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Volume 2, Issue 4 2012

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EDITORIAL BOARD Sooyoun Ahn

W.K. Kim

University of Florida, USA

University of Manitoba, Canada

Walid Q. Alali

M.B. Kirkham

University of Georgia, USA

Kansas State University, USA

Kenneth M. Bischoff

Todd Kostman

NCAUR, USDA-ARS, USA

University of Wisconsin, Oshkosh, USA

Debabrata Biswas

Y.M. Kwon

University of Maryland, USA

University of Arkansas, USA

Claudia S. Dunkley

Maria Luz Sanz

University of Georgia, USA

MuriasInstituto de Quimica Organic General, Spain

Lawrence Goodridge

Melanie R. Mormile

Colorado State University, USA

Missouri University of Science and Tech., USA

Leluo Guan

Rama Nannapaneni

University of Alberta, Canada

Mississippi State University, USA

Joshua Gurtler

Jack A. Neal, Jr.

ERRC, USDA-ARS, USA

University of Houston, USA

Yong D. Hang

Benedict Okeke

Cornell University, USA

Auburn University at Montgomery, USA

Divya Jaroni

John Patterson

Oklahoma State University, USA

Purdue University, USA

Weihong Jiang Shanghai

Toni Poole

Institute for Biol. Sciences, P.R. China

FFSRU, USDA-ARS, USA

Michael Johnson

Marcos Rostagno

University of Arkansas, USA

LBRU, USDA-ARS, USA

Timothy Kelly

Roni Shapira

East Carolina University, USA

Hebrew University of Jerusalem, Israel

William R. Kenealy

Kalidas Shetty

Mascoma Corporation, USA

University of Massachusetts, USA

Hae-Yeong Kim Kyung Hee University, South Korea Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012

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EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA Cesar Compadre University of Arkansas for Medical Sciences, USA

MANAGING and LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR Jessica C. Shabatura Fayetteville, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, USA

Philip G. Crandall University of Arkansas, USA

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 4 - 2012

TABLE OF CONTENTS BRIEF COMMUNICATIONS 275 Attachment of E. coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal Microscopy

J. A. Neal, E. Cabrera-Diaz, and A. Castillo

ARTICLES 246

Developing an in vitro Method for Determining Feed Soluble Protein Degradation Rate by Mixed Ruminal Microorganisms W. L. Crossland, L. O. Tedeschi, T. R. Callaway, P. J. Kononoff, and K. Karges

253 Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal Contents

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

280 Lack of Effect of Feeding Lactoferrin on Intestinal Populations and Fecal Shedding of Salmonella 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

291 Effect of Cooking on Selected Nutritional and Functional Properties of red amaranths Md. A. A. Mamun, R. Ara, H. U. Shekhar, A. T. M.A. Rahim, and Md. L. Bari

297 Evaluation of the Ruminal Bacterial Diversity of Cattle Fed Diets Containing Citrus Pulp Pellets

Broadway, 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

Introduction to Authors 315 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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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 Department of Animal Science, Texas A&M University, College Station, TX 77843-2471 2 Food and Feed Safety Research Unit, USDA-ARS, College Station, TX 77845 3 Department of Animal Science, University of Nebraska, Lincoln 68583 4 Dakota Gold Research Association, Sioux Falls, SD 57104-4506

1

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 determination. 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

Agric. Food Anal. Bacteriol. 2: 246-252, 2012

Correspondence: L. O. Tedeschi, luis.tedeschi@tamu.edu

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INTRODUCTION The field of ruminant nutrition commonly attempts to fractionate feed proteins based on their physicochemical properties and fractional ruminal degradation rates (kd). This provides a structure for ration balancing and decision-making programs commonly 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 difference or assigned tabular kd values (Schwab et al., 2003). The in situ technique is the most commonly used method for determining protein degradability

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 adjusted for bacterial protein.

MATERIALS AND METHODS Feeds Four corn-milling coproducts produced by Poet LLC (Sioux Falls, SD) were utilized to determine the kd

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 variable (120 to 400 %/h) (Sniffen et al., 1992). A direct comparison of neutral detergent fiber (NDF) fermentation between in vitro and in situ techniques suggested 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 correlations exist between these techniques (Lopéz et al., 1998). Several factors may affect the fermentability 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 suggested 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 alternatives that still closely mimic the rumen environment. The major point of concern for in vitro ruminal fermentation approaches is the accumulation of fermentation end products (e.g. VFA and lactate) and the decrease of pH; however, this can be overcome by adding adequate buffering salts to the fermentation mixture (Hungate, 1950). Because soluble protein contained in feeds is rapidly degraded to am-

of their soluble protein. These feeds were used because of their diverse protein fractions, different processing methods, and importance to the cattle industry. 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 lessen 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, resulting 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 ruminant 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 intrinsic variation among sub-samples to obtain a rep-

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 accumulation such as ammonia and AA are confounded by

resentative feed. Composite feed samples were then sent to Cumberland Valley Analytical Service (Hagerstown, MD) for chemical analysis in accordance with the AOAC (2000).

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In vitro fermentation and sample collection 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 combination of Fd + Md in which corn-milling coproducts 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 bacterial protein (C1 was Rf + Md, of which Rf was mixed

for analyses. The 0-h samples were collected from the bottles, immediately following inoculation. Fermented samples were collected by removing 4 mL from each treatment via needle and syringe. Samples were transferred to micro centrifuge tubes and centrifuged at 10,000 × g for 5 min to remove cellular 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 determination

with Md to account for any pre-existing nitrogen and bacterial protein in the inoculate and C2 was comprised 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 mortar and pestle to pass a 2 mm screen (0.60 g), transferred 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 buffer 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 medium 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

Ammonia concentrations were determined by the method of Chaney and Marbach (1962) and were performed in duplicate. Bacterial protein was determined 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 nonprotein 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.

Orion 3-Star bench top pH meter (Thermo Fisher Scientific, Inc.) recorded and 12 mL of filtered inoculate 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)

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-

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Enumeration and statistical analysis The TRT3 was calculated as shown in Eq. [1]. It was used to compute the ammonia net balance (production 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 soluble protein of a feed.

TRT3ij = TRT1ij – (TRT2ij – C2) – (C1 – C2) – C2

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[1]

Figure 1. Schematic representation of the correction for feed (Fd), rumen fluid (Rf), and buffer media (Md) contribution to ammonia and bacteria protein during the incubation period

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 effects and feeds (BPX-DDG, BRAN, GERM, and HPDDG) as random blocks for the whole plot and time of incubation (0, 1, 3, 6, 12, 24, and 48 h) as the repeated measure. The average between duplicates within feeds and treatments were used. The interaction 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

cubation since the feeds were composites.

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 analysis because we assumed the only source of variation of ammonia production would be the anaerobic in-

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 protein degradation is greater than the rate of carbo-

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 accumulation at around 6 h. Our in vitro ammonia concentration pattern is in agreement with the ammonia concentration 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 bacteria, which are estimated to account for less than 10% of the known rumen bacterial species (Krause

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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 3000

Ammonia, nmol

2500 2000

Rumen fluid and buffer media

1500

Buffer media alone

1000

Adjusted for bacterial protein

500 0 0

10

20

30

40

50

-500 Incubation time, h

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, likely 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 System, Tedeschi et al., 2010; and the CPM Dairy model, Tedeschi et al., 2008) the protein A (NPN) + B2 (soluble protein) fractions of DDGS and GERM comprises

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 fraction 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.

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 originate from the protein B2 fraction. These nutrition

may be accounted for by using different fermentation 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

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Evaluation of the methodology The methodology described herein was based on the hypothesis that bacterial uptake of protein

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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 addition, not all soluble protein, soluble oligopeptides,

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 methods that determine protein degradation rates.

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 averaged 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.

For the purposes of this type of in vitro study, incubation times should be limited to 12 h due to interference 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 HPDDG. 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., protein was not being degraded to ammonia, but was

AOAC. 2000. Official Methods of Analysis of AOAC International. (17th ed.) Association of Official Analytical Chemists, Arlington, VA. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Broderick, G. A. 1978. In vitro procedures for estimating rates of ruminal protein degradation and proportions of protein escaping the rumen undegraded. J. Nutr. 108:181-190.

Broderick, G. A. 1987. Determination of protein degradation rates using a rumen in vitro system containing inhibitors of microbial nitrogen metabolism. Br. J. Nutr. 58:463-475.

Chaney, A. L., and E. P. Marbach. 1962. Modified reagents for determination of urea and ammonia.

Clin. Chem. 8:130-132.

Fox, D. G., L. O. Tedeschi, T. P. Tylutki, J. B. Russell, M. E. Van Amburgh, L. E. Chase, A. N. Pell, and T. R. Overton. 2004. The Cornell Net Carbohydrate and Protein System model for evaluating herd nutrition and nutrient excretion. Anim. Feed Sci. Technol. 112:29-78.

Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analysis: Apparatus, reagents, procedures,

being synthesized for microbial protein.

