AFAB Volume 3 Issue 3 by cielo shabatura

ISSN: 2159-8967 www.AFABjournal.com

Volume 3, Issue 3 2013

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EDITORIAL BOARD Sooyoun Ahn

W.K. Kim

University of Florida, USA

University of Georgia, USA

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

North Dakota State University, USA

Hae-Yeong Kim Kyung Hee University, South Korea Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 3 - 2013

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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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TABLE OF CONTENTS BRIEF COMMUNICATIONS 186 Vibrio Densities in the Intestinal Contents of Finfish from Coastal Alabama J.L. Jones, R.A. Benner Jr., A. DePaola, and Y. Hara-Kudo

ARTICLES 176

Antimicrobial Activity of Red Clover (Trifolium pratense L.) Extract on Caprine Hyper-Ammonia-Producing Bacteria M. D. Flythe, B. Harrison, I. A. Kagan, J. L. Klotz, G. L. Gellin, B. M. Goff, G. E. Aiken

230 Suitability of Various Prepeptides and Prepropeptides for the Production and Secretion of Heterologous Proteins by Bacillus megaterium or Bacillus licheniformis S. Saengkerdsub, R. Liyanage, J. O. Lay Jr.

REVIEW 195 Utility of Egg Yolk Antibodies for Detection and Control of Foodborne Salmonella P. Herrera, M. Aydin, S. H. Park, A. Khatiwara and S. Ahn

218 Potential for Dry Thermal Treatments to Eliminate Foodborne Pathogens on Sprout Seeds T. Hagger and R. Morawicki

Introduction to Authors 252 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors. Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 3 - 2013

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Antimicrobial Activity of Red Clover (Trifolium pratense L.) Extract on Caprine Hyper Ammonia-Producing Bacteria M. D. Flythe1,2, B. Harrison1, I. A. Kagan1,3 J. L. Klotz1,2, G. L. Gellin1, B. M. Goff3, G. E. Aiken1,3

USDA, Agricultural Research Service, Forage-Animal Production Research Unit; Lexington, Kentucky 40546 2 University of Kentucky, Department of Animal and Food Sciences, Lexington, Kentucky 40546 3 University of Kentucky, Department of Plant and Soil Sciences, Lexington, Kentucky 40546

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, nor exclusion of others that may be suitable.

ABSTRACT One of the inefficiencies in rumen fermentation is the catabolism of feed amino acids and peptides by hyper ammonia-producing bacteria (HAB). The HAB can be controlled through selective inhibition with antimicrobials. In vitro ammonia production by mixed goat rumen bacteria was inhibited by red clover (Trifolium pratense L.) phenolic extract. One component of the extract was the isoflavone, biochanin A. When the biochanin A concentration was 30 ppm, amino acid fermentation and ammonia production decreased. The effect of biochanin A was tested on a cultured caprine HAB isolate (Peptostreptococcus spp.). The growth of the HAB was not inhibited by biochanin A alone, even if the concentration was as much as 200 ppm. However, the addition of sterile rumen fluid (5%) caused growth inhibition at 2 ppm biochanin A. To determine what component in the rumen fluid acted synergistically with biochanin A, the growth experiment was repeated with either a mixture of volatile fatty acids (VFA) or the supernatant of the bacteriocinproducing Streptococcus bovis HC5 (5% v/v) in place of the rumen fluid. The combination of biochanin A and S. bovis supernatant caused growth inhibition, but VFA had no effect. These results are consistent with the hypothesis that biochanin A potentiates the activity of other heat-stable antibacterial compounds that are present in the rumen environment, and that the spectrum of activity could depend on the inhibitors present. Red clover extract and its components represent plant-based feed additives that could be used to control ammonia production in goats and other ruminants. Keywords: Ammonia, feed efficiency, ionophores, plant secondary metabolite, rumen Agric. Food Anal. Bacteriol. 3: 176-185, 2013

Correspondence: Michael D. Flythe, michael.flythe@ars.usda.gov Tel: +1 -859- 421-5699

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INTRODUCTION Most of the nitrogen in plants is incorporated into protein. However, non-protein nitrogen (NPN) is also present, and must be considered when the nutritional value of a feed is assessed. All animals can utilize amino acids, but ruminants are especially suited to utilize NPN, such as ammonia and urea, because the rumen flora can assimilate them into microbial protein (Polan, 1988). Unfortunately, rumen microorganisms also depolymerize feed protein, catabolize peptides, and deaminate amino acids. In many cases, the rate and extent of ruminal deamination exceeds assimilation, and the animal cannot absorb all excess NPN. This problem is both a nutritional inefficiency and a source of environmental pollution (Tedeschi et al., 2003). Many rumen bacteria are proteolytic, but the proteolytic bacteria are not necessarily responsible for amino acid deamination (Rychlik and Russell, 2000). The hyper ammonia-producing bacteria (HAB) are a phylogenetically diverse group of species, which are characterized by the ability to rapidly deaminate amino acids and produce ammonia. HAB species were first discovered in the rumen of dairy cattle in the 1980â&#x20AC;&#x2122;s (Russell et al. 1988, Chen and Russell 1989). Since that time, they have been isolated from deer (Atwood et al. 1998), sheep (Atwood et al., 1998; Eschenlauer et al., 2002; Wallace et al., 2003), and goats (Flythe and Andries, 2009). The rumen is both an organ and a complex ecological habitat (Hungate, 1960). However, it is also a natural fermentation, and to a certain degree, it can be manipulated as if it were an industrial fermentation (Russell and Rychlik, 2001). Antimicrobials can be added to ruminant diets to alter rumen fermentation via selective inhibition of the microorganisms that promote ammonia production, methanogenesis, and other wasteful processes (Van Nevel and Demeyer, 1977; Tedeschi et al., 2003). Many of the antimicrobial compounds used as feed additives are synthesized by microorganisms. For example, the ionophore monensin is produced by the soil bacterium, Streptomyces cinnamonensis (Van Nevel and Demeyer, 1977). However, numerous antimicrobial

compounds are also produced by plants, some constitutively (Osbourn, 2006) and some in response to biotic (Hammerschmidt, 1999) or abiotic stresses (Saviranta et al., 2009). Botanical antimicrobials shown to decrease rumen ammonia include pure compounds such as cinnamaldehyde and eugenol (Busquet et al., 2005; Cardozo et al., 2006), as well as mixtures of essential oils (McIntosh et al., 2003; Benchaar et al., 2008), and other types of plant extracts. This latter category includes a phenolic extract of red clover (Trifolium pratense), which inhibited the growth and ammonia production of the bovine HAB, Clostridium sticklandii (Flythe and Kagan, 2010). The primary components of the red clover extracted by Flythe and Kagan (2010) were isoflavones, one of which (biochanin A) was active against C. sticklandii. Other phenolic compounds have been found to decrease ammonia production by mixed rumen microorganisms in vitro (Getachew et al., 2009). These results indicate that phenolic compounds from legumes might be used to control ammonia production in the rumen. However, because the previous studies employed bacteria of bovine origin, the relevance to goat production is uncertain. Khan and colleagues (2011) showed that Egyptian clover (Trifolium alexandrinum) extracts exhibited a broad spectrum of activity against phylogenetically diverse human bacteria. The broad spectrum of activity suggests that clover extract might inhibit amino acid degradation by the diverse rumen microbial community. The experiments described here were initiated to determine: 1) the effects of red clover phenolic extract and biochanin A on in vitro ammonia production by mixed rumen bacteria from goats, and 2) that biochanin A is one of the inhibitory compounds by assessing its effect on a pure caprine HAB.

MATERIALS AND METHODS Animals and diet The University of Kentucky Institutional Animal Care and Use Committee approved all animal procedures (protocol number 2009-0520). Kiko goat weth-

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ers (n=8, 2 y, 40 to 50 kg) were maintained at the University of Kentucky’s Research Farm. Mixed rumen microorganisms were obtained from non-fistulated goats, as described below. Rumen fluid for sterile additions to media was obtained from a rumenfistulated goat in the same herd. Access to pasture was unrestricted. The botanical composition of the pasture was estimated at the beginning of the study by observing the predominant species at 1 m intervals along 37 m transects. A total of 6 transects were used and were spaced approximately 11 m apart. The predominant species were: tall fescue (Lolium arundinaceum (Schreb.) Darbysh., 44.4%), orchardgrass (Dactylis glomerata L., 14.9%), white clover

mg KH2PO4, 480 mg NaCl, 480 mg Na2SO4, 64 mg CaCl2·2H2O, 100 mg MgSO4·7H2O, 600 mg cysteine hydrochloride, trace minerals and vitamins as previously described (Russell et al., 1988). An initial pH of 6.5 was obtained by adding NaOH. The broth was autoclaved (121˚C, 103 kPa, 20 min) to remove O2 and cooled under O2-free CO2. The buffer (4.0 g Na2CO3) was added before dispensing and autoclaving again for sterility. The amino acid substrate, Casamino acids (Fisher BioReagents, Fair Lawn, NJ), was prepared separately and added aseptically (15 mg mL-1 final concentration). Streptococcus bovis HC5 was obtained from the culture collection of James B. Russell, Cornell University, Ithaca, NY. It is a

(Trifolium repens L., 14.9%), Kentucky bluegrass (Poa pratensis L., 8.9%), red clover (Trifolium pratense L., 3.3%), bermudagrass (Cynodon dactylon (L.) Pers., 1.4%) and broad-leaf weeds (12.6%). The goats were supplemented with 1.0 kg head-1d-1 orchardgrass/ alfalfa hay (18% crude protein, as fed), and 0.25 kg head-1d-1 supplement (16% crude protein; Kalmbach Feeds, Upper Sandusky, OH). Water and a mineral mixture (Southern States Cooperative, Richmond, VA) were provided free choice. The composition of the mineral mixture, as reported by the manufacturer, was (crude protein 1.75%, calcium 22.0%, phosphorus 5.0%, NaCl 21.0%, magnesium 3.0%, sulfur 0.25%, iodine 40 ppm, copper 400 ppm, cobalt 15 ppm, selenium 32 ppm, zinc 3000 ppm, manganese 2000 ppm, vitamin A 300000 IU/lb., vitamin D 25000 IU/lb., vitamin E 200 IU/lb). The supplements were non-medicated. The herd had never received ionophores or antibiotic feed supplements.

bacteriocin-producing strain that was isolated from the bovine rumen (Mantovani and Russell, 2002). The Streptococcus bovis medium was based on previously described media (Mantovani and Russell, 2002), and contained (per liter): 240 mg K2HPO4, 240 mg KH2PO4, 480 mg NaCl, 480 mg (NH4)2SO4, 64 mg CaCl2·2H2O, 100 mg MgSO4·7H2O, 600 mg cysteine hydrochloride, 0.5 g yeast extract, 1.0 g Trypticase (Fisher BioReagents, Fair Lawn, NJ). Glucose was prepared as an anaerobic stock and added aseptically (4.0 mg mL-1 final concentration).

In vitro ammonia production with red clover extract or biochanin A

The isolation and characterization of the caprine HAB culture (Peptostreptococcus spp. BG1) used in these experiments were reported previously (Fly-

Three goats (described above) were sampled for each experiment. The rumen samples were obtained by gastric intubation. Briefly, a speculum was inserted into the mouth, and a sterilized tube (0.25 inch O.D., Tygon®, U.S. Plastics, Lima, OH) was inserted through the speculum. A sample (5-8 ml) of rumen fluid was aspirated into the tube with a handheld pump (Drummond, Broomall, PA) that was connected to the distal end of the tube. The tube was removed and the sample was immediately ex-

the and Andries 2009). Briefly, BG1 is most closely related to Peptostreptococcus anaerobius. It grows with amino acids as the sole energy source and produces ammonia and VFA as products. The HAB medium contained (per liter): 240 mg K2HPO4, 240

pressed into a Hungate tube, sealed, sparged with CO2, and transported to the laboratory under a CO2 headspace. The microorganisms in the rumen samples were harvested by centrifugation (25600 x g, 10 min, 27˚C), then washed and re-suspended in HAB

Bacterial strains and media composition

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medium. This suspension was diluted to an optical density (OD, absorbance 600 nm) of 1.0, using a Biowave II spectrophotometer (Biochrom, Cambridge, UK). The suspension was used to inoculate (10%) enrichment tubes containing HAB medium with Casamino Acids (15 mg mL-1). The enrichment tubes were amended with either red clover extract (obtained from plants allowed to wilt 24 h, described by Flythe and Kagan 2010) or biochanin A (Indofine, Hillsborough, NJ), as indicated. Biochanin A concentrations in the red clover extracts were calculated from the concentrations determined by high-performance liquid chromatography for extracts from the same tissue (Flythe and Kagan, 2010). Controls for each

during transport to the laboratory. The feed particles and microorganisms were removed by centrifugation (25600 x g, 5 min, 27˚C). The supernatant was transferred to a serum bottle and sparged with O2-free CO2 until the serum bottle was stoppered, sealed and autoclaved (121˚C, 103 kPa, 20 min). The sterile rumen fluid was not inhibitory to the HAB in the absence of biochanin A (data not shown). VFA were added to the HAB medium to emulate the concentrations used in typical nonselective rumen fluid media (Caldwell and Bryant, 1966). It contained (per liter): acetic acid (1.26 mL), propionic acid (0.45 mL), butyric acid (0.22 mL valeric acid (0.07 mL), isovaleric acid (0.07 mL), isobutyric acid (0.07 mL),

treatment were included. The enrichment tubes were incubated (39˚C, 48 h), and sampled at 0 and 48 h. Supernatant samples were clarified by centrifugation and frozen (-20˚C). The supernatant samples were later thawed and the ammonia concentrations were determined by the phenolic acid/hypochlorite method (Chaney and Marbach, 1962).

2-methylbutyric acid (0.07 mL). The initial pH was adjusted to 6.5 by addition of NaOH. The VFA additions were not inhibitory to the caprine HAB at this pH value (data not shown). Streptococcus bovis HC5 was grown (16 h) in Streptococcus bovis medium as previously described (Mantovani et al., 2001). The supernatants were clarified by centrifugation, and added to HAB medium with a tuberculin syringe at 1, 2.5, 5, 7.5, and 10% volume to volume (v/v). S. bovis did not grow in the HAB medium because there was no fermentable carbohydrate. The 5% v/v amendment was used to test synergy with biochanin A because it was subinhibitory to all of the caprine HAB strains (data not shown).

Pure culture growth experiments The HAB medium was amended with Casamino acids (15 mg mL-1). Overnight Peptostreptococcus spp. BG1 culture was used as the inoculum (10%). All incubations were conducted in a shaking incubator (150 rpm, 39˚C). Growth was determined by OD (absorbance 600 nm) at 16, 24 and 48 h. The treatments included biochanin A with or without: sterile rumen fluid, VFA, or the supernatant of a bacteriocin-producing Streptococcus bovis. Biochanin A was dissolved in acetone and added with a Hamilton syringe to achieve the concentrations indicated (200 to 0.2 ppm). The acetone carrier was not inhibitory when less than 200 µL was added to 10 mL media (data not shown). Sterile rumen fluid was added to the tubes when indicated (10% v/v). The rumen fluid (750 mL) was obtained from a rumen-fistulated goat that was maintained in the same herd as the goats sampled for mixed rumen microorganisms. Atmospheric oxygen was excluded by endogenous gas production

Replication and statistical analysis The in vitro ammonia production experiments with mixed rumen microorganisms were performed in triplicate with rumen fluid obtained from three different goats. The means are reported. Statistical differences from the controls (the same mixed rumen microorganism suspensions with no biochanin A or clover extract) were determined with paired Student’s t-tests. P values less than 0.05 were considered significant, and indicated with asterisks on the figures. Pure culture growth experiments were performed in triplicate, and there was no variation in the results that are reported.

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Figure 1. Effect of red clover (Trifolium pratense) phenolic extract on ammonia production by mixed microorganisms from the rumen of a goat (n=3). The 48 h enrichments were performed (39°C) in HAB medium with Casamino acids (15 mg mL-1) as the substrate. Clover extract was added at 0 h to achieve the biochanin A concentration indicated on the horizontal axis. The difference between initial ammonia concentrations and the 48 h ammonia concentrations are shown on the vertical axis. Means of triplicate experiments are shown. Differences from the control (no addition) were determined by Student’s t-tests, and the asterisk indicates P < 0.05.

Ammonia production (mM)

0 00

-10

Clover extract (ppm Biochanin A) RESULTS Effects of red clover extract and biochanin A on ammonia production by mixed rumen microorganisms When mixed rumen microorganisms from goats were used to inoculate media rich in free amino acids (Casamino acids, 15 mg mL-1), but lacking red clover extract, the average ammonia concentration was 39 mM after 48 h (Figure 1). The addition of red clover phenolic extract decreased the ammonia concentrations in tubes inoculated with the same mixed rumen microorganisms. However, the ammonia concentration was not significantly less than the control until enough extract was added to achieve a 180

biochanin A concentration of 20 ppm (70 µM). At 20 ppm biochanin A, the mean ammonia concentration was 17 mM in the amino acid enrichments, and these concentrations were significantly less than the controls (P < 0.05). The experiment was repeated with mixed rumen microorganisms from the same goats, but the media were amended with pure biochanin A rather than red clover extract (Figure 2). The mean ammonia concentration in the controls after 48 h was 39 mM. When ≤ 15 ppm biochanin A was added, the ammonia concentration was not different from the controls. When 30 ppm biochanin A was added the mean ammonia concentration was 20 mM. These concentrations were significantly less than those of the controls (P < 0.05).

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Figure 2. Effect of biochanin A on ammonia production by mixed microorganisms from the rumen of a goat (n=3). The 48 h enrichments were performed (39°C) in HAB medium with casamino acids (15 mg mL-1) as the substrate. Biochanin A was added at 0 h to achieve the concentration indicated on the horizontal axis. The difference between initial ammonia concentrations and the 48 h ammonia concentrations are shown on the vertical axis. Means of triplicate experiments are shown. Differences from the control (no biochanin A added) were determined by Student’s t-tests, and the asterisk indicates P < 0.05.

Ammonia production (mM)

00 -10

Biochanin A (ppm) Growth inhibition of a caprine HAB culture by biochanin A, sterile rumen fluid, VFA, and the supernatant of a bacteriocin-producer When the caprine ruminal HAB, BG1, was grown in HAB media with amino acids (Casamino acids, 15 mg mL-1) as a substrate, the cultures reached stationary phase in less than 48 h, as determined by OD (data not shown). The addition of biochanin A (0.2, 2.0, 20, or 200 ppm) had no effect on viable cell number at 48 h when no rumen fluid was included (Figure 3). The addition of sterile rumen fluid (10% v/v) from a rumen-fistulated goat did not prevent growth when no biochanin A was present. However, the growth of BG1 was inhibited by the combination of

sterile rumen fluid and 2, 20, or 200 ppm biochanin A. To elucidate the antimicrobial agent in the rumen fluid, a VFA mixture and a bacteriocin were tested. Neither the VFA mixture nor the addition of Streptococcus bovis HC5 supernatant (5% v/v) inhibited the growth in the absence of biochanin A. The addition of the VFA mixture to culture tubes did not change the inhibitory concentration of biochanin A. However, BG1 did not grow in the presence of S. bovis HC5 supernatant when the biochanin A concentration was 2 ppm or greater. The S. bovis HC5 supernatant inhibited the culture when it was included at greater than 10% v/v (data not shown). The pure culture experiments were performed three times with identical results.

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Figure 3. Effect of biochanin A on the growth of the hyper ammonia-producing bacterium, Peptostreptococcus spp. BG1. The culture was grown in HAB medium with Casamino acids (15 mg mL-1) as the substrate. Biochanin A was added to achieve the concentration indicated on the horizontal axis. The medium was supplemented with no addition (orange bars), rumen fluid (green bars), a volatile fatty acid mixture (hatched bars) or the supernatant of a bacteriocin-producing Streptococcus bovis (blue bars). The stationary phase optical density was taken after 24 h incubation (39°C). The experiment was repeated three times with identical results.

