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Measurements of Fitness and Competition in Commensal Escherichia coli and E. coli O157:H7 Strains

Lisa M. Durso,^1, David Smith,² and Robert W. Hutkins^1*

Department of Food Science and Technology,¹ Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, Nebraska²

Received 3 May 2004/ Accepted 1 July 2004

	ABSTRACT

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Although the main reservoirs for pathogenic Escherichia coli O157:H7 are cattle and the cattle environment, factors that affect its tenure in the bovine host and its survival outside humans and cattle have not been well studied. It is also not understood what physiological properties, if any, distinguish these pathogens from commensal counterparts that live as normal members of the human and bovine gastrointestinal tracts. To address these questions, individual and competitive fitness experiments, indirect antagonism assays, and antibiotic resistance and carbon utilization analyses were conducted using a strain set consisting of 122 commensal and pathogenic strains. The individual fitness experiments, under four different environments (rich medium, aerobic and anaerobic; rumen medium, anaerobic; and a minimal medium, aerobic) revealed no differences in growth rates between commensal E. coli and E. coli O157:H7 strains. Indirect antagonism assays revealed that E. coli O157:H7 strains more frequently produced inhibitory substances than commensal strains did, under the conditions tested, although both groups displayed moderate sensitivity. Only minor differences were noted in the antibiotic resistance patterns of the two groups. In contrast, several differences between commensal and O157:H7 groups were observed based on their carbon utilization profiles. Of 95 carbon sources tested, 27 were oxidized by commensal E. coli strains but not by the E. coli O157:H7 strains. Despite the observed physiological and biochemical differences between these two groups of E. coli strains, however, the O157:H7 strains did not appear to possess traits that would confer advantages in the bovine or extraintestinal environment.

	INTRODUCTION

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Free-living Escherichia coli bacteria are generally found in two environments (51). Their primary habitat is the intestines of warm-blooded animals. Their secondary habitat is water, soil, and sediment. The primary habitat of the animal host provides a warm nutrient-rich environment that supports vigorous growth and reproduction of E. coli. In contrast, the secondary environment is generally cool, with wide fluctuations in temperature. It is also usually nutrient limiting, such that growth rates of E. coli are typically low. In theory, an individual E. coli bacterium can be expected to spend part of its life in the primary habitat, after which it is excreted into the secondary habitat, where it and its progeny either survive until they colonize a new host or die. In order to be successful, therefore, any specific E. coli clone must be able to outcompete not only other microorganisms but also other E. coli strains in either or both of these habitats (51).

While most E. coli strains are harmless inhabitants of the large intestine (5), some E. coli strains cause disease in humans, with over 60 serotypes able to produce Shiga-like toxins (31). Strains of enterohemorrhagic E. coli, such as E. coli O157:H7, are of particular concern due to their ability to cause serious food-borne disease. Although this serotype appears to be rather widespread in the animal environment, one of the main sources of this serotype is considered to be foods of animal origin, especially beef (12, 38, 40, 41, 46, 50, 59). Cattle, like humans, serve as gastrointestinal hosts for E. coli (5, 6), including E. coli O157:H7, and cattle are generally considered to be a reservoir for E. coli O157:H7 (10, 25, 33, 41) Indeed, E. coli O157:H7 can readily be isolated from the rumen and colon of experimentally inoculated calves and is thought to colonize these sites in the adult animal as well (9, 18, 24, 27, 41). As in humans, there can be significant fluctuation in the total number of generic E. coli bacteria and in the number of specific E. coli serotypes occurring at any one time in any particular animal (11, 28, 53, 55). Thus, E. coli O157:H7 can be expected to compete against other commensal E. coli strains within the bovine intestinal tract.

Most natural environments that support the growth or survival of E. coli contain a mixed population comprised of numerous individual clones (5, 6, 30, 55). Success in these environments depends on at least several physiological properties. Strains having enhanced metabolic diversity and capable of utilizing a wide array of carbon sources and other nutrients would be predicted to do especially well in the secondary, nutrient-limiting habitat. Production of, and resistance to, known intraspecific antagonistic agents, such as bacteriocins, would also be useful in enabling one clone to compete successfully against other similar clones (49). Likewise, resistance to antibiotics may also confer competitive advantages on those strains. Finally, all other factors being equal, the strains that are able to grow most rapidly will ultimately be more successful (22, 23).

