Abstract
Spontaneous outbreaks of Clostridium difficile infection (CDI) occur in neonatal piglets, but the predisposing factors are largely not known. To study the conditions for C. difficile colonization and CDI development, 48 neonatal piglets were moved into isolators, fed bovine milk–based formula, and infected with C. difficile 078. Analyses included clinical scoring; measurement of the fecal C. difficile burden, toxin B level, and calprotectin level; and postmortem histopathological analysis of colon specimens. Controls were noninfected suckling piglets. Fecal specimens from suckling piglets, formula-fed piglets, and formula-fed, C. difficile–infected piglets were used for metagenomics analysis. High background levels of C. difficile and toxin were detected in formula-fed piglets prior to infection, while suckling piglets carried about 3-fold less C. difficile, and toxin was not detected. Toxin level in C. difficile–challenged animals correlated positively with C. difficile and calprotectin levels. Postmortem signs of CDI were absent in suckling piglets, whereas mesocolonic edema and gas-filled distal small intestines and ceca, cellular damage, and reduced expression of claudins were associated with animals from the challenge trials. Microbiota in formula-fed piglets was enriched with Escherichia, Shigella, Streptococcus, Enterococcus, and Ruminococcus species. Formula-fed piglets were predisposed to C. difficile colonization earlier as compared to suckling piglets. Infection with a hypervirulent C. difficile ribotype did not aggravate the symptoms of infection. Sow-offspring association and consumption of porcine milk during early life may be crucial for the control of C. difficile expansion in piglets.
Clostridium difficile has been documented as a cause of uncontrolled enteritis outbreaks in neonatal pigs [1], and the predisposing factors for these outbreaks are largely not known. Different species of farm animals can be affected and are thereby potential sources for C. difficile infection (CDI) in humans [2]. In addition, a new hypervirulent type of C. difficile, ribotype 078, originating from pigs, was transmitted to and caused infection in farmworkers [3]. C. difficile belongs to the natural early colonizers of the gastrointestinal tract of pigs, and up to 100% of piglets test positive for the bacterium (increasing their probability of colonization by hypervirulent ribotypes) within 2 days after birth, followed by a rapid decline in the prevalence of C. difficile positivity with increasing age [4, 5]. In neonatal pigs, the C. difficile toxins cause a proinflammatory response manifested by mesocolonic edema and, often, diarrhea [6]. The association between mother and offspring during early life is a critical factor for the subsequent succession of the intestinal commensal bacteria and immunity development [7, 8], and it may be crucial for the passive protection against CDI through the constant supply of protective antibodies in milk against C. difficile toxins [9]. Thus, any disruption of the natural colonization process could enhance the susceptibility to CDI. Antibiotics are known to alter the gut microbiota, allowing the expansion of opportunistic pathogens including C. difficile. However, non–antibiotic-treated piglets may also develop CDI [10]. Because animal models of CDI have been established mainly in gnotobiotic neonatal piglets or in piglets born by cesarean section or maintained in sterile environments [11–13], they are difficult to use in intervention studies. To study the conditions for C. difficile colonization and CDI development, we hypothesized that the types of feeding and rearing environments, the timing of CDI, and the type of antibiotic intervention could affect the predisposition of naturally born neonatal piglets to CDI.
MATERIALS AND METHODS
Animal Experiments
The institutional and national guidelines for the care and use of animals were followed, and the study was approved by the State Office of Health and Social Affairs (Landesamt für Gesundheit und Soziales Berlin; registration G0255/14 and G0269/16). In a series of 4 consecutive infection trials, 48 neonatal piglets were moved into artificial sanitized rearing units (isolators) and received a bovine milk–based formula (approximately 46% lactose, 20% ether extract, and 23% crude protein) [14, 15]. The length of each subsequent trial was adjusted on the basis of experience during the previous trial, to identify the time when the most-severe symptoms occurred. The experimental design of the trials (trials I–IV) is illustrated in Figure 1. The piglets were housed in pairs. The formula was offered to animals every 2 hours between 6:00 am and 10:00 pm. Water was provided ad libitum. The ambient temperature was maintained at 28°C. The lighting program was set to 16 hours of light and 8 hours of darkness. Spores of C. difficile ribotype 078 were produced and purified according to the protocol of Grześkowiak et al [16], and the concentration of inoculum in each trial was verified on chromID agar (Biomerieux, France).
