J. Anim. Sci. 2003. 81:1080-1087
© 2003 American Society of Animal Science
Pre-harvest factors influencing the acid resistance of Escherichia coli and E. coli O157:H7
C. J. Fu,
J. H. Porter,
E. E. D. Felton1,
J. W. Lehmkuhler2 and
M. S. Kerley3
Department of Animal Science, University of Missouri, Columbia 65211
3 Correspondence:
111 ASRC (phone: 573-882-0834; fax: 573-884-7712; E-mail:
kerleym{at}missouri.edu.
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Abstract
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The effects of pH, acetate, propionate, or butyrate concentration, and diet on acid resistance of fecal Escherichia coli and E. coli O157:H7 were determined by in vitro and in vivo experiments. The pH tested was from 4.0 to 8.0, and the VFA concentrations tested were 0 to 100 mM. The E. coli O157:H7 used was strain 505B. In an in vivo study, cattle were fed a grain-based diet, then either not switched or switched to a grain-based diet with 3% added calcium carbonate or two fiber-based diets (soybean hulls or hay). Acid resistance was expressed as viability after acid-shock at pH 2.0 for 1 h and 4 h for fecal E. coli and E. coli O157:H7, respectively. Enumeration methods used were multitube fermentation, agar plate, and petri-film methods. The E. coli O157:H7 was not found in continuous culture inocula or in vivo samples. The viability of fecal E. coli decreased linearly (P < 0.01) as the culture pH increased, and viability of E. coli O157:H7 was highest (P < 0.01) when cultivated at pH 6.0. The viability of fecal E. coli and E. coli O157:H7 showed quadratic responses (P < 0.05) as acetate and butyrate concentrations increased at pH 7.2, with maximal acid resistance at 20 and 12 mM, respectively. As propionate concentration increased, the acid resistance was not different (P > 0.05) for fecal E. coli. Acid resistance of E. coli was induced by acetate and butyrate, even though the environmental pH was near neutral. Similar results were measured in the in vivo study, where viability after acid shock was more dependent on VFA concentration than on pH. Increasing the dietary calcium carbonate concentration also increased (P < 0.05) acid resistance of fecal E. coli. Results from these studies demonstrated that culture pH and VFA affect acid resistance of E. coli.
Key Words: Diet • Escherichia coli • pH • Resistance • Volatile Fatty Acids
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Introduction
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Escherichia coli can become acid resistant if it is grown in a mildly acidic medium in the presence of VFA (Poynter et al., 1986; Diez-Gonzalez et al., 1998; Jarvis and Russell, 2001). Acid-resistant E. coli is a potential pathogen for humans if it contaminates food because of its ability to tolerate the low pH of the gastric stomach. More importantly, the E. coli O157:H7 strain is an important foodborne pathogen that affects humans. E. coli O157:H7 has caused approximately 2.6 infections per 100,000 individuals per year, with nearly 0.5% of those infections resulting in death (CDC, 2001). Furthermore, the infective dose can be as low as ingestion of 10 cells (Armstrong et al., 1996; Shallow et al., 1997), and antibiotic therapy can be ineffective (Neill, 1998).
Diez-Gonzalez et al. (1998) reported that when cattle were switched from a concentrate to a hay diet the colonic E. coli were killed by an acid shock that mimicked the gastric stomach of humans, whereas cattle fed 90% grain diets had large numbers of acid-resistant E. coli in colonic digesta. The forage diets caused the pH to increase and VFA concentration to decrease in the colon. Cray et al. (1998) demonstrated that a 48-h fast increased the E. coli O157:H7 population. Kudva et al. (1997) showed that an undescribed grass diet resulted in a longer time period of E. coli O157:H7 shedding than a concentrate:forage mixed diet. Hovde et al. (1999) demonstrated that the acid resistance of fecal E. coli O157:H7 was unaffected by the cattle diets. Ohya and Ito (1999) indicated that the concentration of VFA in calf feces might be associated with the presence of E. coli O157:H7.
