected cattle and

are frequently positive for E. coli O157:H7 when

sampled (Barham et al., 2002; Cuesta Alonso et al.,

2007; Reicks et al., 2007). It has been shown that the

incidence of E. coli O157:H7 in transport trailers in-

creases the risk of transmission to farms and feedlots

from cattle on these trailers (Cuesta Alonso et al.,

2007). To date however, the washing of trailers has

only been shown to be effective against Salmonella

contamination in swine (Rajkowski et al., 1998), yet it

is an intuitive, feasible solution to prevent some de-

gree of cross-contamination of cattle during a stress-

ful period.


Lairage and holding facilities are further locations

that can impact the prevalence and concentration of

E. coli O157:H7 on hides of cattle, which is an impor-

tant route of entry to the food supply (Arthur et al.,

2007). Studies have shown that the transfer of E. coli

O157:H7 to hides that occurs in lairage at processing

plants accounted for more of the hide and carcass

contamination than did the population of cattle leav-

ing the feedlot (Arthur et al., 2008). Furthermore, the

presence of supershedding cattle in these pens can

increase the spread of E. coli O157:H7 between ani-

mals from different farms or feedlots (Arthur et al.,

2010; Cernicchiaro et al., 2010). The exact role of

lairage and transport/trailers in the spread of E. coli

O157:H7 (and other pathogens) in cattle is unclear,

and is likely time- and animal density-dependent,

and may also be affected by stress.

Stress

While we understand stress intuitively, any dis-

cussion of “stress” in animals is fraught with anthro-

pomorphism and complexity (Rostagno, 2009; Ver-

brugghe et al., 2012). Long-term stress may depress

immune function in cattle (Carroll and Forsberg,

2007; Kelley, 1980; Salak-Johnson and McGlone,

2007), making them more susceptible to coloniza-

tion, but the short term effects of stress from wean-

ing, handling or transport on immune status are un-

known. Catecholamines rise when animals are under

stress, and catecholamines (along with other hor-

mones) have been demonstrated to have an effect

on the microbial population, including pathogens

(Freestone and Lyte, 2010; Lyte, 2010; Walker and

Drouillard, 2012). To date the effect of stress on col-

onization or shedding of E. coli O157:H7 is unclear.

Weaning is stressful to calves, and was shown to

increase colonization with STEC (Herriott et al., 1998)

and E. coli O157:H7 (Chase-Topping et al., 2007). In

other studies however, these researchers demon-

strated that weaning does not affect the likelihood of

shedding (Synge et al., 2003). Interestingly, calving

was seen to reduce the likelihood of E. coli O157:H7

shedding (Synge et al., 2003). Further studies found

that weaning stresses alone did not impact shedding

of E. coli O157:H7 in dairy calves (Edrington et al.,

2011). Other stresses such as movement have been

identified as playing a role in E. coli O157:H7 shed-

ding in Scottish cattle (Chase-Topping et al., 2007),

but this has not been clearly defined in U.S. cattle

systems.

When calves were preconditioned to transport

stress, they were found to be less susceptible to in-

fection from the environment than were calves not

preconditioned to this stressor (Bach et al., 2004).

Cattle with excitable temperaments were less like-

ly to shed E. coli O157:H7 than were “calm” cattle

(Brown-Brandl et al., 2009; Schuehle Pfeiffer et al.,

2009). In studies with pigs, it was found that the so-

cial stress/excitement of mixing penmates increased

fecal shedding of Salmonella (Callaway et al., 2006),

but this has not been shown to date in cattle, how-

ever this implies a potential role of social stresses in

cattle during lairage.

Heat stress (and methods to alleviate it) can have

effects on animal health and productivity (Brown-

Brandl et al., 2003), as well as shedding of E. coli

O157:H7 and Salmonella (Callaway et al., 2006).

Water sprinkling to alleviate heat stress in cattle

increased measures of animal well-being and de-

creased E. coli O157:H7 populations on the hides of

cattle, but did not affect fecal populations (Morrow

et al., 2005). In another study with dairy cattle, re-

searchers found that heat stress had no impact on

STEC shedding, but Salmonella shedding was in-

creased (Edrington et al., 2004). Other researchers

have also found that heat stress did not impact E.

coli O157:H7 shedding in cattle (Brown-Brandl et al.,

2009).

CATTLE WATER AND FEED MANAGE-MENT

Diet and water supplies can be used to reduce

horizontal transmission of STEC between animals on

the same farm or in the same feedlot pen. The un-

derlying biology behind these effects has not been

elucidated to this point, but it has been suggested

that difference could be due to increased fecal pH or


intermediate endproducts of the yeast fermentation

(e.g., vitamins, organic acids, L-lactic acid), however

these suggestions remain hypothetical (Wells et al.,

2009). While the magnitude of the dietary impacts

effects is relatively small, it underlines the point that

dietary composition can potentially significantly im-

pact E. coli O157:H7 populations in the gut of cattle.

Drinking Water treatments

Cattle water troughs can harbor E. coli O157:H7

for long periods of time (Hancock et al., 1998;

LeJeune et al., 2001a; LeJeune et al., 2001b; Mu-

rinda et al., 2004; Polifroni et al., 2012; Wetzel and

LeJeune, 2006), and as many as 25% of cattle farm

water samples have been shown to contain E. coli

O157:H7 (Sanderson et al., 2006). These results sug-

gest that these common-use troughs can be vectors

for horizontal transmission of E. coli O157:H7 within

a group of animals. The organic material in the wa-

ter troughs tends to harbor and protect the STEC,

and modeling research has shown that an increase

in water trough cleaning frequency would increase

the death rate of E. coli O157:H7 (Vosough Ahmadi

et al., 2007) as well as exposure to sunlight (Jenkins

et al., 2011). Chlorination of water supplies has long

been used to reduce bacterial populations in mu-

nicipal water supplies, and this also can be used in

cattle water troughs to reduce E. coli O157:H7 pop-

ulations. However, sunlight and organic material in

the water reduces the effectiveness of chlorination

over time, as has been seen in real world chlorina-

tion studies with cattle water troughs (LeJeune et al.,

2004). Electrolyzed oxidizing (EO) water has been

shown to reduce STEC populations as high as 104

CFU/mL (Stevenson et al., 2004), and can be used as

an in-plant hide cleaning strategy (Bosilevac et al.,

2005). Other treatments such as cinnamaldehyde

and sodium caprylate addition to water supplies

have been shown to reduce STEC populations, but

the effects on palatability are not currently known

(Amalaradjou et al., 2006; Charles et al., 2008).