Scheme of the 4 experiments (I–IV) of Clostridium difficile ribotype 078 infection in neonatal piglets, indicating the beginning of formula feeding (along with placement of the animals into the artificial rearing units), time point of infection with C. difficile 078, antibiotic (clindamycin) treatment, and day of dissection (which occurred on the same day as euthanasia). The control piglets were also fed a formula and received a sham inoculum (deionized water) instead of C. difficile 078. In experiment IV, 6 of 12 animals challenged with C. difficile 078 were pretreated with antibiotic (Ab). CFU, colony-forming units.
Animal groups consisted of formula-fed piglets (FP), formula-fed piglets with CDI (FP-CD) and formula-fed piglets with CDI pretreated with antibiotics (FP-CD-Ab). Clindamycin was used for the FP-CD-Ab group because it is considered to confer a high risk for CDI in humans [17]. Control piglets received a sham inoculum (deionized water). Piglets were monitored daily for behavior (scored as 1 for normal, 2 for depressed, 3 for lethargic, and 4 for moribund), body condition (1, normal; 2, gaunt; and 3, very poor), feed intake (1, normal; 2, diminished; and 3, anorexic) and fecal consistency (1, constipation; 2, normal; 3, moderate; and 4, diarrheic). Fecal specimens were collected daily and stored frozen (at −30°C) until analysis.
At the end of each experiment, animals were sedated with 20 mg of ketamine hydrochloride (Ursotamin; Serumwerk Bernburg, Bernburg, Germany) per kg of body weight and 2 mg of azaperone (Stresnil; Jansen-Cilag, Neuss, Germany) per kg of body weight and euthanized by intracardial injection of 10 mg of tetracaine hydrochloride, mebezonium iodide, and embutramide (T61; Intervet, Unterschleißheim, Germany) per kg of body weight. Findings of postmortem examination of the colon (to detect signs of mesocolonic edema) were scored as 1 for normal, 2 for moderate edema, and 3 for severe edema. Fecal specimens from 5 age-matched suckling piglets and colon tissue specimens from 4 suckling animals were collected for comparison to those from piglets in the experimental trials. To further elucidate the effect of rearing environment or diet on natural colonization with C. difficile, newborn piglets (6/group) were either kept with their mother sows or moved into isolators 24 hours after birth and were fed either sow milk or formula. In this experiment, none of the piglets were infected with C. difficile. Fecal specimens were collected at 1, 2, 3, 5, and 7 days of life for determination of C. difficile status by quantitative real-time polymerase reaction (PCR) analysis targeting the partial C. difficile 16S ribosomal RNA (rRNA) gene as outlined below.
C. difficile, Toxin, and Calprotectin Analyses
Spores of C. difficile in fecal specimens were quantified using chromID agar, and the toxin B (TcdB) level was measured by an enzyme-linked immunosorbent assay (ELISA) kit (tgcBiomics, Bingen, Germany) [4]. The C. difficile burden in noninfected piglets fed formula or sow milk in the last experiment, defined as the log C. difficile 16S rRNA gene copy number, was determined by quantitative real-time PCR analysis [16]. The fecal calprotectin level was measured 72 hours after the challenge, using the Porcine Calprotectin ELISA kit (MyBioSource, San Diego, CA).
PCR Ribotyping
C. difficile 078 colonization of piglets in trials I–IV was verified by PCR ribotyping. Briefly, the DNA from C. difficile colonies grown on chromID agar was extracted and subjected to PCR analysis with specific primers [18, 19]. The amplicons were analyzed by microcapillary gel electrophoresis, using the Agilent 2100 Bioanalyzer system (Agilent; Santa Clara, CA), and the profile of the bands were compared to bands of C. difficile 078. Colonies demonstrating a band profile different from C. difficile 078 were sent for PCR ribotyping by multilocus variable-number tandem-repeat analysis (tgcBiomics).
Metagenomics of Microbiota
Colon digesta from select age-matched animals (3 in the FP group and 3 from the FP-CD group, all from trial III) and 4 noninfected suckling piglets were used for metagenomics analysis of microbial communities. Total DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany). Shotgun metagenomics libraries were generated using the Nextera DNA Library Preparation Kit (Illumina). Libraries were quality-controlled using the D5000 DNA Analysis ScreenTape on a 2200 TapeStation Instrument (Agilent Technologies) and were sequenced on the Illumina NextSeq500 system with two 150–base pair reads. Sequencing reads were demultiplexed based on the used Nextera indices (dual-indexing principle). The FLEXBAR tool [20] was used to remove adapter sequences and discard reads with <100 nucleotides. Quality checked sequences (89 % of the original sequencing reads) were mapped against reference genomes of 13193 different bacterial and archaeal species taken from the National Center for Biotechnology Information (available at: ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/), using the Yara tool [21]. The resulting mapping files were analyzed using the Species Level Identification of Microorganisms from Metagenomes tool [22] to produce taxonomic profiles of each sample. Identified bacterial taxa were used for calculation of ecological indices (Shannon diversity index, evenness) and genus-level comparison between the groups. The metagenomics sequences are available from the European Nucleotide Archive (accession code PRJEB19642).
