Reemergence of Bordetella parapertussis, United States, 2019-2023.

To determine changes in Bordetella pertussis and B. parapertussis detection rates, we analyzed 1.43 million respiratory multiplex PCR test results from US facilities from 2019 through mid-2023. From mid-2022 through mid-2023, Bordetella spp. detection increased 8.5-fold; 95% of detections were B. parapertussis. While B. parapertussis rates increased, B. pertussis rates decreased.

Pertussis outbreak in children hospitalized in Rabat (Morocco).

INTRODUCTION: Cyclical pertussis epidemics primarily affect young infants. This study aims to estimate pertussis prevalence during the ongoing 2023 outbreak at our institution, focusing on affected age groups and clinical presentations. MATERIEL AND METHODS: This retrospective study includes patients admitted to Rabat University Hospital Center from 1st January 2021 to 30th June 2023. Symptomatic patients underwent Multiplex Respiratory Panel PCR testing for respiratory infections. The analysis included cases where RT-PCR identified Bordetella spp., with data analysed using SPSS 15.0. RESULTS: Pertussis cases sharply increased from December 2022, constituting 85.4 % of positive samples. Most cases (78.2 %) occurred in infants under 3 months, presenting symptoms such as coughing (94.5 %) and dyspnoea (94.5 %). Pertussis was suspected in 60 % of RT-PCR confirmed cases. B. pertussis DNA was identified in 81.8 % of cases and B. parapertussis DNA in 18.2 % of cases. CONCLUSION: The study exposes a significant pertussis outbreak affecting predominantly young infants.

Diagnóstico diferencial de Bordetella bronchiseptica por RT-PCR en un niño con tos paroxística sin antecedentes patológicos previos / Differential diagnosis by RT-PCR of Bordetella bronchiseptica in a child without previous pathologic antecedents suffering whooping cough

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A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.

Ellipsometric-based novel DNA biosensor for label-free, real-time detection of Bordetella parapertussis.

Pertussis (or whooping cough) is a contagious disease mainly affecting infants and children and predominantly caused by Bordetella pertussis followed by Bordetella parapertussis. B. parapertussis causes a milder cough but usually symptomatically appears like B. pertussis infection. Thus the epidemiology of illness caused by B. parapertussis is not well understood. In this study, a sensitive and specific method for the rapid diagnosis of B. parapertussis is presented. The covalent immobilization of thiol-terminated DNA oligonucleotides (ss DNA SAM) on a silicon surface by disulfide bond formation is investigated with atomic force microscopy (AFM) and ellipsometry. The measurements indicated an average layer thickness of 5 ± 0.84 nm for 2 µg/µl concentration and 24 h incubation time. This thickness changed to 8.4 ± 0.92 nm for the same concentration (2 µg/µl) by altering the incubation time to 48 h. Ellipsometric data recorded before and after hybridization of B. parapertussis revealed an increase in mean grain area from 91 nm2 to 227 nm2 and a change in the refractive index from 1.489 to 1.648 for 2 µg/µl B. parapertussis, respectively. This change in the refractive index was used to evaluate the amount of adsorbed molecules and their density. The results showed that the density of adsorbed molecules increased from 0.2 to 0.97 g/cm3 after B. parapertussis attachment, respectively. To confirm the hybridization of B. parapertussis to ss DNA SAM, the ds DNA SAM was denatured and the ss DNA SAM surface was reproduced with an average height variation of 6.42 ± 0.75 nm. This showed the stability of the DNA film that can be tuned by varying the concentration and incubation time, thus providing a robust method for the label-free detection of B. parapertussis other than routinely used PCR detection.

Multicenter Clinical Evaluation of the Automated Aries Bordetella Assay.