Fitness describes the overall ability of an organism to survive and reproduce and can be measured in bacteria by calculating growth rate (2, 3, 13). The environments in which a bacterium must survive have been shown to affect fitness values (45), such that the same bacterium can have different fitness values in different environments (15). Natural selection would suggest that, in order for E. coli O157:H7 to have emerged and be maintained in a particular niche, it must be more fit than competing commensal E. coli strains. The aim of this present study, therefore, was to compare general fitness properties of E. coli O157:H7 and commensal E. coli strains. Specifically, we focused on issues of nutrient utilization, competition, and fitness in environments simulating the mammalian gastrointestinal tract, the cattle rumen, and nutrient-rich and nutrient-poor extraintestinal environments. We hypothesized that E. coli O157:H7 would display distinctive patterns of nutrient utilization, competition, and fitness relative to those of commensal E. coli strains and that these differences would reflect the overall ability of O157 strains to compete against the commensal E. coli population.

	MATERIALS AND METHODS

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Bacterial strains.
The strain set used in this study consisted of 81 strains of E. coli O157:H7, 39 commensal E. coli isolates, and two laboratory K-12 strains of E. coli, for a total of 122 strains. Of the E. coli O157:H7 strains, 43 were isolated from infected humans and 32 were isolated from cattle. All had been previously characterized genotypically by octamer-based genome scanning (OBGS [32]) and classified as lineage 1 (44 strains) or lineage 2 (31 strains). Another six strains were obtained from the Karch laboratory in Germany. The commensal strains included 31 E. coli reference strains (37) and 8 cattle strains from the culture collections at the University of Wisconsin and University of Nebraska (17). The E. coli O157:H7 portion of the set was designed to represent the genetic and geographic diversity of this serotype in the United States, while the commensal E. coli portion of the set was designed to represent the entire genetic diversity of the species (54). All cultures were maintained in 15% glycerol stocks at –80°C.

Media.
All strains were routinely maintained on tryptic soy agar (TSA; Difco, Ann Arbor, Mich.) and propagated by aerobic growth at 37°C in tryptic soy broth (TSB). For both individual and competitive fitness experiments three medium conditions were used: TSB, M-9 minimal medium (4), and modified 98-5 medium (42).

Carbon source utilization.
Biolog ES (Biolog, Inc., Hayward, Calif.) plates were used to detect carbon utilization on 95 substrates. Each plate included a negative water control. The Biolog microplate carbon utilization assay is based on oxidation of each carbon source and subsequent detection of the reduced (purple) form of a tetrazolium dye. Each strain was grown in TSB, harvested during log phase via centrifugation (5 min at 8,000 x g), washed, and resuspended to an optical density at 600 nm of 0.1 to 0.2 in 0.85% saline. Samples (150 µl) were inoculated into each well of prewarmed Biolog plates. Plates were incubated at ambient atmosphere for 24 h at 37°C. Plates were scored in a binary fashion as either positive or negative. Data were analyzed in subgroups representing pathogenic versus commensal strains, OBGS group 1 versus group 2 strains (32), human O157:H7 versus bovine O157:H7 strains, and human versus bovine commensal strains.

Bacterial competition assays.
Bacterial competition assays were performed in a 122-by-122 matrix format by an agar diffusion technique. Producer and test strains were grown aerobically overnight in TSB at 37°C. Producer strain cells were centrifuged for 5 min, and the supernatants were held on ice. Test strain cells were inoculated into tempered soft TSA (20 µl of cells into 3 ml of soft agar) and overlaid onto TSA plates. Sterile 6-mm-diameter paper disks were placed on the agar, and 25 µl of each supernatant was inoculated onto each disk. Plates were incubated aerobically overnight at 37°C. Zones of clearing, if present, were recorded in millimeters. All positive tests were repeated.

