Bordetella pertussis

For PCR evaluation of respiratory specimens, we used the Fast-track Diagnostics Respiratory Pathogens 33 multiplex PCR kit (FTD Resp-33 kit) (Fast-track Diagnostics, Sliema, Malta). NP/OP specimens were collected in viral transport medium (universal transport medium [UTM], Copan Diagnostics, Bresica, Italy) and refrigerated at 2°C–8°C for a maximum of 8 hours, or frozen at –80°C prior to nucleic acid extraction. Induced sputum, pleural fluid, and lung aspirate specimens were collected in saline in universal containers and either refrigerated at 2°C–8°C for a maximum of 24 hours, or frozen at –80°C prior to nucleic acid extraction.
Total nucleic acid extraction was performed on respiratory specimens using the NucliSENS easyMAG platform (bioMérieux, Marcy l’Etoile, France). Four hundred microliters of each respiratory specimen (NP specimen in UTM, induced sputum aliquot in normal saline, pleural fluid aliquot, or lung aspirate aliquot) was eluted to a final volume of 60–110 μL nucleic acid. Prior to extraction, induced sputum specimens were digested with 1:1 dithiothreitol and incubated at ambient temperature until any mucus was broken down.
The FTD Resp-33 kit is a real-time PCR arranged in 8 multiplex groups for the detection of the following 33 viruses, bacteria, and fungi: influenza A, B, and C; parainfluenza viruses 1, 2, 3, and 4; coronaviruses NL63, 229E, OC43, and HKU1; human metapneumovirus A/B; human rhinovirus; respiratory syncytial virus A/B; adenovirus; enterovirus, parechovirus; bocavirus; cytomegalovirus; Pneumocystis jirovecii; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Streptococcus pneumoniae; Haemophilus influenzae type b; Staphylococcus aureus; Moraxella catarrhalis; Bordetella pertussis; Klebsiella pneumoniae; Legionella species; Salmonella species; and Haemophilus influenzae species. The K. pneumoniae target was not used in any of the final analyses because of difficulties with assay specificity, as has been found elsewhere [9 (link)]. Positive, negative, and internal extraction controls were included in each run.
Quantitative PCR (qPCR) data were generated through the creation of standard curves using 10-fold serial dilutions of plasmid standards provided by FTD on an approximately quarterly basis at each study site, with calculation of pathogen density (copies/milliliter) from the sample cycle threshold (Ct) values. Because the results for the known standards were highly consistent across laboratories, standard curve data from all sites were pooled to create “standardized” standard curves for each pathogen target; data points beyond 2 standard deviations of the mean were excluded. Quantitative PCR was performed at each site using an Applied Biosystems 7500 (ABI-7500) platform (Applied Biosystems, Foster City, California). Cycling conditions were 50°C for 15 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 8 seconds followed by 60°C for 34 seconds.

Mice were immunized subcutaneously with 250 μg MOG³⁵, ³⁶, ³⁷, ³⁸, ³⁹, ⁴⁰, ⁴¹, ⁴², ⁴³, ⁴⁴, ⁴⁵, ⁴⁶, ⁴⁷, ⁴⁸, ⁴⁹, ⁵⁰, ⁵¹, ⁵², ⁵³, ⁵⁴, ⁵⁵ peptide (AnaSpec, Fremont, CA) emulsified in CFA containing 500 μg Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). On days 0 and 3, each mouse was injected intraperitoneally with 200 ng of purified Bordetella pertussis toxin (Enzo Life Sciences, Farmingdale, NY). Mice were scored daily for the severity of the disease using a five‐point scale: 0, no symptoms; 1, limp tail; 2, limp tail with loss of righting; 3, paralysis of a single hind limp; 4, paralysis of both hind limps; and 5, moribund state or death. Mice were sacrificed on day 14 (peak symptomatic day). Spinal cord tissues were harvested and flash‐frozen and used for mRNA analysis.

Bacteria pellets were lysed in 200 μl of Tris-EDTA buffer (Invitrogen™) with 15 mg.ml⁻¹ of lysozyme (Thermo Fisher Scientific) and 1 mg.ml⁻¹ of proteinase K (Qiagen) for 10 min at 400 rpm, at room temperature. Then, RNA extraction was performed on column using RNeasy® mini kit (Qiagen) including an on-column DNase digestion. A second step of DNase digestion was performed by DNase TURBO™ (Thermo Fisher Scientific). RNA purity was assessed using NanoDrop™ 2000 (Thermo Scientific™). RNA integrity was analyzed by TapeStation 4200 (Agilent) using the High Sensitivity RNA ScreenTape. Only samples with a RINe over 7 were selected. RNA was quantified using Qubit™ 2.0 Fluorometer (Thermo Fisher Scientific) with the Qubit™ RNA high sensitivity kit (Invitrogen™).
Libraries were prepared in triplicate for each sample with the Sciclone® G3 NGSx iQ™ liquid handling workstation (PerkinElmer) by using the Universal RNA-Seq kit (Tecan Genomics, 2021 ) customized for B. pertussis. Briefly, cDNA was synthetized by reverse transcription and mechanically fragmented with the Covaris® M220 Focused-ultrasonicator™ for 90 s at 50 W, 10% duty factor, and 200 burst per cycle, to obtain 400 bp (base pair) cDNA fragments. Fragments were, then, purified using Agencourt® beads, followed by end repair, adaptor ligation, and sense strand selection steps. Ribosomal RNAs (rRNA) were depleted using AnyDeplete technology (Tecan Genomics) with specific B. pertussis rRNA probes. Libraries obtained after PCR amplification (18 cycles) were characterized by TapeStation with D5000 ScreenTape (Agilent), normalized at 10 nM, pooled, diluted at 4 nM and, denatured according to Illumina NextSeq500 protocol A (Illumina, 2022 ). Denatured libraries were diluted at 1.8 pM and 1 μl of PhiX at 20 pM was added as control. Then, three sequencing runs were launched, with 1.3 ml of this mix on a NextSeq® 500 platform (Illumina) using 75-bases single-end protocol on a High Output flow cell (Illumina).
Following base calling steps by sequencer software (Illumina), quality control of the sequencing run was carried out using Sequencing Analysis Viewer (SAV, Illumina, version 2.4.7) regarding reads quality and cluster density. After demultiplexing steps, sequencing QC was performed on raw data with fastqc tool (Babraham Bioinformatics, 2019 ) and visualized using multiQC tool (Ewels et al., 2016 (link)).
Raw data were imported in Array Studio (v11.0) software (OmicSoft Qiagen). Reads were trimmed to remove low quality bases with default parameters and mapped to the NCBI genome of the strain of B. pertussis Tohama I (RefSeq accession number NC_002929.2; Parkhill et al., 2003 (link)). Finally, QC was performed on aligned reads. Count table was generated with default parameters. Transcripts were normalized using RLE (implemented in DESeq2 R package; Love et al., 2014 (link)), those with low abundance (mean normalized expression under 10) were filtered out.