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Journal of Animal Science - Animal Nutrition

Transit effects on fecal Escherichia coli O157 prevalence and coliform concentrations in feedlot cattle

 

This article in JAS

  1. Vol. 92 No. 2, p. 676-682
     
    Received: May 16, 2013
    Accepted: Nov 20, 2013
    Published: November 24, 2014


    1 Corresponding author(s): jdrouill@ksu.edu
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doi:10.2527/jas.2013-6712
  1. C. C. Aperce*,
  2. C. A. Alvarado*,
  3. K. A. Miller*,
  4. C. L. Van Bibber-Krueger* and
  5. J. S. Drouillard 1
  1. Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506-1600

Abstract

Our objectives were to evaluate the effects of transportation and lairage on fecal shedding of Escherichia coli O157 (E. coli O157), total Escherichia coli, and total coliforms in feedlot cattle, and the relationships between E. coli O157 prevalence and total E. coli population. The study was a randomized complete block design with a split-plot including 2 treatments: a nontransported group, which remained in its pen at all times, and a transported group, which was transported for 1 h in a trailer and subsequently unloaded in a different pen. The experiment was repeated on 3 different days (blocking factor) with 20 steers/d (10 steers/treatment, 60 total). Fecal samples were taken pretransport (h 0) and after 4 and 28 h, lairage from freshly voided fecal pats were taken from each animal. One gram of feces was transferred to a PBS tube, serially diluted, and plated onto Petrifilm for enumeration of total coliforms. Another sample (1 g) was added to gram-negative broth containing cefixime, cefsulodin, and vancomycin, and subjected to immunomagnetic separation. Resulting beads were plated onto MacConkey agar with sorbitol, cefixime, and tellurite. Nonsorbitol fermenting colonies were selected and tested for indole production and O157 antigen agglutination. Results were confirmed using an API 20E kit. Prevalence of E. coli O157 was transient across blocks. E. coli O157 prevalence revealed no treatment × sampling time interaction (P = 0.179) or sampling time effect (P = 0.937), but a tendency for a treatment effect (P = 0.092). Numbers of E. coli and other coliforms did not change across blocks. No effect of treatment (P > 0.7) was observed on total E. coli concentrations or total coliforms. However, tendencies for treatment × sampling time interactions were observed on both populations (P < 0.08), as well as a tendency for a sampling time effect on total E. coli (P = 0.087) and an effect on total coliforms (P = 0.004). Prevalence of E. coli O157 was not correlated with the concentration of total E. coli (P = 0.954). Results suggest that shedding of E. coli O157 and coliforms can vary within a period of 29 h. Greater statistical power and pathogen quantification, as well as hide sampling and stress-related measurements, are needed to be able to conclude on the effects of transport stress on E. coli O157 prevalence and the changes undergone in pathogen shedding patterns after transportation.



INTRODUCTION

Foodborne illness from Escherichia coli O157:H7 is a major concern for the food industry. Contamination of food products can occur at slaughter by contact with contaminated hide or feces; therefore, limiting E. coli O157:H7 shedding in cattle is important to prevent outbreaks. Previous studies have demonstrated a relationship between stress and levels of pathogens shed in feces (Freestone and Lyte, 2010). Transport to the abattoir represents a significant stressor for cattle (Grandin, 1997) during which cattle can develop a stress response through the sympathetic adrenal medullary axis and the hypothalamic pituitary adrenal axis, releasing epinephrine, norepinephrine, and cortisol (Matteri et al., 2000) into the gastrointestinal tract and into general circulation.