0.7

Optical Density (600 nm)

0.6 0.5 0.4 0.3 0.2 0.1 0 0

200

Biochanin A conc. (ppm) DISCUSSION Previous work showed that red clover extract, as well as biochanin A, a major component of red clover extract, inhibited the growth and ammonia production of Clostridium sticklandii SR (Flythe and Kagan, 2010). Strain SR is the most commonly used model for HAB (Chen and Russell, 1989; Van Kessel and Russell, 1992; Krause and Russell, 1996; Rychlik and Russell, 2002; Attwood et al., 2006, Xavier and Rus-

Gram-negative bacteria (e.g. Fusobacteria) can also occupy amino acid-fermenting niches (Attwood et al., 1998; Russell, 2005). Structural differences between these taxa cause differences in susceptibility to antimicrobials, which raised the concern that red clover phenolic extract might have a spectrum of activity too narrow to control ammonia production in the rumen. In the current study, mixed rumen microorganisms produced approximately 40 mM ammonia from free

sell, 2009; Flythe, 2009). HAB is a functional category, or guild, rather than a taxon, and includes phylogenetically diverse members. C. sticklandii is a member of Phylum Firmicutes, and has a typical Grampositive cell envelope (Paster et al.,1993). However,

amino acids. Clover extract or pure biochanin A reduced the final ammonia concentration by half. This indicates that red clover phenolic extract was effective against the HAB that were present in the goats at the time of sampling. Biochanin A did not inhibit

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the growth of the caprine HAB in defined media, but isolate BG1 was inhibited by biochanin A concentrations â&#x2030;Ľ 2 ppm when the broth was amended with sterile rumen fluid. These results suggest that biochanin A potentiated, or acted synergistically with, one or more heat-stable, antimicrobial compounds present in the rumen fluid. Others have reported synergistic interactions between biochanin A and other compounds. Morel and colleagues (2003) showed that biochanin A and other isoflavones, although not active against Staphylococcus aureus, potentiated the antibacterial activity of other compounds. The minimum inhibitory concentration (MIC) of berberine against S. aureus

impacts the number and activity of HAB (Rychlik and Russell, 2000). The caprine HAB was not inhibited by biochanin A, even when a mixture of VFA was added to the media. However, one of the HAB strains was inhibited by a combination of biochanin A and a small amount of culture supernatant from a bacteriocin-producing Streptococcus bovis. It is possible that the inhibition of ammonia production by mixed rumen microorganisms was due to a synergy between the isoflavone and native bacteriocins. Furthermore, bacteriocins are membrane-associated, and can be transmitted from both producing cells and sensitive cells, to other sensitive cells (Xavier and Russell, 2009). We propose that a sub-inhibitory

decreased 94% in the presence of the isoflavones. Subsequent work revealed that biochanin A was the most effective of nine phenolic compounds at inhibiting ethidium bromide efflux from Mycobacterium smegmatis cells (Lechner et al., 2008), thus decreasing the MIC of ethidium bromide. Biochanin A also has been found to have a synergistic effect on the activity of the antibiotic ciprofloxacin (Liu et al., 2011). A variety of antimicrobial compounds are present in the rumen. Some of these compounds, like phenolics, are present in feed, but the rumen microorganisms produce other antimicrobial compounds. This latter category includes fermentation acids, which are sometimes called short chain fatty acids or VFA. Acetic acid and other VFA are common bacterial products, and they are known to have antimicrobial activity in fermented foods (Sengun and Karabiyikli 2011), feeds (Flythe and Russell, 2006; Van Immerseel et al., 2006), and industrial fermentations (Dharmagadda et al., 2010). The mechanism of action is intracellular accumulation of the anion, which has been shown to disrupt osmotic homeostasis in amino acid-fermenting bacteria (Flythe and Russell, 2006). The rumen also contains bacteriocins, which are small, ribosome-synthesized peptides. Streptococ-

concentration of bacteriocin was retained on the mixed microorganisms that were separated from the rumen digesta. Then the antimicrobial activity of the bacteriocin was potentiated by the addition of biochanin A.

cus bovis (Mantovani et al., 2001), Butyrivibrio fibrisolvens (Rychlik and Russell, 2002) and Ruminococcus albus (Chen et al., 2004) are among the rumen bacteria that produce bacteriocins. It has been estimated that the presence of bacteriocins in vivo

CONCLUSIONS The host is the most important factor that determines the composition of gastrointestinal microbial community of a goat (Shi et al., 2007). Our results demonstrate that red clover phenolic extract and one of its major components, biochanin A, inhibited ammonia production by uncultivated, mixed goat rumen microorganisms. Furthermore, a hyper ammonia-producing bacterium of caprine origin was inhibited by biochanin A. The mechanism of action appeared to be a synergistic interaction between biochanin A and heat-stable rumen components. Therefore, the spectrum of activity and minimum inhibitory concentrations are likely to be dependent on compounds present in the rumen environment.

ACKNOWLEDGEMENTS The Agricultural Research Service, USDA, supported this work. The authors thank Adam Barnes and Tracy Hamilton for technical assistance.

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Eschenlauer, S.C., N. McKain, N.D. Walker, N.R. McEwan, C.J. Newbold, and R.J. Wallace. 2002. Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen. Appl. Environ. Microbiol. 68:4925-4931. Flythe, M.D., and J.B. Russell. 2006. Fermentation acids inhibit amino acid deamination by Clostridium sporogenes MD1 via a mechanism involving a decline in intracellular glutamate rather than protonmotive force. Microbiology 152:2619-2624. Flythe, M.D. 2009. The antimicrobial effects of hops (Humulus lupulus L.) on ruminal hyper ammonia-pro-

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ducing bacteria. Lett. Appl. Microbiol. 118:242-248. Flythe, M., and K. Andries. 2009. The effects of monensin on amino acid catabolizing bacteria isolated from the Boer goat rumen. Small Rum. Res. 81:178–181. Flythe, M., and I. Kagan. 2010. Antimicrobial effect of red clover (Trifolium pratense) phenolic extract on the ruminal hyper ammonia-producing bacterium, Clostridium sticklandii. Curr. Microbiol. 61:125-131. Getachew, G., A.M. Dandekar, W. Pittroff, E.J. DePeters, D.H. Putnam, S. Goyal, L. Teuber, and S. Uratsu. 2009. Impacts of polyphenol oxidase enzyme expression in transgenic alfalfa on in vitro gas production and ruminal degradation of protein, and nitrogen release during ensiling. Anim. Feed Sci. Technol. 151:44-54. Hammerschmidt, R. 1999. Phytoalexins: What have we learned after 60 years? Ann. Rev. Phytopathol. 37:285-306. Hungate, R.E. 1960. Symposium: selected topics in microbial ecology. I. Microbial ecology of the rumen. Bacteriol. Rev. 24:353-364. Khan, A.V., Q.U. Ahmed, I. Shukla, and A.A. Khan. 2012. Antibacterial activity of Trifolium alexandrinum Linn. leaves extracts against pathogenic bacteria causing tropical diseases. Asian Pac. J. Trop.

Dharmagadda, V. S. S., S. E. Nokes, H. J. Strobel, and M. D. Flythe. 2010. Investigation of the metabolic inhibition observed in solid-substrate cultivation of Clostridium thermocellum on cellulose. Bioresource Technol. 101:6039-6044.

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Lechner, D., S. Gibbons, and F. Bucar. 2008. Plant phenolic compounds as ethidium bromide efflux inhibitors in Mycobacterium smegmatis. J. Antimicrob. Chemother. 62:345-348. Liu, G., J.C. Liang, X.L. Wang, Z.H. Li, W. Wang, N. Guo, X.P. Wu, F.G. Shen, M.X. Xing, L.H. Liu, L. Li, M.Y. Liu, and L. Yu. 2011. In vitro synergy of biochanin A and ciprofloxacin against clinical isolates of Staphylococcus aureus. Molecules 16:6656-6666. Mantovani, H.C., D.K. Kam, J.K. Ha, and J.B. Russell. 2001. The antibacterial activity and sensitivity of Streptococcus bovis strains isolated from the rumen of cattle. FEMS Microbiol. Ecol. 37:223-229. Mantovani, H.C., and J.B. Russell. 2002. Bovicin

bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production. FEMS Microbiol. Ecol. 32:121-128. Rychlik, J.B., and J.B. Russell. 2002. Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production. Appl. Environ. Microbiol. 86:1040-1046. Saviranta, N.M.M., R. Julkunen-Tiitto, E. Oksanen, and R. O. Karjalainen. 2010. Red clover (Trifolium pratense L.) isoflavones: root phenolic compounds affected by biotic and abiotic stress factors. J. Sci. Food. Agric. 90:418-423. Sengun, I. Y., and S. Karabiyikli. 2011. Importance of acetic acid bacteria in food industry. Food Control

HC5, a bacteriocin from Streptococcus bovis HC5. Microbiology 148:3347-3352. McIntosh, F., P. Williams, R. Losa, R.J. Wallace, D. Beever, and C. Newbold. 2003. Effects of essential oils on ruminal microorganisms and their protein metabolism. Appl. Environ. Microbiol. 69:5011-5014. Morel, C., F.R. Stermitz, G. Tegos, and K. Lewis. 2003. Isoflavones as potentiators of antibacterial activity. J. Agric. Food Chem. 51:5677-5679. Osbourn, A.E. 1996. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821-1831. Paster, B.J., J.B. Russell, C.M. Yang, J.M. Chow, C.R. Woese, and R. Tanner. 1993. Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum sp. nov. Int. J. Syst. Bacteriol. 43:107-110. Polan, C. 1988. Update: dietary protein and microbial protein contribution. J. Nutr. 118:242-248. Russell, J.B. 2005. Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth. Anaerobe 11:177-184. Russell, J.B., H.J. Strobel, and G.J. Chen. 1988. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia produc-

22:647-656. Shi, P.J., K. Meng, Z.G. Zhou, Y.R. Wang, Q.Y. Diao, and B. Yao. 2007. The host species affects the microbial community in the goat rumen. Lett. Appl. Microbiol. 46:132-135. Tedeschi, L.O., D.G. Fox, and T. P. Tylutki. 2003. Potential environmental benefits of ionophores in ruminant diets. J. Environ. Qual. 32:1591-1602. Van Immerseel F, J.B. Russell, M.D. Flythe, I. Gantois, L. Timbermont, F. Pasmans , F. Haesebrouck, and R. Ducatelle. 2006. The use of organic acids to combat Salmonella in poultry: A mechanistic explanation of the efficacy. Avian Pathol. 35: 182-188. Van Kessel, J.S., and J.B. Russell. 1992. Energetics of arginine and lysine transport by whole cells and membrane vesicles of strain SR, a monensin-sensitive ruminal bacterium. Appl. Environ. Microbiol. 58:969-975. Van Nevel, C.J., and D.I. Demeyer. 1977. Effect of monensin on rumen metabolism in vitro. Appl. Environ. Microbiol. 34:251-257. Wallace, R.J., N. McKain, N.R. McEwan, E. Miyagawa, L.C. Chaudhary, T.P. King, N.D. Walker, J.H.A. Apajalahti, and C.J. Newbold. 2003. Eubacterium pyruvativorans sp. nov., a novel non-saccharolytic anaerobe from the rumen that ferments pyruvate and amino acids, forms caproate and utilizes acetate and

tion. Appl. Environ. Microbiol. 54:872-877. Russell, J.B., and J.L. Rychlik. 2001. Factors that alter rumen microbial ecology. Science 11:1119-1122. Rychlik, J.L., and J.B. Russell. 2000. Mathematical estimations of hyper-ammonia producing ruminal

propionate. Int. J. Syst. Evol. Microbiol. 53:965-970. Xavier, B., and J.B. Russell. 2009. The ability of nonbacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria. Anaerobe 15:168-172.

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BRIEF COMMUNICATION Vibrio Densities in the Intestinal Contents of Finfish from Coastal Alabama J.L. Jones1*, R.A. Benner Jr.1, A. DePaola1, and Y. Hara-Kudo2 FDA, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, Dauphin Island, AL 2 National Institute of Health Sciences, Division of Microbiology, Setagayaku, Tokyo 158 8501, Japan

ABSTRACT Vibrio vulnificus, Vibrio parahaemolyticus and Vibrio cholerae, are human pathogens ubiquitous in the marine and estuarine environments. Correlation between abundance of these pathogens and increased water temperature is well established, but little is known about their environmental persistence. Previous studies have identified finfish intestines as a potential reservoir of V. vulnificus; however, the data for other pathogenic Vibrios is sparse. The objective of this study was to simultaneously enumerate the three Vibrio spp. of greatest human health concern in finfish intestines collected from the Gulf of Mexico and estuarine sites in Mobile Bay, Alabama. V. vulnificus, V. parahaemolyticus, and V. cholerae levels in fish intestines were enumerated using a microtiter plate most probable number (MPN)-real-time polymerase chain reaction (Rti-PCR) method. Of the 21 finfish samples examined, 62%, 76%, and 19% had detectable levels (≥3 MPN/g) of V. vulnificus, V. parahaemolyticus and V. cholerae, respectively. The highest levels of V. vulnificus (7.63 log MPN/g), V. parahaemolyticus (7.97 log MPN/g), and V. cholerae (4.58 log MPN/g) were found in sheepshead (Archosargus probatocephalus) collected from estuarine sites. There was a greater detection frequency of all three organisms in the estuarine samples, compared to the Gulf of Mexico samples; V. cholerae was only detected in the estuarine samples. Additionally, the levels of V. vulnificus and V. parahaemolyticus were significantly higher in the estuarine samples. This is the first report for simultaneous enumeration of V. vulnificus, V. parahaemolyticus, and V. cholerae in their environmental reservoir of finfish intestines. Keywords: Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae, real-time PCR, MPN, CPC+, finfish, Alabama, intestinal contents Agric. Food Anal. Bacteriol. 3: 186-194, 2013

Correspondence: Jessica L. Jones, Jessica.Jones@fda.hhs.gov Tel: +1 -251-406-8136

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INTRODUCTION Vibrio spp. are ubiquitous in the marine and estuarine environments (Chakraborty et al., 1997). Some of these species, predominately V. vulnificus, V. parahaemolyticus and V. cholerae, are pathogenic to humans (Scallan et al., 2011). The main vector of seafood-related Vibriosis in the United States is the consumption of shellfish, particularly oysters (Iwamoto et al., 2010). Levels of pathogenic Vibrio spp. have been frequently measured in oysters and the environment (Baross and Liston, 1970; Colwell et al., 1977; DePaola et al., 2003; Johnson et al., 2010; Motes et al., 1998). However, when environmental

water temperatures are unfavorable for Vibrio proliferation. However, the data for other pathogenic Vibrios is sparse. In a recent report by our group (Jones et al., 2012), we reported levels of V. vulnificus and V. parahaemolyticus found in fish intestine samples as determined by multiple molecular detection methods. However, in that study, we did not correlate reported levels to species of fish, sampling location, or associated environmental data. These correlations, in conjunction with the previously unreported levels of V. cholerae in the same finfish intestine samples, are the basis of the current report. As such, the objective of this study was to simultaneously enumerate the three Vibrio spp. of greatest

conditions are unfavorable for Vibrio spp. to be detected in oysters, the question of how they persist arises. One theory is that the Vibrios survive in a reservoir such as sediment or finfish intestines. While many studies have been conducted on the microbiota of fish intestines, they have largely focused on aquacultured fish of commercial or research importance (Cantas et al., 2012; Ringo and Olsen, 1999; Silva et al., 2011). The limited available studies conducted on wild fish populations are mainly focused on the microbial diversity within the fish gut (Aiso et al., 1968; Smriga et al., 2010). In some of these studies, Vibrio spp. were identified among the dominant flora (Aiso et al., 1968; Silva et al., 2011; Smriga et al., 2010). A few previous studies have specifically examined finfish intestines for occurrence and/or densities of human pathogenic Vibrios, specifically V. parahaemolyticus, V. alginolyticus, and V. cholerae (Baross and Liston, 1970; DePaola et al., 1994; Jaksic et al., 2002; Senderovich et al., 2010; Sudha et al., 2012); however, only a few studies have enumerated V. vulnificus and V. parahaemolyticus (DePaola et al., 1994; Noorlis et al., 2011). The findings of these previous studies and personal observations (Jones et al., 2012) support the theory that fish guts may be a reservoir for pathogenic Vibrios, but

human health concern in finfish intestines during a period of cooler weather (September to December). This time period was selected because Vibrio infections are rarely reported during these months and detection of pathogenic Vibrios in finfish intestines would further support the theory that this is an important reservoir for V. vulnificus and V. parahaemolyticus while providing preliminary evidence of finfish intestines as a reservoir for V. cholerae. Additionally, insights into environmental factors influencing these levels were examined.

no study has simultaneously enumerated the three Vibrio species of greatest human health concern. Previous studies have clearly identified finfish intestine as a potential reservoir of V. vulnificus (DePaola et al., 1994) during times when surrounding

eromorus cavalla), pin fish (Lagodon rhomboides), red snapper (Lutjanus campechanus), red drum (Sciaenops ocellatus), sea catfish (Arius felis), sheepshead (Archosargus probatocephalus), and Spanish mackerel (Scomberomorus maculates). Half of the

MATERIALS AND METHODS Fish sampling Sampling locations in and around Mobile Bay, Alabama, are identified in Figure 1. Fish were collected by rod and reel between the months of September and December 2008 from three sampling locations: the Gulf of Mexico, Mobile Bay, and Fowl River. Ten finfish species were examined during this study: Atlantic spadefish (Chaetodipterus faber), blue fish (Pomatomus saltatrix), blue runner (Caranx crysos), crevalle jack (Caranx hippos), king mackerel (Scomb-

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Figure 1. Satellite photograph of Mobile Bay, Alabama. Approximate collection sites are noted with yellow stars and site names.

fish species were represented by a single sample (Atlantic spadefish, blue fish, crevalle jack, king mackerel, and Spanish mackerel). Of the species sampled multiple times, only sheepshead were caught at all locations.

Fish intestine preparation for bacterial analysis The entire intestinal tract (posterior to the stomach) was aseptically removed and contents were massaged into a sterile bag. Intestinal contents were diluted 1:10 with phosphate buffered saline 188

(7.65 g sodium chloride, 0.724 g anhydrous disodium phosphate, 0.21 g potassium phosphate, pH 7.4; PBS) and homogenized in a Pulsifier® (Microgen Bioproducts, Camberley, Surrey, United Kingdom) for 30 sec. Serial ten-fold dilutions were made in PBS.

Vibrio enumeration A modified MPN-real-time PCR (Rti-PCR) format was used to enumerate V. vulnificus, V. parahaemolyticus, and V. cholerae. In microtiter plates, 180 µL alkaline peptone water (1% peptone, 1% NaCl, pH 8.5±0.2; APW) were inoculated in triplicate with 20 µL

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of serially diluted samples for a three-“tube” MPN. The microtiter plates were incubated for 18 to 24 h at 35°C. After incubation, an aliquot from each turbid well was tested for V. vulnificus, V. parahaemolyticus, and V. cholerae using the BAX® Real-Time PCR Kit using the manufacturer’s protocol (DuPont Qualicon, Wilmington, DE).

Identification of interfering microflora Interfering microflora are those that produce similar colonies on culture media. To identify these organisms from Vibrio-specific selective medium, aliquots of serially diluted samples were spread onto CPC+ agar. Plates were incubated for 18 to 24 h at 35°C. Individual colonies were selected based on their typical morphology (flat and yellow or flat with a yellow halo). Colonies were streaked for isolation on Marine Agar (Difco, Sparks, MD). Once a purified isolate was obtained, a single colony was inoculated into Marine Broth (Difco) and incubated at 35°C for 48 h. A 100 µL aliquot was removed, heated to 100°C for 10 min, and then put on ice. This was used as template in PCR for 16S rDNA amplification as described below.

attle, WA). Overlapping regions of the edited forward sequences and reverse complement of reverse sequences were aligned to obtain a full sequence (~1400 bp). Organisms were identified by a BLAST (nt) search of the full sequence. A phylogenetic tree was constructed in MegAlign (Lasergene Core Suite, DNASTAR, Madison, WI) using clustal W alignment. Sequences were submitted to GenBank under accession numbers JX999943-JX999946 and JX999948JX999960.

Data analysis Rti-PCR observations were recorded as positive or negative for each well in an MPN series for each Vibrio tested. These observations were used for MPN estimates following standard methods (Blodgett, 2010). MPN estimates were log transformed and Student’s t-test was used to determine significant differences between observed levels, while Pearson’s correlation coefficient was used to determine correlations between Vibrio levels and temperature or salinity.

RESULTS AND DISCUSSION Isolate identification by 16S rDNA sequence PCR conditions were as previously described for amplification of bacterial 16S rDNA (DeLong, 1992). PCR products were examined by 2% agarose gel electrophoresis at a constant voltage of 100V. PCR products from isolates producing an amplicon of the appropriate size were purified using QiaQuick (Qiagen, Valencia, CA). Concentrations of the purified DNA were determined using a Nanodrop spectrophotometer (Thermo Fisher, Pittsburg, PA). Each

Of the 21 finfish samples, 13 (62%), 16 (76%), and 4 (19%) had detectable levels (≥3 MPN/g) of V. vulnificus, V. parahaemolyticus, and V. cholerae, respectively. The highest levels of Vibrios detected were 7.63, 7.97, and 4.58 log MPN/g of V. vulnificus, V. parahaemolyticus, and V. cholerae, respectively (Figure 2). While these rates of detection and levels for V. vulnificus and V. parahaemolyticus were provided in our previous report (Jones et al., 2012), results were not presented in relation to the origin of samples (harvest location, fish species, or environmental parameters at time of collection). This report details

purified PCR product was sent to MCLAB (South San Francisco, CA) for two sequencing reactions, one using the forward primer and the other using the reverse primer for PCR. Chromatographs were viewed and manually edited using FinchTV (Geospiza, Se-

those connections to provide appropriate perspective to the observed data. The current report also includes the first documentation of V. cholerae levels in finfish intestines, to our knowledge. In this study, all of the fish collected from estua-

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Figure 2. Levels of V. vulnificus, V. parahaemolyticus, and V. cholerae in finfish intestines. Water salinity (dark blue circles) and temperature (yellow diamonds) at time of collection are plotted on the secondary Y-axis. Species of fish (along with identification number), collection site, and collection date are provided on the X-axis.

