Longitudinal Follow-Up of the Persistence and Dissemination of EHEC on Cattle Farms in Belgium

Abstract

A longitudinal survey was performed on three cattle herds known to be positive for, respectively, Enterohemorrhagic Eschericia coli (EHEC) O157, O26/O103, and O26 in a slaughterhouse study. This study aimed to investigate the persistence and dissemination of EHEC in beef cattle and beef cattle farms. At each farm, a cohort of 10 animals was sampled, seven times on farm B and eight times on farms A and C, at intervals of approximately 4–6 weeks. In addition, incoming cattle and environmental samples were also examined for the presence of EHEC at each sampling occasion. In 65 (18.8%) out of 345 samples, EHEC was detected, of which 41 were from cohort animals, four from incoming cattle and 20 from environmental samples (cats 3/23; dogs 2/7; feed 4/23, water 2/23, and dust 9/23). On two farms, non-EHEC strains harboring either vtx or eae genes were detected in 21 samples. EHEC was detected at least once in 23 of the cohort animals, with a maximum of four positive sampling occasions. Genetic typing by pulsed-field gel electrophoresis (PFGE) demonstrated that a same strain occurred for several months (up to 11 months) in two of three cattle farms. Among the environmental samples, dust harbored EHEC most frequently. In conclusion, transmission and dissemination of EHEC might have occurred not only in the bovine reservoir but also in the farm environment and in other farm animals.

Introduction

E nterohemorrhagic Eschericia coli (EHEC) are an important group of foodborne pathogens. The pathogenicity is mainly mediated by genes encoding for verocytotoxins (vtx1 and vtx2) and intimin (eae). EHEC can cause a broad spectrum of human illness, ranging from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome (HUS). Humans can acquire EHEC infection through a variety of foods (Buvens et al., 2011), water (Bopp et al., 2003; Hamner et al., 2007), person-to-person (O'Donnell et al., 2002) or animal-to-person contact (Busch et al., 2007). The majority of human illnesses are related to cattle or cattle products, and therefore cattle are considered the primary reservoir. Studies on the involvement of cattle in the spread of EHEC in cattle farms is mainly focused on E. coli O157. These investigations identified interesting, although sometimes conflicting, risk factors (Dargatz et al., 1997; Ellis-Iversen et al., 2009; Faith et al., 1996; Garber et al., 1995; Schouten et al., 2004). In addition, the impact of non-O157 serogroups, potentially circulating simultaneously with E. coli O157 at cattle herds, are less understood. Transient infections can be introduced by incoming cattle, contaminated farm environments such as paddle waterers, feed bunks, and dust. EHEC has been shown to persist for a long time in contaminated environments and therefore be spread to uninfected animals. Many studies assumed that cattle farms harbor populations of predominant EHEC strains rather than several distinguishable strains of the same serogroup (Beutin et al., 1997; Cobbold and Desmarchelier 2000; Jenkins et al., 2002; Oliver et al., 2005).

Therefore, a longitudinal study was performed to investigate the maintenance and dissemination of EHEC strains in cattle and cattle herds. Furthermore, the genetic diversity of EHEC in three cattle herds over time was investigated.

Materials and Methods

Study design

A longitudinal study was conducted on three cattle farms (A, B, and C) from September 2010 to August 2011. The three farms were known to be positive for EHEC (Joris et al., 2011). On each farm, a cohort of 10 Belgian Blue double-muscled bulls of approximately 12 months of age was selected randomly, and samples were collected, respectively, seven times on farm B and eight times on farm A and C, with a time interval of 4–6 weeks. Farm A is 39 km from farm B and 108 km from farm C. Farm B and C are located 90 km apart.

Herd characteristics

Farm A was a mixed farm (i.e., farm raising dairy as well as beef cattle) and raised only a small proportion of bulls (<10%). The mean age of the bulls arriving at the farm was 10 months, all originating from the same cattle herd. All cohort animals were housed in the same pen with no other animals on concrete floor in an open front stable.