The hypothesis was that diet could be used to manipulate the digesta VFA concentration and pH to decrease the acid-resistant population of fecal E. coli. Our objective was to determine the effect of VFA concentration on E. coli and E. coli O157:H7 acid resistance.
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Materials and Methods
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Continuous Culture Study
The continuous culture experiment was designed to examine the effect of pH on acid resistance of E. coli collected from cattle feces. The continuous culture system, control of dilution rate, and operation conditions used have been previously described in detail (Meng et al., 1999; Fu et al., 2001). Fermenter pH was controlled by infusion of different capacity buffers (Slyter, 1990) at pH 6.5, 7.0, and 7.5. The dilution rate was set at 0.045/h. Replicated fermentations were run two times using 12 fermenters (four fermenters randomly assigned to each pH level per run). Combined feces from 20 feedlot steers fed 85% corn-based concentrate diets were used to inoculate the fermenters. For the 0- to 100-mM acetate experiment, feces were collected from a different set of steers at a later time point than the inoculum for all other fermentations. The composite feces were mixed 1:2 with warm (36°C) buffer (Table 1
). After being strained through one layer of cheesecloth, 1,400 mL of fluid was added to each of the 12 fermenters. Each fermenter was fed 20 g of diet DM (95% corn + 5% soybean meal) every 8 h. Incubation time was 7 d, and samples were collected once daily during the last 3 d from each fermentor and frozen (µ80°C) until analyses were performed.
Pure Culture Study
The pure culture experiment was designed to examine the effects of pH and VFA concentration on the acid resistance of a pure strain of E. coli O157:H7 (505B) and E. coli from the continuous culture experiment. The culture medium was tryptic soy broth without dextrose (Difco, No. 862, Sparks, MD). The media pH and VFA concentration were adjusted by adding 4N HCl or 1N NaOH and pure acetate, propionate, and butyrate to the media. The media pH tested were 4.0, 5.0, 6.0, 7.0, and 8.0. The acetate and butyrate concentrations examined were 0, 5, 10, 15, 20, and 25 mM. An additional acetate level experiment (from 0 to 100 mM) was designed because the fecal acetate level is typically above 25 mM. The propionate concentrations were 0, 4, 8, 12, 16, and 20 mM. The pH of media used for studying the effect of VFA was maintained at 7.2 using 1N NaOH. Media (9 mL) was distributed into Hungate tubes that were continuously flushed with O2-free N2. The media was autoclaved at 121°C for 15 min. The inoculating E. coli O157:H7 was strain 505B and donated by another laboratory. The inoculating fecal E. coli was from conserved samples of the continuous culture study. However, the fecal E. coli for the additional test of acetate (0 to 100 mM) and pH (4 to 8) was from a different isolate E. coli of cattle feces. Inoculant (0.1 mL, ~104 fecal E. coli or E. coli O157:H7) was pipetted into the prepared Hungate tubes and cultivated for 24 h. After 24 h, the E. coli number reached about 108/mL, and then 0.1 mL (~107 E. coli) was transferred to identical treatment culture tubes for another 24 h, at which time bacteria were assumed to reach the stationary phase of growth. The culture was immediately enumerated by the methods described below. Each pure culture treatment was repeated six times.
Animal Study
Fifty-four mixed crossbred steers (average initial weight = 541 ± 37 kg) were used in a completely randomized design experiment to examine the effects of diet on acid resistance of fecal E. coli, including E. coli O157:H7. The experimental protocol was approved by the University of Missouri Animal Care and Use Committee. Steers were fed a concentrate diet (Table 2, control) for 58 d. After 58 d, steers were switched to one of four treatment diets. Composition and nutrient profile are presented in Table 2. The steers were fed once daily and had ad libitum access to the feed through the following day’s feeding. The steers were allotted by weight into 12 pens, with four to five steers per pen to achieve similar average pen weights. Three pens were randomly allotted to each treatment. Steers were weighed and rectal grab fecal samples were taken the day before being switched to treatment diets and 5 d after feeding the treatment diets. Two fecal samples were obtained for each animal, one for microbial analysis and the other for testing pH (mixed with deionized H2O at a 1:1 ratio). The samples for microbial analysis were mixed with autoclaved glycerol salt buffer at a 1:1 ratio and stored at µ80°C until microbial analysis.
