Preliminary investigations of the distribution of Escherichia coli O149 in sows, piglets, and their environment

Abstract

Little is known about the sources and kinetics of enterotoxigenic Escherichia coli colonization in pigs during the pre-and post-weaning period. In this study, farrowing pens, sows, and piglets were tested for the presence of E. coli O149 by real-time polymerase chain reaction (PCR) after bacterial culture pre-enrichment on 2 farms, one with a history of post-weaning diarrhea (problem farm — PF) and the other without such a history (non-problem farm — NPF). Unlike those on the PF, the sows from the NPF did not carry E. coli O149 before parturition, although they were colonized to frequencies similar to animals on the PF soon afterwards. Most piglets from the NPF were colonized within a week after birth, whereas only a small proportion of those on the PF were colonized during that period. No difference was observed in the frequency of piglet colonization at the 2 farms either at weaning or during the following week. Post-weaning diarrhea (PWD), which is caused by enterotoxigenic E. coli (ETEC), is a multifactorial disease. The presence of ETEC alone is not always sufficient for the disease to develop. Many other factors are considered to be associated with the occurrence of PWD, including feed type (1,2), feeding regimen (1,3,4), the presence of other infectious agents (3,5), weaning age, and weight (6). Weaning, which is considered to be a major physiological and psychological stress factor, is critical for the disease to occur (7). Although piglets are already colonized with ETEC before weaning (4,8), on many farms, clinical disease occurs only after weaning (1). Both sows (9,10) and the environment (6) could be possible sources of infection for piglets, but results from previous studies have not resolved this issue because of the low sensitivity of ETEC detection methods. This study provides preliminary data based on a sensitive detection method for E. coli O149 in pigs and their environment. The results demonstrate the potential of real-time PCR for future studies on this topic.

Introduction

Escherichia coli O149 is currently the most frequently occurring enterotoxigenic E. coli (ETEC) serotype in Ontario (11). The objective of the present investigation was to use a highly sensitive E. coli O149 detection method (12) to conduct a preliminary case study on the distribution of E. coli O149 in sows, piglets, and their environment during the pre-weaning and post-weaning period on 2 farms with a different history of post-weaning diarrhea (PWD). One farm with known recurrent problems of PWD (problem farm — PF) and one non-problem farm (NPF) with no recent history of PWD but with a comparable herd size were used for the study. The farms were selected on the basis of convenience of location and the owner’s willingness to participate. The main differences in feed and management practices between these 2 farms are shown in Table I. The procedures for this study were approved by the Animal Care Committee of the University of Guelph and conducted in accordance with the guidelines of the Canadian Council of Animal Care.

Table I.

Differences in feed and management practices aimed at controlling post-weaning diarrhea (PWD) on both the non-problem farm (NPF) and the problem farm (PF)

	NPF	PF
Differences in nursery management
Creep feed	No	Yes (44 mg/kg lincomycin)
Weaning age	3 wk	4 wk
Medication in starter feed	None	Zinc oxide (3000 ppm) Lincomycin (44 mg/kg)
Differences in farrowing pen measures
Sow vaccination	None	E. coli toxoid and pilia (5 wk and 3 wk before farrowing)
Drying period of crates	1 d	1 wk
Sow wash	No	Yes
Routine medication	None	Gentamicin (5 mg) IM to piglets (day 3 of age)

^aE. coli toxoid and pili vaccine induce the production of protective maternal antibodies that are passively transferred to piglets, thereby preventing neonatal diarrhea caused by ETEC producing heat-labile toxin or having the K99, K88, 987P, or F41 adherence factors.

