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ANIMAL NUTRITION |
* Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL 62901;
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Pennsylvania State University Extension, Lancaster, PA 17601
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Department of Animal Science, Purdue University, West Lafayette, IN 47906;
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University of Florida, Institute of Food and Agricultural Sciences, Range Cattle Research & Education Center, Ona, FL 33865; and
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# Department of Plant and Soil Sciences, Southern Illinois University, Carbondale, IL 62901
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: pig • digestion • transgenic plant • real-time polymerase chain reaction • DNA
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). In 2001, 26% of the corn, 68% of the soybeans, and 69% of the cotton planted in the United States were GM (GAO, 2002).
The presence of GM crops has created environmental, agronomic, and perhaps most importantly, food safety concerns. Because livestock consume more than 60% of the corn produced in the United States (National Corn Growers Association, 2003), the safety of livestock products as foodstuffs may be a concern. Studies show that dietary DNA components, such as nucleosides and free bases, may be incorporated into animal cells (Condon et al., 1970; Berthold et al., 1995). One major question is whether larger components of DNA could be assimilated into an animal’s genome. Einspanier et al. (2001) reported, "Today, no safe indication for the biological relevance of highly degraded DNA can be provided to prove an interaction with animals’ or consumers’ health."
A variety of studies have attempted to locate transgenic DNA in either ingesta or animal tissues. However, these studies focused on either ingesta (Chowdhury et al., 2003a,b,c) or tissue samples (Jennings et al., 2003a,b), and none of these studies included young swine. Thus, the objective of this demonstration research was to determine if transgenic DNA was present in stomach, ileal, and large intestine ingesta, plasma and white blood cells from the hepatic portal vein and liver and muscle tissue of weanling swine fed a diet containing GM glutamate dehydrogenase (gdhA+) corn. Analyzing samples from 3 locations within the digestive tract and 4 different tissues will give an indication of the digestive fate of the gdhA transgene in weanling swine. The efficiency of real-time quantitative PCR to detect transgenic DNA is also presented.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Diets and Collection Timeline
The study was conducted with a corn variety (gdhA) developed at Southern Illinois University, Carbondale. This corn variety has an Escherichia coli gene encoding glutamate dehydrogenase placed in its genome to facilitate the assimilation of N (Ameziane et al., 2000). Because this variety has not been released for commercial use, the gdhA transgene should not be present in commercial swine diets.
The trial duration was 29 d and consisted of 13 d of prestarter pellets (1.5% lysine; Hog Inc., Greenfield, IL), followed by 7 d of a conventional cornsoybean meal (SBM) starter diet (1.28% lysine), and 8 to 9 d of gdhA+ corn-SBM diet (1.28% lysine; Table 1). The conventional corn-SBM-based diet was formulated to meet or exceed NRC nutrient requirements (NRC, 1998). The gdhA+ diet was formulated to meet or exceed NRC nutrient requirements with the exception of vitamins and Se. Because of a mixing malfunction, the concentration of trace minerals exceeded NRC recommendations during the 8- to 9-d treatment period; however, because of the short timeframe, the authors believe the effect of elevated nutrients to be inconsequential to the results.
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Samples from 0-h control pigs were collected on d 14. One pig from each pen was randomly selected (4 males and 4 females total) and killed before exposure to the gdhA+ diet. Treatment samples were collected at slaughter on d 28 and 29, after the remaining 56 pigs had been fed the gdhA+ corn-SBM diet for 8 and 9 d, respectively. Four pigs per pen (32 pigs total) were randomly selected for sample collection on d 28 and the remaining 3 pigs per pen (24 pigs total) were killed on d 29 after a 24-h fast and a 3-h refeeding period. This fast/refeed protocol insured that the ingesta would be available in the cranial portion of the digestive tract because the stomach of ad libitumfed pigs will occasionally be empty of ingesta (Radcliffe et al. 1998). In this study, all pigs had ingesta samples from all portions of the digestive tract, regardless of whether they were allowed ad libitum feed access (d 28) or fasted and refed (d 29) before sample collection.
