Upload
cielo-shabatura
View
230
Download
10
Tags:
Embed Size (px)
DESCRIPTION
This journal is a peer reviewed scientific forum for the latest advancements in bacteriology research on a wide range of topics including food safety, food microbiology, gut microbiology, biofuels, bioremediation, environmental microbiology, fermentation, probiotics, and veterinary microbiology.
Citation preview
Volume 2, Issue 42012
ISSN: 2159-8967www.AFABjournal.com
242 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 243
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 ShettyUniversity of Massachusetts, USA
EDITORIAL BOARD
244 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA
EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA
Cesar CompadreUniversity of Arkansas for Medical Sciences, USA
Philip G. CrandallUniversity of Arkansas, USA
MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium
TECHNICAL EDITORJessica C. ShabaturaFayetteville, USA
ONLINE EDITION EDITORC.S. ShabaturaFayetteville, USA
ABOUT THIS PUBLICATION
Agriculture, Food & Analytical Bacteriology (ISSN
2159-8967) is published quarterly, beginning with
this inaugural issue.
Instructions for Authors may be obtained at the
back of this issue, or online via our website at
www.afabjournal.com
Manuscripts: All correspondence regarding pend-
ing manuscripts should be addressed Ellen Van Loo,
Managing Editor, Agriculture, Food & Analytical
Bacteriology: [email protected]
Information for Potential Editors: If you are interested
in becoming a part of our editorial board, please con-
tact Editor-in-chef, Steven Ricke, Agriculture, Food &
Analytical Bacteriology: [email protected]
Advertising: If you are interested in advertising with
our journal, please contact us at advertising@afab-
journal.com for a media kit and current rates.
Reprint Permission: Correspondence regarding re-
prints should be addressed Ellen Van Loo, Managing
Editor, Agriculture, Food & Analytical Bacteriology
Ordering Print Copies: print editions of this journal
may be purchased and shipped internationally from
our website order form at www.afabjournal.com
Subscription Rates: Subscriptions are not available
at this time. To be advised when subscriptions plans
are made available, please join our newsletter at
www.afabjournal.com
Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Website: www.AFABjournal.com
EDITORIAL STAFF
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 245
Developing an in vitro Method for Determining Feed Soluble Protein Degradation Rate by Mixed Ruminal MicroorganismsW. L. Crossland, L. O. Tedeschi, T. R. Callaway, P. J. Kononoff, and K. Karges
246
Lack of Effect of Feeding Lactoferrin on Intestinal Populations and Fecal Shedding of Sal-monella typhimurium in Experimentally-Infected Weaned Pigs
D. J. Nisbet, T. S. Edrington, R. L. Farrow, K. G. Genovese, T. R. Callaway, R. C. Anderson, and N. A. Krueger
280
Effect of Cooking on Selected Nutritional and Functional Properties of red amaranthsMd. A. A. Mamun, R. Ara, H. U. Shekhar, A. T. M.A. Rahim, and Md. L. Bari
291
Evaluation of the Ruminal Bacterial Diversity of Cattle Fed Diets Containing Citrus Pulp PelletsBroadway, P. R., T. R. Callaway, J. A. Carroll, J. R. Donaldson, R. J. Rathmann, B. J. Johnson, J. T. Cribbs, L. M. Durso, D. J. Nisbet, and T. B. Schmidt
297
ARTICLES
Attachment of E. coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal MicroscopyJ. A. Neal, E. Cabrera-Diaz, and A. Castillo
275
BRIEF COMMUNICATIONS
Instructions for Authors315
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal ContentsR. S.Pinder, and J.A. Patterson
253
TABLE OF CONTENTS
246 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACT
The objective of this work was to describe a novel in vitro system based on the subtraction of ammonia
pools obtained with and without rumen fluid inoculum to determine the soluble protein fraction of feeds
and their degradability, with adjustments for microbial contributions and bacterial contamination. Four
corn-milling coproducts were used in this study as random factors. The feeds (Fd) were dried distillers grain
(DDG), one high protein (HP-DDG), one containing added solubles (BPX-DDGS), and the corn coproducts
BRAN and GERM, concentrated corn kernel components derived during the processing of HP-DDG. Three
treatments were investigated: Fd was fermented in vitro with rumen fluid (Rf) and buffered media (Md)
(TRT1) or with Md alone (TRT2). Two controls were used without the inclusion of feed: Rf + Md (C1) and
Md alone (C2). The third treatment (TRT3) was calculated as TRT1 – (TRT2 – C2) – (C1 – C2) – C2 to account
for bacteria protein contamination. Feeds were incubated in duplicates for 0, 1, 3, 6, 12, 24, and 48 h and
subsamples of TRT1, TRT2, C1, and C2 were taken to determine ammonia and bacterial protein determi-
nation. The fractional rate of disappearance of soluble protein for BPX-DDGS (0.06 h-1) was less than half
of HP-DDG (0.13 h-1), BRAN (0.13 h-1), and GERM (0.15 h-1). These results suggest that this method may be
used to determine the degradability of the soluble protein fraction of ruminant feeds.
Keywords: fractional rate of degradation, protein assay, soluble protein
Correspondence: L. O. Tedeschi, [email protected]
Developing an in vitro Method for Determining Feed Soluble Protein Degradation Rate by Mixed Ruminal Microorganisms
W. L. Crossland1, L. O. Tedeschi1, T. R. Callaway2, P. J. Kononoff 3, K. Karges4
1Department of Animal Science, Texas A&M University, College Station, TX 77843-24712Food and Feed Safety Research Unit, USDA-ARS, College Station, TX 77845
3Department of Animal Science, University of Nebraska, Lincoln 685834Dakota Gold Research Association, Sioux Falls, SD 57104-4506
Agric. Food Anal. Bacteriol. 2: 246-252, 2012
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 247
INTRODUCTION
The field of ruminant nutrition commonly attempts
to fractionate feed proteins based on their physico-
chemical properties and fractional ruminal degrada-
tion rates (kd). This provides a structure for ration
balancing and decision-making programs common-
ly used by the beef and dairy industries (Lanzas et
al., 2007). Several researchers have expressed the
need to standardize these methods and to account
for protein fractions that are calculated by differ-
ence or assigned tabular kd values (Schwab et al.,
2003). The in situ technique is the most commonly
used method for determining protein degradability
in the rumen (Schwab et al., 2003). Nonetheless, this
method is costly and fails to determine the kd of the
soluble protein fraction, which is known to be vari-
able (120 to 400 %/h) (Sniffen et al., 1992). A direct
comparison of neutral detergent fiber (NDF) fermen-
tation between in vitro and in situ techniques sug-
gested a lag time of 3.5 h less, kd of 0.03 h-1 faster,
and an extent of 6% greater for the in situ method
(Varel and Kreikemeier, 1995), but significant correla-
tions exist between these techniques (Lopéz et al.,
1998). Several factors may affect the fermentabil-
ity of the feeds other than pH alone, including the
removal of fermentation end products and escape
of feed particles. The in situ method has been sug-
gested to simulate the rumen environment better
than other techniques (e.g. in vitro and enzymatic
digestion) (Nocek, 1988). In vitro methods, however,
are more affordable, fast, and less labor intensive al-
ternatives that still closely mimic the rumen environ-
ment. The major point of concern for in vitro ruminal
fermentation approaches is the accumulation of fer-
mentation end products (e.g. VFA and lactate) and
the decrease of pH; however, this can be overcome
by adding adequate buffering salts to the fermenta-
tion mixture (Hungate, 1950). Because soluble pro-
tein contained in feeds is rapidly degraded to am-
monia by rumen bacteria (Nocek and Russell, 1988),
the kd may be calculated from the rate of ammonia
and AA accumulation (Schwab et al., 2003). However,
calculation of protein kd via end product accumula-
tion such as ammonia and AA are confounded by
microbial catabolism (Broderick, 1987). The purpose
of the present work was to describe a novel in vitro
system based on the subtraction of ammonia pools
obtained with and without rumen fluid inoculation to
determine the kd of the soluble protein fraction ad-
justed for bacterial protein.
MATERIALS AND METHODS
Feeds
Four corn-milling coproducts produced by Poet LLC
(Sioux Falls, SD) were utilized to determine the kd
of their soluble protein. These feeds were used be-
cause of their diverse protein fractions, different pro-
cessing methods, and importance to the cattle in-
dustry. Briefly, the first corn-milling coproduct, dried
distillers grain (BPX-DDGS), contains added solubles
and is the result of a low heat processing and drying
method. The low heat method is suggested to less-
en the amount of heat-damaged proteins, which are
typically found in traditional corn-milling coproducts
and are known to be less digestible by ruminants
(Krishnamoorthy et al., 1982). The other corn-milling
coproduct comes from a novel processing method
that physically removes the bran (BRAN) and the
dehydrated germ (GERM) prior to fermentation, re-
sulting in a fourth high-protein-content corn-milling
coproduct (HP-DDG). The solubles from this fourth
corn-milling coproduct are added back to the BRAN
and GERM feed products. Thirty samples (1 kg) of
each corn-milling coproduct (BPX-DDGS, HP-DDG,
BRAN, and GERM) were collected and sent to the ru-
minant nutrition research department at Texas A&M
University (College Station, TX). Thirty sub-samples
(30 g) were taken and combined to obtain 900 g of
a composite feed, respectively, for each corn-milling
coproduct. A composite was used to remove the in-
trinsic variation among sub-samples to obtain a rep-
resentative feed. Composite feed samples were then
sent to Cumberland Valley Analytical Service (Hager-
stown, MD) for chemical analysis in accordance with
the AOAC (2000).
248 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
In vitro fermentation and sample collec-tion
Two treatments were used to measure ammonia
and bacteria protein. The first treatment (TRT1) was
the combination of each corn-milling coproduct
(Fd) with rumen fluid (Rf) and buffered media (Md)
mixture. The second treatment (TRT2) was the com-
bination of Fd + Md in which corn-milling coprod-
ucts were mixed with Md only to account for protein
solubility upon saturation of the feed. Additionally,
two controls (C1 and C2) that did not include feed
incubation were used to measure ammonia and bac-
terial protein (C1 was Rf + Md, of which Rf was mixed
with Md to account for any pre-existing nitrogen and
bacterial protein in the inoculate and C2 was com-
prised of Md that was incubated alone to account
for any endogenous nitrogen contribution from the
Md) to obtain a third calculated treatment (TRT3) as
described below. For each time of incubation, each
treatment was incubated in duplicate per feed (n =
16) and each control was incubated in duplicate (n =
4). Composite feeds were hand-ground using mor-
tar and pestle to pass a 2 mm screen (0.60 g), trans-
ferred into 125 mL Wheaton bottles, and dampened
with 6.0 mL of distilled water to prevent feed particle
scattering. Bottles were flushed with CO2 to create
an in vitro anoxic atmosphere, and 42 mL of a buf-
fer media (Goering and Van Soest, 1970) was added.
Bottles were sealed with butyl rubber stoppers and
incubated at 39°C for 48 h using water bath. The Rf
was collected from four different locations inside the
rumen of a non-lactating Jersey cow, grazing medi-
um quality grass and receiving a balanced salt and
mineral supplement. There was a small contribution
of animal effect to the total variance when prairie hay
was the main forage consumed (Vanzant et al., 1998).
The Rf was thoroughly mixed and filtered through
eight layers of cheesecloth and continuously flushed
with CO2. Ruminal fluid pH was measured using an
Orion 3-Star bench top pH meter (Thermo Fisher
Scientific, Inc.) recorded and 12 mL of filtered inocu-
late was injected via syringe into appropriate bottles.
Seven time points were used to collect fermentation
products (0, 1, 3, 6, 12, 24, and 48 h of fermentation)
for analyses. The 0-h samples were collected from
the bottles, immediately following inoculation. Fer-
mented samples were collected by removing 4 mL
from each treatment via needle and syringe. Sam-
ples were transferred to micro centrifuge tubes and
centrifuged at 10,000 × g for 5 min to remove cel-
lular debris; cell-free supernatants were frozen and
stored at -20°C for further analysis. Microbial mass
pellets were re-suspended in 0.9% NaCl to prevent
cell shattering and frozen and stored at -20°C.
Ammonia and bacterial protein deter-mination
Ammonia concentrations were determined by
the method of Chaney and Marbach (1962) and were
performed in duplicate. Bacterial protein was deter-
mined via the Bradford (1976) method in a microtiter
plate format compared with a bovine serum albumin
(BSA; 1 g/L). The Bradford (1976) method was chosen
due to its reduced interference by reagents and non-
protein components (Kruger, 2002). Bacterial pellets
were lysed with 500 µL of 1 M NaOH and centrifuged
(10,000 × g for 5 min) to allow for the solubilization of
membrane proteins (Sun et al., 2007), and resulting
supernatants were utilized.
Enumeration and statistical analysis
The TRT3 was calculated as shown in Eq. [1]. It was
used to compute the ammonia net balance (produc-
tion or uptake) associated with the degradation of a
feed if ammonia concentrations (µg/mL) from TRT1,
TRT2, C1, and C2 were used. Alternatively, Eq. [1]
was used to calculate the net balance of bacterial
protein associated with the degradation of the solu-
ble protein of a feed.
TRT3ij = TRT1ij – (TRT2ij – C2) – (C1 – C2) – C2 [1]
Where TRT1ij is the buffer media mixed ruminal
fluid fermentation of the ith feed for the jth incubation
time, TRT2ij is the ith feed and buffer media mixture
for the jth incubation time, TRT3ij is the ammonia and
bacterial protein adjusted for rumen fluid and me-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 249
dia, C1 is the rumen fluid and buffer media mixtures,
and C2 is the buffer media measures.
Figure 1 depicts a schematic representation of the
calculation of TRT3. The reason for measuring the
contributions of ammonia and bacterial protein from
TRT2 and C1 was to account for the interaction of Fd
+ Md and Rf + Md, and the stability of the Md during
the incubation period. Thus, at each sampling, the
contribution (i.e. contamination) from Fd, Md, and Rf
were discounted from the values obtained in TRT1.
The ammonia concentration and bacteria protein
data were analyzed as a repeated measures design,
assuming a completely randomized block design
with treatments (TRT1, TRT2, and TRT3) as fixed ef-
fects and feeds (BPX-DDG, BRAN, GERM, and HP-
DDG) as random blocks for the whole plot and time
of incubation (0, 1, 3, 6, 12, 24, and 48 h) as the re-
peated measure. The average between duplicates
within feeds and treatments were used. The interac-
tion between treatment and feed was assumed as
a random effect and the PROC MIXED of SAS (SAS
Inst., Cary, NC) was used.
The fractional rate of ammonia disappearance
(kf, h-1) was obtained for the post-ammonia peak for
TRT3 using the PROC NLIN of SAS version 9.2 (SAS
Inst. Inc., Cary, NC) as shown in Eq. [2]. All replicates
within feeds and treatments were used for this analy-
sis because we assumed the only source of variation
of ammonia production would be the anaerobic in-
cubation since the feeds were composites.
kf = NH 3,t=0 × exp(-kf×t) [2]
Where NH3,t is the ammonia concentration (nM) at
time t and kf is the fractional rate of disappearance
of ammonia, h-1.
RESULTS AND DISCUSSION
There was an interaction between treatments and
time (P < 0.001) as shown in Figure 2. The ammonia
production was greater for TRT1 (Fd + Rf + Md) than
TRT2 and TRT3 with the peak of ammonia accumula-
tion at around 6 h. Our in vitro ammonia concentra-
tion pattern is in agreement with the ammonia con-
centration in the rumen of steers fed 14.2 g urea/h for
6 h (Mizwicki et al., 1980), suggesting that ammonia
release was greater than ammonia uptake (or use)
by the microbes up to 6 h. Aside from the ammonia
produced by obligatory amino acid fermenting bac-
teria, which are estimated to account for less than
10% of the known rumen bacterial species (Krause
and Russell, 1996), there are two reasons ammonia
accumulates in ruminal fermentations. First, some
bacteria ferment amino acids and release NH3 along
with carboxylic or ketoacids. Second, the rate of pro-
tein degradation is greater than the rate of carbo-
Figure 1. Schematic representation of the correction for feed (Fd), rumen fluid (Rf), and buffer me-dia (Md) contribution to ammonia and bacteria protein during the incubation period
250 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
hydrate degradation (Nocek and Russell, 1988). In
agreement with Broderick (1978) and Annison et al.
(1954), a negative value in Figure 2 would suggest
a greater amount of fermentable carbohydrate in
which bacteria used most of the soluble NPN.
There was no difference in the fractional rate of
ammonia disappearance (P = 0.30) across feeds, like-
ly due to the large variation of in vitro incubation by
itself. However, the rate for BPX-DDGS (0.06 h-1) was
less than half of HP-DDG (0.13 h-1), BRAN (0.13 h-1),
and GERM (0.15 h-1). In some nutrition models (e.g.
Cornell Net Carbohydrate and Protein System and
Large Ruminant Nutrition System, Fox et al., 2004;
Tedeschi et al., 2005; Small Ruminant Nutrition Sys-
tem, Tedeschi et al., 2010; and the CPM Dairy model,
Tedeschi et al., 2008) the protein A (NPN) + B2 (solu-
ble protein) fractions of DDGS and GERM comprises
about 70% of the CP. Because the protein A fraction
contains mostly NPN, it would have been used by
microorganisms quickly; therefore, the release of
ammonia due to protein fermentation would origi-
nate from the protein B2 fraction. These nutrition
models assigned the values of 0.06 and 0.08 h-1 to
the kd of protein B2 fraction of DDGS and GERM,
respectively, and 0.12 h-1 to the kd of protein B2 frac-
tion of rice and wheat brans. While kf represents the
disappearance of ammonia post peak of production
and the kd represents the degradation of protein,
both represent the degradation, uptake, and use of
protein and nitrogen by the microbes. The kf is the
greatest fractional rate value that kd can have, and
they tend to be similar when energy is not limiting
the growth of microbes in which what gets degraded
(via kd) is used (via kf) by the microbes.
Evaluation of the methodology
The methodology described herein was based
on the hypothesis that bacterial uptake of protein
may be accounted for by using different fermenta-
tion controls and by measuring bacterial protein.
The TRT2 was performed to account for protein that
is soluble in neutral liquid media and it is important
to correct for this as saturation of feed may release
Figure 2. Average ammonia production (nmol) of corn-milling coproducts fermented in vitro with rumen fluid and buffer media, with buffer media alone, or adjusted for bacterial protein. Negative values indicate microbial protein synthesis
-500
0
500
1000
1500
2000
2500
3000
0 10 20 30 40 50
Am
mo
nia
, n
mo
l
Incubation time, h
Rumen fluid and buffer media
Buffer media alone
Adjusted for bacterial protein
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 251
soluble protein at varying rates, and solubility does
not equal degradation (National Research Council,
2001). The C1 was used to correct for soluble protein
in the rumen inoculate and microbial protein whereas
the C2 was used to account for any protein detected
in the buffering media from the casein, the nitrogen
source in the media (Goering and Van Soest, 1970).
By difference, the resulting ammonia production and
bacterial protein measurements should be a direct
result of the fermentation of the feeds. Although our
intent was not to compare different types of feed
protein, the current technique would have to be
tested across different feeds for consistency. In ad-
dition, not all soluble protein, soluble oligopeptides,
or soluble amino acids are hydrolyzed to ammonia
in the rumen; some escape ruminal degradation
whereas on in vitro situation they are not removed.
Data from Reynal et al. (2007) showed that on aver-
aged across diets, 27, 75, and 93% of soluble amino
acid in soluble protein (>10 kDa), oligopeptides (3
to 10 kDa), and small peptides plus free amino acids
(< 3 kDa) that escaped the rumen were of dietary
origin. Hence, more ammonia can be produced in
vitro than in vivo.
Variation
For the purposes of this type of in vitro study, incu-
bation times should be limited to 12 h due to inter-
ference of the degradation of other protein fractions
and to maintain first order kinetics. Figure 2 shows
24 and 48 h time points to illustrate this point. The
replicates for BRAN had similarly shaped profiles,
but reached very different peaks. The most deviated
replicate fermentations were observed using HP-
DDG. Specifically, a replicate displayed two distinct
peaks early in the fermentation. When profiles were
negative, it was considered that ammonia produced
by TRT2, C1, or C2 was greater than TRT1; i.e., pro-
tein was not being degraded to ammonia, but was
being synthesized for microbial protein.
In conclusion, the current method provided pre-
liminary information for the development of a meth-
od that may be used to determine the degradability
of the soluble protein fraction of ruminant feeds.
Future research and technology may offer valuable
improvements to this method, which could evolve
into a rapid and reliable routine in vitro method. This
methodology should be compared with other meth-
ods that determine protein degradation rates.
REFERENCES
Annison, E. F., M. I. Chalmers, S. B. M. Marshall, and
R. L. M. Synge. 1954. Ruminal ammonia formation
in relation to the protein requirement of sheep: III.
Ruminal ammonia formation with various diets. J.
Agric. Sci. 44:270-273.
AOAC. 2000. Official Methods of Analysis of AOAC
International. (17th ed.) Association of Official Ana-
lytical Chemists, Arlington, VA.
Bradford, M. M. 1976. A rapid and sensitive method
for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye bind-
ing. Anal. Biochem. 72:248-254.
Broderick, G. A. 1978. In vitro procedures for esti-
mating rates of ruminal protein degradation and
proportions of protein escaping the rumen unde-
graded. J. Nutr. 108:181-190.
Broderick, G. A. 1987. Determination of protein deg-
radation rates using a rumen in vitro system con-
taining inhibitors of microbial nitrogen metabo-
lism. Br. J. Nutr. 58:463-475.
Chaney, A. L., and E. P. Marbach. 1962. Modified re-
agents for determination of urea and ammonia.
Clin. Chem. 8:130-132.
Fox, D. G., L. O. Tedeschi, T. P. Tylutki, J. B. Russell,
M. E. Van Amburgh, L. E. Chase, A. N. Pell, and T.
R. Overton. 2004. The Cornell Net Carbohydrate
and Protein System model for evaluating herd
nutrition and nutrient excretion. Anim. Feed Sci.
Technol. 112:29-78.
Goering, H. K., and P. J. Van Soest. 1970. Forage
fiber analysis: Apparatus, reagents, procedures,
and some applications. Agric. Handbook. No. 379.
ARS, USDA, Washington, DC. 1-20 p.
Hungate, R. E. 1950. The anaerobic mesophilic cel-
lulolytic bacteria. Bacteriological Reviews. 14:1-49.
Krause, D. O., and J. B. Russell. 1996. An rRNA ap-
252 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
proach for assessing the role of obligate amino
acid-fermenting bacteria in ruminal amino acid-
fermenting bacteria in ruminal amino acid deami-
nation. Appl. Environ. Microbiol. 62:815-821.
Krishnamoorthy, U., T. V. Muscato, C. J. Sniffen, and
P. J. Van Soest. 1982. Nitrogen fractions in selected
feedstuffs. J. Dairy Sci. 65:217-225.
Kruger, N. J. 2002. The Bradford Method for Protein
Quantitation. Pages 15-21 in The Protein Protocols
Handbook. J. M. Walker, ed. Humana Press.
Lanzas, C., L. O. Tedeschi, S. Seo, and D. G. Fox.
2007. Evaluation of protein fractionation systems
used in formulating rations for dairy cattle. J. Dairy
Sci. 90:507-521.
Lopéz, S., M. D. Carro, J. S. González, and F. J. Ove-
jero. 1998. Comparison of different in vitro and in
situ methods to estimate the extent and rate of
degradation of hays in the rumen. Anim. Feed Sci.
Technol. 73:99-113.
Mizwicki, K. L., F. N. Owens, K. Poling, and G. Bur-
nett. 1980. Timed ammonia release for steers. J.
Anim. Sci. 51:698-703.
National Research Council. 2001. Nutrient Require-
ments of Dairy Cattle. (7th ed.) National Academy
Press, Washington, DC.
Nocek, J. E. 1988. In situ and other methods to es-
timate ruminal protein and energy digestibility: a
review. J. Dairy Sci. 71:2051-2069.
Nocek, J. E., and J. B. Russell. 1988. Protein and
energy as an integrated system. Relationship of
ruminal protein and carbohydrate availability to
microbial synthesis and milk production. J. Dairy
Sci. 71:2070-2107.
Reynal, S. M., I. R. Ipharraguerre, M. Liñeiro, A. F.
Brito, G. A. Broderick, and J. H. Clark. 2007. Oma-
sal flow of soluble proteins, peptides, and free
amino acids in dairy cows fed diets supplemented
with proteins of varying ruminal degradabilities. J.
Dairy Sci. 90:1887-1903.
Schwab, C. G., T. P. Tylutki, R. S. Ordway, C. Sheaffer,
and M. D. Stern. 2003. Characterization of proteins
in feeds. J. Dairy Sci. 86:E88-E103.
Sniffen, C. J., J. D. O’Connor, P. J. Van Soest, D. G.
Fox, and J. B. Russell. 1992. A net carbohydrate
and protein system for evaluating cattle diets: II.
Carbohydrate and protein availability. J. Anim. Sci.
70:3562-3577.
Sun, Z. H., Z. L. Tan, S. M. Liu, G. O. Tayo, B. Lin, B.