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rine environments (Mobile Bay and Fowl River sites) were positive for V. vulnificus and V. parahaemolyticus; this is slightly higher than a previous report of an 82% detection rate of V. vulnificus (DePaola et al., 1994). The only samples with V. vulnificus and V. parahaemolyticus levels below the limit of detection were collected at the Gulf of Mexico site with high salinities (>30 ppt). These results are consistent with earlier investigations that found 10 to 12% of fish from full oceanic salinity (>30 ppt) sites were positive for V. vulnificus or V. parahaemolyticus (DePaola et al., 1994; Jaksic et al., 2002). As the current study reports similar detection rates of V. vulnificus and V. parahaemolyticus in fish intestine samples as previ-

mal (25.7°C) and salinity (10.3 ppt) was slightly below optimal for V. parahaemolyticus, but still within the optimal range for V. vulnificus. Mean V. parahaemolyticus levels were 3.79 log MPN/g (range of 0.56 to 6.18) and mean V. vulnificus levels were 4.31 log MPN/g (range of 3.38 to 5.38) during this sampling trip. A significant negative correlation with salinity and V. vulnificus and V. parahaemolyticus levels was observed (P<0.005). Environmental temperatures (11.2°C) at Fowl River in December were sub-optimal for Vibrio growth. Mean levels recorded at this sampling site and period were 3.55 log MPN/g (range of 1.63 to 4.63) and 1.89 log MPN/g (range of 0.96 to 2.38) for V. parahae-

ously reported, we feel this validates the application of the microtiter plate MPN-Rti-PCR methodology to this sample type. When detectable levels were present in fish intestines collected from the high salinity site, Gulf of Mexico, they were lower than levels detected at the two, lower salinity, estuarine sites. V. vulnificus levels were 2.83, 4.63, and 7.20 mean log MPN/g from the Gulf of Mexico, Fowl River, and Mobile Bay sites, respectively. Similarly, V. parahaemolyticus was detected at 4.17, 5.43, and 7.67 mean log MPN/g from the Gulf of Mexico, Fowl River, and Mobile Bay sites, respectively. The difference in levels of V. vulnificus and V. parahaemolyticus between the high salinity and estuarine sites is statistically significant (P<0.01). A previous study of V. vulnificus in finfish also found lower prevalence and densities in Gulf of Mexico fish than estuarine fish (DePaola et al., 1994). A previous study enumerating V. parahaemolyticus in finfish intestines from market fish in Malaysia also found similar levels (Noorlis et al., 2011). The highest levels of V. vulnificus (6.18 to 7.63 log MPN/g) and V. parahaemolyticus (6.63 to 7.97 log MPN/g) were detected in samples from Mobile Bay (Figure 2). Water temperature (24.7°C) and salinity (13.7 ppt) were within the optimal ranges for V. vulni-

molyticus and V. vulnificus, respectively (Figure 2). V. vulnificus levels were approximately 2 logs lower with the colder temperatures. In contrast, V. parahaemolyticus, levels were similar (0.24 logs less) to the September Fowl River collection (25.7°C and 10.3 ppt). Although the levels were generally lower than during the warmer sampling from the same site, levels well over the LOD (0.48 log MPN/g) were still observed in the finfish intestines. Additionally, no significant correlation between V. vulnificus or V. parahaemolyticus levels and temperature was observed. Only 4 of the 21 finfish intestinal samples collected contained detectable (≥3 MPN/g) levels of V. cholerae; all of which were obtained from the estuarine sites. Three of the samples with detectable levels of V. cholerae (0.48 to 0.87 log MPN/g) were found during the Fowl River September collection, when the water temperature was 25.7°C and salinity 10.3 ppt. This was the collection date when the salinity was the lowest, so it is not unexpected to see a greater prevalence of V. cholerae, as V. cholerae prefers lower salinity environments (Thomas et al., 2006). The fourth sample containing V. cholerae was the December Fowl River collection when the water temperature was 11.2°C and salinity 14.2 ppt. This sample contained the highest level of V. cholerae

ficus (>20°C and 5 to 15 ppt) and V. parahaemolyticus (>15°C and 15 to 25 ppt) during this collection (DePaola et al., 2003; McLaughlin et al., 2005; Motes et al., 1998; Wright et al., 1996). During the Fowl River collection in September, temperature was also opti-

(4.58 log MPN/g), but only one of the three fish from this collection had detectable levels. These data suggest that surrounding water salinity influences the V. cholerae levels in finfish intestines, but as only four samples had detectable levels of V. cholerae, no

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Figure 3. Phylogenetic tree of non- V. vulnificus strains isolated on CPC+. Strains with accession names provided were included as reference for relatedness of unknown isolates.

statistical significance could be assigned. It is important to note that 42°C is the optimal incubation temperature for V. cholerae, (DePaola et al., 1988) but 35°C was used in this study to facilitate detection by the BAX® Real-Time PCR V. parahaemolyticus/V. vulnificus/V. cholerae Kit, which may lead to an underestimation of these populations. Currently, no reports of total V. cholerae den-

nificus of fish examined (DePaola et al., 1994). In the current study, we also found the highest levels of V. vulnificus (7.63 log MPN/g) in sheepshead. The same sheepshead sample also contained the highest level of V. parahaemolyoticus (7.97 log MPN/g) and a different sheepshead harbored the highest level of V. cholerae (4.58 log MPN/g) observed in this study. Other species of fish in this study with high

sities in fish intestines are available for comparison to the results presented here. A previous study in the Mobile Bay area determined the intestinal content of sheepshead contained the highest levels (7.1 log MPN/g) of V. vul-

levels of Vibrios were Atlantic spadefish and pinfish (>4 log MPN/g of V. vulnificus and >6 log MPN/g of V. parahaemolyticus). Samples collected from the Gulf of Mexico site were spread plated on CPC+ to identify organisms,

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other than V. vulnificus, with typical colony appearance. Finfish from the Gulf of Mexico September collection (red snapper, red drum, sheepshead, and blue runner) and October collection (red snapper, red drum, and sea catfish) yielded isolates typical of V. vulnificus, but these could not be confirmed using species-specific PCR (BAX® Real-Time PCR V. parahaemolyticus/V. vulnificus/V. cholerae Kit). Partial 16S sequencing was done to identify these organisms. Of the 17 isolates, 6 were identified as V. sinaloensis, 2 as V. shilonii, 1 as V. campbellii, 1 as V. harveyi, while 6 could only be matched to the Vibrio genus (Figure 3). Additionally, one strain was identified as Morganella morganii (Figure 3). This high-

ACKNOWLEDGEMENTS

lights the need for confirmatory testing of typical isolates, even from a medium as selective as CPC+. This is the first report of identification of competing microflora on V. vulnificus-specific media.

marine environments of Washington State. Appl. Microbiol. 20:179-186. Blodgett, R. 2010. Most probable number from serial dilutions. Bacteriological Analytical Manual online: 9th. http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm109656.htm Cantas, L., J. R. Sorby, P. Alestrom, and H. Sorum. 2012. Culturable gut microbiota diversity in zebrafish. Zebrafish. 9:26-37. Chakraborty, S., G. B. Nair, and S. Shinoda. 1997. Pathogenic Vibrios in the natural aquatic environment. Rev. Environ. Health 12:63-80. Colwell, R. R., J. Kaper, and S. W. Joseph. 10-281977. Vibrio cholerae, Vibrio parahaemolyticus, and other Vibrios: occurrence and distribution in Chesapeake Bay. Science 198:394-396. DeLong, E. F. 6-15-1992. Archaea in coastal marine environments. Proc. Natl. Acad. Sci. U. S. A 89:5685-5689. DePaola, A., G. M. Capers, and D. Alexander. 1994. Densities of Vibrio vulnificus in the intestines of fish from the U.S. Gulf Coast. Appl. Environ. Microbiol. 60:984-988. DePaola, A., M. L. Motes, and R. M. McPhearson. 1988. Comparison of APHA and elevated temperature enrichment methods for recovery of Vibrio

CONCLUSIONS This is the first report, to our knowledge, using an MPN-Rti-PCR method to simultaneously enumerate the levels of human pathogenic Vibrios in fish intestines and associate those levels with surrounding water temperature and salinity. The levels of V. vulnificus, V. parahaemolyticus, and V. cholerae in finfish intestines reported here support the theory that finfish serve as a reservoir for potentially pathogenic Vibrios in estuarine and marine environments. The greatest frequency of detection and highest levels of V. vulnificus, V. parahaemolyticus, and V. cholerae were found in fish from estuarine areas favorable for shellfish production, posing the possibility of transmission via fish feces. Additionally, this study identified non-pathogenic species, such as V. shilonii, V. sinaloensis, and V. harveyi on selective plating media, emphasizing the need for isolate confirmation, even from selective media.

A part of this study was supported by a Research on Food Safety in Health and Labour Science Research Grant in Japan.

REFERENCES Aiso, K., U. Simidu, and K. Hasou. 1968. Microflora in the digestive tract of inshore fish in Japan. J. Gen. ral Microbiol. 52:361-364. Baross, J. and J. Liston. 1970. Occurrence of Vibrio parahaemolyticus and related hemolytic Vibrios in

cholerae from oysters: collaborative study. J. Assoc. Off Anal. Chem. 71:584-589. DePaola, A., J. L. Nordstrom, J. C. Bowers, J. G. Wells, and D. W. Cook. 2003. Seasonal abundance of total and pathogenic Vibrio parahaemolytiAgric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 3 - 2013

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cus in Alabama oysters. Appl. Environ. Microbiol. 69:1521-1526. Iwamoto, M., T. Ayers, B. E. Mahon, and D. L. Swerdlow. 2010. Epidemiology of seafood-associated infections in the United States. Clinical Microbiology Reviews 23:399-411. Jaksic, S., S. Uhitil, T. Petrak, D. Bazulic, and L. G. Karolyi. 2002. Occurrence of Vibrio spp. in sea fish, shrimps and bivalve molluscs harvested from Adriatic Sea. Food Control 13:491-493. Johnson, C. N., A. R. Flowers, N. F. Noriea, III, A. M. Zimmerman, J. C. Bowers, A. DePaola, and D. J. Grimes. 2010. Relationships between environmental factors and pathogenic Vibrios in the Northern

Griffin. 2011. Foodborne illness acquired in the United States-major pathogens. Emerg. Infect. Dis. 17:7-15. Senderovich, Y., I. Izhaki, and M. Halpern. 2010. Fish as reservoirs and vectors of Vibrio cholerae. PLoS. One. 5:e8607Silva, F. C., J. R. Nicoli, J. L. Zambonino-Infante, S. Kaushik, and F. J. Gatesoupe. 2011. Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparus aurata) and intestinal contents in goldfish (Carassius auratus). FEMS Microbiol. Ecol. 78:285296. Smriga, S., S. A. Sandin, and F. Azam. 7-1-2010.

Gulf of Mexico. Appl. Environ. Microbiol. 76:70767084. Jones, J. L., Y. Hara-Kudo, J. A. Krantz, R. A. Benner, Jr., A. B. Smith, T. R. Dambaugh, J. C. Bowers, and A. DePaola. 2012. Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus. Food Microbiol. 30:105-111. McLaughlin, J. B., A. DePaola, C. A. Bopp, K. A. Martinek, N. P. Napolilli, C. G. Allison, S. L. Murray, E. C. Thompson, M. M. Bird, and J. P. Middaugh. 10-6-2005. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. N. Engl. J. Med. 353:1463-1470. Motes, M. L., A. DePaola, D. W. Cook, J. E. Veazey, J. C. Hunsucker, W. E. Garthright, R. J. Blodgett, and S. J. Chirtel. 1998. Influence of water temperature and salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica). Appl. Environ. Microbiol. 64:1459-1465. Noorlis, A., F. M. Ghazali, Y. K. Cheah, T. C. Tuan Zainazor, J. Ponniah, R. Tunung, J. Y. H. Tang, M. Nishibuchi, Y. Nakaguchi, and R. Son. 2011. Prevalence and quantification of Vibrio species and Vibrio parahaemolyticus in freshwater fish at hypermarket level. Int. Food Research J. 18:689-695. Ringo, E. and R. E. Olsen. 1999. The effect of diet on

Abundance, diversity, and activity of microbial assemblages associated with coral reef fish guts and feces. FEMS Microbiol. Ecol. 73:31-42. Sudha, S., P. S. Divya, B. Francis, and A. A. Hatha. 2012. Prevalence and distribution of Vibrio parahaemolyticus in finfish from Cochin (south India). Vet. Ital. 48:269-281. Thomas, K. U., N. Joseph, O. Raveendran, and S. Nair. 2006. Salinity-induced survival strategy of Vibrio cholerae associated with copepods in Cochin backwaters. Mar. Pollut. Bull. 52:1425-1430. Wright, A. C., R. T. Hill, J. A. Johnson, M. C. Roghman, R. R. Colwell, and J. G. Morris, Jr. 1996. Distribution of Vibrio vulnificus in the Chesapeake Bay. Appl. Environ. Microbiol. 62:717-724.

aerobic bacterial flora associated with intestine of Arctic charr (Salvelinus alpinus L.). J. Appl. Microbiol. 86:22-28. Scallan, E., R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, M. A. Widdowson, S. L. Roy, J. L. Jones, and P. M. 194

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REVIEW Utility of Egg Yolk Antibodies for Detection and Control of Foodborne Salmonella P. Herrera1, M. Aydin2, S. H. Park3, A. Khatiwara4# and S. Ahn5 Food Safety and Inspection Service, 2736 Lake Shore Drive, Waco, TX 72708 Molecular Biosciences Graduate Program, Arkansas State University, Jonesboro, AR 72401 3 Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR 72704 4 Cell and Molecular Biology Program, Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701 5 Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611 1

Current address: Food & Drug Administration/Center for Food Safety and Applied Nutrition, College Park, MD 20740

ABSTRACT Foodborne Salmonella spp. continue to be problematic in food production and processing. This is partly due to their ability to survive and adapt to a wide range of environments and colonize and infect various hosts. Consequently not only the ability to detect low numbers of Salmonella spp. in these various niches is important but also quantitation is emerging as an important consideration for assessing effectiveness of control measures and better profiling of potential problematic areas for control in food production and processing. Immunoassays offer promise for both detection and quantitation as well as high-throughput analyses. Although both monoclonal and polyclonal antibodies have been utilized for immunoassays, polyclonal antibodies offer additional versatility and ease of production, which makes them more attractive for routine use. In this review, recent advances in the use of immunological approaches, particularly egg yolk antibodies are discussed and compared. Finally the potential for feed grade egg yolk antibodies as a therapeutic agent to be added to feed is discussed along with ongoing limitations and possible solutions using clay materials as carriers. Keywords: Immunological approaches, egg yolk antibodies, foodborne, Salmonella Agric. Food Anal. Bacteriol. 3: 195-217, 2013

Correspondence:Soohyoun Ahn, sahn82@ufl.edu Tel: +1 -352-392-1991 Ext. 310.

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INTRODUCTION Salmonella spp. are members of the family Enterobacteriaceae and individual species are differentiated primarily on basis of the host they are generally attributed to associate with on a regular frequency (Bhunia, 2008; Pui et al., 2011; Li et al., 2013). Salmonellosis is one of the most common foodborne diseases in the United States, with Salmonella enterica serovars Enteritidis (S. Enteritidis), S. Typhimurium and S. Heidelberg being some of the more common isolates found on a wide range of food as well as associated with foodborne disease and/or isolated from humans (St. Louis et al., 1988; Ricke et

Many of these interventions have proven to be relatively effective but success is not always universal and can depend on several factors. One of the primary reasons for limited effectiveness of certain intervention measures is that Salmonella spp. possess the capability with a wide range of gene systems to survive environmental changes including decreases in pH and water activity, sudden increases in temperature, and other fairly drastic changes that can commonly occur in food production (Juven et al., 1984; Ha et al., 1998a,b; Durant et al., 1999a,b, 2000a,b,c; Kwon and Ricke, 1998, 1999; Kwon et al., 2000; Ricke, 2003b; Carrique-Mas et al., 2007; Dunkley et al., 2009b; Milillo and Ricke 2010; Milillo et al., 2011; Pet-

al. 2001; Committee on Salmonella, 2002; Mumma et al., 2004; Patrick et al., 2004; Braden, 2006; CDC, 2006; Boyen et al., 2008; Hanning et al., 2009; Foley et al., 2011; Scallan et al., 2011; Finstad et al., 2012; Howard et al., 2012; Koo et al., 2012; Li et al., 2013). The annual cost of salmonellosis due to medical costs and lost production has been estimated to be several billion dollars (Frenzen et al., 1999; Bhunia, 2008; Scallan et al., 2011). The Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture has identified it as a pathogen of importance and has consequently implemented a testing regime in meat/poultry processing facilities to aid in the surveillance and control of Salmonella (FSIS-USDA, 2006). In many food animal production systems such as poultry, Salmonella can readily become established in the gastrointestinal tract and be easily disseminated into the surrounding environment. General persistence of Salmonella can also influence all aspects of food production and processing (Murray, 2000; Park et al., 2008; Dunkley et al., 2009a). Consequently, a wide range of intervention strategies to reduce Salmonella contamination has taken into account not only the particular food production or processing system but any restriction on what can be

kar et al., 2011; Sirsat et al., 2009, 2010, 2011). In addition, the fact that not all Salmonella serovars genetically respond equally to certain stresses such as low pH further complicates intervention approaches (González-Gil et al., 2012; Shah et al., 2012a). Therefore, the combination of a complex genetic system coupled with the high adaptability of Salmonella to a range of stressors have limited the ability to not only predict the persistence of a particular Salmonella serovar, but also detect and develop the appropriate control measures to minimize its dissemination (Ricke, 2003b; Ricke et al., 2005, 2013b; Spector and Kenyon, 2012). However, the development of rapid detection approaches, next generation sequencing methods and other approaches such as metabolomics offer opportunities to more specifically target foodborne Salmonella in many of the corresponding food production systems (Maciorowski et al., 2005, 2006; Jarquin et al., 2009; Kwon and Ricke, 2011; Park et al. 2013; Ricke et al., 2013b). The objectives of this review are to examine the prevalence of certain Salmonella serovars in specific food production systems and to discuss immunological Salmonella detection systems with an emphasis on polyclonal egg yolk antibodies. Although several Salmonella serovars are problematic for food

applied from a government regulatory standpoint (Ricke and Pillai, 1999; Ricke, 2003a,b; Ricke et al., 2005; Maciorowski et al., 2004; O’Bryan et al., 2008; Sirsat et al., 2009; Vandeplas et al., 2010; Cox et al., 2011; Ricke et al., 2012a,b; Siragusa and Ricke, 2012).

production, our review on the prevalence of Salmonella in food production systems will be focused on Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) as an example since this serovar can represent the complexity in disseminating Salmonella

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through a host animal and its associated food products and the ensuing difficulty of developing appropriate intervention measures. In our review on immunological Salmonella detection systems, the application of polyclonal egg yolk antibodies as a potential intervention in the form of feed grade antibodies will also be discussed in addition to the discussion on their utility for incorporation into Salmonella detection systems.

