Bordetella

IS1002 has not been exploited before as a template in PCR detection. We developed a specific IS1002 PCR (Table 1) to improve our ability to recognize the correct Bordetella species, and to combine it with IS481, and IS1001 specific PCRs. Addition of Phocine Herpes Virus (PhHV) as internal control acts to monitor the extraction as well as the efficiency of amplification [17 (link)].
We investigated standard laboratory strains of Bordetella (kindly provided by Dr. Frits Mooi and Kees Heuvelman, Laboratory for Vaccine Preventable Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands), which are shown in Table 2. Since detection of B. pertussis is of highest concern, we investigated the performance of the newly developed PCR on 100 clinical samples that were previously positive. To verify the specificity of the PCR we investigated 20 clinical respiratory tract samples that were suspect for other pathogens than Bordetella. Prior to PCR, laboratory strains were diluted and boiled to release DNA (equivalent to approximately 5 cells/μl). Clinical samples were extracted using EasyMAG (Biomerieux, Grenoble, France).
PCRs for detection of IS481, IS1001, and IS1002 were performed in a reaction mixture of 25 μl containing 0.5 μM of IS481 and IS1001 primers, 0.8 μM and 0.6 μM of IS1002 Forward and Reverse primer, respectively, and 0.2 μM of PhHV primers. Probes (Table 1) were added with concentrations of 0.14, 0.14, 0.16, and 0.08 μM for respectively IS481, IS1001, IS1002, and the internal control in PCR reaction mix (Sigma -Aldrich (E3004), Munich, Germany). Nine μl of template DNA was added. Amplification was carried out on an ABI 7500 Real-Time PCR system (Applied Biosystems (ABI), Nieuwerkerk a/d IJsel, The Netherlands). The temperature profile included initial denaturation of 4 min. at 94°C, followed by 50 cycles of 94°C for 15 sec., and 60°C for 1 min. Cycle treshold (Ct) values were determined automatically using the ABI SDS software.

We began computation of putative panorthologs for each set of genomes using NCBI BLASTP (release 2.2.16) to analyze all genes in all genomes for sequence similarity. We kept for later processing all BLAST hits within an E-value threshold of 1. These hits include each gene's self hit. We stored the E-value, bit score and alignment length for each hit. When running BLASTP, we used default parameters except for setting the E-value threshold and for setting the maximum number of hits to keep.
We next identified homologs as those gene pairs that had BLAST hits in both directions within a given scaled bit score threshold. We scaled the bit scores by the bit score of the self hit of the query gene. That is, scaledBitScore(A->B) = bitScore(A->B)/bitScore(A->A). This method has been used previously to identify conserved homologs among bacterial genomes and has been shown to be more stringent than criteria based solely on reciprocal best matches using E values [17] (link).
We then formed homolog families by including two genes in a family if they had been identified as homologs. Note that not all pairs of genes in a family need to be identified as homologs. For example, if A and B are homologs, and B and C are homologs, then A and C will be in the same family even if A and C have not been identified as homologs. Finally we identified the putative panorthologs as being the genes from homolog families with exactly one gene from each genome. For each set of genomes we kept the largest set of panorthologs found by computing the putative panorthologs while varying the scaled bit score threshold from .1 to .9 in .1 increments.
The following scaled bit score thresholds were used for genome sets A–E depicted in Fig. 1, followed by the number of putative panorthologs identified at that threshold: group A: threshold = 0.7, 4141 panorthologs; group B: 0.7, 3758, group C: 0.4, 2203, group D: 0.3, 902, group E: 0.2, 581. To produce groups d and e, the five Bordetella genomes were first analyzed by this method (0.5, 1592) as well as the five Xanthomonas genomes (0.5, 2450). The intersections of these Bordetella and Xanthomonas panortholog sets with groups b and c were used to produce groups d and e, respectively.

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.

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committees at The University of Georgia at Athens, GA (A2022 04-001-Y1-A0 Bordetella-Host Interactions, A2022 04-025-Y1-A0 Breeding Protocol, and A2022 04-022-Y1-A0 Neonatal Models of Bordetella infection, transmission, and immunity). Mice were consistently monitored for signs of distress over the course of the experiments to be removed from the experiment and euthanized using carbon dioxide inhalation to prevent unnecessary suffering.

The samples were applied to culture media using standard techniques. Culture media included Bordetella Agar (Difco^TM Bordet Gengou Agar Ref#248200, Becton Dickinson, Le Pont de Claix, France (BD) with 15% sheep blood), BBL^TM Columbia Agar with 5% sheep blood (Ref# 211124, BD) and BBL^TM Columbia colistin nalidixic acid (CNA) agar with 5% sheep blood (Ref 212104, BD) in order to provide appropriate media for a variety of aerobic bacterial species.
The inoculated agar plates were incubated at 36–38 °C and examined for bacterial growth after 24, 48, and 72 h. Bacterial growth was semiquantitatively assessed. Degree of contamination was defined as negligible (<10 colony forming units (CFU)), moderate (10–50 CFU), or severe (>50 CFU). If bacterial growth was identified, bacterial species were further classified using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF, Microflex LT and MALDI Biotyper Identification-Software 3.1, Bruker Daltonik GmbH, Bremen, Germany; Library: Bruker Taxonomy Tree (8599 Spectra)).