In conclusion, the current method provided preliminary information for the development of a method that may be used to determine the degradability of the soluble protein fraction of ruminant feeds.

and some applications. Agric. Handbook. No. 379.

ARS, USDA, Washington, DC. 1-20 p.

Hungate, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriological Reviews. 14:1-49.

Krause, D. O., and J. B. Russell. 1996. An rRNA ap-

Variation

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proach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acidfermenting bacteria in ruminal amino acid deamination. Appl. Environ. Microbiol. 62:815-821.

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Lanzas, C., L. O. Tedeschi, S. Seo, and D. G. Fox. 2007. Evaluation of protein fractionation systems used in formulating rations for dairy cattle. J. Dairy Sci. 90:507-521.

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Sun, Z. H., Z. L. Tan, S. M. Liu, G. O. Tayo, B. Lin, B. Teng, S. X. Tang, W. J. Wang, Y. P. Liao, Y. F. Pan, J. R. Wang, X. G. Zhao, and Y. Hu. 2007. Effects of dietary methionine and lysine sources on nutrient digestion, nitrogen utilization, and duodenal amino acid flow in growing goats. J. Anim. Sci. 85:3340-3347.

Tedeschi, L. O., A. Cannas, and D. G. Fox. 2010. A nutrition mathematical model to account for dietary supply and requirements of energy and nutrients for domesticated small ruminants: The development and evaluation of the Small Ruminant

Lopéz, S., M. D. Carro, J. S. González, and F. J. Ovejero. 1998. Comparison of different in vitro and in situ methods to estimate the extent and rate of degradation of hays in the rumen. Anim. Feed Sci. Technol. 73:99-113.

Mizwicki, K. L., F. N. Owens, K. Poling, and G. Burnett. 1980. Timed ammonia release for steers. J. Anim. Sci. 51:698-703.

National Research Council. 2001. Nutrient Requirements of Dairy Cattle. (7th ed.) National Academy Press, Washington, DC. Nocek, J. E. 1988. In situ and other methods to estimate ruminal protein and energy digestibility: a review. J. Dairy Sci. 71:2051-2069.

Nocek, J. E., and J. B. Russell. 1988. Protein and energy as an integrated system. Relationship of ruminal protein and carbohydrate availability to microbial synthesis and milk production. J. Dairy Sci. 71:2070-2107.

Reynal, S. M., I. R. Ipharraguerre, M. Liñeiro, A. F. Brito, G. A. Broderick, and J. H. Clark. 2007. Omasal flow of soluble proteins, peptides, and free amino acids in dairy cows fed diets supplemented with proteins of varying ruminal degradabilities. J. Dairy Sci. 90:1887-1903.

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Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal Contents R.S.Pinder 1,2 and J.A. Patterson1 1 Animal Science Dept, Purdue University, West Lafayette, IN 47907-1026 2 Current address; 7855 South 600 East, Brownsburg, IN 46112

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 acetate 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 utilizing 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

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Correspondence: J. A. Patterson, jpatters@purdue.edu Tel: +1 -765-494-4826 Fax: +1-765-494-9347

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INTRODUCTION During fermentation of plant carbohydrates in the rumen of ruminants extensive degradation occurs with considerable cross-feeding and generation of metabolities that serve as substrates for other ruminal organisms (Ricke et al., 1996; Weimer et al., 2009). During this process certain ruminal microorganisms (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 consequent reduction of fermentation efficiency and microbial yield (Wolin and Miller, 1983). Although sev-

utilizing carbohydrates and H2/CO2, [isolates H3HH and A10 (Boccazzi and Patterson, 2011; Jiang et al., 2012a,b; Pinder and Patterson, 2011), Acetitomaculum ruminis (Greening and Leedle, 1989); Eubacterium limosum (Genthner et al., 1981), and Syntrophococcus sucromutans (Krumholz and Bryant, 1986), only E. limosum has been tested to determine substrate 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 reported herein was to determine if isolate A10 was capable

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, respectively; Thauer et al., 1977], and 2) methanogens have a higher affinity for H2 than acetogens (CordRuwisch et al., 1988). However, other factors affect the competition between these two groups of microorganisms because acetogenesis predominates over methanogenesis in several habitats [e.g., termite guts (Brauman et al., 1992), rodent ceca (Prins and Lankhorst, 1977), and human intestines (Lajoie et al. (1988)].

Breznak and Blum (1991) suggested that mixotrophy, 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

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 labeling 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 intracellular hydrogenase activity and NAD(H) concentrations in isolate A10 during the sequential utilization of glucose and H2/CO2 was obtained.

and H2 could consume H2 regardless of carbohydrate concentration, a non-mixotrophic species may cease H2 consumption if carbohydrate concentrations exceed threshold levels. Of the five acetogenic bacteria isolated from ruminal contents and capable of

results) from a filter-sterilized stock solution (15% w/v). After inoculation (1% v/v from an overnight culture), 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-

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MATERIALS AND METHODS Organism and cultivation medium Isolate A10, a previously described and ruminal isolate (Boccazzi and Patterson, 2011) was used in all experiments. This organism was maintained and (for most experiments) grown in acetogenic medium (Pinder and Patterson, 2011). Glucose was added at the appropriate concentration (as described in the

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ing (180 rpm) for appropriate time periods as described in the results.

For mass spectrometry experiments, NaH13CO3 replaced unlabeled Na2CO3 on an equivalent carbon basis. Moreover, to further reduce the presence of unlabeled CO2, the medium was gassed with oxygen-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 sterilization, glucose was added to a final concentration of 0.05 % from a filter-sterilized glucose stock solution (15% w/v). The flasks were inoculated (1% v/v) with an overnight culture grown in the same medi-

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 uninoculated 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

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).

pivalic acid (internal standard) and 40 μl of metaphosphoric acid (25% w/v in H2O). Concentrations of volatile fatty acids in the culture supernatant were determined using a Hewlett Packard 5890 gas chromatograph (Hewlett Packard Co., Palo Alto, CA) fitted with a glass column packed with GP 60/80 carbopack C / 0.3% Carbowax M / 0.1 % H3PO4 (Supelco, Inc.; Bellefonte, PA). The injector and detector temperatures were set at 200°C while the column temperature was set at 135°C. Formate concentrations in the culture supernatant were determined using a formate dehydrogenase assay (Schaller and Triebig, 1983). The pH of the culture was measured immediately after sampling for 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.

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 resuspending 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 determined 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 atmosphere in the bottles was determined by injecting 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

Determination of NAD(H) content Twenty liters of anaerobic acetogen medium, supplemented 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 centrifuged (10,000 x g, 7 min, 4°C). The cell pellet was

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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 alkaline 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 determined 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 chromatography/mass spectrometry techniques. Essentially, 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., Bellefonte, 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 increased to 240°C at a rate of 15°C/min The volatized 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 double-labeled acetate was calculated as follows: Total mass = mass 60 + mass 61 + mass 62.

Unlabeled acetate (CH3COOH) = (mass 60 / total 256

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 inoculated 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 approximately 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 continuously gassed with CO2, and used immediately. Hydrogenase activity of the cell lysate was determined as described by Ragsdale and Ljungdahl (1984). An aliquot of the cell extract was injected into serumstoppered 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 atmosphere of 100 % H2. The absorbance at 604 nm, was measured over 10 minutes. The change in absorbance 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).

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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.

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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.

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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 column. Enzymes used in formate, NAD+ and NADH assays were obtained from Boeringer Mannheim (Indianapolis, IN). All chemicals used were of reagent grade.

RESULTS Results presented in this section are typically from one experiment although the experiment was duplicated 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 pattern when grown in glucose-supplemented medium and an atmosphere of H2/CO2 (Figure 1). This diauxic pattern was observed regardless of the initial concentration of glucose (0.025 to 27 mM). The maximum 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 substrates (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 exhausted (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 protein 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 concentration 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 significant burst of growth, without a lag phase, was observed (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 incubation (Figure 4). These results were observed regardless 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 consuming H2, the consumption of H2 ceased until the additional glucose was exhausted (data not shown).

When non-metabolizable glucose analogues (2-deoxyglucose 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 period of H2 consumption (Figure 6). However, formate alone (50 mM as HCOONa) did not support growth of isolate A10 (data not shown).

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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 incubation, 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 nonmetabolizable glucose analogs.

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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.

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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 percentage of uninoculated bottles because, over time, there was a decrease in pressure of the atmosphere 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).

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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 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 formate concentration of the culture supernatant was determined using a formate dehydrogenase asssay.

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Product formation

Acetate was the major final product of isolate A10 (Figure 7). The final fermentation stoichiometry of acetate production from glucose was 2.2 acetates per glucose fermented. Cultures growing solely on H2/CO2, produced acetate to final concentrations 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

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 glucose to growth on H2/CO2. Non-denaturing, anaerobic polyacrylamide gel electro-phoresis revealed that the hydrogenase activity migrated as one band (data not shown).

