Animals and feeding regimes
Animals
Twenty-three German Blackhead Mutton ewes (entering the 2nd and 3rd lactation, age: 2–3 years, weight: 85–100 kg) were synchronized with Chronogest CR intravaginal sponges (20 mg, flugestone acetate, MSD Santé Animale, Beaucouzé, Cedex, France), and mated naturally with three rams from the same breed. At day 39 post-mating, ewes were transabdominal scanned for pregnancy by using a diagnostic scanner, out of the 23 ewes, nineteen became pregnant.
The 19 clinically healthy pregnant ewes were randomly divided into two groups: control group (n = 9) and Mg group (n = 10). A sample size calculation was not performed The animals belong to the Federal Research Institute for Animal Health, Institute of Farm Animal Genetics, Friedrich-Loeffler Institute (FLI), Mecklenhorst, Germany. The ewes were housed in the facilities of the Institute. At the end of the experiments the animals were released. An owner consent was not required.
Feeding regimen
In brief, the animals were group-fed under surveillance of the personnel. Throughout the experimental period, there were no significant refusal. The ewes received grass silage (3 kg per ewe and day, 32% dry matter, DM) and concentrate (500 g per ewe and day, 89% DM). While the control group was fed a common concentrate available for sheep (Raiffeisen Schafe S2 lose, Agravis Niedersachsen-Süd GmbH, Hannover, Germany) containing 0.29% Mg (as fed), the pellets for the Mg group had been additionally supplemented with magnesium oxide to a final content of 0.51% (as fed). After parturition (48 h post-partum), the amount of concentrate was increased to 1200 g per ewe and day (as fed). Ante-partum, the estimated daily Mg intake was 2.97 g per ewe in the control group (0.21% of DM) and 4.19 g per ewe in the Mg group (0.30% of DM). Postpartum, the estimated daily Mg intake was 4.96 g per ewe in the control group (0.24% of DM) and 7.74 g per ewe in the Mg group (0.38% of DM). Ewes were allowed to adjust to this diet for 2 weeks before starting the experiment (adaptation period). The groups were housed separately with water available ad libitum. No anthelmintic treatment was applied for these animals during the experimental period.
Blood collection
Blood was obtained by jugular vein puncture into K2E (EDTA), sodium heparin, and Clot Activator Tube (CAT) vacutainer tubes (BD Vacutainer systems, Roborough, UK) at five time points: d 30 a.p., d 14 a.p., d 1 p.p., d 14 p.p. and d 30 p.p. at 08:00 before the morning feeding. Serum was separated by centrifugation of clotted blood (3000 g, 10 min, 4 °C) and stored in aliquots at − 20 °C for further analysis.
Serum variables
Total serum calcium levels were measured using a commercial kit (Labor + Technik LT-SYS, Labor + Technik Eberhard Lehmann GmbH, Berlin, Germany) spectrophotometrically (Uvikon XL UV-Visible, Scanning spectrophotometer, Biotek Instruments Inc., Winooski, VT, USA). Total Mg, was determined by using commercial kits (Labor + Technik LT-SYS, Labor + Technik Eberhard Lehmann GmbH) in the diagnostic laboratory of the Clinic for Swine, Small Ruminants and Forensic Medicine, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany. Serum cortisol levels were estimated using ABNOVA® Sheep cortisol ELISA kits (Abnova, Taoyuan, Taiwan) in accordance with the manufacturer’s instructions.
