ADAPTATION OF NUCLEIC ACID EXTRACTION METHODS FOR ANIMAL FEEDS

K.G. MACIOROWSKI', S.D. PILLAI and S.C. RICKEZ

Poultry Science Department Texas A&M University

College Station, Ix 77843-2472

Accepted for Publication September 26, 2001

ABSTRACT

Animal feeds contain a wide variety of microorganisms that may cause animal and human health problems. It would be desirable to rapidly detect as many of these organisms as possible. The objective of this study was the evaluation of nucleic acid extraction protocols to determine an optimal sample processing strategy for DNA isolation from diflerent animal feeds. DNA extraction methods were applied to selected animal feeds to determine which could consistently provide DNA visible by gel electrophoresis. Comparable recovery of DNA extraction protocols was achieved after 20 g of each feed samples were mixed with 0.2% peptone containing 0.01 % Triton X-100. For 9 diflerent feeds visible DNA could be consistently detected for each feed indicating that a detergent-based dilution step prior to DNA extraction recovered suficient DNA regardless of the feed matrix.

INTRODUCTION

Animal feed contains a diverse microflora that is acquired from multiple sources in the environment, including dust from the air, soil, water and insects as well as other sources. This becomes a concern when some of these organisms that come in contact with animal feeds are also pathogenic to animal and human hosts. Consequently the feed becomes a carrier for the organism leading to a multitude of problems associated with dissemination of the organism throughout the animal population. Specific instances include mycotoxins in feeds that

' Current address: Dr. Kenneth G. Maciorowski, Deparhnent of Agriculture and Natural Resources, 1200 North DuPont Highway, Delaware State University, Dover, DE 19901.

* Address for correspondence: Dr. Steven C. Ricke, Poultry Science Department, 2472 TAMU, College Station, TX 77843-2472, TEL: (979) 862-1528; FAX: (979) 845-1921; E-mail: srickeQpoultry . tamu .edu

Journal of Rapid Methods and Automation in Microbiology 9 (2001) 217-227. All Righrs Reserved. OCopyrighi 2001 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 217

218 K.G. MACIOROWSKI, S.D. PILLAI and S.C. R I C E

decrease animal productivity and negatively influence poultry economics and consumption of Salmonella-contaminated feed by food animals that can potentially be linked to the human population (Tabib et al. 1981; Williams 1981). Given the myriad of bacterial and fungal populations present in animal feeds that may cause animal and human health problems it would be desirable to screen for as many of these organisms as possible. However, diversity studies by culture approaches would require selective media for individual bacterial species as well as differential media for distinguishing fungal colonies from bacteria (Ha et al. 1995a, b; Ricke et al. 1998).

Molecular methods can be used to assess microbial community structure at the genetic level without recovery of viable cells capable of growth in selective media. However, one of the primary problems associated with application of molecular methods to complex organic environments such as animal feeds is the recovery of sufficient DNA in an intact form. Most of the DNA extraction protocols available for environmental microflora have been developed for soil. Two general approaches for DNA recovery have involved either separation of bacterial cells from soil particles followed by lysis to recover the bacterial DNA or direct lysis and release of bacterial DNA while still attached to the soil particle (Faegri et al. 1977; Torsvik and Goksoyr 1978; Ogram el al. 1987; Holben et al. 1988; Steffan et al. 1988; Pillai et al. 1991; Picard et al. 1992; Zhou et al. 1996; Krsek and Wellington 1999). Similar comparisons of extraction methods for community DNA in animal feeds and comparable matrices have not been made because DNA extraction has only been evaluated as part of protocols for polymerase chain reaction (PCR) amplification and detection of single bacterial species such as Salmonella. Pillai et al. (1994) used centrifugation and resuspension of the bacterial cell pellet for poultry cecal samples while Cohen et al. (1994) utilized a DNA extraction method procedure on poultry fecal material that included lysozyme-proteinase K digestion and extraction with a phenol-chloroform-isoamyl alcohol mixture. Maciorowski er al. (1998; 2000) combined feed samples with a 2% peptone - 0.01 % Triton X- 100 liquid extraction followed by either centrifugation and resuspension in 2% peptone or extraction buffer prior to PCR detection of Salmonella.

Improved extraction methods for nucleic acids from animal feeds and ingredients would provide the means for more consistent DNA detection protocols for a wide variety of organisms across varying feed matrices. Our overall goal is to determine which sample processing strategy has the best potential to be adapted for the isolation of DNA from animal feeds. Our first objective in this study was to use pure culture Escherichia coli and selected feed samples to evaluate two direct extraction DNA protocols combined with different purification steps for DNA recovery on gel electrophoresis. A second objective was to assess these two DNA extraction protocols after pretreatment of the feed samples with a detergent containing solution.