Bacteria use hormone-like compounds, or autoinducers (AI; Hughes and Sperandio, 2008), to communicate with each other. This phenomenon is known as quorum sensing. Binding of AI to adrenergic receptors on bacterial membranes (Chen et al., 2006; Kendall and Sperandio, 2007) triggers a cascade of phosphorylation activating the locus of enterocyte effacement (LEE) genes, flagella regulon motility genes, and Shiga toxin genes (Hughes and Sperandio, 2008). Recent studies have shown that communication through this system is not limited to signaling molecules produced by prokaryotic organisms, and that norepinephrine and epinephrine were able to substitute AI 3 (Clarke and Sperandio, 2005) and increase E. coli O157:H7 motility, adherence, and virulence. Furthermore, norepinephrine releases iron sequestered by lactoferrin in the lumen of the gut, thus making iron available for bacterial growth (Freestone et al., 2007; Freestone and Lyte, 2008).

Based on these mechanisms, we hypothesized that transported animals will be at greater risk for E. coli O157 colonization than nontransported animals and that the increase in pathogenic bacteria could also induce a shift in concentrations of total E. coli and coliforms. Therefore, the objectives of this experiment were to evaluate effects of transportation and lairage on fecal shedding of E. coli O157, total E. coli, and total coliforms in feedlot cattle, and the relationships between E. coli O157 prevalence and total E. coli population.


MATERIALS AND METHODS

Procedures for this study were approved by the Kansas State University Institutional Animal Care and Use Committee.

Study Design

The study was a randomized complete block design with a split-plot. Sixty crossbred beef steers (527 ± 110 kg initial BW) were randomly selected to be used in this experiment. Animals were present at the feedlot before the experiment and were accustomed to their housing arrangements and penmates. Steers were fed a finishing diet based on dry-rolled corn, corn silage, and corn steep liquor (Table 1) once a day at 0830 h and had ad libitum access to water in their respective pens.


View Full Table | Close Full ViewTable 1.

Composition of the diet (dry basis) fed to feedlot steers subjected or not to 1-h transport

 
Finishing diet
Ingredient, %
    Dry-rolled corn 74.20
    Corn silage 12.00
    Steep corn liquor 8.00
    Vitamin/mineral premix1 3.63
    Feed additive premix2 2.16
Nutrient composition, %
    DM 72.23
    CP 14.00
    NDF 11.36
    Crude fat 0.007
    Ca 0.70
    P 0.45
1Formulated to provide 0.1 mg Co, 10 mg Cu, 0.6 mg I, 60 mg Mn, 0.25 mg Se, 60 mg Zn, and 2640 IU vitamin A and 11 IU vitamin E/kg of diet DM.
2Provided 300 mg of Rumensin (Elanco Animal Health, Greenfield, IN), 90 mg tylosin (Elanco) per animal daily in a ground corn carrier.

The experiment was repeated 3 times (blocking factor) on consecutive days in August 2011. The summer time frame was chosen to increase the likelihood of high E. coli O157 prevalence in the cattle. A new set of 10 steers was used for both groups for each replication (20 steers/block) to avoid the potential for adaptation to transport stress. The 2 treatments (10 steers/treatment) included a nontransported group and a transported group. On experiment day, steers from the transported group were loaded into a trailer at 0800 h, before feeding, transported for 1 h, and unloaded on return to the research facility into a clean new concrete-surfaced pen (36 m2) with an overhead shade covering approximately 50% of the pen. They had ad libitum access to water but feed was withheld until 0830 h the following morning, thus mimicking preslaughter lairage. Neighbor pens were empty to prevent contact with other animals. Nontransported steers remained in their pens at all times throughout the experiment.

Collection of Fecal Samples

Fecal samples were taken from each animals pretransport (h 0), and at 4 and 28 h post-transport (h 5 and h 29) from freshly voided fecal pats. A new plastic spoon was used to collect each sample. Spoons with feces were placed in individual Whirl-Pak bags (Nasco, Ft. Atkinson, WI) and kept on ice until they were transported to the Preharvest Food Safety Laboratory at Kansas State University.

Microbial Analyses

Total Escherichia coli and Coliform Counts.