Vaccination
At d 14 a.p., the ewes in both groups were injected s/c with 1 mL of a commercial vaccine against Mycobacterium avium paratuberculosis (MAP), strain 316 F, (Gudair®, CZ Veterinaria, S. A, Pontevedra, Spain). Whole blood samples were collected immediately before the injection (0) and 24 h following the vaccination (1), to assess vaccination-associated changes in the composition of blood leukocytes (neutrophils, lymphocytes, CD4+ and CD8+ cells, monocytes, and monocyte subsets) see below. At d 0, d 7 and d 21 post vaccination serum samples were taken and transferred to the diagnostic laboratory of the Clinic for Swine, Small Ruminants and Forensic Medicine University of Veterinary Medicine Hannover, Foundation, Hannover, Germany, to determine the level of MAP-specific antibodies (MAP Abs) using a commercial diagnostic indirect ELISA (Cattletype® MAP Ab, Indical bioscience, Leipzig GmbH, Germany) in accordance with the manufacturer’s instructions. Results are given in optical densities (OD). Mean values (MV) of the ODs for the negative (NC) and the positive Control (PC) were calculated. The ratio of sample OD to mean OD of the positive control (S/P) was calculated according to the following equation:
$$ \frac{S}{P}= OD\ Sample- MV\ OD\ NC\div MV\ OD\ PC- MN\ OD\ NC $$
As suggested by the manufacturer, an S/P ratio of ≥0.4 was considered positive.
Total leukocyte counts
Whole heparinised blood (50 μL) was mixed with 450 μL Turk’s solution, and 20 μL were applied to a Neubauer chamber. Leukocytes were counted microscopically (Nikon microscope ECLIPSE 80i). Fractions of neutrophils, lymphocytes and monocytes among blood leukocytes were determined flow cytometrically (Figure S1). For this purpose, 100 μL whole heparinised blood was mixed with 500 μL distilled water (DW) for 3 s, followed by addition of 500 μL double concentrated phosphate buffered saline (2xPBS). After centrifugation (517 x g, 4 min, 8 °C), the supernatant was discharged and the cell pellet was resuspended in PBS. In the case of the remaining erythrocytes, the hypotonic lysis step was repeated until complete erythrolysis. The last cell pellet was suspended in 100 μL PBS (2 μg/mL propidium iodide) and the cell suspension was measured by flow cytometry (BD Accuri C6 flow cytometer®, Becton Dickinson Inc., Holdrege, NE, USA). For each sample 20,000 events were collected. After setting regions to identify propidium iodide-negative (viable) cells (Figure S1-A), identification of singlets among viable leukocytes was performed in a forward scatter area (FSC-A) versus FSC-height density plot A (Figure S1-B). Afterwards, granulocytes (neutrophils), lymphocytes and monocytes were determined among the singlets based on their characteristic forward scatter area (FSC-A) and side scatter area (SSC-A) properties (Figure S1-C). The percentages of the major leukocyte subpopulations (lymphocytes, monocytes and granulocytes) were determined. The obtained percentages were multiplied with the total leukocyte counts to obtain total numbers of these cell types among leukocytes.
Neutrophil phagocytic activity in whole blood samples
In vitro phagocytosis was performed as previously described [60] with some modifications. Fresh heparinised whole blood (100 μL) was incubated with heat-killed, FITC-labelled Staphylococcus aureus (0.5 × 109 bacteria in 400 μL PBS) (Institute of Microbiology, University of Veterinary Medicine, Hannover, Germany). In 1 mL vials; this bacteria suspension was added to 100 μL blood to achieve a bacteria/neutrophil ratio of 50:1. The needed volume of bacteria suspension was calculated based on total numbers of neutrophils/mL blood. Mixtures were incubated for 30 min (37 °C, 5% CO2). Blood samples with added PBS (same volume as the bacteria suspension) served as controls. Thereafter, blood/bacteria mixtures were subjected to a hypotonic lysis step by adding 500 μL D. W for 3 s followed by adding 500 μL 2xPBS. The mixture was centrifuged (517 x g, 4 min, 8 °C) and the cell pellet was resuspended in 1 mL PBS (2 μg/ml PI). Neutrophil phagocytic activity was defined flow cytometrically as the percentage of green fluorescent granulocytes (cells were identified based on forward/side scatter characteristics) among viable (PI negative) granulocytes after excluding eosinophils on FITC-fluorescence vs SSC-A density plots (Figure S2-A-D). The mean cellular phagocytic capacity was defined as the geometric mean green fluorescence of phagocytosis-positive (green fluorescing) granulocytes.