The intracellular concentration of NAD+ increased

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 incubation. 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). Formate production was greatest (2 mole of formate per mole of glucose fermented) when the organism was incubated in bottles containing acetogen medium with glucose and a H2/CO2 atmosphere. Formate 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 organic 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-

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.

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 production during glucose consumption is a previously

have suggested that the higher affinity of methanogens for hydrogen may explain the dominance of methanogenesis over acetogenesis in H2-limited environments such as the rumen. However, these theories cannot explain why acetogenesis predominates

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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 designed to explain the relatively low numbers of acetogens in the rumen but rather to gather information that may explain the low rate of chemolithoautotrophic acetogenesis observed in ruminal contents.

Thauer et al. (1977) and Cord-Ruwisch et al. (1988)

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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 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 acetate concentration of the culture supernatant was determined using gas chromatography techniques.

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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.

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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 acetate produced was unlabeled (CH3COOH; ●), singly labeled (CH313COOH; ■) or double labeled (13CH313COOH;♦). The relative abundance of unlabeled, single- or double label acetate was determined by GC/MS.

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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 (approximately 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.

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Figure 11. Intracellular concentration of NAD(H) in isolate A10. Cells were grown in batch cultures 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.

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over methanogenesis in certain habitats. This conundrum 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-utilizing 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 contents 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

CO2 growth commenced (even in the absence of exogenous glucose), a determination of the mixotrophic 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 glucose is exhausted, isolate A10 is most likely not a mixotroph.

The doubling time of isolate A10 growing on glucose (approximately 2.1 h) is much less than that reported for E. limosum (7.1 h; Genthner and Bryant, 1987). Unfortunately, comparisons with other acetogens isolated from ruminal contents are not possible because the doubling time of these organisms grow-

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 acetogens 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 pfennigii is the only ruminal acetogen isolated thus far that is incapable of utilizing sugars for growth (Krumholtz and Bryant, 1986). However, information of the ability 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 capabilities has been tested is E. limosum. Glucose was used as the organic energy substrate for these studies because preliminary experiments suggested that similar results would be obtained whether glucose, maltose, cellobiose or xylose were used as the organic substrate (data not shown).

The growth pattern of isolate A10 grown in bottles 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

ing on glucose has not been reported. The doubling 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 glucose 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 glucose. 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 glucose PTS system, glucose kinase activity was detected regardless of growth on glucose or H2/CO2.

However, the lag period between the end of glucose consumption and the initiation of H2/CO2 consumption could be construed to suggest that H2/CO2 utilization 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

ruminal contents, namely, E. limosum (Genthner and Bryant, 1987) and isolate H3HH (Boccazzi and Patterson, 2011). Because glucose-dependent growth was relatively fast (compared to H2/CO2-dependent growth), coupled with the long lag time before H2/

(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-

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tion of hydrogenase activity has been reported for a number of organisms, including Escherichia coli, Proteus 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

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, intracellular production of NADH is sufficient to meet needs.

However, during the time period when the cells are switching from one substrate to another, NADH utilization is greater than production, causing the precipitous 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.

dependent on the pyridine nucleotides for activity. However, the hydrogenase is capable of reducing methyl viologen, an analog of ferredoxin. These characteristics are similar to those of most hydrogenases isolated thus far (Adams et al. 1981). Our findings 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 possible (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

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 characterization (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 determine 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). Chemolithoautotrophic 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. Experiments specifically demonstrating the activities of key enzymes of the acetyl-CoA pathway (i.e. carbon monoxide dehydrogenase) will be needed for unambiguous proof. Notwithstanding, the data presented herein provides strong evidence that isolate A10 is a true acetogen.

The combination of the data on diauxic growth patterns, acetate labeling, hydrogenase induc-

enzymes including glyceraldehyde-3-phosphate dehydrogenase, a key enzyme in glucose catabolism.

The high concentration of NAD+ could be interpreted as an attempt to make this reaction as thermodynamically feasible as possible in order to process

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

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this is a general statement about all carbohydrates.

These findings are of significance because they suggest that the inability to use carbohydrates and H2/ CO2 mixotrophically may, in part, explain why acetogens 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 isolate 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 consequence, have a higher H2 threshold (Breznak and

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-

Blum, 1991; Zinder, 1993), these factors are probably of secondary importance to the regulation of H2 utilization by carbohydrates in isolate A10, and probably to other non-mixotrophic acetogens.

The appearance of single labeled (carboxyl) acetate (and lack of double labeled acetate) during the period of formate consumption suggests that formate provided the methyl carbon of acetate during this time period. In these experiments, formate was unlabeled (HCOOH) and disappeared from the culture 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 strictest 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 capability, temperature preference, and guanine-plus-

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 apparently exerts a strong repressive influence on the utilization of H2 by these organisms. However, owing to the diversity of acetogens, the possibility still exists of locating and introducing non-ruminal mixotrophic acetogens into the ruminal ecosystem to successfully compete for H2 against methanogens.

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 carbohydrates as growth substrates, S. termitida and C. pfen-

L. L. Barton (eds), Academic Press, Inc. New York, NY. Boccazzi, P., and J.A. Patterson. 2011. Using hydrogen limited anaerobic continuous culture to isolate low hydrogen threshold ruminal acetogenic bacte-

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Balch, W. E., and R. S. Wolfe. 1976. A new approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol.

32:781-791.

Bhatnagar, L., M. K. Jain and J. G. Zeikus. 1991. In: “Variations in Autotrophic Life.” J. M. Shively and

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Brauman, A., M. D. Kane, M. Labat and J. A. Breznak.

1992. Genesis of acetate and methane by gut bacteria of nutritionally diverse termites. Science 257:1384-1386.

Braun, K., and G. Gottschalk. 1981. Effect of molecular hydrogen and carbon dioxide on chemoorganotrophic growth of Acetobacterium woodii and Clostridium aceticum. Arch. Microbiol.

128:294-298.

Breznak, J. A., and J. S. Blum. 1991. Mixotrophy in the termite gut acetogen, Sporomusa termitida.

Arch. Microbiol. 156:105-110.

Cord-Ruwisch, C., H.-J. Seitz and R. Conrad. 1988.

Kerby, R., and J. G. Zeikus. 1987. Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum. J. Bacteriol. 169:20632068.

Klingenberg, M. 1983. Nicotinamide-adenine dinucleotides and dinucleotide phosphates. End-point UV-methods. In: “Methods of Enzymatic Analysis, 3rd edition.” H. U. Bergmeyer (ed.), Verlag Chemie, Weinheim, Germany.

Krasna, A. I. 1980. Regulation of hydrogenase activity in Enterobacteria. J. Bacteriol. 144:1094-1097.

Krumholz, L. R., and M. P. Bryant. 1985. Clostridium pfennigii sp. nov. uses methoxyl groups of monobenzenoids and produces butyrate. Int. J. Sys.

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor. Arch. Microbiol. 149:350-357.

Drake, H. L. 1992. Acetogenesis and Acetogenic bacteria. In: Lederberg, J., Ed. “Encyclopedia of Microbiology. Academic Press, Inc. San Diego, CA. Genthner, B. R. S. , C. L. Davis and M. P. Bryant. 1981.

Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol-and H2-CO2utilizing species. Appl. Environ. Microbiol. 42:1219.

Genthner, B. R. S., and M. P. Bryant. 1987. Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii. Appl. Environ. Microbiol. 53:471-476.

Greening, R. C., and J. A. Z. Leedle. 1989. Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen. Arch. Microbiol. 151:399-407.

Hungate, R. E. 1967. Hydrogen as an intermediate in the rumen fermentation. Arch. Microbiol. 59:158164.

Jiang, W., R.S. Pinder, J.A. Patterson, and S.C. Ricke.

2012a. Sugar phosphorylation activity in ruminal acetogens. J. Environ. Health Sci., Part A 47:843-

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Lajoie, S. F., S. Bank, T. L. Miller, and M. J. Wolin.

1988. Acetate production from hydrogen and [13C] carbon dioxide by the microflora of human feces.

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Le Bloas, P., N. Guilbert, P. Loubiere, and N. D. Lindley. 1993. Growth inhibition and pyruvate overflow during glucose metabolism of Eubacterium limosum are related to a limited capacity to reassimilate CO2 by the acetyl-CoA pathway. J. Gen. Microbiol. 139:1861-1868.

Miller, T. L., and M. J. Wolin. 1973. Formation of hydrogen and formate by Ruminococcus albus. J. Bacteriol. 116:836-846.

Pine, L., and H. A. Barker. 1954. Tracer experiments on the mechanism of acetate formation from carbon dioxide by Butyribacterium rettgeri. J. Bacteriol. 68:216-226.

Pinder, R.S., and J.A. Patterson. 2011. Isolation and initial characterization of plasmids in an acetogenic ruminal isolate. Agric. Food Anal. Bacteriol.

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Pinder, R. S., J.A . Patterson,. C. A. O’Bryan, P. G. Crandall, and S. C. Ricke. 2012. Dietary fiber content influences soluble carbohydrate levels in ruminal fluids. J. Environ. Health Sci., Part B 47:710-

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Ragsdale, S. W. 1991. Enzymology of the acetyl-CoA pathway of CO2 fixation. Crit. Rev. Biochem. and Mol. Biol. 26:261-300.