One gram of feces was transferred to a tube containing PBS (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, and 1.8 mM of KH2PO4 adjusted to pH 7.4). The PBS tube was serially diluted, plated on Petrifilm, and incubated for 24 h at 37°C for enumeration of total coliforms and E. coli. Most E. coli bacteria produce glucuronidase and will appear as blue colonies on Petrifilm, whereas the other coliforms will appear red. E. coli O157 does not produce β-glucuronidase, and thus is enumerated along with other coliforms.

Escherichia coli O157 Isolation.

One gram of feces was transferred to 9-mL gram-negative broth (Difco-BD, Franklin Lakes, NJ) with 0.05 mg/L cefixime, 10 mg/L cefsulodin, and 8 mg/L vancomycin (GNccv). The GNccv tubes were incubated at 40°C for 6 h. After incubation, tubes were subjected to immunomagnetic separation using serotype-specific beads for E. coli O157 (Invitrogen Dynal AS, Oslo, Norway). Beads were resuspended in 200 μL of PBS and plated onto two MacConkey sorbitol plates (CT-SMAC) containing cefixime (0.05 mg/L) and potassium tellurite (2.5 mg/L). Up to 6 nonsorbitol fermenting colonies from the CT-SMAC plate were selected and inoculated into 5 mL Tryptic soy broth. Colonies were grown overnight at 37°C and tested for indole production. Indole-positive colonies were plated onto SMAC and further tested for O157 antigen agglutination. Colonies positive for indole production and antigen agglutination were confirmed as E. coli O157 by Gram staining and API 20E (Biomerieux, Durham, NC).

Statistical Analyses

This experiment followed a split-plot design with a whole-plot factor in a randomized complete block design. The experiment was replicated on 3 consecutive days corresponding to our blocking factor. Treatment (no transport or transport) was a fixed effect randomly assigned to each experimental unit (EU) within a block and was considered as the whole-plot factor. Animals receiving the same treatment were housed and transported as a group, consequently the group of 10 steers was considered as our EU and individual animal as a subsample (10 subsamples/EU and 2 EUs/replication). Fecal samples were collected from each EU at different time points (0, 5, and 29 h), which were considered as the subplot factor after verifying that the covariance between experimental units at h 0 and h 5 was similar to the covariance between experimental units at h 5 and h 29. E. coli O157 prevalence was expressed as the number of positive samples over the number of animals for the specific treatment, and the specific sampling time. Data were analyzed using a GLIMMIX procedure of SAS 9.2 (SAS Inst. Inc., Cary, NC) with replication and interactions between replication × treatment and replication × treatment × sampling time as random effects. Total coliforms and E. coli data were logtransformed and analyzed as continuous variables using the MIXED procedure of SAS with replication and interactions between replication × treatment and replication × treatment × sampling time as random effects. Subsample (individual animals) was also considered as a random effect nested within replication and treatment. To assess the effect of E. coli O157 on total E. coli, a MIXED procedure of SAS was used with E. coli O157, treatment and sampling time as fixed effects. Replication, treatment, subsample nested within replication and treatment and interactions between replication × treatment and replication × treatment × sampling time × E. coli O157 were considered as random effects. Generated least square means with Pdiff option were used to compare means. For all models, P-values < 0.05 were considered statistically significant and P-value < 0.1 were considered statistical trends.


RESULTS

Prevalence of E. coli O157 in fecal samples varied with replications. In the first replication, 13.3% of the cattle were found positive for E. coli O157, against 5% in the second replication and 28.3% in the third one. Overall, we observed no interaction between sampling time and treatment (P = 0.179), no sampling time effect (P = 0.937), and a tendency for a treatment effect on E. coli O157 prevalence (Fig. 1; P = 0.092). Prevalence of E. coli O157 in the transported group did not differ across the 3 sampling times (10, 3.3, and 16.7%, respectively; P > 0.1). Nontransported group also showed no statistical difference in prevalence across the 3 sampling times (17%, 33%, and 13%; P > 0.1).

Figure 1.
Figure 1.