Antibiotic resistance.
Antibiotic susceptibility tests were performed for all 122 strains according to the National Committee for Clinical Laboratory Standards (NCCLS) standard procedures. Briefly, premeasured antimicrobial disks were applied to the surface of Mueller-Hinton agar plates, previously seeded with each strain. Included were three beta-lactam penicillins, five expanded-spectrum cephalosporins, five aminoglycosides, two sulfas, cephalothin, cefoxitin, aztreonam, trimethoprim, tetracycline, chloramphenicol, nalidixic acid, ciprofloxacin, and imipenem. Plates were inverted and incubated overnight at 37°C in an aerobic incubator. Zones of clearing were recorded in millimeters, and sensitivity or resistance was determined using standard NCCLS interpretation charts. All experiments were performed in duplicate.

Individual fitness assays.
The entire strain set was randomly arrayed, in triplicate, in 96-well plates (Costar 3598). The wells were then filled with 200 µl of sterile TSB medium and incubated overnight at 37°C. Experiments were performed by addition of 1% of the overnight cultures into fresh plates filled with 200 µl of the appropriate medium. Plates were incubated under the appropriate conditions, and cell densities were determined by optical density measurement (Sunrise Microplate Reader; Phoenix Research Products, Hayward, Calif.) at a wavelength of 620 nm. Each well was read twice, and the values were averaged. Each of the three sets of plates was run in duplicate, for a total of six measurements per strain. Data were exported into an Excel file, and calculations were performed using Microsoft Excel software. Optical density measurements were converted to cell number by using a standard curve. Fitness was measured as maximum growth rate, with growth rate calculated as [(R2 – R1)/T)]*2.303, where R equals the log of the converted optical density reading at a wavelength of 620 nm and T equals time in hours. Reported growth rates represent the average of the maximum growth rate for all replications. Results were compared using the two-tailed Student t test. Experiments were run in four experimental conditions, simulating four different environments. These included aerobic growth in TSB, aerobic growth in M-9, anaerobic growth in TSB, and anaerobic growth in modified 98-5 rumen medium. Medium used for the anaerobic experiments was allowed to equilibrate in an anaerobic environment for 24 h before being inoculated. All anaerobic manipulations and incubations were performed in an anaerobic chamber (model 1025; Forma Scientific, Marietta, Ohio).

	RESULTS

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Carbon utilization.
Carbon source utilization tests showed that 28 of the 95 substrates were oxidized by 100% of the E. coli strains tested. An additional 30 substrates were used by at least 85% of the strains within each group. Eight of these substrates were reduced by at least 85% of the strains from each subgroup, except for the K-12 strains, which means that either one or both of the K-12 strains did not oxidize these eight substrates. Ten of the 95 substrates were oxidized by less than 25% of the strains from each subgroup. The results for the remaining 27 substrates are listed in Table 1. Of particular interest were D-saccharic acid, D-sorbitol, D-serine, D-galactonic acid -lactone, and glycolic acid, which were oxidized by nearly 80% of the commensal strains but by less than 20% of the O157:H7 strains. Fewer differences were observed between the OBGS lineage 1 and lineage 2 strains and between cattle and human E. coli O157:H7 strains, with dulcitol and glycyl-L-aspartic acid showing the greatest differences.

Producer results can be further analyzed by subgroup. The commensal strains were active against other commensal strains and both OBGS lineage 1 and OBGS lineage 2 strains. In contrast, although the O157:H7 strains produced inhibitory substances that were active against both commensal and O157:H7 strains, there were more than three times as many positive tests in which an E. coli O157:H7 strain produced a substance which was active against OBGS lineage 1 strains as those in which such a strain produced a substance active against OBGS lineage 2 strains.

When the results are examined from the perspective of which strains were more frequently inhibited, the results for the two main subgroups are similar, with 50% of the sensitive test strains being commensal or K-12 E. coli strains and 50% being E. coli O157:H7. It was noted that 16 commensal strains and two K-12 strains were responsible for 51 and 33 sensitive tests, respectively, compared to 44 E. coli O157:H7 strains being responsible for the other 84 sensitive tests. Seven commensal E. coli strains and 18 E. coli O157:H7 strains were sensitive to more than one producer strain. These results can also be analyzed by subgroup. Of interest is that both commensal and O157:H7 E. coli strains are more likely to be inhibited by a substance produced by an E. coli O157:H7 strain than by a substance produced by a commensal strain.