DNA EXTRACTION METHODS FOR ANIMAL FEEDS 219

MATERIALS AND METHODS

Escherichia coli and Feed Sources

The Eschenchia coli pure culture used in this study was an F,, HS (pFamp) R (male-specific coliphage host; U.S. Environmental Protection Agency, 2000). A 50-lb (22 kg) bag of each of eight animal feed samples was acquired from a local commercial feed store. The feeds included game bird finisher, milk replacer, and pullet starter (manufactured by Farmland Industries, Kansas City, MO), fish meal, range breeder meal, emu starter, swine finisher and turkey starter. Soybean meal (SBM) was acquired from the research farm at Texas A&M University. For this study, approximately 10 kg were sub- sampled from each respective or feed ingredient.

Rapid (1 Day) DNA Direct Extraction Procedure

Community DNA was extracted from feeds as described by Porteous et al. (1994). Briefly, 100 mg of feed sample was mixed with 350 pL of a homogeni- zation solution (250 mM NaC1, 100 mM EDTA, pH 8.0) and vortexed for 30 s. Cells were lysed by sonication at room temperature for 3 min. and 12.5 p L of lysozyme (stock concentration: 8 mglmL; final concentration: 0.28 pg/mL homogenization mixture) was added. The mixture was vortexed for 10 s and incubated at 37C for 60 min. The resulting suspension was mixed with 350 pL of lysis buffer (250 mM NaCl, 100 mM EDTA, and 4% w/v sodium dodecyl sulfate, pH 8.0) and 50 pL of 5 M guanidine isothiocyanate (final concentration: 0.33 M in lysis mixture), vortexed for 10 s and incubated at 68C for 60 min. After incubation, the mixture was centrifuged (12000 X g, 4C) for 15 min. The supernatant was mixed with 750 pL of isopropanol, incubated at -2OC for 60 min and centrifuged (12000 x g, 4C) for 15 min to pellet DNA. The superna- tant was decanted and the pellet was air dried for 15 min before resuspending it in approximately 40 pL of TE buffer (pH 8.0). The resuspended DNA was stored at 4C until electrophoresis could be done.

Polyethylene Glycol Precipitation DNA Extraction

A second DNA extraction protocol for the isolation of DNA from feed matrices was adapted from a procedure developed for microbial extraction from soil (Widmer et al. 1996). Briefly, 100 mg of each feed was mixed with 700 pL of the lysis buffer as described in the previous section. After vortexing for 20 s, 50 pL of 5 M guanidine isothiocyantate was added (final concentration in lysis buffer: 0.36 M). The sample was vortexed, incubated at 68C for 10 min, sonicated for 3 min and incubated for 1 h at 68C, with manual mixing every 15 min during incubation. The samples were centrifuged (13,000 x g) for 5 min

220 K.G. MACIOROWSKI, S.D. PILLAI and S.C. RICKE

at room temperature. The supernatant was removed, mixed with 750 pL of cold isopropanol, incubated at -2OC overnight to precipitate DNA, and centrifuged (13,000 x g, 4C) for 30 min. The pelleted DNA was washed with 70% cold ethanol, centrifuged (13,000 x g) for 15 min, air dried for 15 min and resuspended in 100 pL of TE buffer (pH 8). The solution was mixed with 260 pL of 24% (w/v) polyethylene glycol 8000 (PEG) and 40 pL of 5 M NaCl (final concentration: 0.5 M) and incubated at -2OC for 30 min. The suspensions were centrifuged (13,000 x g, 4C) for 30 min, the supernatants discarded, and the pellets washed with 200 pL of cold 70% ethanol with centrifugation centrifuged (13,000 x g, 4C) for 15 min. The pellets were air dried for 15 min and resuspended in approximately 40 pL of TE buffer (pH 8.0).

Liquid Dilution of Feed in a Detergent-based Solution

Twenty grams of each feed sample were mixed with 100 mL of a 0.2% peptone containing 0.01% Triton X-100 solution (Tabib et al. 1981). The suspension was shaken horizontally (American Scientific, Los Angeles, CA; 150 oscillations/min of a 20 mm stroke length) for 20 min and left for 1 min for the settling of large particles. The liquid fraction was decanted and centrifuged (2600 x g) for 5 min to remove large particles not separated by settling. The supernatant was decanted and centrifuged (16270 x g) for 20 min. After discarding the supernatant, the resulting pellets were washed once with 0.2% peptone and stored at 4C until nucleic acid extraction by one of the methods described in the previous sections was implemented.