Prevalence of Escherichia coli O157 in feces collected at h 0, 5, and 29 from cattle subjected (open bars), or not (solid bars), to 1-h transport. Transported animals (10 steers) were placed into a trailer and transported for 1 h. Upon return to the research facility, animals were unloaded into new, clean concrete-surfaced pens. Nontransported animals (10 steers) remained in their pens at all times throughout the experiment. Fecal samples were taken from each animal of each group pretransport (h 0), and at 4 and 28 h post-transport (h 5 and h 29) from freshly voided fecal pats. The experiment was repeated 3 times using a new set of animals for each replication. Bars without a common superscript letter are different (P < 0.05). Treatment effect P = 0.092; sampling time effect P = 0.937; treatment × sampling time interaction P = 0.179. SEM = 0.063.

 

Concentrations of total E. coli (Fig. 2) and other coliforms (Fig. 3) were evaluated in samples to determine if these populations varied with transportation and lairage. Numbers of E. coli or other coliforms were constant across replications. Tendencies for an interaction between treatment and sampling time (P = 0.087) and a sampling time effect (P = 0.092) were observed on total E. coli, but not for treatment effect (P = 0.771). The nontransported group had total enumerable E. coli numbers (log cfu/g; Fig. 2) of 6.10 at h 0, 5.84 at h 5, and 5.88 at h 29, which were not significantly different (P > 0.1). The transported group enumerable E. coli numbers were not different when comparing h 0 to h 5 (5.92 vs. 5.77 log cfu/g; P = 0.308), tended to be different when comparing h 0 to h 29 (5.92 vs. 6.25 log cfu/g; P = 0.067), and increased from h 5 to h 29 (5.77 vs. 6.25 log cfu/g; P = 0.019). Total coliform concentrations revealed a tendency for a treatment × sampling time interaction (P = 0.060), a sampling time effect (P = 0.004), and no treatment effect (P = 0.717). Coliform counts for the nontransported group (Fig. 3) were not different across the different sampling times (4.77, 4.18, and 4.13 log cfu/g; P > 0.1). Transported cattle had decreased fecal coliform concentrations at h 5 (3.2 log cfu/g; P = 0.001) compared with h 0 (4.57 log cfu/g) but returned to pretransport level of 4.54 log cfu at h 29 (P = 0.924). No correlation was observed between the prevalence of E. coli O157 and concentrations of total E. coli (P = 0.954; Fig. 4).

Figure 2.
Figure 2.

Fecal concentrations of Escherichia coli in feces collected at h 0, 5, and 29 from cattle subjected (open bars), or not (solid bars), to 1-h transport. Transported animals (10 steers) were placed into a trailer and transported for 1 h. Upon return to the research facility, animals were unloaded into new, clean concrete-surfaced pens. Nontransported animals (10 steers) remained in their pens at all times throughout the experiment. Fecal samples were taken from each animal of each group pretransport (h 0), and at 4 and 28 h post-transport (h 5 and h 29) from freshly voided fecal pats. The experiment was repeated 3 times using a new set of animals for each replication. Bars without a common superscript letter are different (P < 0.05). Treatment effect P = 0.771; sampling time effect P = 0.092; treatment × sampling time interaction P = 0.087. SEM = 0.094.

 
Figure 3.
Figure 3.

Fecal concentrations of coliforms other than Escherichia coli in feces collected at h 0, 5, and 29 from cattle subjected (open bars), or not (solid bars), to 1-h transport. Transported animals (10 steers) were placed into a trailer and transported for 1 h. Upon return to the research facility, animals were unloaded into new, clean concrete-surfaced pens. Nontransported animals (10 steers) remained in their pens at all times throughout the experiment. Fecal samples were taken from each animal of each group pretransport (h 0), and at 4 and 28 h post-transport (h 5 and h 29) from freshly voided fecal pats. The experiment was repeated 3 times using a new set of animals for each replication. Bars without a common superscript letter are different (P < 0.05). Treatment effect P = 0.717; sampling time effect P = 0.004; treatment × sampling time interaction P = 0.060. SEM = 0.43.