Ricke, S.C., S. A. Martin, and D. J. Nisbet. 1996. Ecology, metabolism, and genetics of ruminal selenomonads. Crit. Rev. Microbiol. 22:27-56.

Schaller, K.-H., and G. Triebig. 1983. Formate dehydrogenase. In: “Methods of Enzymatic Analysis, 3rd edition.” H. U. Bergmeyer (ed.), Verlag Chemie, Weinheim, Germany.

Schulman, M., D. Parker, L. G. Ljundahl, and H. G. Wood. 1972. Total synthesis of acetate from CO2.

V. Determination by mass analysis of the different types of acetate formed from 13CO2 by heterotrophic bacteria. J. Bacteriol. 109:633-644.

Thauer, R. K., K. Jungermann and K. Decker. 1977.

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Wolin, M. J., and T. L. Miller. 1983. Interactions of microbial populations in cellulose fermentation.

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Wood, H. G. 1952. A study of carbon dioxide fixation by mass determination of the types of C13acetate. J. Biol. Chem. 194:905-931.

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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

Department of Animal Science, 2471 TAMU, Texas A&M University, College Station, TX Current address: Conrad N. Hilton College of Hotel and Restaurant Management, University of Houston, Houston, TX 3 Current address: Department of Public Health, University Center for Agricultural and Biological Sciences, University of Guadalajara, Guadalajara, Mexico 1

2

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 measuring 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 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 of spinach sample. These images demonstrate the ability of pathogens 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 Agric. Food Anal. Bacteriol. 2: 275-279 2012

Correspondence: Alejandro Castillo, a-castillo@tamu.edu Tel: +1 -979-845-3565 Fax: +1-979-862-3475

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INTRODUCTION The number of reported foodborne illness outbreaks associated with fresh produce has increased in the past thirty years (Alkertruse et al., 1996; Hedberg 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; Sivapalasingam et al., 2004; Hanning et al., 2009).

study was to determine how pathogens associate with spinach leaves after washing cut spinach leaves, simulating incorrect washing practices during postharvest processing of spinach.

MATERIALS AND METHODS Source of spinach leaves Fresh spinach leaves typical of leafy greens entering the U.S. food supply were kindly provided by the Winter Garden Spinach Producers Board (Crystal City, TX). The spinach was harvested at approxi-

Bacteria can be introduced to leafy greens at any step from planting to consumption and once they are introduced, their colonization can have a tremendous effect on both the quality and safety of the product. The attachment and colonization of microorganisms on fresh produce have significant public health implications due to the fact that these processes may be related to the inability of sanitizers and decontamination treatments to remove or inactivate 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 penetrate the interior of cut leaf edges or become internalized 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 recent review of literature on the internalization of produce by pathogens (Erickson, 2012) shows how most

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.

studies involving spinach have focused on the internalization 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

EC) and RFP-producing S. Typhimurium (RFP-ST) was harvested, washed in sterile phosphate buffer saline (PBS; EMD Biosciences, Inc. La Jolla, CA) and resuspended in 0.1% peptone water (Becton Dickinson).

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Sources of bacteria and plasmids Isolates from the Texas A&M Food Microbiology Laboratory culture collection were previously transformed by electroporation using the plasmid vectors pEGFP and pDsRed-Express (Clontech Laboratories, 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 microorganisms 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-

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Preparation of inoculum and sample preparation for confocal scanning laser microscopy

RESULTS AND DISCUSSION

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. Samples 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-

For the confocal microscopy study, the spinach leaf provided a thin, relatively flat sample, which produced 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 Figure 1A. The preferential gathering of pathogens to the stomata and cracks in the cuticle are seen in Figure 1B. Fluorescent bacteria allocated in the interior

move loosely attached cells. Strongly attached bacteria 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 observed using a BioRad Radiance 2000MP confocal microscope (Zeiss, Heltfordshire, UK) using an excitation wavelength of 488 nm. The confocal microscopy was conducted at the Image Analysis Laboratory at Texas A&M University (College Station, TX).

of the spinach stem are shown in Figures 2A and 2B. These images demonstrate the ability of pathogens to congregate in areas on the leaf surface as well as internalization within the plant possibly escaping 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 inoculated 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).

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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 colonization mechanisms (Brandl and Mandrell, 2002; Gomes et al., 2009; Kroupitski et al., 2009; Solomon et al., 2002; Xicohtencatl-Cortes et al., 2009). This internalization seems to result in the microorganisms being out of reach of antimicrobial compounds used for washing and disinfecting produce (XicohtencatlCortes 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-

and suggested that E. coli O157:H7 may attach to less favorable attachment sites once all of the preferred initial attachment sites were occupied.

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 penetration of the pathogen into the interior of lettuce leaf.

Takeuchi and Frank (2000) reported similar findings

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.

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 inaccessible to chemical sanitizers but can also be internalized within the plant structure. Therefore, both farmers and processors must realize that chemical sanitizers may not reach all microorganisms when

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 (arrows).

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Swatzberg, D. Granot, and S. Sela. 2009. Internalization of Salmonella enterica in leaves is induced by light and involves chemotaxis and penetration through open stomata. Appl. Environ. Microbiol.

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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 United 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

1

ABSTRACT Two experiments were conducted to evaluate the effect of the iron-binding molecule lactoferrin on reducing 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 randomly 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 following 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 bacterial 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 inoculation, 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 Agric. Food Anal. Bacteriol. 2: 280-290, 2012

Correspondence: Tom S. Edrington, tom.edrington@ars.usda.gov Tel: +1 -979-260-3757 Fax: +1-979-260-9332

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INTRODUCTION Salmonella is the second leading cause of foodborne illness, resulting in an estimated 1.4 million cases every year (Foley and Lynne, 2008). Of these human cases of salmonellosis, 6-9% are associated with the consumption of pork or pork products (Frenzen et al., 1999). Salmonella has been isolated 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,

ing abilities of lactoferrin enable it to scavenge iron within the intestinal tract thereby depriving microorganisms of this critical element and inhibiting their metabolic activities (Naidu et al., 1993). Facilitating iron absorption, stimulation of mucosal differentiation, 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 antimicrobial properties of lactoferrin go beyond simple iron deprivation and include damage of the outer membrane and subsequent permeability alterations (Ellison et al., 1988) and modulation of bacterial motility, aggregation and adhesion (Valenti and An-

rodents, etc.) and activities within the swine production 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 immune 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 development of pre-harvest, “on-farm” intervention strategies is crucial.

Most all bacteria, including the pathogenic bacteria Campylobacter, E. coli and Salmonella, require iron for survival and important intracellular reactions (Naikare et al., 2006; Brock, 1980; Ratledge and Dover, 2000), thus iron-sequestering compounds such as lactoferrin and transferrin provide a primary nonspecific host defense system against microbial infection. A variety of preventative and therapeutic strategies for treating bacterial infections are based upon interfering with microbial iron acquisition and utilization. The immune system likewise exploits the iron requirement of bacteria, utilizing iron withholding as an essential antimicrobial component of the innate

tonini, 2005). Lactoferrin has been shown to inhibit growth of several important bacteria, including Salmonella, 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 administration 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 objective of the current project was to determine whether oral administration of lactoferrin would significantly reduce the populations of Salmonella within the gastrointestinal tract of experimentally-infected pigs.

immune system.

Lactoferrin is a major iron-binding protein present 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-

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

MATERIALS AND METHODS Experiment I Forty crossbred barrows and gilts (avg. BW = 24 kg) were purchased locally and transported to our labo-

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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. Treatments (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 experimental 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-

(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.

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 intakes were recorded and used to calculate an average 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 Typhimurium (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 below. Scour and activity scores (for each pen) were recorded daily following inoculation through necropsy. 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

were utilized. Thirty crossbred piglets (average BW = 6.6 kg), were purchased within one week of weaning and transported to our laboratory facilities. Pigs were weighed, eartagged, rectally swabbed and randomly 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 acclimation, treatments were initiated (d 1) and administered throughout the remainder of the experimental period (13 total days; 10 pigs and 5 pens/treatment).

Treatments were identical to those used in Experiment I with the exception that a non-infected control 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 containing 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 described above on d 13. Body weights were recorded upon arrival and on d 1, 8 and 13 of the experimental

post-challenge, pigs were sedated with an intramuscular 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

period.

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Experiment II A second experiment was conducted, similar to the first, with the exception that much younger pigs

Bacterial Culture Experiment I

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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, respectively) for detection of any wild-type Salmonella. A few pigs were naturally-colonized with a wild-type Salmonella capable of growth on BGANOV, therefore all samples collected following inoculation of pigs were streaked on BGANN. The inoculation strain of Salmonella 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 commercially available spiral plater (Spiral Biotech Autoplate 4000; Advanced Instruments, Inc., Norwood, MA). Black colonies were counted following incubation (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 postinoculation swabs. Following incubation at 37°C for 24 h, BGANN plates containing pink colonies exhibiting typical Salmonella morphology were considered positive.