Gradient density separation of mononuclear cells
Separation of mononuclear cells was performed as described previously [41] with some modifications. A total of 20 mL fresh EDTA blood was diluted 1:1 PBS, layered gently on the top of 15 mL lymphocyte separation medium (Density: 1.077 g/mL, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and centrifuged (1000 x g, 30 min, 4 °C). The interphase containing mononuclear cells (MNC) was collected and placed in a fresh 50 mL tube. This was filled up to 50 mL with PBS and centrifuged (500 x g, 10 min at 4 °C). The supernatant was discharged and the pellet resuspended. Erythrocytes lysis step was performed by adding 20 mL DW, mixing it for 3 s and then adding 20 mL 2xPBS. After centrifuging (250 x g, 10 min at 4 °C) the supernatant was discharged and the pellet was resuspended in 25 mL PBS followed by centrifugation (120 x g, 10 min at 4 °C). The final cell pellet was resuspended and adjusted to 10 × 106/mL in PBS. The purity of the MNC separation was determined flow cytometrically on an SSC-A vs FSC-A density plot (S3-A).
Monocyte subset determination
Separated MNC were placed in a 96 well round bottom plate (2 × 106 MNC /well) and 30 μL of a mixture of two directly conjugated, ovine cross-reactive, monoclonal mouse anti-human antibodies was added: anti-CD14-RPE (BIO-RAD, MCA1568PE, 100 TESTS/mL, final dilution 1:10), anti-CD16-FITC (BIO-RAD, MCA5665F, 0.1 mg/mL, final dilution 1:45). The mixture was incubated for 30 min at 4 °C. Cells were washed twice with 200 μL membrane immunofluorescence (MIF) buffer (PBS + 2.5 g/L bovine serum albumin + 0.1 g/L of NaN3). Afterwards, 100 μL PI was added to exclude the dead cells, after gating MNC according to their FSC-A and SSC-A properties (Figure 3S-A, B, C). Three ovine monocyte subsets were defined flow cytometrically based on their CD14 and CD16 expression: classical monocytes (cM, CD14+/CD16-), intermediate monocytes (intM, CD14+/CD16+), and nonclassical monocytes (ncM, CD14−/CD16+) (Figure 3S-D). Preliminary double staining of cells with concentration-matched isotype controls (IgG2a-PE, BIO-RAD MCA929PE, IgG2a-FITC, BIO-RAD MCA929F) ensured that murine IgG2a antibodies display no unspecific reactivity with sheep monocytes. Total counts of monocyte subsets were calculated by multiplying absolute monocyte counts with monocyte subset percentages obtained after flow cytometric analysis.
Lymphocyte proliferation capacity
Separated MNCs (10 × 106/mL) were labelled with carboxyfluorescein succinimidyl ester (CFSE, 1.5 μg/mL in BPS) (C1157, ThermoFisher Scientific In., Waltham, MA, USA) and incubated for 10 min at 37 °C. The double volume of culture medium (RPMI 1640 medium, SIGMA-Aldrich®, Darmstadt, Germany) supplemented with 10% foetal calf serum (Biochrom AG, Berlin, Germany), and 100 U/mL Penicillin/Streptomycin (Invitrogen GmbH, Karlsruhe, Germany) was added and the cell suspension was centrifuged (500 x g, 10 min at 4οC). The supernatant was discharged, 50 mL PBS was added and the suspension was centrifuged again (500 x g, 10 min at 4 °C). The last cell pellet was resuspended in 4 mL culture medium and adjusted to 3 × 106/mL. Afterwards, the CFSE-labelled MNCs (3 × 105 cells/well) were stimulated with Concanavalin A (ConA, 4 μg/mL, Sigma-Aldrich, Biochemie GmbH, Hamburg, Germany) in 96-well round bottom plates. Each setup was done in duplicate. Set ups without ConA served as controls. Plates were incubated for 4 days (37 οC, 5% CO2 in air). Thereafter, the plates were centrifuged (351 x g, 4 min, 8 °C), the supernatant was removed and the cells were incubated with directly labelled with a murine monoclonal antibody specific for sheep CD4 (anti sheep CD4-Alexa Fluor®-647, BIO-RAD MCA2213A647, 1:160) and a monoclonal antibody cross reactive with sheep CD8 (anti bovine CD8-RPE, BIO-RAD MCA837PE, 1:40). Preliminary staining of cells with concentration-matched isotype controls (IgG2a-PE, BIO-RAD MCA929PE, IgG2a- Alexa-Fluor-647, BIO-RAD MCA MCA929A647) ensured that murine IgG2a antibodies display no unspecific reactivity with sheep lymphocytes. After a 30 min incubation period at 4 °C cells were washed twice with MIF buffer as described above and resuspended in MIF buffer containing 2 μg/ml PI. Labelled cells were analysed flow cytometrically. CFSE fluorescence of viable (PI-negative) mononuclear cells was plotted against the cell size (FSC-A) (Figure S4-A, B). Cells displaying reduced CFSE fluorescence were identified as activated/proliferating cells. (Figure 4S-C). The proliferative capacity of T-cell subsets was determined in CFSE versus CD4-Alexa 647 and CFSE versus CD8-PE density plots, respectively (Figure S4-D, E).