Teng, S. X. Tang, W. J. Wang, Y. P. Liao, Y. F. Pan,
J. R. Wang, X. G. Zhao, and Y. Hu. 2007. Effects
of dietary methionine and lysine sources on nutri-
ent digestion, nitrogen utilization, and duodenal
amino acid flow in growing goats. J. Anim. Sci.
85:3340-3347.
Tedeschi, L. O., A. Cannas, and D. G. Fox. 2010. A
nutrition mathematical model to account for di-
etary supply and requirements of energy and nu-
trients for domesticated small ruminants: The de-
velopment and evaluation of the Small Ruminant
Nutrition System. Small Ruminant Res. 89:174-184.
Tedeschi, L. O., W. Chalupa, E. Janczewski, D. G.
Fox, C. J. Sniffen, R. Munson, P. J. Kononoff, and R.
C. Boston. 2008. Evaluation and application of the
CPM Dairy nutrition model. J. Agric. Sci. 146:171-
182.
Tedeschi, L. O., D. G. Fox, and P. H. Doane. 2005.
Evaluation of the tabular feed energy and protein
undegradability values of the National Research
Council nutrient requirements of beef cattle. Prof.
Anim. Scient. 21:403-415.
Vanzant, E. S., R. C. Cochran, and E. C. Titgemeyer.
1998. Standardization of in situ techniques for ru-
minant feedstuff evaluation. J. Anim. Sci. 76:2717-
2729.
Varel, V. H., and K. K. Kreikemeier. 1995. Technical
note: Comparison of in vitro and in situ digestibil-
ity methods. J. Anim. Sci. 73:578-582.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 253
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Isolate A10, an acetogen isolated from rumen contents, displayed diauxie when incubated with glucose
and H2/CO2 (80:20), regardless of initial glucose concentration (0.025 - 27 mM). Glucose consumption
preceded H2 consumption. Acetate, formate and H2 were detected during growth on glucose. Only ac-
etate was detected during growth on H2/CO2. Regardless of the atmosphere (N2/CO2 or H2/CO2), growth
on glucose occurred at µ max rate of 0.47, while growth on H2/CO2 was slower (µ max rate 0.12). When
glucose was the main organic carbon source, NaH13CO3 the major inorganic carbon source, and H2 the
sole atmospheric gas, unlabeled CH3COOH and HCOOH were detected during growth on glucose. After
glucose was used (during formate consumption), CH313COOH was also detected in the culture supernatant.
Following formate depletion, 13CH313COOH was detected as well. These findings suggest that formate is
utilized as a carbon source for the methyl group of acetate. Hydrogenase activity was lower in cells utiliz-
ing glucose (37 µmol H2 oxidized min-1 mg protein-1) as compared to cells growing on H2/CO2 (260 µmol
H2 oxidized min-1 mg protein-1). Intracellular [NAD+] was high during growth on glucose (14 µM g bacterial
DM-1), and low during growth on H2/CO2 (4 µM g bacterial DM-1). Concurrently, intracellular [NADH] was
low during growth on glucose (4 µM g bacterial DM-1) but higher (15 µM g bacterial DM-1) during the H2/
CO2-dependent growth phase. We conclude that isolate A10 is not capable of mixotrophic growth on
glucose and H2/CO2.
Keywords: acetogen, mixotrophy, H2, acetate, glucose, hydrogenase
Correspondence: J. A. Patterson, [email protected] Tel: +1 -765-494-4826 Fax: +1-765-494-9347
Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal Contents
R.S.Pinder 1,2 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. 2: 253-274, 2012
254 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
INTRODUCTION
During fermentation of plant carbohydrates in
the rumen of ruminants extensive degradation oc-
curs with considerable cross-feeding and genera-
tion of metabolities that serve as substrates for other
ruminal organisms (Ricke et al., 1996; Weimer et al.,
2009). During this process certain ruminal microor-
ganisms (i.e., Ruminococcus albus) release H2 into
the ruminal fluid (Miller and Wolin, 1973). The H2
must be removed from the ruminal environment
to prevent reduced acetate production and conse-
quent reduction of fermentation efficiency and mi-
crobial yield (Wolin and Miller, 1983). Although sev-
eral groups of bacteria are capable of utilizing H2 as
an energy source, only methanogens and acetogens
compete for H2 in the rumen. Methanogens use H2
to reduce CO2 to methane (Bhatnagar et al., 1991)
while acetogens use the same substrates to produce
acetate (Ragsdale, 1991).
Typically, methanogenesis is the primary H2 sink
in the rumen (Hungate, 1967). The predominance
of methanogenesis over acetogenesis in anaerobic
habitats such as ruminal contents could be explained
as follows: 1) methanogenesis is more exergonic
than acetogenesis [ΔGo’ (kJ) of -135.6 and -104.6, re-
spectively; Thauer et al., 1977], and 2) methanogens
have a higher affinity for H2 than acetogens (Cord-
Ruwisch et al., 1988). However, other factors affect
the competition between these two groups of mi-
croorganisms because acetogenesis predominates
over methanogenesis in several habitats [e.g., ter-
mite guts (Brauman et al., 1992), rodent ceca (Prins
and Lankhorst, 1977), and human intestines (Lajoie
et al. (1988)].
Breznak and Blum (1991) suggested that mixotro-
phy, the ability to use two substrates simultaneously,
may influence whether acetogens or methanogens
predominate in certain habitats. Although, a species
capable of mixotrophic growth on carbohydrates
and H2 could consume H2 regardless of carbohydrate
concentration, a non-mixotrophic species may cease
H2 consumption if carbohydrate concentrations ex-
ceed threshold levels. Of the five acetogenic bac-
teria isolated from ruminal contents and capable of
utilizing carbohydrates and H2/CO2, [isolates H3HH
and A10 (Boccazzi and Patterson, 2011; Jiang et al.,
2012a,b; Pinder and Patterson, 2011), Acetitomacu-
lum ruminis (Greening and Leedle, 1989); Eubacte-
rium limosum (Genthner et al., 1981), and Syntropho-
coccus sucromutans (Krumholz and Bryant, 1986),
only E. limosum has been tested to determine sub-
strate preferences (Genthner et al., 1987). Because
the concentration of carbohydrates in ruminal fluid
is sufficient for growth by acetogens (Pinder et al.,
2012), it was important to determine the mixotrophic
nature of ruminal acetogens.
The primary objective of the research report-
ed herein was to determine if isolate A10 was capable
of utilizing glucose and H2/CO2 mixotrophically. The
growth of isolate A10 on glucose was relatively rapid
and occurred before detectable H2/CO2-dependent
growth. Thus we could not unequivocally conclude
that isolate A10 was unable to utilize glucose and H2/
CO2 mixotrophically based on growth and substrate
consumption patterns alone. Therefore, the label-
ing pattern of acetate produced by isolate A10 when
grown in the presence of glucose and NaH13CO3
was used to determine the mixotrophic character of
isolate A10. Finally, information regarding the intra-
cellular hydrogenase activity and NAD(H) concentra-
tions in isolate A10 during the sequential utilization
of glucose and H2/CO2 was obtained.
MATERIALS AND METHODS
Organism and cultivation medium
Isolate A10, a previously described and ruminal
isolate (Boccazzi and Patterson, 2011) was used in
all experiments. This organism was maintained and
(for most experiments) grown in acetogenic medium
(Pinder and Patterson, 2011). Glucose was added at
the appropriate concentration (as described in the
results) from a filter-sterilized stock solution (15%
w/v). After inoculation (1% v/v from an overnight cul-
ture), the atmosphere of the bottles was flushed and
pressurized to 200 kPa with H2/CO2 (80:20 ratio). All
bottles were incubated at 39°C with vigorous shak-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 255
ing (180 rpm) for appropriate time periods as de-
scribed in the results.
For mass spectrometry experiments, NaH13CO3
replaced unlabeled Na2CO3 on an equivalent car-
bon basis. Moreover, to further reduce the presence
of unlabeled CO2, the medium was gassed with ox-
ygen-free 100 % N2, rather than the normally used
oxygen-free 100 % CO2, during preparation. Twenty
ml of prereduced acetogenic medium were added
to 170-mL anaerobic nephelometry flasks. After ster-
ilization, glucose was added to a final concentration
of 0.05 % from a filter-sterilized glucose stock solu-
tion (15% w/v). The flasks were inoculated (1% v/v)
with an overnight culture grown in the same medi-
um. The atmosphere of the flasks was replaced with
a 100% H2 atmosphere, then pressurized to 200 kPa.
The bottles were incubated at 39°C with vigorous
shaking (180 rpm).
Quantitation of cell mass, substrates and products
Optical density of cultures was determined by
measuring absorbance of the culture at 660 nm
with a Spectronic 70 spectrophotometer (Bausch &
Lomb, Inc., Rochester, NY ). Bacterial dry matter was
determined by centrifuging (10,000 x g, 10 min 4°C)
the cultures, washing once with 0.9% NaCl, and re-
suspending the pellet in 1 mL of distilled H2O. The
suspension was placed in aluminum weighing pans
and dried overnight at 105°C. Cells were lysed by
addition of NaOH (1 N final conentration) followed
by boiling for 10 min. Total cell protein was deter-
mined using a bicinchonic acid kit (Pierce Chemical
Co., Rockford, IL).
At appropriate time points, the gas volume in
culture bottles was measured manometrically (Balch
and Wolfe, 1976). The H2 concentration of the at-
mosphere in the bottles was determined by inject-
ing 1 mL of the atmosphere into a Varian 3700 gas
chromatograph (Varian Associates, Palo Alto, CA)
fitted with a thermal conductivity detector and a 100
cm stainless steel column packed with Carbosphere
80/100 (Supelco, Inc.; Bellefonte, PA). The carrier gas
was nitrogen. The H2 headspace volume (in mL) was
obtained by multiplying the atmospheric volume
times the H2 concentration in the culture bottles.
The H2 volume of treatment bottles were converted
to a percentage of uninoculated bottles because
there was an 11% decrease in gas content of unin-
oculated bottles over the length of the incubation
period. The H2 content of uninoculated bottles was
254.85 mL at the start of incubation but decreased to
227.68 mL by the end of the incubation period (96 h).
Once gas samples had been obtained, 1 mL of
culture was collected, and immediately centrifuged
(14,000 x g, 15 min, RT). One hundred and forty µl
of supernatant was combined with 20 µl of 100 mM
pivalic acid (internal standard) and 40 µl of meta-
phosphoric acid (25% w/v in H2O). Concentrations
of volatile fatty acids in the culture supernatant were
determined using a Hewlett Packard 5890 gas chro-
matograph (Hewlett Packard Co., Palo Alto, CA) fit-
ted with a glass column packed with GP 60/80 carbo-
pack C / 0.3% Carbowax M / 0.1 % H3PO4 (Supelco,
Inc.; Bellefonte, PA). The injector and detector tem-
peratures were set at 200°C while the column tem-
perature was set at 135°C. Formate concentrations
in the culture supernatant were determined using a
formate dehydrogenase assay (Schaller and Triebig,
1983). The pH of the culture was measured imme-
diately after sampling for volatile fatty acids with an
Ag combination electrode connected to a Fisher
Accumet pH meter (Fisher Scientific, 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 uninoculated control bottles.
Determination of NAD(H) content
Twenty liters of anaerobic acetogen medium, sup-
plemented with glucose (0.2 g/L) and continuously
bubbled with H2/CO2 (approx. 100 mL/min), were
inoculated with 250 mL of an overnight culture of
isolate A10 and incubated at 39°C. At appropriate
times, 500 mL of culture were collected and centri-
fuged (10,000 x g, 7 min, 4°C). The cell pellet was
256 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
resuspended in 3 mL of Tris buffer (50 mM Tris-Cl,
pH 7.6). The cell suspension was divided into three
1-mL fractions. One fraction (A) was acidified with
0.5 ml of 0.33 N HCl, while a second fraction (B) was
alkalinized with 0.5 mL of 0.33 N NaOH. The third
fraction was used to determine the dry matter and
protein content of the cultures. The acid and alka-
line fractions were incubated for 10 minutes at 65°C.
Once cooled, both fractions were neutralized (to pH
7) with either 1 N HCl or 1 N NaOH. NAD+ was de-
termined in fraction A extracts while the fraction B
extracts were used to determine NADH content as
described by Klingenberg (1983).
Mass spectrometry
The 13C/12C ratio of volatile fatty acids in
culture supernatants were determined by gas chro-
matography/mass spectrometry techniques. Es-
sentially, the individual volatile fatty acids were
separated with a Hewlett Packard 5890 series II gas
chromatograph (Hewlett Packard Co., Palo Alto, CA)
fitted with a DBWax column (Supelco, Inc., Belle-
fonte, PA). The injector temp. was 230°C and the
detector temp. was 210°C. The column temperature
started and was held at 50°C for 0.1 min, then in-
creased to 240°C at a rate of 15°C/min The vola-
tized compounds were directed into a Finnigan 4000
mass spectrometer set to obtain electron impact
spectra. All samples were ionized at 70 eV and at a
temperature of 250°C. An electrode multiplier set at
approximately 1200 volts was used as the detector.
The ion stream was scanned for ions with mass from
41 to 150 AMU and a spectrum constructed for each
peak that eluted from the gas chromatograph. The
spectrum of each peak was compared to spectrums
of authentic volatile fatty acid standards in order to
establish the identity of the compound in each peak.
The concentration of unlabeled, single- and dou-
ble-labeled acetate was calculated as follows:
Total mass = mass 60 + mass 61 + mass 62.
Unlabeled acetate (CH3COOH) = (mass 60 / total
mass) x mM acetate.
Single labeled acetate (13CH3COOH or
CH313COOH) = (mass 61 / total mass) x mM acetate.
Double labeled acetate (13CH313COOH) = (mass
62 / total mass) x mM acetate.
Hydrogenase activity
Three liters of acetogenic medium were in-
oculated with 30 mL of an overnight culture of isolate
A10. The energy substrates were glucose (0.2 g/L)
and H2/CO2 (80:20 ratio, bubbled through at approx-
imately 100 mL/min). At appropriate time points (3,
6, 12, 24, and 48 h), 40 mL of culture were removed,
and centrifuged (10 min, 7,000 x g, RT). The pellet
was resuspended in 1 mL of anaerobic Tris buffer (50
mM Tris-Cl, pH 7.6). Cells were lysed under anaerobic
conditions with a French Press (Aminco, Inc., Urbana,
IL). The cell lysate was collected into tubes continu-
ously gassed with CO2, and used immediately. Hy-
drogenase activity of the cell lysate was determined
as described by Ragsdale and Ljungdahl (1984). An
aliquot of the cell extract was injected into serum-
stoppered tubes (10 x 100 mm) that contained 2 ml
of the assay mixture (100 mM Tris-Cl, pH 7.6; 3.2 mM
dithiothreitol and 10 mM methyl viologen) and an at-
mosphere of 100 % H2. The absorbance at 604 nm,
was measured over 10 minutes. The change in ab-
sorbance over time was converted to enzyme activity
using an extinction coefficient for methyl viologen of
13,900 M-1 cm-1. One unit of hydrogenase activity is
defined as 2 µmol of methyl viologen reduced min-1
which is equivalent to 1 µmol of H2 oxidized min-1
(Ragsdale and Ljungdahl, 1984). The specific activity
of hydrogenase was calculated after determination
of the protein content of the cell lysate.
Reagents
NaH13CO3 (13C content: > 99 atom %) was
obtained from Aldrich Chemical Co (Milwaukee, WI).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 257
Figure 1. Growth of isolate A10 in acetogen media ± glucose and ± H2/CO2. Cells (0.1 mL of an overnight culture) were inoculated into 120-mL serum bottles containing 10 ml of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Following inoculation, the bottles were flushed and pressurized with either H2/CO2 (80:20, closed symbols) or H2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, three bottles from each treatment were randomly selected and the optical density (absorbance at 660 nm) of an aliquot of the culture in each bottle was measured. Data presented in this Figure as well as Figures 6, 8-11 originated from the same cultures.
258 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Figure 2. Effect of time of glucose addition on growth of isolate A10. Cells of isolate A10 were inoculated into 120-mL serum bottles containing 10 mL of acetogen medium. After inoculation, the bottles were flushed and pressurized with H2 + CO2 (80:20) to 200 kPa. At either 0 h (■) or 48 h (♦)of incubation, glucose (final concentration 5.5 mM) was introduced into the bottles. As a control, some bottles did not receive glucose (●). At appropriate time points, three bottles from each treatment were randomly selected to determine the optical density of the culture.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 259
H2, H2/CO2 (80:20 ratio), CO2, N2, and N2/CO2 (80:20
ratio) were obtained from Airco, Inc (Indianapolis,
IN). Traces of oxygen present in these gases were
removed by passing through a heated copper col-
umn. Enzymes used in formate, NAD+ and NADH
assays were obtained from Boeringer Mannheim (In-
dianapolis, IN). All chemicals used were of reagent
grade.
RESULTS
Results presented in this section are typically from
one experiment although the experiment was dupli-
cated at least once with similar results. Each data
point represents the mean from at least two (in many
cases three) individual cultures.
Cell growth
Isolate A10 displayed a typical diauxic growth pat-
tern when grown in glucose-supplemented medium
and an atmosphere of H2/CO2 (Figure 1). This diauxic
pattern was observed regardless of the initial con-
centration of glucose (0.025 to 27 mM). The maxi-
mum growth rate of cells growing on glucose was
0.47, regardless of the atmosphere (H2/CO2 or N2/
CO2) present in the serum bottles. A limited amount
of growth was observed (final OD approximately
0.2 A660 units) in the absence of added energy sub-
strates (glucose or H2) indicating that isolate A10 was
capable of some growth solely supported by media
components. Growth of ruminal acetogenic isolates
is stimulated by yeast extract, a component of the
medium used (B. Morvan and G. Fonty, personal
communication).
Immediately after the glucose supply was ex-
hausted (typically within 6 h of inoculation), the cells
entered a phase during which the OD of the cultures
declined (usually 0.05 to 0.1 A660 units). Bacterial pro-
tein declined during this phase as well, suggesting
that the decrease in OD was due to bacterial lysis
(data not shown) and not just changes in cell size or
shape.
If the atmosphere of the bottles contained N2/
CO2, the OD of the cultures declined for at least 70
h. Conversely, if the atmosphere contained H2/CO2,
the cells reinitiated growth at a much slower pace (µ
h-1 between 0.06 and 0.12 ). Growth supported by
H2/CO2 typically could be detected 18 to 24 h after
inoculation and concluded approximately 72 h after
inoculation, regardless of the initial glucose concen-
tration in the medium (0.025 - 27 mM). Cessation of
H2/CO2-dependent growth was not due to substrate
exhaustion, as considerable amounts of H2 could be
detected after growth ceased. However, the rate
and extent of growth on H2/CO2 decreased as the
amount of glucose initially present in the medium
increased. If glucose was added to cells that had
been growing on H2/CO2 for 48 h, a brief but signifi-
cant burst of growth, without a lag phase, was ob-
served (Figure 2). When non-metabolizable glucose
analogues (2-deoxyglucose or a-methyl glucoside)
were added to the same type of cells (growing on
H2/CO2), growth ceased (Figure 3).
Substrate consumption
Glucose was utilized within the first 6 h of incuba-
tion (Figure 4). These results were observed regard-
less of the growth substrate (i.e., glucose and/or H2/
CO2) used for the inoculum. Hydrogen consumption
did not begin until approximately 18 h of incubation,
regardless of the initial concentration of glucose
(Figure 5). If glucose was added to cells consum-
ing H2, the consumption of H2 ceased until the ad-
ditional glucose was exhausted (data not shown).
When non-metabolizable glucose analogues (2-de-
oxyglucose or a-methyl glucoside) were added, H2
consumption ceased and did not restart (data not
shown).
Formate (produced during glucose utilization)
consumption began immediately after the glucose
supply was exhausted but continued into the peri-
od of H2 consumption (Figure 6). However, formate
alone (50 mM as HCOONa) did not support growth
of isolate A10 (data not shown).
260 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Figure 3. Effect of non-metabolizable glucose analog on H2/CO2-dependent growth of isolate A10. The cultures were grown in 170-mL anaerobic nephelometry flasks containing 20 mL of acetogenic medium and an atmosphere of H2/CO2 pressurized to 200 kPa. After 48 h of incuba-tion, 2-deoxyglucose (♦), or α-methyl glucoside (●) were added to a final concentration of 1 mM. The controls (□) did not receive any additions. Optical density of the cultures was measured by determining the absorbance at 660 nm. The arrow represents the time of addition of the non-metabolizable glucose analogs.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 261
Figure 4. Glucose consumption by isolate A10 in acetogen media ± glucose and ± H2/CO2. Cells (0.1 mL of an overnight culture) were inoculated into 120-ml serum bottles containing 10 mL of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Following inoculation, the bottles were flushed and pressurized with either H2/CO2 (80:20, closed symbols) or N2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, three bottles from each treatment were randomly selected and the glucose content of the culture supernatant was measured using a glucose oxidase kit.
262 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Figure 5. H2 production and consumption by batch cultures of isolate A10 in acetogen media ± glucose and ± H2/CO2. Cells (0.1 mL of an overnight culture) were inoculated into 120-mL serum bottles containing 10 mL of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Once inoculated, the bottles were flushed (30 sec) and pressurized with either H2/CO2 (80:20, closed symbols) or N2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, 3 bottles from each treatment were randomly selected and the H2 content of the headspace in the bottles was measured as detailed in materials and methods section. The values are expressed as a percent-age of uninoculated bottles because, over time, there was a decrease in pressure of the atmo-sphere in all the bottles (including uninoculated controls). The H2 content of the uninoculated bottles was 254.85 mL at the start of incubation but decreased to 227.68 mL by the end of the inoculation period (96 h).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 263
Figure 6. Formate production and consumption by batch cultures of isolate A10 in acetogen media. Cells (0.1 mL of an overnight culture) were inoculated into 120-mL serum bottles con-taining 10 mL of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Following inoculation, the bottles were flushed and pressurized with either H2/CO2 (80:20, closed symbols) or N2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, three bottles from each treatment were randomly selected and the formate concentration of the culture supernatant was determined using a formate dehydrogenase asssay.
264 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Product formation
Acetate was the major final product of iso-
late A10 (Figure 7). The final fermentation stoichi-
ometry of acetate production from glucose was 2.2
acetates per glucose fermented. Cultures growing
solely on H2/CO2, produced acetate to final concen-
trations of up to 69 mM.
Cultures of isolate A10 growing on glucose (0.05%
v/v) alone did not cause drastic changes in the pH of
the culture medium (Figure 8). However, cultures of
isolate A10 growing on H2/CO2 drastically lowered
the culture medium pH and the rate of pH decrease
correlated to acetate production. The final pH of
cultures growing on H2/CO2 was typically between
5.5 and 5.6.
When isolate A10 was grown in acetogen medium
with glucose (0.05 g/L), NaH13CO3 as the major source
of inorganic carbon and H2 as the sole component
of the atmosphere, unlabeled acetate (CH3COOH)
was produced during growth on glucose (Figure 9).
Single label acetate (CH313COOH) was not detected
until 24 h of incubation, while double label acetate
(13CH313COOH) did not appear until 48 h of incuba-
tion. These findings demonstrate that isolate A10 is
capable of chemolithoautotrophic acetogenesis.
Formate, was produced during the early stages of
growth regardless of the substrate (Figure 6). For-
mate production was greatest (2 mole of formate
per mole of glucose fermented) when the organism
was incubated in bottles containing acetogen me-
dium with glucose and a H2/CO2 atmosphere. For-
mate production by cells growing solely on glucose
was much less (1 mole formate per mole glucose
fermented). Formate was unlabeled during growth
of isolate A10 on glucose as the major source of or-
ganic carbon and NaH13CO3 as the major source of
inorganic carbon, suggesting that glucose was the
carbon source used for formate production.
Hydrogen production was detected during glu-
cose-dependent growth, (Figure 5), regardless of
the atmosphere (H2/CO2 or N2/CO2) in the culture
bottles. The stoichiometry of H2 production was 0.5
mole H2 per 1 mole of glucose consumed. H2 pro-
duction during glucose consumption is a previously
unknown characteristic of this organism. As with
exogenous H2, the H2 produced during growth on
glucose was consumed after glucose consumption
ceased.
Hydrogenase activity was detected in cell lysates
of isolate A10, regardless of the substrate (glucose
or H2/CO2) utilized (Figure 10). This activity increased
from 38 to 262 µmol H2 oxidized min-1 mg bacterial
protein-1 as the cells switched from growth on glu-
cose to growth on H2/CO2. Non-denaturing, anaer-
obic polyacrylamide gel electro-phoresis revealed
that the hydrogenase activity migrated as one band
(data not shown).
The intracellular concentration of NAD+ increased
during growth on glucose and peaked (14 µM / g
of bacterial DM) at 4 h of incubation (Figure 11),
which corresponded to the late log phase of growth
on glucose. Once glucose was utilized, intracellular
levels of NAD+ declined to approximately 4 µM g of
bacterial DM-1. Intracellular concentrations of NADH
decreased to 4 µM g of bacterial DM-1 during growth
on glucose, but increased to 15 µM g of bacterial
DM-1 during growth on H2/CO2.
DISCUSSION
One of the focal points of the interest in acetogens
and acetogenesis has been the possibility of using
acetogens (instead of methanogens) as H2 utilizers in
ruminal contents. However, very little is understood
about the physiology of acetogens isolated from
ruminal contents and even less is understood about
the factors that constrain the rate of acetogenesis in
ruminal contents. These experiments were not de-
signed to explain the relatively low numbers of ace-
togens in the rumen but rather to gather information
that may explain the low rate of chemolithoauto-
trophic acetogenesis observed in ruminal contents.