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 (incidence of positive luminal content and tissue samples) was subjected to Chi-square analysis using the PROC FREQ procedure. Daily rectal swab culture results (positive or negative), activity and scour scores were analyzed using the PROC MIXED procedure for repeated measures with treatment, day and treatment x day interaction included in the model. For some samples, Salmonella was recovered only from enriched specimens or not at all indicating that concentrations 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 statistical analysis. Results were considered statistically significant at the 0.05 level for type-one error.

RESULTS Experiment I

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 wildtype 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

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 wildtype Salmonella. All samples from the first collection 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

swabs and necropsy samples additionally plated on BGANNR. Spiral plating of luminal content samples was done on XLD + novobiocin and XLD + rifampicin. All enrichment procedures were identical to those used in Experiment I described above.

pigs were culture negative on this medium (data not shown).

Rectal swabs collected over the 4-d post-inoculation period were mostly positive in all treatment groups, including the non-infected control pigs. Di-

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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.

Item

Treatment

Control

1X

5X

NI Cont

P>F

d1

100

100

100

100

1

d2

90

90

80

80

0.85

d3

100

90

100

80

0.26

d4

100

90

80

20

0.0002

Overall

97.5

92.5

90

70

0.001

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

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

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

90

90

90

.

1

Rectal Swabs (% positive)a

Luminal contents Direct plate [cfu/g (log10)] Stomach

% positive after enrichment

Tissue % positive after enrichment

a

284

Rectum

By day post-inoculation

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rect streaking of the swab onto the agar was conducted on d17 â&#x20AC;&#x201C; 19 to get an indication of Salmonella concentrations in the feces. A positive swab via direct plating would be indicative of a higher concentration of Salmonella being shed by the animal, compared to a swab requiring enrichment to test culture positive. All direct swabs were negative in the non-infected control treatment, therefore only the direct plating data for the infected-control, 1X and 5X treatments were analyzed and no differences (P > 0.10) observed (data not shown). Following 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

1X treatment, which had concentrations numerically higher in contents from the ileum, spiral colon, cecum and rectum. All stomach content samples were negative in all treatments. Following enrichment, luminal content samples were not different (P > 0.10) among treatments. Tissue samples were also not different (P > 0.10) among treatments, with the exception 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

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 experimental period and were surprised by the number 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. Obviously, 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 Salmonella is transmitted among pigs and the short time duration required for fecal shedding following exposure. We did not serogroup any of the isolates from these animals to determine if the recovered Salmonella 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 determine if the whey-protein concentrate influenced growth, however, as all of these pigs were Salmonella-positive at some point in the experiment the decision was made not to necropsy this group.

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 interaction for activity or scour scores (P > 0.10), nor were significant differences observed when data was combined across days (data not shown).

Necropsy results are presented in Table 1. Concentrations 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

perimental diets pre-challenge. Post-challenge, no differences (P > 0.10) were observed for shedding of the wild-type strain (direct plated and enriched samples), while a trend (P < 0.10) was observed for the inoculated strain following direct plating (prevalence

Experiment II The majority of pigs were Salmonella-positive during the pre-screening process, therefore we attempted 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-challenge 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 wildtype Salmonella following direct plating or after enrichment during the seven days of feeding the ex-

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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 experimentallyinfected 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.

Item

Treatment

Control

Rectal swabs (% positive)

1X

P>F

5X

nov

nnr

nov

nnr

nov

nnr

nov

nnr

Pre-challenge - overall (n=70/trt) Direct plate

7.1

.

5.7

.

2.9

.

0.51

.

52.3

.

54.3

.

52.9

.

0.98

.

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

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

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

50

50

50

50

40

40

0.87

0.87

Enriched Post-challenge - overall (n=40/trt)

Luminal contents Concentration [cfu/g (log10)] Stomach

% positive w/enrichment

Tissue (% positive w/enrichment) Stomach

286

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

100

100

100

100

100

100

1

1

Liver

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in 1X and 5X treatments numerically higher than control pigs). No differences were observed for Salmonella 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

(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 supplementation on growth performance of weaned piglets 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 together, we reasoned that administration of lactoferrin to pigs may reduce the gut populations and fecal shedding of Salmonella.

Due to some facility constraints and pig availabil-

in the 5X treatment exhibited a numerical increase in BW gain compared to control animals following inoculation 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 temperatures than the control and 1X animals (data not shown). No treatment x day interactions were observed 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 experimental period (data not shown).

Oral administration of lactoferrin has been reported to provide host protection against various diseases 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 immune responses to pathogens, their components, as well as mucosal immunity in the intestines and that

ity, the pigs in the first experiment were older and larger than we considered ideal for this experimentation. We hypothesized that lactoferrin treatment had the best chance of success in a younger animal with an immature or under-developed gut microbiota 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 experiments, indicating that the experimental challenges were similarly effective in the older and younger pigs and that at these ages, differences in the gut microbial 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 Salmonella. 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-

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 irondependent bacteriostatic effect on this pathogen

ministered the challenge strain of Salmonella Typhimurium. 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

DISCUSSION

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lactoferrin treatment in reducing Salmonella populations 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 provided by the lactoferrin. A lower dose, more realistic of what the pigs might be exposed to in a production 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 animal except those with a very immature or disturbed

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 research, 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 adaptations bacteria make in order to compete with iron-sequestering compounds such as lactoferrin.

gut microflora. As pigs in both experiments were weaned and eating well, we expected that a larger challenge would be necessary to establish Salmonella within the gut and produce concentrations that could subsequently be detected in the luminal contents and gut tissues at necropsy several days postinoculation.

Similar to our research, Sarelli and co-workers (2003) evaluated lactoferrin for preventing E. coli diarrhea 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 lactoferrin. The authors hypothesized that continual feeding 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 protected piglets against lethal shock induced by intravenously administered E. coli LPS (endotoxin) with significantly less mortality compared to the control

Some strains of bacteria adapt to the iron-deprived conditions by producing their own high affinity iron chelators called siderophores, which compete directly 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 availability (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 contributes to the innate immune system of the host by interfering with microbial virulence (adhesion, internalization). 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 experiments is difficult at best. It is unclear if the inocu-

treatment.

Others have reported a beneficial effect of lactoferrin and lactoperoxidase system (LP-s) on experimentally-induced E. coli diarrhea in calves with improvements in mortality, occurrence of severe di-

lated Salmonella (Exp. I) or the naturally-colonized Salmonella (Exp. II) infected the mucosal surface of the gastrointestinal tract or merely populated the luminal contents throughout. However, we would suspect that a lactoferrin-response such as this would

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only be effective or measurable at much lower populations of Salmonella.

While the void of treatment differences in this research is disappointing, it would be premature to dismiss lactoferrin as a potential pre-harvest intervention. 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 lactoferrin to recently weaned pigs prior to Salmonella challenge, either administered in a lower oral dose or via exposure to Salmonella-positive pigs.

Ellison, R.T., III, T.J. Giehl, and F.M. LaForce. 1988.

Damage of the outer membrane of enteric gramnegative bacteria by lactoferrin and transferrin. Infect. Immun. 56:2774-2781.

Foley, S.L., and A.M. Lynne. 2008. Food animalassociated Salmonella challenges: Pathogenicity and antimicrobial resistance. J. Anim. Sci. 86(14 Suppl):E173-87.

Frenzen, P.D., J.C. Buzby, and T. Roberts. 1999. An updated estimate of the economic costs of human illness due to foodborne Salmonella in the United States. In: Proc. of the 3rd Int. Symp. on the Epidemiology and Control of Salmonella in Pork, Washington, D.C., p. 215-218.

ACKNOWLEDGEMENTS

Ajello, M., R. Greco, F. Giansanti, M.T. Massucci, G. Antonini, and P. Valenti. 2002. Anti-invasive activity of bovine lactoferrin towards group A streptococci. Biochem. Cell Biol. 80:119-124.

Bessler, H.C., I.R. de Oliveira, and L.G. Giugliano.

2006. Human milk glycoproteins inhibit the adherence of Salmonella typhimurium to HeLa cells. Microbiol. Immunol. 50:877-882.

Blecha, F., D.S. Pollmann, and D.A. Nichols. 1983.

Weaning pigs at an early age decreases cellular immunity. J. Anim. Sci. 56:396-400.

Brock, J.H. 1980. Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn infant. Arch. Dis. Child.

55:417-421.

Crosa, J.H. 1989. Genetics and molecular biology of siderophore-mediated iron transport in bacteria.

Microbiol. Rev. 53:517-530.

Gislason, J., I. Suhasini, T.W. Hutchens, and B. Lonnerdal. 1993. Lactoferrin receptors in piglet small intestine: Lactoferrin binding properties, ontogeny, and regional distribution in the gastrointestinal tract. J. Nutr. Biochem. 4:528-533.

