Developing and Evaluating a Weaned Piglet Model of Enterotoxigenic Escherichia Coli Infection

Abstract

Enterotoxigenic Escherichia Coli (ETEC) remains the main pathogen associated with post-weaning diarrhea in piglets. Here, we introduce a reliable method to develop an ETEC-infected weaned piglet model. For the first 24 h post-birth, neonatal piglets were separated from dams so that they did not receive colostrum. Afterwards, piglets were returned to the care of their mothers, until weaning (21-day-old). On days 25, 26 and 27 post-birth, piglets were orally challenged with 1011 Colony-Forming Units (CFU) of virulent ETEC strain incubated in colony-forming antigen medium and delivered in chitosan-coated capsules. In all infected piglets, severe diarrhea and typical body weight loss were observed post-infection. To simplify the work, for the newly developed method, the number of challenging ETEC cells was lowered to 1010 CFU/head and the incubation medium was changed to tryptic soy. These modifications influenced neither the diarrheal defecation ratio nor the body weight loss in piglets. To induce experimentally ETEC infection in future work, we recommend that 10¹⁰ CFU/head of ETEC cell grown in tryptic soy broth be delivered in chitosan-coated capsules to colostrum restricted piglets, because this methodology stably caused 100% diarrheal defecation and growth reduction.

Keywords: Enterotoxigenic Escherichia coli; Heat-labile toxin; Infection model; Post-weaning diarrhea; Weaned pig

Introduction

Pathogenic Escherichia coli infection remains a major problem in the swine industry. Pathogenic strains of E. coli recovered from intestinal tracts of animals have been categorized as enterotoxigenic, enteropathogenic, enterohemorrhagic, and necrotoxigenic [1]. In particular, Enterotoxigenic E. Coli (ETEC) is the main pathogen associated with post-weaning diarrhea in piglets [2,3]. To date, antimicrobials are mainly used to prudently prevent and/or treat post-weaning porcine diarrhea worldwide. Nonetheless, alternative agents to cure or prevent ETEC infection are being actively evaluated [2]. To discover preventive or therapeutic agents against ETEC infection, researchers generally use experimental infection methods [2,4]. However, piglets experimentally infected with pathogenic E. coli do not always have symptoms. Previous work speculated that lacking symptoms in experimental infection was caused by a low pH in the stomach [5]. For example, in 58 previous studies, the mean diarrheal defecation ratio was merely 68% [4]. Diarrheal defecation is the best parameter to evaluate the effectiveness of agents; therefore, to minimize the number of experimental animals to a humane level, a stable method with 100% diarrheal defecation is deemed necessary.

Recently, Matsumoto et al. [6] reported that MUC4 genotype and/or pig breed-line are the controlling factors of susceptibility to ETEC infection, and that regulation of these factors could induce 100% diarrheal defecation in weaned piglets. Separately, we have reported an improved method to experimentally induce Shiga-toxin-producing E. coli (STEC) infection [7]. The aforementioned method caused the typical pathological symptom of porcine edema disease in almost all piglets [7, 8]. In the present study, to induce 100% diarrheal defecation in weaned piglets, we took a different approach than that of Matsumoto et al [6]. Indeed, we used our own developed STEC method [7] (i.e., colostrum depletion and chitosan capsule inoculation) and applied it to cause ETEC in weaned piglets. In Experiment 1, the maximal challenging dose [1011 colony-forming units (CFU)/head] was prepared using a ETEC strain incubated in a Colony-Forming Antigen (CFA) broth, which was the most suitable medium for the expression of ETEC adhesion factors [9]. In Experiment 2, to simplify the methodology, we used Tryptic Soy (TS), which is a general and basic broth medium that can be purchased from several commercial suppliers; in addition, using it, helped lower the challenging dose to 10¹⁰ CFU/head.

Materials and Methods

Challenging Strain

The ETEC strain 8185 isolated from pig diarrhea, kindly donated by Dr. Ikuko Koike from Swine Management Consultation (Kanagawa, Japan), was used in the present study. This strain was positive of several virulence genes (estB, elt1, ast1 and faeG). As phenotypes, we detected heat-liable toxin (LT) and F4 with the commercial kits (VET-RPLA and Toxigenic E. coli Pilli Antisera, Denka Seiken, Tokyo, Japan). The characteristics of this strain are shown in Table S1.

The ETEC strain was cultured either in CFA medium broth (1% of casamino acids, 0.15% of yeast extract, 0.05% of MgSO₄, 0.0005% of MnCl₂, pH 7.4) (Experiment 1) or in TS medium broth (Difco, Detroit, MI, USA) (Experiment 2), under an aerobic condition at 37°C. Four h post-incubation, ETEC cells in logarithmic growth phase were harvested by centrifugation (1,450×g, 15 min, 4°C). Cell slurries were prepared immediately prior to use.