Molecular methods offer superior sensitivity and specificity and reduce testing turnaround time from days to hours for detection of Bordetella pertussis and Bordetella parapertussis In this study, we evaluated the performance of the automated PCR-based Aries Bordetella Assay, which detects both B. pertussis and B. parapertussis directly from nasopharyngeal swab specimens. The limits of detection (LoDs) were 1,800 CFU·ml-1 for B. pertussis and 213 CFU·ml-1 for B. parapertussis The assay detected 16/18 unique B. pertussis/B. parapertussis strains. Of 71 potentially cross-reacting organisms, 5 generated false positives in 1/6 replicates; none of 6 additional Bordetella spp. were erroneously detected. Specimens were stable at 20 to 25°C for at least 10 h, at 4 to 8°C for 10 days, and at temperatures not exceeding -70°C for 6 months. Of 1,052 nasopharyngeal specimens from patients with suspected pertussis, 3.0% (n = 32) were B. pertussis positive and 0.2% (n = 2) were B. parapertussis positive. Combining these data with Aries Bordetella Assay data from 57 nasopharyngeal samples with previously confirmed B. pertussis or B. parapertussis data and with data from 50 contrived B. parapertussis samples, the proportions of positive and negative agreement of the respective Aries assays with the reference assays were 97.1% and 99.0% for B. pertussis and 100% and 99.7% for B. parapertussis The Aries Bordetella Assay provides accurate detection and distinction of B. pertussis and B. parapertussis infections within 2 h. (This study has been registered at ClinicalTrials.gov under registration no. NCT02862262.).

Pertussis and parapertussis in children and adults with a persistent cough: an observational study.

PURPOSE: We aimed to determine the prevalence, symptoms and course of pertussis and parapertussis among patients at any age with a cough of unknown aetiology that had lasted for ≥ 7 days and to assess the diagnostic value of the symptoms included in the World Health Organisations' (WHO) clinical case definition of pertussis. METHODS: Patients were enrolled between the 23 April 2012 and 31 December 2014 at 25 general practitioner (GP) centres and three paediatric hospitals. Pertussis was confirmed by culture and/or polymerase chain reaction (PCR) and/or quantitative serology. Parapertussis was confirmed by culture and/or PCR. RESULTS: Altogether, 549 patients were recruited. Of them, 22 (4.0%; 95% CI 2.5-6.0) had pertussis (predominately diagnosed by positive serology 17/22) and 7 (1.3%; 95% CI 0.5-2.6) had parapertussis. Patients with pertussis were more likely to have inspiratory whooping and posttussive emesis than those with a cough of another/unknown aetiology. However, the presence or absence of these two symptoms did not definitively confirm or exclude pertussis. The sensitivity and specificity of the WHO's clinical definition was 0.77 and 0.38, respectively. CONCLUSIONS: The prevalence of pertussis and parapertussis among patients with a persistent cough of unknown aetiology in Estonia is low. As clinical symptoms alone cannot be used to distinguish pertussis, we recommend that laboratory testing for pertussis is performed in all patients with a persistent cough regardless of age.

Nasopharyngeal pertussis toxin IgA antibodies in the diagnosis of pertussis in Australian community patients.

Nasopharyngeal aspirate (NPA) Bordetella pertussis-specific IgA antibody assay using whole-cell (WC) antigen has previously been shown to have promise in the diagnosis of patients with suspected pertussis. Recently, the use of WC assays in serum have been replaced by pertussis toxin (PT) because of specificity concerns. In this study, PT and WC B. pertussis-specific IgA antibody was assayed in 491 NPAs. Specimens also had molecular testing for the presence of B. pertussis and B. parapertussis as per the usual laboratory protocol. Positive concordance of the two serological assays was 51.2%, negative concordance was 67.5% and total concordance was 75.8%. 99 of 119 discordant specimens were resolved by utilising the B. pertussis polymerase chain reaction (PCR) result and clinical status, and yielded a sensitivity of 57.6% and a specificity 97.7% for WC, with 90.2% and 93.1%, respectively, for the PT assay (p < 0.00025 and 0.025-0.01). In contrast, the sensitivity of PCR was only 19.1% in this cohort. We conclude that specificity is not a significant issue for mucosal pertussis-specific IgA assays using WC, but the superior sensitivity of the PT assay favours the latter method. This assay, combined with PCR assays, should significantly improve the diagnosis of pertussis cases.

Bordetella parapertussis outbreak in Southeastern Minnesota and the United States, 2014.

