Microb Ecol (2008) 55:553–560 DOI 10.1007/s00248-007-9300-8
ORIGINAL ARTICLE
Effects of Exogenous Melatonin and Tryptophan on Fecal Shedding of E. Coli O157:H7 in Cattle Tom S. Edrington & Todd R. Callaway & Dennis M. Hallford & Liang Chen & Robin C. Anderson & David J. Nisbet
Received: 27 March 2007 / Accepted: 29 June 2007 / Published online: 15 September 2007 # Springer Science + Business Media, LLC 2007
Abstract Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer decreasing to very low levels in the winter. We hypothesize that this seasonal variation is a result of physiological responses within the host animal to changing day-length. To determine the effects of melatonin (MEL) on fecal shedding of E. coli O157:H7 in cattle, eight crossbred beef steers identified as shedding E. coli O157:H7, were allotted to treatment: control or MEL (0.5 mg/kg body weight (BW); 1×) administered orally daily for 7 days. After a 5-day period of no treatment, a second MEL dose (5.0 mg/kg BW; 10×) was administered daily for 4 days. Fecal samples were collected daily for qualification of E. coli O157:H7. No differences (P>0.10) were observed in the percentage of E. coli O157:H7 positive fecal samples in steers receiving the 1× MEL dose, however the 10× dose decreased (P=0.05) the percentage of fecal samples E. coli O157:H7 positive. Serum MEL concentrations were higher in the 1×, but not 10×, treated animals compared to control animals. Although it is difficult to explain, this may be a result of decreasing day-length increasing serum melatonin concen-
T. S. Edrington (*) : T. R. Callaway : R. C. Anderson : D. J. Nisbet Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA e-mail:
[email protected] D. M. Hallford : L. Chen Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003, USA
trations that may have masked any treatment effect on serum melatonin. In a second similar experiment, a second group of cattle (heifers and steers) were administered tryptophan (TRP) over a 17-day experimental period (5 g/head/day for 10 days followed by 10 g/head/day for 7 days). Tryptophan had no effect (P>0.20) on the percentage of fecal samples positive for E. coli O157. Serum TRP (P0.20), concentrations were elevated in TRP-treated animals. The decrease in the number of positive fecal samples observed in the first experiment, may be related to gastrointestinal MEL, affected by the 10×, but not 1× MEL dose.
Introduction Cattle are considered the primary reservoir for Escherichia coli O157:H7 [11, 16]. Although other routes of transmission have been identified (animal contact at petting zoos, apple cider, swimming water, drinking water, vegetables), a link to cattle or products exposed to or contaminated with cattle manure have often been implicated [11, 16, 36]. Historically, ground beef has been linked to more human outbreaks of E. coli O157:H7 than any other source [16]. Previous estimates reported E. coli O157:H7 may have cost the U.S. beef industry approximately $2.7 billion over the past 10 years, as a result of lost demand, impact on beef prices, product recalls, and costs associated with the implementation of food safety intervention strategies [23]. The prevalence of fecal shedding of E. coli O157:H7 in cattle is typically low or undetectable in the winter months, increases in the spring, and peaks in the summer or early fall before decreasing again [2, 12, 18, 44]. Not surpris-
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ingly, human outbreaks of E. coli O157:H7 mirror the seasonal shedding patterns in cattle, occurring predominantly in the summer months [4, 36]. It is interesting to note that although the seasonal shedding of this pathogen is well documented and generally accepted, research in this area is limited. Ambient temperature is generally implied as the most reasonable explanation for the seasonality of E. coli O157:H7, although other factors such as rainfall and insect populations [33, 37] have been suggested as potential contributors. However, if ambient temperature is the cause, then it would be expected that the prevalence of E. coli O157:H7 would be greater or at least the fecal shedding period would extend longer into the fall in the southern climates, which is not the case. In research conducted at our laboratory in College Station, Texas, we have observed a marked seasonal decline in fecal shedding of E. coli O157: H7 in naturally colonized cattle, although ambient temperatures were comparable to summer temperatures in the northern