Individual ADG, pen ADFI, and G:F were calculated for the 56 treatment pigs over a 7-d feeding of the gdhA corn-based diet. Pigs were weighed individually, but feed intake was determined on a pen basis (7 pigs/pen).
Sample Collection Protocol
Pigs were injected intramuscularly with ketaset (22 mg/kg of BW from a 100 mg/mL solution; Aveco, Longmont, CO) and acepromazine (1.1 mg/kg of BW from a 10 mg/mL solution; Fort Dodge, Overland Park, KS) and then masked with halothane. Blood from the hepatic portal vein was collected into EDTA-coated Vacutainer tubes (10-mL; Becton Dickinson, Fisher Scientific, St. Louis, MO), centrifuged (518.4 x g, 8° C, 10 min) to provide 4 mL of plasma and 2 mL of white blood cells, and stored at –80° C.
Once blood was drawn, pigs were killed via exsanguination. Liver and 10th rib LM samples were collected into labeled Ziploc bags and immediately placed in a –20° C freezer. Ingesta from the stomach, distal ileum, and large intestine was collected, and duplicate sub-samples consisting of 1.5 mL of ingesta and 2 mL of a glycerine solution (20% glycerine, 80% distilled water) were collected into 4-mL Corning cryogenic tubes (Fisher Scientific, Pittsburgh, PA). Subsample tubes were flash frozen in either dry ice and ethyl alcohol or liquid nitrogen and stored at –80° C. This variance was due to economic and sourcing problems with the cooling materials.
Strict sanitation protocols were observed during all sample collection. Gloves were changed between every sample, and table covers were changed as they became soiled or after 2 pigs, whichever occurred first. All dissection instruments were dipped in DNA Away (Fisher Scientific, Pittsburgh, PA) and rinsed thoroughly with saline to prevent cross contamination. Samples were collected in a room separate from where pigs were housed to prevent feed dust from potentially contaminating samples.
Extraction Protocol
The DNA was extracted from 200 to 240 mg of stomach, ileal, and large intestine ingesta samples using the QIAamp DNA Stool Mini Kits (Qiagen, Valencia, CA). The QIAamp DNA Blood Mini Kit was used to purify DNA from 200 µL of plasma and white blood cells isolated from the hepatic portal vein. The DNeasy Tissue Kit was used to isolate DNA from liver and muscle tissue samples using 22.0 or 25.0 mg of tissue, respectively. Tissue was sampled after removing 2 layers from the outside of the liver and muscle samples with a single use, sterile scalpel blade to prevent contamination from any DNA that may have come in contact with the outside of the sample. Forceps were washed in tap water, soaked in DNA Away for 45 s, and rinsed with distilled water (18 MOhm) between samples. In preliminary tests, the optimal lysing time to elicit maximum DNA yield from liver and muscle tissue was determined to be 3.5 h in a 55° C rocking waterbath platform.
The DNA was extracted from gdhA+ corn samples, a commercially available corn that did not contain the gdhA transgene (negative assay control), and positive diet samples (containing gdhA+ corn) using the QI-Aamp DNA Stool Mini Kits.
During extractions, the risk of contamination was minimized. All extractions took place at a location separate from the PCR and gel electrophoresis stations. Bench paper was changed between sample sets, and gloves were changed frequently, even if no actual contact with sample material was made. All pipette tips were autoclaved (20 min, 122° C, 1.1 kg/cm2). Sterile centrifuge tubes (Fisherbrand, Fisher Scientific, Pittsburgh, PA) that were certified RNase- and DNase-free were used. Once extracted, the samples were stored at –20° C.
PCR Protocol
Samples were analyzed using PCR with primers designed to amplify a 490-bp region of the plasmid vector. This region bridged the ligation site between a maize ubiquitin intron and the gdhA gene (Figure 1). Amplifying this region ensured that the target DNA originated from the plasmid and not from ubiquitin maize DNA or DNA from E. coli inhabiting the digestive tract. Preliminary tests were conducted to insure the primers were functioning properly. The sequence obtained from gdhA+ corn was identical to the anticipated sequence with the exception of 16 bp at the site of ligation. It is possible that these extraneous base pairs were inserted during the creation of the plasmid. The primers resulted in a product sequence that bridged the ligation site between the maize ubiquitin and gdhA genes, indicating that the primers were amplifying DNA of plasmid origin. The primer sequences were: forward, 5'-TTGGATGATGGCATAT GCAGCA-3'; and reverse, 5'-AAGGTTTGTTCAAAGCCGAGGA-3'.