Fasting

Cattle can be fasted for up to 48 h before and dur-

ing their transport to slaughter, which can affect the

prevalence of E. coli O157:H7 (Pointon et al., 2012).

Ruminal and intestinal VFA concentrations limit the

proliferation of E. coli because of toxicity of the VFA

to the bacteria (Hollowell and Wolin, 1965; Russell

and Diez-Gonzalez, 1998; Wolin, 1969). This has cre-

ated the demand for the use of organic acids/VFA

as methods to alter the ruminal fermentation and to

reduce pathogen populations in the gut (Ohya et al.,

2000; Prohaszka and Baron, 1983; Van Immerseel et

al., 2006). However, fasting causes levels of VFA to

decline rapidly (Harmon et al., 1999).

Fasting increased E. coli, Enterobacter and total

anaerobic bacterial populations throughout the in-

testinal tract of cattle (Buchko et al., 2000b; Greg-

ory et al., 2000), and increased Salmonella and E.

coli populations in the rumen (Brownlie and Grau,

1967; Grau et al., 1969). More recent research has

demonstrated that fasting can cause “apparently

E. coli (O157:H7) negative animals to become posi-

tive” (Kudva et al., 1995). Fasting made calves more

susceptible to colonization by inoculated E. coli

O157:H7 (Cray et al., 1998). Cattle fasted for 48 h

prior to slaughter contained significantly greater

E. coli populations throughout the gut than cattle

fed forage (Gregory et al., 2000). In contrast, it was

demonstrated that a fasting period had no effect

on E. coli O157:H7 shedding (Harmon et al., 1999).

When culled dairy cows were reconditioned through

feeding high energy diets for 28 d before harvest,

the prevalence of E. coli O157:H7 declined from

14% to 6% (Maier et al., 2011). In general, studies

examining the intestinal environment have repeat-

edly indicated that low pH and high concentrations

of short chain VFA result in lower STEC populations

(Bach et al., 2002a; Bach et al., 2005b; Cobbold and

Desmarchelier, 2004; Pointon et al., 2012; Shin et al.,

2002). Thus the bulk of research supports the con-

cept that fasting increases shedding or population

concentrations, or makes cattle more susceptible to

colonization due to decreased short chain VFA and

increased pH in the gastrointestinal tract. Because

feed withdrawal and/or starvation results in de-

creased VFA concentrations in the gut, it has been


suggested that this shift plays a role in the effects of

transport and/or lairage on the shedding of STEC.

Feed types

The first dietary practice shown in early studies

to significantly increase the risk of STEC shedding

among heifers was feeding corn silage (Herriott et

al., 1998). In adult cows, the inclusion of animal by-

products in the diet (currently discontinued) was

shown to increase STEC shedding (Herriott et al.,

1998). Other studies linked feeding whole cotton-

seed with reduced E. coli O157 shedding (Garber et

al., 1995; Hancock et al., 1994). Fecal samples from

cattle fed dry rolled corn, high-moisture corn and

wet corn gluten feed did not contain different popu-

lations of generic E. coli, or extreme acid-resistant E.

coli during a limit-feeding period (Scott et al., 2000).

However, feces from cattle fed wet corn gluten ad

libitum contained significantly higher concentrations

of extreme acid resistant E. coli (resistant to an acid

shock simulating passage through the human stom-

ach) than did feces of cattle fed dry-rolled or high

moisture corn (Scott et al., 2000).

Barley is often fed to cattle and is ruminally fer-

mented more rapidly than corn by the commensal

microbial population. More starch is fermented in

the lower gut of corn-fed cattle than in barley-fed

cattle, resulting in barley-fed cattle having higher fe-

cal pH and lower VFA concentrations compared with

corn-fed animals (Bach et al., 2005a; Berg et al., 2004;

Buchko et al., 2000a). Barley feeding was linked (al-

beit at a low correlation) to increased E. coli O157:H7

shedding (Dargatz et al., 1997); and in experimen-

tal infection studies barley feeding was again asso-

ciated with increased shedding of E. coli O157:H7

by feedlot cattle (Buchko et al., 2000a). Survival of

E. coli O157:H7 in manure from corn-and barley fed

cattle is approximately equal, therefore simple sur-

vival in the feces is not responsible for the increased

prevalence of E. coli O157:H7 in barley-fed cattle

(Bach et al., 2005b).

Distiller’s grains

The industrial fermentation of corn to produce

ethanol has increased more than 4-fold between

2001 and 2007, and its use doubled by 2010 (Rich-

man, 2007). Thus, an economic incentive to increase

the utilization of distillers grains (DG) by-product

feeds in the cattle industry has increased dramati-

cally in recent years, especially given DG’s role as

cost-effective feed supplements for finishing and

lactating cattle (Firkins et al., 1985). The inclusion

DG into cattle rations has been shown to be an ef-

fective replacement for common feedstuffs and has

demonstrated an increased daily gain in beef cattle

(Al-Suwaiegh et al., 2002) and milk yield and feed ef-

ficiency in dairy cows (Kleinschmit et al., 2006). This

improvement is likely due to the fact that DG alters

the population structure and function of the micro-

bial ecosystem of the rumen and throughout the

gastrointestinal tract (Callaway et al., 2010a; Durso

et al., 2012; Williams et al., 2010b). Cattle fed 40%

corn wet distiller’s grains (WDG) were very different

than the fecal populations in cattle fed a non DG-

containing diet, and populations of generic E. coli

were higher in their feces (Durso et al., 2012), and in

previous studies the survival of E. coli O157:H7 in fe-

ces was increased by increasing levels of DG supple-

mentation (Varel et al., 2008).

Unfortunately, research has suggested a potential

association between DG feeding and an increased

prevalence and fecal shedding of the foodborne

pathogen E. coli O157:H7 in cattle (Jacob et al.,

2008a; Jacob et al., 2008b; Yang et al., 2010). Distill-

ers grains were shown to increase the shedding of

E. coli O157:H7 in cow-calf operations in Scotland

(Synge et al., 2003). Other researchers found that

feeding a related product (brewers grain) to cattle

was also associated with increased E. coli O157

shedding, and increased the odds of shedding by

more than 6-fold (Dewell et al., 2005). The individual

animal prevalence of feedlot cattle shedding E. coli

O157 on d 122 (but not d 136) was higher in cattle

fed 25% wet distiller’s grain compared to control di-

ets lacking WDG (Jacob et al., 2008b), but the pen-

level shedding was unaffected by WDG feeding.