Antibiotic sensitivity assays revealed that most E. coli strains were sensitive to the majority of antibiotics tested. However, 17 of the 81 E. coli O157:H7 strains and 11 of the 40 commensal strains were resistant to antibiotics (Table 2). Resistance to streptomycin, sulfamethoxazole, and tetracycline was most common. It is interesting that, when E. coli O157:H7 resistance patterns were examined by OBGS subgroup, a greater percentage of OBGS lineage 2 than of lineage 1 strains were resistant.

	DISCUSSION

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Data presented here support the hypothesis that E. coli O157:H7 strains, as a group, have a different carbon-source utilization pattern from that of commensal E. coli strains. There were 19 carbon sources that were differentially oxidized by E. coli O157:H7 than by commensal E. coli strains. When E. coli O157:H7 was first described, extensive biochemical analysis were performed on this new clone in search of phenotypic characteristics that would distinguish it from nonpathogenic E. coli strains (1, 44, 59). In addition to sorbitol fermentation, which was widely exploited for screening and identification of the new pathogen (34), raffinose, dulcitol, and rhamnose were also initially considered as potential discriminatory substrates (43). The use of the phenotypic microarray plates allowed us to expand the range of substrates examined and process samples in a high-throughput manner. Among the 19 carbon sources identified using the Biolog plates were sorbitol and dulcitol, as expected. It is important to note that the Biolog plates measure substrate oxidation and not sugar fermentation. This can be seen most dramatically in the sorbitol results for the 19% of the E. coli O157:H7 strains that were positive for sorbitol oxidation on the Biolog ES plate but negative for sorbitol fermentation when grown on sorbitol-MacConkey agar. E. coli O157:H7 strains were more likely to use dulcitol, sucrose, and L-galactonic acid -lactone than were their commensal E. coli counterparts. All other differentially used carbon sources were more frequently used by commensal E. coli than by E. coli O157:H7 strains. Thus, within the limits of the 95 carbon sources tested in this assay, distinct patterns of carbon source utilization exist for E. coli O157:H7 and commensal E. coli strains. These observations may be due to stochastic events or, alternatively, may be a result of exposure to different selective pressures.

In general, the commensal E. coli strains oxidized more substrates than the E. coli O157:H7 strains did. This was unexpected, considering the larger genome of the E. coli O157:H7 strains (7, 29, 39). Sequence analysis has revealed that E. coli O157:H7 codes for 1,632 proteins not found in E. coli K-12, only 131 of which are associated with virulence functions (29). We had expected that some of these non-virulence-associated E. coli O157:H7-specific proteins might serve to enhance the metabolic flexibility of the pathogenic strains. Our results showed, however, that E. coli O157:H7 strains were metabolically less diverse, based on carbon utilization patterns, than were the commensal strains tested.

Differences were also observed in the intraspecific competition patterns of E. coli O157:H7 and commensal E. coli strains. Intraspecific competition, especially the production of bacteriocins, has been well studied. Early work (47, 48) showed that a large percentage of E. coli strains both produce and are resistant to at least one bacteriocin and that both commensal and O157:H7 E. coli cells are capable of producing colicins (35, 36, 48). Despite extensive research on E. coli to characterize the production of and sensitivity to colicins under mitomycin C-induced laboratory conditions (19, 21, 35, 36, 47-49), little is known about the patterns of interaction under more naturalized settings or whether E. coli O157:H7 strains, as a group, demonstrate the same intra- and interspecific competition patterns as their commensal counterparts do. We therefore decided to characterize the nature of intraspecific E. coli interactions under noninduced conditions. Data from our experiments show that E. coli O157:H7 and commensal E. coli appear to have different competition patterns, with E. coli O157:H7 more likely to both produce and be sensitive to inhibitory agents when grown under standard laboratory conditions.

The inhibitory interactions observed in these experiments most likely represent bacteriocin-mediated interactions. Phage-mediated interactions were ruled out based on the zone morphology and the inability to propagate plaques. In addition, zones of clearing were still observed even after the supernatant was boiled before inoculation. Since many of the bacteriocin proteins are small (8), they are not easily denatured by heat. Finally, most of the bacteriocin plasmids in E. coli also contain genes coding for lysis of proteins, which are expressed during bacteriocin production (56). This would explain the zones of clearing around the disks despite the fact that live producer cells were also inoculated onto the plate. Thus, most of the zone-of-clearing interactions were suspected to have been due to colicin production by the producer strain, although the substances responsible for the clearing were not further characterized.