regions of the United States. Based on these observations, we examined published reports on E. coli O157:H7 prevalence in cattle and found a higher correlation for day-length and fecal shedding of E. coli O157:H7 than ambient temperature [15]. Further research examined the effect of artificial lighting on the prevalence of E. coli O157 in feedlot cattle during a period of decreasing daylength [15]. Fecal prevalence of E. coli O157 remained constant in cattle in the lighted pens whereas prevalence was lower in control pens [15]. After removal of the lights, prevalence returned to levels similar to the control pens. As all other conditions were similar among pens, this supports our hypothesis that day-length and not ambient temperature is a more viable explanation; specifically that physiological responses within the animal in response to changing daylength are responsible for the seasonal shedding patterns of E. coli O157:H7. The effects of day-length on animal behavior and physiology are well documented [29, 34] and are thought to be mediated, at least in part, by hormones produced by the pineal and thyroid glands. Pineal melatonin is secreted in response to changing day-length with low serum concentrations in the summer that increase through the fall and peak in the winter, inverse to the seasonal patterns of E. coli O157:H7 prevalence. In addition, the gastrointestinal tract (GIT) produces a significant amount of melatonin, far exceeding pineal production [5, 6], which may play a role in the seasonality of E. coli O157:H7 shedding. Based on our new hypothesis, we conducted an experiment designed to examine the effects of exogenous melatonin on fecal shedding of E. coli O157:H7 in naturally colonized feedlot cattle. Based on the results of this first experiment, we subsequently examined the effect of tryptophan (a melatonin precursor) administered to cattle naturally colonized with and shedding E. coli O157:H7.
T.S. Edrington et al.
Materials and Methods Melatonin Experiment Eight crossbred beef steers, previously identified as shedding E. coli O157:H7 (rectal grab samples at the feedlot and confirmed at our laboratory after purchase using the immunomagnetic separation technique described below), were transported from a commercial feedlot in the southwestern United States to our laboratory facilities in College Station, Texas. The steers were housed in an outdoor, dirt pen and maintained on a high concentrate diet (80% concentrate, 20% grass hay) fed at 2.5% of BW daily each morning after sample collection. Water and salt were available for ad libitum consumption. After a 4-day adjustment period, steers were randomly assigned to one of two treatments (four steers per treatment): control (empty gelatin capsule only) or melatonin (Sigma-Aldrich, St. Louis, MO), dosed orally in a gelatin capsule. In the first phase of the experimental period, steers received a low dose of melatonin (0.5 mg/kg BW) daily for 7 days followed by a 5-day period of no treatment. During the second phase, steers were administered a high melatonin dose (5.0 mg/kg BW) daily for 4 days, followed by a 10-day period of no treatment. Steers were run through a squeeze chute daily (0700 h) for sample collection and treatment administration. Fecal samples were collected via rectal retrieval using a sterile palpation sleeve for qualification (sample positive or negative) of E. coli O157:H7 daily and quantification of generic E. coli on days 0, 7, 14, 21, and 28 as described below. Blood samples were collected via jugular venipuncture on days 0, 2, 4, 6, 10, 13, 16, 18, and 21 for determination of serum melatonin concentrations using a double antibody radioimmunoassay (RIA). The RIA used components of a commercial kit (melatonin direct RIA; Buhlmann Laboratories AG, Schonenbuch, Switzerland) and the procedure was as described by the manufacturer. Melatonin was determined in two assays having an average within-assay coefficient of variation (CV) of 14% and a between-assay CV of 4%. BWs were recorded at the initiation of the experimental period and weekly thereafter. Tryptophan Experiment Sixteen crossbred beef steers and heifers (different from above animals) were housed, handled, and sampled as above. Over a 2-week adaptation period, before treatment administration and sampling, all cattle were gradually adjusted from a 100% roughage diet to a 80% concentrate, 20% grass hay diet. Once during the adjustment period and again before initiation of the experiment, cattle were confirmed as shedding E. coli O157:H7 as above. Animals were randomly assigned to one of the two treatments