Histopathologic Analysis and Tight Junction Protein Analysis
Colon samples from select age-matched animals (4 from the FP group, 4 from the FP-CD group, and 4 from the FP-CD-Ab group) and 4 noninfected suckling animals were stained with hematoxylin-eosin and then histopathologically scored according to the protocol of Whelan et al [23]. Scoring was based on the following characteristics: infiltration (scored as 0 for none, 1 for minimal, 2 for mild, 3 for moderate, and 4 for severe), degree of infiltration (0, none; 1, mucosal; 2, mucosal and focal submucosal; 3, mucosal and submucosal; and 4, transmural), epithelial surface damage (0, none; 1, focal denudation; 2, extensive denudation; 3, erosion; and 4, ulceration), crypt epithelial damage (0, none; 1, sporadic crypt abscesses; and 2, multiple crypt abscesses), and hyperplasia (0, none; 1, minimal; 2, mild; 3, moderate; and 4, severe). Alcian blue (pH 2.5)–periodic acid Schiff staining was used for visualization and counting of goblet cells. Samples were analyzed in a light microscope.
Colon samples from 4 mice in the FP-CD group and 4 suckling animals were used for tight junction protein analysis by immunohistochemical staining and Western blot. Primary antibodies were rabbit anti-claudin 3 and mouse anti-claudin 4, and secondary antibodies were goat anti-rabbit Alexa Fluor-488 (for claudin 3) and goat anti-mouse Alexa Fluor-594 (for claudin 4). Nuclei were stained with 4′,6-diamidino-2-phenylindole. Images were taken with a DMI6000 B microscope (Leica, Heidelberg, Germany). Western blot analyses of claudin 3 and 4 protein expression were performed with primary antibodies to claudin 3, 4, and β-actin (Life Technologies, Durham, NC) and binding peroxidase-conjugated secondary antibodies (goat anti-mouse and anti-rabbit; Cell Signaling Technology, Danvers, MA). Densitometry of the specific signals was performed using ImageLab software. Measured signals for claudin 3 and 4 were expressed in relation to the β-actin bands of the same sample.
Statistics
Normally distributed data were analyzed by 1-way analysis of variance with the Tukey honest significant difference post hoc comparisons. Nonnormally distributed data were analyzed by Kruskal-Wallis or Mann-Whitney U tests, when appropriate. The Friedman test was used to analyze differences between the challenge groups. Correlation analyses were assessed by the nonparametric Spearman correlation analysis procedure. Microbiome data were analyzed by principle component analysis, using the Canoco statistical package [24], and by 1-way analysis of variance with the Tukey honest significant difference post hoc test for group comparisons. Differences with P values of ≤ .05 were considered statistically significant. Statistical analyses were performed using SPSS software, version 21.0 (SPSS, Chicago, IL).
RESULTS
Clinical Symptoms and Gross Lesions
In all infection trials, the FP and FP-CD groups (regardless of dose and age at infection) developed diarrhea 1 day after being transferred to isolators, which lasted in most cases for 1 day, and no differences in clinical signs or performance were observed (Table 1). Mesocolonic edema, gas-filled distal small intestines, and ceca were observed 3 days after infection in all animals (control and infection) in trials II and III and in infected animals in trial IV and shortly after 7 days in infected animals in trial I. Signs of infection were absent in suckling piglets (Figure 2A–D).