Pen floor fecal sample prevalence of E. coli O157 was

significantly higher across a 12 week finishing period


in cattle fed 25% DDG and either 15% or 5% corn

silage compared with cattle fed 0% DDG and 15%

corn silage (Jacob et al., 2008a). However, follow-up

studies found no differences in E. coli O157:H7 fe-

cal shedding in cattle fed DG (Edrington et al., 2010;

Jacob et al., 2009), with no indications of why this

difference in results was observed. In a further study

utilizing both dry and wet-distillers grains, research-

ers found that higher levels (40% of the ration) of DG

inclusion did increase fecal E. coli O157:H7 shedding

(Jacob et al., 2010). When cattle were fed 40% wet

DG, they had higher populations of E. coli O157:H7

as well as higher pH values and lower concentra-

tions of L-lactate (Wells et al., 2009). Further studies

found that the DG-associated increase in fecal E. coli

O157:H7 populations could be mitigated by reduc-

ing WDG concentrations to 15% or less for 56 d prior

to slaughter (Wells et al., 2011). When corn or wheat

DDG were supplemented into cattle on a primarily

barley-based diet, there was no difference in impact

of DDG supplementation, likely because barley in-

clusion had already increased the E. coli O157:H7

populations through some complementary mecha-

nism (Hallewell et al., 2012). Interestingly, research-

ers found that the numbers of E. coli O157:H7 were

greater in fecal in vitro incubations that contained

corn DG than with wheat DG (Yang et al., 2010).

Grain form

Other scientists have examined the form of corn

included in cattle rations can impact E. coli O157:H7.

In feedlot cattle, steam-flaked grains increased E.

coli O157 shedding in feces compared to diets com-

posed of dry-rolled grains (Fox et al., 2007). This

difference was theorized to be due to dry rolling al-

lowing the passage of more starch to the hindgut

where it was fermented to produce VFA thereby kill-

ing E. coli O157 (Fox et al., 2007). This theory is sup-

ported by the fact that post-ruminal starch infusion

increased generic E. coli populations in the lower

gut numerically (Van Kessel et al., 2002). However, to

date studies have shown no effect on E. coli O157:H7

populations of increasing starch concentrations in

the diet (Nagaraja, T. G., personal communication)

or by increasing fecal starch concentrations (Depen-

busch et al., 2008).

Forage feeding

Escherichia coli can and does thrive in the lower

gut of animals fed forage diets (Hussein et al., 2003a;

Hussein et al., 2003b; Jacobson et al., 2002). Com-

paring grain-fed to forage-fed cattle indicates that

more E. coli (including O157:H7) are present in the

feces of cattle fed grain diets. The effects of high

grain or high forage diets on the duration or shed-

ding of fecal E. coli O157:H7 populations in experi-

mentally inoculated calves have been examined. In

these studies the calves that consistently shed the

highest concentrations of E. coli O157:H7 were fed

a high concentrate (grain) diet (Tkalcic et al., 2000).

Ruminal fluid collected from steers fed a high-forage

diet allowed E. coli O157:H7 to proliferate to higher

populations in vitro than did ruminal fluid from high-

grain fed steers (Tkalcic et al., 2000). This was pos-

sibly due to differences in VFA concentrations be-

tween the ruminal fluids.

Other researchers found that feeding forage ac-

tually increased the shedding of E. coli O157:H7 in

cattle (Van Baale et al., 2004). When cattle were fed

forage E. coli O157:H7 was shed for 60 d compared

to 16 d for cattle on a grain-based diet (Van Baale et

al., 2004). Studies examining the effects of forage

on survival of E. coli O157:H7 in manure found that

low quality forages caused a faster rate of death of

E. coli O157 populations (Franz et al., 2005), indicat-

ing a possible role of forage chemical or secondary

plant components (such as tannins, see below) in fe-

cal shedding (Min et al., 2007). Feces from cattle fed

grain had higher VFA concentrations and lower pH

which allowed E. coli O157:H7 populations to survive

longer than feces from grass-fed cattle (Lowe et al.,

2010). Other studies have found that feeding forage

rich secondary compounds such as sainfoin, might

be a method to manipulate fecal populations of E.

coli O157:H7 to a limited extent (Aboaba et al., 2006;

Berard et al., 2009).

Although E. coli O157:H7 populations are gen-

erally lower in cattle fed forage diets, it must be


emphasized that STEC are still isolated from cattle

solely fed forage, so forage feeding should not be

viewed as a magic bullet (Hussein et al., 2003b; Thran

et al., 2001). Many outlets have claimed that grass-

fed cattle contain fewer pathogens than do cattle

fed grain; however this has not been demonstrated

scientifically. Researchers have found no difference

in food safety parameters of beef from grass-fed cat-

tle versus grain fed cattle (Zhang et al., 2010). Fur-

thermore, research into organic versus conventional

rearing systems have demonstrated no difference in

the incidence of E. coli O157:H7 shedding (Jacob et

al., 2008c; Reinstein et al., 2009).

Dietary shifts

A sudden shift from grain to hay appears to cause

a severe, widespread disruption in the gut microbial

flora population, much like an earthquake in a mac-

robiological environment (Fernando et al., 2010).

Thus the effects of rapid dietary shifts on the micro-

bial population in regards to E. coli O157:H7 popula-

tions have been examined. Early studies investigat-

ing (generic) E. coli and dietary effects indicated that

a sudden decrease in hay intake by cattle increased

fecal E. coli populations (Brownlie and Grau, 1967).

Other studies using experimentally infected sheep

found a sudden switch from an alfalfa pellet diet or

a corn/alfalfa ration to a poor-quality forage diet

increased E. coli O157:H7 shedding (Kudva et al.,

1995; 1997).

Cattle fed feedlot-type ration contained (generic)