Enumeration and Confirmation.
The stationary phase culture and thawed fecal samples were serially diluted with a peptone water solution. The E. coli and E. coli O157:H7 enumeration and confirmation in continuous culture study were done following the protocol of the Bacterial Analytical Manual of FDA and most probable number (MPN) method (Hitchins et al., 1998). The pure culture bacteria were enumerated (cfu/mL) by Luria-Bertani agar plate (Difco, No. 2445207) methods. For the in vivo study, the E. coli was enumerated (cfu/mL) by a petrifilm method according to the manufacture’s instruction (3M, Petrifilm, St. Paul, MN). The presence of O157 and H7 antigens was tested with the RIM E. coli O157:H7 Latex Test kit according to the manufacture’s procedures (Remel, Lenexa, KS).
Acid Shock
The serially diluted samples were pipetted into an aerobic acid shock medium (5 g of yeast extract and 10 g of trypticase per liter; Diez-Gonzalez and Russell, 1999) that had been adjusted to pH 2.0 with 6N HCl. Acid-shocked cultures were incubated for 1 or 4 h at 37°C for the E. coli (continuous culture and feces) and E. coli O157:H7, respectively. The cell survival was determined by the MPN method referenced above. The acid resistance is the viability of fecal E. coli and E. coli O157:H7 after the acid shock and calculated using the following equation:
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Statistical Analyses
The in vitro data were analyzed using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) with linear and quadratic responses tested by the CONTRAST statement. The linear and quadratic responses were considered significant when P < 0.05. The dietary treatment effects of in vivo data were also analyzed using the GLM procedure of SAS, and pen was used as the analysis unit. However, the dietary shift effects within each treatment were paired comparison using the t-test means procedure of SAS.
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Results and Discussion
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The effects of pH on the population and acid resistance of E. coli and E. coli O157:H7 are shown in Tables 3 and 4. The population of fecal E. coli grown in pure culture increased (P < 0.01) quadratically as pH increased, with the greatest population occurring at pH 8.0. The population of E. coli O157:H7 also increased (P < 0.01) quadratically as pH increased, with the highest population occurring at pH 6.0. The acid resistance of fecal E. coli grown in pure culture showed a quadratic (P < 0.01) response when pH was increased from 4.0 to 8.0, with the highest viability occurring at pH 5.0 to 6.0. The same numerical pattern was observed for E. coli O157:H7, but no statistical difference (P > 0.05) existed (Table 3). The population of fecal E. coli grown in continuous culture increased (P < 0.01) linearly as pH increased. The pH had no effect on population counts after acid shock, but the percentage viable was decreased linearly (P < 0.01) as pH increased (Table 4). The E. coli O157:H7 was not detected in the continuous culture study. These results were in agreement with previous studies (Goodson and Rowbury, 1989; Benjamin and Datta, 1995; Diez-Gonzalez and Russell, 1999) that indicated that lower pH could induce the acid resistance of E. coli. Interestingly, the pure culture fecal E. coli and E. coli O157:H7 had a numerically higher acid resistance at pH 5.0 and 6.0 than at pH 4.0. Although statistically different, the population difference before acid shock does not fully explain the acid-resistance data. A simple explanation would be that pH 4.0 is lethal to E. coli and E. coli O157:H7. Current feeding practices that use concentrate diets make the animal feces only mildly acidic, with pH values typically around 6.5 (Scott et al., 1999). Our results led us to conclude that acid resistance for both pathogenic and nonpathogenic E. coli could be induced at this pH. These results supported the conclusion that high concentrate diets could make the fecal E. coli acid resistant in ruminant animals (Diez-Gonzalez et al., 1998).