Melatonin and E. coli O157
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(control or tryptophan) with sex (three heifers/treatment) equally represented. As in the above experiment, treatments were administered in two phases. In the first phase (days 0– 10), cattle received daily via oral bolus, an empty gelatin capsule (control) or a capsule containing 5 g tryptophan (Sigma-Aldrich, St. Louis, MO) and during the second phase (days 11–17), the tryptophan dose was increased to 10 g/head/day. Fecal samples were collected daily throughout the 17-day experimental period for qualification of E. coli O157 and on days 8 and 17 for quantification of generic E. coli. Blood samples were collected for determination of serum melatonin and tryptophan concentrations on days 7 and 14. Serum melatonin was determined as described above and tryptophan concentrations determined using gas chromatography and an amino acid analysis kit purchased from Phenomenex (Torrence, CA) according to the manufacturer’s recommended procedures. Bacterial Culture and Isolation E. coli O157:H7 culture and isolation was conducted on all samples within 2 h of collection using an immunomagnetic separation technique. Briefly, 10 g of feces was enriched in 90 mL of tryptic soy broth for 2 h at room temperature before incubating for 6 h at 37°C. After incubation, 20 μl of anti-E. coli O157 antibody-labeled paramagnetic beads (Neogen, Lansing, MI) were added to 1 mL volumes of the above enrichments, mixed and washed. Fifty microliters of the resulting suspension was spread-plated on CHROMagar™ O157 (DRG International, Mountain Side, NJ) plates (containing novobiocin 10 μg/mL and potassium tellurite 2.5 μg/mL) and incubated overnight (37°C). Pink colonies exhibiting typical E. coli O157 morphology were resuspended in phosphate-buffered saline (PBS, pH 6.5) and confirmed as E. coli O157:H7 (melatonin experiment) using the Reveal® microbial screening test (Neogen, Lansing, MI) or as E. coli O157 (tryptophan experiment) using the DrySpot™ E. coli O157 test (Key Scientific Products, Round Rock, TX) according to the manufacturer’s instructions [14].
Generic E. coli was quantified by serially diluting 1 g of feces in sterile phosphate-buffered saline before spread plating on CHROMagar™ E. coli (DRG International, Mountain Side, NJ) plates and incubating (37°C, 24 h). Colonies exhibiting typical E. coli color and morphology were manually counted. Statistical Analyses Daily fecal shedding data of E. coli O157:H7 was obtained from all animals in the experiments, however because of the sporadic nature of shedding and the number of animals used in these experiments, the data was insufficient to analyze by day. Therefore, data was pooled across days within a phase or collection period of the experiment (no treatment, low melatonin treatment, high melatonin treatment, low tryptophan dose, etc.) and the percentage of positive fecal samples (by phase) subjected to chi-square analysis using the Proc Freq procedure. Concentrations of generic E. coli and serum concentrations of melatonin and tryptophan were analyzed using the Proc Mixed procedure for repeated measures with treatment, day, and the treatment×day interaction included in the model. The covariance structure utilized was the antedependence structure. The random effect of animal within treatment accounted for the correlations among repeated observations on the same animal. BW change was analyzed using ANOVA appropriate for a completely randomized design. Differences among means were considered significant at a 5% level of significance. All data were analyzed using SAS version 8.02 (SAS Inst., Cary, NC, USA).
Results Melatonin Experiment The number and percentage of fecal samples culture positive for E. coli O157:H7 is presented collectively for each phase of the experiment by treatment in Table 1. No
Table 1 Proportion of fecal samples positive for E. coli O157:H7 during administration of exogenous melatonin for 8 days (low melatonin; 0.5 mg melatonin/kg BW) and again for 4 days (high melatonin; 5.0 mg melatonin/kg BW) after a 5-day period of no treatment Treatment
Days sampled
P>F
Treatment Control
Low melatonin No treatment High melatonin No treatment
0 to 8 9 to 14 15 to 19 20 to 28
Melatonin
n
Percent
n
Percent
23/50 15/28 13/20 9/22
46 54 65 41
27/50 13/28 7/20 13/22
54 46 35 59
n: number of positive fecal samples for the days sampled.