Gomez, H.F., T.J. Ochoa, L.G. Carlin, and T.G. Cleary.

2003. Human lactoferrin impairs virulence of Shigella flexneri. J. Infect. Dis. 187:87-95.

Lee, N.Y., K. Kawai, I. Nakamura, T. Tanaka, H. Kumura, and K. Shimazaki. 2004. Susceptibilities against bovine lactoferrin with microorganisms isolated from mastitic milk. J. Vet. Med. Sci. 66:1267-1269.

Lee, W.J., J.L. Farmer, M. Hilty, and Y.B. Kim. 1998.

The protective effect of lactoferrin feeding against endotoxin lethal shock in germfree piglets. Infect.

Immun. 66:1421-1426.

Lonnerdal, B., and S. Iyer. 1995. Lactoferrin: molecular structure and biological function. Annu. Rev. Nutr. 15:93-110.

Masson, P.L., J.F. Heremans, and E. Schonne. 1969.

Lactoferrin, an iron-binding protein in neutrophilic leukocytes. J. Exp. Med. 130:643-658.

Naikare, H., K. Palyada, R. Panciera, D. Marlow, and A. Stintzi. 2006. Major role for FeoB in Campylobacter jejuni ferrous iron acquistition, gut colonization, and intracellular survival. Infect. Immun.

Di Mario, F., G. Aragona, N. Dal Bo, G.M. Cavestro, L. Cavallaro, V. Iori, G. Comparato, G. Leandro, A. Pilotto, and A. Franze. 2003. Use of bovine lactoferrin for Helicobacter pylori eradication. Dig. Liver Dis. 35:706-710.

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This research was funded in part by Wyeth Pharmaceuticals, Inc., Collegeville, PA.

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Ochoa, T. J., and T. G. Cleary. 2009. Effect of lactoferrin on enteric pathogens. Biochemie 91:30-34.

Pakkanen, R., and J. Aalto. 1997. Growth factors and antimicrobial factors of bovine colostrum. Int. Dairy J. 7:285-297.

Petschow, B. W., R. D. Talbott, and R. P. Batema.

1999. Ability of lactoferrin to promote the growth of Bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level. J. Med. Microbiol. 48:541-549.

Ratledge, C., and L. G. Dover. 2000. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol.

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van der Strate, B. W., L. Beljaars, G. Molema, M. C. Harmsen, and D. K. Meijer. 2001. Antiviral activities of lactoferrin. Antiviral Res. 52:225-239.

Van Leeuwen, P., S.J. Oosting, J.M.V.M. Mouwen, and M.W.A. Verstegen. 2000. Effects of a lactoperoxidase system and lactoferrin, added to a milk replacer diet, on severity of diarrhea, intestinal morphology and microbiology of digesta and faeces in young calves. J. Animal Physiol Animal Nutr.

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Vorland, L.H. 1999. Lactoferrin: a multifunctional glycoprotein. APMIS 107:971-981.

Wakabayashi, H., M. Takase, and M. Tomita. 2003.

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Schryvers, A. B., R. Bonnah, R. H. Yu, H. Wong, and M. Retzer. 1998. Bacterial lactoferrin receptors.

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Wang, Y., T. Shan, Z. Xu, J. Liu, and J. Feng. 2006.

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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 Center for Advanced Research in Sciences Department of Biochemistry and Molecular Biology 3 Institute of Nutrition and Food Science, University of Dhaka, Dhaka-1000, Bangladesh.

1

2

ABSTRACT Previously, we reported on the prevalence of microorganisms and pathogens in raw red amaranth, effectiveness of sanitizers in reducing the pathogenic bacteria and impact of cooking in eliminating the microbiological 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 cytotoxicity 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

Agric. Food Anal. Bacteriol. 2: 291-296 2012

INTRODUCTION Vegetables and their products are usually valued for their nutrient content but they are now also regarded as rich sources of non-starch polysaccharides, collectively referred to as dietary fiber. Dietary fiber includes polysaccharides, oligosaccharides, Correspondence: Md. Latiful Bari, latiful@univdhaka.edu Tel: 8802-9661920-59 Ext 4721 Fax: 8802-8615583

and associated plant substances that are resistant to digestion and adsorption in the human small intestine 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 composition and physicochemical properties of dietary fiber produce different beneficial physiological effects including laxation, and blood cholesterol and glu-

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cose attenuation (AACC, 2001). In addition, the fiber component of the diet is nutritionally important because of their properties such as bulk density, hydration 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 composition, 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-

Since consumers typically cook or stir fry red amaranths samples with spices and consume them, an experiment was designed to see the impact of cooking 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 biosafety cabinet to facilitate cooling down to room temperature.

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 between 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 eliminate the pathogenic bacteria; however, these eating habits may result in consuming less nutritional content.

Therefore, in this study the impact of cooking on proximate content, and functional properties including fat binding capacity, glucose binding capacity and cytotoxicity of red amaranths are reported.

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 Nutrition and Food Science laboratory of University of Dhaka and had been used for the last 15 years. Analyses 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 DIGEST SYSTEMK-437 equipped with a Buchi Scrubber, B-414, samples were distilled in Buchi Distillation 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.

MATERIALS AND METHODS Sample collection Commercial red amaranth samples were purchased from 20 different market of Dhaka City, Bangladesh and composite mixture were prepared within 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

292

Nutritional Quality Analysis 1) Proximate content:

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.

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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 (Ferris 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

National Labnet Company, Woodbridge, NJ, USA) for 20 min and supernatant was collected. Concentrations 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:

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 (eppendorf, 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 Instruments Inc. Taipei, Taiwan) for 1 min to disperse the sample. The contents were left at ambient temperature 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 decanted, 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.

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 primary 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 sterile artificial seawater and pH was adjusted at 8.5 using 0.1 N NaOH under constant aeration for 48 h. After hatching, active nauplii free from egg shells were collected from the brighter portion of the hatching chamber and used for the assay. Red amaranth extract 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. After overnight incubation, the nauplii were counted.

Vincristine sulfate (0.5 mg/mL; an anticancer drug) was considered as a positive control.

Statistical analysis 2) Sugar Binding Capacity (SBC) SBC of raw and cooked red amaranth was measured 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 solution for 2 hours at room temperature. The content was then centrifuged at 3,500 rpm (Z383K, HERMLE-

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 significance.

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Table 1. The proximate content of Red amaranth after treating with different water disinfectants followed by cooking (g/100 g edible portion)1

Nutrient

Fresh

Cl2 treated cooked

Cl2 treated raw

SP treated raw

SP 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

Results 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 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

01

d

GlucStn

A

02

n

03

Conc

Absorbed glucose in 10mL solution (mg)

GBC (%)

(mg/mL)

Residual glucose in 10 mL solution (mg)

0.953

2.000A

-

-

-

GlucSol

0.847A

3.774A

37.742A

0

-

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.

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Table 4. Mortality of Brine shrimp (Artemiasalina) nauplii at different concentrations of cooked red amaranth extract Sample No.

Sample Name

Dose [mg/ml]

No. of nauplii present after incubation

Mortality [%]

01

Positive control (Vincristine sulfate)

0.1

0

100

02

Negative control (artificial sea water)

-

10

0

0.01

10

0

03

Red amaranths

0.1

7

30

RESULTS AND DISCUSSION The nutritional properties of proximate content were examined and were presented in Table 1. No significant differences were found in proximate contents 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 recorded 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 micronutrients even though microbiologically safe.

The functional properties including fat binding capacity, sugar binding capacity and in vitro cytotoxicity test was conducted and the results were presented in Table 2, 3 and 4. The fat binding capacity and sugar binding capacity were 96% and 129%, respectively 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 vitamin 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 amaranths are also good sources of essential amino acids 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 immune systems (Imura and Okada 1998).

The results of this work and the previous work results demonstrated that cooking could completely reduce the risk of microbial pathogen without significant loss of nutritional quality except for vitamin C.

ACKNOWLEDGEMENTS This is an intra-collaborative work between Center 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.

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AOAC 2000. Association of Official Analytical Chemists (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. Content and composition of dietary fiber in some Bangladeshi vegetables. Diab. Endocr. J. 29:61-66.

Imura, K., and A. Okada. 1998. “Amino acid metabolism 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 Bangladeshi foods prepared by customary cooking process. Dhaka Univ. J. Biol. Sci. 17:9-16.

Rahman, F., K. Fatema, A. T. M. A. Rahim, and L. Ali. 2011. Glucose, insulin and non esterified fatty acid responses to Ladies Finger and Pointed Gourd in type 2 diabetes mellitus. Asian J. Clin. Nutr. 3:2532.

Wang, J.C., and Kinsella, J. E. 1976. Functional properties of novel proteins: alfalfa leaf protein. J. Food Sci. 41:286-292.