SALMONELLA ENTERITIDIS AND EGG PRODUCTION

these types of molting procedures to interrupt the first cycle of egg production by causing a cessation and retrenchment of the reproductive tract so that it could enter a period of rest and rejuvenation prior to ending the molt period to start a second egg laying cycle (Bell, 2003; Berry, 2003; Ricke, 2003a; Ricke et al., 2010; 2013a). Generally speaking, induced molting was considered economically advantageous by not only enabling an extension in egg production but also potentially increasing eggs produced by older laying hens (Keshavarz and Quimby, 2002; Bell, 2003; Berry, 2003; Holt, 1999, 2003; Ricke et al., 2013a). The advantages of induced molting became evident by the fact that 60 percent of the estimated

The difficulty of limiting Salmonella in food production systems is especially evident with particular serovars that can be entrenched in food production ecosystems and become continually problematic during all phases of production. Consequently as mores cases are documented these serovars become characteristically identified with a particular food product. Historically, eggs contaminated with S. Enteritidis strains have represented one of the more prevalent exposure routes in human outbreaks. For example, in the year 1999, estimates of the number of human salmonellosis cases ranged from 800,000 to 4 million (Angulo and Swerdlow, 1999). For the past two decades, the number of cases of gastroenteritis due to S. Enteritidis infections has increased markedly in the United States and Europe and still remains a serious food safety issue (Humphrey, 1999; Guard-Petter, 2001; Patrick, 2004; Mumma et al., 2004; Schroeder et al., 2006; CDC, 2010; Norberg et al., 2010; Howard et al., 2012; Ricke et al., 2010, 2013a). One of the primary problems with S. Enteritidis is that it can easily become systemic and invade multiple organ systems in susceptible hosts such as laying hens (Guard-Petter, 2001; Holt, 1999, 2003; Shah et al., 2012b). It became evident that periodic clusters

240 million laying hens nationwide and 90 percent in California were force-molted and the practice became more popular (Bell, 1987; 2003; Holt, 1999; 2003). Feed deprivation-based molt induction, however, proved to be problematic as it disrupted the activity of protective intestinal microflora and allowed S. Enteritidis to colonize the gastrointestinal tract of birds and become pathogenic in them (Thiagarajan et al., 1994; Durant et al., 1999a; Ricke, 2003a; Dunkley et al., 2007a, 2009a; Golden et al., 2008; Shah et al., 2012b; Ricke et al., 2013a). Depending on microecological conditions created by the removal of feed in the gastrointestinal tract, S. Enteritidis after initial colonization could initiate the expression of several key regulatory and functional virulence genes and through a series of steps become more pathogenic leading to systemic invasion of multiple organ systems including the reproductive tract (Thiagarajan et al., 1994; Holt, 1999; Humphrey et al., 1999; Ricke, 2003a; Dunkley et al., 2007a; Norberg et al., 2010; Shah et al., 2012b; Ricke et al., 2010; 2013a). Because of this invasiveness into the reproductive tract organs including the ovaries, S. Enteritidis possessed the potential ability to contaminate eggs by transovarian transmission following establishment in the

of contaminated eggs produced by laying hens were attributed to specific management practices such as feed withdrawal-based molting (Durant et al., 1999a; Holt, 1999, 2003; Ricke, 2003a; Ricke et al. 2013a). Traditionally, the commercial egg industry used

intestinal tract (Thiagarajan et al., 1994; Holt, 1999; Humphrey et al., 1999). Administration of particular antibiotics, followed by introduction of probiotic cultures, has been demonstrated to limit S. Enteritidis in susceptible birds (Seo et al., 2000; Holt, 2003);

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however, due to the emergence of antibiotic resistant microorganisms, there are public health concerns that such practices may lead to dissemination of antibiotic resistant pathogens and an increase of untreatable human disease (Jones and Ricke, 2003). Several alternative means for limiting Salmonella establishment in laying hens and occurrence in table eggs have been examined, including vaccination of the hens and treatments designed to remove bacteria from the surfaces of egg shells (Kinner and Moats, 1981; Holley and Proulx, 1986; LeClair et al., 1994; Kuo et al., 1996, 1997a,b,c;, 2000 McKee et al., 1998; Knape et al., 1999, 2001; Holt, 2003; Howard et al., 2012; Ricke et al., 2012a; 2013a). The most

technologies, utility of egg yolk polyclonal antibodies, and their potential applications for foodborne Salmonella.

extensively examined approach has been focused on modifying the molt induction process such that it no longer is invoked by removal of feed but instead is done via an alteration on the diet. A number of different dietary amendments have been examined over the years that have involved an alteration of dietary components such as sodium or calcium and these have been summarized elsewhere (Bell, 2003; Berry, 2003; Park et al., 2004b,c; Ricke et al., 2010; 2013a). Increasing the dietary level of zinc has been shown to initiate molt in laying hens and although experimental infection studies indicated that S. Enteritidis establishment could be limited by these diets, potential management issues and other issues precluded their practical use (McCormick and Cunningham, 1984a,b; Cunningham and McCormick, 1985; Berry and Brake, 1985; Goodman et al., 1986; Berry et al., 1987; Alodan and Mashaly, 1999; Bar et al., 2003; Park et al., 2004a,b,c,d; Moore et al., 2004; Ricke et al., 2004). Adding dietary ingredients containing high levels of fiber sources such as alfalfa, wheat middlings, and guar gum, were also demonstrated in a wide range of studies to have the ability to induce molt, alter gastrointestinal fermentation, shift microbial populations, and limit S. Enteritidis in laying hens (Seo et al., 2001; Holt, 2003; Hume et al.,

figuration of ELISA, primary antibodies (also called capture antibodies) are immobilized to a solid matrix such as a microtiter plate, and subsequently capture the target antigen from enriched samples. Target antigens can include either the whole microorganism of interest or a more purified isolated protein from the target microorganism such as toxins. Secondary antibodies (also called detection antibodies) that are conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase bind directly to alternative site(s) on the target antigen and function in the detection of the bound antigen. Conversely, the antigen can be initially bound to the solid plate followed by adding primary antibodies that bind directly to the antigen and finally the secondary chromogenic antibodies that bind to the primary antibodies. In this approach, it is critical that the antigens or target microorganisms bind on the solid phase equally to ensure that any difference detected during the assay is strictly due to differences in the antigen-antibody binding relationship. Successful antigen-antibody binding is detected when the enzyme conjugated to the detection antibodies reacts with added chromogenic substrate and generates a detectable color reaction. The presence of an antigen can be detected and quantified

2003; Ricke, 2003a; Woodward et al., 2005; McReynolds et al., 2005, 2006; Dunkley et al., 2007a,b; Donalson et al., 2008; Gutierrez et al., 2008; Ricke et al., 2010; 2013a). The remainder of this review will discuss general concepts for immunological assay

by reading the generated color reaction with a spectrophotometer, and the amount of antigen is correlated to the intensity of the color (Clark and Engvall, 1980; Maggio, 1980; Yolken, 1982; Butt, 1984; Blake and Gould, 1984; O’Sullivan, 1984). The ELISA plat-

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IMMUNOLOGICAL METHODS – GENERAL CONCEPTS Enzyme-Linked Immunosorbent Assay (ELISA) based immunoassays are extensively reviewed elsewhere and will only briefly be described here (Clark and Engvall, 1980; Maggio, 1980; Yolken, 1982; Butt, 1984; Blake and Gould, 1984; O’Sullivan, 1984; Maciorowski et al., 2006). In a typical sandwich con-

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form offers several advantages in comparison to cultural detection methods, which include much shorter total assay times than cultural methods (1 to 2 days including pre-enrichment versus 4 to 7 days) and the possibility for automation to further reduce the total assay time and manual labor input. Consequently this offers the opportunity for enhanced throughput to allow large number of samples to be simultaneously analyzed. Another key advantage of ELISA-based immunological approaches over cultural methods is the increased specificity due not only to the affinity between antibodies and the respective antigens they have been generated against but the ability to amplify detection sensitivity via enzyme re-

test (Ziprin et al., 1990; Corrier et al., 1995; Nisbet et al., 1996a,b; Durant et al., 1997b; Young et al., 1997). To test these respective antibodies for the detection sensitivity and quantification of the corresponding bacteria, the cultures were grown anaerobically in batch culture containing a nutrient rich broth both as pure cultures and in a mixed culture. The detection limits for the ELISA were 104 cells per mL for all three bacteria with no cross reactivity between the antibodies and bacteria. In addition, when cells enumerated by selective plating were compared to the numbers of cells estimated by ELISA, the resulting enumeration from the respective method demonstrated linearity as the cell numbers were propor-

actions and use of chromogenic, colorimetric or fluorometric agents (Maggio, 1980; Yolken, 1985; 1988).

Large quantities of antibodies can be produced by fusing an activated B lymphocyte to an immortal cell line to form a hybridoma (Kimball, 1983). Antibodies produced by hybridoma cells are monoclonal, and they can react to specific determinant of a particular antigen of interest (Kimball, 1983). This specificity has advantages for detection of Salmonella in immunological assays and also offers a certain level of uniformity that is highly reproducible (Kimball, 1983). Their development, generation and application has been documented for a wide variety of microorganisms over the years (Macario, and Conway de Macario, 1985; Hazlewood et al., 1986; Brooker and Stokes, 1990; Bhunia et al., 1991; Bhunia and Johnson, 1992; Di Padova et al., 1993; Chaiyaroj et al., 1995; Corthier et al., 1996; Young et al., 1997; Nannapaneni et al., 1998a,b; Geng et al., 2006; Zhang et al., 2006; Heo et al., 2007, 2009) and only their utility for foodborne Salmonella will be briefly discussed here. Durant et al., (1997a) assessed the

tionally increased (r2 above 0.9). Durant et al. (1997a) concluded that monoclonal antibodies generated for each of these microorganisms could be used to quantitate these microorganisms in mixed cultures as well as potentially in vivo. As the authors pointed out, however, reliable quantification would require the antigens be consistently and proportionally expressed under all environmental conditions. While monoclonal antibodies can provide high specificity in bacterial detection, this specificity can also limit their usefulness in simultaneous detection of multiple strains as an antibody made toward the antigen of one strain may not react to another closely related strain and therefore may require different screening methods to retrieve multiple monoclonal antibodies for each strain (Mierendorf and Dimond, 1983). Furthermore the creation, culturing and the purification of antibodies from hybridomas can be both time-consuming and expensive (Kimball, 1983). Polyclonal antibodies derived from the sera of immunized animals offer an alternative to avoid some of these limitations in monoclonal antibodies. Detection of microorganisms based on the immune response of an animal host is long-standing basis for generating fairly specific antibody probes for differentiating among genera, species and even at

specificity of mouse monoclonal antibodies made to probiotic bacteria Veillonella and Enterococcus avium previously isolated from a continuous culture of chicken cecal contents versus antibodies generated to a S. Typhimurium poultry isolate in an ELISA

the strain level (Mims¸ 1976; Dawson and Cresswell, 1988). The essential process for antigen preparation and animal immunization has been described much more extensively in previous reviews (Mims¸ 1976; Amos, 1988; Dawson and Cresswell, 1988; Cook and

SOURCES OF ANTIBODIES

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Trott, 2010). Briefly, the antibody-producing animal host is introduced to either the whole organism or selected antigens from that target organism that have been purified to some extent. When this entity is introduced into the animal via oral or intramuscular routes, it mounts an immune response, which in turn generates highly specific antibody populations as exposure to the antigen is continued mostly via repeated booster administration. These antibodies are referred to as polyclonal antibodies due to the presence of different subpopulations of antibodies, which are specific for the particular antigen but each identifies different epitopes associated with the respective antigen (Mims¸ 1976; Amos, 1988; Dawson

of avian antibodies have been recognized for several decades and their use offers many advantages compared to mammalian antibodies. The major serum antibody in chicken is immunoglobulin G (IgG or IgY) which is transported into the egg in a manner similar to the placental transfer of IgG in mammals (Coleman, 2000). The protection against pathogens that the relatively immunocompetent newly hatched chick possesses is through transmission of antibodies from the mother via the egg (Kovacs-Nolan and Mine, 2004). The yolk of immunized chickens is a rich and inexpensive source of a variety of high value proteins including polyclonal antibodies (Cook, 2004; Kovacs-

and Cresswell, 1988). Polyclonal antibodies have been developed for a wide range of microorganisms (Archer and Best, 1980; Minnich et al., 1982; Archer, 1984; Mårtensson et al., 1984; Aleixo et al., 1985; Singleton et al., 1985; Maciorowski et al., 2006; Zhang et al., 2006). Polyclonal antibodies are generally recovered from the serum after bleeding the animal immunized over a sufficient time to the antigen in question; however, bleeding can be avoided altogether in laying hens where similar types of antibodies to those generated in the animal sera can also be deposited in the egg yolks. The following sections describe the use and development of polyclonal antibodies with particular emphasis on egg yolk antibodies.

A relatively recent development has been the ability to recover specific antibodies from eggs of hens that have been immunized to a specific antigen or microorganism. These antibodies are similar to the antibodies recovered from serum that are used for immunoassays but are deposited in the egg

Nolan and Mine, 2004; Cook and Trott, 2010). Kovacs-Nolan and Mine (2004) pointed out that a hen typically producing 50 to 100 mg IgY per egg and 5 to 7 eggs in one week compared to 200 mg per bleed (40 mL) in a mammal will equate to 40, 000 mg antibody in a year for a hen versus 1,400 for the mammalian source. Cook and Truitt (2010) summarized studies that demonstrated that the concentration of antibody appears to be independent of the rate of lay or size of the egg and the amount deposited could not be increased. In addition to avoiding the bleeding of the animal altogether, egg yolk antibodies can be retrieved daily without bleeding the hen (Karlsson et al., 2004; Kovacs-Nolan and Mine, 2004). Only IgG (IgY) and not IgM is selectively deposited in the yolk and its levels are similar to those found in serum, and the antibody activity is stable in eggs stored from 4 to 6 weeks and storage can be extended by spray drying whole eggs and holding at 21°C or lower (Brandly et al., 1946; Patterson, et al., 1962a,b; Malkinson, 1965; Orlans, 1967; Brambell, 1970; Faith and Clem, 1973; Rose and Orlans, 1981; Ricke et al., 1988; Cook and Trott, 2010; Sun et al., 2013). In addition, Cook and Truitt (2010) pointed out that although thermal (steam) exposure such as that experienced during

yolk and biologically are intended to be used by the hatchling as a source of passive immunity to potential threats it may encounter early in its life (Malkinson, 1965; Kramer and Cho, 1970; Kowalczyk et al., 1985; Anton, 1998; Hamal et al., 2006). The benefits

feed pelleting can cause loss of their biological activity this can be alleviated to some extent by the addition of certain carbohydrates which offered some protection. Applications of egg yolk antibodies in the food animal industry have been two-fold,

EGG YOLK ANTIBODIES AS A POLYCLONAL ANTIBODY SOURCE

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namely as therapeutic agents to food animals for disease prevention and growth promotion and as a source of polyclonal antibodies for diagnostic detection of microorganisms (Lösch et al., 1986; Pimentel, 1999; Mine and Kovacs-Nolan, 2002; Tini et al., 2002; Cook, 2004; Karlsson et al., 2004; Kovacs-Nolan and Mine, 2004; Berghman et al., 2005; Schade et al., 2005; Trott et al., 2009; Kim et al., 2013). These two applications will be addressed in more detail in the following sections.

EGG YOLK ANTIBODIES FOR DETECTION ASSAYS

titate two strains (Selenomonas ruminantium strain D versus strain GA192) in a biculture by comparing microscopic enumerations of individual cells versus the immunoassay response (Ricke and Schaefer, 1990a). Since the strains were metabolically similar this proved to be significant that they could be distinguished by an ELISA assay with polyclonal antibodies and suggested that surface antigens may differ to some extent (Ricke and Schaefer, 1990a). As the authors suggested the issue remains as to whether antibodies made to strains maintained in the laboratory for several years would react with similar strains occurring currently found in the rumen or if the respective epitopes evolves such that

Egg yolk antibodies have been used as the source of polyclonal antibodies for detection assays for several microorganisms. In a series of studies hens immunized to rumen bacteria produced specific antibodies that could be used in immunoassays to differentiate them (Ricke et al., 1988). By week 2 of post immunization cross reactivity for most rumen bacteria tested was reduced except for Streptococcus bovis antibodies which still exhibited considerable cross reactivity with heterologous antigens of the other rumen bacteria tested. Ricke et al. (1988) speculated that this sustained cross reactivity may be related to Streptococcus bovis being a Gram positive organism with an extensive capsule that masks some of the surface antigens that might be important for eliciting an optimal immune response in the bird (Lancefield , 1933; Dain et al., 1956; Deibel, 1964; Cheng et al., 1976; Russell and Robinson, 1984; Ricke et al., 1988; Jones et al., 1991; Holt, 1994; Herrera et al., 2009, 2012). Further studies demonstrated that specific egg yolk antibodies could be generated against individual strains of Selenomonas ruminantium, a common isolate from the rumen of ruminant animals fed a variety of diets that has had multiple strains isolat-

the resulting reactivity is either altered or lost entirely. Better structural characterization and purification of the specific epitopes that are binding to the egg yolk antibodies would be helpful to alleviate this uncertainty. There has been considerable evidence that laying hens could produce specific antibodies to Salmonella spp. when the birds are exposed to Salmonella colonization and infection. For example, in a series of studies it has been demonstrated that serovar S. Enteritidis under certain circumstances can easily colonize and infect susceptible laying hens (Durant et al., 1999a; Ricke, 2003a; Woodward et al., 2005; McReynolds et al., 2005; 2006; Dunkley et al., 2007a, 2009a; Norberg et al., 2010; Ricke et al., 2010; 2013a). In these infected hens S. Enteritidis can usually be recovered from egg contents either due to penetration of the egg shell or via internal deposition directly into the yolk from an infected reproductive tract in the laying hen (Humphrey et al., 1989, 1991; Humphrey, 1999; Gast and Beard, 1990a; Gast, 1993; Guard-Petter, 2001). It follows that immune responses would be elicited from S. Enteritidis colonization and infection, resulting in antibodies that could be detected in the serum (Gast and Beard, 1990b). It was also shown that birds

ed and characterized over the years (Kingsley and Hoeniger, 1973; Brooker and Stokes, 1990; Ricke and Schaefer, 1990a,b, 1991, 1996a,b,c; Ricke et al., 1996; Patterson et al., 2010). In a study involving egg yolk antibodies, the authors were able to quan-

either experimentally infected with S. Enteritidis or naturally infected deposited detectable serogroup D specific antibodies in their yolks (Gast and Beard, 1991). More recently, Gürtler and Fehlhaber (2004) demonstrated that multiple immunizations with S.

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Enteritidis would increase the quantity of anti- S. Enteritidis antibody in the egg yolk. Therefore, it would appear that specific antibodies suitable for detection purposes could be generated in the eggs of hens immunized to the target Salmonella spp. Biswas et al. (2010) compared the specificity of egg yolk antibodies generated after immunization of laying hens with purified antigens from S. Enteritidis and S. Typhimurium to that of antibodies produced against whole cells of common Salmonella serovars (S. Enteritidis, S. Typhimurium, S. Anatum, S. Arizona, S. Javiana, S. Muenchen, S. Newport, S. Rubsilaw, and S. Texas along with Escherichia coli as a control) prepared by inactivation in 10% forma-

with the S. Enteritidis strains not surprisingly being the most cross reactive. The next most specific heterologous reactions occurred with the flagellar antibodies followed by the LPS antibodies which were clearly highly cross reactive. For S. Typhimurium antibodies the least cross reactivity occurred with the flagellar antibody and as expected the most cross reactivity that was detected occurred with the other S. Typhimurium strains while there was much less cross reactivity detected with the non-S. Typhimurium serovars. The S. Typhimurium OMP and LPS antibodies were generally much more cross reactive with all Salmonella serovars and even cross reacted to some extent with the inactivated E. coli bacterial

lin phosphate buffered saline solutions. Egg yolk antibodies had been previously produced in laying hens immunized against purified fimbriae, flagella and lipopolysaccharide (LPS) from a specific S. Enteritidis strain and flagella, LPS and outer membrane proteins (OMP) from a specific S. Typhimurium strain. When serial dilutions of the individual antibodies were titrated against their respective homologous (Salmonella antibody reacted against the same serovar) inactivated whole cell Salmonella serovar in an ELISA test, the anti-S. Enteritidis antibodes generally showed higher sensitivity than the S. Typhimurium antibodies. For S. Enteritidis antibodies the highest activity occurred with the fimbrial antibody. The next highest activity detected was the homologous reaction that occurred with the LPS followed by the reaction of inactivated whole cells with the flagellar antibody. For S. Typhimurium antibodies the highest activity occurred with the flagellar antibody. The next highest activity was the homologous reactions that occurred with the OMP followed by the reaction of inactivated whole cells with the LPS antibody. When serially diluted aliquots of the individual antibodies were titrated against the respective heterologous (Salmonella antibody reacted against

cells. Biswas et al. (2010) concluded that these particular Salmonella egg yolk antibodies had two potential applications. The very specific antibodies such as the S. Enteritidis fimbriae antibodies would be highly useful for detection of S. Enteritidis and potentially with further refinement could even be used to delineate detectable differences among different strains. The antibodies which displayed the most cross reactivity might actually be better suited for administration in animal diets as feed grade antibodies to limit Salmonella colonization since they would react with most Salmonella serovars fairly equally. Ideally, when used in passive immunity applications these highly cross reactive antibodies would be able to limit colonization of almost any Salmonella serovar that the animal host would come in contact with and potentially help to repress dissemination of foodborne Salmonella within flocks of birds or herds of animals. It would be critical to determine the resilience of these antibodies as they traversed the gastrointestinal tract and eventually reached the environment. If they proved to be relatively fragile some sort of carrier could potentially be devised that would serve as a potential means to protect the antibody during passage through the

different serovars) inactivated whole cell Salmonella serovars the anti-S. Enteritidis antibodes were also generally more specific than the S. Typhimurium antibodies. For S. Enteritidis antibodies the least cross reactivity occurred with the fimbrial antibody

more harsh elements of specific environments such as the highly acidic stomach. These aspects will be discussed in the following sections.