 
Figure 4.
Figure 4.

Fecal concentrations of total Escherichia coli (open bars) in relation to Escherichia coli O157 prevalence (solid bars) in feces collected at h 0, 5, and 29 from cattle subjected, or not, to 1-h transport. Transported animals (10 steers) were placed into a trailer and transported for 1 h. Upon return to the research facility, animals were unloaded into new, clean concrete-surfaced pens. Nontransported animals (10 steers) remained in their pens at all times throughout the experiment. Fecal samples were taken from each animal of each group pretransport (h 0), and at 4 and 28 h post-transport (h 5 and h 29) from freshly voided fecal pats. The experiment was repeated 3 times using a new set of animals for each replication. E. coli O157 prevalence effect on total E. coli, P = 0.954.

 

DISCUSSION

We had hypothesized that transported cattle would be at greater risk for E. coli O157 colonization than nontransported animals and that increases in pathogenic bacteria might also induce shifts in total E. coli and coliforms concentrations. We observed that concentrations of total E. coli and total coliforms did not differ statistically across treatments and replications. Coliforms and E. coli concentrations decreased numerically at h 5 regardless of treatment. The similarity in patterns in transported and nontransported groups suggests that transport was not the causative factor in this change. Variation in population could be attributed to a circadian rhythm; however, h 5 and h 29 samplings occurred at the same time during the day with a 24-h interval. Differences observed between these two sampling times questioned the hypothesis of a circadian rhythm; moreover, animals from the transported group did not have access to feed on the first day of sampling, which could be responsible for the variation in concentration of coliform and total E. coli in that group.

We found no correlations between prevalence of E. coli O157 in feces and concentrations of total E. coli. The binomial nature of pathogen prevalence may not be sufficiently robust to detect this relationship.

Prevalence of E. coli O157 revealed an important variation in shedding from one replication to the other, which was not unexpected due to the transient nature of E. coli O157, although we conducted the experiment during the summer, when expected pathogen prevalence is greatest (Chapman et al., 1997; Hancock et al., 1997). Despite the absence of a significant interaction between sampling time and treatment and of a sampling time effect, we observed a tendency for a treatment effect. Nontransported animals had increased shedding of the pathogen at h 5, whereas transported animals showed a slight but insignificant decrease at that time. Such change in shedding patterns of transported cattle relative to their nontransported counterparts could be the consequence of transport-related stress. Animals under stress tend to defecate and urinate more often than nonstressed animals (Friend, 1991). Increased defecation due to transport stress could induce a rapid washout of the pathogen, depleting numbers by h 5. Another hypothesis is that under stressful conditions, E. coli O157:H7 virulence and attachment is amplified by action of catecholamine on LEE and motility genes (Hughes and Sperandio, 2008), delaying excretion of the pathogen in feces. Likewise, flagellar regulon (FlhDC) encodes for a flagella-mediated motility and has been shown to play a role in the adherence of the pathogen to the epithelium cells (Giron et al., 2002). Greater attachment of the pathogen to the gastrointestinal tract would likely make the bacteria more resistant to shedding in feces. Independent of changes in timing of fecal shedding, the pathogen prevalence was highly transient within a period of 29 h, which implies that pathogen populations can amplify and decay relatively quickly. Such observations underscore the importance of the choice of sampling time to assess the effects of stress on E. coli O157 shedding patterns.

It is important to state that statistical power of this experiment was limited, as only 3 pens of animals were used for each treatment. Increasing the number of days (replicates) or number of loads within a day would have improved statistical power which may have enhanced our ability to detect differences due to treatment. In our design, fecal samples were obtained at only 2 time points post-transport, h 5 and h 29. The 4-h lairage period was chosen arbitrarily based on the time animals commonly spend in the lairage area at abattoirs before harvest. Considering the rapid fluctuation in pathogen populations within feces, collection of all fecal material produced, including in the trailer, could be performed in future studies to portray E. coli O157 prevalence patterns following transport.