E. coli populations that were 1000-fold higher than

cattle fed a 100% good-quality hay diet (Diez-Gon-

zalez et al., 1998). When these cattle were abruptly

switched from a 90% grain finishing ration to a 100%

hay diet, fecal E. coli populations declined 1000-fold

within 5 d (Diez-Gonzalez et al., 1998). However,

it is important to note that in this study no E. coli

O157:H7 were detected. Based on these results

the authors suggested that feedlot cattle could be

switched from high grain diets to hay for 5 days prior

to slaughter to reduce E. coli contamination entering

the abattoir (Diez-Gonzalez et al., 1998). Research

indicated that a brief (5 d) period of hay-feeding did

not impact carcass characteristics; however, when

cattle were fed hay during the final portion of the fin-

ishing period, they had lower dry matter intake and

lost 2.2 lb/head/d (Stanton and Schutz, 2000). Hay

feeding did not significantly impact carcass weight,

dressing percentage, carcass grades, or quality pa-

rameters, but significantly reduced total coliform

counts and (generic) E. coli counts (Stanton and

Schutz, 2000), but the impact was not as large as that

reported by Diez-Gonzalez et al. (1998). Cattle fed

hay for a 48 h period immediately prior to transport

to slaughter did not lose more weight during trans-

port than fasted or pasture fed animals (Gregory et

al., 2000). Cattle with a natural E. coli O157:H7 in-

fection (53%) were divided into two groups and one

was fed grain and the other abruptly switched to

hay, 52% of the grain-fed controls remained E. coli

O157:H7 positive, but only 18% of the hay-fed cattle

continued to shed E. coli O157:H7; but this switch re-

sulted in a BW decrease of 1.25 lb/hd/d compared to

controls (Keen et al., 1999). Other researchers found

that cattle fed a high-concentrate diet and switched

to a diet containing 50/50% corn silage/alfalfa hay

diet had lower E. coli counts (0.3 log10) after just 4

days (Jordan and McEwen, 1998). Cattle that were

fed an 80% barley ration, fasted for 48 h and then

subsequently switched to 100% alfalfa silage did

not exhibit any change in E. coli O157:H7 shedding

(Buchko et al., 2000b). However, when these same

animals were again fasted for 48 h and re-fed alfalfa

silage, the prevalence of E. coli O157:H7 shedding

increased significantly (Buchko et al., 2000b). Re-

searchers found that experimentally-infected cattle

fed hay shed E. coli O157:H7 significantly longer

than did grain-fed cattle (42 d vs. 4 d), but E. coli

O157:H7 populations shed were similar between

diets (Hovde et al., 1999). Cattle abruptly switched

from a finishing diet that contained wet corn gluten

feed to alfalfa hay for 5 d showed an increase in co-

lonic pH and total E. coli populations decreased ap-

proximately 10-fold (Scott et al., 2000).

Conversely, it was found that when cattle were

switched from forage-type diets to a high grain fin-

ishing ration, fecal and ruminal generic E. coli con-

centrations increased (Berry et al., 2006). In another


study slightly outside of the “normal” dietary switch

structure, switching cattle from pasture to hay for 48

h prior to slaughter significantly reduced the E. coli

population throughout the gut (Gregory et al., 2000).

Gregory et al., found that hay feeding increased in-

testinal Enterococci populations that are capable of

inhibiting E. coli populations in a fashion similar to

that of a competitive exclusion culture. However,

the effects of high grain versus forage diets were not

examined in this New Zealand-based study (Greg-

ory et al., 2000). Based on their data, the authors

concluded, “the most effective way of manipulating

gastro-intestinal counts of E. coli was to feed hay”

(Gregory et al., 2000).

Collectively, these results emphasize that while

dietary manipulations such as shifting cattle from

a high grain to forage ration could be a power-

ful method to reduce E. coli/STEC populations in

cattle prior to harvest, the mechanism remains un-

known and the effect is very inconsistent. It appears

that a factor in this inconsistency involves forage

quality and type, but this remains a hypothesis. It

does appear that the presence of endproducts of

fermentation (e.g., VFA) and some secondary plant

compounds in forages play some role in pathogen

population levels. While a dietary switch to forage

in feedlots is not advocated due to feasibility, weight

loss and other logistical issues, other high fiber feed-

stuffs (e.g., soy hulls, cottonseed meal) or feedstuffs

rich in phenolics or essential oils (see below), may be

a more feasible alternative strategy to decrease in E.

coli O157:H7 populations.

Tannins, phenolics, and essential oils

Plants contain phenolic and polyphenolic com-

pounds, such lignin and tannins, that can affect the

microbial ecosystem of the gastrointestinal tract

through antimicrobial action (Berard et al., 2009;

Cowan, 1999; Hristov et al., 2001; Jacob et al., 2009;

Patra and Saxena, 2009). It is theorized that some of

these compounds may penetrate biofilms and have

an anti-quoroum-sensing effect, which may play a

role in STEC colonization (Edrington et al., 2009b;

Kociolek, 2009; Sperandio, 2010). Tannins have been

demonstrated to significantly inhibit the growth of E.

coli O157:H7 in vitro and generic E. coli populations

in cattle (Berard et al., 2009; Cueva et al., 2010; Min

et al., 2007; Wang et al., 2009). Other researchers

have found that the phenolic acids that comprise lig-

nin also demonstrated antimicrobial activity against

E. coli O157:H7 in fecal slurries, and highly lignified

forages showed a reduced period of E. coli O157:H7

shedding compared with cattle fed only corn silage

(Wells et al., 2005). Phenolic compounds in cranber-

ry extract and sorrel are also effective against E. coli

O157:H7 growth in vitro (Caillet et al., 2012; Fullerton

et al., 2011), also the anthocyanins/proanthocyani-

dins from lowbush blueberries demonstrated in vitro

potential to inhibit E. coli O157:H7 growth (Lacombe

et al., 2012).

Essential oils are most often associated with aro-

matic compounds in various plants used as spices

or extracts (Barbosa et al., 2009). Many of these es-

sential oils exhibit antimicrobial acitivity (Dusan et

al., 2006; Fisher and Phillips, 2006; Kim et al., 1995;

Pattnaik et al., 1996; Reichling et al., 2009; Turgis et

al., 2009), often through the mode of action of dis-

solving bacterial membranes (Di Pasqua et al., 2007;

Turgis et al., 2009). As a result, many plant prod-

ucts have been used for centuries for the preserva-

tion and extension of the shelf life of foods (Dab-

bah et al., 1970). Essential oils have been proposed

as potential modifiers of the ruminal fermentation

(Benchaar et al., 2008; Benchaar et al., 2007; Boadi

et al., 2004; Patra and Saxena, 2009) and to reduce

E. coli O157:H7 in the live animal via in vitro studies

(Benchaar et al., 2008; Jacob et al., 2009). Some es-

sential oils have been shown to penetrate biofilms

and kill E. coli O157:H7 (Pérez-Conesa et al., 2011),

which could potentially play a role in reducing colo-

nization in the rumen and/or terminal rectum.

Seaweed (Tasco)

Brown seaweed (Tasco-14) is a feed additive that

has been included in cattle diets to improve carcass

quality characteristics and shelf life, increase anti-

oxidants and to improve ruminal fermentation ef-

ficiency (Anderson et al., 2006; Braden et al., 2007;


Leupp et al., 2005). In vitro studies have indicated

that Tasco-14 can reduce populations of E. coli and

Salmonella (Callaway, unpublished data), and more

recent results have linked this antipathogen activity

to presence of phlorotannins in the brown seaweed

(Wang et al., 2009). The phlorotannin anti- E. coli

activity was greater than that found in other studies

with terrestrial tannin sources (Min et al., 2007; Wang

et al., 2009). Studies in vivo found that Tasco-14

feeding reduced fecal and hide prevalence of E. coli

O157 in cattle (Braden et al., 2004). Because Tas-

co-14 is currently available in the market place, this

is a product that can be included in cattle rations;

however the extent of anti-pathogen activity in vivo

is still not clear, therefore the cost of addition must

be weighed carefully by the producer.