Clinical Scoring and Macroscopic Lesions in Piglets on the Day of Euthanasia
| Variable | Behavior | Body condition | Feed intake | Fecal score | Temperature, °C | Weight, kg | Mesocolonic Edema |
|---|---|---|---|---|---|---|---|
| Trial I | |||||||
| Control (n = 4) | 2.00 ± 0 | 3.00 ± 0 | 1.50 ± 0.58 | 4.00 ± 0 | 38.05 ± 0.17 | 1.89 ± 0.26 | 1.00 ± 0 |
| 104 CFU C. difficile (n = 3) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.63 ± 0.55 | 2.55 ± 0.36 | 2.00 ± 0 |
| 106 CFU C. difficile (n = 4) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 39.05 ± 0.10 | 2.68 ± 0.13 | 2.00 ± 0 |
| Trial II | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.10 ± 0.22 | 38.93 ± 0.24 | 2.03 ± 0.13 | 1.80 ± 0.45 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.40 ± 0.89 | 39.02 ± 0.39 | 2.19 ± 0.27 | 1.67 ± 0.52 |
| Trial III | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 1.83 ± 0.41 | 38.65 ± 0.31 | 1.66 ± 0.26 | 2.83 ± 0.41 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.42 ± 0.47 | 1.47 ± 0.26 | 2.50 ± 0.84 |
| Trial IV | |||||||
| Ab plus 108 CFU C. difficile (n = 5) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.20 ± 0.96 | 1.24 ± 0.26 | 1.00 ± 0 |
| 108 CFU C. difficile (n = 6) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.42 ± 0.29 | 1.28 ± 0.32 | 2.17 ± 0.75 |
| Variable | Behavior | Body condition | Feed intake | Fecal score | Temperature, °C | Weight, kg | Mesocolonic Edema |
|---|---|---|---|---|---|---|---|
| Trial I | |||||||
| Control (n = 4) | 2.00 ± 0 | 3.00 ± 0 | 1.50 ± 0.58 | 4.00 ± 0 | 38.05 ± 0.17 | 1.89 ± 0.26 | 1.00 ± 0 |
| 104 CFU C. difficile (n = 3) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.63 ± 0.55 | 2.55 ± 0.36 | 2.00 ± 0 |
| 106 CFU C. difficile (n = 4) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 39.05 ± 0.10 | 2.68 ± 0.13 | 2.00 ± 0 |
| Trial II | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.10 ± 0.22 | 38.93 ± 0.24 | 2.03 ± 0.13 | 1.80 ± 0.45 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.40 ± 0.89 | 39.02 ± 0.39 | 2.19 ± 0.27 | 1.67 ± 0.52 |
| Trial III | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 1.83 ± 0.41 | 38.65 ± 0.31 | 1.66 ± 0.26 | 2.83 ± 0.41 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.42 ± 0.47 | 1.47 ± 0.26 | 2.50 ± 0.84 |
| Trial IV | |||||||
| Ab plus 108 CFU C. difficile (n = 5) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.20 ± 0.96 | 1.24 ± 0.26 | 1.00 ± 0 |
| 108 CFU C. difficile (n = 6) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.42 ± 0.29 | 1.28 ± 0.32 | 2.17 ± 0.75 |
Data are means (± SDs). In trial I, euthanasia was performed 7 days after infection; in trial II, 3 days; in trial III, 3 days; and in trial IV, 3 days.
Abbreviations: Ab, antibiotic; C. difficile; Clostridium difficile; CFU, colony-forming units.
Clinical Scoring and Macroscopic Lesions in Piglets on the Day of Euthanasia
| Variable | Behavior | Body condition | Feed intake | Fecal score | Temperature, °C | Weight, kg | Mesocolonic Edema |
|---|---|---|---|---|---|---|---|
| Trial I | |||||||
| Control (n = 4) | 2.00 ± 0 | 3.00 ± 0 | 1.50 ± 0.58 | 4.00 ± 0 | 38.05 ± 0.17 | 1.89 ± 0.26 | 1.00 ± 0 |
| 104 CFU C. difficile (n = 3) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.63 ± 0.55 | 2.55 ± 0.36 | 2.00 ± 0 |
| 106 CFU C. difficile (n = 4) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 39.05 ± 0.10 | 2.68 ± 0.13 | 2.00 ± 0 |
| Trial II | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.10 ± 0.22 | 38.93 ± 0.24 | 2.03 ± 0.13 | 1.80 ± 0.45 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.40 ± 0.89 | 39.02 ± 0.39 | 2.19 ± 0.27 | 1.67 ± 0.52 |
| Trial III | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 1.83 ± 0.41 | 38.65 ± 0.31 | 1.66 ± 0.26 | 2.83 ± 0.41 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.42 ± 0.47 | 1.47 ± 0.26 | 2.50 ± 0.84 |
| Trial IV | |||||||
| Ab plus 108 CFU C. difficile (n = 5) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.20 ± 0.96 | 1.24 ± 0.26 | 1.00 ± 0 |
| 108 CFU C. difficile (n = 6) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.42 ± 0.29 | 1.28 ± 0.32 | 2.17 ± 0.75 |
| Variable | Behavior | Body condition | Feed intake | Fecal score | Temperature, °C | Weight, kg | Mesocolonic Edema |
|---|---|---|---|---|---|---|---|
| Trial I | |||||||
| Control (n = 4) | 2.00 ± 0 | 3.00 ± 0 | 1.50 ± 0.58 | 4.00 ± 0 | 38.05 ± 0.17 | 1.89 ± 0.26 | 1.00 ± 0 |