Pigs

The pregnant sows (Landrace × Large White), used in the present work, which were impregnated by Duroc boars and purchased from a commercial pig farm, were the same as those previously reported [7]. A qualified veterinarian (Dr. Nakanishi) investigated beforehand the pathogenic permeation of the farm used in the present study. No enteropathogens such as hemolytic E. coli, Salmonella sp., Clostridium perfringens, Lawsonia intracellularis, Brachyspira hyodysentariae, porcine epidemic diarrhea virus, rotavirus and parasites were detected in the feces of weaning piglets of this farm. The screening methods for these enteropathogens have been described elsewhere [3]. The health statuses of the introduced sows were checked by the veterinarian, and who also assessed the retrospective statuses of stable deliveries. Sows were vaccinated against atrophic rhinitis and swine erysipelas [ARBP/Swine Erysipelas-combined vaccine (inactivated), Nisseiken, Tokyo, Japan)], Japanese encephalitis, porcine parvovirus infection, Getah virus infection (“Kyotobiken” swine abnormal birth 3 combo live vaccine; Kyotobiken, Kyoto, Japan), and porcine reproductive and respiratory syndrome (Ingelvac PRRS MLV, Boehringer Ingelheim Animal Health Japan, Tokyo, Japan). For all experiments, sows were individually housed in fallowing pens at the KYODOKEN Institute (Fukuchiyama, Kyoto). No LT genes were detected in the feces of any of the sows. Throughout the study, the sows were fed a commercial diet (Bree-Meal Maxim, Feed One, Yokohama, Japan). After delivery and for the first 24 h, neonatal piglets received no colostrum and instead, were fed artificial milk (Meiji Hohoemi, Meiji, Tokyo, Japan), as previously described [7]. Afterwards, all piglets were returned to their own dams and, during the nursing period, received maternal milk only. All piglets were weaned at 21 days of age, put together in experimental groups and transferred to concrete pens with brooders. Throughout the weaning period, the piglets were fed ad libitum a commercial feed for weaned piglets (SDS No. 1; Feed One). The experimental diet was free of intestinal microbiota modifiers, such as antimicrobials and probiotics. The nutrient composition of the aforementioned diet (g/kg), expressed on a dry-matter basis (906g/kg), was as follows: crude protein, 220g; crude fat, 46g; crude fiber, 9g; and crude ash, 63g.

Experiment 1: ETEC Challenge Using the Maximal Dose

Of 10 healthy piglets delivered by one sow, six were used for Experiment 1. After weaning, piglets were divided into two groups (n=3). One group was considered the non-infection control (group C1; mean body weight, 5.3 kg; one male and two females) and the ETEC-infected group (group LT1; mean body weight, 6.0 kg; two males and one female). Four days post-acclimatization, the piglets (25 days old) in LT1 group were orogastrically inoculated the ETEC preparation in chitosan-coated capsules (Sansho, Tokyo, Japan). These capsules were resistant to gastric digestion and hence, viably delivered ETEC to the small intestine [5]. The ETEC capsules containing approximately 1011 CFU were given to piglets for three consecutive days [i.e., days post-infection (dpi) 0, 1 and 2]. The precise numbers of challenging ETEC cells were 7.2×1010, 7.8×1010, and 1.0×1011 CFU/head on dpi 0, 1, 2, respectively. Separately, the same type of capsules but filled with saline were given to the C1 piglets. Individual body weights and feed intake of each group were measured on dpi 0, 3, 5 and 9. In addition, fecal samples from all piglets were simultaneously collected. The fecal samples were stored at -80 °C until DNA extraction.

Experiment 2: ETEC Challenge Using a Lower Dose

Two sows delivered 11 healthy piglets each. Of these, six were used for Experiment 2. After weaning, piglets were divided into two groups. One group was considered the non-infection control (group C2; mean body weight, 6.1 kg; one male and two females) and the ETEC-infected group (group LT2; mean body weight, 3.6 kg; one male and two females). Four days post-acclimatization, the piglets in LT2 group were orogastrically inoculated ETEC in the same manner as in Experiment 1 (i.e., colostrum depletion and chitosan capsule inoculation), with the following exceptions. Unlike in Experiment 1, the ETEC cell numbers were 6.3×10⁹, 9.1×10⁹, and 8.1×10⁹ CFU/head on dpi 0, 1, 2, respectively. Individual body weights and feed intake of each group were measured on dpi 0, 3 and 9. We omitted dpi 5 measurement, because we considered measurement of this point was unnecessary to assess the successively of ETEC infection. As in Experiment 1, fecal samples from all piglets were simultaneously collected. Fecal samples were stored at -80 °C until DNA extraction.

Clinical Observations

Throughout the study, the condition of fresh feces was individually evaluated once daily (dpi 1-9). The criteria used for feces scoring have been previously described [5]. Briefly, the criteria of fecal score were described as follows, 0: normal, 1: loose stool, 2: moderate diarrhea, 3: severe diarrhea.