Thauer et al. (1977) and Cord-Ruwisch et al. (1988)
have suggested that the higher affinity of methano-
gens for hydrogen may explain the dominance of
methanogenesis over acetogenesis in H2-limited en-
vironments such as the rumen. However, these theo-
ries cannot explain why acetogenesis predominates
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 265
Figure 7. Acetate production by batch cultures of isolate A10 in acetogen media ± glucose and ± H2/CO2. Cells (0.1 mL of an overnight culture) were inoculated into 120-ml serum bottles con-taining 10 mL of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Following inoculation, the bottles were flushed and pressurized with either H2/CO2 (80:20, closed symbols) or N2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, three bottles from each treatment were randomly selected and the acetate concentration of the culture supernatant was determined using gas chromatography techniques.
266 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Figure 8. pH of batch cultures of isolate A10 in acetogen media ± glucose and ± H2/CO2. Cells (0.1 mL of an overnight culture) were inoculated into 120-mL serum bottles containing 10 mL of acetogen medium supplemented with 5.5 mM glucose (squares) or no added glucose (circles). Following inoculation, the bottles were flushed and pressurized with either H2/CO2 (80:20, closed symbols) or N2/CO2 (80:20, open symbols) to 200 kPa. The bottles were incubated at 37°C with vigorous shaking. At appropriate time points, three bottles from each treatment were randomly selected and the pH of the culture was determined.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 267
Figure 9. Acetate production by isolate A10 grown in acetogen medium where glucose was the primary organic carbon source and NaH13CO3 was the primary inorganic carbon source. The ac-etate produced was unlabeled (CH3COOH; ●), singly labeled (CH3
13COOH; ■) or double labeled (13CH313COOH;♦). The relative abundance of unlabeled, single- or double label acetate was determined by GC/MS.
268 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Figure 10. Hydrogenase activity of crude cell extracts of isolate A10. Cells were grown in 3 L batch cultures of acetogenic medium with 0.2 g of glucose L-1 and bubbled with H2 + CO2 (ap-proximately 100 mL / min). Cells were anaerobically harvested and ruptured as described in the materials and methods section. The hydrogenase activity (●) was determined using a methyl viologen assay system described by Ragsdale and Ljungdahl (1984). Bacterial protein (■) was determined as described in the materials and methods section.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 269
Figure 11. Intracellular concentration of NAD(H) in isolate A10. Cells were grown in batch cul-tures containing acetogen media amended with glucose (1.1 mM) and continuously bubbled with H2 + CO2 (approximately 100 mL min-1). The intracellular concentrations of NAD+ (□), NADH (■), and optical density (absorbance at 660 nm) (●) of the cultures were determined as described in the Materials and Methods section.
270 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
over methanogenesis in certain habitats. This conun-
drum suggests that other factors (either organismal
or environmental) are responsible. After Breznak
and Blum (1988) suggested that the ability to grow
mixotrophically on H2/CO2 and carbohydrates may
explain why acetogens are the predominant H2-uti-
lizing organisms in termite gut contents, we decided
to explore the possibility of the opposite being true
in acetogens isolated from ruminal contents, that is,
the inability of acetogens isolated from ruminal con-
tents to use H2 in the presence of organic substrates
(carbohydrates) may explain why acetogenesis is not
a significant H2 sink in the rumen.
During the initial characterization, Boccazzi and
Patterson (2011) determined that isolate A10 was
able to use carbohydrates (e.g., glucose, maltose
and cellobiose) for growth in addition to H2/CO2.
This observation is not surprising because aceto-
gens isolated from many environments including
other species isolated from ruminal contents (i.e.,
E. limosum, A. ruminis, S. sucromutans and isolate
H3HH), have similar capabilities. Clostridium pfenni-
gii is the only ruminal acetogen isolated thus far that
is incapable of utilizing sugars for growth (Krumholtz
and Bryant, 1986). However, information of the abil-
ity of acetogens isolated from ruminal contents to
mixotrophically utilize carbohydrates and H2/CO2 is
limited. Up to this time, the only acetogen isolated
from ruminal contents in which mixotrophic capa-
bilities has been tested is E. limosum. Glucose was
used as the organic energy substrate for these stud-
ies because preliminary experiments suggested that
similar results would be obtained whether glucose,
maltose, cellobiose or xylose were used as the or-
ganic substrate (data not shown).
The growth pattern of isolate A10 grown in bot-
tles containing glucose-supplemented acetogen
medium and a H2/CO2 atmosphere showed a typical
diauxic growth curve. Similar growth patterns have
been observed with other acetogens isolated from
ruminal contents, namely, E. limosum (Genthner and
Bryant, 1987) and isolate H3HH (Boccazzi and Pat-
terson, 2011). Because glucose-dependent growth
was relatively fast (compared to H2/CO2-dependent
growth), coupled with the long lag time before H2/
CO2 growth commenced (even in the absence of ex-
ogenous glucose), a determination of the mixotro-
phic capabilities of isolate A10 based on analysis of
the growth curve alone was not possible. However,
based on the observation that chemolithotrophically
produced acetate does not appear until after glu-
cose is exhausted, isolate A10 is most likely not a
mixotroph.
The doubling time of isolate A10 growing on glu-
cose (approximately 2.1 h) is much less than that re-
ported for E. limosum (7.1 h; Genthner and Bryant,
1987). Unfortunately, comparisons with other aceto-
gens isolated from ruminal contents are not possible
because the doubling time of these organisms grow-
ing on glucose has not been reported. The dou-
bling time of isolate A10 during H2/CO2 - dependent
growth was variable but ranged between 8.3 and
16.7 h. The doubling time of isolate A10 growing
on H2/CO2 is in range with that (2 to 36 h) reported
for other acetogens (Boccazzi and Patterson, 2011).
The growth rate and extent of growth on H2/CO2
were both negatively affected by the quantity of glu-
cose initially present in the medium. For example,
the doubling time of isolate A10 between 12 and 72
h was 16. 7 h when 5 mM glucose was added to the
medium versus 8.3 h for cultures with no added glu-
cose. Similar results were observed with E. limosum
(Genthner and Bryant, 1987).
The lack of a lag phase before the initiation of
glucose consumption was not unexpected because
data obtained by Jiang et al., (2012a) demonstrated
that while isolate A10 possesses an inducible glu-
cose PTS system, glucose kinase activity was de-
tected regardless of growth on glucose or H2/CO2.
However, the lag period between the end of glucose
consumption and the initiation of H2/CO2 consump-
tion could be construed to suggest that H2/CO2 utili-
zation is not a constitutive function of isolate A10. A
similar lag phase between glucose consumption and
H2/CO2 utilization has been observed in E. limosum
(Genthner and Bryant, 1987) and C. thermoaceticum
(Kerby and Zeikus, 1983). That H2/CO2 utilization is
an inducible characteristic is supported by the 7-fold
increase in hydrogenase activity as the organism
switched from glucose to H2/CO2 utilization. Induc-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 271
tion of hydrogenase activity has been reported for a
number of organisms, including Escherichia coli, Pro-
teus vulgaris and Citrobacter freundii (Krasna, 1980).
Regulation of H2/CO2 utilization may be construed to
suggest that isolate A10 prefers carbohydrates over
H2/CO2. The observation that H2/CO2 utilization and
growth ceased when non-metabolizable glucose
analogs were added to the cultures, also supports
this conclusion. Thus, utilization of H2/CO2 may be a
strategy that the organism uses to obtain energy and
carbon during periods of carbohydrate starvation.
The hydrogenase of isolate A10 is not inhibited
by analogs (e.g. Procion HE3-B) of NAD+ (data not
shown) which indicates that this hydrogenase is not
dependent on the pyridine nucleotides for activ-
ity. However, the hydrogenase is capable of reduc-
ing methyl viologen, an analog of ferredoxin. These
characteristics are similar to those of most hydroge-
nases isolated thus far (Adams et al. 1981). Our find-
ings of H2 production by isolate A10 during growth
on glucose coupled with the observation that there
was only one hydrogenase band in electrophoresis
gels could suggest that isolate A10 has a reversible
hydrogenase. Reversible hydrogenases are vectorial
H2 tranlocators, and if the hydrogenase is engaged
in H2 production, simultaneous H2 uptake is not pos-
sible (Adam et al., 1981).
Our observation that the NAD+ concentration in
isolate A10 was greater during growth on glucose,
as compared to growth on H2/CO2, is similar to data
obtained with E. limosum (Le Bloas et al., 1993). In
contrast, the intracellular NADH concentration of
isolate A10, which was relatively high during growth
on H2/CO2 and growth on glucose, is considerably
different than that of E. limosum. The pronounced
differences in NAD+ concentration between cells
growing on glucose and cells growing on H2/CO2,
would suggest that fundamental changes in cell
metabolism occur as isolate A10 switches from one
substrate to the other. NAD+ is a coenzyme to many
enzymes including glyceraldehyde-3-phosphate de-
hydrogenase, a key enzyme in glucose catabolism.
The high concentration of NAD+ could be interpret-
ed as an attempt to make this reaction as thermo-
dynamically feasible as possible in order to process
as much glucose as possible. NADH concentration
was lowest as the cells shifted from glucose to H2/
CO2 - dependent growth. This finding suggests that
during glucose or H2/CO2 consumption, intracellu-
lar production of NADH is sufficient to meet needs.
However, during the time period when the cells are
switching from one substrate to another, NADH uti-
lization is greater than production, causing the pre-
cipitous decline in intracellular NADH concentration.
Further work will be needed to determine the source
of NADH during glucose or H2/CO2 utilization, the
destination (i.e., intracellular or extracellular) of the
reducing equivalents of NADH, and the importance
of the dramatic shifts in NADH concentration.
Drake (1992) proposed “that an anaerobe [which]
grows chemolithoautotrophically and forms acetate
as its sole product is extremely good evidence that
the organisms is indeed an acetogen”. Initial char-
acterization (Boccazzi, and Patterson, 2011) and the
results of the present study have established that
isolate A10 possesses these two characteristics.
However, one of the more compelling tests to deter-
mine if a bacterial species is an acetogen has been
to study the fixation of 13CO2 or 14CO2 into acetate
(Wood, 1952; Pine and Barker, 1954; Schulman et al.,
1972; Kerby and Zeikus, 1983). Chemolithoautotro-
phic acetogenesis by isolate A10 was demonstrated
by production of both single- and double-labeled
acetate during growth in the presence of NaH13CO3.
Nevertheless, these results cannot unequivocally
prove that isolate A10 uses the acetyl-CoA pathway
for chemolithoautotrophic acetate synthesis. Experi-
ments specifically demonstrating the activities of
key enzymes of the acetyl-CoA pathway (i.e. carbon
monoxide dehydrogenase) will be needed for unam-
biguous proof. Notwithstanding, the data present-
ed herein provides strong evidence that isolate A10
is a true acetogen.
The combination of the data on diauxic growth
patterns, acetate labeling, hydrogenase induc-
tion, and intracellular [NAD+] and [NADH] indicates
that: isolate A10 is incapable of mixotrophic growth
on glucose and H2/CO2. Further, because diauxic
growth by isolate A10 is observed with H2/CO2 and
either maltose or cellobiose, one may conclude that
272 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
this is a general statement about all carbohydrates.
These findings are of significance because they sug-
gest that the inability to use carbohydrates and H2/
CO2 mixotrophically may, in part, explain why aceto-
gens isolated from ruminal contents are unable to
compete against methanogens for H2 produced in
the rumen. Other experiments (Pinder et al., 2012)
show that the concentration of cellobiose in ruminal
fluid should be above the growth thresholds of iso-
late A10 for these substrates and thus isolate A10
may grow preferentially on soluble carbohydrates in
the rumen. Although acetogens do not obtain as
much energy from H2 as methanogens and as a con-
sequence, have a higher H2 threshold (Breznak and
Blum, 1991; Zinder, 1993), these factors are probably
of secondary importance to the regulation of H2 utili-
zation by carbohydrates in isolate A10, and probably
to other non-mixotrophic acetogens.
The appearance of single labeled (carboxyl) ac-
etate (and lack of double labeled acetate) during the
period of formate consumption suggests that for-
mate provided the methyl carbon of acetate during
this time period. In these experiments, formate was
unlabeled (HCOOH) and disappeared from the cul-
ture supernatant at the same time that CH313COOH
appeared in the culture supernatant. Based on these
observations we conclude that formate was being
used as the carbon source for the methyl group of
acetate, in agreement with previous data (reviewed
by Ragsdale,1991). On account that formate did not
support growth of isolate A10, formate most likely
is used as a methyl source (interchangeably with
CO2) but not as an energy source. Thus, in the strict-
est sense isolate A10 is not capable of mixotrophic
growth with formate and H2/CO2, however, it does
co-metabolize formate.
Drake (1992) alluded to the physiological diversity
of acetogens by pointing out that the morphology,
staining properties, motility, spore-forming capa-
bility, temperature preference, and guanine-plus-
cytosine (G+C) content vary considerably among
acetogenic species. The diversity of acetogens also
includes substrate specificity and preference. For
example, while many acetogens can utilize carbohy-
drates as growth substrates, S. termitida and C. pfen-
nigii cannot (Breznak and Blum, 1991; Krumholz and
Bryant, 1985). Furthermore, in contrast to the data
presented here and by Genthner et al. (1981), some
acetogens are mixotrophs. For example, A. woodii is
capable of mixotrophic growth on H2/CO2 and either
fructose, glucose, or lactate (Braun and Gottschalk,
1981), and S. termitida can grow mixotrophically
on H2/CO2 and lactate and methanol (Breznak and
Blum, 1991). The diversity of acetogens demands
careful attention of generalized statements about
acetogens but offers the possibility of locating an
acetogen capable of flourishing in ruminal contents.
The results presented suggest that utilization of
acetogens isolated from ruminal contents as a sub-
stantial H2 consuming group in the rumen is unlikely
even when methanogens are inhibited. Although all
acetogens isolated (thus far) from ruminal contents
are able to consume considerable amounts of H2,
the presence of carbohydrates in ruminal fluid ap-
parently exerts a strong repressive influence on the
utilization of H2 by these organisms. However, owing
to the diversity of acetogens, the possibility still ex-
ists of locating and introducing non-ruminal mixotro-
phic acetogens into the ruminal ecosystem to suc-
cessfully compete for H2 against methanogens.
REFERENCES
Adams, M. W. W., Mortenson, L. E. and H.-S. Chen.
1981. Hydrogenase. Biochim. Biophys. Acta.
594:105-176.
Balch, W. E., and R. S. Wolfe. 1976. A new approach
to the cultivation of methanogenic bacteria: 2-mer-
captoethanesulfonic acid (HS-CoM)-dependent
growth of Methanobacterium ruminantium in a
pressurized atmosphere. Appl. Environ. Microbiol.
32:781-791.
Bhatnagar, L., M. K. Jain and J. G. Zeikus. 1991. In:
“Variations in Autotrophic Life.” J. M. Shively and
L. L. Barton (eds), Academic Press, Inc. New York,
NY.
Boccazzi, P., and J.A. Patterson. 2011. Using hydro-
gen limited anaerobic continuous culture to isolate
low hydrogen threshold ruminal acetogenic bacte-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 273
ria. Agric. Food & Analytical Bacteriol. 1:33-44.
Brauman, A., M. D. Kane, M. Labat and J. A. Breznak.
1992. Genesis of acetate and methane by gut
bacteria of nutritionally diverse termites. Science
257:1384-1386.
Braun, K., and G. Gottschalk. 1981. Effect of mo-
lecular hydrogen and carbon dioxide on chemo-
organotrophic growth of Acetobacterium woodii
and Clostridium aceticum. Arch. Microbiol.
128:294-298.
Breznak, J. A., and J. S. Blum. 1991. Mixotrophy in
the termite gut acetogen, Sporomusa termitida.
Arch. Microbiol. 156:105-110.
Cord-Ruwisch, C., H.-J. Seitz and R. Conrad. 1988.
The capacity of hydrogenotrophic anaerobic bac-
teria to compete for traces of hydrogen depends
on the redox potential of the terminal electron ac-
ceptor. Arch. Microbiol. 149:350-357.
Drake, H. L. 1992. Acetogenesis and Acetogenic
bacteria. In: Lederberg, J., Ed. “Encyclopedia of
Microbiology. Academic Press, Inc. San Diego, CA.
Genthner, B. R. S. , C. L. Davis and M. P. Bryant. 1981.
Features of rumen and sewage sludge strains of
Eubacterium limosum, a methanol-and H2-CO2-
utilizing species. Appl. Environ. Microbiol. 42:12-
19.
Genthner, B. R. S., and M. P. Bryant. 1987. Additional
characteristics of one-carbon-compound utiliza-
tion by Eubacterium limosum and Acetobacterium
woodii. Appl. Environ. Microbiol. 53:471-476.
Greening, R. C., and J. A. Z. Leedle. 1989. Enrich-
ment and isolation of Acetitomaculum ruminis,
gen. nov., sp. nov.: acetogenic bacteria from the
bovine rumen. Arch. Microbiol. 151:399-407.
Hungate, R. E. 1967. Hydrogen as an intermediate in
the rumen fermentation. Arch. Microbiol. 59:158-
164.
Jiang, W., R.S. Pinder, J.A. Patterson, and S.C. Ricke.
2012a. Sugar phosphorylation activity in ruminal
acetogens. J. Environ. Health Sci., Part A 47:843-
846.
Jiang, W., R.S., Pinder, and J.A. Patterson. 2012b. In-
fluence on growth conditions and sugar substrate
on sugar phosphorylation activity in acetogenic
bacteria. Agric. Food Anal. Bacteriol. 2:94-102.
Kerby, R., and J. G. Zeikus. 1987. Anaerobic catabo-
lism of formate to acetate and CO2 by Butyribac-
terium methylotrophicum. J. Bacteriol. 169:2063-
2068.
Klingenberg, M. 1983. Nicotinamide-adenine dinu-
cleotides and dinucleotide phosphates. End-point
UV-methods. In: “Methods of Enzymatic Analysis,
3rd edition.” H. U. Bergmeyer (ed.), Verlag Che-
mie, Weinheim, Germany.
Krasna, A. I. 1980. Regulation of hydrogenase activ-
ity in Enterobacteria. J. Bacteriol. 144:1094-1097.
Krumholz, L. R., and M. P. Bryant. 1985. Clostridium
pfennigii sp. nov. uses methoxyl groups of mono-
benzenoids and produces butyrate. Int. J. Sys.
Bacteriol. 35:454-456.
Lajoie, S. F., S. Bank, T. L. Miller, and M. J. Wolin.
1988. Acetate production from hydrogen and [13C]
carbon dioxide by the microflora of human feces.
Appl. Environ. Microbiol. 54:2723-2727.
Le Bloas, P., N. Guilbert, P. Loubiere, and N. D. Lind-
ley. 1993. Growth inhibition and pyruvate overflow
during glucose metabolism of Eubacterium limo-
sum are related to a limited capacity to reassimi-
late CO2 by the acetyl-CoA pathway. J. Gen. Mi-
crobiol. 139:1861-1868.
Miller, T. L., and M. J. Wolin. 1973. Formation of hy-
drogen and formate by Ruminococcus albus. J.
Bacteriol. 116:836-846.
Pine, L., and H. A. Barker. 1954. Tracer experiments
on the mechanism of acetate formation from car-
bon dioxide by Butyribacterium rettgeri. J. Bacte-
riol. 68:216-226.
Pinder, R.S., and J.A. Patterson. 2011. Isolation and
initial characterization of plasmids in an aceto-
genic ruminal isolate. Agric. Food Anal. Bacteriol.
1:186-192.
Pinder, R. S., J.A . Patterson,. C. A. O’Bryan, P. G.
Crandall, and S. C. Ricke. 2012. Dietary fiber con-
tent influences soluble carbohydrate levels in
ruminal fluids. J. Environ. Health Sci., Part B 47:710-
717.
Prins, R. A., and A. Lankhorst. 1977. Synthesis of
acetate from CO2 in the cecum of some rodents.
FEMS Micro. Lett. 1:255-258.
Ragsdale, S. W., and L. G. Ljundahl. 1984. Hydrog-
274 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
enase from Acetobacterium woodii. Arch. Micro-
biol. 139:361-365.
Ragsdale, S. W. 1991. Enzymology of the acetyl-CoA
pathway of CO2 fixation. Crit. Rev. Biochem. and
Mol. Biol. 26:261-300.
Ricke, S.C., S. A. Martin, and D. J. Nisbet. 1996. Ecol-
ogy, metabolism, and genetics of ruminal sele-
nomonads. Crit. Rev. Microbiol. 22:27-56.
Schaller, K.-H., and G. Triebig. 1983. Formate de-
hydrogenase. In: “Methods of Enzymatic Analysis,
3rd edition.” H. U. Bergmeyer (ed.), Verlag Che-
mie, Weinheim, Germany.
Schulman, M., D. Parker, L. G. Ljundahl, and H. G.
Wood. 1972. Total synthesis of acetate from CO2.
V. Determination by mass analysis of the different
types of acetate formed from 13CO2 by heterotro-
phic bacteria. J. Bacteriol. 109:633-644.
Thauer, R. K., K. Jungermann and K. Decker. 1977.
Energy conservation in chemotrophic anaerobic
bacteria. Bact. Rev. 41:100-180.
Weimer, P.J., J.B. Russell, and R.E. Muck. 2009. Les-
sons from the cow: What the ruminant animal can
teach us about consolidated bioprocessing of cel-
lulosic biomass. Bioresource Technol. 100:5323-
5331.
Wolin, M. J., and T. L. Miller. 1983. Interactions of
microbial populations in cellulose fermentation.
Federation Proc. 42:109-113.
Wood, H. G. 1952. A study of carbon dioxide fixa-
tion by mass determination of the types of C13-
acetate. J. Biol. Chem. 194:905-931.
Zinder, S. H. 1993. Physiological ecology of metha-
nogens. In: “Methanogenesis. Ecology, Physiol-
ogy, Biochemistry and Genetics”. J. G. Ferry (ed.),
Chapman and Hall. New York, NY.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 275
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Foodborne illness outbreaks associated with fresh produce have significantly increased. Researchers
must investigate sources of these pathogens as well as new modes of transmission including internalization
within plant vascular systems. Confocal scanning laser microscopy (CSLM) was used to observe the location
of Escherichia coli O157:H7 and Salmonella on and within fresh spinach leaves. Sections of leaves measur-
ing 1 cm2 and stems measuring 0.5 cm2 were inoculated in a suspension of green fluorescent protein (GFP)
E. coli O157:H7 and red fluorescent protein (RFP) Salmonella transformed by electroporation to express
and at initial levels of 106 to 107 CFU/cm2. Samples were washed before preparing for CSLM, therefore, all
microorganisms visualized were assumed to be strongly attached. Both pathogens were found attached to
the surface, cut edges and within tissue layers. Internalization was determined on leaves and stems by tak-
ing multiple images of the same sample at different layers. Fluorescent cells not seen on the surface layer
of the sample appeared in the interior of spinach sample. These images demonstrate the ability of patho-
gens to congregate in areas on the leaf surface as well as internalization within the plant possibly escaping
chemical decontamination treatments.
Keywords: Confocal microscopy, spinach, E. coli O157:H7, Salmonella
Correspondence: Alejandro Castillo, [email protected]: +1 -979-845-3565 Fax: +1-979-862-3475
BRIEF COMMUNICATION
Attachment of Escherichia coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal Microscopy
J. A. Neal1,2, E. Cabrera-Diaz1,3, and A. Castillo1
1Department of Animal Science, 2471 TAMU, Texas A&M University, College Station, TX2Current address: Conrad N. Hilton College of Hotel and Restaurant Management, University of Houston, Houston, TX
3Current address: Department of Public Health, University Center for Agricultural and Biological Sciences, University of Guadalajara, Guadalajara, Mexico
Agric. Food Anal. Bacteriol. 2: 275-279 2012
276 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
INTRODUCTION
The number of reported foodborne illness out-
breaks associated with fresh produce has increased
in the past thirty years (Alkertruse et al., 1996; Hed-
berg and Osterholm, 1994; Sivapalasingam et al.,
2004). This increase can be attributed not only to
changes in consumption patterns but also changes
in production and processing technologies, new
sources of produce as well as the manifestation of
pathogens such as Salmonella and Escherichia coli
O157:H7 that have not been previously associated
with raw produce (Burnett and Beuchat, 2001; Siv-
apalasingam et al., 2004; Hanning et al., 2009).
Bacteria can be introduced to leafy greens at any
step from planting to consumption and once they
are introduced, their colonization can have a tre-
mendous effect on both the quality and safety of the
product. The attachment and colonization of micro-
organisms on fresh produce have significant public
health implications due to the fact that these pro-
cesses may be related to the inability of sanitizers
and decontamination treatments to remove or in-
activate human pathogens (Beuchat, 2002; Frank,
2001). Bacteria attach to fruits and vegetables in
pores, indentations and natural irregularities on the
produce surface where there are protective binding
sites as well as cut surfaces, puncture wounds, and
cracks in the surface (Sapers, 2001; Seo and Frank,
1999).