| 104 CFU C. difficile (n = 3) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.63 ± 0.55 | 2.55 ± 0.36 | 2.00 ± 0 |
| 106 CFU C. difficile (n = 4) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 39.05 ± 0.10 | 2.68 ± 0.13 | 2.00 ± 0 |
| Trial II | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.10 ± 0.22 | 38.93 ± 0.24 | 2.03 ± 0.13 | 1.80 ± 0.45 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.40 ± 0.89 | 39.02 ± 0.39 | 2.19 ± 0.27 | 1.67 ± 0.52 |
| Trial III | |||||||
| Control (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 1.83 ± 0.41 | 38.65 ± 0.31 | 1.66 ± 0.26 | 2.83 ± 0.41 |
| 108 CFU C. difficile (n = 6) | 1.00 ± 0 | 1.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 38.42 ± 0.47 | 1.47 ± 0.26 | 2.50 ± 0.84 |
| Trial IV | |||||||
| Ab plus 108 CFU C. difficile (n = 5) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.20 ± 0.96 | 1.24 ± 0.26 | 1.00 ± 0 |
| 108 CFU C. difficile (n = 6) | 2.00 ± 0 | 1.00 ± 0 | 2.00 ± 0 | 4.00 ± 0 | 37.42 ± 0.29 | 1.28 ± 0.32 | 2.17 ± 0.75 |
Data are means (± SDs). In trial I, euthanasia was performed 7 days after infection; in trial II, 3 days; in trial III, 3 days; and in trial IV, 3 days.
Abbreviations: Ab, antibiotic; C. difficile; Clostridium difficile; CFU, colony-forming units.
Macroscopic lesions of the study piglets. A, Healthy colon from a suckling piglet. B and C, Colons from a formula-fed piglet (B) and a formula-fed piglet infected with Clostridium difficile (C) with visible mesocolonic edema. D, Colon from a formula-fed piglet infected with C. difficile and treated with antibiotic, with intestinal liquid and gas.
C. difficile and Toxin Levels
High levels of C. difficile and TcdB were detected in fecal specimens from all piglets before infection and from the control FP group during trials I and II. In addition, suckling piglets had lower C. difficile numbers, and TcdB could not be detected. In infected animals, C. difficile and toxin concentrations peaked 48 hours after infection. In the FP group, C. difficile but not TcdB concentrations declined below the detection limit after 10 days during trial I (Supplementary Figures 1 and 2). The concentrations of C. difficile and TcdB correlated positively in the 4 trials (Spearman rho = 0.484 and P ≤ .01 for the pooled C. difficile and TcdB data). PCR ribotyping revealed C. difficile 078 colonization in infected piglets 20 hours after infection, whereas this ribotype was undetectable in animals from the FP group. However, 2 other toxigenic ribotypes, 014/020 and 005, were identified in piglets from both the control and infection groups (Supplementary Figure 3). The abundance of these naturally colonizing C. difficile bacteria was significantly higher in the FP groups as compared to that in suckling littermates and littermates kept in isolators and fed sow milk (Supplementary Table 1).
Microbial Ecology Measures
The number of identified bacterial genera, bacterial diversity, and bacterial evenness were lower (P ≤ .05) in the FP and FP-CD groups, compared with age-matched suckling counterparts (Figure 3A). Drastic shifts in the abundance of bacterial genera were observed, with significantly higher proportions of Bacteroides, Prevotella, Bifidobacterium, Lachnoclostridium, and Oscillibacter species in suckling animals and higher proportions of Escherichia, Roseburia, Ruminococcus, Shigella, and Streptococcus species in the FP and FP-CD groups (Figure 3B and 3C and Supplementary Table 2).
A, Influence of formula feeding (FP) and formula feeding plus infection with Clostridium difficile ribotype 078 (FP-CD) on the number of bacterial species, Shannon diversity index, and distribution of bacterial species as revealed by the evenness index, compared with age-matched and noninfected suckling piglets (SP). *P < .05 for comparisons to the FP and FP-CD groups. B and C, The FP and FP-CD groups were associated with different distributions of bacterial genera (B), which was further revealed by principal component analysis (C) illustrating the association of FP (orange circles) and FP-CD (red circles) groups with different bacterial genera as compared to suckling piglets (green circles). The length of, direction of, and angle between arrows are an approximation of correlations between variables or variables and canonical axes. Percentages on axes 1 and 2 indicate the variability described through the canonical axes.