The present E. coli O157 analysis was qualitative and not quantitative; in such conditions, the potential for presence of supershedder animal(s) in the nontransported group cannot be excluded. Supershedders are defined as cattle shedding more than 103 to 104 cfu/g of feces, and their incidence in a pen has been shown to increase the prevalence of E. coli O157 for the whole pen (Cobbold et al., 2007; Stephens et al., 2009). To overcome this limitation, quantifying E. coli O157 present in feces of individual animals would be useful.

Another measurement that would be beneficial to our design is the pathogen prevalence on the hides of cattle. Hides have been shown to be a main vector for carcass contamination (McGee et al., 2004; Arthur et al., 2009). Animal hides, with transport space limitations, are likely to be contaminated with feces from others and disseminate the pathogen.

We hypothesized that handling, loading, transport, unloading, and confronting cattle with unfamiliar housing conditions (i.e., new pens) would induce stress responses in the cattle used in our experiment. We made no attempt to quantify stress response to confirm this, and thus cannot exclude the possibility that our model failed to induce the desired stress response. Assessment of stress response typically requires blood sampling and subsequent characterization of the secretion of cortisol, epinephrine, norepinephrine, or other stress-related compounds. Handling of animals to obtain these samples arguably would induce some degree of stress, potentially masking effects of our desired treatments. Analysis of cortisol levels in feces has been reported as a reliable indicator of stress (Mostl and Palme, 2002), and may have been a useful addition to the present experiment. On average, cortisol metabolite can be detected in feces about 12 h following a stress event, and concentrations in feces parallel those of circulating cortisol immediately after induction of stress (Palme et al., 2000). Our intent was to minimize exposure to stress in the nontransported group, but we cannot exclude the possibility that these animals were stressed by the mere presence of humans in their pens waiting to collect fresh fecal pats.

Previous studies have been performed to assess the impact of transportation on prevalence of E. coli O157 in cattle, but none of these studies compared shedding patterns of the pathogen in transported animals vs. non-transported animals. These studies evaluated E. coli O157 prevalence in fecal samples collected at the feedyard and after transport in the lairage pens or right after slaughter. Some authors observed an increase in E. coli O157 following transportation (Bach et al., 2004; Dewell et al., 2005; Arthur et al., 2007; Dewell et al., 2008), whereas others observed no significant effect of transport (Barham et al., 2002; Minihan et al., 2003; Fegan et al., 2009) on pathogen shedding.

Differences in detection techniques used can account for part of the discrepancy in the results. Animal genetics, age, gender, and management history have been shown to influence stress perception (Stanger et al., 2005). These criteria should be taken into consideration when comparing results from studies because they confer a unique capacity on each animal to cope with stress. Multiple factors are involved in transport stress (Nielsen et al., 2011), and variation in each of these factors could affect findings. Over the course of their journey, cattle are subjected to feed and water depravation (Warriss et al., 1995); psychological stressors, such as handling, novelty, and disruption of their social organization (Grandin, 1997); repression of their basic behaviors, such as lying down (Munksgaard et al., 2005); and they may be exposed to large bacterial loads (Avery et al., 2004) from other animals and the surrounding environment. Variations in the effects of transport on E. coli O157 prevalence in fecal samples are thus to be expected.

In conclusion, our results suggest that shedding patterns for E. coli O157 can vary within a period of 29 h, but no significant change in shedding patterns due to transport were revealed. Collection of fecal samples from a greater number of experimental units would increase statistical power of the experiment. In addition, considering pathogen concentration in the samples instead of prevalence may also help explain the effect of transport on E. coli O157 shedding. Moreover, sampling of the hides and measurement of animal stress-related components should further improve our capacity to accurately portray the effect of transport stress on E. coli O157 prevalence and the changes undergone in pathogen shedding patterns after transportation. Change in shedding patterns could have important ramifications for beef safety.

 

References

Footnotes


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