Virus and cells
The TGEV strain Purdue 46-MAD (kindly provided by Dr. C. Schwegmann-Wessels, Institut für Virologie, Tierärztliche Hochschule Hannover) was used in this study. To prepare TGEV stocks, a stable mycoplasma-free swine testicle (ST) cell line supporting the growth of TGEV was used. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; PAN-Biotech, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal calf serum (Hyclone), and 1% penicillin/streptomycin (Biochrom, Berlin, Germany) at 37°C with 5% CO2. Stock virus was propagated in ST cells to a titer of 1.00E + 07 plaque-forming unit (PFU)/mL.
Animals and experimental design
German landrace piglets (n = 60) of both sexes from a TGEV-free herd (Leibniz Institut für Nutztierbiologie, Dummerstorf, Germany) were weaned at the age of 21 days and were randomly assigned to three different dietary groups (20 animals each). Diets either contained no additional ZnO (Znlow: 50 mg Zn/kg diet), or were supplemented with analytical grade ZnO (>98% purity) to contain 150 mg Zn/kg diet (Znmed) or 2,500 mg Zn/kg diet (Znhigh). The Znlow diet represents the regular feed of the animals and the Znlow group, therefore, represented the control group for this experiment. At 26 days of age, animals were moved to a containment facility (Bundesinstitut für Risikobewertung, Berlin, Germany) where they were randomly allocated to 6 pens per group. The piglets were fed the respective diet ad libitum in a pelleted form, water was also provided ad libitum by nipple drinkers. The pens were of equal size (2.8 m2), equipped with a feeding automate with 5 feeding places and a nipple drinker. The floors were covered with rubber mate and a red light lamp was placed above to provide additional heat. Room temperature was kept at 25 ± 1°C with humidity of 50 - 60% and constant air volume exchange. The pens were thoroughly cleaned by brushing the floors and walls with consecutive flushing with lukewarm water in the mornings. Superficial flushing of the floors was additionally performed in the late afternoon. At day 28 of age, all piglets were challenged orally with 2 mL TGEV with a titer of 1.0E+07 PFU/mL. In each group, half of the piglets (n = 10 per group) were sacrificed 1 day post infection (dpi) in order to examine acute infection, since piglets reportedly display strong symptoms of gastroenteritis within 20 h post-infection [42], and the other half (n = 10 per group) were sacrificed 18 dpi to see the effect of Zn on adaptive immune response.
Clinical follow-up and sampling
The study was approved by the local animal welfare authority (Landesamt für Gesundheit und Soziales, Berlin, Germany) under the registration number G 0116/12. Animals were clinically examined on arrival. Blood samples were collected at 0, 4, 7, 11, 14 and 18 dpi. Sera were used to determine TGEV-specific antibodies. Piglets were monitored daily for rectal temperature and body weight was recorded once weekly. Fecal scores (from 1 to 5, where 1 means watery and 5 dry and hard stool) were also recorded daily up to 12 dpi. Fecal swabs were taken before and then daily after infection for the detection of virus shedding. At necropsy (both 1 and 18 dpi), 10 – 15 cm long samples from the descending duodenum, jejunum (approximately 100 cm distal to the duodeno-jejunal flexure), ileum (distal 15 cm), spleen and jejunal mesenteric lymph nodes were taken to determine gene expression profiles. Furthermore, defined pieces of the jejunum of the same length were washed with 25 mL PBS and intestinal fluid was collected to detect adaptive immune response (sIgA). Additionally, 15 cm of mid jejunum (1 dpi) were removed, cut open, and rinsed with and transported in cooled saline solution (0.9% NaCl, 1 mM CaCl2). Jejunal tissue was stripped off the muscle layer and explants were mounted in Ussing chambers for electrophysiological analysis as described in detail below. Part of each intestinal tissue sample was additionally fixed in buffered 4% formalin.
Confirmation of Zn status
Trace element status of the pigs was determined in serum and liver tissue as described previously [18]. Briefly, organs were freeze dried, incinerated and hydrolysed in concentrated hydrochloric acid. Serum samples were hydrolysed directly. Trace element concentration was determined by atomic absorption spectrometry in an AAS vario 6 spectrometer (Analytik Jena, Jena, Germany).
Real-time quantitative RT-PCR (qRT-PCR) analysis
Total RNA was extracted from 20 mg of jejunum sample or 200 μL of serum using a Nucleo-Spin® RNA II Kit for Tissue (Macherey & Nagel, Düren, Germany) following the manufacturer’s instructions. Reverse transcription (RT) was performed using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. PCR reactions were performed in a total volume of 25 μL in an iCycler iQ5® detection system (Bio-Rad Laboratories, München, Germany). Melt curve analysis and agarose gel electrophoresis were performed after completion of each assay to confirm specificity of the amplification. For the following target genes: Zn transporters SLC30A1 (ZnT1), SLC30A2 (ZnT2), SLC30A5 (ZnT5), SLC39A4 (ZIP4), and metallothionein (MT1), quantitative real-time RT-PCR was performed using the one-step QRT-PCR master mix kit (Brilliant®II SYBR®Green, Agilent Technologies, Santa Clara, USA) as described previously [18].
TGEV genome copy numbers were quantified using a TaqMan fluorescent quantitative (q) PCR assay as previously shown [25]. Expression levels of IFN-α, IL-6, OAS, and TNF-α were calculated using the Delta-Delta-Ct-Method calculation [43]. Four commonly used reference genes (β-2 microglobulin, RPL13, RPL19 and SDHA) were selected for normalization of gene expression. A mean expression value (normalization factor) for the four reference genes was calculated to enable normalization of gene expression data for all the genes of interest. Samples from Znmed group were used the references for the calculations. For the expression of Zn transporters ZnT1, ZnT2, ZnT5, ZIP4 and MT1, standard curves were generated using serial dilutions of pooled RNA (within a range from 5–200 ng/μL) from 20 samples to convert Ct values into arbitrary values. These values were then normalized using the mean values of the house-keeping genes and then used for statistical comparisons. The names of genes, primer sequences, annealing temperature, and references are listed in Additional file 1: Table S1.
