Cultures of U. diversum
The strain of U. diversum ATCC 49782 and 44 field isolates were supplied by the Mycoplasma Laboratory of the Biomedical Sciences Institute, University of São Paulo. Some strains were isolated from cows with granulomatous vulvovaginitis, and others were isolated from the semen of healthy bulls. The bacteria were obtained from the following four states: 19 isolates in São Paulo (farms 1, 2, 3, 4 and 5), 2 isolates from Mato Grosso do Sul (farm 6), 1 isolate from Minas Gerais (farm 7) and 22 isolates from Bahia (farms 8, 9, 10, 11) (Figure S1 in Supplementary file). All microorganisms were cultured in 5 mL of UB broth at 37 °C for 2 days [2]. After growth, the cultures were stored at − 80 °C for the study. The isolates were first submitted to genetic analysis and, after the genetic groups were defined, and representative profiles of each group were evaluated for pathogenicity.
DNA extraction
DNA extraction was performed using the Nucleospin DNA-tissue commercial kit (Macherey-Nagel, Düren, Germany, 2016), following the manufacturer’s recommendations.
Primers design for MLST and virulence genes
Based on descriptions from the literature, seven housekeeping genes were selected for the MLST scheme - ftsH, rpL22, valS, thrS, rpoB, polC [9] and the ureA gene [2] - while other genes were selected for virulence analysis - phospholipase D (pld) (gudiv_472), triacylglycerol lipase (tgl) (gudiv_748), hemolysin (hlyA) (gudiv_91), MIB-MIP system (mib,mip) (gudiv_161/gudiv_162), surface molecules, such as multiple banded antigens (mba) (gudiv_653), and variable surface antigen lipoprotein (VsA) (gudiv_179) and ribose ABC transporter (tABC) (gudiv_307) [2]. After selection of loci for MLST and virulence factors, primers were designed. Through the Manatee database (https://manatee.igs.umaryland.edu) it was possible to access the categorized genes of U. diversum, strain ATCC 49782. Genomic sequences of interest were obtained and possible primer sequences were chosen at random and their analyses were started through the sites https://www.idtdna.com/calc/analyzer and https://www.bioinformatics.org/sms/rev_comp.html. After checking important parameters for constructing primers (size, GC content, melting temperature and annealing, formation of secondary structures), the similarity performed by BLAST was validated (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the specificity of the primers for U. diversum. Forward and reverse primers for each of the selected genes (housekeeping and virulence) are described in Table 1.
Polymerase chain reaction and electrophoresis
Amplifications were performed with a total volume of 25 μL, with 1 μL of DNA, 10x PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl), 1.5 mM MgCl2; 200 μM dNTP, 50 pmol of each primer and 1.5 U of Taq DNA polymerase (Invitrogen®, Brazil). The reactions for ftsH, polC, rpL22, urea, valS and all virulence genes occurred with initial denaturation of 94 °C for 5 min followed by 35 thermal cycles each consisting of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 1 min, concluding with a final extension at 72 °C for 5 min. While for the rpoB and thrS genes, there was initial denaturation of 94 °C for 5 min, followed by 35 thermal cycles each consisting of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, concluding with a final extension of 72 °C for 5 min. The targeted products were analyzed by 2% agarose gel electrophoresis, stained with 2.5 μL ethidium bromide (10 mg / mL), visualized and photographed under UV light. A marker was used as standard to evaluate the size of amplified fragments - 100 bp DNA Ladder (Invitrogen®, Brazil).
PCR product purification and sequencing for MLST technique
After electrophoresis, the total volume of the remaining PCR reaction was precipitated with 500 μL of 65% isopropanol, followed by centrifugation at 19,000 xg for 5 min, washing with 250 μL of 70% alcohol and further centrifugation at 19,000 xg for 5 min. The tubes were inverted for 1 h to dry at room temperature, then resuspended with 20 μL ultrapure water and the primers diluted to 5 pmol. Sanger sequencing reactions were performed at the Biomedical Sciences Institute, Federal University of Sao Paulo according to the protocol for the MegaBACE 1000, using the DYEnamic ET Dye Terminator Kit (with Thermo Sequenase™ II DNA Polymerase) code US81090. The sequences obtained were analyzed by Sequence Analyzer software using the Cimarron Caller Base 3.12.
Allele, sequence type, and clonal complex assignment
The sequencing data were analyzed using nonredundant database (NRDB) comparison tools (https://pubmlst.org/analysis) to assign alleles. Distinct alleles were identified by comparing the sequences of the same gene, and these were assigned arbitrary numbers. The combination of the seven identified alleles formed the allelic profile, which was used to determine the sequence type (ST), and assigned an arbitrary number. Each observed variation, of at least one allele, generated a new ST [9, 26]. Clonal complexes (CC) were formed by different STs that shared, at least, three loci with one other member in the group. A clonal complex must include, at least, two STs to be defined as a new clonal group. STs that were not in any CC, were ST singletons [26].