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EGG YOLK ANTIBODIES FOR THERAPEUTIC APPLICATIONS Feed grade antibodies derived from the egg yolks of immunized hens have the advantage of being easily accessible, inexpensive, and a rich source of polyclonal antibodies (Li et al., 1998; Cook, 2004; Cook and Trott, 2010). Because of the ability of laying hens to produce large quantities of egg yolk antibodies on a relatively ongoing basis they have been promoted and tested as potential feed grade prophylactic agents (Cook, 2004; Cook and Trott, 2010). They have been administered as potential inhibitors of the enzyme uricase to reduce nitrogen

vived and all of the survivors gained weight. In contrast, control piglets that were treated with egg-yolk powder that did not contain the specific antibodies had severe diarrhea, were dehydrated, lost weight and several died within 48 hours (Marquardt, 1999). Oral administration of egg yolk antibodies has also been successfully used for the prevention of bacterial infections in other animal models. It has been used to prevent rotavirus infections in mice (Ebina et al., 1996), Escherichia coli infections in rabbits (O’Farrelly et al., 1992) and piglets (Wiedemann et al., 1990), and caries in rats (Hamada et al., 1991). Egg yolk antibodies have also been developed for attempts to prevent establishment of foodborne

emissions in poultry due to the excess production of uric acid in the manure by microorganisms (Kim and Patterson, 2003; Kim et al. 2013). The ability to generate specific antibodies in fairly large quantities has also proven advantageous for therapeutic prevention of microbial pathogen colonization. Incorporating feed grade egg yolk antibodies into animal diets has been examined extensively to attempt to limit pathogenic diarrhea causing Escherichia coli in swine, and limit Salmonella establishment in calves and mice, as well as Campylobacter, Clostridium, and Salmonella in poultry (Wiedemann et al., 1991; Peralta et al., 1994; Yokoyama et al., 1998; Marquardt et al., 1999; Sahin et al., 2001; Fulton et al., 2002; Owusu-Asiedu et al., 2002; 2003; Kassaify, and Mine, 2004; Wilkie et al., 2006; Rahimi et al., 2007; Chalghoumi et al., 2009a; Al-Aldawari et al., 2013). Chicken egg-yolk antibodies when administered orally have been used for passive immunization against infectious diarrheal diseases in animals (Marquardt, 1999). Therefore, these antibodies offer a practical means of controlling certain intestinal diseases (scours) caused by microorganisms such as enterotoxigenic Esherichia coli in early weaned pigs. Studies have demonstrated that egg-yolk antibodies obtained from hens immunized with a strain of

pathogens that commonly colonize food animals. Campylobacter jejuni is one of the major foodborne disease causing microorganisms that also happens to be very well adapted to the ecological conditions prevalent in the poultry gastrointestinal tract (Horrocks et al., 2009; Pendleton et al., 2013). In an attempt to isolate antibodies that could limit C. jejuni colonization Al-Adwani et al. (2013) generated chicken egg-yolk-derived antibodies (IgY) in laying hens against the five different C. jejuni colonization-associated cell surface proteins. These proteins were produced in sufficient quantities by first expressing the respective protein in E. coli and subsequently purifying the proteins for intramuscular injection as a water-oil mixture in combination with Freund’s complete adjuvant into C. jejuni-free free laying hens. Eggs were collected upto 10 weeks post-immunization and egg yolks were lyophilized for eventual purification and quantitation of specific egg yolk antibodies reactive to each of the C. jejuni proteins. After characterizing specificity and reactivity of the individual egg yolk antibodies generated against the specific cell surface proteins they demonstrated that several of these egg antibodies limited attachment of C. jejuni to chicken hepatocellular carcinoma cells and concluded that these were candidate egg yolk

enterotoxigenic Esherichia coli, K88, were highly effective in protecting 3 to 14 day-old piglets against the pathogenic effects of this organism. Pigs fed egg-yolk with anti- enterotoxigenic Esherichia coli antibodies only had transient diarrhea, nearly all sur-

antibodies with potential to reduce C. jejuni colonization in chickens. In a series of studies, previously developed antiSalmonella spp. egg yolk antibodies that were simultaneously directed against Salmonella Enteriti-

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dis and Salmonella Typhimurium (Chalghoumi et al. (2008; 2009b) were administered as feed additives in the form of freeze dried egg yolk powder to broiler chicks for determining whether the egg yolk antibodies would prevent cecal colonization of both Salmonella serovars (Chalghoumi et al., 2009a). The birds were simultaneously infected with these Salmonella serovars three days after initial feeding of the respective dietary treatments with or without the antibodies and over the 28 day trial, birds were removed on days 7, 14, 21 and 28 days, sacrificed and cecal contents sampled for the presence of the Salmonella serovars using a quantitative real-time polymerase reaction assay. When monitoring bird feed

VEHICLES FOR THERAPEUTIC EGG YOLK ANTIBODIES â&#x20AC;&#x201C; POTENTIAL OF CLAY PARTICLES

intake and growth rate the authors noted that experimentally infecting with the Salmonella serovars negatively decreased performance parameters while inclusion of egg yolk antibodies enhanced these performance indicators even though they did not reach the same levels of the uninoculated control birds or birds fed antibodies and not inoculated with Salmonella. However, none of the treatments containing anti-Salmonella specific antibodies reduced the cecal levels of Salmonella Chalghoumi et al. (2009a) concluded that any growth benefit associated with the feeding of the antibodies were due to properties of the antibodies other than their anti-Salmonella function since the inclusion of the egg yolk antibody preparations containing anti-Salmonella antibodies did not statistically reduce the levels of Salmonella in the infected birds compared to the positive control that did not receive anti-Salmonella antibodies. The authors also speculated that the concentration of the antiSalmonella antibodies may have been too low to be effective perhaps due to their becoming denatured and degraded during their transit through the chicken gastrointestinal tract. Overcoming this loss of activity remains a potential obstacle for further practical application of therapeutic antibodies and

maximum antibacterial effect with minimal dosage (Cook and Trott, 2010). Ideally a carrier should be inexpensive, non-toxic, easy to handle, and readily available. Previous research suggests that clay minerals may be ideal carriers for this purpose (Herrera et al., 2004). Clay minerals are products of the chemical and physical change involved in the erosion of rocks (Millot, 1979). In clays the elements silicon, aluminum, oxygen, iron, magnesium, sodium, and potassium are arranged in a regular crystalline structure. During the process of weathering, water carries away or introduces new elements into the crystalline matrix, altering its chemical composition. Thus clay minerals represent a chemically diverse class of materials. Clays have a high affinity for and readily adsorb a diverse array of organic compounds, including nucleic acids and proteins. Multiple studies have researched the binding and activity of proteins bound to clay particles (Sanjay and Sugunan, 2005; Lee et al., 2003). Lee et al. (2003) described the binding and activity of insecticidal proteins from Bacillus thuringiensis subsp. israelensis. Equilibrium adsorption of the insecticidal toxins was rapid and concentration-dependent. Adsorption was pH dependent suggesting that binding was due to electrochemical adsorption. However,

suggests the need to develop protective measures to ensure that specific antibodies can survive as they travel through the intestinal tract prior to reaching their target microorganism.

attempts to desorb the toxins from the clay released only 2 to 12% of the absorbed toxins signifying the clay had a high affinity for the toxins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) revealed that adsorption did not alter the

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The disadvantage in using antibodies clinically to treat gastrointestinal disease is their instability under gastrointestinal conditions. To be therapeutically useful, the antibodies have to avoid denaturation by stomach acid and proteolytic digestion by the intestinal enzymes (Cook and Trott, 2010). Secondly, they must be transported to the intestines in sufficient concentration in order to exert their therapeutic effects. What is needed is a method to protect and deliver the antibody to the intestinal tract to achieve

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structure of the toxin proteins. In fact the insecticidal activity of the bound proteins was greater than that of the unbound proteins. The study also suggested that adsorption of the toxins to clay could protect them from environmental degradation. The insecticidal activity of the bound proteins in non-sterile water after 45 d was greater than that of free proteins as 63% versus 25%, respectively. Sanjay and Sugunan (2005) described the binding of Îą-amylase on acid activated montmorillonite. Isothermal analysis, x-ray diffraction, and nuclear magnetic resonance revealed that the enzyme was bound to the clay by both electrochemical adsorption and covalent binding. The ability of the bound enzyme to hydrolyze

antibodies from gastrointestinal conditions allowing them to exert their antibacterial effects in the lower intestine.

starch was subsequently investigated. The bound enzyme retained enzymatic activity and exhibited a greater stability over a wider range of pH and temperature compared to free enzyme. There are procedures which can be used to attach antibodies and other proteins to silicate surfaces (Pierce Biotechnology, Inc., 2006; Zhao et al., 2004). One such procedure involves derivatizing the surface of the adsorbant with primary amines (-NH2) using an aminosilane reagent. The amines are then reacted to the heterobifunctional crosslinker Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (Sulfo-SMCC), resulting in a maleimideactivated surface. This crosslinker is able to react with sulfhydryl groups on antibodies. The antibodies are subsequently treated in order to cleave disulfide bonds (-S-S-) and reduce the sulfurs to sulfhydryls (-SH). The antibodies and silica surface can be reacted with each other to form a crosslinked composite material. While these procedures are effective, they are also time consuming and expensive. However, caution must be observed when activating the antibodies in preparation for crosslinking, as the chemical reaction used in activation can also denature the protein, rendering it non-functional. The binding of antibodies to clay should be relatively quick and in-

trol Salmonella in food animals. Egg yolk antibodies have several advantages over mammalian antibodies from sera of immunized host animals; (1) they are less expensive; (2) they can be collected in larger amounts and higher concentration; (3) their collecting procedure is simpler and do not require animal bleeding. Egg yolk antibodies also have a great potential as a therapeutic agent to prevent infectious diarrheal diseases in food animals, even though there are limitations to overcome to use these antibodies for the therapeutic applications such as finding a dependable carrier and avoiding denaturation. With developing stable and dependable carriers of these antibodies, it is expected egg yolk antibodies will be used in various applications in detection of other pathogens and control of infectious diseases in food animals.

CONCLUSIONS Salmonella have been a major foodborne pathogen problem in food production and processing systems and therefore the sensitive detection and control strategies for Salmonella are highly desirable for food safety. Use of egg yolk antibodies has been studied as a component of immunoassays for Salmonella detection and as a therapeutic agent to con-

ACKNOWLEDGEMENTS We thank the Cell and Molecular Biology (CEMB) program at the University of Arkansas in Fayetteville, AR, for supporting a graduate student assistantship to S. Park.

expensive due to the high surface area, cationic exchange capacity and the availability of the clay materials. Presumably, the immunogenic activity of the antibodies would not be affected by the adsorption process. Finally, the clay materials should protect the Agric. Food Anal. Bacteriol. â&#x20AC;˘ AFABjournal.com â&#x20AC;˘ Vol. 3, Issue 3 - 2013

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zinc in poultry nutrition, immunity, and reproduction. Biological Trace Element Res. 101:147-163. Park, S.Y.,W.K. Kim, S.G. Birkhold, L.F. Kubena, D.J. Nisbet, and S.C. Ricke, 2004c. Induced moulting issues and alternative dietary strategies for the egg industry in the United States. World’s Poultry Science J. 60:196-209. Park, S.Y., W.K. Kim, S.G. Birkhold, L.F. Kubena, D.J. Nisbet, and S.C. Ricke. 2004d. Using a feed grade zinc propionate to achieve molt induction in laying hens and retain postmolt egg production and quality. Biological Trace Element Res. 101:165-179. Patrick, M.E., P.M. Adcock, T.M. Gomez, S.F. Altekruse, B.H. Holland, R.V. Tauxe, and D.L. Swerdlow.

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2004. Salmonella Enteritidis infections, United States, 1985-1999. Emerg. Infect. Dis. 10:1-7. Patterson, J.A., V.I. Chalova, R.B. Hespell, and S.C. Ricke. 2010. Dilution rates influence ammonia assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture. J. Appl. Microbiol. 108:357-365. Patterson, R., J. S. Youngner, W.O. Weigle, and F. J. Dixon. 1962a. The metabolism of serum proteins in the hen and chick and secretion of serum proteins by the ovary of the hen. J. Gen. Physiol. 45:501-513. Patterson, R., J. S. Youngner, W.O. Weigle, and F. J. Dixon. 1962b. Antibody production and transfer to egg yolk in chickens. J. Immunol. 89:272-278. Pendleton, S., I. Hanning, D. Biswas, and, S.C. Ricke. 2013. Evaluation of whole genome sequencing as a genotyping tool for Campylobacter jejuni in comparison with pulsed-field gel electrophoresis and flaA typing. Poultry Sci. 92:573–580. Peralta, R.C., H. Yokoyama, Y. Ikemori, M. Kuroki, and Y. Kodama. 1994. Passive immunization against experimental salmonellosis in mice by orally administratered hen egg-yolk antibodies specific for 14- kDa fimbriae of Salmonella Enteritidis. J. Med. Microbiol. 41:29–35.

body. Int. J. Poult. Sci. 6:230–235. Ricke, S.C. 2003a. The gastrointestinal tract ecology of Salmonella Enteritidis colonization in molting hens. Poultry Sci. 82:1003-1007. Ricke, S.C. 2003b. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poultry Sci. 82:632-639. Ricke, S.C. and S.D. Pillai. 1999. Conventional and molecular methods for understanding probiotic bacteria functionality in gastrointestinal tracts. Crit. Reviews Microbiol. 25:19-38. Ricke, S.C. and D.M. Schaefer. 1996a. Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride. Appl. Microbiol. Biotechnol. 46:169-175. Ricke, S.C. and D.M. Schaefer. 1996b. Nitrogen-limited growth response of ruminal bacterium Selenomonas ruminantium strain D to methylamine addition in a minimal medium. J. Rapid Methods Automation Microbiol. 4:297-306. Ricke, S.C. and D.M. Schaefer. 1996c. Glucose fermentation and growth of Selenomonas sputigena on a minimal medium. J. Rapid Methods Automation Microbiol. 4:173-181. Ricke, S.C. and D.M. Schaefer. 1991. Growth inhi-

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MINI-REVIEW Potential for Dry Thermal Treatments to Eliminate Foodborne Pathogens on Sprout Seeds T. Hagger1 and R. Morawicki1 1

Food Science Department, 2650 Young Ave., University of Arkansas, Fayetteville, AR

ABSTRACT Consumption of raw sprouts have been associated with several outbreaks of foodborne diseases. Contaminated seeds used to produce sprouts are the main source of pathogenic microorganisms that multiply during the sprouting process, which is favored by high moisture contents and temperatures in the optimal range for microbial growth. The current intervention recommended by the Food and Drug Administration to decrease seeds’ microbial load is the use of 20,000-ppm calcium hypochlorite, which produces a reduction of around 3-logarithmic cycles for Escherichia coli and Salmonella. Therefore, there is a need for new procedures to further reduce or eliminate microorganisms in seeds used for the preparation of sprouts. One potential treatment is the application of dry thermal treatments which have been used for decades to reduce plant pathogens from seeds to eliminate pathogenic E. coli and Salmonella in seeds used for sprout production while preserving seed vigor and viability. This review will discuss the potential for dry heat treatment of seeds from the Brassicaceae and Leguminosae families to reduce contaminated pathogenic microorganisms. Keywords: Pathogens, dry heat, sprout seeds Agric. Food Anal. Bacteriol. 3: 218-229, 2013

INTRODUCTION oodborne disease outbreaks associated with vegetables and vegetable processing continue to be one of major sources of public health and economic concerns associated with food systems (Hedberg et al., 1999; Sewell and Farber, 2001; SivavapalasCorrespondence: Ruben Morawicki, rmorawic@uark.edu Tel: +1 -479-575-2980

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ingham et al., 2004; Hanning et al., 2008; 2009). Although most of the primary bacterial pathogens and viruses responsible for the majority of the foodborne illnesses have been historically identified with a particular animal food source, such as poultry for Salmonella and Campylobacter (Ricke, 2003b; Park et al., 2008; Dunkley et al., 2009; Horrocks et al., 2009; Foley et al., 2011; Finstad et al., 2012; Ricke et al, 2013), beef for pathogenic E. coli (Anderson et al., 2009; Callaway et al., 2013), and retail deli meats for

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Listeria (Lungu et al., 2010; Crandall et al., 2011; Milillo et al, 2012) most of these same pathogens can contaminate vegetable crops as well. The fact that many vegetable products are consumed raw further increases the risk as pathogens are capable of growing on these raw products (Escartin et al., 1989; Asplund and Nurmi, 1991; Abdul-Raouf et al., 1993; Hedberg et al., 1999; Nutt et al., 2003a,b). It has also been shown that supernatants derived from centrifugation of some raw vegetables after mechanical stomaching will not only support growth but increase virulence in pathogens such as Salmonella (2003a,b; 2004). Part of the difficulty in assessing the impact of

SPROUT PRODUCTION AND FOODBORNE DISEASE

potential foodborne pathogen contamination is the multitude of routes and sources that these pathogens can originate from at the production as well as the retail side (Sagoo et al., 2003; Gibson and Ricke, 2012; Neal et al., 2012b). Routes and sources of contamination include manure used to fertilize fields, aerosols from contaminated wastes, contaminated irrigation water, contaminated wash water used during processing, and sick humans, who handle the produce, just to name the more extensively studied sources (Beuchat and Ryu, 1997; Pillai and Ricke, 2002; Islam et al., 2004; Fonseca and Ravishankar, 2007; Jay et al., 2007). Raw sprouts represent one aspect of vegetable commodity production that has proven to be somewhat difficult to construct consistently effective and comprehensive food safety protocols for their application during production. Sprouts are produced from seeds that are germinated in high moisture environments at temperatures that are optimal for the development of pathogenic bacteria. Contaminated seeds are the main source of microorganisms during sprouts production, and certainly, the fact that sprouts can be consumed raw is a primary issue that promotes food borne illnesses. However, there are limited interventions available that have been shown effective to eliminate patho-

isms have presented significant challenges to the sprout industry. Recommendations were introduced in 1999 by the Food and Drug Administration to treat sprout seeds with bleach (calcium hypochlorite) to reduce pathogenic loads; however, the recommended treatment has limited effectiveness (Brooks et al,. 2001; Fett, 2002; Montville and Schaffner, 2004). In addition, gastrointestinal illnesses from the consumption of raw sprouts continue. The Centers for Disease Control and Prevention (2012) reported a very recent multi-state outbreak of a Shiga-toxin producing strain of Escherichia coli (STEC) associated with clover sprouts with a hospitalization rate of more than 25% of those reportedly affected. This most recent outbreak did not result in any known cases of hemolytic uremic syndrome (HUS) or deaths. However, another strain of STEC on fenugreek sprouts in Germany (in 2011) was responsible for approximately 50 deaths and 850 cases of HUS. The increasing popularity of sprouts and the sprouting conditions that permit a single pathogenic cell to grow to an infective dose is very problematic, particularly for immune-compromised individuals that are highly susceptible to severe complications from foodborne illness. There is no single treatment method, including the Food and Drug Administration -recommended

gens while retaining seeds vigor and germination rate. This review will offer discussion on raw sprouts as a source of foodborne pathogens, current control measures, and the potential for dry heat as an alternative treatment of seeds.

chemical treatment that completely destroys all Salmonella and E. coli on contaminated seeds (Montville and Schaffner, 2005) therefore, the purpose of this review is to discuss safe, non-chemical treatments of sprout seeds that could be made commer-

Sprouts are primarily consumed raw in the United States and are derived from numerous types of seeds, including beans, radishes, and alfalfa. Nutritionally, they are considered a good source of amino acids, oligopeptides, fiber, vitamins, trace elements and minerals as well as phytochemicals with purported health benefits (Marton, et al., 2010). In the United States, sprouts are produced commercially and by individuals using home sprouters. As the popularity of sprouts has increased, the outbreaks of foodborne illness related to pathogenic microorgan-

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Figure 1. Sprout Production Process (Adapted from NACMCF, 1999)

cially-feasible and would potentially alleviate health risks associated with sprout consumption.