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The DNA sequence from positive bands was identical to the sequence obtained in the preliminary primer tests. This sequence was further tested for gene homologies using the National Center for Biotechnology Information BLAST Web site (www.ncbi.nim.nih.gov/BLAST/Blast.cgi). The DNA was identified as the maize ubiquitin and E. coli gdhA gene coding for NADP-specific glutamate dehydrogenase. Thus, the bands indicating a positive result were in fact indicative of the transgenic region of interest.
A random subsample of each day’s extractions underwent spectrophotometry to determine the DNA concentration. Spectrophotometric analysis was performed using an Eppendorf BioPhotometer and disposable Eppendorf UVette cuvets (Eppendorf, Hamburg, Germany). Average spectrophotometry readings were as follows: gdhA+ corn, 20.15 ± 6.46 ng/µL; conventional corn, 85.35 ± 16.21 ng/µL; gdhA+ diet, 32.79 ± 4.70 ng/µL; ileal ingesta, 7.78 ± 3.37 ng/µL; large intestine ingesta, 32.39 ± 13.08 ng/µL; stomach ingesta, 7.66 ± 2.22 ng/µL; muscle, 75.8 ± 15.88 ng/µL; liver, 401.57 ± 103.04 ng/µL; plasma, 9.95 ± 1.95 ng/µL; and white blood cells, 28.44 ± 22.07ng/µL. Because 2 µL of DNA was used, liver samples were diluted to 10% of the original concentration, and muscle samples were diluted to 50% so the final quantities in each PCR reaction were < 200 ng. Because the largest quantity of DNA allowed by the protocol was used to maximize the likelihood of detecting the transgene, samples were not diluted to a constant concentration. Extraction differences between corn sources cannot be explained.
Samples were run on an Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany) with a 105° C lid. The cycling conditions were: an initial cycle at 95° C for 15 min, 31 replications at 95° C for 45 s, 66° C for 45 s, 72° C for 2 min 20 s, and a single final elongation at 72° C for 5 min. All samples of the same type underwent PCR at the same time. Four PCR reactions containing gdhA+ corn DNA acted as positive assay controls, whereas 2 reactions containing conventional corn DNA and 2 reactions containing distilled water in place of any DNA acted as negative assay controls.
To establish the level of detection (LOD) for conventional PCR followed by gel electrophoresis, dilutions containing the target DNA were created and analyzed (Figure 2). Because this protocol attempted to detect transgenic DNA in the presence of nontransgenic DNA (i.e., DNA from ingesta and various tissues), 500 ng of conventional corn DNA was added to each 20 µL LOD reaction. However a recovery assay, or known concentrations of target DNA added to known negative samples, was not conducted.
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Because of the much larger number of pigs than used in comparable studies (Chowdhury et al., 2003a,b; Jennings et al., 2003a), sample analysis was not replicated during conventional PCR. It is expected that any variation due to analysis would be reflected in the larger number of samples similarly to the variation seen by replication of fewer samples.
Real-Time PCR
Real time PCR was performed using QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) and analyzed on the DNA Engine Opticon 2 (MJ Research, Waltham, MA). Optimum cycling conditions were determined to be as follows: 95° C for 15 min, 36 replications at 95° C for 45 s, 62° C for 45 s, 72° C for 2 min, plate read, and a final elongation at 72° C for 5 min with a melting curve analysis from 55 to 95° C read every 0.2° C and held for 1 s. Each 50-µL reaction contained 25 µL SYBR Green PCR Master Mix (including HotStarTaq DNA polymerase), 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer, 1 µL of ultrapure RNase-free water, and 20 µL template DNA.