Although several studies have demonstrated that
human bacterial pathogens have the ability to pen-
etrate the interior of cut leaf edges or become in-
ternalized within lettuce tissue (Seo and Frank, 1999;
Solomon et al., 2002; Takeuchi and Frank, 2001;
Takeuchi et al., 2000; Wachtel et al., 2002), studies
on spinach, particularly those aimed at simulating
postharvest operations, are less obtainable. A re-
cent review of literature on the internalization of pro-
duce by pathogens (Erickson, 2012) shows how most
studies involving spinach have focused on the inter-
nalization of pathogens during growth. The scenario
where spinach is subjected to a postharvest wash
where pathogens may be transferred to the leaves
has not been profusely studied. The purpose of this
study was to determine how pathogens associate
with spinach leaves after washing cut spinach leaves,
simulating incorrect washing practices during post-
harvest processing of spinach.
MATERIALS AND METHODS
Source of spinach leaves
Fresh spinach leaves typical of leafy greens en-
tering the U.S. food supply were kindly provided by
the Winter Garden Spinach Producers Board (Crys-
tal City, TX). The spinach was harvested at approxi-
mately 45 days and placed in coolers with an internal
temperature of 4°C for 6 h and transported 250 miles
to the Texas A&M Food Microbiology Laboratory,
College Station, TX, where it was stored at 4°C for
up to 24 h. In the laboratory, spinach leaves were
manually sorted to remove leaves that were bruised,
cut or had decay. Spinach leaves were not washed or
decontaminated in any manner before the spinach
was obtained for this study.
Sources of bacteria and plasmids
Isolates from the Texas A&M Food Microbiology
Laboratory culture collection were previously trans-
formed by electroporation using the plasmid vectors
pEGFP and pDsRed-Express (Clontech Laborato-
ries, Inc., Mountain View, CA) to express GFP or RFP
and resistance to ampicilin. The GFP plasmid was
inserted in the strain of E. coli O157:H7, which had
been isolated from cattle fecal samples, whereas the
RFP plasmid was inserted into S. Typhimurium ATCC
13311. Three days prior to the experiment the mi-
croorganisms were resuscitated by two consecutive
transfers to tryptic soy broth (TSB; Becton Dickinson,
Sparks, MD) and incubated at 37°C for 24 h. A 24 h
TSB culture of GFP-producing E. coli O157:H7 (GFP-
EC) and RFP-producing S. Typhimurium (RFP-ST) was
harvested, washed in sterile phosphate buffer saline
(PBS; EMD Biosciences, Inc. La Jolla, CA) and resus-
pended in 0.1% peptone water (Becton Dickinson).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 277
Preparation of inoculum and sample preparation for confocal scanning laser microscopy
A bacterial cocktail was prepared consisting of 1
mL each of GFP-EC .and RFP-ST. The cocktail then
was added to 8 mL 0.1% peptone water to produce
a suspension containing 7.0 to 8.0 log CFU/mL. Sam-
ples consisting of 4 spinach leaf pieces measuring
1 cm2 and 4 stem samples measuring 0.5 cm2 were
placed in the cocktail and stored in an incubator at
37°C for 24 h to promote growth. The spinach leaf
and stem samples then were washed twice in 0.1%
peptone water. This wash had been validated to re-
move loosely attached cells. Strongly attached bac-
teria were those not removed by washing, and were
verified by plate counting on tryptic soy agar (Becton
Dickinson) supplemented with 100 µg/mL ampicillin
(TSA + Amp). The washed spinach samples were ob-
served using a BioRad Radiance 2000MP confocal
microscope (Zeiss, Heltfordshire, UK) using an exci-
tation wavelength of 488 nm. The confocal microsco-
py was conducted at the Image Analysis Laboratory
at Texas A&M University (College Station, TX).
RESULTS AND DISCUSSION
For the confocal microscopy study, the spinach
leaf provided a thin, relatively flat sample, which pro-
duced meaningful images. Internalization of E. coli
O157:H7 and Salmonella was seen on leaves and
stems by taking multiple images of the same sample
at different layers. Fluorescent cells not seen on the
surface layer of the sample appeared in the interior
images of the same sample. Microorganisms near
the cut surface of a spinach leaf can be seen in Fig-
ure 1A. The preferential gathering of pathogens to
the stomata and cracks in the cuticle are seen in Fig-
ure 1B. Fluorescent bacteria allocated in the interior
of the spinach stem are shown in Figures 2A and 2B.
These images demonstrate the ability of patho-
gens to congregate in areas on the leaf surface as
well as internalization within the plant possibly es-
caping chemical decontamination treatments. One
possible reason for the congregation of pathogens
in specific areas on the leaf surface may be due in
part to high hydrophobic leaf surfaces allowing
surface water to accumulate in depressions of leaf
veins suggesting that more free water is available
Figure 1. Confocal scanning laser microscopy (CSLM) photomicrographs of spinach leaves inocu-lated with GFP-expressing E. coli O157:H7 and RFP- expressing Salmonella. (A) Pathogens lined along the cut edge of the spinach leaf (arrows). (B) Pathogens at the stomata and cracks (arrows).
278 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
to pathogens at these locations. The accumulation
of bacteria in the stomata or intercellular spaces of
lettuce and spinach has been reported in different
studies and seems to be induced by light and colo-
nization mechanisms (Brandl and Mandrell, 2002;
Gomes et al., 2009; Kroupitski et al., 2009; Solomon
et al., 2002; Xicohtencatl-Cortes et al., 2009). This in-
ternalization seems to result in the microorganisms
being out of reach of antimicrobial compounds used
for washing and disinfecting produce (Xicohtencatl-
Cortes et al., 2009).
In addition, lesions on lettuce and spinach leaves
provide sites for internalization of microorganisms
where they may be protected from adverse condi-
tions and provide a higher availability of substrates
(Brandl, 2008). Seo and Frank (1999) described the
preferential attachment of E. coli O157:H7 to cut
edges rather than intact surfaces and the penetra-
tion of the pathogen into the interior of lettuce leaf.
Takeuchi and Frank (2000) reported similar findings
and suggested that E. coli O157:H7 may attach to
less favorable attachment sites once all of the pre-
ferred initial attachment sites were occupied.
CONCLUSIONS
From our findings, it is apparent that pathogens
such as E. coli O157:H7 and Salmonella can not only
lodge themselves onto exterior locations inacces-
sible to chemical sanitizers but can also be inter-
nalized within the plant structure. Therefore, both
farmers and processors must realize that chemical
sanitizers may not reach all microorganisms when
washing leafy greens, such as spinach. Efforts must
be taken to reduce the overall microbial load of the
produce and begins with preventing contamination
by implementing Good Agricultural Practices.
Figure 2. Confocal scanning laser microscopy (CSLM) photomicrographs showing GFP-expressing E. coli O157:H7 and RFP-expressing Salmonella in the interior of spinach stems. (A) Pathogens throughout stem fissures (arrows). (B) Pathogens lodged within crevices in the stem interior (ar-rows).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 279
REFERENCES
Alkertruse, S. F., and D. L. Swerdlow. 1996. The
changing epidemiology of foodborne disease.
Am. J. Med. Sci. 311:23-29.
Beuchat, L. R. 2002. Ecological factors influencing
survival and growth of human pathogens on raw
fruits and vegetables. Microbes and Infect. 4:413-
423.
Brandl, M. T. 2008. Plant lesions promote the rapid
multiplication of Escherichia coli O157:H7 on post-
harvest lettuce. Appl. Environ. Microbiol. 74:5285-
5289.
Brandl, M. T., and R. E. Mandrell. 2002. Fitness of Sal-
monella enterica serovar Thompson in the cilantro
phyllosphere. Appl. Environ. Microbiol. 68:3614-
3621.
Burnett, S. L., and L. R. Beuchat. 2001. Human patho-
gens associated with raw produce and unpasteur-
ized juices, and difficulties in decontamination. J.
Ind. Microbiol. Biotechnol. 27:104-110.
Erickson, M. C., 2012. Internalization of fresh pro-
duce by foodborne pathogens. Annul. Rev. Food
Sci. Technol. 3:283–310.
Frank, J. F. 2001. Microbial attachment to food and
food contact surfaces. Adv. Food. Nutri. Res.
43:319-370.
Gomes, C., P. Da Silva, R. G. Moreira, E. Castell-
Perez, E. A. Ellis, and M. Pendleton. 2009. Under-
standing E. coli internalization in lettuce leaves for
optimization of irradiation treatment. Int. J. Food
Microbiol. 135:238-247.
Hanning, I. B., J. D. Nutt, and S. C. Ricke. 2009. Sal-
monellosis outbreaks in the United States due to
fresh produce: sources and potential intervention
measures. Foodborne Path. Dis. 6: 635-648.
Hedberg, C. W., and M. T. Osterholm. 1994. Chang-
ing epidemiology of food-borne diseases: a Min-
nesota perspective. Clin. Infec. Dis. 18: 671-682.
Kroupitski, Y., D. Golberg, E. Belausiv, R. Pinto, D.
Swatzberg, D. Granot, and S. Sela. 2009. Internal-
ization of Salmonella enterica in leaves is induced
by light and involves chemotaxis and penetration
through open stomata. Appl. Environ. Microbiol.
75:6076-6086.
Sapers, G. M. 2001. Efficacy of washing and sani-
tizing methods for disinfection of fresh fruit and
vegetable products. Food Technol. Biotechnol.
39:305-311.
Seo, K. H., and J. F. Frank. 1999. Attachment of Esch-
erichia coli O157:H7 to lettuce leaf surface and
bacterial viability in response to chlorine treatment
as demonstrated by using confocal scanning laser
microscopy. J. Food Prot. 62:3-9.
Sivapalasingam, S., C. R. Friedman, L. Cohen, and
R. V. Tauxe. 2004. Fresh produce: a growing cause
of outbreaks of foodborne illness in the United
States, 1973 through 1997. J. Food Prot. 67: 2342-
2353.
Solomon, E. B., S. Yaron, and K. R. Matthews. 2002.
Transmission of Escherichia coli O157:H7 from
contaminated manure and irrigation water to let-
tuce plant tissue and its subsequent internaliza-
tion. Appl. Environ. Microbiol. 68:397-400.
Takeuchi, K., and J. F. Frank. 2000. Penetration of
Escherichia coli O157:H7 into lettuce tissues as
affected by inoculum size and temperature and
the effect of chlorine treatment on cell viability. J.
Food Prot. 63:434-440.
Takeuchi, K., and J. F. Frank. 2001. Quantitative de-
termination of the role of lettuce leaf structures in
protecting Escherichia coli O157:H7 from chlorine
disinfection. J. Food Prot. 64:147-151.
Takeuchi, K., C. M. Matute, A. N. Hassan, and J. F.
Frank. 2000. Comparison of the attachment of
Escherichia coli O157:H7, Listeria monocytogenes,
Salmonella Typhimurium, and Pseudomonas fluo-
rescens to lettuce leaves. J. Food Prot. 63:1433-
1437.
Wachtel, M. R., L. C. Whitehand, and R. E. Mandrell.
2002. Association of Escherichia coli O157:H7 with
preharvest leaf lettuce upon exposure to contami-
nated irrigation water. J. Food Prot. 65:18-25.
Xicohtencatl-Cortes, J., E. S. Chacón, Z. Saldaña, E.
Freer, and J. A. Giron. 2009. Interaction of Esch-
erichia coli O157:H7 with leafy green produce. J.
Food Prot. 72:1531-1537.
280 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Two experiments were conducted to evaluate the effect of the iron-binding molecule lactoferrin on re-
ducing gut populations and fecal shedding of Salmonella Typhimurium in experimentally-infected weaned
pigs. For each experiment, crossbred barrows and gilts were purchased locally and transported to our
laboratory facilities. All pigs were fed a ground starter diet available for ad libitum consumption and ran-
domly assigned to pen (2 pigs/pen) and treatment (10 pigs/treatment; 5 pens/treatment): Control [1.25
g whey protein concentrate (WPC)/kg BW (body weight)/d); 1X lactoferrin [0.25 g lactoferrin (LF) + 1.0 g
WPC/kg BW/d]; and 5X LF (1.25 g LF/kg BW/d). Experimental treatments were fed prior to inoculation via
oral gavage with Salmonella Typhimurium. Rectal swabs (collected daily for 4 days) for quantification of the
challenge Salmonella strain and scour and activity scores, and body temperatures recorded daily follow-
ing inoculation. Five days post-challenge, pigs were euthanized and tissue and luminal content samples
aseptically collected from the stomach, ileum, cecum, spiral colon and rectum. Additional tissue samples
were collected from the ileo-cecal lymph nodes, spleen, tonsil, and liver. Quantitative and qualitative bac-
terial culture was conducted for the challenge strain of Salmonella. No treatment differences (P > 0.10)
were observed for daily fecal shedding or luminal concentrations of Salmonella in either experiment. The
percentage of tissue samples Salmonella positive was not significantly different among treatments with the
exception of liver tissue in Experiment I, which was lower (P < 0.05) in the 1X and 5X treatments compared
to control pigs. Body weights and BW change were not affected (P > 0.10) by treatment. Following inocu-
lation, body temperatures, scour and activity scores were not different when examined by day or when data
was combined across days. Future research should evaluate increasing the duration of feeding and/or the
levels of lactoferrin fed in conjunction with a more subtle Salmonella challenge.
Keywords: Salmonella, lactoferrin, pigs
Correspondence: Tom S. Edrington, [email protected]: +1 -979-260-3757 Fax: +1-979-260-9332
Lack of Effect of Feeding Lactoferrin on Intestinal Populations and Fecal Shedding of Salmonella Typhimurium
in Experimentally-Infected Weaned Pigs
D. J. Nisbet1, T. S. Edrington1, R. L. Farrow1, K. G. Genovese1, T. R. Callaway1, R. C. Anderson1, N. A. Krueger1
1United States Department of Agriculture, Agriculture Research Service, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, 2881 F&B Road, College Station, TX 77845 USA
Agric. Food Anal. Bacteriol. 2: 280-290, 2012
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 281
INTRODUCTION
Salmonella is the second leading cause of
foodborne illness, resulting in an estimated 1.4 mil-
lion cases every year (Foley and Lynne, 2008). Of
these human cases of salmonellosis, 6-9% are asso-
ciated with the consumption of pork or pork prod-
ucts (Frenzen et al., 1999). Salmonella has been iso-
lated throughout all stages of the pork production
cycle and has received considerable attention, not
only from a food safety standpoint, but additionally,
because Salmonella can cause clinical infection in
swine. Salmonella positive pigs are thought to arise
from one of two general factors, inputs (pigs, feed,
rodents, etc.) and activities within the swine produc-
tion process (mixing of animals, transport, housing,
and other management factors). Early weaning (<
21 d of age) which has gained in popularity, results
in an immature digestive tract (Shields et al., 1980)
and perhaps more importantly, a decrease in im-
mune system function (Blecha et al., 1983), both of
which would favor Salmonella colonization in these
animals. To respond to the challenge of providing a
safe pork product for the consumer, improve swine
health, and maintain a safe environment, the devel-
opment of pre-harvest, “on-farm” intervention strat-
egies is crucial.
Most all bacteria, including the pathogenic bac-
teria Campylobacter, E. coli and Salmonella, require
iron for survival and important intracellular reactions
(Naikare et al., 2006; Brock, 1980; Ratledge and Do-
ver, 2000), thus iron-sequestering compounds such
as lactoferrin and transferrin provide a primary non-
specific host defense system against microbial infec-
tion. A variety of preventative and therapeutic strate-
gies for treating bacterial infections are based upon
interfering with microbial iron acquisition and utiliza-
tion. The immune system likewise exploits the iron
requirement of bacteria, utilizing iron withholding as
an essential antimicrobial component of the innate
immune system.
Lactoferrin is a major iron-binding protein pres-
ent in multiple body fluids and found in particularly
high concentrations in both human and porcine milk
(Gislason et al., 1993; Vorland, 1999). The iron-bind-
ing abilities of lactoferrin enable it to scavenge iron
within the intestinal tract thereby depriving microor-
ganisms of this critical element and inhibiting their
metabolic activities (Naidu et al., 1993). Facilitating
iron absorption, stimulation of mucosal differen-
tiation, and modulation of mucosal immunity have
been suggested as possible functions of lactoferrin
within the gastrointestinal tract (Lonnerdal and lyer,
1995). Additional research indicates that the antimi-
crobial properties of lactoferrin go beyond simple
iron deprivation and include damage of the outer
membrane and subsequent permeability altera-
tions (Ellison et al., 1988) and modulation of bacterial
motility, aggregation and adhesion (Valenti and An-
tonini, 2005). Lactoferrin has been shown to inhibit
growth of several important bacteria, including Sal-
monella, E. coli, Listeria, Streptococcus and Shigella
(Weinberg, 1995; Lonnerdal and Iyer, 1995; Pakkanen
and Aalto, 1997; Weinberg, 2001; Lee et al., 2004).
Other research has demonstrated that oral admin-
istration of lactoferrin decreases bacterial infections
within the gastrointestinal tract while at the same
time increasing populations of beneficial bacteria
such as Lactobacillus and Bifidobacteria with low iron
requirements (Petschow et al., 1999; Weinberg, 2001;
Tomita et al., 2002; DiMario et al., 2003; Teraguchi
et al., 2004; Sherman et al., 2004). Thus, based on
the antimicrobial activities of lactoferrin, the objec-
tive of the current project was to determine whether
oral administration of lactoferrin would significantly
reduce the populations of Salmonella within the gas-
trointestinal tract of experimentally-infected pigs.
MATERIALS AND METHODS
Experiment I
Forty crossbred barrows and gilts (avg. BW = 24 kg)
were purchased locally and transported to our labo-
ratory facilities. Upon arrival all pigs were weighed,
eartagged and a rectal swab collected for culture
of wild-type Salmonella. All pigs were housed in
environmentally-controlled isolation rooms (10 pigs/
room) for one week and maintained on a pelleted
282 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
commericial pig starter feed available for ad libitum
consumption. The following week, pigs were moved
to another part of the same building and randomly
assigned to pen (2 pigs/pen) where they remained
for the remainder of the experimental period. Treat-
ments (detailed below) were randomly assigned to
pen, therefore a few pigs were moved to ensure
similar sex and BW distribution among treatments.
Two days following movement into the experimen-
tal pens, adaptation from the pelleted to a ground
meal feed was initiated. All pigs were fed a 50/50
mix of pelleted and meal feed for four days, 25/75
pellets and meal for 2 days and 100% meal feed for
three days prior to initiation of the experimental di-
ets. One day prior to the start of the experiment, all
pigs were weighed and given new eartags.
Experimental treatments (10 pigs/treatment; 5
pens/treatment) consisted of: Control [1.25 g whey
protein concentrate (WPC)/kg BW/d); 1X lactoferrin
[0.25 g lactoferrin (LF) + 1.0 g WPC/kg BW/d]; 5X LF
(1.25 g LF/kg BW/d); and Non-infected Control (1.25
g WPC but not inoculated with Salmonella). Feed in-
takes were recorded and used to calculate an aver-
age daily feed intake per treatment. Based on the
average feed intakes, diets were mixed to provide
the amounts above of the experimental compounds
per pig each day. Body weight and feed intake were
recorded weekly and the feed adjusted accordingly.
Experimental treatments were fed for a total of 20
days. On day 15 of the experimental diets, all pigs
were inoculated via oral gavage with Salmonella Ty-
phimurium (2.6 x 1010 in 20 mL TSB). Rectal swabs
were collected daily for 4 days for quantification of
the challenge Salmonella strain as described be-
low. Scour and activity scores (for each pen) were
recorded daily following inoculation through nec-
ropsy. Body temperature was recorded daily for each
pig following inoculation using the ThermoFlash®
electronic thermometer (PRO-IR ZH-36 Veterinary
thermometer; Synergy USA, Miami, FL). Five days
post-challenge, pigs were sedated with an intra-
muscular injection of a cocktail containing Ketaset,
Telazol (Ft. Dodge Laboratories, Kansas City, MO)
and Xylazine (Phoenix Scientific, St. Joseph, MO)
prior to administration of a lethal dose of Euthasol
(Delmarva Laboratories, Midlothian, VA). Tissue and
luminal content samples were aseptically collected
from the stomach, ileum, cecum, spiral colon and
rectum. Additional tissue samples were collected
from the ileo-cecal lymph nodes, spleen, tonsil, and
liver. All tissue and content samples were cultured as
described below immediately following collection.
Non-infected control pigs were not euthanized for
reasons discussed below.
Experiment II
A second experiment was conducted, similar to
the first, with the exception that much younger pigs
were utilized. Thirty crossbred piglets (average BW
= 6.6 kg), were purchased within one week of wean-
ing and transported to our laboratory facilities. Pigs
were weighed, eartagged, rectally swabbed and ran-
domly assigned to pen (2 pigs/pen). All pigs were
provided a pig starter ration (ground) and water for
ad libitum intake. Following analysis of initital BW, a
few pigs were moved to assure equal distribution of
BW among treatments. Animals were provided a 4
day adjustment period to acclimate to pens and diet
and determine feed intakes. Following this acclima-
tion, treatments were initiated (d 1) and adminis-
tered throughout the remainder of the experimental
period (13 total days; 10 pigs and 5 pens/treatment).
Treatments were identical to those used in Experi-
ment I with the exception that a non-infected con-
trol treatment was not included due to the ease in
which pigs in this treatment acquired Salmonella in
the first experiment. On day 8 of the experiment, all
pigs were orally inoculated with 8 mL of TSB con-
taining 5.6 x 109 cfu Salmonella Typhimurium. Rectal
swabs, body temperature, activity and scour scores
were collected daily for 5 days following inoculation.
All animals were euthanized and necropsied as de-
scribed above on d 13. Body weights were recorded
upon arrival and on d 1, 8 and 13 of the experimental
period.
Bacterial Culture
Experiment I
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 283
Rectal swabs were collected using a foam-tipped
swab (ITW Texwipe, Mahwah, NJ). Swabs taken prior
to inoculation were incubated in 9 mL tetrathionate
broth (37° C, 24 h), followed by a second enrichment
[100 µL to 5 mL of Rappaport-Vassiliadis (RV) R10
broth; 42° C, 24 h], before spread plating on brilliant
green agar (Oxoid Ltd., Hampshire, UK) containing
novobiocin (BGANOV; 20 µg/mL) and novobiocin plus
naladixic acid (BGANN; 20 and 25 µg/mL, respective-
ly) for detection of any wild-type Salmonella. A few
pigs were naturally-colonized with a wild-type Sal-
monella capable of growth on BGANOV, therefore all
samples collected following inoculation of pigs were
streaked on BGANN. The inoculation strain of Salmo-
nella was enumerated in luminal contents by direct
plating from a mixture of 1 g contents in 10 mL of
tryptic soy broth (TSB) onto XLD agar using a com-
mercially available spiral plater (Spiral Biotech Auto-
plate 4000; Advanced Instruments, Inc., Norwood,
MA). Black colonies were counted following incuba-
tion (37° C, 24 hours). An additional 1 g of luminal
content or tissue sample was enriched (qualitative
culture) in 10 mL of tetrathionate broth, transferred
to RV and plated as described above for the post-
inoculation swabs. Following incubation at 37°C for
24 h, BGANN plates containing pink colonies exhibit-
ing typical Salmonella morphology were considered
positive.
Experiment II
Upon arrival all pigs were naturally-colonized
with Salmonella capable of growth on BGANOV and
BGANN, therefore fecal swabs were collected daily
throughout the entire experimental period and
plated on BGANOV to monitor shedding of the wild-
type Salmonella and any response to experimental
treatments. Due to the presence of this Salmonella,
the inoculated strain of Salmonella Typhimurium
was made resistant to rifampicin (25 µL/mL; prior to
administration to the pigs) and all post-inoculation
swabs and necropsy samples additionally plated on
BGANNR. Spiral plating of luminal content samples
was done on XLD + novobiocin and XLD + rifam-
picin. All enrichment procedures were identical to
those used in Experiment I described above.
Statistical Analysis
All data were analyzed using SAS Version 9.1.3
(SAS Inst. Inc., Cary, NC, USA). Quantitative culture
data from the luminal contents (log-transformed),
body weight and temperature data were subjected
to analysis of variance appropriate for a completely
randomized design. Qualitative culture data (inci-
dence of positive luminal content and tissue sam-
ples) was subjected to Chi-square analysis using the
PROC FREQ procedure. Daily rectal swab culture re-
sults (positive or negative), activity and scour scores
were analyzed using the PROC MIXED procedure for
repeated measures with treatment, day and treat-
ment x day interaction included in the model. For
some samples, Salmonella was recovered only from
enriched specimens or not at all indicating that con-
centrations were below our limit of detection (< 20
cfu/g of contents). Due to the inherent assumption
that these samples were below the limit of detection
(rather than assumed to be truly zero), we assigned a
value of 1.0 cfu/g to all quantitative data prior to sta-
tistical analysis. Results were considered statistically
significant at the 0.05 level for type-one error.
RESULTS
Experiment I
All pigs were pre-screened for Salmonella three
times prior to initiation of the experimental diets
using rectal swabs. The first and second collections
were plated on BGANOV for detection of any wild-
type Salmonella. All samples from the first collec-
tion were negative while five pigs were Salmonella
positive in the second collection (serogroups B and
C2). The second and third collections were plated on
BGANN to determine its suitability for detecting the
challenge strain of Salmonella post-inoculation. All
pigs were culture negative on this medium (data not
shown).