Although sprout-related illnesses are predominantly attributed to Salmonella spp. and enterohemorrhagic E. coli (serotype O157:H7), there have been outbreaks involving other STECs, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Aeromonas hydrophila (Bari et al., 2010). The seeds are considered the primary source of contamination with exponential growth of the microorgan-

days (Hu et al., 2004). Although contamination during sprouting, irrigation, and post-harvesting is possible, the majority of cases of foodborne-illness are attributed to contaminated seeds which is likely from the use of contaminated irrigation water or fertilizer, fecal contamination from animals (residing in neighboring fields or wild animals with access to the seed fields), or inadequate hygiene practices during seed collection (National Advisory Committee on Microbiological Criteria for Foods (NACMCF) 1999). Additionally, treatment of the sprouts after germination is impractical due to the delicate nature of the sprouts. Treatment of seeds is considered more effective due to the lower microbial load and potential problems

isms throughout sprouting due to the conditions (temperature and moisture) maintained during the sprouting process (Figure 1), which can permit a single viable pathogen cell to grow to an infective dose of 2 to 3 log colony forming units (CFU)/g in only 2

with bacteria located within the sprout tissues (Penas et al., 2010) therefore elimination of the pathogens on the seed and the maintenance of sterilized conditions for growth are the most favorable preventative methods.

PATHOGENIC BACTERIA ON SPROUTS

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Table 1. Classifications and common names of sprout seeds (Adapted from Sproutpeople, n.d.) Family

Common Names

Amaranthaceae

Amaranth

Amaryllidaceae

Garlic chive, leek, onion

Brassicaceae/Cruciferae

Broccoli, cabbage, mustard, tatsoi, arugula, mizuna, garden cress, radish

Chenopodiaceae

Quinoa

Compositae

Sunflower

Cucurbitaceae

Pumpkin

Gramineae/Poaceae

Oats, barley, millet, rye, wheat, spelt, triticale, corn

Leguminosae

Peanut, garbanzo bean, soybean, lentil, alfalfa, black bean, pinto bean, pea, clover, fenugreek, adzuki bean, mung bean

Linaceae

Flax

Pedaliaceae

Sesame

Polygonacea

Buckwheat

Rosaceae

Almond

Umbelliferae

Celery, dill

SOURCES OF SPROUTS

rates of treated seeds should be evaluated on a case-by-case basis (Bari et al., 2010).

Sprouts are derived from a wide variety of seeds including grains, beans, nuts, and various brassica vegetables (Table 1). Although alfalfa and mung bean sprouts are among the more well-known varieties, there are numerous other legumes, such as peanuts, soybeans, lentils, and peas that are consumed as sprouts (Sproutpeople, n.d.). Other seed sources are eclectic and range from onion to almond to sunflower; thus, sprout consumption is diverse which generates additional challenges in seed treatments since some seeds may be more susceptible to certain treatments. For example, mung bean seed coats are tough and considered relatively heat resistant thus less affected by heat treatment than other seeds (Bari et al., 2010). Consequently, germination

CURRENT ANTIMICROBIAL TREATMENTS The current recommendations by the Food and Drug Administration for reducing pathogenic loads on sprouts is to treat the seeds (prior to sprouting) with 20,000 ppm Ca(OCl)2 or other approved antimicrobial agents (Food and Drug Administration, 1999). The recommended treatment does not completely eliminate the pathogenic load with a mean reduction of only 2.81 log CFU/g and 3.21 log CFU/g for E. coli and Salmonella, respectively (Fett, 2002; Food and Drug Administration, 1999; Montville, and Schaffner 2004). Based on a case study of an outbreak of

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Salmonella Typhimurium on treated and untreated sprout seeds, Brooks et al. (2001) concluded that the risk of infection is only reduced but not completely eliminated with the Food and Drug Administration -recommended treatment. In addition, there is significant waste generated with chlorinated water posing ecological and economic challenges for sprout producers (Bari et al., 2010). Lastly, the majority of raw sprout consumers are demographically classified as health-conscience individuals that are generally averse to chemically-treated products; thus, there is significant interest in effective, safe, and natural approaches to the removal of E. coli and Salmonella. Since simply washing the seeds or the sprouts with

mic reduction of all pathogens (Kim et al., 2006, Nei, et al. 2011, Pandrangi et al., 2003). There are other potential chemical antimicrobial treatments that have been evaluated in other food model systems or against pure cultures (Aldrich et al., 2011; Ganesh et al., 2012; Muralli et al., 2012; Neal et al., 2012c). Irradiation, ultrasound, and pressure have also been examined under a variety of conditions for various food systems but were generally inadequate methods to eradicate inoculated pathogens without combining with other treatments (Penas et al., 2010; Kim et al., 2006; O’Bryan et al., 2008). Overall, combining hurdles (or treatments), often a physical and a chemical treatment, is considered

sterilized water is insufficient to remove the pathogens, antimicrobial treatment is the only mechanism to reduce the microbial load. Various physical and chemical treatments have been applied to seeds to enhance the chlorine efficacy, reduce the effective chlorine dose, or to provide an alternative to the use of chlorine altogether, but the results have been inconsistent. This may be partly attributed to bacteria embedded deep in crevices, naturally occurring on the surface of certain seeds or generated by damage during handling that are not readily removed with traditional antimicrobial washes (Takeuchi and Frank, 2000; 2001; Enomoto et al. 2002; Solomon et al., 2002; Takeuchi et al., 2002; Wachtel et al., 2002; Feng et al., 2007; Brandl, 2008; Gomes et al., 2009; Kroupitski et al., 2009; Erickson, 2012; Neal et al., 2012a). Gandhi et al., (2001) used a green fluorescent protein expressing Salmonella Stanley to demonstrate this microorganism’s ability to penetrate alfalfa sprout tissue. In addition, extreme conditions can dramatically reduce seed viability so germination rates limit the extent of treatment. Sodium bicarbonate, trisodium phosphate, acetic acid, hydrogen peroxide, ethanol, commercial peroxyacetate solutions, and acidic electrolyzed water (a solution comprised of less than 80 mg/L free chlorine with a

more effective than isolated treatments; however, although combinatorial methods can dramatically reduce microbial loads, most are incapable of fully eliminating Salmonella and E. coli (Penas et al., 2010; Beuchat and Scouten, 2002; Ricke, 2003b; Ricke et al., 2005; Sirsat et al., 2009). Furthermore, selection of antimicrobials must proceed with caution as there is potential for cross protection among different antimicrobials which renders the combinations less effective (Kwon et al., 2000; Sirsat et al., 2010). This has led to the concept of using genomic screening tools to better predict when cross protection might occur by identifying which gene(s) or gene families are shared for the respective microorganism to successfully resist multiple antimicrobials (Sirsat et al., 2010). Genomic analysis based on transcriptome microarrays have been applied to assess potential genetic responses in foodborne pathogens such as Salmonella and Listeria to either external antimicrobial combinations or intrinsic food properties generated during food processing (Milillo et al., 2011, 2012; Sirsat et al., 2011; Chalova et al., 2012). Combination treatments are also generally cumbersome and, in some cases, expensive so practical usage would be very limited.

high oxidation-reduction potential and a pH range of 2.3 to 2.7) are some of the chemical treatments evaluated over the past decade with varying results but no single method is able to achieve the Food and Drug Administration recommended 5-logrith-

THERMAL ANTIMICROBIAL TREATMENTS

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The application of heat (also known as “thermotherapy”) to eradicate various phytopathogens from seeds is an agronomic practice that has existed for

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Table 2. Treatment conditions used in various studies to determine the most effective hot water treatment of seeds

Temp

Time

References

2 d, 5 d, 10 d, 15 d, 20 d

Feng et al., 2007; Beuchat and Scouten, 2002; Neetoo and Chen, 2011; Hu et al., 2004

24 h, 4 d, 8 d, 12 d, 15 d

Neetoo and Chen, 2011; Bang et al., 2011

12 h, 24 h, 3 d, 6 d, 12 d

Neetoo and Chen, 2011

2 h, 5 h, 10 h, 15 h, 24 h

Neetoo and Chen, 2011; Bang et al., 2011

15 min, 60 min, 3 h, 6 h, 12 h

Beuchat and Scouten, 2002; Neetoo and Chen, 2011

10 min, 60 min, 2 h, 4 h, 8 h

Beuchat and Scouten, 2002; Neetoo and Chen, 2011

(oC)

over a century and is used to reduce the losses attributed to diseased plants (Baker, 1962). The basic premise is that the seed can withstand slightly greater thermal treatments than the host pathogens (fungal, bacterial, or viral) thereby eliminating pathogens but retaining germination capabilities of the seed (Jensen, 1888; Grondeau and Samson, 1994). In modern horticulture, treatment of seeds by hot water, dry heat, and hot, moist air is still a practical, ecological alternative to chemical treatments (Gilbert, 2009; Forsberg et al., 2005; Miller and Lewis-Ivey, 2005). The use of prolonged dry heat as opposed to hot water or steam to reduce plant pathogen loads has been primarily used over the last 30 years (Luthra, 1953). However, in India, the practice of controlling loose smut (a fungal infection) in wheat by exposure to solar radiation was successfully implemented as early as 1929 (Luthra, 1953). In spite of the lengthier

sonable that heat treatment of sprout seeds to kill bacteria that are pathogenic to humans is a viable and acceptable alternative to chemicals. Although, outbreaks are not as common in Japan since most sprouts are consumed in cooked dishes hot water treatments of sprout seeds is a relatively common practice in Japan (Bari et al., 2010). However, in the United States, sprouts are primarily consumed raw so the efficacy of the method to eliminate pathogenic microorganisms remains under scrutiny (Bari et al., 2010). Although several studies have evaluated hot water treatment of seeds as a solitary treatment or in combination with other hurdles (Table 2) (Bari et al., 2010; Enomoto et al., 2002; Kim et al., 2006) there is still limited and conflicting data regarding the use of dry heat on sprout seeds. One of the initial studies demonstrated that dry heat treatment was a highly promising technique for mung bean seeds (Hu et al.,

treatment times, the application of dry heat generally causes less damage to the seed than hot water (Grondeau and Samson, 1994; Feng et al., 2007). Since thermotherapy of all types is still used in horticulture to control plant pathogens, it is rea-

2004). Even three days post-sprouting, the authors demonstrated that the levels of inoculated E. coli O157:H7 and Salmonella remained non-detectible if seeds were treated at 55Â°C for four and five days, respectively.

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Although studies that have attempted to treat alfalfa seeds with dry heat have been confounded by the thermostability of Salmonella, there is evidence that the method is still a viable option. Neetoo and Chen (2011) were able to successfully reduce both pathogens to non-detectible levels by treating the seeds at 65°C for 10 days while maintaining germination rates greater than 90%. The lengthy treatment was necessary due to the heat tolerance of Salmonella; E. coli was eliminated after only 2 days. In contrast to this response, Feng et al. (2007) demonstrated that holding alfalfa seeds at 55°C for eight days was insufficient to eliminate Salmonella with exponential growth of the pathogen observed during the three-day sprouting period. The same study demonstrated, however, that a six day treatment at 55°C very effectively controlled E. coli. The differences in the results of the two studies are likely attributable to the extent of heat treatment (55°C for eight days versus 65°C for 10 days); thus, further studies are warranted to determine optimum conditions. Additionally, there was no evidence presented by Neetoo and Chen (2011) that damaged bacteria would not recover and still grow on the sprouts since they only evaluated the pathogenic load of the seeds. Interestingly, a recent study by Bang et al. (2011) demonstrated that humidity control (i.e. RH of 23%) during heating can also permit longer heating times and higher temperatures with minimal effects on germination; therefore, the implementation of humidity control during dry heating may ensure complete elimination of the more recalcitrant bacteria. Although the use of dry heat alone as a means to control bacterial pathogens has not been extensively evaluated, dry heat has been combined with several different treatments with relatively good outcomes including various chemical (sodium hypochlorite, chlorine dioxide, ethanol, phytic acid, oxalic acid) and physical (radiation, pressure) hurdles (Bang et al, 2011; Kim et al, 2010; Neetoo and Chen, 2011; Bari et al, 2009). In general, the primary interest in using combinatorial techniques is to reduce the length of time necessary to treat the seeds with the dry heat. For example, although Neetoo and Chen (2011) were able to control pathogens with dry heat 224

alone, they also demonstrated that high hydrostatic pressure applied after heat treatment reduced the effective dry heat application time from 10 days to 12 hours. Although the combinatorial technique was effective, the need to implement pressure treatments would be significantly more laborious for seed farmers and would require additional equipment and supplies. Ideally, dry heat treatments alone would be a preferred method, but the efficacy of treatments needs to be systematically evaluated and optimized for each seed type to reduce losses in germination and retain non-detectible levels of pathogens in sprouts.

CONCLUSIONS Sprouts continue to be a source for foodborne disease outbreaks when consumed raw. Microbial contamination including foodborne pathogens occurs early in sprout production primarily via contaminated seeds which as they sprout, support microbial growth due to the favorable moisture and temperature conditions. To reduce microbial levels on seed the Food and Drug Administration recommends application of 20,000-ppm calcium hypochlorite that will lead to a 3-logarithmic reduction of Escherichia coli and Salmonella. To further reduce or eliminate microorganisms and foodborne pathogens in seeds that serve as a source of raw sprouts for human consumption will require interventions that are more effective. Dry thermal treatments have been used for decades to reduce plant pathogens from seeds and offer a potential treatment to eliminate pathogenic E. coli and Salmonella in seeds. However, preservation of seed vigor and viability must be retained and unfortunately some strains of foodborne pathogens can be somewhat heat resistant. Overcoming this will probably require combining dry heat treatment with some additional antimicrobial treatments to achieve synergism in the form of a multiple hurdle intervention approach and thus a more effective reduction in foodborne pathogen levels. Designing optimal multiple hurdle intervention strategies will require not only testing under conditions similar to

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the seed environment that the foodborne pathogen is associated with but a better understanding of the biology and genetic responses of the microorganism in the presence of the antimicrobials being used as potential interventions.

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Issues of Food Animal Derived Foods, S.C. Ricke and F.T. Jones (eds.) University of Arkansas Press, Fayetteville, AR. Sirsat, S.A., A. Muthaiyan, and S.C. Ricke. 2009. Antimicrobials for pathogen reduction in organic and natural poultry production. J. Appl. Poultry Res. 18:379-388. Sivavapalasingham, S., C. R. Friedman, L. Cohen, and R.V. Tauxe. 2004. Fresh Produce: A growing cause of outbreaks of foodborne illness in the United States, 1973 through 1997. J. Food Prot. 67:2342–2353. Solomon, E. B., S. Yaron, and K. R. Matthews. 2002. Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internaliza¬tion. Appl. Environ. Microbiol. 68:397-400. Sproutpeople. Sprouting seed taxonomy. http:// sproutpeople.org/sprouts/grow/taxonomy.html. Takeuchi, K., and J. F. Frank. 2001. Quantitative de¬termination of the role of lettuce leaf structures in protecting Escherichia coli O157:H7 from chlorine disinfection. J. Food Prot. 64:147-151. Takeuchi, K., and J. F. Frank. 2000. Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability. J. Food Prot. 63:434-440. Takeuchi, K., C. M. Matute, A. N. Hassan, and J. F. Frank. 2000. Comparison of the attachment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas fluorescens to lettuce leaves. J. Food Prot. 63:1433-1437. Wachtel, M. R., L. C. Whitehand, and R. E. Mandrell. 2002. Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contami¬nated irrigation water. J. Food Prot. 65:18-25. Weiss, A., and W.P. Hammes. 2003. Thermal seed treatment to improve the food safety status of sprouts. J. Appl. Bot. 77:152-155.

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Suitability of Various Prepeptides and Prepropeptides for the Production and Secretion of Heterologous Proteins by Bacillus megaterium or Bacillus licheniformis S. Saengkerdsub1,2, Rohana. Liyanage3, Jackson Oliver Lay Jr.3 1

Center for Food Safety, and Department of Food Science, University of Arkansas, Fayetteville, AR, 72704 2 Department of Poultry Science, University of Arkansas, Fayetteville, AR 72701 3 State Wide Mass Spectrometry Facility, Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701

ABSTRACT Secretion of heterologous proteins using Bacillus species is a well-established system, but inefficient translocation of the proteinacross the plasma membrane is a problem with these expression hosts. A recent study demonstrated that the prepropeptide of Staphylococcus hyicus lipase enhanced the secretion of heterologous proteins in B. subtilis. In the study reported here, prepropeptides of S. hyicus lipase, B. subtilis nprE, and B. megaterium nprM were investigated by using hydrolase from T. fusca (Tfh) and E. coli alkaline phosphatase PhoA as models. The results indicate that the secreted Tfh and PhoA activities were lower when the proteins fused with propeptides, compared to those without propeptides. Only propeptide S. hyicus lipase protected Propionibacterium acnes linoleic acid isomerase from proteolytic degradation and did not impede the translocation. However, no activity of isomerase was detectable. Linoleic acid isomerase was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometryand it was discovered that the propeptide was still attached with secreted protein. In addition, the results of the propeptide S. hyicus lipase fused with B. subtilis nprE demonstrated that enzymatic activities were interfered with by the attached propeptide S. hyicus lipase. Keywords: Heterologous protein, Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, prepropeptide, Staphylococcus hyicus Agric. Food Anal. Bacteriol. 3: 230-248, 2013

INTRODUCTION Heterologous protein production in bacterial systems, particularly Escherichia coli, is economical due Correspondence: Suwat Saengkerdsub, saengsuwat@yahoo.com

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to the ability of bacteria to grow rapidly and yield high cell concentrations on inexpensive substrates (Terpe, 2006). However, inclusion body formation, incorrect protein folding, and inefficient bond formation may occur during intracellular production in E. coli (Schallmey et al., 2004) . Using Bacillus species as the host is an alternative way to produce heterolo-

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gous proteins that are secreted into the surrounding medium and avoids the problems associated with E. coli (Pohl and Harwood, 2010). Bacillus subtilis is widely used in industry to produce enzymes that are naturally produced by it as well as by closely related species and these enzymes are released into the growth medium in commercially relevant quantities (Harwood and Cranenburgh, 2008). However when used for production of heterologous proteins there may be problems with translocating the foreign protein across the plasma membrane, release of the protein from the cell envelope into the medium, or with the correct folding of the protein (Bolhuis et al, 1999; Kouwen et al., 2010;

B-14212. Prepropeptides from S. hyicus lipase, B. subtilis nprE, and B. megaterium nprM were evaluated for promoting heterologous secretion by using hydrolase from Thermobifida fusca and E. coli alkaline phosphatase PhoA as models. The prepropeptide from S. hyicus lipase was chosen because it had been previously demonstrated to be successful for enhancing E. coli alkaline phosphatase PhoA secretion in B. subtilis (Kouwen et al., 2010) while B. subtilis nprE, and B. megaterium nprM are the major extracellular proteases in B. subtilis, and B. megaterium, respectively.

Puohiniemi et al., 1992; Saunders et al., 1987). Proteins that are destined to act outside of a cell are taken from the site of synthesis to the cell membrane with the help of pre-peptide helper proteins (von Heijne 1990a; von Heijne 1990b). Most proteins are translocated across the membrane in an unfolded state, the signal peptide is cleaved and the protein then folded (Tjalsma et al., 2004; van Wely et al., 2001). Many proteins are also produced with a propeptide that acts as a chaperone to assist in folding and release from the plasma membrane (Sarvas et al., 2004; Shinde and Inouye, 2000; Takagi et al., 2001). Since these signal proteins have been optimized for specific proteins in particular bacteria they may not function as efficiently for the production of heterologous proteins (Kouwen et al., 2010), leading to a search for better secretion signals for heterologous proteins in organisms such as B. subtilis (Brockmeier et al., 2006). A recent study demonstrated that the prepropeptide of Staphylococcus hyicus lipase enhanced the secretion of heterologous proteins in B. subtilis (Kouwen et al., 2010) . Bacillus megaterium in contrast to B. subtilis has the advantage of highly stable, freely replicating plasmids (Vary, 1994). In addition, a recent study developed expression plasmids for maximizing heter-

MATERIALS AND METHODS

ologous protein production (Stammen et al., 2010). In this study we investigated the suitability of several prepeptides and prepropeptides for the heterologous production and secretion of proteins by B. megaterium YYBm1 and Bacillus licheniformis NRRL

duction hosts. Antibiotics were used at the following concentrations: ampicillin, 100 μg/mL and tetracycline 10 μg/mL.

Plasmids and bacterial growth conditions Plasmids used in this study are listed in Table 1. All B. megaterium and B. licheniform is transformed with plasmids were grown in baffled shake flasks at 30 or 37°C in Luria-Bertani (LB) medium (BD, Franklin Lakes, NJ), TM medium (Takara Bio Inc. USA, Mountain View, CA), 2SY medium (Takara Bio Inc. USA), Modified Plasma Broth (MPB) medium (Chiang et al., 2010), or Modified Super Rich (MSR) medium (Chiang et al., 2010) at 200 rpm. Recombinant expression of genes under transcriptional control of the xyloseinducible promoter was induced by the addition of 0.5% (w/v) xylose at OD578 of 0.4.