Detection of DNA

DNA was detected by electrophoresis on 1.2% agarose gels containing 0.5 pg/mL ethidium bromide, using Tris-borate EDTA (TBE) as the running buffer. After electrophoretically separating the DNA for 60 min at 100 V, bands were visualized by ultraviolet light using a transilluminator. Electrophoresis gels were photographed with a digital camera connected to imaging software.

RESULTS AND DISCUSSION

DNA extraction methods were applied to the feeds to determine which could consistently provide extracted DNA visible by gel electrophoresis. The E. coli F,, HS (pFamp)R (male-specific coliphage host) was chosen as a representative pure culture bacterial strain for this study because it had been demonstrated from a previous study that male-specific coliphages could be detected in a wide variety of similar animal feeds using this strain as the host

DNA EXTRACTION METHODS FOR ANIMAL FEEDS 22 1

(Maciorowski et al. 2001). For direct DNA extraction studies on feeds, three feeds (fresh fish meal, soybean meal, and turkey starter) were chosen to represent variations in protein content and particle size. Two DNA extraction methods were adapted that had been used previously for soil and plant samples. The l-day community DNA method described by (Porteus et al. 1994) is considered faster because it involves only a centrifugation step and consequently minimizes extraction time and potential DNA shearing. A second DNA extraction protocol was adapted from a procedure developed for DNA extraction from soil (Widmer et al. 1996). This approach combined initial lysis with a PEG precipitation step for the isolation of more purified DNA from feed matrices.

DNA direct extraction from either E. coli pure culture or the three animal feeds with the community DNA extraction procedure is shown in Fig. 1. The community DNA extraction procedure yielded more visible bands of DNA from 3 replicates of E. coli in pure culture, whereas only 1 replicate of E. coli yielded detectable DNA with the modified PEG procedure. The extra steps in the PEG protocol may have washed away some DNA, leaving the remaining DNA undetectable with ethidium bromide. Negative controls consisting of double distilled water (DDI) were not contaminated with DNA for the l-day community DNA extraction protocol as indicated by the lack of visible DNA bands in the respective lanes. DNA extracted by the procedure of Porteus er al. (1994) could be detected for fishmeal, swine, and turkey starter. Although more DNA was visible for fishmeal than the other two feeds, the pattern indicating that some shearing of the DNA during isolation may have occurred.

Comparison of DNA extraction from either E. coli pure culture or three animal feeds with the modified PEG procedure (Widmer et al. 1996) is shown in Fig. 2. Negative DDI controls were not contaminated with DNA for the PEG protocol as indicated by the lack of visible DNA in the respective lanes. The PEG extraction procedure yielded visible bands of DNA from all 3 replicates of E. coli in pure culture and visible bands of DNA were apparent from all three animal feeds. As with the l-day procedure more DNA was visible for fishmeal than the other two feeds, the pattern indicating that some shearing may have also occurred with the PEG procedure.

The alternative strategy to achieve DNA recovery from organic matrices has involved initial separation of the bacterial cells from the matrix followed by lysis to recover the bacterial DNA. Detergent-based solutions are often used for detaching bacteria from solid matrices (Pate1 2000). Previously, it had been shown that combining animal feeds with solutions containing a high concentra- tion of the nonionic detergent Triton X-100 was required to achieve maximal counts of bacteria and fungi (Stewart et al. 1977; Tabib et a1. 1981). This approach was modified in the current study by combining 20 g feed samples with 0.2% peptone containing Triton X-100 followed by low and high speed

222 K.G. MACIOROWSKI. S.D. PILLAI and S.C. IUCKE

FIG. 1 . DNA RECOVERY FROM AN ESCHERICHIA COLI PURE CULTURE OR THREE ANIMAL FEEDS USING TWO DNA DIRECT EXTRACTION PROTOCOLS

Positive control: 0.1 g bacterial pellet from 100 mL overnight culture (TSB, 37C) E. coli Famp Samples: 0.1 g feed (or DDI) directly extracted by the following procedures: Community: Porteous et al. (1994) PEG: modified Widmer er al. (1996) Gel loaded: 10 pL DNA extract or DDI + 3 pL loading dye

L, DI 5 molecular weight marker (Novel Experimental Technology, San Diego, CA) 1 Community E. coli 2 Community E. coli 3 Community E. coli 4 PEG E. coli 5 PEG E. coli 6 PEG E. coli 7 Community DDI 8 Community DDI 9 Community DDI

1 1 Community fishmeal 12 Community fishmeal 13 Community fishmeal 14 Community swine finisher 15 Community swine finisher 16 Community swine finisher 17 Community turkey starter 18 Community turkey starter 19 Community turkey starter