To determine DNA concentration, all samples were initially analyzed with an Eppendorf BioPhotometer at A260 = 50 ng of dsDNA/µL using disposable Eppendorf UVette cuvets. The average amount of template DNA in the PCR reaction was as follows: 317 ng of conventional corn DNA, 270 ng of gdhA+ corn DNA, 300 ng of diet DNA, 153 ng of stomach ingesta DNA, and 156 ng of ileal ingesta DNA. These were the maximum quantities allowable by the extraction concentrations and were below the 500 ng/reaction maximum threshold recommended by the SYBR Green PCR Handbook (Qiagen, 2003).
To establish absolute quantification, a standard curve was created. Positive corn DNA underwent conventional PCR and gel electrophoresis. The target DNA was purified from the gel using Zymoclean Gel DNA recovery kit and underwent spectrophotometry (Eppendorf BioPhotometer at A260 = 50 ng of dsDNA/µL) to determine concentration. A series of dilutions was created. The quantities of the standard reactions were: 990 pg, 99 pg, 0.99 pg, 9.9 fg, 0.099 fg, 0.99 ag, 9.9 zg, and 99 yg. Standards were run in duplicate during every real-time PCR run.
Treatment and 0-h control stomach ingesta samples from pens 1 to 4 were run in duplicate at one time, and samples from pens 5 to 8 were run in duplicate at a separate time. Ileal ingesta samples were likewise analyzed in 2 separate runs. Known standards and positive and negative assay controls were analyzed with every run.
The cycle threshold line, C(t), was used for quantification. The C(t), was set above the background noise of known negative and assay control samples in a manner that provided the tightest fit of the replicate standards onto the standard curve. Due to differences in background noise, different C(t) lines were used for stomach and ileal ingesta samples, although the same C(t) line was used for both runs within sample type so that each sample type had one standard curve regardless of the run. The point at which sample signal intensity crossed the C(t) line was designated as the C(t) value. Sample C(t) values were averaged and CV were calculated. All samples with CV less than 5% were considered acceptable, and the average C(t) value was used for quantification. Those samples that expressed C(t) values with CV greater than 5% were considered unacceptable and declared nondeterminant, and not included in the pen average C(t) values that were reported.
A one-sample t-test was used to analyze the difference in the concentration of transgenic to total DNA between diet and stomach ingesta. The t-test was conducted using the Statistical Package for the Social Sciences Version 10.0 (SPSS, SPSS Inc., Chicago IL), and the model included the effects of pen and sex. A 
2 analysis was conducted to compare the effect of day of sample collection on the nondeterminant or negative stomach samples.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Using conventional PCR, detection levels of 0.99 fg of target DNA per 20 µL reaction were attained, even when 500 ng of conventional corn DNA were included in the reaction (Figure 3
). Because the quantity of template DNA added to each reaction never exceeded 400 ng per 20 µL reaction, 500 ng of conventional corn DNA is well above the quantity of nontarget DNA present in the experimental reactions.
The 490 bp region of the gdhA plasmid was amplified from the gdhA+ corn diet, showing transgenic DNA was detectable in the diet. Furthermore, this transgenic fragment was detected in 40 of 56 stomach ingesta samples from pigs fed the gdhA+ corn diet (Table 2). The transgenic fragment was not detected in the stomach ingesta from any control-fed pigs. During analysis of stomach ingesta, the lanes corresponding to the gdhA+ corn were positive for this amplicon, whereas the 2 lanes corresponding to water and the 2 lanes corresponding to the conventional corn did not show a band, supporting the absence of false positives or false negatives.
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). The transgenic fragment was not detected in large intestine ingesta, plasma, white blood cells, liver, or muscle of any pigs (data not shown). The gdhA+ corn was positive for the amplicon during analysis of each of these samples, and the conventional corn and the water were expectedly negative.
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). Because the protocol using the real-time thermocycler used a total of 36 cycles, the theoretical level of detection was determined by using a C(t) value of 36 in conjunction with the standard curve. The level of detection was estimated to be 52.46 ag per 50 µ L reaction.