DNA manipulation for the construction of plasmids The synthetic oligonucleotides used in this work are listed in Table S1 in the supplemental material. E. coli 10G (Lucigen, Middleton, WI) was used for all cloning purposes. B. megaterium YYBm1 and B. licheniformis NRRL B-14212 were used as protein pro-

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Table 1. Plasmids used in this study Plasmid

Description

Source

pSSBm97

PxylA-(-35+rbs+)-prepeptideyocH-tfh

Stammen et al., (2010)

pPlip

prepeptide of S. hyicus lipA lipase inserted into BsrGI and SpeI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptidelipA-tfh

This study

pPPlip

prepropeptide of S. hyicus lipA lipase inserted into BsrGI and SpeI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptidelipA-propeptidelipA-tfh

This study

pPnprE

prepeptide of B. subtilis nprE neutral protease inserted into BsrGI and SpeI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptidenprE-tfh

This study

pPPnprE

prepropeptide of B. subtilis nprE neutral protease inserted into BsrGI and SpeI sites of pSSBm97; This study PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprE-tfh

pPnprM

prepeptide of B. megaterium nprM neutral protease inserted into BsrGI and SpeI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptidenprM-tfh

This study

pPPnprM

prepropeptide of B. megaterium nprM neutral protease inserted into BsrGI and SpeI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprM-tfh

This study

pCR®4-TOPO

Plasmid used for cloning PCR product

Invitrogen

pA97

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pSSBm97; This study PxylA-(-35+rbs+)-prepeptideyocH-PhoA

pAPL

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPlip; This study PxylA-(-35+rbs+)-prepeptidelipA-PhoA

pAPPL

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPPlip; This study PxylA-(-35+rbs+)-prepeptidelipA-propeptidelipA-PhoA

pAPE

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPnprE; This study PxylA-(-35+rbs+)-prepeptidenprE-PhoA

pAPPE

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPPnprE; This study PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprE-PhoA

pAPM

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPnprM; This study PxylA-(-35+rbs+)-prepeptidenprM-PhoA

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Table 1. (Continued)

Plasmid

Description

Source

pAPPM

E. coli phoA alkaline phosphatase inserted into SpeI and EagI sites of pPPnprM; This study PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprM-PhoA

pL97

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pSSBm97; PxylA-(-35+rbs+)-prepeptideyocH-pal

This study

pLPL

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPlip; PxylA-(-35+rbs+)-prepeptidelipA-pal

This study

pLPPL

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPPlip; PxylA-(-35+rbs+)-prepeptidelipA-propeptidelipA-pal

This study

pLPE

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPnprE; PxylA-(-35+rbs+)-prepeptidenprE-pal

This study

pLPPE

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPPnprE; PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprE-pal

This study

pLPM

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPnprM; PxylA-(-35+rbs+)-prepeptidenprM-pal

This study

pLPPM

P. acnes linoleateisomerase inserted into SpeI and EagI sites of pPPnprM; PxylA-(-35+rbs+)-prepeptidenprE-propeptidenprM-pal

This study

pEPL

B. subtilis nprE inserted into SpeI and EagI sites of pPlip; This study PxylA-(-35+rbs+)-prepeptidelipA-nprE

pEPPL

B. subtilis nprE inserted into SpeI and EagI sites of pPPlip; PxylA-(-35+rbs+)-prepeptidelipA-propeptidelipA-nprE

This study

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The basic expression plasmid of this study was pSSBm97. The oligonucleotides lipF, lipR, and lipR2 were used as primers for the PCR to amplify pre- and prepropeptide S. hyicus lipA lipase by using oligonucleotide PL1 to Pu12 as the template. For pre- and prepropeptide B. subtilis nprE neutral protease amplification, the oligonucleotide nprEF, nprER, nprER2 were used as primers and B. subtilis genomic DNA was used as the template. The oligonucleotides nprMF, nprMR, nprMR2 were used as primers for the PCR to amplify the pre- and prepropeptide B. megaterium nprM gene by using B. megaterium DSM319 as the template. Insertion of all PCR products via the corresponding BsrGI and SpeI restriction sites led to

Localization of heterologous protein expression

pPL, pPPL, pPE, pPPE, pPM, and pPPM, respectively. The phoA gene was amplified from E. coli K12 by using primers PhoF and PhoR. The resulting PCR product was cloned into pCR®4-TOPO and later into pSSBm97, pPL, pPPL, pPE, pPPE, pPM, and pPPM after SpeI-EagI digestion, resulting in construction of pA97, pAPL, pAPPL, pAPE, pAPPE, pAPM, pAPPM, respectively. For P. acnes linoleate isomerase, the new DNA sequence was designed by JCat software (http://www. jcat.de/) (Grote et al., 2005) and was synthesized by Integrated DNA Technologies, Coralville, IA. The linoleate isomerase fragment was flanked by SpeI-EagI restriction sites, was digested with these respective enzymes, and subsequently inserted into pSSBm97, pPL, pPPL, pPE, pPPE, pPM, and pPPM after SpeIEagI digestion, creating the plasmids pL97, pLPL, pLPPL, pLPE, pLPPE, pLPM, pLPPM, respectively. The oligonucleotides enprEF and enprER were used as nprE gene amplification by using B. subtilis genome as the template and PCR product was cloned into pCR®4-TOPO. Insertion of the nprE PCR products via the corresponding SpeI and EagI restriction sites in plasmids pPL and pPPL led to pEPL and pEPPL, respectively. Protoplast B. megaterium YYBm1 cells were transformed with the appropriate

Cells of B. megaterium were grown at 30°C at 200 rpm. Cells were separated from the growth medium by centrifugation. The secreted proteins in the growthmedium were collected for SDS-PAGE, gels were stained with Coomassie Brilliant Blue, and the respective protein bands were identified by matrixassisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS).

expression plasmids using a polyethylene glycol-mediated procedure described by Christie et al.(2008) while plasmids were transferred into B. licheniformis NRRLB-14212 by electroporation as described in Xue et al. (1999).

sured as described by Stammen et al. (2010). The enzymatic release of p-nitrophenol was photometrically detected at 410 nm. One enzyme unit was defined as the amount that caused the release of 1 μmol pnitrophenol per minute under the given assay condi-

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The subcellular localization of heterologous protein production was determined using the protocol described in Kouwen et al. (2010). After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membraneand detected with 6X his tag antibody (Abcam, Cambridge, MA) and horseradish peroxidase–anti-rabbit immunoglobulin G conjugates.

Proteomics

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) After Sephadex LH-20 cleanup, the extract was mixed with a 1 M solution of dihydroxybenzoic acid (DHB) in 90% methanol in a 1:1 ratio, and 1 µL of the mixture was spotted onto a ground stainless steel MALDI target for MALDI analysis using the dry droplet method. A Bruker Reflex III MALDI-TOF-MS (Billerica, MA) equipped with a N2 laser (337 nm) was used in the MALDI analysis, and all the data were obtained in positive ion reflectron TOF mode.

Enzymatic activity measurements The hydrolase activity of the enzyme Tfh was mea-

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Figure 1. Secretion of active Tfh. Strain YYBm1 without plasmid was used as a negative control. Samples were taken 6 hours after the xylose supplement. The activities are expressed in U/mL. a,b,c Means within the same sample measurements with unlike superscripts (P < 0.05). Error bars indicate standard deviations between enzyme activities detected for each construct. 3.0000

Tfh activity (U/ml)

2.5000

2.0000

1.5000

1.0000 b 0.5000

b, c

0.0000 YYBm1

SSBm97

PPL

PPE

PPM

tions. The enzymatic activity was calculated using a molar absorption coefficient of 15,000 M-1 cm-1. Alkaline phosphatase (PhoA) activity was carried out as described by Darmon et al. (2006). Briefly, PhoA activity was determined by measuring the rate of p-nitrophenyl phosphate hydrolysis. Aliquots of fractions were mixed with freshly prepared substrate and incubated at room temperature for 10 to 30 min, and the reactions were stopped by addition of 2 M NaOH. The PhoA activity, expressed in U/ml/unit of optical density at 600 nm (OD600), was determined by

Protease activity was measured as described by Mansfeld and Ulbrich-Hofmann (2007). One unit of proteolytic activity was defined as the amount of enzyme yielding an increase of 0.001 per min in the optical density at 275 nm at 30°C under standard reaction conditions. Linoleate isomerase activity was carried out as described by Peng et al. (2007) . The preparation of fatty acid methyl esters (FAME) was described in Lewis et al. (2000) and FAME was detected by GC with tridecanoic acid as the internal standard. Analyses of the FAME were performed

measuring changes in the optical density at 405 nm as a function of the time of incubation (in minutes) and the OD600. To do this, the following formula was used: [2/3 × (OD405× 352)]/(t × OD600), where t is the time of incubation.

with a Hewlett Packard 5890 GC equipped with a 50 m x 0.32 mm internal diameter cross-linked HP5 methyl silicone (0.17 µm film thickness) fused-silica capillary column and flame ionization detector.

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Figure 2. Secretion of active PhoA. Strain YYBm1 without plasmid was used as a negative control. Samples were taken 6 hours after the xylose supplement. The activities are expressed in U/mL/OD600. a,b Means within the same sample measurements with unlike superscripts (P < 0.05). Error bars indicate standard deviations between enzyme activities detected for each construct.

PhoA activity (U/ml/OD600)

3000

a 2500 a a a

2000 a

1500

1000

500 b

YYBm1

A97

0 APL

Statistical analyses Where standard deviations are presented in the respective figures, these values represent the average of at least triplicate measurements. The statistical tests for treatment effects were performed using an analysis of variance (ANOVA) procedure of Statistica 9.1 Analytical Software (SAS Institute, Cary, NC). Means were further separated using least-significant difference multiple comparisons.

RESULTS Fusion of Tfh and PhoA with prepropeptides from S. hyicus lipase, B. subtilis nprE, and B. megaterium nprM

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APPL

APE

APPE

APM

APPM

Kouwen et al. (2010) demonstrated that the prepropeptide of S. hyicus lipase enhanced the secretion of PhoA in B. subtilis. The prepropeptides of B. subtilis nprE and B. megaterium nprM were chosen because both are major extracellular proteases in these organisms. In this study, all plasmids originated from pSSBm97 (Stammen et al., 2010). Plasmid pSSBm97 contains an optimal the -35 region and the ribosome-binding site, enhancing heterologous production in B. megaterium YYBm1 (Stammen et al., 2010). The secreted amounts of active Tfh were assessed by measuring hydrolase activity in growth medium of cells containing the seven different prepropeptides (Figure 1). Analysis of the medium fractions showed that the highest Tfh activity was detected in growth medium of cells carrying yocH as the prepeptide (pSSBm97), while the levels of Tfh activity were lower

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Figure 3. Secretion of linoleate isomerase in B. megaterium. Cells carrying plasmids were cultured in LB at 37°C and were collected at 6 hours after xylose induction. Proteins from 3 mL of cell-free supernatants from B. megaterium cultures carrying plasmids were precipitated with trichloroacetic acid. Samples were used for SDS-PAGE and Western blotting. The 6X His tag antibody was used to detect the proteins.

Figure 4. Total cell samples of linoleate isomerase in B. megaterium. Cells carrying plasmids were cultured in LB at 37°C and were collected at 6 hours after xylose induction. Proteins from 50 μL of cells were collected by centrifugation. Samples were used for SDS-PAGE and Western blotting. The 6X His tag antibody was used to detect the proteins.

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Figure 5. Secretion and subcellular localization of linoleate isomerase in B. megaterium. Cells carrying pLPPL were cultured in LB at 37°C and were collected at 6 hours after xylose induction. The sample was divided into medium, total cells, cell walls, protoplasts. Protoplasts were incubated for 30 min in the presence of 1 mg/mL of trypsin with or without 1% Triton X-100. Samples were used for SDS-PAGE and Western blotting, and 6X His tag antibody was used to detect the proteins. (Note: medium fraction was from 6 mL culture while other fractions were from 75μL culture).

Figure 6. Secretion of linoleate isomerase in B. megaterium. Cells carrying pLPPL were cultured in LB, 2SY, TM, MPB, and MSR media at 37°C and were collected at 6 hours after xylose induction. Proteins from 6 mL of cell-free supernatants from B. megaterium cultures carrying plasmids were precipitated with trichloroacetic acid. Samples were used for SDS-PAGE and Western blotting. The 6X His tag antibody was used to detect the proteins. (Note: all samples were originated from 6 mL culture).

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(P < 0.05) in the medium of cells expressing other prepropeptides. Surprisingly, the levels of active Tfh in the medium of cells expressing prepropeptide S. hyicus lipase and B. subtilis nprE were significantly lower than those cells containing only prepeptides. For secreted PhoA levels in B. megaterium, seven prepropeptides were fused with PhoA at the N-terminus. Analysis of the medium fractions showed that the levels of PhoA of cells expressing prepropeptides were lower than cells expressing only their pair prepeptides (Figure 2); however, the levels between their pair prepeptides and prepropeptides were not significantly different. In this case, the lowest level of active PhoA in the medium fraction was cells carry-

the other cells with prepropeptides, indicating that these proteins were less stable. To observe the efficiency of translocation across the protoplast membrane, the cells expressing prepropeptide S. hyicus lipase fused with linoleate isomerase were divided into medium, total cell, cell wall, protoplast, protoplast incubated with trypsin, and protoplast incubated with trypsin and Triton X-100 fractions. The proteins from medium, total cell, cell wall, and protoplast fractions were all the same size (Figure 5), representing no processing of translocated linoleate isomerase after crossing the membrane. The protein in protoplasts was degraded when adding trypsin, suggesting that the protein was effec-

ing yocH as prepeptide (pA97).

Linoleate isomerase in P. acnes is the enzyme that isomerizes the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (Deng et al., 2007). The conjugated linoleic acids have been demonstrated to exhibit anticancer, anti-diabetic, and immune-enhancing properties (Pariza, 2004). This gene was expressed in E. coli BL21 (DE3) as the host; unfortunately, the recombinant enzyme formed an inclusion body (Deng et al., 2007). Secreted protein production might solve the formation of insoluble protein inclusion bodies from intracellular accumulations (Schallmey et al., 2004). The major goal of this investigation was extracellular linoleate isomerase production by B. megaterium and B. licheniformis. By using the 6X His tag antibody detection, only cells expressing prepropeptide S. hyicus lipase produced linoleate isomerase (Figure 3). To analyze whether the lack of P. acnes linoleate isomerase production after fusions with prepropeptides, except with prepropeptide S. hyicus lipase, was due to a failure in secretion or to instability of

tively translocated across the protoplast membrane. In order to maximize linoleate isomerase production, the cells carrying pLPPL were cultured under various conditions. The results from the 6X His tag antibody detection showed that MPB enhanced more linoleate isomerase production than other media (Figure 6). In addition, a different set of forms of linoleate isomerase larger than the major band could represent an aggregated form of linoleate isomerase or linoleate isomerase bound to other proteins. In addition to the major protein from cells culturing in MSR medium, minor bands of lower mass reacted with the antibody, suggesting that they represented shortened species of the fusion protein. By culturing at 37°C in MPB medium, the supernatant of cells expressing prepropeptide S. hyicus lipase exhibited smear bands, indicating proteolytic degradation, compared to supernatant from cells cultured at 30°C (Figure 7). The 6X His tag antibody detection showed that the linoleateisomerase was initially detectable 4 hours and increased production up to 8 hoursafter xylose addition (Figure 8). After 8 hours induction, the amounts of protein were reduced due to possible proteolytic degradation. To observe linoleate isomerase activity, B. megaterium YYBm1 carrying pLPPL was cultured in MPB

the protein, the total cell samples were probed with the 6X his tag-specific antibody (Figure 4). Only a distinct band from B. megaterium carrying pLPPL was observed, suggesting that the protein was reasonably stable. No corresponding protein occurred in

medium and the supernatant was collected at 8 hours after xylose addition. The supernatant was incubated with linoleate as described in Peng et al. (2007). FAME was prepared as described in Lewis et al. (2000); however, no activity was detected.

Linoleate isomerase production in B. megaterium and B. licheniformis

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Figure 7. Secretion of linoleate isomerase in B. megaterium. Cells carrying pLPPL were cultured in MPB medium at 30 and 37°C and were collected at 6 hours after xylose induction. Proteins from 4.5 mL of cell-free supernatants from B. megaterium cultures carrying plasmids were precipitated with trichloroacetic acid. Samples were used for SDS-PAGE and Western blotting. The 6X His tag antibody was used to detect the proteins. (Note: both samples were originated from 4.5 mL culture).

Figure 8. Secretion of linoleate isomerase in B. megaterium. Cells carrying pLPPL were cultured in MPB medium at 30°C and were collected at 0, 4, 6, 8, 10, 15, and 20 hours after xylose induction. Proteins from 4.5 mL of cell-free supernatants from B. megaterium cultures carrying plasmids were precipitated with trichloroacetic acid. Samples were used for SDS-PAGE and Western blotting. The 6X His tag antibody was used to detect the proteins. (Note: all samples were originated from 4.5 mL culture).

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Figure 9. Secretion of active protease. Strain YYBm1 without plasmid was used as a negative control. Samples were taken 6 hours after the xylose supplement. The activities are expressed in U/mL. a,b Means within the same sample measurements with unlike superscripts (P < 0.05). Error bars indicate standard deviations between enzyme activities detected for each construct. 70

Protease activity (U/mL)

b b

0 YYBm1

EPL

EPPL

Protease production in B. megaterium

Since no activity was found when B. megaterium YYBm1 was used as the host, B. licheniformis and B. subtilis were chosen due to their abilities to secrete large quantities of extracellular enzymes (Schallmey et al., 2004). The plasmids L97 to pLPPL were transferred into B. licheniformis NRRL B-14212 by electroporation; however, plasmid pLPPE transformation was unsuccessful. Unfortunately, no plasmid transformations were successful in B. subtilis. B. licheni-

Since prepropeptide S. hyicus lipase fusion impeded Tfh (Figure 1) and PhoA activities (Figure 2) but supported secreted linoleate isomerase production (Figure 3), B. subtilis nprE gene was chosen for the further evaluation of this propeptide on secreted protein in B. megaterium. By measuring active protease in the medium fractions collected from

formis carrying these plasmids were cultured in LB medium at 37°C and the supernatant was collected at 6 hours after xylose supplement; unfortunately, no protein was detected by using the 6X His tag antibody.

B. megaterium expressing pre- and prepropeptide S. hyicus fused with nprE gene, the results showed that the secreted NprE did not require propeptide for translocation; however, its activity was inhibited by the propeptide fusion (Figure 9). In addition, the

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Figure 10. A typical peptide mass fingerprint of trypsin digested proteins from B. megaterium carrying pLPPL.

results from the secreted NprE proteins detected by the 6X His tag antibody showed that cells expressing prepropeptide S. hyicus lipase produced a larger form than proteins from cells expressing prepeptide (data not shown).

the peptide analysis, the results demonstrated that propeptide S. hyicus lipase was still attached with the linoleate isomerase (Figure 11).

DISCUSSION Peptide analysis by MALDI-TOF MS B. megaterium carrying pLPPL was cultured in MPB medium at 30°C and the sample was collected at 8 hours after xylose induction. The major protein band in SDS-PAGE was excised and digested with trypsin. Subsequently, the peptide sample was analyzed by MALDI-TOF MS. From the peptide mass fingerprint obtained from the MALDI-TOF MS (Figure 10), the intense peaks were selected and subjected to MS/ MS ion search. The MS/MS data were analyzed both by running MASCOT (http://www.matrixscience.

In this study, two enzymes,Tfh and PhoA, were chosen to be model proteins to study the influence of prepropeptides on export efficiency in B. megaterium. Tfh has been shown to be successful for generating secreted protein in B. megaterium YYBm1 up to 7,200 U per liter (Stammen et al., 2010). PhoA from E. coli was the other model protein because it contains two disulfide bonds which is one of the limitations for heterologous production in Bacillus species (Kouwen and van Dijl, 2009). From Tfh and PhoA activity measurement, the re-

com) as well as manual analysis in order to identify the protein. MS/MS spectra were searched with GPS software using 95% confidence interval threshold (P < 0.05), with which a minimum Mascot score of > 61 was considered imperative for further analysis. From

sults showed that prepeptide yocH and prepeptide nprE were the best choices for secreted protein production. Currently, every secreted heterologous protein requires prepeptide optimization (Brockmeier et al., 2006). In some cases, the efficiency of het-

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Figure 11. Amino acid sequences of prepropeptide S. hyicus lipase and P. cenes linoleate isomerase in plasmid LPPL. The underlines show the amino acid sequences obtained from MALDI-TOF MS results.