Rectal swabs collected over the 4-d post-inocu-
lation period were mostly positive in all treatment
groups, including the non-infected control pigs. Di-
284 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Treatment
Item Control 1 X 5 X NI Cont P > F
Rectal Swabs (% positive)a
d 1 100 100 100 100 1
d 2 90 90 80 80 0.85
d 3 100 90 100 80 0.26
d 4 100 90 80 20 0.0002
Overall 97.5 92.5 90 70 0.001
Luminal contents
Direct plate [cfu/g (log10)]
Stomach 1 1 1 . 1
Ileum 2.4 3.4 2.7 . 0.31
Spiral colon 2.7 3.7 2.8 . 0.14
Cecum 3.2 3.7 3.2 . 0.23
Rectum 2.4 3.2 2.4 . 0.17
% positive after enrichment
Stomach 50 40 60 . 0.67
Ileum 90 80 100 . 0.33
Spiral colon 90 80 70 . 0.54
Cecum 100 90 90 . 0.59
Rectum 60 80 50 . 0.37
Tissue
% positive after enrichment
Ileo-cecal lymph nodes 100 90 100 . 0.36
Spleen 10 30 20 . 0.54
Tonsil 80 80 70 . 0.83
Liver 70 10 30 . 0.02
Stomach 60 60 90 . 0.24
Ileum 80 100 90 . 0.33
Spiral colon 100 100 100 . 1
Cecum 90 90 90 . 1
Rectum 90 90 90 . 1aBy day post-inoculation
Table 1. Daily fecal shedding, luminal content populations of Salmonella (CFU/g log10) and Salmonella positive tissue and luminal content samples in pigs experimentally-infected with Salmonella Typhimurium and fed diets containing 1.25 g whey protein concentrate (WPC)/kg BW(body weight)/d = (Control); 0.25 g lactoferrin (LF) + 1.0 g WPC/kg BW/d (1X); 1.25 g LF/kg BW/d (5X); or 1.25 g WPC but not inoculated with Salmonella (NI Cont) – Experiment I.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 285
rect streaking of the swab onto the agar was con-
ducted on d17 – 19 to get an indication of Salmo-
nella concentrations in the feces. A positive swab
via direct plating would be indicative of a higher
concentration of Salmonella being shed by the ani-
mal, compared to a swab requiring enrichment to
test culture positive. All direct swabs were nega-
tive in the non-infected control treatment, therefore
only the direct plating data for the infected-control,
1X and 5X treatments were analyzed and no differ-
ences (P > 0.10) observed (data not shown). Fol-
lowing enrichment, no treatment differences (P >
0.10) were observed on each of the first three days
post-inoculation, but by day 4 and when daily rectal
swab data was combined and examined across days,
non-infected controls had fewer Salmonella positive
swabs (P < 0.01; Table 1). We certainly expected the
non-infected controls to have a lower prevalence of
Salmonella-positive fecal swabs throughout the ex-
perimental period and were surprised by the num-
ber of positive animals early on in the experiment.
Although the non-infected control pigs were housed
in the same room as infected-animals, they were not
able to have any animal to animal contact. Obvious-
ly, contamination of these pigs could have occurred
via workers, air-movement, or other factors, however,
finding 100% of these pigs Salmonella-positive one
day following inoculation of the other pigs, was not
expected and highlights the ease in which Salmo-
nella is transmitted among pigs and the short time
duration required for fecal shedding following expo-
sure. We did not serogroup any of the isolates from
these animals to determine if the recovered Salmo-
nella was the same as used to infect pigs in the other
treatments as this information would be of limited
value. Non-infected controls were included to de-
termine if the whey-protein concentrate influenced
growth, however, as all of these pigs were Salmonel-
la-positive at some point in the experiment the deci-
sion was made not to necropsy this group.
Necropsy results are presented in Table 1. Con-
centrations of the challenge-strain of Salmonella
were not statistically different among treatments
throughout the GIT, although the 5X treatment had
populations more similar to controls than did the
1X treatment, which had concentrations numerically
higher in contents from the ileum, spiral colon, ce-
cum and rectum. All stomach content samples were
negative in all treatments. Following enrichment, lu-
minal content samples were not different (P > 0.10)
among treatments. Tissue samples were also not dif-
ferent (P > 0.10) among treatments, with the excep-
tion of liver tissue, which was lower (P < 0.05) in the
1X and 5X treatments compared to control pigs.
Body weights and BW change were not affected
(P > 0.10) by treatment, although the 5X pigs gained
2.4 kg more than infected-control animals (data not
shown). Following inoculation, body temperatures
were not different when examined by day or when
data was combined across days. A trend (P < 0.10)
was observed on d 18 and when data was combined
across days, however the differences were slight and
do not suggest treatment effects (data not shown).
There was not a significant treatment x day interac-
tion for activity or scour scores (P > 0.10), nor were
significant differences observed when data was com-
bined across days (data not shown).
Experiment II
The majority of pigs were Salmonella-positive
during the pre-screening process, therefore we at-
tempted to examine the effect of the experimental
treatments on the wild-type Salmonella strains as
well as the experimentally-inoculated strain. Table 2
presents the prevalence of Salmonella positive rectal
swabs (pre-challenge for the wild strain; post-chal-
lenge for all Salmonella) as well as necropsy results.
To distinguish the two types of Salmonella, samples
were plated on BGANOV for the wild-type Salmonella
and BGANNR for Salmonella Typhimurium (challenge
strain). No differences (P > 0.10) were observed in
the prevalence of rectal swabs positive for the wild-
type Salmonella following direct plating or after en-
richment during the seven days of feeding the ex-
perimental diets pre-challenge. Post-challenge, no
differences (P > 0.10) were observed for shedding of
the wild-type strain (direct plated and enriched sam-
ples), while a trend (P < 0.10) was observed for the
inoculated strain following direct plating (prevalence
286 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Treatment
Control 1 X 5 X P > F
Item nov nnr nov nnr nov nnr nov nnr
Rectal swabs (% positive)
Pre-challenge - overall (n=70/trt)
Direct plate 7.1 . 5.7 . 2.9 . 0.51 .
Enriched 52.3 . 54.3 . 52.9 . 0.98 .
Post-challenge - overall (n=40/trt)
Direct plate 27.5 25 40 50 32.5 40 0.49 0.07
Enriched 87.5 87.5 87.5 90 92.5 92.5 0.71 0.76
Luminal contents
Concentration [cfu/g (log10)]
Stomach 1 1 1 1 1 1 1 1
Ileum 1.2 1.2 1.4 1.1 1.8 1.7 0.37 0.14
Spiral colon 1.8 1.5 2.4 2.1 2.3 2.3 0.48 0.16
Cecum 1.9 1.6 1.4 1.3 1.8 1.6 0.54 0.75
Rectum 1.4 1.4 1.8 1.3 1.8 1.8 0.54 0.34
% positive w/enrichment
Stomach 10 10 0 0 30 30 0.13 0.13
Ileum 60 60 80 80 80 80 0.51 0.51
Spiral colon 100 100 100 100 100 100 1 1
Cecum 100 100 70 70 90 90 0.13 0.13
Rectum 100 100 100 100 90 90 0.37 0.37
Tissue (% positive w/enrichment)
Stomach 50 50 50 50 40 40 0.87 0.87
Ileum 90 90 90 90 90 90 1 1
Spiral colon 100 100 100 100 100 100 1 1
Cecum 80 80 100 100 100 100 0.18 0.18
Rectum 90 90 100 100 90 90 0.59 0.59
Ileo-cecal lymph nodes 60 60 90 90 70 70 0.3 0.3
Spleen 10 10 40 30 30 20 0.3 0.54
Tonsil 50 50 30 30 20 20 0.35 0.35
Liver 100 100 100 100 100 100 1 1
Table 2. Daily fecal shedding, luminal content populations of Salmonella (CFU/g log ) and Salmonella positive tissue and luminal content samples in pigs both naturally and experimentally-infected and fed diets containing 1.25 g whey protein concentrate (WPC)/kg BW (body weight)/d = (Control); 0.25 g lactoferrin (LF) + 1.0 g WPC/kg BW/d (1X); or 1.25 g LF/kg BW/d (5X). Naturally-occurring and experimentally-infected strains of Salmonella were plated on brilliant green agar containing novobiocin (nov) and novobiocin plus naladixic acid and rifampicin (nnr), respectively – Experiment II.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 287
in 1X and 5X treatments numerically higher than con-
trol pigs). No differences were observed for Salmo-
nella Typhimurium following enrichment of the rectal
swabs. Concentrations of Salmonella in the luminal
contents throughout the GIT were not different (P >
0.10) among treatments for either the wild-type or
inoculated Salmonella strains, nor was prevalence
different (P > 0.10) following enrichment of content
samples. Similarly, the prevalence of positive tissue
samples following enrichment were not different
among treatments for either Salmonella type.
Body weights and BW change were similar (P >
0.10) among treatments throughout the experiment
(data not shown). Similar to the first experiment, pigs
in the 5X treatment exhibited a numerical increase in
BW gain compared to control animals following in-
oculation with the challenge strain. No differences (P
> 0.10) in body temperature were observed pre- or
post-challenge, however there was a tendency (P =
0.09) for pigs in the 5X treatment to have higher tem-
peratures than the control and 1X animals (data not
shown). No treatment x day interactions were ob-
served for activity or scour scores, therefore data was
combined and presented as pre- and post-challenge
and across all days. Neither activity nor scour scores
were statistically different pre- or post-challenge or
when data was combined across the entire experi-
mental period (data not shown).
DISCUSSION
Oral administration of lactoferrin has been report-
ed to provide host protection against various dis-
eases in animals and humans, including infections,
cancers and inflammations (Tomita et al., 2002).
Teraguchi and colleagues (2004) concluded that oral
lactoferrin enhances the systemic or peripheral im-
mune responses to pathogens, their components, as
well as mucosal immunity in the intestines and that
these responses may contribute to elimination of
the pathogens and/or a reduction of the symptoms.
Lactoferrin binds to Salmonella Typhimurium and
bovine lactoferrin has been shown to have an iron-
dependent bacteriostatic effect on this pathogen
(Naidu et al., 1993; Ochoa and Cleary, 2009). Both
bovine and human lactoferrin inhibit the adherence
and invasion of Salmonella to tissue culture cells
(Bessler et al., 2006). Wang and co-workers (2006)
reported a beneficial effect of lactoferrin supple-
mentation on growth performance of weaned pig-
lets and concluded the use of lactoferrin to improve
nonspecific immunity and strengthen host defenses
would be a good method of protecting weaned pigs
from infections and stress due to weaning. Taken to-
gether, we reasoned that administration of lactofer-
rin to pigs may reduce the gut populations and fecal
shedding of Salmonella.
Due to some facility constraints and pig availabil-
ity, the pigs in the first experiment were older and
larger than we considered ideal for this experimen-
tation. We hypothesized that lactoferrin treatment
had the best chance of success in a younger animal
with an immature or under-developed gut microbio-
ta where Salmonella had less competition from other
microbes and was therefore more likely to flourish.
However, as pigs can be exposed to Salmonella at
all stages of the pork production cycle, the decision
was made to examine the effect of lactorferrin in the
larger animals. Whether or not this was the reason for
the lack of treatment effects in the first experiment
is unknown. The percentage of positive rectal swabs
and luminal contents were similar in the two experi-
ments, indicating that the experimental challenges
were similarly effective in the older and younger pigs
and that at these ages, differences in the gut micro-
bial ecosystem were negligible in terms of affecting
the challenge strain of Salmonella.
The second experiment was conducted virtually
identical to the first with the exception that we used
much younger pigs and had the added bonus that
the pigs were “naturally-colonized” with Salmonel-
la. In theory, this should provide for a more realistic
evaluation of the treatments, however to ensure all
pigs were similarly infected, animals were also ad-
ministered the challenge strain of Salmonella Ty-
phimurium. No effects of treatment were observed
on either the naturally-colonized or experimental
strain of Salmonella.
The lack of any observable benefits due to the
288 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
lactoferrin treatment in reducing Salmonella popula-
tions or the severity of infection in these experiments
is disappointing but may be explained by one or a
combination of factors discussed below. The most
plausible explanation is that the challenge doses
(109 and 1010 cfu Salmonella) utilized were such that
they simply overwhelmed any beneficial effects pro-
vided by the lactoferrin. A lower dose, more realistic
of what the pigs might be exposed to in a produc-
tion setting, may have provided a better test for the
lactoferrin treatments examined. However, in our
experience with experimental inoculation, the lower
doses are generally cleared quickly in any age ani-
mal except those with a very immature or disturbed
gut microflora. As pigs in both experiments were
weaned and eating well, we expected that a larger
challenge would be necessary to establish Salmo-
nella within the gut and produce concentrations that
could subsequently be detected in the luminal con-
tents and gut tissues at necropsy several days post-
inoculation.
Similar to our research, Sarelli and co-workers
(2003) evaluated lactoferrin for preventing E. coli di-
arrhea in weaned pigs. They reported no significant
effect on occurrence of diarrhea, fecal E. coli counts,
or weight gain in pigs dosed twice daily with lacto-
ferrin. The authors hypothesized that continual feed-
ing of the lactoferrin in the feed may provide more
protection than the twice-daily dosing regimen they
used and likewise suggested that the massive dose
of E. coli administered to the pigs may have simply
overwhelmed any protective effect exerted by the
lactoferrin and that future research should employ
challenges similar to what would be encountered by
the pigs in commercial production settings. Contrary
to these findings and our own reported herein, Lee
and co-workers (1998) reported oral lactoferrin pro-
tected piglets against lethal shock induced by intra-
venously administered E. coli LPS (endotoxin) with
significantly less mortality compared to the control
treatment.
Others have reported a beneficial effect of lac-
toferrin and lactoperoxidase system (LP-s) on ex-
perimentally-induced E. coli diarrhea in calves with
improvements in mortality, occurrence of severe di-
arrhea and duration of diarrhea observed (Still et al.,
1990). A combination of lactoferrin and LP-s given
orally decreased E. coli counts in the intestine and
feces of calves and likewise reduced the severity of
diarrhea (van Leeuwen et al., 2000). In the current re-
search, diarrhea was observed in pigs during both
experiments following Salmonella inoculation, but
contrary to the research by Still and van Leeuwen, no
beneficial effects of lactoferrin were observed on the
incidence or severity of diarrhea.
A second explanation for the lack of a treatment
effect in this research may be explained by the ad-
aptations bacteria make in order to compete with
iron-sequestering compounds such as lactoferrin.
Some strains of bacteria adapt to the iron-deprived
conditions by producing their own high affinity iron
chelators called siderophores, which compete di-
rectly with lactoferrin for iron (Crosa, 1989). Bacteria
may also synthesize specific lactoferrin receptors to
bind and extract iron from lactoferrin directly, as a
method to adapt to lactoferrin reduced iron avail-
ability (Schryvers et al., 1998). Either or both of these
adaptations may help explain the lack of treatment
effect on Salmonella in the current research.
A direct bactericidal activity independent of iron
acquisition has been proposed for lactoferrin, in
which the peptide lactoferricin is reported to have
a broad antimicrobial activity against several gram
negative bacteria (Wakabayashi et al., 2003). Other
reports (van der Strate et al., 2001; Ajello et al., 2002;
Gomez et al., 2003) suggest that lactoferrin contrib-
utes to the innate immune system of the host by in-
terfering with microbial virulence (adhesion, internal-
ization). Neutrophils provide a source of lactoferrin
in external fluids (Masson et al., 1969) in response to
microbial challenge and are thought to augment the
innate immune response against microbial infection
at the mucosal surface. Determining whether or not
lactoferrin produced this type of response in our ex-
periments is difficult at best. It is unclear if the inocu-
lated Salmonella (Exp. I) or the naturally-colonized
Salmonella (Exp. II) infected the mucosal surface of
the gastrointestinal tract or merely populated the lu-
minal contents throughout. However, we would sus-
pect that a lactoferrin-response such as this would
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 289
only be effective or measurable at much lower popu-
lations of Salmonella.
While the void of treatment differences in this re-
search is disappointing, it would be premature to
dismiss lactoferrin as a potential pre-harvest inter-
vention. It is likely that the large challenge dose used
in this research simply overwhelmed any protective
benefits offered by the lactoferrin. Future research
should examine the protective effects of feeding lac-
toferrin to recently weaned pigs prior to Salmonella
challenge, either administered in a lower oral dose
or via exposure to Salmonella-positive pigs.
ACKNOWLEDGEMENTS
This research was funded in part by Wyeth Phar-
maceuticals, Inc., Collegeville, PA.
REFERENCES
Ajello, M., R. Greco, F. Giansanti, M.T. Massucci, G.
Antonini, and P. Valenti. 2002. Anti-invasive activity
of bovine lactoferrin towards group A streptococ-
ci. Biochem. Cell Biol. 80:119-124.
Bessler, H.C., I.R. de Oliveira, and L.G. Giugliano.
2006. Human milk glycoproteins inhibit the adher-
ence of Salmonella typhimurium to HeLa cells. Mi-
crobiol. Immunol. 50:877-882.
Blecha, F., D.S. Pollmann, and D.A. Nichols. 1983.
Weaning pigs at an early age decreases cellular
immunity. J. Anim. Sci. 56:396-400.
Brock, J.H. 1980. Lactoferrin in human milk: its role
in iron absorption and protection against enteric
infection in the newborn infant. Arch. Dis. Child.
55:417-421.
Crosa, J.H. 1989. Genetics and molecular biology of
siderophore-mediated iron transport in bacteria.
Microbiol. Rev. 53:517-530.
Di Mario, F., G. Aragona, N. Dal Bo, G.M. Cavestro,
L. Cavallaro, V. Iori, G. Comparato, G. Leandro, A.
Pilotto, and A. Franze. 2003. Use of bovine lacto-
ferrin for Helicobacter pylori eradication. Dig. Liver
Dis. 35:706-710.
Ellison, R.T., III, T.J. Giehl, and F.M. LaForce. 1988.
Damage of the outer membrane of enteric gram-
negative bacteria by lactoferrin and transferrin. In-
fect. Immun. 56:2774-2781.
Foley, S.L., and A.M. Lynne. 2008. Food animal-
associated Salmonella challenges: Pathogenicity
and antimicrobial resistance. J. Anim. Sci. 86(14
Suppl):E173-87.
Frenzen, P.D., J.C. Buzby, and T. Roberts. 1999. An
updated estimate of the economic costs of human
illness due to foodborne Salmonella in the United
States. In: Proc. of the 3rd Int. Symp. on the Epide-
miology and Control of Salmonella in Pork, Wash-
ington, D.C., p. 215-218.
Gislason, J., I. Suhasini, T.W. Hutchens, and B. Lon-
nerdal. 1993. Lactoferrin receptors in piglet small
intestine: Lactoferrin binding properties, ontoge-
ny, and regional distribution in the gastrointestinal
tract. J. Nutr. Biochem. 4:528-533.
Gomez, H.F., T.J. Ochoa, L.G. Carlin, and T.G. Cleary.
2003. Human lactoferrin impairs virulence of Shi-
gella flexneri. J. Infect. Dis. 187:87-95.
Lee, N.Y., K. Kawai, I. Nakamura, T. Tanaka, H. Kumu-
ra, and K. Shimazaki. 2004. Susceptibilities against
bovine lactoferrin with microorganisms isolated
from mastitic milk. J. Vet. Med. Sci. 66:1267-1269.
Lee, W.J., J.L. Farmer, M. Hilty, and Y.B. Kim. 1998.
The protective effect of lactoferrin feeding against
endotoxin lethal shock in germfree piglets. Infect.
Immun. 66:1421-1426.
Lonnerdal, B., and S. Iyer. 1995. Lactoferrin: molecu-
lar structure and biological function. Annu. Rev.
Nutr. 15:93-110.
Masson, P.L., J.F. Heremans, and E. Schonne. 1969.
Lactoferrin, an iron-binding protein in neutrophilic
leukocytes. J. Exp. Med. 130:643-658.
Naikare, H., K. Palyada, R. Panciera, D. Marlow, and
A. Stintzi. 2006. Major role for FeoB in Campylo-
bacter jejuni ferrous iron acquistition, gut colo-
nization, and intracellular survival. Infect. Immun.
74:5433-5444.
Naidu, S.S., U. Svensson, A.R. Kishore, and A.S. Nai-
du. 1993. Relationship between antibacterial activ-
ity and porin binding of lactoferrin in Escherichia
coli and Salmonella typhimurium. Antimicrob.
290 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Agents Chemother. 37:240-245.
Ochoa, T. J., and T. G. Cleary. 2009. Effect of lactofer-
rin on enteric pathogens. Biochemie 91:30-34.
Pakkanen, R., and J. Aalto. 1997. Growth factors
and antimicrobial factors of bovine colostrum. Int.
Dairy J. 7:285-297.
Petschow, B. W., R. D. Talbott, and R. P. Batema.
1999. Ability of lactoferrin to promote the growth
of Bifidobacterium spp. in vitro is independent of
receptor binding capacity and iron saturation lev-
el. J. Med. Microbiol. 48:541-549.
Ratledge, C., and L. G. Dover. 2000. Iron metabo-
lism in pathogenic bacteria. Annu. Rev. Microbiol.
54:1319-1323.
Sarelli, L., M. Heinonen, T. Johansson, K. Heinonen,
and H. Saloniemi. 2003. Lactoferrin to prevent ex-
perimental Escherichia coli diarrhea in weaned
pigs. Int. J. Appl. Res. Vet. Med. 1:303-310.
Schryvers, A. B., R. Bonnah, R. H. Yu, H. Wong, and
M. Retzer. 1998. Bacterial lactoferrin receptors.
Adv. Exp. Med. Biol. 443:123-133.
Sherman, M.P., S.H. Bennett, F.F. Hwang, and C. Yu.
2004. Neonatal small bowel epithelia: enhancing
anti-bacterial defense with lactoferrin and Lacto-
bacillus GG. Biometals 17:285-289.
Shields, R. G., Jr., K. E. Ekstrom, and D. C. Mahan.
1980. Effect of weaning age and feeding method
on digestive enzyme development in swine from
birth to ten weeks. J. Anim. Sci. 50:257-265.
Still, J., P. Delahaut, P. Coppe, A. Kaeckenbeeck, J.P.
Perraudin. 1990. Treatment of induced enterotoxi-
genic colibacillosis (scours) in calves by the lacto-
peroxidase system and lactoferrin. Annals Vet. Res.
21:143-152.
Teraguchi, S., H. Wakabayashi, H. Kuwata, K. Yamau-
chi, and Y. Tamura. 2004. Protection against infec-
tions by oral lactoferrin: evaluation in animal mod-
els. Biometals 17:231-234.
Tomita, M., H. Wakabayashi, K. Yamauchi, S. Teragu-
chi, and H. Hayasawa. 2002. Bovine lactoferrin and
lactoferricin derived from milk: production and ap-
plications. Biochem. Cell Biol. 80:109-112.
Valenti, P., and G. Antonini. 2005. Lactoferrin: an im-
portant host defence against microbial and viral
attack. Cell. Mol. Life Sci. 62:2576-2587.
van der Strate, B. W., L. Beljaars, G. Molema, M. C.
Harmsen, and D. K. Meijer. 2001. Antiviral activities
of lactoferrin. Antiviral Res. 52:225-239.
Van Leeuwen, P., S.J. Oosting, J.M.V.M. Mouwen,
and M.W.A. Verstegen. 2000. Effects of a lactoper-
oxidase system and lactoferrin, added to a milk
replacer diet, on severity of diarrhea, intestinal
morphology and microbiology of digesta and fae-
ces in young calves. J. Animal Physiol Animal Nutr.
83:15-23.
Vorland, L.H. 1999. Lactoferrin: a multifunctional gly-
coprotein. APMIS 107:971-981.
Wakabayashi, H., M. Takase, and M. Tomita. 2003.
Lactoferricin derived from milk protein lactoferrin.
Curr. Pharm. Des. 9:1277-1287.
Wang, Y., T. Shan, Z. Xu, J. Liu, and J. Feng. 2006.
Effect of lactoferrin on the growth performance, in-
testinal morphology, and expression of PR-39 and
protegin-1 genes in weaned piglets. J. Anim. Sci.
84:2636-2641.
Weinberg., E.D. 1995. Acquisition of iron and oth-
er nutrients in vivo. In: Virulence Mechanisms of
Bacterial Pathogens, 2nd ed., pp. 79-93, Roth, J.
A., Bolin, C. A., Brogden, K. A., Minion, F. C. and
Wannemuehler, M. I. et al., (eds), American Society
for Microbiology, Washington, D.C.
Weinberg, E. D. 2001. Human lactoferrin: a novel
therapeutic with broad spectrum potential. J.
Pharmac. Pharmacol. 53:1303-1310.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 291
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Previously, we reported on the prevalence of microorganisms and pathogens in raw red amaranth, effec-
tiveness of sanitizers in reducing the pathogenic bacteria and impact of cooking in eliminating the micro-
biological risk. However, information on the impact of cooking on nutritional and functional properties has
not been addressed in detail. In this study the impact of cooking on nutritional quality including proximate
content, and functional properties including fat binding capacity, glucose binding capacity and cytotoxic-
ity of raw and cooked red amaranths was examined. It was found that cooking did not have any significant
impact on these nutritional and functional properties. Therefore, these study results along with previous
study results demonstrated that cooking could reduce the microbiological risk of these vegetables and still
remain safe for human consumption without losing any nutritional and functional properties except vitamin
C.