Histopathological Scores, Tight Junction Protein Levels, and Calprotectin Levels
In the 16 selected animals, histological examination revealed cellular infiltrations and a loss of goblet cells in the colon of animals from the FP and FP-CD groups but not in suckling animals (Figure 4). The sum of histopathological scores was lower in suckling piglets as compared to the FP and FP-CD groups (P ≤ .05 for both comparisons). There was a trend toward a lower sum of scores for suckling piglets versus the FP-CD-Ab group (P = .059; Figure 5 and Supplementary Table 3). Immunohistochemical staining demonstrated lower claudin 3 and 4 expression in the FP-CD group as compared to suckling animals (Figure 6A). This observation was confirmed by detection of lower expression levels of both claudins in the intestinal epithelium of animals from the FP-CD group, compared with that of suckling piglets (P ≤ .05), as assessed by Western blotting (Figure 6B and 6C). The level of calprotectin measured in fecal specimens collected 72 hours after infection in trial I did not differ between the study groups (P = .171 for comparison between the control group and the groups infected with 104 or 106 colony-forming units). However, the calprotectin level correlated positively with the toxin level (Spearman rho = 0.708; P = .05; n = 8), and there was a trend toward a positive correlation between the calprotectin level and C. difficile burden in fecal specimens (Spearman rho = 0.634; P = .091; n = 8).
Microscopic lesions in colons from study piglets. Arrows indicate crypt damage, loss of goblet cells, or infiltration. Tissues were stained with hematoxylin-eosin, for detection of infiltration and crypt cell damage, and with alcian blue, for analysis of goblet cells. FP, tissues from formula-fed animals; FP-CD, tissues from a formula-fed, Clostridium difficile–infected animals; FP-CD-Ab, tissues from formula-fed, C. difficile–infected animals treated with antibiotics; SP, healthy tissues from suckling piglets.
Mean sum of microscopic lesion scores in the large intestine of 16 piglets from different feeding and rearing environments and challenged with Clostridium difficile and/or treated with antibiotic. The sum of microscopic lesions was calculated based on the scores for infiltration, degree of infiltration, epithelial surface damage, crypt epithelial damage and for hyperplasia. FP, formula-fed piglets (n = 4); FP-CD, formula-fed piglets infected with C. difficile (n = 4); FP-CD-Ab, formula-fed, C. difficile–infected piglets treated with antibiotic (n = 4); SP, suckling piglets (n = 4). *P ≤ .05, by 1-way analysis of variance and the Tukey honest significant difference post hoc test, for comparisons to the FP and FP-CD groups.
Immunohistological appearance of claudins in piglet colon tissues. A, Claudin 3 (CLDN3), claudin 4 (CLDN4), and merged proteins (merge), respectively, in the colon tissues of suckling piglets (SP) and formula-fed Clostridium difficile–infected piglets (FP-CD). B and C, Results of a Western blot analysis of claudin 3 (B) and claudins 4 (C) in colon tissues of SP and FP-CD piglets. The relative expression levels of both claudins were analyzed by densitometry. Data represent the percentage of the densitometry value for controls. *P ≤ .05, by 1-way analysis of variance, compared to the FP-CD group.
DISCUSSION
We assessed the conditions for C. difficile colonization and CDI development by feeding naturally born neonatal piglets with milk formula and challenging the animals with the hypervirulent C. difficile ribotype 078 or antibiotics. The data clearly demonstrate that artificial rearing and formula feeding are important factors that predispose neonatal piglets to C. difficile colonization. In our studies, FP animals were characterized by earlier colonization with toxigenic C. difficile and infection development, as assessed by postmortem examination; C. difficile and toxins could already be detected at postpartum day 1, while in suckling piglets C. difficile was detectable at day 2 and toxins at day 4 after birth [4]. Interestingly, already at the age of 2 days, FP animals carried about 3 times more C. difficile than suckling piglets [4], suggesting that the type of feeding may influence early C. difficile colonization. The only source of nutrients for newborn piglets is milk, which contains numerous bioactive and essential compounds, such as growth factors, microbial antigens, and host antibodies, such as those directed against certain pathogens and contributing to passive immunization in offspring [25, 26]. On the contrary, standard milk replacers for suckling piglets are based on bovine milk containing higher amounts of lactose, foreign proteins, and antigens and has lower fat content than sow milk. They also lack beneficial microbes, which may delay the naturally occurring de novo colonization of the infant gut. These differences are challenging and have been reported to affect the activities of digestive enzymes and microbiota, the immune response, and barrier function, leading to intestinal dysbiosis in piglets [14, 15].