Faecal worm eggs count
Rectal faecal samples (10–15 g) were collected from individual animals at d 14 a.p, 1 p.p., 14 p.p and 30 p.p., and transported to the diagnostic laboratory of the Clinic for Swine, Small Ruminants and Forensic Medicine, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
Faecal samples were examined using saturated Nacl as a flotation method [61]. With this method roundworms were differentiated microscopically according to the size and shape of the eggs. The roundworm species with eggs of the same shape (e.g. Haemonchus contortus, Teladorsagisa circumcinta, Trichostrongylus colubriformis and others) were not differentiated.
Briefly, 10 g faecal samples were suspended in D.W, sieved through a grid into a beaker in a volume of 250 ml and allowed to stand for 30 min. 2 mL of the sediment was resuspended in 9 mL saturated Nacl and centrifuged (90 x g/3 min). Three drops were taken from the surface of the liquid and placed in glass slide which covered with cover slip and examined under a microscope (10X), results were expressed as eggs per gram.
Sodium silicate solution was used for sedimentation of egg of liver fluke worms (Fasciola spp.). For this method 2 mL of the sediment was resuspended in 9 mL sodium silicate, 200 μL of methylene blue 3% were added and mixed well, afterwards the tubes were centrifuged (800 x g/10 min). Three drops were taken from the surface of the liquid and placed in glass slide which then was covered with cover slip and examined under the microscope (10x). The results of this investigation were negative, since eggs of liver fluke worms were not found.
Eosinophils count
Blood was obtained by jugular vein puncture into K2E (EDTA) vacutainer tubes (BD Vacutainer, Belliver Industrial Estate, Plymouth, UK) in parallel with the collection of the faecal samples. The percentage of lymphocytes, neutrophils, monocytes and eosinophils were determined microscopically (counting 200 leukocytes in thin Giemsa May-Grunwald-stained blood smears). Eosinophils total counts were calculated by multiplying the percentage of eosinophils among leukocytes with the total numbers of leukocytes determined whith a hematology analyser (Celltac Alpha Nihon Kohden Europe GmbH, Rosbach vor der Höhe, Germany).
Flow cytometric data evaluation
After acquisition, flow cytometric data were analysed with the Acurri BD™ C6 software. The gating strategies to identify cell populations, frequencies of phagocytosis-positive cells, proliferating cells, CD4+ and CD8+ positive cells among proliferating cells, and mean fluorescence intensities of phagocytosis-positive cells are described in Supplementary Figures S1 to S4.
Statistical analysis
The data were expressed as mean ± SEM, n representing the number of animals per group. The unpaired t-test, two-way ANOVA test and Sidak multiple comparisons test (GraphPad Prism 8 Software, San Diego, CA, USA) were used for comparison between the different time points and groups. Correlations between selected variables (faecal worm egg counts and eosinophil numbers) were analyzed by Pearson’s correlation. Differences were considered statistically significant when P < 0.05. All data except faecal egg counts were normally distributed according to Shapiro-Wilk and Kolmogorov-Smirnov tests. Log-transformed faecal egg counts were normally distributed. Two-way ANOVA analysis of faecal egg counts was performed with antilogarithmic values calculated with the EXP function (Microsoft Excel).