Keywords: Red amaranth, cooking conditions, nutritional quality, functional properties
INTRODUCTION
Vegetables and their products are usually valued
for their nutrient content but they are now also re-
garded as rich sources of non-starch polysaccha-
rides, collectively referred to as dietary fiber. Dietary
fiber includes polysaccharides, oligosaccharides,
Correspondence: Md. Latiful Bari, [email protected]: 8802-9661920-59 Ext 4721 Fax: 8802-8615583
and associated plant substances that are resistant
to digestion and adsorption in the human small in-
testine with complete or partial fermentation in the
large intestine (AACC 2001). However, dietary fiber
can be best viewed as a biological entity rather than
a chemically defined component of the diet (FAO,
1998). It is now well known that different composi-
tion and physicochemical properties of dietary fiber
produce different beneficial physiological effects
including laxation, and blood cholesterol and glu-
Effect of Cooking on Selected Nutritional and Functional Properties of Red amaranths
Md. A. A. Mamun1 , R. Ara3, H. U. Shekhar2, A. T. M. A. Rahim3 and Md. L. Bari1
1Center for Advanced Research in Sciences2Department of Biochemistry and Molecular Biology
3Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh.
Agric. Food Anal. Bacteriol. 2: 291-296 2012
292 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
cose attenuation (AACC, 2001). In addition, the fiber
component of the diet is nutritionally important be-
cause of their properties such as bulk density, hydra-
tion capacity, binding properties and fermentibility.
Therefore, experts generally recommend increased
dietary fiber intake by increasing consumption of
grains, legumes, vegetables, and fruits rather than
by taking supplements (Mahmod, 1999).
A number of studies on the content and compo-
sition, and the physiological role of dietary fiber of
Bangladeshi foodstuffs have been conducted in our
laboratory (Huq et al., 2001; Rahim et al., 2008; Khan
et al., 1996; Rahman et al., 2011).
Red amaranth (lalshak) is one of the most popu-
lar vegetables in Bangladesh and is grown in many
homestead gardens and consumed as a type of red
spinach. Usually, there are no processing steps in be-
tween harvest and market, and consumers buy these
vegetables from the local market, and boil or stir fry
them with spices prior to consumption. Therefore,
cooking of these vegetables was observed to elimi-
nate the pathogenic bacteria; however, these eating
habits may result in consuming less nutritional con-
tent.
Therefore, in this study the impact of cooking on
proximate content, and functional properties includ-
ing fat binding capacity, glucose binding capacity
and cytotoxicity of red amaranths are reported.
MATERIALS AND METHODS
Sample collection
Commercial red amaranth samples were pur-
chased from 20 different market of Dhaka City, Ban-
gladesh and composite mixture were prepared with-
in 24 h of collection. Raw vegetable samples were
collected aseptically in sterile polyethylene bags
and transported to the laboratory. Cracked or dirty
red amaranth samples were discarded.
Cooking the samples and cooling down to room temperature
Since consumers typically cook or stir fry red ama-
ranths samples with spices and consume them, an
experiment was designed to see the impact of cook-
ing on nutrient content. Commercial red amaranths
samples were boiled approximately at 90°C for 15
minutes and after boiling, the red amaranth samples
were placed on a sterile perforated tray to drain off
the excessive water and placed in laminar flow bio-
safety cabinet to facilitate cooling down to room
temperature.
Nutritional Quality Analysis
1) Proximate content:
The proximate analyses for red amaranth samples
were done according to the Association of Official
Analytical Chemists (AOAC, 2000) Methods. These
methods were established at the Institute of Nutri-
tion and Food Science laboratory of University of
Dhaka and had been used for the last 15 years. Anal-
yses were performed with homogenate samples in a
repeated manner.
Proximate composition of each sample of each
item was determined in duplicate estimations and
the mean value was recorded. Moisture content
was determined by weight loss after drying of the
sample in an oven at 105°C for 6 h (AOAC, 2000).
The moisture-free samples were charred and heated
to 600°C until a constant weight was achieved, the
residue being quantified as ash (AOAC, 2000). The
protein content was determined by Kjeldahl method
No 984.13 (AOAC, 2000) modified in our laboratory
at a micro scale. After acid digestion in BUCHI DI-
GEST SYSTEMK-437 equipped with a Buchi Scrub-
ber, B-414, samples were distilled in Buchi Distilla-
tion Unit, K-350 (BUCHI Labortechnik AG, Flawil,
Switzerland). Released nitrogen was trapped in 0.1
N sulfuric acid and back titrated with 0.1 N sodium
hydroxide to estimate the total nitrogen which was
converted to protein by multiplying with 6.25.
Since the study sample contained more than 10%
water, they were dried to constant weight at 60 to 70°
C for 16 to 18 hours (overnight) and stocked for fat
estimation. The "Soxhlet” method is recognized by
AOAC as the standard method for crude fat analysis.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 293
The crude fat from the dried sample was estimated
by the semicontinuous solvent extraction procedure
(Soxhlet method), described in method no. 991.36 of
AOAC (2000). The fat was extracted from the dried
sample (5 g) using petroleum ether (40 to 60 boiling
range) as a solvent. The nitrogen free extract (NFE)
was obtained by subtracting the sum of the values
for moisture, protein, fat and ash from 100 (Fer-
ris et.al., 1995). This value was considered as “total
carbohydrate” and was calculated by the following
equation.
Carbohydrate (NFE g %) =
100 − (Protein+ lipid + moisture +ash) g/100 g
Functional Properties Analysis
1) Fat Binding Capacity (FBC)
FBC of was measured using a modified method
of Wang and Kinsella, (1976). Briefly, FBC was initially
carried out by weighing empty centrifuge tubes (ep-
pendorf, 1.5 mL) as well as sample containing tubes.
Samples were mixed with 0.5 ml of soyabean oil on
a vortex mixer (VM-2000, Digisystem Laboratory In-
struments Inc. Taipei, Taiwan) for 1 min to disperse
the sample. The contents were left at ambient tem-
perature for 30 min with intermittent shaking for 5
s every 10 min and centrifuged (Spinwin, Spain) at
4,000 rpm for 25 min. After the supernatant was de-
canted, the tube was weighed again. An eppendorf
tube containing only 0.5mL soyabean oil was also
centrifuged and subsequently discarded to minimize
the error due to having unbound oil in the tube.
FBC was calculated as follows:
FBC (%) =
[Fat (soyabean oil) bound (g)/ initial sample weight
(g)] × 100.
2) Sugar Binding Capacity (SBC)
SBC of raw and cooked red amaranth was mea-
sured by incubating the food extract with glucose
sample. Glucose solution was prepared and was
taken in different test tubes. Five milligram (5.0 mg)
of food extract was incubated in 10mL glucose solu-
tion for 2 hours at room temperature. The content
was then centrifuged at 3,500 rpm (Z383K, HERMLE-
National Labnet Company, Woodbridge, NJ, USA)
for 20 min and supernatant was collected. Concen-
trations of glucose solution in the samples were then
estimated by colorimetric method.
Glucose concentration was calculated as follows
Asample/Astandard×Cstandard
Here,
Asample= Absorbance of supernatant at 520 nm
Astandard = Absorbance of supernatant at 520 nm
Cstandard= Concentration of standard = 2mg/mL
FBC was calculated as follows:
SBC (%) = [Bound glucose (mg)/ initial sample
weight (mg)] × 100.
3) In Vitro Cytotoxicity Study
An in vitro cytotoxicity test was performed using
a Brine Shrimp Lethality Bioassay method. It is a pri-
mary toxicity screening procedure used as an initial
screening of bioactive compounds. Brine shrimps
(Artemiasalina) were hatched using brine shrimp
eggs in a conical shaped vessel (1 L), filled with ster-
ile artificial seawater and pH was adjusted at 8.5 us-
ing 0.1 N NaOH under constant aeration for 48 h. Af-
ter hatching, active nauplii free from egg shells were
collected from the brighter portion of the hatching
chamber and used for the assay. Red amaranth ex-
tract was dissolved in artificial seawater at 0.01 and
0.1 mg/mL concentration and was taken in petri
plates where the active nauplii were inoculated. Af-
ter overnight incubation, the nauplii were counted.
Vincristine sulfate (0.5 mg/mL; an anticancer drug)
was considered as a positive control.
Statistical analysis
All trials were replicated three times. Data were
subjected to analysis of variance using the Microsoft
Excel program (Redmond, Washington DC, USA.).
Significant differences in data were established by
the least-significant difference at the 5% level of sig-
nificance.
294 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Nutrient Fresh Cl2 treated rawCl2 treated cooked
SP treated rawSP treated cooked
Moisture (%) 88.00 89.37 91.33 87.62 90.48
Ash 1.60A 1.38A 0.98B 1.58A 1.17B
Protein 5.30A 5.04B 3.65B 5.30A 3.92B
Fat 0.10A 0.18A 0.30B 0.15A 0.75B
Total CHO 5.00A 4.03A 3.74B 5.35A 3.68B
1Results are expressed as mean of duplicated estimation of each sample after duplicate extraction. The mean values with different letters across rows are significantly (P < 0.05) different, while means value with the same letter are not significantly different
Table 1. The proximate content of Red amaranth after treating with different water disinfectants followed by cooking (g/100 g edible portion)1
Table 2. Fat Binding Capacity of cooked Red amaranth extract
No Sample Initial Sample Wt. (gm) Final Wt. (gm) Total Fat Bound (gm) FBC (%)
01 Red amaranth 0.062 0.135 0.06 96.77
Table 3. Sugar Binding Capacity of cooked Red amaranth extract
No Sample Absorbance at 520nm
Conc
(mg/mL)
Residual glu-cose in 10 mL solution (mg)
Absorbed glucose in 10mL solution
(mg)
GBC (%)
01 GlucStnd 0.953A 2.000A - - -
02 GlucSoln 0.847A 3.774A 37.742A 0 -
03 Red amaranth 0.714A 3.125B 31.247A 6.495A 129.90
The mean values with different letters in columns are significantly (P < 0.05) different, while mean values with the same letter are not significantly different.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 295
RESULTS AND DISCUSSION
The nutritional properties of proximate content
were examined and were presented in Table 1. No
significant differences were found in proximate con-
tents in raw and cooked red amaranth (Table 1). The
average moisture content was 88% in raw and 90% in
cooked red amaranth. Protein and fat content in raw
red amaranth was 5.30 and 5.00 g/100 g respectively
and in cooked samples these values were 3.65 and
3.74 g/100g respectively. However, in our previous
paper, vitamin C content in raw red amaranths was
recorded as 14.2 mg/100g, and after cooking, the
vitamin C content was reduced significantly and re-
corded as 1.5 mg/100g, which is approximately 90%
lower than fresh one. This finding suggested that
eating habits could lead to a lower intake of micro-
nutrients even though microbiologically safe.
The functional properties including fat binding
capacity, sugar binding capacity and in vitro cytotox-
icity test was conducted and the results were pre-
sented in Table 2, 3 and 4. The fat binding capacity
and sugar binding capacity were 96% and 129%, re-
spectively in the cooked red amaranth (Table 2, and
3). In vitro cytotoxicity bioassay results showed that
the number of dead brine shrimp nauplii increased
at higher concentrations (Table 4).
Red amaranth provides a good source of vita-
min A, K, B6, and C, riboflavin, folate, calcium, iron,
magnesium, phosphorous, potassium, zinc, copper,
and manganese. It is moderately high in oxalic acid
which inhibits the absorption of calcium and zinc,
so it should be consumed in moderation. Red ama-
ranths are also good sources of essential amino ac-
ids including arginine, cystine and tyrosine that are
required by infants and growing children. During the
past five decades, studies have revealed arginine
to be useful in a variety of applications e.g. among
body builders, athletes and those with weakened im-
mune systems (Imura and Okada 1998).
The results of this work and the previous work re-
sults demonstrated that cooking could completely
reduce the risk of microbial pathogen without signifi-
cant loss of nutritional quality except for vitamin C.
ACKNOWLEDGEMENTS
This is an intra-collaborative work between Cen-
ter for Advanced Research in Sciences (CARS) and
departments of the University of Dhaka. The authors
express their sincere gratitude to the Department of
Biochemistry and Molecular biology; and Institute of
Food Science and Nutrition, for their technical and
all-out support and cooperation in this work.
REFERENCES
Association of American Cereal Chemists (AACC).
2001. The definition of dietary fiber. Cereal Food
World 46:112-116.
Table 4. Mortality of Brine shrimp (Artemiasalina) nauplii at different concentrations of cooked red amaranth extract
Sample No.
Sample NameDose
[mg/ml]No. of nauplii present
after incubationMortality [%]
01Positive control (Vincristine
sulfate)0.1 0 100
02Negative control (artificial sea
water)- 10 0
03 Red amaranths0.01 10 0
0.1 7 30
296 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
AOAC 2000. Association of Official Analytical Chem-
ists (AOAC), 17th edition.
FAO. Carbohydrates in Human Nutrition. Report of
a joint FAO/WHO expert consultation, Rome,1997.
FAO Food and Nutrition Paper 66, Rome, 1998.
Huq, F., K. Fatema, and A.T.M.A. Rahim 2001. Con-
tent and composition of dietary fiber in some Ban-
gladeshi vegetables. Diab. Endocr. J. 29:61-66.
Imura, K., and A. Okada. 1998. “Amino acid metabo-
lism in pediatric patients”.
Khan, M.R., S.A. Mamun, A. Hasin, U. F. Choudhury,
and F. Ahmed. 1996. Effect of different dosages of
ispagula husk on serum lipid profile. Dhaka Univ. J.
Biol. Sci. 5:61-68.
Mahmod, F. 1999. Dietary fiber of some Bangladeshi
foods and meals: A compositional analysis. INFS,
University of Dhaka. Bangladesh.
Rahim, A.T.M.A., I. Jerin, and S. M. M. Rahman. 2008.
Total dietary fiber and retention factors of Bangla-
deshi foods prepared by customary cooking pro-
cess. Dhaka Univ. J. Biol. Sci. 17:9-16.
Rahman, F., K. Fatema, A. T. M. A. Rahim, and L. Ali.
2011. Glucose, insulin and non esterified fatty acid
responses to Ladies Finger and Pointed Gourd in
type 2 diabetes mellitus. Asian J. Clin. Nutr. 3:25-
32.
Wang, J.C., and Kinsella, J. E. 1976. Functional prop-
erties of novel proteins: alfalfa leaf protein. J. Food
Sci. 41:286-292.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 297
www.afabjournal.comCopyright © 2012
Agriculture, Food and Analytical Bacteriology
ABSTRACTVariations to dietary components cause shifts in the ruminal microflora that can affect animal health and
productivity. However, the majority of these changes, especially in terms of quantitative changes, have not
been elucidated. Therefore, the objective of this study was to analyze the diversity of bacterial populations
in the rumen of cattle fed various amounts of citrus pulp pellets (CPP). Heifers (n=18; 298.7±5.1 kg) were
randomly assigned to 1 of 3 treatment diets (n=6/diet) containing CPP (0, 10, or 20%). Using bacterial tag-
encoded FLX amplicon pyrosequencing (bTEFAP), the ruminal microbiota was examined to understand
how different concentrations of CPP affected ruminal microbial ecology. The Firmicutes:Bacteroidetes ratio
tended to increase (P = 0.07) in heifers fed CPP compared to controls. Specifically within the Firmicutes,
Butyrivibrio and Carnobacterium populations increased in number with increasing amounts of CPP in the
diet. In contrast, a linear decline (P = 0.009) in the population of Dialister and Catonella occurred with in-
creasing CPP concentrations. Bacteria in the genera of Prevotella and Eubacterium were observed to be
the predominant bacteria that populated the rumen (34% and 6%, respectively) in control heifers. An in-
crease (P = 0.04) in the proportion of Bacilli bacteria in the ruminal microflora was associated with increases
in dietary CPP. Overall, there were relatively few changes observed in ruminal microbial populations, thus
highlighting the functional flexibility of the rumen and demonstrating that feeding CPP at rates up to 20%
does not adversely impact ruminal microbial ecology. The lack of major changes in ruminal microflora may
possibly be due to a lack of essential oils in the CPP utilized in the current study which may play a greater
role in the alteration of ruminal microbial populations and may also explain the lack of an apparent effect
in the current study as compared to previously reported studies.
Keywords: Bacterial diversity, orange pulp, citrus pulp, rumen, nutrition, rumenocentesis, pyrosequenc-ing, nutrition
Correspondence: Todd R. Callaway, [email protected]: +1 -979-2609374 Fax: +1-979-260-9332
Evaluation of the Ruminal Bacterial Diversity of Cattle Fed Diets Containing Citrus Pulp Pellets
P. R. Broadway1, T. R. Callaway2, J. A. Carroll3, J. R. Donaldson4, R. J. Rathmann1, B. J. Johnson1, J. T. Cribbs1, L. M. Durso5, D. J. Nisbet2, and T. B. Schmidt6
1Department of Animal and Food Science, Texas Tech University, Lubbock, TX2Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, USDA, College Station, TX
3Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX 4Department of Biological Sciences, Mississippi State University, Mississippi State, MS
5Agroecosystem Management Research Unit, Agricultural Research Service, USDA, Lincoln, NE6Department of Animal Science, University of Nebraska, Lincoln, NE
Mandatory Disclaimer: “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 no approval of the product, or
exclusion of others that may be suitable.” USDA is an equal opportunity provider and employer
Agric. Food Anal. Bacteriol. 2: 297-308, 2012
298 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
INTRODUCTION
While much research has been performed over
the course of the past half-century investigating the
composition of the ruminal microbial ecosystem
(Hungate, 1947), quantitative information on the mi-
crobial community and how it is affected by diet is
still lacking. Assumptions about the role and impor-
tance of bacterial species in the rumen have been
based on correlations between culture-dependent
population estimates and fermentation end prod-
uct (e.g., VFA, NH3, and CH4) accumulation (Bryant
and Robinson, 1962). Development of molecular
methodologies and technologies has progressed
in recent years, and pyrosequencing is now used to
evaluate the microbial diversity and composition of
ruminant intestinal ecosystems (Callaway et al., 2010;
Dowd et al., 2008), allowing for detailed information
to be obtained in relation to changes in specific mi-
crobial population changes.
Dietary components play a significant role in the
health and well-being of cattle and can impact food
safety (Krause et al., 2003; Wells et al., 2009). Cit-
rus peel and pulp are by-product feedstuffs that are
commonly fed to cattle and have a good nutritive
value for ruminants (Arthington et al., 2002). Citrus
peel and pulp have been included as low-cost ra-
tion ingredients at concentrations of 5 - 16% in dairy
and beef cattle rations for many years (Arthington et
al., 2002); and these products have a good nutritive
value for ruminants (6.9% CP; TDN, 82%; NEm, 1.9
Mcal/kg; NEg, 1.3 Mcal/kg).
Recent studies have indicated that the addition
of > 1% orange peel and pulp to mixed ruminal
fluid fermentations reduced populations of E. coli
O157:H7 and Salmonella typhimurium (Nannapa-
neni et al, 2008; Callaway et al., 2008). Further stud-
ies have demonstrated that feeding orange peel
and pulp reduced intestinal populations of Salmo-
nella and E. coli O157:H7 in experimentally inocu-
lated sheep (Callaway et al., 2011a,b). In the present
study, a tag bacterial diversity amplification pyrose-
quencing method (bTEFAP; Dowd et al., 2008) was
utilized to evaluate the ruminal microbial diversity
in cattle that were fed diets containing 0, 10 or 20%
citrus pulp pellets (CPP; 1:1 replacement of steam-
flaked corn). It was hypothesized that there would be
a significant shift in the gastrointestinal population
of cattle in response to CPP feeding and this may
possibly explain some of its reported antipathogenic
effects. Studies have indicated that diet composition
can also impact shedding of foodborne pathogenic
bacteria such as E. coli O157:H7 in cattle (Wells et al.,
2009; Jacob et al., 2008).
Citrus fruits contain a variety of compounds, most
notably essential oils in the peel that exert antimi-
crobial activity and can alter the microbial ecology of
the gastrointestinal tract (Viuda-Martos et al., 2008;
Friedly et al., 2009), and essential oils were hypoth-
esized to be responsible for the anti-pathogen effect
in these studies. When feeding citrus pulp pellets
that contained little or no essential oils, research-
ers found that citrus pulp feeding had no effect on
experimentally-infected Salmonella populations in
swine (Farrow et al., 2012). However, the collateral
effects of citrus pulp on the ruminal microbial eco-
system, and ultimately, animal health, productivity
and food safety remain unclear.
MATERIALS AND METHODS
All procedures involving live animals were ap-
proved (#10085-11) by the Texas Tech University Ani-
mal Care and Use Committee.
Animals
English x Continental heifers (n = 18) were sourced
from auction barns in the central Texas area. Cattle
arrived in two semi-truck loads on July 21 and 23,
2011 and processed 24 h after arrival. Initial process-
ing of both groups included: 1) body weight (BW)
measurement, 2) individual identification by ear tag;
3) vaccination with an IBR-BVD-PI3-BRSV modified
live virus vaccine (Vista 5, Intervet Schering Plough
Animal Health, DeSoto, KS); 4) vaccination with a
Clostridial bacterin-toxoid (Vision 7 with SPUR, Inter-
vet Schering Plough Animal Health, DeSoto, KS); 5)
antihelmitic treatment and (Ivomec injectable, Me-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 299
rial, Duluth, GA); and 6) metaphylactic treatment
(Micotil, Elanco Animal Health, Greenfield, IN).
Fourteen d after arrival, cattle were implanted with
Component TE-IH with Tylan (Elanco Animal Health,
Greenfield, IN) and also re-vaccinated with Vista 5
(Intervet Schering Plough Animal Health, DeSoto,
KS).
Experimental Design
The initial processing body weights for the two
loads were 188.7 + 18.0 kg and 225.2 + 22.2 kg, re-
spectively. Heifers two standard deviations from the
load average for BW that appeared temperamental,
lame, unthrifty, or appeared to have excessive Bos
indicus influence were eliminated prior to the start
of the trial. A total of 18 heifers were utilized for
the study. A completely randomized block design
was imposed. Heifers were blocked by body weight
nested within respective load. Treatments included:
1) control (CTRL) diet containing 0% dried citrus pulp
pellets (CPP); 2) 10% CPP = diet containing 10% CPP;
and 3) diet containing 20% CPP. The dried CPP were
guaranteed to contain no more than 1.5% lime (Texas
Citrus Exchange, Mission, TX). The diets containing
CPP were formulated to be exchanged with steam
flaked corn on a 1:1 basis. Diets were formulated
to meet or exceed NRC (1996) recommendations for
nutrients (Table 1). Cattle were fed a 63% concen-
trate starter diet from d 0 to d 28, a 73% concentrate
transition ration from d 28 to d 42, and an 83% con-
centrate diet from d 42 to d 56.
Starter Diets2 Transition Diets3 Finishing Diets4
Ingredients, %5 CTRL 10% CPP20% CPP
CTRL10% CPP
20% CPP
CTRL10% CPP
20% CPP
Steam-flaked corn 46.7 36.7 26.7 56.4 46.4 36.4 65.9 55.9 45.9
Dried citrus pulp 0.0 10.0 20.0 0.0 10.0 20.0 0.0 10.0 20.0
Alfalfa hay, ground 24.0 24.0 24.0 17.5 17.5 17.5 11.0 11.0 11.0
Cottonseed hulls 13.0 13.0 13.0 9.5 9.5 9.5 6.0 6.0 6.0
Cottonseed meal 7.7 7.7 7.7 7.7 7.7 7.7 7.7 7.7 7.7
Molasses 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Tallow 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Supplement pre-mix6 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Urea 0.40 0.40 0.40 0.50 0.50 0.50 0.63 0.63 0.63
Limestone 0.20 0.20 0.20 0.45 0.45 0.45 0.75 0.75 0.75
Table 1. Formulated composition of treatment diets1
1Treatment diets: CTRL = control diet containing 0% dried citrus pulp pellets; 10% CPP= diet containing 10% dried
citrus pulp pellets; 20% CPP = diet containing 20% dried citrus pulp pellets.2The starter diet was fed from d 0 to d 28.3The transition diet was fed from d 28 to d 42.4The finishing diet was fed from d 42 to d 56.5Dry matter basis6Supplement for the diet contained (DM basis): 66.383% cottonseed meal; 0.500% Endox® (Kemin Industries, Inc., Des
Moines, IA); 0.648% dicalcium phosphate; 10% potassium chloride; 4.167% ammonium sulfate; 15.000% salt; 0.002% co-
balt carbonate; 0.196% copper sulfate; 0.083% iron sulfate; 0.003% ethylenediamine dihydroiodide; 0.333% manganese
oxide; 0.125% selenium premix (0.2% Se); 0.986% zinc sulfate; 0.010% vitamin A (1,000,000 IU/g); 0.157% vitamin E (500
IU/g); 0.844% Rumensin (176.4 mg/kg; Elanco Animal Health, Indianapolis, IN); and 0.563% Tylan (88.2 mg/kg; Elanco
Animal Health). Concentrations in parenthesis are expressed on a 90% DM basis.