Overall, zones of clearing were observed in only 1% of the assays. As expected, this number is lower than the observations of Riley and Wertz that between 10 and 50% of all E. coli strains are producers of bacteriocins (49). This difference can be attributed to the fact that Riley and Wertz used mitomycin C to induce colicin production, whereas we performed our experiments under noninduced conditions. Within the positive tests, E. coli O157:H7 strains were much more likely to produce inhibitory substances than their commensal counterparts were. In 96% of the positive tests the producer strain was an E. coli O157:H7 strain, compared to only 4% of the tests showing a commensal strain as a producer. These results are in contrast to those of DeAlwis and Thomlinson (14), who examined colicin activity of commensal and pathogenic E. coli isolates from pigs and found that the commensal strains produced a broad spectrum of colicins affecting the pathogenic strains but that the pathogenic strains produced only a few colicins, with a narrow spectrum. Our results showed that the E. coli O157:H7 supernatants were most commonly active against multiple strains, compared to only a single instance in which the commensal E. coli supernatant was active against more than one other strain. Furthermore, E. coli O157:H7 strains appear to have inhibited other E. coli O157:H7 strains as often as they inhibited K-12 and commensal E. coli strains. Thus, there does not appear to be any targeting of nonpathogenic strains by members of the E. coli O157:H7 group.

From the perspective of which strains were sensitive to inhibitory substances, both E. coli O157:H7 and commensal E. coli strains appeared to be moderately sensitive. In fact, the two K-12 strains were responsible for 33 of the 84 instances in which strains other than E. coli O157:H7 were inhibited. Since the K-12 strains were probably not very representative of wild commensal E. coli strains, the K-12 results were separated from those of other commensal E. coli strains. Thus, with the K-12 strains removed from the commensal category, 50% of the inhibited strains were E. coli O157:H7 strains, while only 30% of the inhibited strains were commensal E. coli strains. Therefore, the E. coli O157:H7 strains, as a group, were more likely than the commensal strains to be involved in production of and sensitivity to intraspecific inhibitory agents.

Minor differences were also observed in antimicrobial resistance patterns between the E. coli O157:H7 and commensal E. coli strains, with the former strains, in general, being more frequently resistant than the latter. Schroeder et al. (52) recently screened 361 E. coli O157:H7 strains for resistance to 13 antimicrobials also used in this study. Our overall susceptibility rates for E. coli O157:H7 strains of 61% are similar to the 63% overall susceptibility rates obtained by Schroeder's group. In most cases, strains in our study that demonstrated resistance were multiply resistant, such that one strain was resistant to more than one antibiotic. Our data also showed that commensal E. coli and E. coli O157:H7 populations carried resistance to a variety of antibiotics that involve different resistance mechanisms.

In both the primary and secondary habitats, different E. coli strains must compete with each other for (often limited) resources. The strains that are most fit would be expected to outcompete those that are less fit. We therefore reasoned that, unless emergence was due entirely to stochastic processes, there must be at least one niche in either the primary or secondary environments where the O157:H7 clone can outcompete other E. coli strains—that is, where the E. coli O157:H7 clone is more fit than its commensal E. coli competitors. To address this hypothesis, we designed a high-throughput 96-well assay to measure fitness of commensal and O157:H7 E. coli strains via growth rate, based on optical density readings over time. Fitness of O157:H7 E. coli and commensal E. coli strains was determined under four different conditions that mimic the environments that these organisms encounter in the primary, secondary, and laboratory environments. Our results showed that not only were there no significant differences in individual fitness between the O157:H7 and commensal E. coli strains tested but the growth rates of these groups were virtually identical in all four environments tested. These results were surprising, especially in light of the larger genome of the E. coli O157:H7 strains and their more frequent production of antagonistic agents—both of which could be expected to increase the metabolic load and thus decrease the fitness of the E. coli O157:H7 strains compared to that of the commensal E. coli strains (20, 26, 49, 56). Also, we had expected to find one environment where the E. coli O157:H7 strains were more fit than their commensal counterparts. However, the fact that E. coli O157:H7 and commensal E. coli strains have similar fitness values in the four environments tested here does not exclude the possibility that these strains would show a fitness differential in other environments. In fact, bacteria have been shown to have different fitness values in different environments, and environmental fitness has been shown to be influenced by a variety of factors, including metabolic load, stress, and nutrient availability (16, 15, 45, 57).