Farm B was also a mixed farm, however, the proportion dairy and beef cattle wasequally distributed. Bulls, purchased at cattle markets, were also entering the farm at the age of 12 months. All cohort animals were housed in the same open front stable on concrete floor in a group of approximately 30 animals.

Farm C was a beef-producing farm, and approximately 12-month-old bulls were purchased at livestock markets. All cohort animals were housed on straw bedding in different pens, with a maximum of eight animals per pen.

Sampling methods

At each visit, animals were sampled by taking fecal samples using rectal, sterile, single-use examination gloves. At each sampling occasion, incoming cattle, who arrived at the farm during the sampling interval, were also sampled. These incoming cattle were housed with the cohort cattle. When handling of the animals was possible, rectal swabs were taken from dogs and cats, if present on the farm. Environmental samples, including feed present in the feeding-troughs, water from the paddle waterers, and dust, were also collected at each farm visit in the pens of the cohort cattle. Dust samples, including spiderwebs, were collected with sterile swabs premoistured with tryptone soy broth (TSB; Oxoid, Ltd., London, UK) from the barriers and walls of the pen. All samples were transported to the laboratory under cooled conditions and processed immediately.

Isolation and characterization of E. coli belonging to the serotypes O26, O103, O111, and O145

The optimized isolation method of Possé (2008) was used (Joris et al., 2011). Briefly, samples (25 g for feces, 10 g for feed or water samples, and 1 g for dust and cobweb samples) were diluted 1/10 in TSB (Oxoid) supplemented with 8 mg L⁻¹ novobiocin (Sigma-Aldrich, St. Louis, MO), 16 mg L⁻¹ vancomycin (Sigma-Aldrich), 2 mg L⁻¹ rifampicin (Sigma-Aldrich), 1.0 mg L⁻¹ potassium tellurite (Sigma-Aldrich), and 1.5 g L⁻¹ bile salts (Oxoid) in filter stomacher bags and enriched for 24 h at 42°C. After 6 and 24 h of incubation, 100 μL was plated onto the differential agar medium for O26, O111, O103, and O145 (Possé et al., 2008). After 24 h of enrichment, a serotype-specific immunomagnetic separation (IMSs; Invitrogen, Paisley, UK) was performed prior to plating. The differential agar plates were incubated at 37°C for 24 h. To all individual rectal swabs from cats and dogs, 40 mL of TSB was added. These samples were further processed as described above.

Following incubation, up to three suspected colonies were subcultured on trypton soy agar (TSA; Oxoid) during 24 h at 37°C (Possé et al., 2008). The isolates were further examined for the presence of virulence genes by a multiplex polymerase chain reaction (PCR), applying the primers for vt1, eae, and hlyA described by Fagan et al. (1999) and for vt2 described by Paton and Paton (1998). Serogroup was confirmed by serogroup-specific PCR (Possé et al., 2007).

Isolation and characterization of E. coli O157

To isolate E. coli O157, samples were diluted in modified TSB (mTSB; Oxoid), supplemented with 20 mg L⁻¹ novobiocin. After incubation for 6 h at 42°C, IMS using O157O157-specific beads (Invitrogen, Grand Island, NY) was performed according the manufacturer's recommendations. One hundred microliters were plated onto sorbitol-MacConkey agar (Oxoid) supplemented with cefixime (0.05 mg L⁻¹) and potassium tellurite (2.5 mg L⁻¹ ; Dynal, Oslo, Norway; CT-SMAC) and incubated for 24 h at 42°C. Up to three suspected colonies were transferred to TSA, incubated for 24 h at 37°C, and consequently serologically identified with the O157 latex agglutination test kit (Oxoid). The isolates were further confirmed by biochemical tests and with a O157-specific PCR (Cobbaut et al., 2008) and examined with the multiplex virulence PCR, described above.