300 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Feeding, Weighing, and Health Moni-toring Practices
During the study period, cattle were housed in 3
m wide x 9.1 m pipe feedlot pens with a dirt floor
and concrete aprons around water troughs and feed
bunks. Cattle were fed once daily between 0700 and
0800 h. Prior to feeding, bunks were monitored to
determine orts, based upon adjustments in feed de-
livery for each pen, and adjustments were made to
ensure ad libitum access to feed (target of 0 to 0.454
kg orts prior to feeding). For days when heifers were
transitioned to new diet, diet was offered at 95% of
the previous day’s delivery.
Daily orts were collected from each diet during the
experimental period. Ort samples were composited
weekly/treatment and a subsample was placed in a
forced-air oven at 100º C for 24 h for assessment of
dry matter (DM) content. These weekly DM values
were utilized to calculate the average DM value for
each diet during the experimental period. In addi-
tion, another weekly composited subsample stored
at -20C until the conclusion of the study, at which
time the samples were analyzed by Servi-Tech Labo-
ratories (Amarillo, TX).
Rumenocentesis
On d 56 of the trial, heifers (n = 18; 6 heifers/dietary
treatment; 298.7±5.1 kg) were randomly selected for
collection of ruminal fluid via rumenocentesis (Nor-
dlund and Farrett, 1994). Heifers were restrained in
a hydraulic chute, a 10 x 10 cm area located 12 to
15 cm caudoventral to the costochondral junction of
the last rib on a line parallel with the top of the stifle
was clipped and disinfected (betadine scrub and a
70% ethanol wipe). After disinfection, a 1.6-mm (o.d)
x 130-mm (16 gauge) stainless steel needle was in-
serted into the ventral rumen using a 25-mL syringe,
and a minimum of 5 mL of rumen fluid was aspirated.
Samples were frozen and stored prior to analysis.
DNA Extraction
Rumen fluid samples were homogenized, and a
200 mg aliquot was used for DNA extraction using
the Qiagen DNA Stool Kit (Qiagen, Valencia, CA). To
ensure complete cell lysis, samples were treated with
sterile 5 mm steel beads (Qiagen, Valencia, CA) and
500 µl volume of sterile 0.1 mm glass beads (Scientif-
ic Industries, Inc., NY, USA) in a Qiagen Tissue Lyser
(Qiagen, Valencia, CA), run at 30 Hz for 5 min prior
to precipitation and purification. DNA samples were
diluted to a final concentration of 20 ng/µL as de-
termined by a Nanodrop spectrophotometer (Nyxor
Biotech, Paris, France).
Tag-Encoded FLX Amplicon Pyrose-quencing (bTEFAP) Analysis.
A 20 ng (1 µl) aliquot of each DNA sample
was used for a 25 µL PCR reaction. The 16S univer-
sal rDNA Eubacterial primers 104F (5’- GGC GVA
CGG GTG AGT AA) and 530R (5’-CCG CNG CNG
CTG GCA C), Archaea selective primers A349F (5’
GYG CAS CAG KCG MGA AW) and A806R (5’ GGA
CTA CVS GGG TAT CTA AT), and 18s rDNA fungal
funSSUF (5’ TGG AGG GCA AGT CTG GTG) and
funSSUR (5’ TCG GCA TAG TTT ATG GTT AAG)
were used for PCR amplification using Hot Star Taq
Plus Master Mix Kit (Qiagen, Valencia, CA) under the
following conditions: 94°C for 3 min., followed by 30
cycles of 94°C for 30 s; 55°C for 40 s and 72°C for 1
min.; and a final elongation step at 72°C for 5 min.
Following PCR, all amplicon products from different
samples were mixed in equal volumes and purified
using Agencourt Ampure beads (Agencourt Biosci-
ence Corporation, MA, USA) (Dowd et al., 2008).
bTEFAP FLX Massively Parallel Pyrose-quencing
In preparation for FLX sequencing (Roche, Nutley,
NJ), the PCR products’ sizes and concentrations were
analyzed using a Bio-Rad Experion Automated Elec-
trophoresis Station (Bio-Rad Laboratories, Hercules,
CA) and a TBS-380 Fluorometer (Promega Corpora-
tion, Madison, WI). A 9.6 x 106 sample of double-
stranded DNA molecules/µL with an average size of
625 bp were combined with 9.6 million DNA capture
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 301
Name Primer sequence ( 5’-3’ )
454-F30 GCCTCCCTCGCGCCATCAGCGCACTACGTGTGCCAGCMGCNGCGG
454-F31 GCCTCCCTCGCGCCATCAGCGCAGCTGTTGTGCCAGCMGCNGCGG
454-F32 GCCTCCCTCGCGCCATCAGCGCATACAGTGTGCCAGCMGCNGCGG
454-F33 GCCTCCCTCGCGCCATCAGCGCATCTATAGTGCCAGCMGCNGCGG
454-F34 GCCTCCCTCGCGCCATCAGCGCATTGGTGGTGCCAGCMGCNGCGG
454-F35 GCCTCCCTCGCGCCATCAGCGCCAGAAAAGTGCCAGCMGCNGCGG
454-F36 GCCTCCCTCGCGCCATCAGTGTGACGTACGTGCCAGCMGCNGCGG
454-F37 GCCTCCCTCGCGCCATCAGTGTGTGCATAGTGCCAGCMGCNGCGG
454-F38 GCCTCCCTCGCGCCATCAGTGTGTCCTCAGTGCCAGCMGCNGCGG
454-F39 GCCTCCCTCGCGCCATCAGTGTGCATCACGTGCCAGCMGCNGCGG
454-F40 GCCTCCCTCGCGCCATCAGTGTGCCTAGAGTGCCAGCMGCNGCGG
454-F41 GCCTCCCTCGCGCCATCAGTGTACATAGTGTGCCAGCMGCNGCGG
454-F42 GCCTCCCTCGCGCCATCAGTGTACATTGAGTGCCAGCMGCNGCGG
454-F43 GCCTCCCTCGCGCCATCAGTGTACATTGTGTGCCAGCMGCNGCGG
454-F44 GCCTCCCTCGCGCCATCAGTGTACCAACAGTGCCAGCMGCNGCGG
454-F45 GCCTCCCTCGCGCCATCAGTGTACCAACTGTGCCAGCMGCNGCGG
454-F46 GCCTCCCTCGCGCCATCAGTGTACCAATCGTGCCAGCMGCNGCGG
454-F47 GCCTCCCTCGCGCCATCAGTGTACCAGATGTGCCAGCMGCNGCGG
454-F48 GCCTCCCTCGCGCCATCAGTGTACCCATAGTGCCAGCMGCNGCGG
454-F49 GCCTCCCTCGCGCCATCAGTGTACAGGGTGTGCCAGCMGCNGCGG
454-F50 GCCTCCCTCGCGCCATCAGTGTACCTATCGTGCCAGCMGCNGCGG
linkerB-1100R GCCTTGCCAGCCCGCTCAGGGGTTNCGNTCGTTG
Table 2. Primer sequences utilized for fecal and ruminal samples during bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)
302 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
beads, and then amplified by emulsion PCR. After
bead recovery and bead enrichment, the bead-at-
tached DNAs were denatured with NaOH, and se-
quencing primers (Table 2) were annealed. A two-
region 454 sequencing run was performed on a 70 ×
75 GS Pico Titer Plate by using a Genome Sequencer
FLX System (Roche, Nutley, NJ). Following sequenc-
ing, all failed sequence reads, low quality sequence
ends (Avg Q25), short reads < 150 bp and tags and
primers were removed. Sequence collections were
then depleted of any non-bacterial, or non-archaeal
ribosome sequences, sequences with ambiguous
base calls, sequences with homopolymers > 5 bp in
length, and chimeras using B2C2 (Gontcharova et
al., 2010) as has been described previously (Handl
et al., 2011; Callaway et al., 2010; Bailey et al., 2010;
Pitta et al., 2010). To determine the predicted iden-
tity of microorganisms in the remaining sequences,
sequences were queried using Kraken (www.kraken-
blast.com) against a highly curated custom database
of high quality 16S bacterial and archaeal sequences
derived and manually curated from NCBI, and fun-
gal identities similarly were predicted using a highly
curated database of fungal small subunit sequences.
Using a NET analysis pipeline, the resulting BLASTn
outputs were compiled and data reduction analysis
was applied as described previously (Handl et al.,
2011; Callaway et al., 2010; Bailey et al., 2010; Pitta
et al., 2010).
Bacterial Diversity Data Analysis
To determine the identity of bacteria in the re-
maining sequences, sequences were denoised and
assembled into OUT clusters at 96.5% identity. The
sequences were then queried using a distributed
.NET algorithm that utilizes Blastn+ (KrakenBLAST;
www.krakenblast.com) against a database of high
quality 16S bacterial sequences. Using a .NET and
C# analysis pipeline, the resulting BLASTn+ outputs
were compiled and data reduction analysis per-
formed as described previously (Bailey et al., 2010;
Pitta et al., 2010; Andreotti et al., 2011).
Bacterial identification
Based upon the above BLASTn+ derived se-
quence identity, (percent of total length query
sequence which aligns with a given database se-
quence) the bacteria and archaea were classified at
the appropriate taxonomic levels based upon the
following criteria. Sequences with identity scores, to
known or well characterized 16S sequences, greater
than 97% identity (< 3% divergence) were resolved
at the species level, between 95% and 97% at the
genus level, between 90% and 95% at the family and
between 85% and 90% at the order level, 80 and 85%
at the class and 77% to 80% at phyla (Stackebrandt
and Goebel, 1994; Handl et al., 2011). After resolv-
ing based upon these parameters, the percentage
of each bacterial and archael ID was individually
analyzed for each sample providing relative abun-
dance information within and among the individual
samples based upon relative numbers of reads with-
in each. Evaluations presented at each taxonomic
level, including percentage compilations, represent
all sequences resolved to their primary identification
or their closest relative (Bailey et al., 2010; Suchodol-
ski et al., 2009; Andreotti et al., 2011).
Statistical Analysis
A completely randomized design was utilized in
this study with each aspirated sample containing ru-
men content being the experimental unit within 1 of
3 treatment groups based on diet. Statistics were
performed using JMP 6.0 (SAS Institute, Cary, NC).
Significance levels were predetermined as P < 0.05
and differences were separated accordingly. Trends
were determined as 0.05 < P < 0.10.
RESULTS AND DISCUSSION
Until recently, progress in understanding what role
bacterial species play in animal health and produc-
tivity has been unclear due to the need to culture
bacteria from the gastrointestinal tract. Pyrose-
quencing (bTEFAP) is not limited to detecting organ-
isms via culture methods and can be used to define
what constitutes a “healthy” or “normal” ruminal
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 303
microbial ecosystem profile. This molecular tech-
nology is also capable of quantitatively correlating
populations of bacterial species with traditional ani-
mal production parameters. As ruminal and intesti-
nal bacterial populations in food animals are further
quantified, researchers should be able to correlate
microbial populations and/or nutrient-utilization/
production guilds with production parameters such
as Residual Feed Intake (RFI), growth, milk produc-
tion and animal health.
Collectively, 22 bacterial phyla were represented
in the total sample set, but only 6 phyla were present
in all heifers examined. The majority of the isolates
were from the Bacteroidetes and Firmicutes phyla,
which together represented 91% of the bacterial
community. Among the Firmicutes, class Clostridia
predominated, with a lesser percent (< 11%) being
class Bacilli. Interestingly, the proportion of the Ba-
cilli bacterial community increased (P = 0.04) with in-
creasing CPP concentrations (Figure 1).
The gastrointestinal microbial population of cattle
is dominated by strict anaerobes. In other reports,
facultative anaerobes have been reported to oc-
cur in numbers at least 100-fold less than the strict
anaerobes (Drasar and Barrow, 1985); this is sup-
ported by the present results in which the predomi-
nant ruminal genera were Prevotella, Eubacterium,
Ruminococcus, Clostridium, and Roseburia (Table
3). At the genus level, the most common 25 genera
accounted for 79 - 83% of the total bacterial popu-
lations (Table 3). To improve understanding of the
role of the microbial ecosystem in ruminant nutrition,
molecular methodologies of population determina-
tion must be correlated with functional data and
approaches that address end-product production
from a quantitative perspective of animal production
(Dahllof, 2002). Data from this study is presented at
the genus level because genera shifts in ruminal pro-
portions are more representative of changes at the
functional guild level, which most closely describes
impacts at the level of the host animal. Collectively,
our data indicate that there is a large breadth of mi-
Figure 1. Effects1 of replacing concentrate with 0%, 10% or 20% CPP in cattle rations on the population of Bacilli class bacteria in ruminal fluid. Error bars represent standard deviations.
1 A increase (P = 0.04) in the percentage of bacilli bacteria was seen with the addition of CPP to the ration.
0
5
10
15
20
25
30
0 10 20
Bac
illi
(% o
f to
t al
bac
teri
al p
op
ula
tio
n)
CPP (% of ration)
304 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Rank Bacterial GeneraAll Diets 0% CPP 10% CPP 20% CPP
Mean Mean Std. Dev. Mean Std. Dev. Mean Std. Dev.
1 Prevotella 34.00 38.42 4.14 29.04 3.94 34.55 6.43
2 Eubacterium 6.71 4.75 3.43 9.11 9.11 6.27 3.60
3 Ruminococcus 5.22 5.04 1.83 4.70 1.66 5.91 2.66
4 Clostridium 4.89 3.63 1.54 5.47 2.57 5.57 1.77
5 Roseburia 4.05 3.45 1.94 4.25 2.63 4.45 2.30
6 Butyrivibrio 3.98 2.92 1.15 3.64 1.77 5.39 2.83
7 Dialister 3.01 4.22 3.74 2.89 2.76 1.91 1.74
8 Carnobacterium 1.98 1.26 1.93 1.76 2.58 2.91 6.24
9 Catonella 1.90 3.11 4.44 1.83 3.04 0.74 0.80
10 Olsenella 1.76 2.49 2.53 1.24 1.39 1.56 1.52
11 Haemophilus 1.41 1.27 1.47 2.35 3.33 0.61 0.56
12 Lachnospira 1.27 1.04 0.74 1.70 1.12 1.09 0.70
13 Lactobacillus 1.26 0.90 0.55 1.95 2.53 0.93 0.84
14 Tannerella 1.22 2.13 1.49 0.94 0.85 0.58 0.50
15 Paludibacter 1.03 0.12 0.16 2.52 2.59 0.45 1.04
16 Acidaminococcus 1.01 1.17 0.66 1.05 0.62 0.80 0.34
17 Oribacterium 0.92 0.91 0.77 1.06 0.82 0.77 0.84
18 Pseudomonas 0.86 0.01 0.02 0.29 0.66 2.28 5.59
19 Selenomonas 0.81 0.55 0.31 0.54 0.18 1.34 1.27
20 Bacteroides 0.81 1.12 0.59 0.68 0.32 0.63 0.49
21 Moryella 0.77 0.88 0.61 0.69 0.35 0.72 0.62
22 Syntrophococcus 0.76 0.52 0.40 0.73 0.99 1.02 1.36
23 Bacillus 0.58 0.66 1.62 0.01 0.03 1.07 1.85
24 Succinivibrio 0.54 0.10 0.10 0.60 0.65 0.92 1.92
25 Acetivibrio 0.54 0.61 0.99 0.78 1.03 0.23 0.31
Total 81.27 81.28 79.83 82.71
Table 3. Most common genera (as a % of the total bacterial population) of bacteria identified from ruminal fluid of cattle (n = 6/diet) fed a ration where the concentrate component was replaced with 0, 10 or 20% citrus pulp pellets. The genera identified are ordered by most abun-dant sequences.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 305
crobial diversity in the rumen of cattle, but that a few
genera predominate population-wise, most notably
Prevotella and Eubacterium (comprising 34% and
6%, respectively). Prevotella have been previously
reported by Stevenson and Weimer, (2007) to be
the predominant bacteria in the rumen as they have
the ability to utilize a plethora of nutrients to sustain
growth and survival. Eubacterium species have been
found to ferment pyruvate and amino acids and are
one of the most important bacteria in the rumen of
animals on high protein diets because it possesses
the ability to ferment pyruvate and amino acids (Wal-
lace et al., 2003; Leng and Nolan, 1984). Modest
changes were observed in several genera in relation
to the different concentrations of citrus pulp fed,
however none of the genera involved in these shifts
accounted for more than 5% of the total bacterial
community. Butyrivibrio and Carnobacterium popu-
lations increased linearly with increasing CPP, where-
as Dialister and Catonella proportions decreased (P
= 0.009). Butyrivibrio is a common ruminal bacte-
rium that is involved in fiber and carbohydrate deg-
radation (Cotta and Zeltwanger, 1995; Fernando et
al., 2010). Carnobacterium is a bacterium that has
been previously studied as a competitive inhibitor of
foodborne pathogens (Lewus et al., 1991). Tanner-
ella is a periodontal pathogen (Sharma, 2010) that
was isolated in this study at 1.22% of the total bacte-
rial community, but this pathogen has not been re-
ported in cattle previously at this level. Populations
decreased with increased levels of CPP in the diet
(2.33, 0.94, and 0.58% for 0, 10, and 20% CPP, respec-
tively), but this change was not significant (P > 0.05).
When examined at the species level, a total of 844
unique bacterial species were detected, with 615
species from the 0% CP, and 633 and 514 species in
10 and 20% CPP diets, respectively, and 380 bacte-
rial species were found in all three diets (data not
shown). A range of 75 - 76% of all assigned clones
in all diets were accounted for by the most predomi-
nate 27 species; thus the reduction in species rich-
ness observed in the 20% CPP diet reflects a loss of
minority community members rather than a dramatic
shift in the composition of the microbiome. Propor-
Table 4. Most common genera (as a % of the total bacterial population) of Archaea identified from ruminal fluid of cattle (n = x/diet) fed ration where the concentrate component was re-placed with 0, 10 or 20% citrus pulp pellets. The genera identified are ordered by most abun-dant sequences.
Archaeal GeneraAll Diets
Mean
0% CPP
Mean Std. Dev.
10% CPP
Mean Std. Dev.
20% CPP
Mean Std. Dev.
Methanobrevibacter 84.46 88.68 1.13 88.80 1.94 75.91 2.24
Methanosphaera 15.50 11.28 4.92 11.13 4.46 24.08 8.63
Methanimicrococcus 0.02 0.03 0.04 0.03 0.08 0.00 0.00
Methanobacterium 0.01 0.00 0.00 0.01 0.03 0.00 0.00
Total 99.99 99.99 99.99 99.99
306 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
tions of Prevotella multisaccharivorax, Roseburia
hominis, Butyvibrio fibrosolvens and Ruminococcus
flavefaciens all increased, ranging from a 50% to a
400% increase, though these increases were not sig-
nificant (P > 0.05). However, these increases corre-
sponded with proportional decreases in Tannerella,
Dialster sp., Bacteroides sp. and Catonella morbi.
Of the 298,022 sequences assigned to the domain
Archaea, all but 9 clones were observed to be in the
Euryarchaeota phylum. Within the Euryarcheota,
30 species from four genera were represented in
this study. As expected, the predominant genus
in ruminal fluid samples was Methanobrevibacter,
which accounted for an average of 84% of all Ar-
chaeal isolates (Table 4). Methanobrevibacter are
often isolated from the intestinal tracts of ruminants
and monogastrics (Hook et al., 2011). No differences
(P > 0.05) were noted in the genera populations be-
tween 0 and 10% CPP diets, though 20% CPP diets
contained fewer Methanobrevibacter along with in-
creased populations of Methanosphaera (Table 4).
Much of the increased Methanosphaera population
could be attributed to Methanosphaera stadtmanae,
which reduces methanol to produce methane (Fricke
et al., 2006); this archaeon has been previously iso-
lated from the rumen of cattle (Whitford et al., 2001).
In the present study, few significant changes were
noted in the ruminal microbial community from feed-
ing up to 20% CPP. This lack of impact may be due
to the lack of essential oils in the CPP, as essential
oils have been suggested to be the active ingredi-
ents responsible for altering the microbial commu-
nity (Viuda-Martos et al., 2008; Friedly et al., 2009).
Thus future studies involving CPP should focus on
including forms of citrus products that contain more
of the essential oils to alter the microbial community
of the rumen in an attempt to improve performance
characteristics, animal health, and food safety.
REFERENCES
Andreotti, R., A. A. Perez de Leon, S. E. Dowd, F. D.
Guerrero, K. G. Bendele, and G. A. Scoles. 2011.
Assessment of bacterial diversity in the cattle tick
Rhipicephalus (Boophilus) microplus through tag-
encoded pyrosequencing. BMC Microbiol. 11:6.
Arthington, J. D., W. E. Kunkle, and A. M. Martin.
2002. Citrus pulp for cattle, in The Veterinary Clin-
ics of North America - Food Animal Practice, G.
Rogers and M. Poore, Editors. W. B. Saunders
Company: Philadelphia, PA. p. 317-328.
Bailey, M. T., S. E. Dowd, N. M. A. Parry, J. D. Galley,
D. C. Schauer, and M. Lyte. 2010. Stressor expo-
sure disrupts commensal microbial populations in
the intestines and leads to increased colonization
by Citrobacter rodentium. Infect Immun. 78:1509-
19.
Bryant, M.P. and I.M. Robinson. 1962. Some nutri-
tional characteristics of predominant culturable
ruminal bacteria. J. Bacteriol. 84:605-614.
Callaway, T. R., J. A. Carroll, J. D. Arthington, C. Pratt,
T. S. Edrington, R. C. Anderson, M. L. Galyean, S.
C. Ricke, P. Crandall, and D. J. Nisbet. 2008. Citrus
products decrease growth of E. coli O157:H7 and
Salmonella Typhimurium in pure culture and in fer-
mentation with mixed ruminal microorganisms in
vitro. Foodborne Path. Dis. 5:621-627.
Callaway, T. R., J. A. Carroll, J. D. Arthington, T.
S. Edrington, M. L. Rossman, M. A. Carr, N. A.
Krueger, S. C. Ricke, P. Crandall, and D. J. Nisbet.
2011a. Escherichia coli O157:H7 populations in ru-
minants can be reduced by orange peel product
feeding. J. Food Prot. 74:1917-1921.
Callaway, T. R., J.A. Carroll, J. D. Arthington, T. S.
Edrington, R. C. Anderson, M. L Rossman, M. A.
Carr, K. J. Genovese, S. C. Ricke, P. Crandall, and D.
J. Nisbet. 2011b. Orange peel pellets can reduce
Salmonella populations in ruminants. Foodborne
Path. Dis. 8:1071-1075.
Callaway, T. R., S. E. Dowd, T. S. Edrington, R. C. An-
derson, N. Krueger, N. Bauer, P. J. Kononoff, and
D. J. Nisbet. 2010. Evaluation of the bacterial di-
versity in the rumen and feces of cattle fed diets
containing levels of dried distiller’s grains plus sol-
ubles using bacterial tag-encoded FLX amplicon
pyrosequencing (bTEFAP). J Anim Sc. 88:3977-
3983.
Cotta, M. A. and R. L. Zeltwanger. 1995. Degrada-
tion and utilization of xylan by the ruminal bacteria
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 307
Butyrivibrio fibrisolvens and Selenomonas rumi-
nantium. Appl. Environ. Microbiol. 61:4396-4402.
Dahllof, I., 2002. Molecular community analysis of
microbial diversity. Curr. Opin. Biotechnol. 13:213-
217.
Dowd, S.E., T. R. Callaway, R. D. Wolcott, Y. Sun, T.
McKeehan, R. G. Hagevoort, and T. S. Edrington.
2008. Evaluation of the bacterial diversity in the
feces of cattle using bacterial tag-encoded FLX
amplicon pyrosequencing (bTEFAP). BMC Micro-
biol. 8:125-132.
Drasar, B. S. and P. A. Barrow, 1985. Intestinal Mi-
crobiology, in Intestinal Microbiology, Aspects of
Microbiology, B.S. Drasar and P.A. Barrow, Editors.
Amer. Soc. Microbiol. Press: Washington, DC. p.
19-40.
Farrow, R. L., T. S. Edrington, N. A. Krueger, K. J.
Genovese, T. R. Callaway, R. C. Anderson, and D.
J. Nisbet. 2012. Lack of effect of feeding citrus by-
products in reducing Salmonella in experimentally
infected weanling pigs. J. Food Prot. 75:573-575.
Fernando, S. C., H. T. Purvis, II, F. Z. Najar, L. O.
Sukharnikov, C. R. Krehbiel, t. G. Nagaraja, B. A.
Roe, and U. DeSilva. 2010. Rumen microbial pop-
ulation dynamics during adaptation to a high-grain
diet. Appl. Environ. Microbiol. 76:7482-7490.
Fricke, W. F., H. Seedorf, A. Henne, M. Kruer, H.
Liesegang, R. Hedderich, G. Gottschalk, and R. K.
Thauer. 2006. The genome sequence of Metha-
nosphaera stadtmanae reveals why this human in-
testinal archaeon is restricted to methanol and H2
for methane formation and ATP synthesis. J. Bact.
188:642-658.
Friedly, E. C., P. G. Crandall, S. C. Ricke, M. Roman,
C. A. O’Bryan, and V. I. Chalova., 2009. In vitro anti-
listerial effects of citrus oil fractions in combination
with organic acids. J. Food Sci. 74:M67-M72.