Citrus products

Orange peel and citrus pulp have excellent nutri-

tional characteristics for cattle and have been includ-

ed as low-cost ration ingredients in dairy and beef

cattle rations for many years (Arthington et al., 2002).

Citrus fruits contain a variety of compounds, includ-

ing essential oils and phytophenols that exhibit an-

timicrobial activity against foodborne pathogens

(Friedly et al., 2009; Mkaddem et al., 2009; Nannapa-

neni et al., 2008; Viuda-Martos et al., 2008). Other

studies have found that limonids from grapefruit

may play a role in inhibiting secretion and intercel-

lular communication by E. coli O157:H7 (Vikram et

al., 2010).

Research has demonstrated that the addition of >

1% orange peel and pulp reduced populations of E.

coli O157:H7 and Salmonella Typhimurium in mixed

ruminal fluid fermentations in the laboratory (Calla-

way et al., 2008; Nannapaneni et al., 2008). Further

studies have demonstrated that feeding orange peel

and pulp reduced intestinal populations of diarrhea-

genic E. coli in weaned swine (Collier et al., 2010).

In ruminants, researchers demonstrated that feeding

of orange peel and citrus pellets (a 50/50 mixture)

at levels up to 10% DM reduced artificially inoculat-

ed populations of E. coli O157:H7 and Salmonella

Typhimurium in sheep (Callaway et al., 2011a; b).

When studies were performed using only dried pel-

leted orange peel, the reduction in pathogen popu-

lations disappeared (Farrow et al., 2012), likely due

to the inactivation of essential oils (limonene and

terpeneless fraction) during the pelleting process.

Continuing studies have demonstrated that orange

oils offer a potential method for reducing both STEC

and Salmonella on beef carcasses as well (Pendleton

et al., 2012; Pittman et al., 2011). To date, orange

peel feeding has not been examined in large-scale

feeding studies, but retains promise as a potential

on farm strategy to reduce the burden of pathogens

on the farm, reducing environmental contamination

and re-infection.

Organic acids

Organic acids have been used in animal nutrition

to modify the ruminal fermentation by providing

some members of the microbial ecosystem a com-

petitive advantage, and by inhibiting other species

(Grilli et al., 2010; Martin and Streeter, 1995; Nisbet

and Martin, 1993; Piva et al., 2007). Some organic

acids (such as lactate, acetate, propionate, malate)

have been shown to have antimicrobial activity

against E. coli O157:H7 (Harris et al., 2006; Sagong

et al., 2011; Vandeplas et al., 2010; Wolin, 1969).

These acids have been used on hide and carcass

washes to reduce pathogen populations, but only

recently has interest in using organic acids to reduce

pathogens in live animals received interest (Callaway

et al., 2010b; Nisbet et al., 2009). Preliminary results

do show some success in inhibiting pathogens in the

lower intestinal tract of animals (unpublished data),

however, further research needs to be performed to

be able to release the appropriate organic acid and

concentration in the appropriate intestinal location

to reduce populations of E. coli O157:H7 in cattle.

Ractopamine

ß-agonists, such as ractopamine, are used in

cattle to improve animal performance and carcass

leanness. In vitro, ractopamine showed no effect on

growth parameters of E. coli O157:H7 (Edrington et


al., 2006c); but when used in sheep, the fecal shed-

ding and cecal populations of E. coli O157:H7 were

increased (Edrington et al., 2006c). When feedlot

cattle were fed ractopamine, the numbers of cattle

shedding E. coli O157:H7 were decreased (Edring-

ton et al., 2006b). In a follow-up study, researchers

demonstrated a negligible effect of ß-agonist (rac-

topamine and zilpaterol) treatment on fecal shed-

ding of E. coli O157:H7 in cattle (Edrington et al.,

2009a; Paddock et al., 2011). Taken as a whole, these

results indicate that the effects of ß-agonist feeding

are minimal or non-existent. Interestingly however,

in an in vitro swine model norepinephrine was shown

to increase E. coli O157:H7 adherence (Green et al.,

2004), though further research is obviously needed

to determine if this applies to cattle colonization.

Ionophores

Ionophores, such as monensin and lasalocid, are

antimicrobial compounds included in most feedlot

and dairy rations to inhibit gram-positive bacteria,

thereby improving feed:gain ratios and production

efficiency (Callaway et al., 2003). Because these feed

additives affect the gram-positive portion of the mi-

crobial population, possibly giving gram-negative

bacteria (such as E. coli) a competitive advantage,

they have been investigated as to their role in the

spread of E. coli O157:H7 in cattle. Because E. coli

O157:H7 has a true gram-negative membrane physi-

ology ionophores did not affect the growth of this

pathogen in vitro when added at concentrations up

to 3 fold higher than those normally found in the ru-

men (Bach et al., 2002b; Van Baale et al., 2004).

Early studies demonstrated a marginal increase of

STEC shedding by heifers fed ionophores (Herriott et

al., 1998), but other studies found no effect (Dargatz

et al., 1997). Further studies examining the effect of

ionophoric feed additives (monensin, lasalocid, laid-

lomycin and bambermycin) on E. coli O157:H7 dem-

onstrated no effect of these additives in vitro (Edring-

ton et al., 2003b), or on fecal shedding or intestinal

populations in experimentally-inoculated lambs in a

short-term (12 d) trial (Edrington et al., 2003a). In an

in vivo study using cattle, it was found that cattle fed

a forage ration that included monensin shed E. coli

O157:H7 for a shorter period of time than forage-fed

cattle not supplemented with monensin, but monen-

sin had no effect on shedding when cattle were fed

a corn-based ration (Van Baale et al., 2004). In an in

vitro study, it was found that monensin and the co-

approved antibiotic tylosin (tylan) treatment reduced

E. coli O157:H7 populations up to 2 log10 CFU/mL

in ruminal fermentations from cows fed forage, but

this did not extend to E. coli O157:H7 populations

in ruminal fluid from cows fed corn (McAllister et al.,

2006). These researchers later found that the inclu-

sion of monensin and tylosin did not alter fecal shed-

ding of experimentally-inoculated E. coli O157:H7

when included in barley (grain)-based diet fed to

cattle (McAllister et al., 2006). These results suggest

there may a potential interaction between diet and

ionophore inclusion in the effects on E. coli O157:H7

populations. Further studies found that monensin

decreased E. coli O157:H7 prevalence when fed at

44 mg/kg of feed, compared to the typical 33 mg/kg

dosing (Paddock et al., 2011).

