The experiment was conducted upon receiving the permission granted from the Local Ethics Committee in Kraków (No. 37, 30 May 2016).
Animals and feeding
Studies were carried out on 18 foals representing Polish Pony (Konik, Polish Konik) horses. This primitive horse breed is genetically and phenotypically closely related to its wild ancestor, the Tarpan Horse (Euroasian wild horse) [17].
All foals with mares were kept in the same stable in individual boxes (size 2.15 × 3.50 m) on permanent straw bedding at the Experimental Station of the University of Agriculture in Krakow. All animals were clinically healthy throughout the experimental period. Mares of 5 to 17 years of age, and 270 to 340 kg live body weight, were not vaccinated during pregnancy. Foal birth weight was 27-35 kg, weight loss on the first day of life was <1.5%.The horses had all been used by university students in the teaching program. No horses were used for equestrian purposes. Inclusion criteria consisted of foals born from healthy mares with no placentitis, normal gestational period, uneventful birth, and having normal physical and neurological examination findings. The foals had to successfully stand and nurse within 2 hours of birth, and remain clinically healthy during the study period.
Mares were fed ad libitum with hay (Lolium 40%, Trifolium L.20%) with addition of oat in the amount of 1.5 kg/mare per day (according to Institute of Physiology and Animal Nutrition standards, 1997) [18]. Foals were fed only with colostrum and mother’s milk ad libitum, without additional supplementation. Water was offered from automatic water drinkers (flow ~10 l/min).
Experiment design
320 days of pregnancy, birth alarm (Abfohlsystem, Jan Wolters) was placed in the labia, and mares were moved to box stalls inside a stable, lit with natural light (Supplementary Figure S1). During the experiment, the foals were kept with their mothers in individual boxes, and were leaving the stalls with the mothers for pasture. The following physiological parameters determined the selection of animals for the experiments:
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- blood counts in foals on the first day of life (hematocrit> 40%, hemoglobin> 13 g / dL, the level of erythrocytes 9.5 x 106 / µl, leukocyte level> 6 x 103 / µl)
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- the quality of the colostrum before the first suckling (immunoglobulin concentration> 30 g / dL, > 20% BRIX)
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- evaluation of the foal's viability in the first 5 minutes. after birth (obtaining min. 8 pts on the Apgar scale)
Foals were randomly assigned into two groups:
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- Experimental Group (E; n=9) - foals that received the multicomponent bacterial immunomodulator; and
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- Control Group (C; n=9) - foals that did not receive the multicomponent bacterial immunomodular or any other pharmacological/additive component that might influence their immune.
.For immunostimutalion used in the present study, multi-component bacterial immunomodulator. It consisted of a mixture of inactivated gram-positive and gram-negative bacteria e.g: Escherichia coli (123 mg/ml), Staphylococcus aureus (74 mg/ml), Streptococcus zooepidermicus (24.6 mg/ml), Streptococcus equi (24.6 mg/ml), Streptococcus equisimilis(24.6 mg/ml), Streptococcus agalactiae (24.6 mg/ml), Streptococcus dysgalactiae (24.6 mg/ml), Pasteurellamultocida (123 mg/ml), and Erysipelothrixinsidiosa (49 mg/ml) as well as pork spleen extract (10 mg/ml). On day 35 and day 40 after birth, the foals from the experimental group received intramuscular (m.pectoralisdescendens) injection of 5 ml of multi-component bacterial immunomodulator.
Blood sampling and analysis
Blood samples were collected from foals by jugular venipuncture. Blood samples were obtained from foals until 60 days of age according to the following scheme: before the first suckling, at 1st, 3rd, 5th, 10th, 20th, 30th, 40th, 50th and 60th day of age. Three mililitres of blood were collected into TEMPUS (Applied Biosystems, Tempus™ Blood RNA Tubes) tubes with RNA stabilizing factor. Samples were stored at -20ºC till further processing. Three mililitres of blood were collected into tubes with EDTAk2. The collected samples were centrifuged at5000 rpm for 5 minutes and plasma were stored in -20°C till further analysis.
Isolation of RNA
Isolation of RNA was carried out using TEMPUS SPIN (Ambion) according to the manufacturer’s protocol (Supplementary). 1 µg RNA was transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the protocol.
A “No-RT” (non-reverse transcriptase) control was used for selected RNA samples to analyse DNA contamination in RNA samples.
Gene expression analyses
Gene expression analyses were performed in theIllumina Eco (Illumina) system using TaqMan®MGB probes (Table 1). Every sample was analysed in triplicate in the final volume of 10 µl (Supplementary Table S1). Amplification was performed according to the following protocol: polymerase activation at 95oC (2 min) and 40 cycles: 95oC for 15s and 60oC for 1 min. SDHA and HPRT genes were used as housekeeping genes (Table 1).
Table 1
Probes used for amplification of TLR genes and housekeeping genes.
Gen | Full name of the gene | Access number GenBank | Taqman gene expression assay ID | Dye |
TLR4 | Toll-like receptor 4 | NC_009168.2 | Ec03468993_m1 | FAM |
SDHA | succinate dehydrogenase complex subunit A | XM_001490889 | Ec03470479_m1 | VIC |
HPRT | Hypoxantine phosphoribosyl transferase | AY372182.1 | Ec03470217_m1 | VIC |
In addition, an analysis of blood morphotic parameters was performed (Supplementary material).
Immunoglobulins and cytokins level
Immunoglobulins and cytokins level were measured using ELISA assay in Spectramax Plus 384 Microplate Reader (Molecular Devices) using 96-well plates coated with monoclonal antibodies against equine IgG2 (GR106527, Genorise) IgM (GR106524, Genorise), IL-6 (GR106001, Genorise), and IL-10 (GR106003, Genorise) (Genorise Scientific, Devon-Berwyn, PA, USA) (Supplementary Table S3). 100 µl of plasma per well were added. Each well was washed three times with Assay Buffer after each step. 100 µl of working dilution of Detection Antibody were added and incubate 1 hour. After wash 100 µl of working dilution of Conjugate were added to each well and incubated for 20 minutes. After the wash, 100 µl of Substarte Solution were added to each well, and incubated for 20 minutes. After the wash, 50 µl of Stop Solution were added to each well. Duplicate wells were used for each sample. Plates were read using microplate reader set to 450 nm. Results were calculated based on standard curve. High standard concentration of 80 ng /ml were prepared, vortexed for 15 seconds, and allowed to sit for 5 minutes. A seven-point standard curve was generated using 2-fold serial dilutions in the Assay Buffer.
Statistical analysis
Data are presented as means ± standard error. The data were analysed using SAS 9.4 software (SAS Institute INC., USA). The Shapiro-Wilk test was considered the best test to check the normality of the distribution of a random variable. Because the data did not have normal distribution, Kruskal-Wallis test was used, with immunostimulant as effects and for interactions between group and time post-immunization on relative antibody concentration and cytokine and mRNA expressions. The relationships between all parameters (TLR4 mRNA expresion level, concetration of IL-10, IL-6 ang IgG2, IgM) were analyzed using nonparametric Spearman's rank correlation test. The value ranges from 0.0 to 0.5, from 0.5 to 1.0, from -0.5 to 0.0 and from -1.0 to -0.5 indicate weak positive, strong positive, weak negative and strong negative correlation, respectively.