Materials and methods

Five sows and their piglets were selected from each farm on the basis of the presence of positive homozygous or heterozygous genotype for the genetic markers of F4-specific receptors (13). Fecal and skin surface samples were collected from the sows and samples were collected from the farrowing pen environment after cleaning just before the sows were transferred to the pens (week 0), within a week after the sows farrowed (week 1), and at weekly intervals thereafter (weeks 2 and 3 on both farms and also week 4 on the PF only). Fecal swabs of 3 randomly selected piglets per litter were collected on weeks 1, 2, and 3 (NPF, PF) and week 4 (PF only). At weaning, each litter was divided into 2 equal groups. Group 1 was moved to a weaning pen and regular management practices were followed. The piglets of all 5 litters in Group 1 were kept together in the same weaning pen without piglets from other litters. The second group (Group 2) from each litter was kept separately in each farrowing pen while the sows were removed on the day of weaning. Fecal swabs were collected from all the piglets on the day of weaning (day 0), as well as on day 3 and day 5 after weaning. On either day 3 or day 5, one representative piglet from each group suffering from diarrhea, if present, was euthanized and submitted to the Animal Health Laboratory of the University of Guelph for postmortem investigation.

Samples taken from the surface of the farrowing pen and from the skin surface of sows were collected aseptically, using 18-oz. Whirl-Pak Speci-Sponge Environmental Surface Sampling Bags (Nasco, Fort Atkinson, Wisconsin, USA) after soaking the sponge in sterile Bacto EC Medium (Difco, Franklin Lakes, New Jersey, USA). One sample was collected per pen and time point by systematically rubbing the sponge on the lower rails, sides, and floor of the pen. The surface samples of skin were collected by rubbing the sponge on the sows’ mammary glands, the dorsal surface of the hindquarters, and the sides of the abdomen. Fecal samples were collected directly from the sow’s rectum by digital manipulation and stored in a sterile container. Fecal swabs were collected from piglets using BBL CultureSwab Liquid Stuart (Becton, Dickinson and Company, Sparks, Maryland, USA). Samples were stored immediately at 4°C and processed within 2 h of collection. All the samples were enriched for E. coli by overnight incubation at 37°C with shaking in the following volumes of Bacto EC Medium (Difco): each sponge in 75 mL of broth, 1 g of feces in 25 mL of broth, and each fecal swab in 10 mL of broth. Uninoculated Bacto EC Medium broth was handled and incubated in a similar manner as a negative control for each series of enrichment. In addition, cultures were performed on blood agar before enrichment for the fecal swabs collected during and after weaning.

Deoxyribonucleic acid (DNA) was extracted from the enriched cultures using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A negative control consisting of sterile water was co-extracted and tested for each series of extraction. DNA extracts were stored at −20°C until they were used. The E. coli O149 specific real-time polymerase chain reaction (PCR) was performed using the primers O149-sp2(L) and O149-sp2(R) and the LightCycler 1.2 Real-Time PCR System (Roche Applied Science, Indianapolis, Indiana, USA) with the LightCycler FastStart DNA Master SYBER Green I kit (Roche) (12).

Ten E. coli isolates were systematically recovered from each culture made before enrichment of the fecal swabs collected at and after weaning. The colonies were differentiated as O149 or non-O149 E. coli on the basis of hemolysis and classical PCR using the primers O149-sp2(L) and O149-sp2(R) (250 nM each). The 25-μL reaction mixture consisted of PCR buffer 1× (Promega, Madison, Wisconsin, USA), dNTPs 200 μM each, MgCl₂ 1.5 mM, and 2.5 U of Taq polymerase (Promega). The thermal cycles consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturing at 94°C for 1 min, annealing at 57°C for 1 min and elongation at 72°C for 1 min, followed by a final elongation at 72°C for 7 min.

Results

The PCR results for detection of E. coli O149 on farrowing pen surfaces, skin surfaces, and feces of sows are presented in Table II. There was a noticeable difference in E coli carriage in the fecal and skin samples on the PF and the NPF at day 0. However, no consistent difference in the frequency of colonization of sows and contamination of farrowing pens was observed between the 2 farms from week 1 postpartum onwards. The kinetics of E. coli O149 colonization in piglets during the pre-weaning period are also shown in Table II. Piglets on both farms were colonized with E. coli O149 within the first week of life. Considerably more piglets were colonized on the NPF than on the PF during the first 2 weeks of life. This difference between the farms disappeared during the piglets’ third week of life.

Table II.