DNA EXTRACTION METHODS FOR ANIMAL FEEDS 223

FIG. 2. DNA RECOVERY FROM AN ESCHERICHIA COW PURE CULTURE OR THREE ANIMAL FEEDS USING A DIRECT PEG EXTRACTION PROTOCOL

Positive control: 0.1 g bacterial pellet from 100 mL. overnight culture (TSB, 37C) E. coli Famp Samples: 0.1 g feed (or DDI) directly extracted by the following procedures: PEG: modified Widmer et al. (1996) Gel loaded: 10 pL DNA exaact or DDI + 3 WL loading dye

4 PEG DDI 5 PEG DDI 6 PEG DDI 7 PEG fishmeal 8 PEG fishmeal 9 PEG fishmeal

L, D15 molecular weight marker (Novel Experimental Technology, San Diego, CA) I PEG E. coli 2 PEG E. coli 3 PECi E. coli

I 1 PEG swine finisher 12 PEG swine finisher 13 PEG swine finisher 4 PEG turkey starter 5 PEG turkey starter 6 PEG turkey starter

224 K.G. MACIOROWSKI, S.D. PILLAI and S.C. RICKE

centrifugation steps. Seven different animal feeds and two commonly used feed ingredients were chosen to represent a variety of different matrices, concentra- tions of macronutrients such as protein and fat, and particle sizes (Maciorowski et al. 2000). The comparison of DNA extracted by either the PEG procedure or the community DNA protocol for 9 different feeds after treatment with the Triton X-100 solution is shown in Fig. 3. In all cases, visible DNA could be detected for each feed indicating that a preliminary step of dilution and centrifugation prior to DNA extraction appears to recover sufficient DNA among the different feeds as well as between the two DNA extraction methods.

This study combined a detergent-based dilution step with two procedures originally designed for community DNA extraction from soils and plants to develop a protocol that could effectively extract DNA from animal feed for molecular analysis. The major difference between the two procedures involved the length of incubation reserved for DNA precipitation and post incubation treatment. The l-day protocol requires only 1 h for DNA precipitation, while the PEG protocol requires an overnight step. While the l-day protocol would shorten the total assay time for pathogen detection considerably, 60 min at -2OC may not be sufficient to recover sufficient DNA consistently from different feeds. Consistency of recoverable DNA for either extraction approach appears to be retained when an initial detergent based bacterial detachment step is done prior to DNA extraction. Development of reliable DNA preparation protocols for molecular diversity studies in animal feeds will lead to a greater understand- ing of feed bacterial and fungal population dissemination and survival in animal feeds.

ACKNOWLEDGMENTS

This research was supported the Texas Higher Education Coordinating Board’s Advanced Technology Program (Grant # 999902- 163, TEXO8239 project, and Hatch grant H8311 administered by the Texas Agricultural Experiment Station. K.G.M. was supported by the Pilgrim’s Pride endowed graduate fellowship and the Heep Foundation Internship.

DNA EXTRACTION METHODS FOR ANIMAL FEEDS

FIG. 3. DNA RECOVERY FROM NINE ANIMAL FEEDS USING TWO DNA EXTRACTION PROTOCOLS IN COMBINATION W H AN INITTIAL

LIQUID DILUTION STEP Samples: 0.1 g pellet from liquid extract of 20 g feed extracted by: Community: Porteous et al. (1994) PEG: modified Widmer el af. (1996) Gel loaded: 10 FL DNA extract or DDI + 3 FL loading dye

L, D1S molecular weight marker (Novel Experimental Technology, San Diego, CA) 1 PEG Fishmeal 2 PEG Game bird starter 3 PEG Milk replacer 4 PEG Pullet starter S PEG Range breeder 6 PEG Emu starter 7 PEG - SBM 8 PEG Swine finisher 9 PEG Turkey starter

11 Community fishmeal 12 Community game bird starter 13 Community milk replacer 14 community pullet starter 15 Community range breeder 16 Community emu starter 17 Community SRM L 8 Community swine finisher 19 Community turkey starter

22s

226 K.G. MACIOROWSKI, S.D. PILLAI and S.C. R I C E

REFERENCES

COHEN, N.D., MCGRUDER, E.D., NEIBERGS, H.L., BEHLE, R. W., WALLIS, D.E. and HARGIS, B.M. 1994. Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella. Poultry Sci. 73, 354-357.

FAEGRI, A., TORSVIK, V.L. and GOKSOYR, J . 1977. Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil Biol. Biochem. 9, 105-1 12.