0.31 0.56 0.05 0.31
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Serum melatonin (pg/ml)
80 70 60 50 40
Control
**
Melatonin
30 20 10 0 No Trt (d 0)
Low Mel No Trt High Mel (d2, 4, 6) (d10, 13) (d 16, 18)
No Trt (d 21)
Treatment/collection days
Figure 1 Serum melatonin concentrations in feedlot steers administered two doses of melatonin (Low Mel=0.5 mg/kg BW daily for 8 days; High Mel=5.0 mg/kg BW daily for 4 days) or not treated (No Trt). Bars represent average of collection days; if more than one, in parentheses. Statistical significance: **P0.20) or during the subsequent 5 days of no treatment (P>0.20). However, when the high dose of melatonin was administered for 4 days, a decrease (P=0.05) in the percentage of positive fecal samples was observed in the melatonin (35%) compared to the control (65%) treatment. Numerically, more fecal samples from the melatonin-treated steers were E. coli O157:H7 positive over the final 10 days of the experimental period after the administration of the high melatonin dose, however, this was not significantly different (P>0.20) from the controls. Fecal generic E. coli concentrations were quantified before treatment administration and weekly thereafter. No treatment differences (P>0.20) were observed at any of the collection times (data not shown) or when data was pooled across collections, averaging 5.7 and 5.6 cfu/g feces (log10) for control and melatonin treatments, respectively. Serum melatonin concentrations are presented by day in Fig. 1, although a treatment by day interaction was not detected (P>0.20). No differences in concentrations were observed on day 0 of the experimental period before
treatment administration. An increase (P=0.03) in serum melatonin was observed in the melatonin-treated animals during administration of the low dose that diminished (P> 0.20) during the subsequent period of no treatment. A substantial increase in serum melatonin concentrations were observed during the administration of the 10× melatonin dose, however, a similar increase was also observed in the control treatment presumably in response to decreasing day-length, and the treatment differences were not significant (P>0.20). No treatment differences were observed after melatonin administration on day 21 of the experimental period. Steers in the control treatment gained an average of 15 kg over the 28-day experimental period, whereas melatonin-treated steers maintained their BW (data not shown). BW change over the course of the 28-day experiment was not different (P>0.20) among treatments, however, with four steers per treatment, we may have lacked the statistical power to detect any real treatment differences. Tryptophan Experiment The effect of tryptophan treatment on the incidence of fecal shedding of E. coli O157 is presented in Table 2. No differences (P>0.20) in the percentage of fecal samples positive for E. coli O157 were observed during administration of the low or high tryptophan treatments or when data was pooled across the 17-day experimental period. Serum tryptophan was examined on day 7 of the low tryptophan treatment phase of the experiment and on day 17, the last day of four successive days of high tryptophan treatment. An increase (P=0.02) in serum tryptophan concentration was observed in the treated cattle on day 7 and when data was pooled across the 17-day experimental period. A similar numerical increase (P=0.07) was observed on day 17 in treated compared to control animals (Fig. 2a). No significant differences were observed in serum melatonin concentrations on day 7 or 17 or when data was pooled across the experimental period (Fig. 2b).
Table 2 Proportion of fecal samples positive for E. coli O157 after administration of two levels of oral tryptophan (low tryptophan, 5 g/head/day, days 0–7; high tryptophan, 10 g/head/day, days 10–17) Tryptophan dose
Days sampled
P>F
Treatment Control
Low (5 mg/head/day) High (10 mg/head/day) Low and high
0 to 7 8 to 17 0 to 17
n: number of positive fecal samples for the days sampled.
Tryptophan
n
Percent
n
Percent
49/88 42/48 91/136
56 88 67
49/87 44/49 93/136
56 90 68
0.93 0.72 0.79
Melatonin and E. coli O157
Serum Tryptophan (nmol/ml)
a
557 Control
45 40 35 30 25 20 15 10 5 0
Treated
*
**
day 7
day 17
Average
day 7
day 17
Average
**
b Serum Melatonin (pg/ml)
40 35 30 25 20 15 10 5 0
Figure 2 Serum concentrations of tryptophan (a) and melatonin (b) in cattle administered oral tryptophan (low tryptophan, 5 g/head/day, days 0–7; high tryptophan, 10 g/head/day, days 10–17). Statistical significance: *P