Whooping cough is traditionally ascribed to Bordetella pertussis; however, Bordetella parapertussis can cause a similar clinical syndrome. This study describes an outbreak of B. parapertussis in Southeastern Minnesota and the United States (US) in 2014. This was a retrospective analysis of Mayo Clinic and Mayo Medical Laboratories patients who tested positive for B. parapertussis from 2012 to 2014. The medical records of Mayo Clinic patients who tested positive in 2014 were reviewed for demographic information, presenting symptoms, disease course, and vaccination history. In Southeast Minnesota, 81% of the 31 patients who tested positive for B. parapertussis in 2014 were found to be positive from October through December. Their mean age was 5.9 years. Five reported "exposure to pertussis." Two pairs of siblings were affected. Patients reported having had symptoms for an average of 2.6 weeks before nasopharyngeal specimen collection for B. parapertussis testing. Cough was the primary symptom reported. Forty percent reported posttussive vomiting, 40% coryza, 32% apnea/sleep disturbance, and 12% sore throat. All were current with pertussis vaccination. Based on the review of national data, an outbreak occurred nationally in the Northeast and Midwest US over the same time period. In 2014, there was an outbreak of B. parapertussis in Southeastern Minnesota and likely other parts of the US. The presenting illness was similar to that of B. pertussis. All patients were vaccinated against pertussis, suggesting that pertussis vaccination is ineffective against B. parapertussis.

Comparison of molecular detection methods for pertussis in children during a state-wide outbreak.

A state-wide pertussis outbreak occurred in Washington during the winter-spring months of 2012, concurrent with respiratory viral season. We compared performance characteristics of a laboratory-developed pertussis PCR (LD-PCR for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii) and rapid multiplex PCR (RM-PCR) for respiratory viruses (FilmArray™, BioFire, B. pertussis data unblinded following FDA approval post outbreak). We analyzed three cohorts of patients using physician testing orders as a proxy for clinical suspicion for pertussis or respiratory viruses: Cohort 1, tested by LD-PCR for pertussis pathogens only by nasopharyngeal swab; Cohort 2, by RM-PCR for respiratory viruses only by mid-nasal turbinate swab; and Cohort 3, by both methods. B. pertussis was detected in a total of 25 of the 490 patients in Cohort 3 in which LD-PCR detected 20/25 (80 %) cases and the RM-PCR detected 24/25 (96 %; p = 0.2). Pertussis pathogens were detected in 21/584 (3.6 %) of samples from Cohort 1 where clinicians had a relatively strong suspicion for pertussis. In contrast, B. pertussis was detected in only 4/3071 (0.1 %) specimens from Cohort 2 where suspicion for pertussis was lower (p < 0.001 for comparison with Cohort 1). In summary, the two laboratory methods were comparable for the detection of B. pertussis.

Incidence and Diagnosis of Pertussis in South African Children Hospitalized With Lower Respiratory Tract Infection.

BACKGROUND: The incidence of pertussis in children in low- and middle-income countries is poorly described. This study aimed to prospectively investigate the incidence of pertussis in South African children hospitalized with lower respiratory tract infection (LRTI). METHODS: Children hospitalized with LRTI in Cape Town, South Africa were enrolled over 1 year. Clinical data were collected. A nasopharyngeal (NP) swab and induced sputum (IS) were taken, and polymerase chain reaction specific for Bordetella pertussis (IS481+/hIS1001-) and Bordetella parapertussis (IS1001+) was performed. RESULTS: A total of 460 children with median age 8 [interquartile range (IQR), 4-18] months were studied. B. pertussis was detected in 17 (3.7%) while total Bordetella spp. were identified on 23 (5.0 %) of 460 NP. Adding IS testing increased the identification of B. pertussis to 32 of 460 cases (7.0%; 95% confidence interval, 4.8%-9.7%); P = 0.028 and total Bordetella to 41 of 460 (8.9%; 95% confidence interval, 4-10%); P = 0.020. Shorter duration of symptoms [median 2 (IQR, 2-3) days versus 5 (IQR, 3-7) days; P = 0.0008] was associated with detection of B. pertussis on IS versus NP. B. pertussis was detected in 15.8% (n=3/19) of HIV-infected children, 10.9% (n = 10/92) of HIV exposed uninfected and 5.4% (n = 19/349) of HIV-unexposed uninfected children. Risk of B. pertussis decreased with each additional dose of diphtheria, tetanus and acellular pertussis vaccine [0 doses = 17.9%; 1 dose = 7.0%; 2 doses = 6.9%; and >3 doses = 6.2%]. CONCLUSIONS: Pertussis is common in South African children hospitalized with LRTI particularly if HIV exposed or infected but decreases sequentially with vaccination doses. Polymerase chain reaction on IS specimen provides confirmation earlier than NP while increasing overall diagnostic yield.