Enumeration of E. coli O157 and non-O157 from bovine feces

Prior to enrichment, a 100-μL aliquot was plated onto one CT-SMAC and one differential agar plate (quantification limit 10² CFU/g). This plating was performed using a Spiral plater (Eddy Jet, Farmingdale, NY). After incubation at 37°C for 24 h, the suspected colonies, according to the definition of Possé et al. (2008) were counted on both agar plates, and up to three suspected colonies from each plate were transferred to TSA. The isolates were examined for the presence of virulence genes and serotyped as described above.

Pulsed-field gel electrophoresis (PFGE)

Up to three isolates (originating from the isolation and, if the case, from enumeration) per sample were genotyped. Therefore, subtyping was performed by PFGE separation of XbaI-digested genomic DNA in accordance with the PulseNet Europe protocol (Ribot et al., 2006) usingBioNumerics^® (Applied Maths, Sint-Martens-Latem, Belgium) software to group isolates based on dice similarity (1% tolerance; 1% optimization).

Results

In cattle herd C, five cohort animals were sold or slaughtered sooner than expected and could therefore not be sampled during the entire study period. The number of EHEC-positive samples among cohort animals, incoming cattle, cats, dogs, feed, water, and dust are listed in Table 1. Overall, 45 of 248 (18.1%) fecal samples tested positive for EHEC. Besides cattle, cats (n=3) and dogs (n=2) also harbored EHEC in their rectum. In water and feed samples, EHEC could be detected, respectively, two and four times. Contaminated water was found on two farms and occurred simultaneously with the detection of at least one animal shedding EHEC in the corresponding pen. Remarkably, nine dust samples, including spider webs (39.1%), were positive for EHEC. Overview of the shedding patterns of EHEC by the cohort animals are illustrated in Table 2. Serogroup O157 was dominant in herd A, although serogroup O26 was isolated from one cohort animal at one sample occasion. In herd B, serogroup O26 was also predominant, although O111 was isolated from two cohort animals at the final sampling occasion. The environmental samples harbored the same EHEC serogroup as isolated in the cohort cattle (Tables 2 and 3). In cattle herd C, serogroup O26 was predominant in the first four sampling occasions and shifted to O111 in the following sampling periods, although O111 was previously isolated at sampling time point 3. In addition, animal 3 on farm C was shedding two serogroups, namely O111 and O157, at sampling occasion 6.

Table 1.

Herd Size and the Number of EHEC-Positive Samples per the Number and Sources of Samples Tested

		No. of EHEC-positive samples/no. of samples tested
Farm	Herd size	Cohort	Incoming cattle	Cats ^a	Dogs ^b	Feed	Water	Dust	Total
A	259	19/80	1/8	1/8	0/0	2/8	0/8	4/8	27/120 (22.5%)
B	52	12/70	2/17	1/7	2/7	2/7	1/7	3/7	24/122 (19.7%)
C	78	10/65	1/8	1/8	0/0	0/8	1/8	2/8	14/105 (13.6%)
Total		41/215 (19.1%)	4/33 (12.1%)	3/23 (13.0%)	2/7 (28.6%)	4/23 (17.4%)	2/23 (8.7%)	9/23 (39.1%)	65/347 (18.7%)

All different cats.

Only one dog sampled.

EHEC, enterohemorrhagic Eschericia coli.

Table 2.

Shedding Patterns and Genotypic Diversity of EHEC by the Cohort Cattle

Serogroup/pulsotype.

EHEC, enterohemorrhagic Eschericia coli; NT, not typeable; not sampled, indicated by striped cell.

Table 3.

Serogroup and PFGE Type of EHEC-Positive Samples from Incoming Cattle, Cats, Dogs, and Farm Environment

Farm	Incoming cattle	Cats	Dogs	Feed	Water	Dust
A	O157/P8 (1)^*	O157/NT (6)	0	O157/P8 (2)	0	O157/P8 (1)
				O157/P8 (6)		O157/P8 (2)
						O157/P8 (5)
						O157/NT (4)
B	O26/P7 (7)	O26/P7 (6)	O26/P7 (3)	O26/P7 (1)	O26/P7 (6)	O26/P7 (1)
	O26/P7 (7)		O26/P7 (6)	O26/P7 (6)		O26/P7 (3)
						O26/P7 (6)
C	O26/P10 (8)	O26/P10 (4)	0	0	O26/NT (8)	O111/P6 (6)
						O26/NT (8)

Serogroup/pulsotype (sampling time point).