Phylogenetic analysis
The phylogenetic tree was constructed by Molecular Evolutionary Genetics Analysis 6 (MEGA) using the neighbor-joining method with 1000 bootstraps from the concatenated sequences of housekeeping genes. The sequence was concatenated using UNION, a tool of the European Molecular Biology Open Software Suite (EMBOSS), (http://www.bioinformatics.nl/emboss-explorer/). Then, the sequence was aligned by MAFFT 7 [2].
Isolation of bovine monocytes / macrophages
Fifty milliliters of peripheral blood was collected from the abdominal vein of a cow in EDTA vacutainer tubes and diluted 1:1 in PBS (pH 7.4) [32]. Then, 10 mL of blood was added to 3 mL of Ficoll-Histopaque (density: 10771 g/mL, Sigma-Aldrich, Brazil), centrifuged at 400 xg at 4 °C for 20 min, and the layer of peripheral blood mononuclear cells (PBMC) was removed, washed in PBS 1X and centrifuged again at 400 xg for 10 min. For monocyte isolation, PBMC was resuspended in solution A (5 mL of RPMI 1640 medium containing 10% fetal bovine serum) and mixed in 5 mL solution B (5 mL RPMI 1640 medium + 4.75 mL Percoll + 0.325 mL 10X PBS). This suspension was centrifuged at 400 xg for 30 min at 20 °C and monocytes were removed between the interface of both solutions. To evaluate cell viability, 0.1% Trypan Blue (viability > 90%) was used; the cells were counted in a Neubauer chamber, adjusted concentration 4 x 105cells / mL and grown in a 5% CO2 chamber at 37 °C. After 24 h, the monocytes / macrophages were infected.
Preparation of inoculum and infection of bovine monocytes / macrophages by U. diversum
Six strains (ATCC 49782, BA78, GOTA, S8, 198 and 805), representative of different genetic groups, were grown in UB broth, centrifuged at 300 xg for 30 min, resuspended in PBS and the culture suspension was quantified in 96-well microplates to obtain the inoculum based on a Colorimetric Change Unit (CCU). Monocytes / macrophages (4 x 105cells / mL) were infected with 105 ureaplasma / mL for GOTA, 198, 805 and 104 ureaplasma / mL for ATCC 49782, BA78 and S8. Cells treated with LPS representing the positive control and PBS were added to the cells as negative control. After 6 h of infection, the supernatant was removed, 50 μL of trypsin was added to the wells, followed by 50 μL of fetal bovine serum. Finally, the cells were resuspended in 200 μL of RNAlater™ (Invitrogen, Brazil) and stored at − 80 °C for later use.
Gene expression
The mRNA of cells infected with ureaplasma was extracted according to manufacturer’s instructions for PicoPure kit (Applied Biosystems, Brazil), with DNAse treatment and RNA elution in a 11 μL kit elution solution. The cDNA was obtained by reverse transcription (RT) from the mRNA, using the SuperScript® III Reverse Transcriptase kit (Applied Biosystems, Brazil). The cDNA was used in a custom StepOnePlus Real-Time PCR System (Applied Biosystems, Brazil) with SYBR Green (Qiagen-SABioscience, Brazil) to determine the gene expression of TNF-α, IL-1β, IL-6, IL − 10 and IL-17 (Qiagen-SABioscience, Brazil), following the cycle: 50 °C for 10 min; 95 °C for 10 min; and 45 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min. The melting curve was evaluated at the end of the reaction to observe the amplification specificity. The data was analyzed using the 2-∆∆CT [39]. Standardization was performed based on GAPDH expression.
Expression heat map
The relative expression data of the TNF-α, IL-1β, IL-6, IL-10 and IL-17 cytokines, induced by different strains of U. diversum, were analyzed using the heatmapper platform (http: // www. Heatmapper.ca/expression/) and represented as a heatmap. The unsupervised hierarchical grouping was performed using the average distance and the Euclidean distance as metrics.
Statistical analysis
In this study, three independent experiments were carried out for each of the six strains of U. diversum. To compare cytokine gene expression of different strains, the Kruskal-Wallis non-parametric test was used, followed by Dunn’s post-hoc test, which makes paired comparisons. All analyses were performed using the GraphPad-Prism 6.0 software (GraphPad software, San Diego, CA-USA). Statistically significant differences were found with P values equal to or less than 0.05, using IC 95% for relative expression of cytokines for the different strains.