Role of Atypical Pathogens and the Antibiotic Prescription Pattern in Acute Bronchitis: A Multicenter Study in Korea.

The role of atypical bacteria and the effect of antibiotic treatments in acute bronchitis are still not clear. This study was conducted at 22 hospitals (17 primary care clinics and 5 university hospitals) in Korea. Outpatients (aged ≥ 18 yr) who had an acute illness with a new cough and sputum (≤ 30 days) were enrolled in 2013. Multiplex real-time polymerase chain reaction (RT-PCR) was used to detect five atypical bacteria. A total of 435 patients were diagnosed as having acute bronchitis (vs. probable pneumonia, n = 75), and 1.8% (n = 8) were positive for atypical pathogens (Bordetella pertussis, n = 3; B. parapertussis, n = 0; Mycoplasma pneumoniae, n = 1; Chlamydophila pneumoniae, n = 3; Legionella pneumophila, n = 1). Among clinical symptoms and signs, only post-tussive vomiting was more frequent in patients with atypical pathogens than those without (P = 0.024). In all, 72.2% of the enrolled patients received antibiotic treatment at their first visits, and ß-lactams (29.4%) and quinolones (20.5%) were the most commonly prescribed agents. In conclusion, our study demonstrates that the incidence of atypical pathogens is low in patients with acute bronchitis, and the rate of antibiotic prescriptions is high.

Widespread Bordetella parapertussis Infections-Wisconsin, 2011-2012: Clinical and Epidemiologic Features and Antibiotic Use for Treatment and Prevention.

BACKGROUND: During October 2011-December 2012, concurrent with a statewide pertussis outbreak, 443 Bordetella parapertussis infections were reported among Wisconsin residents. We examined clinical features of patients with parapertussis and the effect of antibiotic use for treatment and prevention. METHODS: Patients with polymerase chain reaction results positive for B. parapertussis reported during October 2011-May 2012 were interviewed regarding presence and durations of pertussis-like symptoms and receipt of azithromycin treatment. Data regarding acute cough illnesses and receipt of azithromycin prophylaxis among parapertussis patient household members (HHMs) were also collected. Using multivariate repeated measures log-binomial regression analysis, we examined associations of treatment receipt by the HHM with the earliest illness onset and prophylaxis receipt among other HHMs with the presence of any secondary cough illnesses in the household. RESULTS: Among 218 patients with parapertussis, pertussis-like symptoms were frequently reported. Illness durations were significantly shorter among patients with treatment initiated 0-6 days after cough onset, compared with nonrecipients (median durations: 10 vs 19 days, P = .002). Among 361 HHMs from 120 households, compared with nonrecipients, prompt prophylaxis of HHMs was associated with no secondary cough illnesses (relative risk: 0.16; 95% confidence interval, .04-.69). CONCLUSIONS: Bordetella parapertussis infection causes pertussis-like illness that might be misclassified as pertussis if B. parapertussis testing is not performed. Prompt treatment might shorten illness duration, and prompt HHM prophylaxis might prevent secondary illnesses. Further study is needed to evaluate antibiotic effectiveness for preventing parapertussis and to determine risks and benefits of antibiotic use.

Bordetella parapertussis outbreak in Bisham, Pakistan in 2009-2010: fallout of the 9/11 syndrome.