Prepeptide sequence M K E T K H Q H T F S I R K S A Y G A A S V M V A S C I F V I G GG V A E A Propeptide sequence NDSTTQTTTPLEVAQTSQQETHTHQTPVTSLHTATPEHVDDSKEAT PLPEKAESPKTEVTVQPSSHTQEVPALHKKTQQQPAYKDKTVPESTI ASKSVESNKATENEMSPVEHHASNVEKREDRLETNETTPPSVDREFS HKIINNTHVNPKTDGQTNVNVDTKTIDTVSPKDDRIDTAQPKQVDV PKENTTAQNKFTSQASDKKPT Linoleate isomerase sequence MSISKDSRIAIIGAGPAGLAAGMYLEQAGFHDYTILERTDHVGGKCH SPNYHGRRYEMGAIMGVPSYDTIQEIMDRTGDKVDGPKLRREFLHE DGEIYVPEKDPVRGPQVMAAVQKLGQLLATKYQGYDANGHYNKVH EDLMLPFDEFLALNGCEAARDLWINPFTAFGYGHFDNVPAAYVLKY LDFVTMMSFAKGDLWTWADGTQAMFEHLNATLEHPAERNVDITRI TREDGKVHIHTTDWDRESDVLVLTVPLEKFLDYSDADDDEREYFSKII H Q Q Y M V D A C L V K E Y P T I S G Y V P D N M R P E RL G H V M V Y Y H R W A D D P H QIITTYLLRNHPDYADKTQEECRQMVLDDMETFGHPVEKIIEEQTW YYFPHVSSEDYKAGWYEKVEGMQGRRNTFYAGEIMSFGNFDEVCH YSKDLVTRFFV

erologous protein secretion with each prepeptide depends on each strain. By comparing between two strains of B. lichniformis, most prepeptides, except SacC, showed related secretion efficiency (Degering et al., 2010). Surprisingly, the Tfh and PhoA activities in medium of three-pair propeptide addition were lower compared to the cells proteins expressing without propeptides. Linoleate isomerase is the enzyme for modifying linoleic acid to conjugated linoleic acids which are

demonstrated propeptide lipase was necessary for human growth hormone translocation process at S. carnosus membrane and Kouwen et al. (2010) demonstrated that propeptide S. hyicus lipase significantly enhanced the secretion of PhoA by B. subtilis. In general, heterologous proteins are vulnerable to wall-associated proteases during the slow protein-folding process (Braun et al., 1999). The results in this study demonstrated that the propeptide protected linoleate isomerase from proteolytic degra-

anti-carcinogenic compounds (Deng et al., 2007). This enzyme formed inclusion bodies when expressing in E. coli BL21 (DE3) (Deng et al., 2007) . The goal of this investigation was to secrete active linoleate isomerase in B. megaterium. Sturmfels et al. (2001)

dation in B. megaterium cells. Demleitner and Gotz (1994) reported that the second half of this propeptide maintained lipase stability in S. carnosus cells and proposed that the heterologous protein without propeptide was degraded in intracellular or at the

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membrane site during translocation. Kouwen and van Dijl (2009) proposed that the propeptide might function like a chaperone to form protein correctly, and therefore the enzyme was not degraded inside the cells. Meens et al. (1997) concluded that the propeptide facilitates the release of unfolded proteins from the translocase and/or passage through the cell wall, protecting them from proteases at the membrane-cell wall interface. In this study, MPB medium appeared to maximize linoleate isomerase production, similar to Rluc production in B. subtilis (2011). When the host cells Bacillus subtilis grew on the MSR medium, production of recombinant protein Rluc was unsuccessful. A de-

InhA was reported (Eppinger et al, 2011); however, the propeptide removal was not found in the study reported here.

tectable amount of Rluc was found when B. subtilis growing on MPB medium. It should be noted that the composition of the MSR and MPB media are similar except that glucose in the former is substituted with casamino acid in the latter. It suggests that the nitrogen source instead of carbon is favorable for the production of Rluc by B. subtilis. The results of linoleate isomerase activity, NprE activity, NprE detection by the 6X His tag antibody, peptide analysis of by MALDI-TOF MS showed that the propeptide was still attached with the enzymes and might inhibit enzyme activities. In S. hyicus, the prepeptide is cleaved after the propeptide-enzyme complex is released into the culture medium (Sarvas et al., 2004). The propeptide is subsequently removed by a metalloprotease, and full enzymatic activity is achieved (Sarvas et al., 2004; Yabuta et al., 2001). Ayora et al.(1994) demonstrated that ShpII, a neutral metalloprotease in S. hyicus, is necessary for the propeptide removal. The propeptide-PhoA complex produced by B. subtilis 168 (trpC2) showed that the propeptide was removed when the complex was released into the medium (Kouwen et al., 2010); however, the propeptide-OmpA complex produced by B. subtilis DB104 (his, nprR2, nprE18, ΔaprA3) was unprocessed after releasing into the medium

the proteins fused with propeptides, compared to those without propeptides. Although propeptide S. hyicus lipase protected linoleic acid isomerase from proteolytic degradation and did not impede translocation, no activity of isomerase was detectable.

(Meens et al., 1997). NprE is an extracellular major metalloprotease in B. subtilis (He et al., 1991) and might remove the propeptide from the propeptide-PhoA complex. From B. megaterium DSM319 genome analysis, an extracellular metalloprotease

Bolhuis, A., H. Tjalsma, H. E. Smith, A. de Jong, R. Meima, G. Venema, S. Bron, and J. M. van Dijl. 1999. Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis. Appl. Environ. Microbiol. 65:2934 - 2941.

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CONCLUSIONS The goal of this investigation was to secrete active linoleate isomerase in B. megaterium. While the propeptide protected linoleate isomerase from proteolytic degradation in B. megaterium cells,the propeptide was still attached to the enzyme after secretion and possibly inhibited enzyme activities.The activity of secreted Tfh and PhoA were lower when

ACKNOWLEDGEMENTS We would like to thank Simon Stammen, Rebekka Biedendieck, and Dieter Jahn of Institute of Microbiology, Technische Universitat Braunschweig for providing plasmids and B. megaterium YYBm1. We appreciate Robert Story, Center for Food Safety and Department of Food Science, the University of Arkansas for providing Bacillus licheniformis NRRL B-14212.

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Braun, P., G. Gerritse, J. M. van Dijl, and W. J. Quax. 1999. Improving protein secretion by engineering components of the bacterial translocation machinery. Curr. Opin. Biotechnol. 10:376-381. Brockmeier, U., M. Caspers, R. Freudl, A. Jockwer, T. Noll, and T. Eggert. 2006. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J. Mol. Biol. 362:393-402. Chiang, C.-J., P. T. Chen, and Y.-P. Chao. 2010. Secreted production of Renilla luciferase in Bacillus subtilis. Biotechnol. Prog. 26:589-594. Christie, G., M. Lazarevska, and C. R. Lowe. 2008.

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Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria. Appl. Environ. Microbiol. 63:2814-2820. Pariza, M. W. 2004. Perspective on the safety and effectiveness of conjugated linoleic acid. Am. J. Clin. Nutr. 79:1132S-1136S. Peng, S. S., M.-D. Deng, A. D. Grund, and R. A. Rosson. 2007. Purification and characterization of a membrane-bound linoleic acid isomerase from Clostridium sporogenes. Enzyme Microb. Technol. 40:831-839.

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Table S1. Oligonucleotides used in this study

Primer

Sequence 5’ to 3’

PL1

TTC ACG TTT TTC CAC ATT TGA AGC ATG ATG TTC AAC AGG TGA CAT CTC ATT TTC TGT TGC

PL2

TTT ATT TGA TTC AAC CGA CTT TGA TGC TAT CGT TGA CTC TGG TAC CGT TTT ATC CTT ATA

PL3

CGC CGG TTG TTG CTG TGT TTT TTT ATG TAA CGC AGG TAC TTC CTG TGT ATG CGA TGA AGG

PL4

TTG AAC TGT CAC TTC GGT TTT TGG TGA CTC TGC TTT TTC AGG TAA AGG TGT TGC TTC TTT

PL5

AGA GTC ATC AAC ATG TTC AGG TGT TGC AGT ATG TAA TGA TGT AAC AGG TGT TTG ATG TGT

PL6

ATG TGT TTC TTG CTG CGA CGT TTG AGC GAC TTC TAG TGG TGT CGT TGT TTG TGT TGT CGA

PL7

ATC ATT TGC CTC TGC CAC GCC CCC ACC GAT GAC AAA TAT ACA TGA TGC GAC CAT AAC CGA

PL8

CGC GGC ACC ATA AGC CGA CTT ACG GAT AGA AAA TGT GTG

PL9

TTG ATG TTT TGT TTC TTT CAT GAC CTT GTG TTC TCC TCC TCT

PL10

TGT TGG TTT TTT GTC GCT CGC TTG TGA TGT AAA TTT ATT TTG TGC CGT TGT ATT TTC TTT

PL11

AGG AAC GTC GAC TTG TTT CGG TTG CGC CGT ATC TAT TCT GTC ATC TTT CGG TGA AAC GGT

PL12

GTC TAT CGT TTT CGT ATC AAC ATT AAC GTT TGT TTG TCC ATC CGT TTT TGG ATT TAC GTG

PL13

CGT ATT ATT GAT GAT TTT ATG GCT AAA TTC ACG GTC CAC TGA TGG

PL14

CGG TGT TGT CTC ATT AGT CTC CAA TCT ATC TTC ACG TTT TTC CAC

PU1

AGA GGA GGA GAA CAC AAG GTC ATG AAA GAA ACA AAA CAT CAA CAC ACA TTT TCT ATC CGT

PU2

AAG TCG GCT TAT GGT GCC GCG TCG GTT ATG GTC GCA TCA TGT ATA TTT GTC ATC GGT GGG

PU3

GGC GTG GCA GAG GCAAAT GAT TCG ACA ACA CAA ACA ACG ACA CCA CTA GAA GTC GCT CAA

PU4

ACG TCG CAG CAA GAA ACA CAT ACA CAT CAA ACA CCT GTT ACA TCA TTA CAT ACT GCA ACA

PU5

CCT GAA CAT GTT GAT GAC TCT AAA GAA GCA ACA CCT TTA CCT GAA AAA GCA GAG TCA CCA

PU6

AAA ACC GAA GTG ACA GTT CAA CCT TCA TCG CAT ACA CAG GAA GTA CCT GCG TTA CAT AAA Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 3 - 2013

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Table S1. (Continued)

Primer

Sequence 5’ to 3’

PU7

AAA ACA CAG CAA CAA CCG GCG TAT AAG GAT AAA ACG GTA CCA GAG TCA ACG ATA GCA TCA

PU8

AAG TCG GTT GAA TCA AAT AAA GCA ACA GAA AAT GAG ATG

PU9

TCA CCT GTT GAA CAT CAT GCT TCA AAT GTG GAA AAA CGT GAA

PU10

GAT AGATTG GAG ACT AAT GAG ACA ACACCG CCA TCA GTG GAC CGT GAA TTT AGC CAT AAA

PU11

ATC ATC AAT AAT ACG CACGTA AAT CCA AAA ACG GAT GGA CAA ACA AAC GTT AAT GTT GAT

PU12

ACG AAA ACG ATA GAC ACC GTT TCACCG AAA GAT GAC AGA ATA GAT ACG GCGCAACCG AAA

lipF

TAT ATGTAC AAT GAA AGAAAC AAA ACA TCA ACA CAC AT

lipR

TAT AAG ATC TACTAG TAT TTG CCT CTG CCA CGC C

lipR2

TAT AAG ATC TACTAGTTGTTG GTT TTT TGTCGC TCG

nprEF

TAT ATGTAC AAT GGG TTT AGG TAA GAA ATT GTC TG

nprER

TAT AAG ATC TACTAGTAGCAG CCT GAA CAC CT

nprER2

TAT AAG ATC TACTAG TAT GTT CTACTT TAT TTT GCT GTT TTA AAA

nprMF

TAT ATGTAC AATGAAAAAGAAAAAACAGGCTTTAAAGG

nprMR

TAT AAG ATC TACTAG TATG TGC AAA AGC AAA TGA TGA AG

nprMR2

TAT AAG ATC TACTAG TAGG TTT CGCTGC CGG C

phoF

TAT AAG ATC TACTAG TCG GGCTGCTCAGGGCGA TAT

phoR

TAT ACG GCC GTT AGT GAT GGT GAT GGT GGT GTT TCA GCC CCA GAG CGG C

enprEF

TAT AAG ACT T ACT AGT GCC GCCGCC ACT GGA AGC

enprER

TAT A CGG CCGTTA GTG ATG GTG ATG GTG GTGCAATCCAACAGC ATT CCA GGC

Restriction sites are highlighted in bold letters.

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VOLUME 3 ISSUE 1 REVIEW 17

Greenhouse Gas Emissions from Livestock and Poultry C. S. Dunkley and K. D. Dunkley

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

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

ARTICLES 6

Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration R. S. Pinder, and J. A. Patterson

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

Introduction to Authors 79

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VOLUME 3 ISSUE 2 ARTICLES 94

Consumers’ Interest in Locally Raised, Small-Scale Poultry in Georgia E. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, and S. C. Ricke

129 Isolation and Initial Characterization of Acetogenic Ruminal Bacteria Resistant to Acidic Conditions

P. Boccazzi and J. A. Patterson

145 Linoleic Acid Isomerase Expression in Escherichia coli BL21 (DE3) and Bacillus spp S. Saengkerdsub

REVIEW 103 Current and Near-Market Intervention Strategies for Reducing Shiga Toxin-Producing Escherichia coli (STEC) Shedding in Cattle.

T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, and D. J. Nisbet

121 Potential for Rapid Analysis of Bioavailable Amino Acids in Biofuel Feed Stocks D. E. Luján-Rhenals, and R. Morawicki

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rial board members and should be sent to submit@ afabjournal.com. There will be no page charges for solicited review papers but the solicitation must originate from the editor-in-chief or one of the editors. Requests from authors will automatically be classified as unsolicited review papers. The running head above the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews AFAB welcomes opportunities to publish papers from symposia, scientific conference, and/or meetings in their entirety. Conference organizers need simply to contact AFAB at submit@afabjournal.com and a rapid decision is guaranteed. If in agreement, the conference organizers must guarantee delivery of a set number of peer reviewed manuscripts within a specified time and submitted in the same format as that described for unsolicited review papers. Conference papers must be prepared in accordance with the guidelines for review articles and are subject to peer review. The conference chair must decide whether or not they wish to serve as Special Issue Editor and conduct the editorial review process. If the conference chair/organizer chooses to serve as special issue editor, this will involve review of the papers and, if necessary, returning them to the authors for revision. The conference organizer then submits the revised manuscripts to the journal editorial office for further editorial examination. Final revisions by the author and recommendations for acceptance or rejection by the chair must be completed by a mutually agreed upon date between the editor and the conference organizer. Manuscripts not meeting this deadline will not be included in the published symposium proceedings but if submitted later can still be considered as unsolicited review papers. Although offprints and costs of pages are the same as for all other papers, the symposium chair may be asked to guarantee an agreed upon number of hard copies to be purchased by conference attendees. If the decision is not to publish the symposium as a special issue, the individual authors retain the right to submit their papers for consideration for the journal as ordinary unsolicited review manuscripts. 254

Book Reviews AFAB publishes reviews of books considered to be of interest to the readers. The editor-in-chief ordinarily solicits reviews. Book reviews shall be prepared in accordance to the style and form requirements of the journal, and they are subject to editorial revision. No page charges will be assessed solicited reviews while unsolicited book reviews will be assigned the regular page charge rate.

Opinions and Current Viewpoints The purpose of this section will be to discuss, critique, or expand on scientific points made in articles recently published in AFAB. Drafts must be received within 6 months of an article’s publication. Opinions and current perspectives do not have page limits. They shall have a title followed by the body of the text and references. Author name(s) and affiliation(s) shall be placed between the end of the text and list of references. If this document pertains to a particular manuscript then the author(s) of the original paper(s) will be provided a copy of the letter and offered the opportunity to submit for consideration a reply within 30 days. Responses will have the same page restrictions and format as the original opinion and current viewpoint, and the titles shall end with “Opinions.” They will be published together. Letters and replies shall follow appropriate AFAB format and may be edited by the editor-in-chief and a technical editor. If multiple letters on the same topic are received, a representative set of opinions concerning a specific article will be published. A disclaimer will be added by the editorial staff that the opinion expressed in this viewpoint is the authors alone and does not necessarily represent the opinion of AFAB or the editorial board.

COPYRIGHT AGREEMENT The copyright form is published in AFAB as space permits and is available online (www.afabjournal.com).

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AFAB grants to the author the right of re-publication in any book of which he or she is the author or editor, subject only to giving proper credit to the original journal publication of the article by AFAB. AFAB retains the copyright to all materials accepted for publication in the journal. If an author desires to reprint a table or figure published from a non-AFAB source, written evidence of copyright permission from an authority representing that source must be obtained by the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS Authors will be requested to provide the names and complete addresses including emails of five (5) potential reviewers who have expertise in the research area and no conflict of interest with any of the authors. Except for manuscripts designated as Rapid Communication each reviewer has two (2) weeks to review the manuscript, and submit comments electronically to the editorial office. Authors have three (3) weeks to complete the revision, which shall be returned to the editorial office within six (6) weeks after which the authors risk having their manuscript removed from AFAB files if they fail to ask the editorial office for an extension by email. Deleted manuscripts will be reconsidered, but they will have to be processed as new manuscripts with an additional processing fee assessed upon submission. Once reviewed, the author will be notified of the outcome and advised accordingly. Editors handle all initial correspondence with authors during the review process. The editor-in chief will notify the author of the final decision to accept or reject. Rejected manuscripts can be resubmitted only with an invitation from the editor or editor-in chief. Revised versions of previously rejected manuscripts are treated as new submissions.

PRODUCTION OF PROOFS Accepted manuscripts are forwarded to the editorial office for technical editing and layout. The manuscript is then formatted, figures are reproduced, and author proofs are prepared as PDFs. Author proofs of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and checked against the typed manuscript, because the responsibility for proofreading is with the author(s). Corrections must be returned by e-mail. Changes sent by e-mail to the technical editor must indicate page, column, and line numbers for each correction to be made on the proof. Corrections can also be marked using “track changes” in Microsoft Word or using e-annotation tools for electronic proof correction in Adobe Acrobat to indicate necessary changes. Author alterations to proofs exceeding 5% of the original proof content will be charged to the author. All correspondence of proofs must be agreed to by the editorial office and the author within 48 hours or proof will be published as is and AFAB will assume no responsibility for errors that result in the final publication.

PUBLICATION CHARGES AFAB has two publication charge options: conventional page charges and rapid communication. The current charge for conventional publication is $25 per printed page in the journal. There is no additional charge for the publication of pages containing color images, micrographs or pictures. For authors who wish to have their papers processed as a rapid communication, authors will pay the rapid communication fee when proofs are returned to the editorial office in addition to twice the conventional page charges. Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one week and $100 per journal page.

HARD COPY OFFPRINTS If you are wishing to obtain a physical hard copy of the AFAB journal, offprints are available in any quantity at an additional charge: $100/page for black-white and $150/page for color prints. You may order your offprints at any time after publication on our website. Scientific conference organizers may be expected to agree to a set number of offprints as a part of their agreement with AFAB.

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MANUSCRIPT CONTENT REQUIREMENTS Preparing the Manuscript File Manuscripts must be written in grammatically correct English. AFAB offers a fee based language service upon request (language@afabjournal.com). Manuscripts should be typed double-spaced, with lines and pages numbered consecutively. All documents must be submitted in Microsoft Word (.doc or .docx, PC or Mac). All special characters (e.g., Greek, math, symbols) should be inserted using the symbols palette available in this font. Tables and figures should be placed in separate sections at the end of the manuscript (not placed in the text). Failure to follow these instructions will cause delays of the processing and review of the manuscript.

Title Page At the very top of the title page, include a title of not more than 100 characters. Format the title with the first letter of each word capitalized. No abbreviations should be used. Under the title, the authors names are listed. Use the author’s initials for both first and middle names with a period (full-stop) between initials (e.g., W. A. Afab). Underneath the authors, a list affiliations must be listed. Please use numerical superscripts after the author’s names to designate affiliation. If an authors address has changed since the research was completed, this new information must be designated as “Current address:”. The corresponding author should be indicated with an asterisk e.g., * Corresponding author. The title page shall include the name and full address of the corresponding author. Telephone and e-mail address must also be provided for the corresponding author, and emailaddresses must be provided for all authors.

at the beginning of the manuscript. In vivo, in vitro and bacterial names must be italicized (obligatory). Authors must avoid single sentence paragraphs and merge such paragraphs appropriately. Authors must not begin sentences with “Figure or Table shows…” as these are inanimate objects and cannot “show” anything. When number are reported in text or in 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 256

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.

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. 258

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. 86 (Abstr.)

Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the 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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