Enzyme-linked immunosorbent assay (ELISA)
Blood samples were collected on 0, 4, 7, 11, 14 and 18 dpi, and TGEV-specific IgG antibodies were determined using a commercial ELISA (INGEZIM TGEV 2.0, Ingenasa). Intestinal wash fluid was collected on 1 and 18 dpi and mucosal sIgA antibodies in the supernatants were also measured using a commercial ELISA (Pig IgA ELISA Kit, Bethyl Laboratories, Montgomery, USA) following the manufacturer’s instructions.
Electrophysiology
Jejunal specimens were mounted in modified Ussing chambers to carry out impedance measurements as described previously [44]. In brief, one-path impedance spectroscopy was performed to determine epithelial (Repi) and subepithelial (Rsub) contributions to the transepithelial resistance (TER). Resistances of bath solution without tissue as well as electrode offsets were recorded prior to each experiment and subtracted from experimental data. Preparations were allowed to equilibrate for 45 min and Repi was measured as previously described [45].
As pathogen-induced secretory diarrhea is caused by excessive chloride secretion with accompanied osmotically driven water flux, forskolin (Calbiochem®, Merck, Darmstadt, Germany; final concentration 10 μM), a secretagogue agent, was added basolaterally in supplemented Ringer’s solution (113.6 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 21 mM NaHCO3, 0.6 mM NaH2PO4, 2.4 mM Na2HPO4, 10 mM D(+)-glucose, 2.5 mM glutamine, 10 mM D(+)-mannose, 0.5 mM β-OH-butyrate, 50 mg/l piperacillin, 4 mg/l imipenem; pH 7.4 when equilibrated with carbogen) in order to trigger a chloride secretory response. For testing the sodium-coupled and therefore electrogenic glucose absorption via SGLT1, glucose (Roth, Karlsruhe, Germany; final concentration 10 mM) was added apically in glucose-free Ringer’s solution (113.6 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 1.2 mM CaCl2, 21 mM NaHCO3, 0.6 mM NaH2PO4, 2.4 mM Na2HPO4; pH 7.4 when equilibrated with carbogen). After administration of effectors, changes in short-circuit current (∆ISC) were recorded.
Permeability to fluorescein (332 Da) represents a measure for the paracellular leakiness/tightness of the epithelial layer and was ascertained in Ussing chamber experiments under voltage clamp conditions. After equilibrating jejunal specimens in supplemented Ringer’s solution, fluorescein (Sigma-Aldrich, St. Louis, USA) was added apically (final concentration, 50 μM). At 30 and 90 min post administration, basolateral samples were replaced with Ringer’s solution. Fluorescein concentrations were determined with a fluorometer at 525 nm (Infinite M200, Tecan, Crailsheim, Germany) and permeabilities were calculated.
Morphometry
Formalin-fixed tissue sections were stained with hematoxylin and eosin (H&E) using standard staining protocols and analyzed using the freehand line selection tool of Image J (Rasband, ImageJ, NIH, Bethesda, Maryland; http://rsb.info.nih.gov/ij/).
The surface area of the jejunal mucosa was assessed as the ratio of mucosal-to-serosal surface area from the lengths of apical epithelial as well as muscular mucosa linings in equivalent fields of view of adjacent sections. Crypt/villus height and density were determined from five sections per piglet, each ~750 μm in width.
Immunofluorescence staining
In order to highlight caspase-3-mediated apoptotic events in jejunal tissue, formalin-fixed tissue slices were rehydrated (xylene, increasing ethanol series), heated at 95 °C in citrate buffer (10 mM citric acid, pH 6.0) for 15 min, and then washed in phosphate-buffered saline (PBS). Tissue slices were then incubated in blocking solution (6% goat serum + 1% BSA in PBS) for 30 min at room temperature (RT) before incubation with rabbit anti-cleaved caspase-3 (Cell Signaling Technology, Cambridge, UK) at a 1:250 dilution in blocking solution for 60 min at RT. After several washings, tissue slices were incubated with goat anti-rabbit F(ab')2 conjugated with DyLight™488 (Jackson ImmunoResearch, Newmarket, UK) at a 1:500 dilution and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/mL; Roche, Mannheim, Germany) for 60 min at RT in the dark. After washing, sections were embedded using ProTaqs Mount Fluor (Biocyc, Luckenwalde, Germany).
Images were taken with an inverted Zeiss LSM 510 META confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). Digital images were processed using Fiji imaging software [46] and Zeiss LSM 510 META software.
Statistical analyses
Calculations were performed with SPSS® Version 21 (IBM, Armonk, USA) and GraphPad Prism 5 (GraphPad Software Inc, La Jolla, USA). Two-factorial mixed models were applied to calculate residuals for all variables (fixed factor: diet, time, diet*time) and a random effect (animal). Data from gene expression were used for the one-factorial model (fixed factor: diet). F-Test was applied for fixed effects and interaction effects with subsequent LSD post hoc test. Electrophysiology and morphometric data are expressed as means ± standard error of the mean (SEM), and statistical analyses were carried out using a one-way ANOVA with Tukey HSD post hoc test. Significances are depicted as: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; tendencies are given as 0.05 ≤ P ≤ 0.1.