The typical clinical symptoms in piglets with CDI often include pasty-to-watery diarrhea, anorexia, growth retardation, dehydration, and death [27, 28]. However, clinical symptoms in pigs often do not correlate with C. difficile and toxins, which makes the diagnosis of CDI difficult [27]. In our studies, both suckling piglets and formula-fed (control, infected, and infected and antibiotic treated) animals showed no visual clinical signs of disease, although C. difficile and toxins could be detected at high levels in feces throughout the trial. Single episodes of diarrhea in study piglets were rare and lasted 1 day and could be associated with animal stress and adaptation. Piglets with a diagnosis of severe mesocolonic edema during postmortem examination had no diarrhea before. Microscopic lesions were absent in suckling piglets but present in formula-fed (control, infected, and infected and antibiotic treated) animals, suggesting an inflammatory process. In humans, the symptoms of CDI are clearly defined and include mild-to-severe life-threatening diarrhea, abdominal pain, and, often, fever [13]. The infection is confirmed by endoscopy, aiming at detection of pseudomembranes, and by ELISA, to detect toxins in feces. CDI in humans is almost exclusively related to antibiotic therapy [29–31], but whether this is also true in the pig is yet not clear. However, piglets of antibiotic-treated sows tend to have greater levels of C. difficile and toxins in their feces as compared to piglets from untreated sows [4]. The use of antibiotics drastically shifts the microbiome, as well, and creates a niche for opportunistic bacteria, including C. difficile. Here, we found that treatment of study piglets with clindamycin before C. difficile infection diminished the C. difficile and toxin loads as compared to levels in C. difficile–infected piglets alone. By treating the animals with clindamycin, we expected the disease to be exaggerated because of the stimulating effect of sublethal antibiotic concentrations on toxin synthesis in C. difficile [32]. However, the colon of clindamycin-treated C. difficile–infected piglets was free from CDI, while the foregut was filled with gas and water, suggesting infection by opportunistic pathogens yet to be characterized.
The 2 major exotoxins, TcdA and TcdB, modulate the intestinal epithelial cell physiology and disrupt the barrier function by inactivating Rho subfamily proteins involved in the formation of cytoskeleton, leading to the loss of tight junctions and epithelial integrity [33]. Additionally, the toxins can cause rapid immune response, as well as inflammation and mesocolonic edema [1,34]. Here, quantification of toxin in piglet fecal specimens involved TcdB only, since more CDI outbreaks are associated with C. difficile producing either both toxins or TcdB only, rather than TcdA only [35]. During intestinal inflammation, activated neutrophils produce calprotectin [34, 36]. In the first trial, we measured the level of calprotectin in fecal specimens obtained 72 hours after infection. Although the calprotectin concentration did not differ between the study groups, it correlated positively with the toxin level, and there was a trend toward a positive correlation with the C. difficile concentration, indicating an important role of toxins in gut inflammation. Moreover, the observed infiltration of neutrophils in the formula-fed control and infected piglets could possibly have been due to the action of calprotectin. We also found a lower abundance of the tight junction proteins claudin 3 and 4 in formula-fed C. difficile–infected piglets as compared to suckling piglets. Such observations would again suggest inflammation possibly due to the detrimental effect of C. difficile toxins targeting tight junction proteins and gut tissues. Similar findings related to the toxins and loss of tight junction proteins have been reported previously in human cell lines [37, 38].