HA, S.D., PILLAI, S.D. and RICKE, S.C. 1995a. Growth response of Salmonella spp. to cycloheximide amendment in media. J. Rapid Methods Automation Microbiology 4, 77-85.

HA, S.D., PILLAI, S.D., MACIOROWSKI, K.G. and RICKE, S.C. 1995b. Cycloheximide as a media amendment for enumerating bacterial populations in animal feeds. J. Rapid Methods Automation Microbiology 4, 95-105.

HOLBEN, W.E., JANSSON, J.K., CHELM, B.K. and TIEDJE, J.M. 1988. DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54, 703-7 1 1.

KRSEK, M. and WELLINGTON, E.M.H. 1999. Comparison of different methods for the isolation and purification of total community DNA from soil. J. Microbiol. Meth. 39, 1-16.

MACIOROWSKI, K.G., PERA, J . , PILLAI, S.D. and RICKE, S.C. 1998. Application of gene amplification in conjunction with a hybridization sensor for rapid detection of Salmonella spp. and fecal contamination indicators in animal feeds. J. Rapid Methods Automation Microbiology 6, 225-238.

MACIOROWSKI, K.G., PILLAI, S.D. and RICKE, S.C. 2000. Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp. in animal feeds. J. Appl. Microbiol. 89, 710-718.

MACIOROWSKI, K.G., PILLAI, S.D. and RICKE, S.C. 2001. Presence of bacteriophages in animal feed as indicators of fecal contamination. J . Environ. Sci. Health B36, 699-708.

OGRAM, A., SAYLER, G.S. and BARKAY, T. 1987. The extraction and purification of microbial DNA from sediments. J. Microbiol. Meth. 7, 57- 66.

PATEL, P. 2000. A review of analytical separation, concentration and segregation techniques in microbiology. 1. Rapid Methods Automation Microbiology 8, 227-248.

PICARD, C., PONSONNET, C., PAGET, E., NESME, X. and SIMONET, P. 1992. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol. 58, 2717-2722.

DNA EXTRACTION METHODS FOR ANIMAL FEEDS 221

PILLAI, S.D., JOSEPHSON, K.L., BAILEY, R.L., GERBA, C.P. and PEPPER, I.L. 1991. Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences. Appl. Environ. Microbiol. 57, 2283-2286.

PILLAI, S.D., RICKE, S.C., NISBET, D.J., CORRIER, D.E. and DELOACH, J.R. 1994. A rapid method for screening for Salmonella typhimurium in a chicken cecal microbial consortium using gene amplifica- tion. Avian Dis. 38, 598-604.

PORTEOUS, L.A., ARMSTRONG, J.L., SEIDLER, R.J. and WATRUD, L.S. 1994. An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis. Curr. Microbiol. 29, 301-307.

RICKE, S.C., PILLAI, S.D., NORTON, R.A., MACIOROWSKI, K.G. and JONES, F.T. 1998. Applicability of rapid methods for detection of Salmonella spp. in poultry feeds: A review. J. Rapid Methods Automation Microbiology 6, 239-258.

STEFFAN, R.J., GOKSQYR, J. , BEJ, A.K. and ATLAS, R.M. 1988. Recovery of DNA from soils and sediments. Appl. Environ. Microbiol. 54,

STEWART, R.G., WYATT, R.D. and ASHMORE, M.D. 1977. The effect of various antifungal agents on aflatoxin production and growth characteristics of Aspergillus parasiticus and Aspergillusflavus in liquid medium. Poultry Sci. 56, 1630-1635.

TABIB, Z., JONES, F.T. and HAMILTON, P.B. 1981. Microbiological quality of poultry feed and ingredients. Poultry Sci. 60, 1392-1397.

TORSVIK, V.L. and GOKSOYR, J. 1978. Determination of bacterial DNA in soil. Soil Biol. Biochem. 10, 7-12.

U.S. Environmental Protection Agency. 2000. Method 1601 : Male-specific (F+) and somatic coliphage in water by two-step enrichment procedure. Draft, April 2000. Office of Water, Washington, D.C.

WIDMER, F., SEIDLER, R.J. and WATRUD, L.S. 1996. Sensitive detection of transgenic plant marker gene persistence in soil microcosms. Molec.

WILLIAMS, J.E. 1981. Salmonellas in poultry feeds - a worldwide view. Part

ZHOU, J., BRUNS, M.A. and TIEDJE, J.M. 1996. DNA recovery from soils

2908-291 5.

Ecol. 5, 603-613.

I: Introduction. World’s Poultry Sci. J. 37, 6-19.

of diverse composition. Appl. Environ. Microbiol. 62, 316-322.