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Of the 56 stomach ingesta samples collected from pigs fed the gdhA+ corn diets, 50 had quantifiable amounts of the target DNA (avg 1.56 fg), 4 had quantities of target DNA below the limit of detection, and 2 were considered nondeterminant because they had CV greater than 5% (Table 2). Of the 6 stomach ingesta samples declared negative or nondeterminant, 5 of them were collected on d 28, when pigs had ad libitum access to feed. Only 1 negative sample was collected on d 29, when pigs underwent a 24-h fast and 3-h refeed. Using 2 analysis, these results are not significantly different from the random model (P > 0.05; data not shown). The numerical difference, however, poses the possibility that DNA in the GI tract of ad libitumfed pigs might have undergone greater degradation before sample collection than those pigs killed immediately after fasting and refeeding.
None of the stomach ingesta samples from any of the 0-h controls had quantifiable amounts of target DNA. All of the samples that tested negative or nondeterminant using real-time PCR also tested negative using conventional PCR. However, several samples that had tested negative using conventional PCR tested positive with real-time PCR, reflecting the greater sensitivity with real-time PCR.
Variations in extraction efficiency resulted in different amounts of template DNA. Because the starting quantity was not standardized, resulting quantities of target DNA were expressed as a proportion of starting DNA. As expected, the concentration of target DNA was greater in the gdhA+ corn samples than in the diet samples. Interestingly, the mean proportion of target DNA in stomach ingesta (1.83 x 10–6%) differed from the concentration in the diet sample (3.42 x 10–6%, P < 0.001).
As expected, the concentration of the transgenic DNA relative to total genomic DNA was greater for gdhA+ corn (1.85 x 10–5%) than for diet (3.42 x 10–6%), stomach (1.82 x 10–6%), or ileal (2.14 x 10–6%) samples. The concentration of the transgenic DNA relative to total genomic DNA differed between diet and stomach samples (P < 0.001). Of 56 ileal ingesta samples collected from pigs fed the gdhA+ corn diets, only one sample had quantifiable amounts of target DNA (2.137 fg). This sample corresponded with the single positive result obtained using conventional PCR.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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The number of animal controls in this experiment may not have been adequate; nonGM-fed animals were not collected simultaneously with treatment animals. This limited our opportunity to evaluate the potential physiologic changes during maturation, which might have altered the findings. We were able to detect the presence of 990 ag of GM DNA when confounded with 500 ng of conventional corn DNA. Takai et al. (1998) reported average dust contamination in swine units of 2.19 mg/m3. With a ventilation rate of 10 cfm, the total presumable feed derived dust accumulation would approach 284 g during the 9-d GM feeding time period. This level of contamination approaches our LOD and could result in finding the GM gene of interest in nonGM-fed animals; hence these data were not acquired.
The positive results obtained from the stomach ingesta samples of gdhA+ diet-fed pigs were not unexpected. Chowdhury et al. (2003a) successfully amplified both intrinsic corn genes and the transgenic cry1Ab gene from stomach ingesta of growing pigs fed a Bt isoline or Bt11 corn diets, respectively. Because the major source of porcine deoxyribonuclease is secreted from the pancreas into the small intestine (White et al., 1977; Yen, 2001), stomach ingesta would have limited enzymatic exposure, reducing the likelihood of DNA digestion. Recent in vitro work by Wilcks et al. (2004) showed that DNA experiences slower degradation in the stomach compared with the rate of degradation occurring in duodenal and jejunal ingesta. The results of this study are in agreement with this finding.
The fact that not every stomach sample was positive for the transgenic DNA may reflect degradation occurring from the mechanical disruption of mastication, acid secretion and/or enzymatic actions from saliva and stomach. The deoxyribonuclease (DNase) I-specific mRNA has been extracted from the submaxillary gland (Mori et al., 2001), suggesting that some DNA degradation is likely to occur early in the digestive process.