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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 Department of Animal and Food Science, Texas Tech University, Lubbock, TX Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, USDA, College Station, TX 3 Livestock Issues Research Unit, Agricultural Research Service, USDA, Lubbock, TX 4 Department of Biological Sciences, Mississippi State University, Mississippi State, MS 5 Agroecosystem Management Research Unit, Agricultural Research Service, USDA, Lincoln, NE 6 Department of Animal Science, University of Nebraska, Lincoln, NE 1

2

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

ABSTRACT Variations 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 tagencoded 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 increasing 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 increase (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, pyrosequencing, nutrition Agric. Food Anal. Bacteriol. 2: 297-308, 2012 Correspondence: Todd R. Callaway, todd.callaway@ars.usda.gov Tel: +1 -979-2609374 Fax: +1-979-260-9332

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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 microbial community and how it is affected by diet is still lacking. Assumptions about the role and importance of bacterial species in the rumen have been based on correlations between culture-dependent population estimates and fermentation end product (e.g., VFA, NH3, and CH4) accumulation (Bryant and Robinson, 1962). Development of molecular methodologies and technologies has progressed

citrus pulp pellets (CPP; 1:1 replacement of steamflaked 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 antimicrobial 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-

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 microbial 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). Citrus 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 ration 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 (Nannapaneni et al, 2008; Callaway et al., 2008). Further studies have demonstrated that feeding orange peel and pulp reduced intestinal populations of Salmonella and E. coli O157:H7 in experimentally inocu-

esized to be responsible for the anti-pathogen effect in these studies. When feeding citrus pulp pellets that contained little or no essential oils, researchers 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 ecosystem, and ultimately, animal health, productivity and food safety remain unclear.

lated sheep (Callaway et al., 2011a,b). In the present study, a tag bacterial diversity amplification pyrosequencing 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%

live virus vaccine (Vista 5, Intervet Schering Plough Animal Health, DeSoto, KS); 4) vaccination with a Clostridial bacterin-toxoid (Vision 7 with SPUR, Intervet Schering Plough Animal Health, DeSoto, KS); 5) antihelmitic treatment and (Ivomec injectable, Me-

298

MATERIALS AND METHODS All procedures involving live animals were approved (#10085-11) by the Texas Tech University Animal 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 processing of both groups included: 1) body weight (BW) measurement, 2) individual identification by ear tag; 3) vaccination with an IBR-BVD-PI3-BRSV modified

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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).

The initial processing body weights for the two loads were 188.7 + 18.0 kg and 225.2 + 22.2 kg, respectively. Heifers two standard deviations from the load average for BW that appeared temperamental,

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% concentrate starter diet from d 0 to d 28, a 73% concentrate

lame, unthrifty, or appeared to have excessive Bos indicus influence were eliminated prior to the start

transition ration from d 28 to d 42, and an 83% concentrate diet from d 42 to d 56.

Experimental Design

Table 1. Formulated composition of treatment diets1

Starter Diets2

Transition Diets3

Finishing Diets4

Ingredients, %5

CTRL

10% CPP

20% CPP

CTRL

10% CPP

20% CPP

CTRL

10% 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 premix6

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

1

Treatment 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.

2

The starter diet was fed from d 0 to d 28.

3

The transition diet was fed from d 28 to d 42.

4

The finishing diet was fed from d 42 to d 56.

5

Dry matter basis

6

Supplement 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% cobalt 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.

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Feeding, Weighing, and Health Monitoring 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 delivery 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 addition, another weekly composited subsample stored at -20C until the conclusion of the study, at which time the samples were analyzed by Servi-Tech Laboratories (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 (Nordlund 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 inserted 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 300

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 (Scientific 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 determined by a Nanodrop spectrophotometer (Nyxor Biotech, Paris, France).

Tag-Encoded FLX Amplicon Pyrosequencing (bTEFAP) Analysis.

A 20 ng (1 µl) aliquot of each DNA sample was used for a 25 µL PCR reaction. The 16S universal 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 Bioscience Corporation, MA, USA) (Dowd et al., 2008).

bTEFAP FLX Massively Parallel Pyrosequencing In preparation for FLX sequencing (Roche, Nutley, NJ), the PCR products’ sizes and concentrations were analyzed using a Bio-Rad Experion Automated Electrophoresis Station (Bio-Rad Laboratories, Hercules, CA) and a TBS-380 Fluorometer (Promega Corporation, Madison, WI). A 9.6 x 106 sample of doublestranded DNA molecules/µL with an average size of 625 bp were combined with 9.6 million DNA capture

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Table 2. Primer sequences utilized for fecal and ruminal samples during bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)

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

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301

beads, and then amplified by emulsion PCR. After bead recovery and bead enrichment, the bead-attached DNAs were denatured with NaOH, and sequencing primers (Table 2) were annealed. A tworegion 454 sequencing run was performed on a 70 × 75 GS Pico Titer Plate by using a Genome Sequencer FLX System (Roche, Nutley, NJ). Following sequencing, 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

Based upon the above BLASTn+ derived sequence identity, (percent of total length query sequence which aligns with a given database sequence) 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 resolving based upon these parameters, the percentage

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 identity of microorganisms in the remaining sequences, sequences were queried using Kraken (www.krakenblast.com) against a highly curated custom database of high quality 16S bacterial and archaeal sequences derived and manually curated from NCBI, and fungal 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).

of each bacterial and archael ID was individually analyzed for each sample providing relative abundance information within and among the individual samples based upon relative numbers of reads within 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; Suchodolski et al., 2009; Andreotti et al., 2011).

Bacterial Diversity Data Analysis To determine the identity of bacteria in the remaining 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 performed as described previously (Bailey et al., 2010; Pitta et al., 2010; Andreotti et al., 2011).

Bacterial identification 302

Statistical Analysis A completely randomized design was utilized in this study with each aspirated sample containing rumen content being the experimental unit within 1 of 3 treatment groups based on diet. Statistics were performed using JMP 6.0 (SAS Institute, Cary, NC).

Significance levels were predetermined as P < 0.05 and differences were separated accordingly. Trends were determined as 0.05 < P < 0.10.

RESULTS AND DISCUSSION Until recently, progress in understanding what role bacterial species play in animal health and productivity has been unclear due to the need to culture bacteria from the gastrointestinal tract. Pyrosequencing (bTEFAP) is not limited to detecting organisms via culture methods and can be used to define what constitutes a “healthy” or “normal” ruminal

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microbial ecosystem profile. This molecular technology is also capable of quantitatively correlating populations of bacterial species with traditional animal production parameters. As ruminal and intestinal 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 production 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,

is dominated by strict anaerobes. In other reports, facultative anaerobes have been reported to occur in numbers at least 100-fold less than the strict anaerobes (Drasar and Barrow, 1985); this is supported by the present results in which the predominant 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 populations (Table 3). To improve understanding of the role of the microbial ecosystem in ruminant nutrition, molecular methodologies of population determination must be correlated with functional data and approaches that address end-product production

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 Bacilli bacterial community increased (P = 0.04) with increasing CPP concentrations (Figure 1).

The gastrointestinal microbial population of cattle

from a quantitative perspective of animal production (Dahllof, 2002). Data from this study is presented at the genus level because genera shifts in ruminal proportions 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.

Bacilli (% of total bacterial population)

30 25 20 15 10 5 0 0

10

20

CPP (% of ration) 1

A increase (P = 0.04) in the percentage of bacilli bacteria was seen with the addition of CPP to the ration.

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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 abundant sequences.

All Diets Rank

Bacterial Genera

0% CPP

Mean

Mean

Std. Dev.

10% CPP Mean

Std. Dev.

20% CPP 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

81.27

81.28

Total

304

79.83

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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 (Wallace et al., 2003; Leng and Nolan, 1984). Modest changes were observed in several genera in relation

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). Tannerella is a periodontal pathogen (Sharma, 2010) that was isolated in this study at 1.22% of the total bacterial community, but this pathogen has not been reported 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, respectively), 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

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 populations increased linearly with increasing CPP, whereas Dialister and Catonella proportions decreased (P = 0.009). Butyrivibrio is a common ruminal bacterium that is involved in fiber and carbohydrate deg-

10 and 20% CPP diets, respectively, and 380 bacterial 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 predominate 27 species; thus the reduction in species richness 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 replaced with 0, 10 or 20% citrus pulp pellets. The genera identified are ordered by most abundant sequences.

Archaeal Genera

All Diets Mean

0% CPP Mean

Std. Dev.

10% CPP

20% CPP

Mean Std. Dev.

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

99.99

99.99

Total

99.99

99.99

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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 significant (P > 0.05). However, these increases corresponded 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-

Rhipicephalus (Boophilus) microplus through tagencoded pyrosequencing. BMC Microbiol. 11:6.

Arthington, J. D., W. E. Kunkle, and A. M. Martin.

2002. Citrus pulp for cattle, in The Veterinary Clinics of North America - Food Animal Practice, G. Rogers and M. Poore, Editors. W. B. Saunders Company: Philadelphia, PA. p. 317-328.

Bailey, M. T., S. E. Dowd, N. M. A. Parry, J. D. Galley, D. C. Schauer, and M. Lyte. 2010. Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization

by Citrobacter rodentium. Infect Immun. 78:150919.