Dissection and Histopathologic Observation

All piglets in both experiments were euthanized by exsanguination under deep sedation with an intraperitoneal injection of sodium pentobarbital (Somnopentyl, Kyoritsu, Tokyo, Japan; 32.4 mg/kg B.W.). Afterwards, the whole intestines were removed and screened for abnormalities with the naked eye.

In Experiment 1, micro-abnormalities were observed in the intestines of ETEC-infected piglets. The small intestine was dislodged and cut into eight segments of equal length [10]. Segments S2, S5 and S8 from the small intestines were considered as stemming from the 1) jejunum, 2) jejunum and ileum complex and 3) ileum, respectively [10]. The middle portions of ceca were also collected. Each intestinal segment was longitudinally incised and fixed in 10% (v/v) phosphate-buffered formalin. Formalin-fixed intestinal tissues were further cut into cross sections of an approximate length of 10 mm. Each intestinal sub-segment was embedded in paraffin wax. To visualize any histopathological abnormality under a light microscopy, micro-sections 3-μm thick were prepared and stained with hematoxylin and eosin. The histopathologic study in Experiment 2 was omitted because remarkable abnormality was not detected in Experiment 1.

Detection of Gene elt1

DNA was extracted from fecal samples of pigs on dpi 0, 3, 5 and 9 (Experiment 1) and dpi 0, 3 and 9 (Experiment 2), respectively. The DNA extraction method was the same as that previously described [11]. The excretion of ETEC was measured in the feces by Taqman quantitative real-time PCR for detection of gene elt1, as described elsewhere [12].

Validation of Methods

To validate Experiment 2, an additional experiment was carried out.

Four colostrum-restricted piglets delivered from one sow were readied. The experimental design was the same as that of Experiment 2, and it was as follows. After weaning, piglets were divided into two groups. One group was considered the non-infection control (group C3; mean body weight: 7.2 kg; one male and one female) and the ETEC-infected group (group LT3; mean body weight: 6.1 kg; one male and one female). Four days post-acclimatization, the piglets in group LT3 were orogastrically inoculated ETEC in the same manner as in Experiment 2 (i.e., colostrum depletion and chitosan capsule inoculation). The ETEC cell numbers were 1.4×1010, 9.9×109, and 1.4×1010 CFU/head on dpi 0, 1, 2, respectively. Individual body weights and the feed intake of each group were measured on dpi 0, 3 and 9. Fecal samples from all piglets were simultaneously collected. Fecal samples were stored at -80 °C until DNA extraction. Clinical observation and detection of gene elt1 in the feces are described in above sections.

Statistical Analyses

Depending on the results of the F-test, either the Student’s or Welch’s t-test was used to analyze differences in body weight gain between groups in Experiments 1 and 2. The Mann–Whitney U-test was used to analyze the differences in ETEC numbers in the feces and clinical and histopathological scores between groups. In all statistical analyses, differences between means were considered significant if P<0.05. These body, clinical and pathological parameters were analyzed using STATCEL (OMS, Saitama, Japan), an add-in application for Microsoft Excel© (Seattle, WA, USA). Values are given as the means ± the standard deviations.

Results

Experiment 1

In general, and unlike control (C1) piglets, ETEC-infected (LT1) piglets experienced significant reductions in their body weights from dpi 0 to 5 (Table 1) (P<0.05). In addition, reductions in body weights caused “no calculation” in the feed conversion ratio during this period. Nonetheless, from dpi 5 onwards, reductions in body weight stopped and LT1 piglets gained weight and thus, body weight gain differences between the groups became unclear. However, the feed conversion ratio of LT1 group improved at a later infection stage (dpi 5-9). At first, LT1 group was not experiencing substantial diarrheal defecation in unison with body weight reductions; however, between dpi 4 and 9 it reached 100% (Table 1), before decreasing in severity between dpi 6 and 9. In total, LT1 piglets had diarrhea for an average of 5.3 days. Regarding the ETEC-infected group, as expected, no cells were detected in feces of any of the piglets on dpi 0 (prior to the ETEC challenge). However, following inoculation, ETEC cells were readily detected in the feces of LT1 piglets in dpi 3, with a mean number of 7.68 log CFU/g. Unexpectedly, no ETEC cells were detected in the feces of LT1 piglets on dpi 5 onwards. Visual inspection with the naked eye detected no abnormalities in the small and large intestine samples. However, some abnormalities were detected after histopathological observation was conducted under a light microscope (Table 2, Figure 1). In particular, the scores of lymphocyte infiltration and abnormalities in the crypts increased significantly with ETEC infection.

    

    

    Figure 1: Representative photomicrographs of the cecum of ETEC infected piglets. (a) Normal mucosa was observed in C1 piglets, ×200; (b) Lymphocyte infiltration was observed in the lamina propria of LT1 piglets, ×200; (c) Normal mucosa was observed in C1 piglets, ×100; (d) Crypt disappearance was observed in the mucosa of LT1 piglets, ×100.