Number of samples positive with the E. coli O149 specific real-time PCR for environmental, skin, and fecal samples on both the problem farm (PF) and the non-problem farm (NPF)

Samples	Farm	Week
		0	1	2	3	4
Farrowing pen surface	NPF	1/5	4/5	3/5	4/5	NA
	PF	2/5	2/5	4/5	2/5	3/5
Sows’ skin surface	NPF	0/5	0/5	2/5	3/5	NA
	PF	3/5	2/5	2/5	1/5	3/5
Sows’ fecal sample	NPF	0/5	4/5	1/5	2/5	NA
	PF	4/5	3/5	4/5	2/5	1/5
Piglets’ fecal swab	NPF	—	10/15	9/15	22/43	NA
	PF	—	3/15	3/15	7/15	17/42

The data are shown as the number of samples positive for E. coli O149 identified by real-time polymerase chain reaction (PCR) over the total number of samples tested after enrichment.

NA — not applicable (piglets were already weaned at that time).

The results of the real-time PCR for the detection of E. coli O149 in the piglets during the days after weaning are presented in Table III. Although approximately half of the piglets were excreting E. coli O149 on the basis of PCR testing at any time point after weaning regardless of experimental group or farm, all bacterial cultures on blood agar remained negative for E. coli O149. Post-weaning diarrhea (PWD) could not be confirmed in any of the 4 diarrheic piglets investigated.

Table III.

Number of piglets positive for E. coli O149 by real-time PCR during the weaning period on both the problem farm (PF) and the non-problem farm (NPF)

Farm		Day
		0	3	5
NPF	Total	22/43 (51%)	25/43 (58%)	23/41 (56%)
	Group 1	9/20 (45%)	12/20 (60%)	10/19 (53%)
	Group 2	13/23 (57%)	13/23 (57%)	13/22 (59%)
PF	Total	17/42 (41%)	26/42 (62%)	28/40 (70%)
	Group 1	8/19 (42%)	14/19 (74%)	12/18 (67%)
	Group 2	9/23 (39%)	12/23 (52%)	16/22 (73%)

Group 1 piglets were taken from the farrowing pen to the weaning pen. Group 2 piglets were kept in the farrowing pen after weaning while the sows were removed from the pen. The data are shown as the number of animals positive for E. coli O149 over the total number of animals that were tested after enrichment. The number in parentheses represents the percentage of animals that were positive for E. coli O149.

The recovery rate of E. coli O149 from both the skin and feces of the sows before farrowing correlated well with the PWD history of the 2 farms. Despite this marked initial difference, the sow colonization was similar on both farms within a week of farrowing. Sows on the NPF may have picked up E. coli O149 from the environment and became colonized readily after entering the contaminated farrowing pens. They may also have been colonized by E. coli O149 shed by piglets. Shorter sampling intervals would help to clarify the respective colonization dynamics of sows and piglets with regard to farrowing. Nevertheless, our results suggest that the initial frequency of carriage or shedding by sows at the time of birth did not influence the frequency of colonization of piglets at the time of weaning.

We also observed a difference between the farms in the kinetics of E. coli O149 in piglets at the pre-weaning stage. Most of the piglets from the NPF were colonized by E. coli O149 very early, while only a few from the PF were colonized during this period. This could be related to a number of differences in management practices between the NPF and the PF, such as feed composition, creep feeding, immunization of sows against ETEC, and antimicrobial use on the PF (6,14–18). Finally, separating the piglets at weaning into 2 groups with different levels of environmental contamination (contaminated farrowing pens versus freshly cleaned and disinfected weaning pens) and social stress (same pen with known pen mates versus new pen environment and mixing with unknown piglets) had no apparent influence on the frequency of colonization and none of the animals in either group developed PWD.

Conclusions

This study provides preliminary data based on a sensitive detection method for E. coli O149 in pigs and their environment. To our knowledge, this is the first time that the kinetics of E. coli O149 at the pre-weaning stages have been studied and our results demonstrate the potential of the real-time PCR used here for future studies on this topic. Differences were observed between the farms in sow colonization before farrowing and in the kinetics of colonization of piglets during the suckling period, although there were no longer any differences detected at weaning. Some of the differences in management practices at the 2 farms, which reflected additional measures taken to control PWD at the problem farm (PF), could be related to the differences observed. More extensive investigations are required with multiple farms and replicates to confirm our findings and assess the role of each of these factors.