The underlying hypothesis for the research presented here is that there are indeed physiological and ecological differences between E. coli O1457:H7 and other E. coli strains and that we would observe differences in fitness between E. coli O157:H7 and commensal E. coli strains. The alternative hypothesis, that these two groups of E. coli were physiologically the same, shared identical ecological niches, and had identical fitness values in primary and secondary habitat environments, was considered unlikely for several reasons. First, according to the tenets of natural selection, genetic differences between two individuals that affect viability or reproductive success will increase or decrease the fitness of an individual relative to others in the population. Thus, different genotypes can be expected to have different fitness values (22, 23). Differences in growth rates have previously been demonstrated for E. coli in a variety of environments (15, 57).

The complete sequencing of two E. coli O157:H7 strains (29, 39) as well as a K-12 strain (7) and the enteropathogenic CFT073 strain (58) shows that E. coli O157:H7 strains are 270 kb larger than the enteropathogenic E. coli strain and 859 kb larger than the K-12 strain (29). While the K-12 strain most likely differs somewhat from its "wild" counterparts, it nonetheless provides us with a framework to examine some of the underlying genetic differences between E. coli O157:H7 and other E. coli strains. It is commonly accepted that genetic load may affect fitness. That is, if all other factors are equal, organisms with more DNA to copy will grow slower and thus be at a disadvantage against their competitors that have smaller genomes. This suggested that, in order for E. coli O157:H7 to be an efficient competitor (compared to other E. coli clones), these "extra" E. coli O157:H7-specific genes must also be important for the organism. That is, they must affect fitness values.

Finally, of the 1,632 E. coli O157:H7-specific proteins, only 131 were associated with virulence factors (29). If E. coli O157:H7 were transmitted directly from human to human, like its close relative Shigella sp., then the presence of the virulence genes alone may have been sufficient to account for the emergence of E. coli O157:H7—the virulence genes would have provided a fitness advantage to E. coli O157:H7, compared to its nonvirulent commensal E. coli counterparts, and allowed the pathogenic clone to outcompete other E. coli clones. Instead, epidemiological evidence has shown that E. coli O157:H7 outbreaks have been most commonly linked to a primary food or water source, with secondary infections from person to person recorded only rarely. Additionally, if the virulence genes allowed E. coli O157:H7 to be reintroduced to cattle from a sick human, then the virulence genes alone may have been sufficient to account for the emergence of E. coli O157:H7. This pattern of transmission, however, has not been observed. Therefore, if the E. coli O157:H7-specific virulence genes alone are not sufficient to account for the emergence of the E. coli O157:H7 clone, then there may have been other differences between E. coli O157:H7 and other E. coli strains which gave the pathogen an advantage and allowed it to emerge. We suggest that it is likely that some of the remaining 1,501 E. coli O157:H7-specific proteins would influence how E. coli O157:H7 interacts in the primary and secondary habitats and would ultimately affect overall fitness values.

	ACKNOWLEDGMENTS

 
We thank Helge Karch and Andrew Benson for kindly providing strains, Heidi Baumert and Jordan Bowers for assistance with competition assays, and Grace Vizcaino for assistance with antimicrobial resistance assays. We also thank Kari Shoaf for her critical review of the manuscript.

This research was supported by the Nebraska Agricultural Experiment Station, State of Nebraska Legislative Bill 1206 research funds, and a USDA Graduate Needs Fellowship to L.M.D.

	FOOTNOTES

 
* Corresponding author. Mailing address: University of Nebraska, Department of Food Science and Technology, 338 FIC, Lincoln, NE 68583-0919. Phone: (402) 472-2820. Fax: (402) 472-1693. E-mail: rhutkins1{at}unl.edu.

Paper no. 14659, Journal Series Nebraska Agricultural Experiment Station, Lincoln, Nebr.

Present address: Roman L. Hruska U.S. Meat Animal Research Center, Agricultural Research Service, United States Department of Agriculture, Clay Center, NE 68933.

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