PFGE, pulsed-field gel electrophoresis; EHEC, enterohemorrhagic Eschericia coli.

In 23 of the cohort animals, EHEC colonies were detected in at least one of the fecal samples (Table 2). In 11 animals, the temporal pattern of EHEC shedding was transient; i.e., shedding occurs at only one sampling time point. However, shedding of EHEC could have been missed as the current isolation procedures from cattle feces are hampered by their inability to detect small numbers of EHEC from this complex and highly variable matrix. EHEC belonging to the same serogroups were detected in six, two, and one animals at r2, 3, and 4 sampling occasions respectively. One animal in herd C shed two serogroups, i.e., O157 and O111, simultaneously (sampling occasion 6). Four animals shed EHEC belonging to two or three serogroups at different time points in the study period.

In only two fecal samples, the concentration of EHEC O157 and O26 on, respectively, farm A and C was above the threshold for quantification (10² CFU/g), namely, 2.9 10⁴ and 1.8 10³ CFU/g feces.

PFGE typing of 79 EHEC isolates resulted in 11 distinguishable PFGE types; however, 19 isolates were not typeable by PFGE. One genotype (P7) was common on farm B and C. On farms with more than one PFGE type per serogroup, a maximum of three PFGE types was detected (Table 2). On two farms, one PFGE type predominated, namely P8 on farm A and P7 on farm B. All EHEC isolates from environmental samples, cats, dogs, and new entering cattle belonged to the farm-specific predominant PFGE type on farms A and B (Table 3).

Conclusion

The purpose of this longitudinal study was to examine the temporal dissemination, predominance, and persistence of EHEC O157 and non-O157 in three cattle herds over a period of 11 months. The cohort groups were comprised of male cattle, since beef production in Belgium is largely based on Belgian Blue double-muscled bulls, slaughtered at the age of 20–24 months. Considering on-farm presence and transmission of EHEC, four groups of samples were considered: cohort cattle, incoming cattle, cats/dogs, and the farm environment. Transmission within and between these groups might contribute to the persistence of EHEC in cattle farms.

Seven cohort animals did not shed EHEC at any sampling time point, although they were housed in pens with EHEC shedding animals. Noteworthy was that shedding of EHEC occurs intermittently. Nevertheless, sequential sampling with a monthly interval has been shown to be a good way to deal with intermittent shedding (Hancock et al., 1997). Only two animals of herd A tested positive for EHEC O157 on two or more consecutive sampling time points, whereas in nine of 23 (39%) animals, EHEC was not isolated at consecutive sampling occasions, with a maximum of four negative samples taken between positive isolations (Table 2). These findings are in accordance with a longitudinal study on VTEC O157 shedding patterns (Smith et al., 2010). In that study, 26.4% of animals were positive more than once (up to four sampling occasions) with a maximum of four negative samples between positive isolations. To date, it is unknown what triggers shedding after a non-shedding period. It could be questioned whether this could be ascribed to re-shedding or to re-infection.