Pertussis or whooping cough is a highly contagious community disease mainly caused by Bordetella pertussis and B. parapertussis. We report a minor outbreak of whooping cough (2009-2010) in symptomatic subjects from Bisham, near Swat, Khyber Pukhtoonkhawa province, Pakistan. Interestingly, our results show that all the culture-positive isolates (n = 21) collected from children (average age 3·46 years), were identified as B. parapertussis after routine identification tests and PCR IS481, IS1001 and IS1002. Furthermore, in the affected patients, none had received immunization with diphtheria-pertussis-tetanus (DTPw) vaccine. Therefore, the possibility of the re-emergence of the disease due to limitation of basic health services as a result of the political unrest due to the 9/11 situation is also examined. Moreover, we discuss the importance of vaccinating both adults and children with DTPwPaw vaccine containing both organisms for better protection.

Incidence and factors predicting whooping cough due to parapertussis diagnosis among patients referred to general practitioners, Poland, 2009-2011.

Parapertussis leads to similar symptoms as pertussis, both being caused by bacteria from the genus Bordetella. Poland does not routinely diagnose nor conduct surveillance for parapertussis. We estimated parapertussis incidence and determined predictors of parapertussis diagnosis in the Polish population. Between July 2009 and April 2011, we conducted a prospective cohort study among patients attending 78 general practices. We included patients aged ≥ 3 years, with cough lasting >2 weeks, interviewed patients and collected a nasopharyngeal swab. We confirmed cases by real-time PCR. We estimated parapertussis incidence rates by dividing the number of cases by the summed person-time of observation in respective practices. We assessed predictors of PCR-confirmed parapertussis by comparing cases with patients testing negative. Using logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (95%CI). We identified 78 cases among 1,231 patients meeting inclusion criteria. The incidence rate was 39/100,000 person-years (95%CI 31-49). The highest rates (140/100,000; 95%CI 74-239), were among children 3-5 years of age and the lowest (24/100,000; 95%CI 13-40) among persons aged 20-39 years of age. Boys aged 3-5 years (7.1; 2.1-25.3) and women aged >40 years (4.1; 1.4-11.7) or living in crowded households (4.3; 1.4-12.9) or contacting persons with prolonged cough (2.3; 1.1-4.5) were more likely to be diagnosed. Our results suggest that laboratory diagnosis could be prioritized for children in the preschool age and women aged over 40 who were referred to their GP with prolonged cough. In the absence of vaccine, post-exposure prophylaxis for close contacts of parapertussis cases could an adequate preventative measure.

Bordetella parapertussis survives inside human macrophages in lipid raft-enriched phagosomes.

Bordetella parapertussis is a human pathogen that causes whooping cough. The increasing incidence of B. parapertussis has been attributed to the lack of cross protection induced by pertussis vaccines. It was previously shown that B. parapertussis is able to avoid bacterial killing by polymorphonuclear leukocytes (PMN) if specific opsonic antibodies are not present at the site of interaction. Here, we evaluated the outcome of B. parapertussis innate interaction with human macrophages, a less aggressive type of cell and a known reservoir of many persistent pathogens. The results showed that in the absence of opsonins, O antigen allows B. parapertussis to inhibit phagolysosomal fusion and to remain alive inside macrophages. The O antigen targets B. parapertussis to lipid rafts that are retained in the membrane of phagosomes that do not undergo lysosomal maturation. Forty-eight hours after infection, wild-type B. parapertussis bacteria but not the O antigen-deficient mutants were found colocalizing with lipid rafts and alive in nonacidic compartments. Taken together, our data suggest that in the absence of opsonic antibodies, B. parapertussis survives inside macrophages by preventing phagolysosomal maturation in a lipid raft- and O antigen-dependent manner. Two days after infection, about 15% of macrophages were found loaded with live bacteria inside flotillin-enriched phagosomes that had access to nutrients provided by the host cell recycling pathway, suggesting the development of an intracellular infection. IgG opsonization drastically changed this interaction, inducing efficient bacterial killing. These results highlight the need for B. parapertussis opsonic antibodies to induce bacterial clearance and prevent the eventual establishment of cellular reservoirs of this pathogen.