A fragment of gdhA+ plasmid was detected in 1.79% of ileal ingesta samples and in none of the large intestine ingesta, in contrast to the work by Chowdhury et al. (2003a, b) who reported amplifying sections of transgenic and intrinsic corn genes from duodenal, ileal, cecal, and rectal contents. However, several differences should be noted between these previous studies and the current research. Although Chowdhury et al. (2003a, b) amplified 1 gene fragment of 1,028 bp, the other 8 fragments amplified ranged in size from 103 bp to 437 bp, compared with 490 bp in this study. As suggested by Einspanier et al. (2001), these smaller fragments may have a greater likelihood of being detected because larger fragments may be partially degraded and go undetected. Additionally, grower pigs used in the Chowdhury et al. (2003a, b) studies were fed diets with a greater inclusion of corn for a longer time period. Feed intake, though not reported, might also have been greater, resulting in a greater input of target DNA. Based upon these differences, the digestive ability of a weanling pig vs. a growing pig must be considered. Little is known about the expression of pancreatic DNase at various ages in a pig’s life; however, ribonuclease activity in the intestines of pigs has been shown to decline with age (Baintner and Farkas, 1989). If DNase follows a similar pattern, dietary DNA would have less exposure to DNase in older pigs, and DNA would be more readily amplified from intestinal ingesta of an older pig than a younger pig. This could explain why dietary DNA has been detected in large intestinal ingesta of grower pigs (Chowdhury et al., 2003a,b) but not in the nursery pigs in this study.
The limited work, including this study, suggests a rapid degradation of DNA as it moves through the alimentary tract. The greatest area of degradation seems to be the small intestine. Eighty percent of dietary DNA is degraded between the abomasum and terminal ileum of steers (McAllan, 1980). Likewise in mice, DNA is most rapidly degraded in duodenal and jejunal ingesta with much slower degradation occurring in the stomach and large intestine (Wilcks et al., 2004). The current study showed that in nursery pigs, the greatest degradation of dietary DNA also occurred in the small intestine; target DNA was detected in 89.29% of stomach ingesta but only 1.79% of samples from the terminal ileum.
In the current study, none of the tissue samples, including plasma and white blood cells from the hepatic portal vein, liver samples, and 10th rib muscle samples was positive for the transgenic plasmid DNA. This disagrees with previous work that showed 0.01 to 0.1% of pipette-fed phage M13 DNA could be detected in the blood of mice (Schubbert et al., 1994). However, it has been suggested that unmethylated viral DNA, such as the M13 DNA, can upregulate inflammatory cell activity and stimulate a vigorous immune response, potentially resulting in a greater than normal uptake of M13 DNA into white blood cells (Beever and Kemp, 2000). Nonviral DNA, such as that used in the current study, would be a less likely target for leukocyte engulfment.
In dairy cows, a 199 bp section of the chloroplast gene was amplified from lymphocytes, although this finding was not repeated in a subsequent study (Einspanier et al., 2001). Chloroplast DNA has been amplified from various chicken tissues (Einspanier et al., 2001), and it has been suggested that chloroplast DNA may have been detectable due to its greater abundance within the plant’s genome (Jennings et al., 2003b). The lower copy number genes, such as those incorporated via transgenesis or any single-copy endogenous gene, are less likely to be detected. This theory is supported, since despite finding the chloroplast DNA, the Bt-maize specific gene fragment was not detected in blood, muscle, liver, spleen, or kidney samples in either chickens or cattle (Einspanier et al., 2001). Endogenous and transgenic DNA was not detected in LM samples from finishing pigs fed Round-up Ready or conventional SBM-based diets (Jennings et al., 2003a). Endogenous and transgenic DNA was also not detected in breast muscles of chickens fed YieldGard corn borer corn or conventional corn grain diets (Jennings et al., 2003b). The current study is in agreement with these findings.
One unexpected result obtained through real-time PCR analysis was the difference in concentration of transgenic DNA relative to total genomic DNA when diet samples were compared with stomach ingesta samples. If all dietary DNA were degraded at the same rate, the concentration of transgenic to total DNA should remain unchanged from diet to stomach. Although endogenous DNA was not accounted for in this analysis, these preliminary results raise questions regarding the rate of degradation of transgenic vs. nontransgenic DNA throughout the digestive tract. This is an area that has not been previously addressed and should be considered in future research.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: pigguy{at}siu.edu
Received for publication December 6, 2004. Accepted for publication October 31, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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