CONCLUSIONS

While STEC of many serotypes can be viewed as

a commensal organism in the gastrointestinal tract

cattle, they represent a significant threat to human

consumers and public health. Pre-harvest controls in

cattle hold great potential to reduce STEC dissemi-

nation on farms, in the environment, and entering

the food chain. However, none of the on farm man-

agement-based controls discussed herein will com-

pletely eliminate STEC from cattle and will certainly

not eliminate the need for proper procedures in the

processing plant. Instead the live-animal manage-

ment controls must be installed in a complementary

fashion to reduce pathogens in a multiple-hurdle

approach (Nastasijevic, 2011) that complements the

in-plant interventions as well, so that the reduction

in pathogen entry to the food supply can be maxi-

mized.


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ABSTRACT

A survey was presented to Georgia independent poultry farmers to evaluate current processing options

as well as desired future changes. A total of 82 Georgia farmers participated in the survey, 31 of whom

were raising broilers at the time of the survey. Most of the farmers surveyed who were growing broilers at

the time (81%) processed on-farm, but these were also the farmers who processed less than 1000 birds per

year. The larger independent Georgia farmers processed off-farm in South Carolina and Kentucky, where

there were processors that served small-scale farmers and provided USDA inspection. These out of state

processing trips took place between 4 and 30 times per year for an average of 391 miles round trip. For

farmers’ future needs, similar numbers of farmers wanted only on-farm or only off-farm drop off processing

(22% and 25% respectively), but 40% of the farmers surveyed were open to more than one processing op-

tion. The farmers were also asked to evaluate the importance of several attributes of processing facilities,

and they chose quality of service to be the most important processing facility attribute, followed by cost of

processing, distance from the farm, and USDA inspection.

Keywords: Poultry processing, independent growers, pastured poultry, mobile processing units,

on-farm processing

INTRODUCTION

The U.S. broiler industry produced more than 8.6

billion broilers in 2009, estimated at a retail equiva-

lent of $45 billion (ERS, 2011). Georgia is the number

one broiler production state producing 1.3 billion

Correspondence: Steven C. Ricke, [email protected]: +1-479-575-4678 Fax: +1-479-575-6936

broilers in 2009, accounting for 15% of the total U.S.

broiler production (ERS, 2011). Federal laws exempt

farms processing fewer than 20,000 birds per calen-

dar year from USDA bird-by-bird inspection (USDA,

2006). However, some states have laws that are strict-

er and prohibit this exemption, which is the case in

Georgia. The small-scale poultry industry in Georgia

is limited in growth due to challenges it faces im-

posed by the strict state rules. Without the exemp-

Independent Poultry Processing in Georgia: Survey of Producers’ Perspective

E. J. Van Loo1,2, W. Q. Alali3, S. Welander4, C. A. O’Bryan1, P. G. Crandall1, S. C. Ricke1

1Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR2Present address: Department of Agricultural Economics, Faculty of Bioscience Engineering,

Ghent University, Ghent, Belgium3Center for Food Safety and Department of Food Science & Technology,

University of Georgia, Griffin, GA4Georgia Organics, 200-A Ottley Drive, Atlanta, GA



tion, small farmers who process more than 1,000

birds/year have to meet the federal inspection re-

quirements for commercial processors. Procedures

and practices need to be well-documented and in-

clude documents such as Standard Operating Pro-

cedures and Hazard Analysis Critical Control Point

(HACCP) plans. Complying with these regulations

to meet federal inspection standards is costly but is

manageable for large commercial processors. How-

ever, it is too costly for small farmers and the farmers

may also need help in developing all the required

documentation (Geering, 2011a,b).

The poultry industry in the U.S. is strongly verti-

cally integrated, leaving limited options for off-farm

processing for small-scale poultry farms. At the time

of conducting the survey in Georgia, the only avail-

able processing facilities were owned by the large

poultry companies, and there were no independent

federally inspected processing facilities. Thus, the

Georgia farmers who processed more than 1,000

birds/year had to travel to other states such as South

Carolina and Kentucky to process their birds. This

results in additional travel and accommodation ex-

penses, requires additional time and results in a tre-

mendous reduction in profitability for the grower.

This lengthy transportation also increases the envi-

ronmental impact of the locally grown poultry prod-

ucts and may have negative effects on the animal

welfare which are both important to many of the lo-

cal poultry product consumers.

The local Georgia poultry farmers’ community is

in need of safe and legal processing options, both

on- and off-farm, that provide both easy access to

poultry processing for farms throughout the state

and ensure a safe product for the Georgia consum-

ers. The purpose of this survey was to evaluate pro-

cessing options for the independent local poultry

farms in Georgia for small-scale poultry processing

that would (1) ensure safe products for Georgia con-

sumers, (2) meet farmer needs for various safe pro-

cessing options, both on- and off-farm, (3) provide

easily accessible poultry processing options, (4) lay

the foundation for future growth of pastured poultry

production in Georgia.

MATERIALS AND METHODS

Notice of the survey was posted in the Georgia

Organics print newsletter, and in an electronic news-

letter, as well as by a targeted email to a list of in-

terested parties amassed based on connections

Georgia Organics made at conferences and meet-

ings. The link to the survey was also posted on Geor-

gia Organics’ website. A total of 82 Georgia farm-

ers took the survey between September of 2008

and July of 2010. The survey consisted of questions

about (i) current processing methods; (ii) interest in

other processing options; (iii) importance of different

processing facilities attributes; (iv) demographics of

the farm. Farmers were asked to rate importance of

attributes on a Likert scale from 1 (not important) to

7 (very important). Table 1 contains the questions

and possible answers. Frequency tables, mean val-

ues and standard deviations were determined using

JMP (release 9.0.0: SAS Institute, Inc.).

RESULTS AND DISCUSSION

Current Processing Methods

A total of 82 Georgia farmers participated in the

survey, of which 31 farmers were raising broilers at the

time of the survey totaling 37,642 birds/year. Most

of the farmers who were currently growing broilers

(28 of the 31) processed on-farm but grew only 43%

of the total pastured birds produced by the respon-

dents in Georgia each year (Figure 1, Figure 2). The

majority (60%) of the 28 farmers that processed on-

farm produced less than 500 birds/year.