Gontcharova, V. Y., E.; Wolcott, R. D.; Hollister, E. B.;
Gentry, T. J.; Dowd, S. E., 2010. Black Box Chime-
ra Check (B2C2): a Windows-Based Software for
Batch Depletion of Chimeras from Bacterial 16S
rRNA Gene Datasets. Open Microbiol J. 4:6.
Handl, S., S. E. Dowd, J. F. Garcia-Mazcorro, J. M.
Steiner, J. S. Suchodolski. 2011. Massive paral-
lel 16S rRNA gene pyrosequencing reveals highly
diverse fecal bacterial and fungal communities
in healthy dogs and cats. FEMS Microbiol Ecol.
76:301-310.
Hook, S. E., M. A. Steele, K. S. Northwood, A. G.
Wright, and B. W. McBride. 2011. Impact of high-
concentrate feeding and low ruminal pH on meth-
anogens and protozoa in the rumen of dairy cows.
Microb. Ecol. 62:94-105.
Hungate, R.E., 1947. Studies on cellulose fermen-
tation. III: The culture and isolation of cellulose-
decomposing bacteria from the rumen of cattle. J.
Bact. 53:631-644.
Jacob, M. E., J. T. Fox, J. S. Drouillard, D. G. Renter,
and T. G. Nagaraja. 2008. Effects of dried distill-
ers’ grain on fecal prevalence and growth of Esch-
erichia coli O157 in batch culture fermentations
from cattle. Appl. Environ. Microbiol. 74:38-43.
Krause, D. O., W. J. M. Smith, L. L. Conlan, J. M.
Gough, M. A. Williamson, and C. S. McSweeney.
2003. Diet influences the ecology of lactic acid
bacteria and Escherichia coli along the digestive
tract of cattle: neural networks and 16S rDNA. Mi-
crobiol (U.K.). 149:57-65.
Leng, R. A. and J. V. Nolan. 1984. Nitrogen metabo-
lism in the rumen. J. Dairy Sci. 67:1072-1089.
Lewus, C.B., A. Kaiser, and T. J. Montville. 1991.
Inhibition of food-borne bacterial pathogens by
bacteriocins from lactic acid bacteria isolated from
meat. Appl. Environ. Microbiol. 57:1683-1688.
Nannapaneni, R., A. Muthaiyan, P. G. Crandall, M.
G. Johnson, C.A. O’Bryan, V. I. Chalova, T. R. Cal-
laway, J. A. Carroll, J. D. Arthington, D. J. Nisbet,
and S. C. Ricke. 2008. Antimicrobial activity of
commercial citrus-based natural extracts against
Escherichia coli O157:H7 isolates and mutant
strains. Foodborne Path. Dis. 5:695-699.
Nordlund, K.V. and E. F. Garrett. 1994. Rumenocen-
tesis: A technique for collecting rumen fluid for
the diagnosis of subacute rumen acidosis in dairy
herds. Bovine Practitioner. 28:109.
Pitta, D. W., W. E. Pinchak, S. E. Dowd, J. Osterstock,
V. Gontcharova, E. Youn, K. Dorton, I. Yoon, B. R.
Min, J. D. Fulford, T. A. Wiekersham, and D. P. Ma-
linowski. 2010. Rumen bacterial diversity dynam-
ics associated with changing from bermudagrass
308 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
hay to grazed winter wheat diets. Microb Ecol.
59:511-52.
Sharma, A., 2010. Virulence mechanisms of Tanner-
ella forsythia. Periodontol. 2000, 54:106-116.
Stackebrandt, E. and B.M. Goebel. 1994. Taxonom-
ic Note: A place for DNA-DNA reassociation and
16S rRNA sequence analysis in the present species
definition in bacteriology. Int. J. Syst. Bacteriol .
44:846-849.
Stevenson, D. M., and P.J. Weimer. 2007. Domi-
nance of Prevotella and low abundance of classi-
cal ruminal bacterial species in the bovine rumen
revealed by relative quantification real-time PCR.
Appl. Microbiol Biotechnol. 75:165-174.
Suchodolski, J. S., S. E. Dowd, E. Westermarck, J.
M. Steiner, R. D. Wolcott, T. Spillmann, and J. A.
Harmoinen. 2009. The effect of the macrolide an-
tibiotic tylosin on microbial diversity in the canine
small intestine as demonstrated by massive paral-
lel 16S rRNA gene sequencing. BMC Microbiol.
9:210.
Viuda-Martos, M., Y. Ruiz-Navajas, J. Fernandez-
Lopez, and J. Perez-Alvarez. 2008. Antibacterial
activity of lemon (Citrus lemon L.), mandarin (Cit-
rus reticulata L.), grapefruit (Citrus paradisi L.) and
orange (Citrus sinensis L.) essential oils. J. Food
Safety. 28:567-576.
Wallace, R. J., N. McKain, N. R. McEwan, E. Miya-
gawa, L. C. Chaudhary, T. P. King, N. D. Walker, J.
H. A. Apajalahti, and C. J. Newbold. Eubacterium
pyruvativorans sp. Nov., a novel non-saccharolytic
anaerobe from the rumen that ferments pyruvate
and amino acids, form caproate and utilizes ace-
tate and propionate. Inter. J of Sys. And Evol. Mi-
crobiol. 53:965-970.
Wells, J. E., S. D. Shackelford, E. D. Berry, N. Kalchay-
anand, M. N. Guerini, V. H. Varel, T. M. Arthur, J. M.
Bosilevac, H. C. Freetly, T. L. Wheeler, C. L. Ferrell,
and M. Koohmaraie. 2009. Prevalence and level
of Escherichia coli O157:H7 in feces and on hides
of feed lot steers fed diets with or without wet dis-
tillers grains with solubles. J. Food Prot. 72:1624-
1633.
Whitford, M. F., R. M. Teather, and R. J. Forster. 2001.
Phylogenetic analysis of methanogens from the
bovine rumen. BMC Microbiol. 1:5-11.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 309
VOLUME 2 ISSUE 1
The Story of the Arkansas Association for Food Protection (AAFP) M. Sostrin
4
A Team Approach for Management of the Elements of a Listeria Intervention and Control Program J. N. Butts
6
Development of a Food Defense Workshop and Graduate Certificate in Food Safety and Defense for Working Professionals K. J. K. Getty
15
Human Noroviruses and Food Safety K. E. Gibson and S. C. Ricke
25
Development and Assessment of Success for Retail Food Safety Programming in Indiana R. H. Linton
35
ConAgra Foods’ Salmonella Chester Outbreak In Marie Callender’s Cheesy Chicken and Rice Catalyzing Change: Next Generation of Food Safety J. Menke-Schaenzer
43
CONFERENCE PROCEEDINGS*
REVIEWS*
Instructions for Authors69
Introduction to Authors
Food Safety For a Diverse Workforce; One Size Does Not Fit AllJ. A. Neal, M. Dawson, J. M. Madera
46
Isolation and Initial Characterization of Plasmids in an Acetogenic Ruminal Isolate O. K. Koo, S. A. Sirsat, P. G. Crandall and S. C. Ricke
56
* Arkansas Association for Food Protection (AAFP) Conference, Enhancing Food protection From Farm to Fork,
held on Sept. 28-29, 2011, Springdale, AR.
SPECIAL ISSUE: Arkansas Association for Food Protection (AAFP) Conference
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 311
VOLUME 2 ISSUE 2
Influence on Growth Conditions and Sugar Substrate on Sugar Phosphorylation Activity in Acetogenic BacteriaW. Jiang, R.S. Pinder, and J.A. Patterson
94
A Membrane Filtration Method for Determining Minimum Inhibitory Concentrations of Essential OilsS. J. Pendleton, R. Story, C. A. O’Bryan, P. G. Crandall, S. C. Ricke, and L. Goodridge
88
Effect of Fertilization on Phytase and Acid Phosphatase Activities in Wheat and Barley Cultivated in Bulgaria
V. I. Chalova, I. Manolov, M. Nikolova, and L. Koleva
103
Transfer of Tylosin Resistance Between Enterococcus spp. During Continuous-Flow Culture of Feral or Domestic Porcine Gut Microbes N. Ramlachan, R.C. Anderson, K. Andrews, R.B. Harvey, and D.J. Nisbet
111
Sugar Recovery from the Pretreatment/Enzymatic Hydrolysis of High and Low Specific Gravity Poplar ClonesA. C. Djioleu, A. Arora, E. M. Martin, J. A. Smith, M. H. Pelkki, and D. J. Carrier
121
Culture dependent molecular analysis of bacterial community of Hazaribagh tannery exposed area in BangladeshA. A. Maruf, M. M. Moosa, S. M. M. Rashid, H. Khan, and S. Yeasmin
132
ARTICLES
Evaluation of an Experimental Sodium Chlorate Product, With and WithoutNitroethane, on Salmonella in Cull Dairy CattleN. A. Krueger, T. S. Edrington, R. L. Farrow, R. Hagevoort, R. C. Anderson, G. H. Loneragan,
T. R. Callaway, and D. J. Nisbet
82
BRIEF COMMUNICATIONS
Instructions for Authors149
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
Impact of By-product Feedstuffs on Escherichia coli O157:H7 and Salmonella Typhimurium in Pure and Mixed Ruminal and Fecal Culture in VitroT. R. Callaway, S. Block, K. J. Genovese, R. C. Anderson, R. B. Harvey, and D. J. Nisbet
139
312 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
VOLUME 2 ISSUE 3
Age and Diet Effects on Fecal Populations and Antibiotic Resistance of a Multi-drug Resistant Escherichia coli in Dairy Calves T. S. Edrington, R. L. Farrow, B. H. Carter, A. Islas, G. R. Hagevoort, T. R. Callaway, R. C. Anderson, and D. J. Nisbet
162
Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus
S. Datta, I. G. Shah, A. Akter, K. Fatema, T. H. Islam, A. Bandyopadhyay, Z. U.M. Khan, and D. Biswas
187
Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking ProcessJ. R. Pittman, T. B. Schmidt, A. Corzo, T. R. Callaway, J. A. Carroll, and J. R. Donaldson
195
Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of SweetgumA. C. Djioleu, E. M. Martin, M. H. Pelkki, and D. J. Carrier
175
Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens And Food Spoilage BacteriaN. Murali, G. S. Kumar-Phillips, N. C. Rath, J. Marcy, and M. F. Slavik
209
ARTICLES
Instructions for Authors233
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
Prevalence of foodborne pathogens and effectiveness of washing or cooking in reducing microbiological risk of contaminated Red amaranth Md. A. A. Mamun, H. A. Simul, A. Rahman, N. N. Gazi, and Md. L. Bari
222
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 313
Developing an in vitro Method for Determining Feed Soluble Protein Degradation Rate by Mixed Ruminal MicroorganismsW. L. Crossland, L. O. Tedeschi, T. R. Callaway, P. J. Kononoff, and K. Karges
246
Lack of Effect of Feeding Lactoferrin on Intestinal Populations and Fecal Shedding of Sal-monella typhimurium in Experimentally-Infected Weaned Pigs
D. J. Nisbet, T. S. Edrington, R. L. Farrow, K. G. Genovese, T. R. Callaway, R. C. Anderson, and N. A. Krueger
280
Effect of Cooking on Selected Nutritional and Functional Properties of red amaranthsMd. A. A. Mamun, R. Ara, H. U. Shekhar, A. T. M.A. Rahim, and Md. L. Bari
291
Evaluation of the Ruminal Bacterial Diversity of Cattle Fed Diets Containing Citrus Pulp PelletsBroadway, P. R., T. R. Callaway, J. A. Carroll, J. R. Donaldson, R. J. Rathmann, B. J. Johnson, J. T. Cribbs, L. M. Durso, D. J. Nisbet, and T. B. Schmidt
297
ARTICLES
Attachment of E. coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal MicroscopyJ. A. Neal, E. Cabrera-Diaz, and A. Castillo
275
BRIEF COMMUNICATIONS
Instructions for Authors315
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal ContentsR. S.Pinder, and J.A. Patterson
253
VOLUME 2 ISSUE 4
314 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 315
MANUSCRIPT SUBMISSION
Authors must submit their papers electronically
([email protected]). According to instruc-
tions provided online at our site: www.afabjournal.
com. Authors who are unable to submit electroni-
cally should contact the editorial office for assistance
by email at [email protected].
INSTRUCTIONS TO AUTHORS
• Aerobic microbiology
• Aerobiology
• Anaerobic microbiology
• Analytical microbiology
• Animal microbiology
• Antibiotics
• Antimicrobials
• Bacteriophage
• Bioremediation
• Biotechnology
• Detection
• Environmental microbiology
• Feed microbiology
• Fermentation
• Food bacteriology
• Food control
• Food microbiology
• Food quality
• Food Safety
• Foodborne pathogens
• Gastrointestinal microbiology
• Microbial education
• Microbial genetics
• Microbial physiology
• Modeling and microbial kinetics
• Natural products
• Phytoceuticals
• Quantitative microbiology
• Plant microbiology
• Plant pathogens
• Prebiotics
• Probiotics
• Rumen microbiology
• Rapid methods
• Toxins
• Veterinary microbiology
• Waste microbiology
• Water microbiology
CONTENT OF MANUSCRIPT
We invite you to consider submitting your re-
search and review manuscripts to AFAB. The jour-
nal serves as a peer reviewed scientific forum for to
the latest advancements in bacteriology research
on Agricultural and Food Systems which includes
the following fields:
316 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
With an open access publication model of this
journal, all interested readers around the world can
freely access articles online. AFAB publishes origi-
nal papers including, but not limited to the types
of manuscripts described in the following sections.
Papers that have been, or are scheduled to be, pub-
lished elsewhere should not be submitted and will
not be reviewed. Opinions or views expressed in pa-
pers published by AFAB are those of the author(s)
and do not necessarily represent the opinion of the
AFAB or the editorial board.
MANUSCRIPT TYPES
Full-Length Research Manuscripts
AFAB accepts full-length research articles con-
taining four (4) figures and/or tables or more. AFAB
emphasizes the importance of sound scientific ex-
perimentation on any of the topics listed in the focus
areas followed by clear concise writing that describes
the research in its entirety. The results of experi-
ments published in AFAB must be replicated, with
appropriate statistical assessment of experimental
variation and assignment of significant difference.
Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.
Manuscripts clearly lacking in language will be re-
turned to author without review, with a suggestion
that English editing be sought before the paper is
reconsidered. AFAB offers a fee based language
service upon request. Please contact [email protected] for more information about our fees
and services.
Rapid Communications
Under normal circumstances, AFAB aims for re-
ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-
cation in the next available issue of AFAB. However,
if an author chooses or requires a much more rapid
peer review, the journal editorial office has the capa-
bility to shorten the review timing to one week or less.
Any type of manuscript whether it be a full length
manuscript, brief communication or review paper can
be submitted as a rapid communication. There will be
additional costs for processing and page charges will
be double the normal rate. Authors who choose this
option must select Rapid Communications as the pa-
per type when submitting the paper and the editors
will judge whether a rapid review is possible and let
the author know immediately.
Brief Communications
Brief communications are short research notes giv-
ing the results of complete experiments but are con-
sidered less comprehensive than full-length articles
with three (3) figures and/or tables or less. Manuscripts
should be prepared with the same subheadings as full
length research papers. The running head above the
title of the paper is “Brief Communications.”
Unsolicited Review Papers
Review papers are welcome on any topic listed in
the focus section and have no page limits. Reviews
are assessed the same pages charges as all other
manuscripts. All AFAB guidelines for style and form
apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter
than 20 pages of double spaced text and references
will be considered mini-reviews with the subhead-
ing above the title on the first page. The running
head above the title of the paper is either “Review”
or “Mini-review”.
Solicited Review Papers
Solicited reviews will have no page limits. The
editor-in-chief will send invitations to the authors
and then review these contributions when they are
submitted. Nominations or suggestions for potential
timely reviews are welcomed by the editors or edito-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 317
rial board members and should be sent to submit@
afabjournal.com. There will be no page charges for
solicited review papers but the solicitation must origi-
nate from the editor-in-chief or one of the editors. Re-
quests from authors will automatically be classified as
unsolicited review papers. The running head above
the title of the paper will be “Invited Review.”
Conference and Special Issues Reviews
AFAB welcomes opportunities to publish papers
from symposia, scientific conference, and/or meet-
ings in their entirety. Conference organizers need
simply to contact AFAB at [email protected]
and a rapid decision is guaranteed. If in agreement,
the conference organizers must guarantee delivery
of a set number of peer reviewed manuscripts within
a specified time and submitted in the same format
as that described for unsolicited review papers. Con-
ference papers must be prepared in accordance with
the guidelines for review articles and are subject to
peer review. The conference chair must decide
whether or not they wish to serve as Special Issue
Editor and conduct the editorial review process. If
the conference chair/organizer chooses to serve as
special issue editor, this will involve review of the pa-
pers and, if necessary, returning them to the authors
for revision. The conference organizer then submits
the revised manuscripts to the journal editorial of-
fice for further editorial examination. Final revisions
by the author and recommendations for acceptance
or rejection by the chair must be completed by a
mutually agreed upon date between the editor and
the conference organizer. Manuscripts not meeting
this deadline will not be included in the published
symposium proceedings but if submitted later can
still be considered as unsolicited review papers. Al-
though offprints and costs of pages are the same
as for all other papers, the symposium chair may be
asked to guarantee an agreed upon number of hard
copies to be purchased by conference attendees. If
the decision is not to publish the symposium as a
special issue, the individual authors retain the right
to submit their papers for consideration for the jour-
nal as ordinary unsolicited review manuscripts.
Book Reviews
AFAB publishes reviews of books considered to
be of interest to the readers. The editor-in-chief ordi-
narily solicits reviews. Book reviews shall be prepared
in accordance to the style and form requirements of
the journal, and they are subject to editorial revision.
No page charges will be assessed solicited reviews
while unsolicited book reviews will be assigned the
regular page charge rate.
Opinions and Current Viewpoints
The purpose of this section will be to discuss, cri-
tique, or expand on scientific points made in articles
recently published in AFAB. Drafts must be received
within 6 months of an article’s publication. Opinions
and current perspectives do not have page limits.
They shall have a title followed by the body of the
text and references. Author name(s) and affiliation(s)
shall be placed between the end of the text and list
of references. If this document pertains to a par-
ticular manuscript then the author(s) of the original
paper(s) will be provided a copy of the letter and of-
fered the opportunity to submit for consideration a
reply within 30 days. Responses will have the same
page restrictions and format as the original opinion
and current viewpoint, and the titles shall end with
“Opinions.” They will be published together. Letters
and replies shall follow appropriate AFAB format
and may be edited by the editor-in-chief and a tech-
nical editor. If multiple letters on the same topic are
received, a representative set of opinions concern-
ing a specific article will be published. A disclaimer
will be added by the editorial staff that the opinion
expressed in this viewpoint is the authors alone and
does not necessarily represent the opinion of AFAB
or the editorial board.
COPYRIGHT AGREEMENT
The copyright form is published in AFAB as space
permits and is available online (www.afabjournal.com).
318 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
AFAB grants to the author the right of re-publication
in any book of which he or she is the author or edi-
tor, subject only to giving proper credit to the original
journal publication of the article by AFAB. AFAB re-
tains the copyright to all materials accepted for pub-
lication in the journal. If an author desires to reprint
a table or figure published from a non-AFAB source,
written evidence of copyright permission from an au-
thority representing that source must be obtained by
the author and forwarded to the AFAB editorial office.
PEER REVIEW PROCESS
Authors will be requested to provide the names
and complete addresses including emails of five (5) potential reviewers who have expertise in the research
area and no conflict of interest with any of the authors.
Except for manuscripts designated as Rapid Commu-
nication each reviewer has two (2) weeks to review
the manuscript, and submit comments electronically
to the editorial office. Authors have three (3) weeks
to complete the revision, which shall be returned to
the editorial office within six (6) weeks after which the
authors risk having their manuscript removed from
AFAB files if they fail to ask the editorial office for
an extension by email. Deleted manuscripts will be
reconsidered, but they will have to be processed as
new manuscripts with an additional processing fee as-
sessed upon submission. Once reviewed, the author
will be notified of the outcome and advised accord-
ingly. Editors handle all initial correspondence with
authors during the review process. The editor-in chief
will notify the author of the final decision to accept or
reject. Rejected manuscripts can be resubmitted only
with an invitation from the editor or editor-in chief. Re-
vised versions of previously rejected manuscripts are
treated as new submissions.
PRODUCTION OF PROOFS
Accepted manuscripts are forwarded to the edito-
rial office for technical editing and layout. The manu-
script is then formatted, figures are reproduced, and
author proofs are prepared as PDFs. Author proofs
of all manuscripts will be provided to the correspond-
ing author. Author proofs should be read carefully and
checked against the typed manuscript, because the
responsibility for proofreading is with the author(s).
Corrections must be returned by e-mail. Changes
sent by e-mail to the technical editor must indicate
page, column, and line numbers for each correction
to be made on the proof. Corrections can also be
marked using “track changes” in Microsoft Word or
using e-annotation tools for electronic proof correc-
tion in Adobe Acrobat to indicate necessary chang-
es. Author alterations to proofs exceeding 5% of the
original proof content will be charged to the author. All
correspondence of proofs must be agreed to by the
editorial office and the author within 48 hours or proof
will be published as is and AFAB will assume no re-
sponsibility for errors that result in the final publication.
PUBLICATION CHARGES
AFAB has two publication charge options: conven-
tional page charges and rapid communication. The
current charge for conventional publication is $25 per printed page in the journal. There is no additional
charge for the publication of pages containing color
images, micrographs or pictures. For authors who
wish to have their papers processed as a rapid com-
munication, authors will pay the rapid communication
fee when proofs are returned to the editorial office
in addition to twice the conventional page charges.
Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one
week and $100 per journal page.
HARD COPY OFFPRINTS
If you are wishing to obtain a physical hard copy of
the AFAB journal, offprints are available in any quan-
tity at an additional charge: $100/page for black-white
and $150/page for color prints. You may order your
offprints at any time after publication on our website.
Scientific conference organizers may be expected to
agree to a set number of offprints as a part of their
agreement with AFAB.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 319
MANUSCRIPT CONTENT REQUIREMENTS
Preparing the Manuscript File
Manuscripts must be written in grammatically
correct English. AFAB offers a fee based language
service upon request ([email protected]).
Manuscripts should be typed double-spaced, with
lines and pages numbered consecutively. All docu-
ments must be submitted in Microsoft Word (.doc or
.docx, PC or Mac). All special characters (e.g., Greek,
math, symbols) should be inserted using the sym-
bols palette available in this font. Tables and figures
should be placed in separate sections at the end of
the manuscript (not placed in the text). Failure to fol-
low these instructions will cause delays of the pro-
cessing and review of the manuscript.
Title Page
At the very top of the title page, include a title of
not more than 100 characters. Format the title with
the first letter of each word capitalized. No abbre-
viations should be used. Under the title, the authors
names are listed. Use the author’s initials for both first
and middle names with a period (full-stop) between
initials (e.g., W. A. Afab). Underneath the authors, a
list affiliations must be listed. Please use numerical
superscripts after the author’s names to designate
affiliation. If an authors address has changed since
the research was completed, this new information
must be designated as “Current address:”. The cor-
responding author should be indicated with an aster-
isk e.g., * Corresponding author. The title page shall
include the name and full address of the correspond-
ing author. Telephone and e-mail address must also
be provided for the corresponding author, and email-addresses must be provided for all authors.
Editing
Author-derived abbreviations should be defined
at first use in the abstract and again in the body of
the manuscript. If abbreviations are extensive au-
thors may need to provide a list of abbreviations
at the beginning of the manuscript. In vivo, in vitro
and bacterial names must be italicized (obligatory).
Authors must avoid single sentence paragraphs and
merge such paragraphs appropriately. Authors must
not begin sentences with “Figure or Table shows…”
as these are inanimate objects and cannot “show”
anything. When number are reported in text or in ta-
bles, always put a zero in front of decimal numbers:
“0.10” instead of “.10”.
MANUSCRIPT SECTIONS
Abstract
The abstract provides an abridged version of the
manuscript. Please submit your abstract on a sepa-
rate page after the title page. The abstract should
provide a justification of your work, objectives, meth-
ods, results, discussion and implications of study or
review findings . Your abstract must consist of com-
plete sentences without references to other work or
footnotes and must not exceed 250 words. On the
same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-
ing. Ideally, these keywords should include signifi-
cant words from the title.
Introduction
The introduction should clearly present the foun-
dation of the manuscript topic and what makes the
research or the review unique. The introduction
should validate why this topic is important based on
previously published literature, and the relevance of
the current research. Overall goals and project ob-
jectives must be clearly stated in the final sentence
of the last paragraphs of the introduction.
Materials and Methods
Information on equipment and chemicals used
must include the full company name, city, and state
(country if outside the United States or Province if
in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].
320 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
Variability, Replication, and Statistical Analysis
To properly assess biological systems indepen-
dent replication of experiments and quantification
of variation among replicates is required by AFAB.
Reviewers and/or editors may request additional
statistical analysis depending on the nature of the
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
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012 321
“(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.
322 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012
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.
AFAB Online Edition is Now Available!
www.AFABjournal.com
• Free Access
• Print PDFs
• Flip Through Issues
• Search Article Archives
• Order Reprints
• Submit a Paper
Online Publication: www.AFABjournal.com