Newborn piglets are continuously exposed to microbes from the maternal environment, which enter the gut together with milk. We found that the numbers of identified bacterial genera, bacterial diversity, and bacterial evenness were lower in the FP and FP-CD groups, compared with findings in suckling animals. The FP and FP-CD groups were enriched in Enterobacteriaceae, suggesting an important role of feeding on the gut microbiota profile and, in turn, susceptibility to infections in early life. Similarly, previous reports revealed lower bacterial abundance and activity in the gut of formula-fed piglets as compared to suckling newborn piglets [14] and infants [39]. A phenomenon termed “colonization resistance,” in which C. difficile gets replaced by other bacteria in the developing ecosystem, could thereby contribute to protection of the host from CDI in suckling piglets [40], but such mechanisms have not been clarified in pigs. Importantly, infection with the hypervirulent C. difficile ribotype 078 did not exhibit further aggravation of the disease, while the toxins of C. difficile 078 have been found to have a detrimental impact on the IPEC-J2, as assessed in vitro (Grześkowiak et al, unpublished data). Our PCR ribotyping results demonstrated that C. difficile 078 is able to colonize the piglet gut and that its spores are shed in the feces. Additionally, we isolated 2 naturally occurring C. difficile ribotypes in fecal specimens from the study piglets (ie, ribotypes 014/020 and 005), both of which were positive for TcdA and TcdB. The early gut colonizers in neonatal piglets aged 1–3 days include clostridia, Enterobacteriaceae, Enterococcaceae, Streptococcaceae, and Peptostreptococcaceae organisms, whereas Lactobacillaceae and other organisms become predominant afterward [41]. For example, it has been shown in humans that Clostridium scindens can successfully outcompete C. difficile and prevent or ameliorate CDI [42]. It is possible that the 2 newly isolated C. difficile ribotypes could have successfully outcompeted the growth and toxin production by C. difficile 078, and this hypothesis should be tested in the future. Similar colonization resistance phenomena have been previously demonstrated in neonatal pigs [43] and hamsters [44]. These observations indicate that colonization with commensal organisms, including nontoxigenic C. difficile, could provide protection against CDI. However, the role of other clostridia (that are less or not toxigenic) in preventing CDI should be assessed with care, owing to the potential risk for a toxin gene transfer between the ribotypes and other C. difficile strains [45].
Maternal antibodies against TcdA have been identified in human blood serum [46], which may protect humans [47] and pigs [48] against CDI. Previously, we detected antibodies (immunoglobulin G) against TcdA in serum and milk from lactating sows, which could provide essential protection of the neonate against CDI (Grześkowiak et al, unpublished data). Thus, the milk may play a key role in prevention from infection in suckling piglets as compared to FP animals. Interestingly, studies of infants have shown that formula feeding is associated with marked shift in the intestinal microbiome and a higher prevalence of intestinal colonization with C. difficile, Streptococcaceae organisms, and Escherichia coli as compared to breastfeeding [49]. Although infants are less susceptible to CDI and are often asymptomatic C. difficile carriers, their role as potential reservoir for putative pathogenic ribotypes is currently under discussion [50]. Further insights into factors affecting C. difficile colonization in both piglets and infants are thus of high relevance, in the context of the One Health approach.
Taken together, our results demonstrate that formula feeding predisposes neonatal piglets to intestinal dysbiosis and favors C. difficile colonization. In addition, infection with C. difficile 078 does not exaggerate disease severity. The repertoire of different maternal and environmental factors seems to set the conditions for disease development. However, caution should be undertaken when diagnosing CDI on the basis of clinical symptoms, since, as demonstrated in the present work and stated previously, they do not always correspond to ongoing gut infection; as a result, there is an urgent need for criteria to diagnose CDI in piglets. The nutritional composition of milk replacers for neonatal piglets should be revised to minimize the risk of gut dysbiosis and may also need to be addressed for infant formulas. Finally, further research is necessary to unravel the interrelations between C. difficile, the gut microbiota, and the immune response in formula-fed and suckling piglets.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. We thank Prof K. Männer, Dr L. Scharek-Tedin, Dr S. Kröger, Dr S. Altmeyer, Dr L. Brucker, Dr C. Heide, Mr J. Riedmüller, Ms M. Eitinger, Ms P. Huck, and animal care takers, for help in the trials and sample analyses; and Prof S. Cutting (University of London, Surrey, United Kingdom), Dr H. A. Hong (University of London, Surrey), and Prof O. Højberg (Aarhus University, Tjele, Denmark), for their collaboration.
J. Z. designed the research protocol. Ł. G. and R. P. conducted the research. Ł. G. performed microbiological and immunological analyses. B. M.-V. helped in the trials and performed histological analyses. Ł. G., W. V., J. Z., and R. P. analyzed the data. Ł. G. and R. P. wrote the manuscript. Ł. G., W. V., J. Z., and R. P. had primary responsibility for the final content. T. H. D., K. R., F.-A. H., and A. F. performed metagenomics analyses. J. R., S. A., and B. M.-V. performed the TJ protein analyses. All authors read and approved the final manuscript.
Financial support. This work was supported by the Animal Health and Welfare ERA-Net and the German Research Foundation (grant PI 946/2-1).
Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