The farm environment is important in the epidemiology of EHEC in terms of dissemination from one animal to another and persistence outside the bovine reservoir. EHEC are well adapted to survive in feces, soil, and water, thus posing a risk of transmission to and reinfection of farm animals. The presence of EHEC in two out of 23 (8.7%) paddle waterers occurred in the farms simultaneously with at least one animal that was shedding EHEC. In water troughs, however, prevalence rates up to 26% are reported for E. coli O157 (LeJeune et al., 2004; Van Donkersgoed et al., 2001). It is very likely that infected cattle frequently contaminate their water troughs with feces containing EHEC strains. The fecally contaminated residual water in the water troughs promotes sediment and biofilm formation, allowing EHEC to survive and to proliferate. In contrast with water troughs, fecal contamination occurs less likely in paddle waterers. In addition, there is almost no residual water and sediment formation in the paddles when cattle finish drinking. When taken into account that cattle have to physically depress the valve of paddle waterers to receive water, oral contamination of the drinking water may be more likely than fecal contamination. Moreover, recovery of E. coli O157 from saliva (Keen and Elder, 2002) and from rhinal mirrors (Cobbaut et al., 2008) of cattle supports the latter assumption. Of all environmental samples, dust (including spider webs) harbored EHEC most frequently, namely in 39.1% of the samples. On farm C, EHEC O111 belonging to pulostype 6 was recovered from dust at sampling time point 6, although none of the animals sampled were shedding that particular EHEC strain at the same time. However, not all animals housed in this pen were sampled. Accordingly, sampling of dust has been shown to be a reliable method to detect Salmonella infection on the farm (Davies et al., 2001; Mahe et al., 2008). Moreover, in an outbreak investigation on a cattle farm, the causative strain could be isolated from dust collected in the stables (Buvens et al., 2011; Van den Branden et al., 2007). In addition, a dust sample may also provide an indication of an earlier infection which has become undetectable by fecal monitoring. However, feces may provide a more accurate indication of substantial current infection, which may be more relevant to public health.

In several studies, cats and dogs were indicated as potential shedders of VTEC O157 (Bentancor et al., 2007; Hancock et al., 1998; Kataoka et al., 2010; Schouten et al., 2005). Recently, the presence of cats and dogs was significantly associated with the prevalence of VTEC O157 in cattle farms (Sasaki et al., 2011). Results of this study support these findings. Besides the role of cats and dogs in the dissemination of EHEC in cattle farms through shedding, EHEC present in cattle feces might adhere to their pads and therefore be spread over the entire cattle farm environment.

Genetic typing by PFGE demonstrated that a same strain occurred up to 11 months on herd A and up to 5 months on herd B in cohort cattle, incoming cattle, and environmental samples. These results are in accordance with other studies who have demonstrated the long-term survival of a predominant strain on cattle farms (Beutin et al., 1997; Cobbaut et al., 2011; Cobbold and Desmarchelier, 2001; Schouten et al., 2005). However, in farm B, O111 was isolated at the sampling period instead of O26. Sanderson et al. (2006) suggested that the establishment of a new strain depend on the balance of two factors, namely, the prevalence of the newly acquired strain and the fitness of this strain versus the resident strain to compete in the cattle farm and the bovine gastrointestinal tract. On the other hand, in farm C, different EHEC O26 and O111 types were isolated. Likewise, Fremaux et al. (2006) reported a high genetic diversity on two cattle farms in France, but some of these EHEC types persisted for up to 12 months. While it is possible that a multitude of strains belonging to the same serogroup are present or introduced on a cattle farm, instability of PFGE patterns of EHEC isolates has been reported as a result of clonal turnover (Akiba et al., 2000; Cobbold and Desmarchelier, 2001; Gannon et al., 2002; Liesegang et al., 2000). Karch et al. (1995) observed clonal turnover first in three human long-term shedders of E. coli O157. In these three patients, the PFGE patterns showed a shift between first and last isolates. Recently, Yoshii et al. (2009) demonstrated PFGE profile changes resulting from spontaneous chromosomal deletions in EHEC during passage in cattle. The exact mechanism governing the clonal turnover of EHEC within cattle remains unclear to date.

Transmission and dissemination of EHEC might have occurred not only in the bovine reservoir but also in the farm environment and in other farm animals. Due to the complexity of the EHEC ecology in cattle farms, mitigation strategies will probably require multiple control measures that address the different sources of EHEC exposure of cattle. Further investigations are needed to determine the genetic diversity of EHEC strains within cattle farms over time.

Footnotes

Acknowledgments

We thank Annelies Wachtelaer for her technical assistance. This research was funded by the Belgian Federal Public Service of Health, Food Chain Safety and Environment (RT-07/8-FOODZOON).

Disclosure Statement

No competing financial interests exist.

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