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Table 3. The effects of pH on the log population and viability of Escherichia coli and E. coli O157:H7 after acid shock in the pure culture study
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Table 4. The effects of pH on the log population and viability of fecal Escherichia coli after acid shock in the continuous culture study
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The effects of acetate, propionate, and butyrate level on the population and acid resistance of E. coli and E. coli O157:H7 are shown in Tables 5 to 8. The population of fecal E. coli in pure culture was not influenced (P > 0.05) by the concentration of acetate (0 to 25 mM, Table 5), propionate (Table 7), or butyrate (Table 8). The population of acid-resistant fecal E. coli was quadratically (P < 0.01) influenced by acetate and butyrate, but not propionate. The acid-resistant population was greatest when acetate was 15 or 20 mM, with the population the same at 0 or 25 mM. The acid-resistant population was greatest when butyrate was 12 mM, with higher concentrations yielding populations equivalent to 0 mM butyrate. Because the population of fecal E. coli was similar before acid shock, our conclusion was that the concentration of acetate and butyrate could induce development of acid resistance. This is shown by the viability after acid shock responding quadratically (P < 0.05) to acetate and butyrate concentration. The greatest viability occurred at an acetate concentration of 15 to 20 and a butyrate concentration of 12 mM. The population of E. coli O157:H7 decreased linearly (P < 0.05) as acetate increased and was quadratically (P < 0.01) influenced by butyrate concentration. However, the range in log population counts was from 8.42 to 8.79 and 8.21 to 8.81, so little biological significance was attributed to the effect of acetate and butyrate on E. coli O157:H7 population. Concentrations of 20 mM acetate and 12 mM butyrate resulted in the greatest acid-resistant population and percentage viability, which showed quadratic (P < 0.05) responses as the VFA concentration increased. These results were similar to the acid-resistant fecal E. coli viability responses and also similar to the acid-resistant fecal E. coli response where 15 and 20 mM acetate resulted in the highest acid-resistant E. coli O157:H7 population and viability. The acetate concentration in vivo is typically greater than 25 mM, so a second pure culture experiment was conducted. The fecal E. coli population before or after acid shock was not influenced by acetate concentration (Table 6). These data were in general agreement with the first experiment showing that concentrations less than 25 mM acetate increased acid resistance development. The E. coli O157:H7 populations were reduced quadratically (P < 0.01) after acid shock as acetate increased. The reduced populations after acid shock were most likely due to the population percentage before acid shock, evidenced by the acetate concentration having no effect (P < 0.05) on percentage viability. We concluded that acetate and butyrate could influence development of acid resistance by E. coli or E. coli O157:H7, which was in agreement with other research (Guilfoyle and Hirshfield, 1996; Brudzinski and Harrison, 1998; Diez-Gonzalez and Russell, 1999). However, most of these studies did not determine the dose response of acetate concentration except that of Diez-Gonzalez and Russell (1999), in which they reported the acid resistance of E. coli O157:H7 increased as the acetate concentration increased from 0 to 40 mM, but from 40 to 80 mM, the acid resistance apparently did not change. Based on our results, the acid resistance of fecal E. coli and E. coli O157:H7 increased as the acetate level increased from 0 to 20 mM, and then returned to the control population level. A consideration expressed by Diez-Gonzalez and Russell (1999) was the possibility that the acid resistance of E. coli may be induced only by the undissociated acetic acid. Due to this possibility, the pH in the pure culture VFA experiments was controlled to near neutral (7.2 ± 0.2), mimicking fecal pH. The undissociated acid was only a small proportion of the total acid compared to the dissociated form and the undissociated acetate concentration would have changed only nominally as the acetate concentration was increased since the pH was kept constant. Therefore, effects of VFA concentration on E. coli population or viability after acid shock should not be confounded by the concentration of undissociated acid.