Pastured poultry is actually not a new concept;

until the 1950’s, all poultry was raised outdoors (Sus-

tainable Agriculture Network, 2006). The renewed

interest in pastured poultry has served to highlight

the lack of suitable processing available to small pro-

ducers. With the vertical integration of the industry,

poultry processing is highly consolidated and not

available to independent producers (Heffernan and

Hendrickson, 2002). This lack of processing is a real

barrier and greatly restricts the ability of pastured


Table 1. Farmers survey questions and possible answers

1. Indicate the number of broilers you are currently processing per year at the different processing sites

listed. a) On-farm b) USDA-inspected facility in South Carolina c) USDA-inspected facility in Kentucky d)

Other off-farm processing facility (please describe) e) Not currently processing (enter 0)

2. If you currently process birds off-farm, please answer the following questions: (Enter your response in

numbers only, with no dollar signs. Decimal points are ok.) a) Roundtrip driving miles to the facility b) Num-

ber of trips made per year c) Average # of birds processed per visit c) Approximate processing cost per bird

charged by facility

3. Please indicate the annual number of broilers that you would like to raise under the following processing

scenarios. Please enter your response in numbers only. a) Processing on-farm b) Shared-use facility with USDA

inspection c) Drop Off/Pick Up facility with USDA inspection d) Contract grower

4. Do you want to sell your poultry direct to consumers, under your own farm’s label? a) Yes, I want to di-

rect market my own processed poultry b) No, I want to raise pastured poultry but have someone else sell it to

the consumer c) I want to pursue both options d) Not sure e) I am not interested in raising pastured poultry at

this time.

5. What is the maximum distance you would be willing to drive for processing?

6. Rate the importance of the following items in considering a Georgia based processing facility where 7 =

Very important, and 1 = Not important at all. Distance from farm, Processing price, Quality of service, Cer-

tified organic, USDA Inspection, Marketing services

7. If you were paying a facility to process your chickens for you (Drop off/pick up arrangement), at what

price per bird processed would this service be a) Absolutely too expensive to ever use? b) Getting expensive?

c) Inexpensive? d) A great value for the money? e) So cheap I would not trust what was happening in there.

8. Are you interested in participating in a co-op of producers to explore: a) processing options? b) Other

infrastructure issues? c) What other issues/comments do you have?

9. Where is your farm located? Please provide answers to at least two of the following options. State:

County: City/Town:


Figure 1. Percentages of current place of processing for 31 Georgia (GA) pastured poultry farm-ers

81%

16%3%

On-farm in GA

Off-farm SC

Off-farm KY

Figure 2. Percentage of current poultry processing of 31 Georgia farmers by total broiler pro-cessed per year (total 37,642 broilers/year)

43%

50%

7%

On-farm in GA

Off-farm SC

Off-farm KY


Figure 3. Type of processing preferred by Georgia pastured poultry farmers (n=82) when pre-sented with multiple choice options: 1) on-farm, 2) shared-use/rental facility, 3) drop off/pick up, 4) contract grower facility. Farmers had the choice to select/be interested in some of the 4 or all 4 processing options.

0

10

20

30

40

50

60

On-farm Shared-use Drop-off Contract

# Fa

rmer

s

All 4 methods

Shared-use + drop-off + contract

On-farm + shared-use + drop-off

On-farm + shared-use

Contract + drop-off

Shared + drop-off

On-farm + drop-off

Only this method


poultry producers to sell and market their products;

if a farmer wants to slaughter more than 1,000 birds a

year, they must meet stringent inspection standards

established by the U.S. Department of Agriculture

for large-scale poultry processing operations. Farm-

ers raising up to 1,000 birds per year may slaughter

on their own farms and sell the whole birds on site

directly to consumers, or sell frozen birds directly to

consumers at farmers markets, provided they have

a mobile vehicle license. These on farm processors

generally consist of family members, are able to pro-

cess about 50 to 100 birds per day, and the work is

very labor intensive (Fanatico, 2003).

Some of the Georgia farmers (19% of the 31 grow-

ing broilers at the time of the survey) processed off-

farm in South Carolina and Kentucky, where there

were processors that serve small-scale farmers and

provide USDA inspection (Figure 1). These were the

larger farms that processed more than 1,000 birds/

year each, and raised 57% of the total amount of

birds raised each year (Figure 2). These farmers trav-

eled between 4 and 30 times per year for an aver-

age of 391 miles round trip to processing facilities

out of state. Not only do the long travel times and

distances cost farmers’ time and money, it may also

adversely affect the survival or quality of the birds

themselves. Vecerek et al. (2006) found that mortality

was higher in broilers transported up to 185 mi com-

pared those transported up to 30 mi (0.9% v. 0.2%).

Warriss et al. (1992) examined the effects of time

of transport and found higher mortality for broilers

transported for 9 h as compared to 4 h (0.3% v. 0.2%).

These last findings, when taken together with those

of Bayliss and Hinton (1990), indicate that it is the

time of transport and stationary waiting time that

affects mortality, rather than distance driven. More

bruising has been seen on carcasses with increasing

transport duration (Scholtyssek and Ehinger, 1977).

Ehinger and Gschwindt (1981) found that meat pH

measured post mortem decreased with duration of

transport, and live weight has been found to de-

crease as much as 3% with transport duration of 4.5

h (Scholtyssek et al., 1977). Blood corticosterone is

an indication of stress in broilers and has been found

to be higher in broilers following transport for 4 h

compared to 2 h duration (Freeman, 1984).

Interest in Potential Processing Options and Importance of Different Processing Facilities Attributes

Interest in on-farm and three different off-farm

processing options was evaluated with a multiple

choice question which was answered by all 82 re-

spondents (Figure 3). Off-farm processing options

included: (1) shared-use facility where the farmers

do the processing, and sell their birds under their

own farm label, (2) drop off/pick up facility where the

facility processes as a service to the farmer, and the

farmer sells the birds under their own label and (3)

Table 2. Importance of processing facility attributes to Georgia pastured poultry farmers (average Likert scale value with 1 = Not important and 7 = Very important)

Processing facilities’ attributesMean Likert scale value

St. dev.

Quality of service 6.42 0.96

Processing price 6.09 1.18

Distance from farm 5.95 1.20

USDA inspection 4.76 2.06

Certified organic 3.64 2.04

Marketing services 2.92 1.80


contract grower facility where the farmer sells the

live birds to the facility, and the facility processes and

sells the birds under the facility’s label.

