AFAB-Volume2-Issue4

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

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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


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


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).


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-


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


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


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.

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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



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.


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-


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


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).


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.


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.


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).


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.


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.


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).


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.


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


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.


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.


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.


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.


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.


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-


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


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.

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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


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.


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).


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).


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).


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).


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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



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.


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


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


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-


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.


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


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.


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


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


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.

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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


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.


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.


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.


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.



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


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-

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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.




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



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.


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-


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.


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


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)


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.


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


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)


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.


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


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.

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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

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Human Noroviruses and Food Safety K. E. Gibson and S. C. Ricke

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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*



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


VOLUME 2 ISSUE 2

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Effect of Fertilization on Phytase and Acid Phosphatase Activities in Wheat and Barley Cultivated in Bulgaria

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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

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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

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222


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

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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

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Attachment of E. coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal MicroscopyJ. A. Neal, E. Cabrera-Diaz, and A. Castillo

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Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal ContentsR. S.Pinder, and J.A. Patterson

253

VOLUME 2 ISSUE 4



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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).


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.


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)].


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


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

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