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Table 5. The effects of acetate concentration (0 to 25 mM) on the log population and viability of Escherichia coli and E. coli O157:H7 after acid shock in the pure culture study
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Table 8. The effects of butyrate concentration (0 to 20 mM) on the log population and viability of Escherichia coli and E. coli O157:H7 after acid shock in the pure culture study
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Table 7. The effects of propionate concentration (0 to 20 mM) on the log population and viability of Escherichia coli and E. coli O157:H7 after acid shockin the pure culture study
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Table 6. The effects of acetate concentration (0 to 100 mM) on the log population and viability of Escherichia coli and E. coli O157:H7 after acid shockin the pure culture study
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The effects of dietary modulation on acid resistance of fecal E. coli of feedlot cattle are presented in Table 9. The fecal VFA concentrations were comparable with a previous study, in which the effects of forage:concentrate ratio on cecum-colon VFA level were determined (Siciliano-Jones and Murphy, 1989). There was no E. coli O157:H7 detected in this experiment. The acid resistance of fecal E. coli was affected (P < 0.05) by dietary modulation. As shown in Table 9, after 5 d on treatment diets, the hay diet decreased the fecal E. coli population and viability (P < 0.01) compared with the other three diets. The diet with 3% calcium carbonate induced higher (P < 0.01) E. coli acid resistance, but not population, than that of the other three diets. The soybean hull diet, a high-fiber diet similar to the hay diet, did not change the fecal E. coli viability compared to the control diet. The experimental diets were expected to change the fecal pH; however, only steers fed the hay diet had an increased fecal pH compared with the other dietary treatments (7.08 vs. 6.78, 6.67, and 6.67). In this study, the diets had more affect on changing fecal VFA concentration than on fecal pH. Fecal acetate concentration decreased (P < 0.05) with feeding the hay diet compared to the other diets. The fecal propionate and butyrate level decreased (P < 0.05) in the steers fed hay and increased (P < 0.01) in the steers fed calcium carbonate, respectively. The change in total VFA followed the acetate level pattern because the acetate concentration accounted for over 80% of the total VFA concentration.
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Table 9. The effects of dietary shifts on the fecal volatile fatty acid concentration, log population before and after acid shock, and the acid resistance of fecal Escherichia coli in the in vivo studya
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The E. coli population and viability of the steers fed hay decreased significantly compared to before hay was fed. The E. coli viability decreased in 10 of the 13 animals in this group. This outcome seemed to be caused mainly by VFA concentration instead of by pH since the fecal pH was increased only marginally compared to the calcium carbonate treatment group (7.08 vs. 6.93). The acetate, propionate, and butyrate concentration decreased 18.0% (41.5 vs. 50.6 mM), 67.2% (2.0 vs. 6.1 mM), and 75.9% (0.7 vs. 2.9 mM), respectively, after 5 d on the hay diet compared to before changing the diet. This result was in agreement with Diez-Gonzalez et al. (1998), who indicated that VFA played an important role in inducing acid resistance. In contrast, E. coli viability increased in 12 of the 13 animals in the calcium carbonate diet group and resulted in a significantly (30.3 vs. 9.9%, P < 0.01) increased viability of fecal E. coli. This study was the first experiment to test the effects of calcium carbonate (buffer) on acid resistance of fecal E. coli. The buffer was expected to increase the fecal pH and decrease the acid resistance of E. coli. However, the pH decreased only marginally (6.67 vs. 6.90). Acetate, propionate, and butyrate increased 49.3% (64.5 vs. 43.2 mM), 54.1% (9.4 vs. 6.1 mM), and 55.6% (4.2 vs. 2.7 mM), after 5 d on the treatment diet, respectively. The reasons for the buffer causing the increased viability of fecal E. coli are unknown. One of the possible reasons is that the Ca may make the E. coli more acid resistant by increased membrane stability because Ca is an important stabilizing agent of bacterial cell walls (Madigan et al., 1997). If Ca level influences acid resistance, there would be a need to reevaluate the Ca level in feedlot cattle diets prior to slaughter. As expected for the control group, there were no significant changes on E. coli population, viability, fecal VFA concentration, and fecal pH at the two sampling times. With the group fed soybean hulls, fecal acetate increased (P < 0.01), propionate and butyrate decreased (P < 0.01), total VFA increased (P < 0.05), and pH decreased numerically. There was only a marginal increase in E. coli viability. The failure of steers fed soybean hulls to harbor E. coli populations that responded similarly to acid shock as hay fed steers was unexpected. The reason for this was not evident.