Bryant, M.P. and I.M. Robinson. 1962. Some nutri-

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 between 0 and 10% CPP diets, though 20% CPP diets contained fewer Methanobrevibacter along with increased 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 isolated from the rumen of cattle (Whitford et al., 2001).

In the present study, few significant changes were noted in the ruminal microbial community from feeding 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 ingredients responsible for altering the microbial community (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.

tional characteristics of predominant culturable ruminal bacteria. J. Bacteriol. 84:605-614.

Callaway, T. R., J. A. Carroll, J. D. Arthington, C. Pratt, T. S. Edrington, R. C. Anderson, M. L. Galyean, S. C. Ricke, P. Crandall, and D. J. Nisbet. 2008. Citrus products decrease growth of E. coli O157:H7 and Salmonella Typhimurium in pure culture and in fermentation with mixed ruminal microorganisms in vitro. Foodborne Path. Dis. 5:621-627.

Callaway, T. R., J. A. Carroll, J. D. Arthington, T. S. Edrington, M. L. Rossman, M. A. Carr, N. A. Krueger, S. C. Ricke, P. Crandall, and D. J. Nisbet.

2011a. Escherichia coli O157:H7 populations in ruminants can be reduced by orange peel product feeding. J. Food Prot. 74:1917-1921.

Callaway, T. R., J.A. Carroll, J. D. Arthington, T. S. Edrington, R. C. Anderson, M. L Rossman, M. A. Carr, K. J. Genovese, S. C. Ricke, P. Crandall, and D. J. Nisbet. 2011b. Orange peel pellets can reduce Salmonella populations in ruminants. Foodborne Path. Dis. 8:1071-1075.

Callaway, T. R., S. E. Dowd, T. S. Edrington, R. C. Anderson, N. Krueger, N. Bauer, P. J. Kononoff, and D. J. Nisbet. 2010. Evaluation of the bacterial diversity in the rumen and feces of cattle fed diets containing levels of dried distiller’s grains plus sol-

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Fernando, S. C., H. T. Purvis, II, F. Z. Najar, L. O. Sukharnikov, C. R. Krehbiel, t. G. Nagaraja, B. A. Roe, and U. DeSilva. 2010. Rumen microbial population dynamics during adaptation to a high-grain diet. Appl. Environ. Microbiol. 76:7482-7490.

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VOLUME 2 ISSUE 1 SPECIAL ISSUE: Arkansas Association for Food Protection (AAFP) Conference CONFERENCE PROCEEDINGS* 4 The Story of the Arkansas Association for Food Protection (AAFP) M. Sostrin

6 A Team Approach for Management of the Elements of a Listeria Intervention and Control Program

J. N. Butts

15 Development of a Food Defense Workshop and Graduate Certificate in Food Safety and Defense for Working Professionals K. J. K. Getty

35 Development and Assessment of Success for Retail Food Safety Programming in Indiana R. H. Linton

43 ConAgra Foods’ Salmonella Chester Outbreak In Marie Callender’s Cheesy Chicken and Rice Catalyzing Change: Next Generation of Food Safety J. Menke-Schaenzer

46 Food Safety For a Diverse Workforce; One Size Does Not Fit All J. A. Neal, M. Dawson, J. M. Madera

REVIEWS* 25 Human Noroviruses and Food Safety K. E. Gibson and S. C. Ricke

56 Isolation and Initial Characterization of Plasmids in an Acetogenic Ruminal Isolate O. K. Koo, S. A. Sirsat, P. G. Crandall and S. C. Ricke

Introduction to Authors 69

Instructions for Authors

* Arkansas Association for Food Protection (AAFP) Conference, Enhancing Food protection From Farm to Fork, held on Sept. 28-29, 2011, Springdale, AR.

VOLUME 2 ISSUE 2 BRIEF COMMUNICATIONS 82

Evaluation of an Experimental Sodium Chlorate Product, With and Without Nitroethane, on Salmonella in Cull Dairy Cattle N. A. Krueger, T. S. Edrington, R. L. Farrow, R. Hagevoort, R. C. Anderson, G. H. Loneragan, T. R. Callaway, and D. J. Nisbet

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A Membrane Filtration Method for Determining Minimum Inhibitory Concentrations of Essential Oils S. J. Pendleton, R. Story, C. A. O’Bryan, P. G. Crandall, S. C. Ricke, and L. Goodridge

ARTICLES 94

Influence on Growth Conditions and Sugar Substrate on Sugar Phosphorylation Activity in Acetogenic Bacteria W. Jiang, R.S. Pinder, and J.A. Patterson

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

V. I. Chalova, I. Manolov, M. Nikolova, and L. Koleva

111 Transfer of Tylosin Resistance Between Enterococcus spp. During Continuous-Flow Culture of Feral or Domestic Porcine Gut Microbes

N. Ramlachan, R.C. Anderson, K. Andrews, R.B. Harvey, and D.J. Nisbet

121 Sugar Recovery from the Pretreatment/Enzymatic Hydrolysis of High and Low Specific Gravity Poplar Clones

A. C. Djioleu, A. Arora, E. M. Martin, J. A. Smith, M. H. Pelkki, and D. J. Carrier

132 Culture dependent molecular analysis of bacterial community of Hazaribagh tannery exposed area in Bangladesh

A. A. Maruf, M. M. Moosa, S. M. M. Rashid, H. Khan, and S. Yeasmin

139 Impact of By-product Feedstuffs on Escherichia coli O157:H7 and Salmonella Typhimurium in Pure and Mixed Ruminal and Fecal Culture in Vitro

T. R. Callaway, S. Block, K. J. Genovese, R. C. Anderson, R. B. Harvey, and D. J. Nisbet

Introduction to Authors 149 Instructions for Authors The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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VOLUME 2 ISSUE 3 ARTICLES 162

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

175 Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of Sweetgum A. C. Djioleu, E. M. Martin, M. H. Pelkki, and D. J. Carrier

187 Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus S. Datta, I. G. Shah, A. Akter, K. Fatema, T. H. Islam, A. Bandyopadhyay, Z. U.M. Khan, and D. Biswas

195 Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking Process J. R. Pittman, T. B. Schmidt, A. Corzo, T. R. Callaway, J. A. Carroll, and J. R. Donaldson

209 Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens And Food Spoilage Bacteria

N. Murali, G. S. Kumar-Phillips, N. C. Rath, J. Marcy, and M. F. Slavik

222 Prevalence of foodborne pathogens and effectiveness of washing or cooking in reducing microbiological risk of contaminated Red amaranth

Md. A. A. Mamun, H. A. Simul, A. Rahman, N. N. Gazi, and Md. L. Bari

Introduction to Authors 233 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

312

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VOLUME 2 ISSUE 4 BRIEF COMMUNICATIONS 275 Attachment of E. coli O157:H7 and Salmonella on Spinach (Spinacia oleracea) Using Confocal Microscopy

J. A. Neal, E. Cabrera-Diaz, and A. Castillo

ARTICLES 246

Developing an in vitro Method for Determining Feed Soluble Protein Degradation Rate by Mixed Ruminal Microorganisms W. L. Crossland, L. O. Tedeschi, T. R. Callaway, P. J. Kononoff, and K. Karges

253 Glucose and Hydrogen Utilization by an Acetogenic Bacterium Isolated from Ruminal Contents

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

280 Lack of Effect of Feeding Lactoferrin on Intestinal Populations and Fecal Shedding of Salmonella 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

291 Effect of Cooking on Selected Nutritional and Functional Properties of red amaranths Md. A. A. Mamun, R. Ara, H. U. Shekhar, A. T. M.A. Rahim, and Md. L. Bari

297 Evaluation of the Ruminal Bacterial Diversity of Cattle Fed Diets Containing Citrus Pulp Pellets

Broadway, 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

Introduction to Authors 315 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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We invite you to consider submitting your research and review manuscripts to AFAB. The journal serves as a peer reviewed scientific forum for to the latest advancements in bacteriology research on Agricultural and Food Systems which includes the following fields:

• • • • • • • • • • • • • • • •

Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control

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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 tables, 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 separate page after the title page. The abstract should provide a justification of your work, objectives, methods, results, discussion and implications of study or review findings . Your abstract must consist of complete 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 indexing. Ideally, these keywords should include significant words from the title.

Introduction The introduction should clearly present the foundation 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 objectives must be clearly stated in the final sentence of the last paragraphs of the introduction.

Materials and Methods 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 authors may need to provide a list of abbreviations

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)].

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent 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 commonly 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 experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, 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. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis 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.

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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 reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented 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 parentheses, 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 alphabetically 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 unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications 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 citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section.

Book Chapter: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication.

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 reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section.

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

Volume number:inclusive pages.

Inclusive pages of 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: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX.

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.

Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX.

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.

Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p.

Book: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number of pages.

Examples: Hungate, R. E. 1966. The rumen and its microbes Academic Press, Inc., New York, NY. 533 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 #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently 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. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p.

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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 experimental 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., Microsoft 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. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

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