The expression of chemokines (CCL-2 and CXCL-8) and cytokines (IL-1
During infection
Proinflammatory cytokines, such as interferon-
Little is known about the interaction between
Two vaccines based on native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit) [
This description is based on the MIQE guidelines [
The first-strand cDNA synthesis was carried out by reverse transcription (RT) from total RNA using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen Life Technologies, USA). Briefly, 2
The set of primers and hydrolysis probes used to determine the expression of chemokines (CCL2 and CXCL-8), cytokines (IL-1
Oligonucleotides used in qPCR analysis.
Genes | Sense | Sequences (5′ to 3′) | Product | Efficiency (%) | Reference |
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IL1- |
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GTGCTCAAAACGAAGACGAACC | 110 bp |
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Duvigneau et al. [ |
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CATATTGCCATGCTTTTCCCAGAA | ||||
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IL1- |
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CCAAAGGCCGCCAAGATATAA | 75 bp |
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Arce et al. [ |
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GGACCTCTGGGTATGGCTTTC | ||||
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TLR-4 |
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GCCATCGCTGCTAACATCATC | 108 bp |
|
This study |
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CTCATACTCAAAGATACACCATCGG | ||||
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IL-6 |
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CTGGCAGAAAACAACCTGAACC | 94 bp |
|
Duvigneau et al. [ |
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TGATTCTCATCAAGCAGGTCTCC | ||||
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IL-10 |
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CGGCGCTGTCATCAATTTCTG | 89 bp |
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Duvigneau et al. [ |
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CCCCTCTCTTGGAGCTTGCTA | ||||
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TNF- |
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GCCCTGGTACGAACCCATCTA | 91 bp |
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Arce et al. [ |
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CAGATAGTCGGGCAGGTTGATCTC | ||||
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||||
| |||||
CCL-2 |
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ACCAGCAGCAAGTGTCCTAAAG | 92 bp |
|
Arce et al. [ |
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TCCTGGACCCACTTCTGCTT | ||||
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||||
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CXCL-8 |
|
TTCGATGCCAGTGCATAAATA | 120 bp |
|
Arce et al. [ |
|
TGACAAGCTTAACAATGATTTCTGAA | ||||
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| |||||
GAPDH |
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ACATGGCCTCCAAGGAGTAAGA | 106 bp |
|
Duvigneau et al. [ |
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GATCGAGTTGGGGCTGTGACT | ||||
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||||
| |||||
cyclophilin |
|
TGCTTTCACAGAATAATTCCAGGATTTA | 77 bp |
|
Duvigneau et al. [ |
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GACTTGCCACCAGTGCCATTA | ||||
|
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||||
| |||||
|
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CTCGATCATGAAGTGCGACGT | 114 bp |
|
Duvigneau et al. [ |
|
GTGATCTCCTTCTGCATCCTGTC | ||||
|
|
The cDNA obtained from tracheobronchial nodes of CHG group was amplified using the primers described in Table
Quantitative real-time PCRs were carried out in 96-well plates (LightCycler 480 Multiwell Plates 96, white, -Roche-) in a total volume of 20
NPAPTim, NPAPTit, and blank groups, whose pigs remained alive until the end of the experiment, were compared with each other. CHG group was not included in the comparative analysis because these animals died at a different time point. Descriptive statistics (mean, standard error), normality (Shapiro-Wilk), and homoscedasticity (Bartlett’s test) were determined. The analysis of cytokines expression levels was performed using one-way analysis of variance (ANOVA). Statistical means were contrasted by Tukey test (
All pigs from the NPAPTim and NPAPTit groups survived until the end of experiment, and no clinical symptoms were observed. As could be expected, all pigs from CHG group died after the challenge, showing the typical clinical symptoms of Glässer’s disease: high temperature, limb incoordination, swollen joints, severe dyspnea, and coughing. Clinical signs and histopathological findings have been extensively reported in Frandoloso et al. [
Three reference genes were tested due to different cell composition of the organs compared (brain, lungs, spleen, and lymph nodes). Cyclophilin showed constantly the highest homogeneous level of expression in all organs, with an average quantification cycle (Cq) of
No significant changes in transcript levels of CCL-2 (Figure
Expression of proinflammatory molecules from different tissues in CHG group (mean ± standard error).
Molecules* | Tissue | |||||
---|---|---|---|---|---|---|
Brain | Lung | Spleen | Mediastinal nodes | Tracheobronchial nodes | Mandibular nodes | |
CCL-2 |
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CXCL-8 |
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IL-1 |
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IL-1 |
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IL-6 |
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TNF- |
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IL-10 |
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Relative mRNA expression rates of chemokines and cytokines from different tissues in NPAPTim, NPAPTit and blank groups. *indicates significant differences between groups (
CXCL-8 has been characterized as the main neutrophil activating chemoattractant chemokine [
IL-1
The vaccinated groups showed an IL-6 concentration about five times higher in mediastinal nodes than that of blank group (
Significantly lower values (
The two vaccinated groups showed transcription levels statistically higher than the blank group for IL-10 in spleen and mediastinal and tracheobronchial nodes (
The experimental infection of colostrum-deprived pigs with
The authors declare that they have no conflict of interests with Cycler Probes Master (Roche) and Analytical Software (Roseville, MN, USA).
Research in the laboratory of R. Frandoloso and S. Martínez-Martínez was supported by long-predoctoral fellowships from the Spanish Ministry of Science and Innovation, as well as by Grant AGL-2011-23195 (Spanish Ministry of “Economy and Competitivity”). The authors also acknowledge Estela Costa for the language editing of their paper.