Over the last 3 yr (1998 to 2000) in the United States, there were 4,473 reported cases of infection from E. coli, mainly from E. coli O157:H7, of which 116 developed hemolytic uremic syndrome. Eleven of these cases resulted in death (CDC, 2001). In these cases, 38% were confirmed related to ruminant animal production, which included animal contact (77 cases) and consumption of ruminant products (e.g., ground beef, milk: 1,617 cases). The contact infection cases supported the possible link to development of acid resistance by the pathogen in the animal. It is important to note that other studies showed that infection might have developed from food storage and processing (Waterman and Small, 1998; Glass et al., 1992; Abdul-Raouf et al., 1993). It has long been recognized that the acid resistance of E. coli is triggered by VFA and low pH (Goodson and Rowbury, 1989), but the relative importance of each factor was not determined in a systematic approach until 1999 by Diez-Gonzalez and Russell (1999). They indicated that the concentration of undissociated acetate was critical for inducing acid resistance of E. coli, and it appeared that pH effects were mediated via acetate dissociation. In this study, our data indicated that the acid resistance was affected by pH, acetate, and butyrate levels. The pH alone could induce acid resistance of E. coli O157:H7 even though the VFA concentration was low (<3 mM, in vitro data not shown). This result was in agreement with a previous study (Castanie-Cornet et al., 1999). However, when combining the in vitro and in vivo data together, the pH seems less important than VFA concentration because of the relative constant, near neutral colonic pH of animals.
Kudva et al. (1997) and Hovde et al. (1999) reported that their research contradicted Diez-Gonzalez et al. (1998). Their research did support Diez-Gonzalez et al. (1998) in that cattle fed hay had colonic E. coli populations less resistant to acid shock than animals fed grain. However, in contradiction, they reported that cattle or sheep fed hay (grass or alfalfa) shed fecal E. coli O157:H7 longer than when fed a grain or alfalfa/grain diet and that fecal E. coli O157:H7 acid resistance was not inducible similar to colonic nonpathogenic E. coli. We believe our results provide a plausible explanation for discrepancies in diet effects and agree with Diez-Gonzalez et al. (1998) in that E. coli O157:H7 does respond similarly to E. coli. Our data led us to interpret that concentration of VFA could be a major determinant to the growth environment for E. coli and E. coli O157:H7. Diets fed in the in vivo experiment (control and hay) were likely similar to those fed by Diez-Gonzalez et al. (1998). Our results mirrored their results on development of acid resistance in E. coli. We further speculate that diets fed where contradicting acid resistance responses occurred resulted in colonic/fecal VFA (acetate) concentration greater than the level that minimized inducement of acid resistance in E. coli. We also conclude from our research that E. coli O157:H7 responds similarly to signals inducing acid resistance of fecal E. coli.
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Implications
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Development of acid resistance by pathogenic Escherichia coli can be minimized by manipulating colonic volatile fatty acid concentrations. The optimal acetate concentration seems to be between 25 to 40 mM. Reduced acid resistance may be further achieved by a butyrate concentration near 16 mM. The effect of dietary calcium on development of acid resistance should be further studied.
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Footnotes
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1 Present address: College of Agriculture, Forestry, and Consumer Sciences, Division of Animal and Veterinary Science, G024 Agricultural Sciences Bldg., West Virginia University, P.O. Box 6108, Morgantown 26506. 
2 Present address: Department of Animal Science, University of Wisconsin, Madison 53706.
Received for publication May 3, 2002.
Accepted for publication January 3, 2003.
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Abstract
Introduction