A similar number of farmers wanted only on-farm

processing (22%) or only off-farm drop off process-

ing (24%). However, 40% of the farmers were more

flexible and were open to more than one processing

option. The off-farm drop-off scored the best with

49 farmers (60%) being interested. This was followed

by on-farm processing (43 farmers or 52%), off-farm

rental (27 farmers or 33%), and contract growers (11

farmers or 13%). Overall, off-farm processing (includ-

ing drop-off, shared use and contract growers) was

more popular than on-farm processing. Comparing

the three off-farm processing options, drop-off was

the preferred off-farm processing option. Availabil-

ity of off-farm processing has potential to signifi-

cantly increase the volume of pastured birds raised

in Georgia. Based on the reported volumes that the

farmers would like to produce, an off-farm drop-off

facility has the potential to increase the total small-

scale poultry production from 37,642 birds/year to

232,205 birds/year, a potential 6-fold increase. How-

ever, it may be difficult to find a location for one sin-

gle processing facility to be conveniently located for

all interested farmers.

The farmers evaluated the importance of several

attributes of processing facilities. The farmers re-

ported quality of service to be the most important

processing facility attribute (Table 2), followed by

the processing price, the distance from the farm and

USDA inspection. It was less important if the pro-

cessing plant was certified organic and if the market-

ing services were provided by the processing plant.

Small-scale farmers in Georgia were having diffi-

culties catching up with demand for local or pasture

raised poultry due to the processing regulations in

Georgia. Georgia is not alone in the need to ad-

dress these issues; many other states are innovating

processing solutions to address the chronic need

for small-scale slaughter facilities in rural communi-

ties while protecting and preserving the health and

safety of the food supply. The production of small-

scale poultry producers is insufficient to justify long

trips to processors and additional fees, and thus is

there a need to find a solution in order to keep small

poultry production as a profitable business. Off-farm

processing, in particular drop-off facilities might of-

fer an efficient solution; however, determination of

the location might be an issue as distance to the pro-

cessing facility is an important factor for many farm-

ers. Of course, these services need to be offered at

an affordable processing price and the quality of the

service is very important to the farmers.

The second preferred processing option, on-farm

processing, also offers some opportunities. One in-

novative solution that is already being used in sev-

eral other states is mobile processing. These consist

essentially of small trailers equipped with everything

needed to process poultry, and travel to the farm

for processing, providing necessary infrastructure

and less environmental impact than a fixed facility.

Several mobile processing units have shown to be

successful in other states (eXtension, 2011), such

as Kentucky (Skelton, 2011). These mobile process-

ing units have the advantages compared to a fixed

processing unit including lower processing costs, re-

duced stress on animals, lower capital investment,

and less resistance from municipalities and neigh-

bors (Simon, 2008).

With a rising consumer interest and awareness in

local and sustainable foods, it will be increasingly

important to make local food products more widely

available in the U.S. food market. Different solutions

exist to help the small local farmers with limited re-

sources to gain profits and make the local poultry

production as a viable industry.

ACKNOWLEDGEMENTS

This research was funded by SARE grant LS11-245

(WQA, PGC and SCR) and USDA-NIFSI grant #2008-

51110-04339 (SCR and PGC) and a farm aid grant

(SW and Georgia Organics, Atlanta, GA).

REFERENCES

Bayliss, P.A., and M.H. Hinton. 1990. Transportation


of broilers with special reference to mortality rates.

Appl. Animal Behaviour Sci. 28:93–118.

Economic Research Service. 2011. U.S. broiler indus-

try: background statistics and information. Avail-

able at : http://www.ers.usda.gov/News/broiler-

coverage.htm Accessed 12 January 2012.

Ehinger, F., and B. Gschwindt. 1981. The influence

of different transport duration on meat quality and

physiological traits in broilers of different origins.

Archiv. Geflugelk. 45: 260–265.

eXtension. 2011. Mobile slaughter/processing units

currently in operation. Available at http://www.

extension.org/pages/19781/mobile-slaughterpro-

cessing-units-currently-in-operation Accessed 12

January 2012.

Freeman, B.M. 1984. Transportation of poultry.

World’s Poult. Sci. J. 40: 19–30.

Geering, D. 2011a. In Georgia, local birds may

take scenic route to your plate. Atlanta Maga-

zine. Available at: http://www.atlantamaga-

zine.com/covereddish/localfoods/blogentry.

aspx?BlogEntryID=10241894 Accessed 12 January

2012

Geering, D. 2011b. Small farmers go to great lengths

to process birds. Atlanta Magazine. Available at:

http://www.atlantamagazine.com/covereddish/

localfoods/blogentry.aspx?BlogEntryID=10244161

Accessed 12 January 2012.

Heffernan, W.D., and M. K. Hendrickson. 2002.

Multi-national concentrated food processing and

marketing systems and the farm crisis. Presented

at conference: The farm crisis: how the heck did

we get here? American Association for the Ad-

vancement of Science Symposium: Science and

Sustainability, Boston, Massachusetts, February

2002. Available at: http://www.foodcircles.mis-

souri.edu/paper.pdf Accessed 11 January 2011.

Scholtyssek, S., F. Ehinger, and F. Lohman. 1977. In-

fluence of transport and fasting on the slaughter

quality of broilers. Archiv. Geflugelk. 41:27–30.

Skelton, S. 2011. Kentucky mobile poultry process-

ing unit. Available at http://www.extension.org/

pages/Kentucky_Mobile_Poultry_Processing_Unit

Accessed 18 January 2012.

Simon, K. 2008. Is a mobile slaughterhouse com-

ing to Connecticut? Cutting-edge process could

benefit state farmers and consumers. Available at

http://www.workingtheland.com/feature-mobile-

slaughterhouse.htm Accessed 12 January 2012.

Sustainable Agriculture Network. 2006. Profitable

poultry: raising birds on pasture. Available at:

http://www.sare.org/Learning-Center/Bulletins/

National-SARE-Bulletins/Profitable-Poultry Ac-

cessed 11 January 2011.

USDA. 2006. Guidance for determining whether a

poultry slaughter or processing operation is ex-

empt from inspection requirements of the poul-

try products inspection act. Available at: http://

www.fsis.usda.gov/oppde/rdad/fsisnotices/poul-

try_slaughter_exemption_0406.pdf Accessed 12

January 2012.

Vecerek, V., S. Grbalova, E. Voslarova, B. Janackova,

M. Malena. 2006. Effects of travel distance and the

season of the year on death rates of broilers trans-

ported to poultry processing plants. Poult. Sci.

85:1881–1884.

Warriss, P.D., E.A. Bevis, S.N. Brown, and J.E. Ed-

wards. 1992. Longer journeys to processing plants

are associated with higher mortality in broiler-

chickens. Br. Poult. Sci. 33:201–206.


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data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

Discussion

The discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

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Documents

hangcornell university

university of houston

kirkhamkansas state

kwon university of arkansas

dunkley university of

inaugural issue

crandalluniversity of

rickeuniversity of arkansas