Evaluation of 3 analyte-specific reagents for detection of Bordetella pertussis and Bordetella parapertussis in clinical specimens.

The performance of 3 analyte-specific reagents (ASRs), Elitech Biosciences, EraGen Biosciences, and Focus Diagnostic, was evaluated for detection of Bordetella pertussis (BP) and Bordetella parapertussis (BPP) in nasopharyngeal swab specimens. A total of 104 frozen, leftover clinical specimens obtained from pediatric patients during 2011-2012 were included in this study. Performance was compared to the Bordetella real-time polymerase chain reaction (PCR) laboratory-developed test (LDT). The positive percent agreement for detection of BP by Elitech was 96% (95% confidence interval [CI]: 85.14-99.30); EraGen and Focus was 98% (95% CI: 87.99-99.89) in comparison to LDT PCR assay. The negative percent agreement of Elitech, EraGen, and Focus in comparison to LDT was 96% (95% CI: 85.14-99.30), 92% (95% CI: 79.89-97.41), and 96% (95% CI: 85.14-99.30), respectively. Limit of detection (LOD) for BP was 0.1 CFU/reaction by both Focus and EraGen and 1.0 CFU/reaction by Elitech. However, LOD for BPP was lower by EraGen (0.1 CFU/reaction) compared to Focus (1.0 CFU/reaction) and Elitech (1.0 CFU/reaction). These results demonstrate that all 3 ASRs tested are comparable and reliable for routine clinical diagnosis of pertussis and parapertussis.

Prevalence of asymptomatic Bordetella pertussis and Bordetella parapertussis infections among school children in China as determined by pooled real-time PCR: a cross-sectional study.

BACKGROUND: Studies have documented that older children and adolescents act as a reservoir of Bordetella pertussis infection for young infants who have not yet completed their primary immunization schedule. Asymptomatic pertussis infection has been reported during outbreaks. This cross-sectional study aimed to investigate whether B. pertussis and Bordetella parapertussis can colonize the nasopharynx of healthy school children, using culture and pooled real-time PCR with targets for insertion sequences IS481 and IS1001. METHODS: Nasopharyngeal (NP) swabs were taken from 629 asymptomatic school children aged 7 to 15 y in 4 counties of China during the period July-September 2011. The number of subjects included in each county ranged from 153 to 165. The 4 counties selected are located in the north, south, east, and southwest regions of China. NP swabs were inoculated onto Regan-Lowe agar for isolation of suspected Bordetella organisms. Pooled real-time PCRs were used to detect B. pertussis and B. parapertussis based on the IS481 and IS1001 targets separately. RESULTS: Of the 629 subjects, 2 (0.3%) and 30 (4.8%) were confirmed to be culture-positive and PCR-positive, respectively, for B. pertussis, and 1 (0.2%) and 13 (2.1%) were confirmed to be culture-positive and PCR-positive, respectively, for B. parapertussis. All culture-positive samples were also PCR-positive. Furthermore, positive B. pertussis and B. parapertussis samples were found in all counties. CONCLUSIONS: Our results indicate that asymptomatic B. pertussis infections are common in school children in China, and asymptomatic B. parapertussis infections are more prevalent than previously documented.

Development of real-time PCR assay for differential detection of Bordetella bronchiseptica and Bordetella parapertussis.

Bordetella parapertussis is a causative agent of whooping cough in humans, and B. bronchiseptica is causing wide variety of respiratory infections in mammals, including humans. Specific diagnostic tests are not currently available. Our first objective was to develop a real-time PCR test for the specific detection of B. bronchiseptica based on the previously described end-point PCR, targeting an intergenomic sequence of the fla gene locus, but it has not been reached. However, there is cross-reactivity between B. parapertussis and B. bronchiseptica. Therefore, the targeted region of several clinical isolates of both species was sequenced, and alignment of the sequences allowed the development of a 2-step real-time PCR assay. The first PCR assay detected the DNA of all clinical isolates of both B. bronchiseptica and B. parapertussis tested. The second PCR assay detected only the DNA of B. parapertussis clinical isolates, thereby allowing discrimination between B. parapertussis and B. bronchiseptica.