Polymorphism of repeated regions of pertactin in Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica

Pertactin is an outer membrane protein expressed by Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica that induces protective immunity to Bordetella infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed that B. parapertussis pertactin is invariant, whereas B. pertussis pertactin varies mostly in region I and B. bronchiseptica pertactin varies in both repeated regions I and II, but mostly in region II. These differences may result from specific characteristics of these Bordetella species.     

Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.     

Ultrastructural analysis of the interactions between Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica and human tracheal epithelial cells

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are respiratory pathogens that colonize the respiratory tract of their host after adhesion to the respiratory epithelium. Presently, the intracellular fate of these bacteria in human tracheal epithelial cells was compared by use of transmission electron microscopy. The three species, even when cytotoxic, were taken-up by epithelial cells. Although, some intracellular bacteria appeared morphologically intact and survived a few days inside epithelial cells, most of them appeared quickly degraded, phenomenon which was associated with an intense cell metabolic activity. Even cytotoxic Bordetella species is ultimately killed by human epithelial cells.     

Molecular Epidemiology of Astrovirus Infection in Barcelona, Spain

A 3-year study involving 2,347 gastroenteritis samples was conducted to determine the prevalence, time distribution, and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus-positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8, and HAstV-2. HAstV-5, HAstV-6, and HAstV-7 were not detected during these 3 years. From our serotype data for each age group, we observed that HAstV-1, HAstV-2, and HAstV-3 affected mostly children younger than 3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.     

Moredun Bordetella Medium, an improved selective medium for isolation of Bordetella parapertussis.

Bordetella parapertussis, previously thought to be an obligate human respiratory tract pathogen, has been isolated from sheep. Attempts to assess the prevalence of B. parapertussis in conventionally reared sheep by nasal swabbing proved futile with existing selective media because of extensive overgrowth with Mucor spp. and other nasal commensals. Moredun Bordetella Medium (MBM), which contains cycloheximide and spectinomycin at final concentrations of 0.5 mg/ml and 100 mu g/ml, respectively, was developed as an improved selective medium to isolate B. parapertussis from the nasal cavities of conventionally reared sheep. The selective ability of MBM was evaluated with 200 nasal swabs from conventionally reared sheep, and B. parapertussis was recovered from 31.5% of the samples. MBM facilitated the simple and effective isolation of B. parapertussis from ovine nasal swabs and, in successfully excluding overgrowth with other contaminants, proved superior to other test formulations evaluated and to existing conventional media.     

Evaluation of PCR for Diagnosis of Bordetella pertussis and Bordetella parapertussis Infections

PCR, using primers PIp1 and PIp2, was evaluated for the detection of DNA from Bordetella pertussis in bacterial strains and in nasopharyngeal samples from patients with a cough lasting at least 7 days. The assay could detect DNA from 6 CFU of B. pertussis/10 μl of sample. Results of the PCR assay were compared with those of cultures, a determination of serum antibodies against pertussis toxin and filamentous hemagglutinin, and a clinical evaluation of 2,442 coughing episodes. The overall sensitivity of PCR was 65% (623 of 956), which was higher than the sensitivity of cultures (58%) (P < 0.001). Factors influencing the sensitivity of PCR were the interval between the onset of symptoms and sampling and the vaccination status of the patient. The specificity of PCR was 98% (1,451 of 1,486). The positive and negative predictive values were 95 and 81%, respectively. Parapertussis PCR, using primers BPPA and BPPZ, was positive in 11 of 18 culture-positive cases and was confirmed by serology in another 4 cases. In conclusion, PCR is a valuable complement to cultures and can probably replace cultures for diagnosis of B. pertussis and Bordetella parapertussis infections.     

Real-time LightCycler PCR for detection and discrimination of Bordetella pertussis and Bordetella parapertussis

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.     

Evaluation of real-time PCR for detection of and discrimination between Bordetella pertussis,Bordetella parapertussis,and Bordetella holmesii for clinical diagnosis

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS481 and IS1001 for B. pertussis and B. parapertussis, respectively, with detection by molecular beacons. The real-time PCR for IS481 detects both B. pertussis and Bordetella holmesii, and the real-time PCR for IS1001 detects both B. parapertussis and B. holmesii. By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis, 10 CFU/ml for B. parapertussis, and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.     

Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids.

Pertussis toxin is produced only by strains of Bordetella pertussis. Cloned genes encoding pertussis toxin from B. pertussis were transferred into Bordetella bronchiseptica and Bordetella parapertussis by conjugation. These transconjugants expressed pertussis toxin at levels comparable to those expressed by B. pertussis. The toxin made by these strains was biologically active in the Chinese hamster cell clumping assay, contained all five subunits, and was mostly periplasmic. Toxin expression appeared to be modulated in the same way as are the vir-regulated genes of B. pertussis. Introduction of these plasmids into B. pertussis failed to produce hypertoxigenic strains. Instead, these transconjugants underwent plasmid loss, gene deletions, or conversion to the avirulent phase.     

Evolution of French Bordetella pertussis and Bordetella parapertussis isolates: increase of Bordetellae not expressing pertactin

Bordetella pertussis and Bordetella parapertussis are closely related bacterial agents of whooping cough. Whole-cell pertussis (wP) vaccine was introduced in France in 1959. Acellular pertussis (aP) vaccine was introduced in 1998 as an adolescent booster and was rapidly generalized to the whole population, changing herd immunity by specifically targeting the virulence of the bacteria. We performed a temporal analysis of all French B. pertussis and B. parapertussis isolates collected since 2000 under aP vaccine pressure, using pulsed-field gel electrophoresis (PFGE), genotyping and detection of expression of virulence factors. Particular isolates were selected according to their different phenotype and PFGE type and their characteristics were analysed using the murine model of respiratory infection and in vitro cell cytotoxic assay. Since the introduction of the aP vaccines there has been a steady increase in the number of B. pertussis and B. parapertussis isolates collected that are lacking expression of pertactin. These isolates seem to be as virulent as those expressing all virulence factors according to animal and cellular models of infection. Whereas wP vaccine-induced immunity led to a monomorphic population of B. pertussis, aP vaccine-induced immunity enabled the number of circulating B. pertussis and B. parapertussis isolates not expressing virulence factors to increase, sustaining our previous hypothesis.     

Nucleotide sequence homology to pertussis toxin gene in Bordetella bronchiseptica and Bordetella parapertussis.

Multiple strains of Bordetella parapertussis and B. bronchiseptica were examined for the presence of nucleotide sequences which hybridized with a cloned 4.5-kilobase (kb) fragment of B. pertussis DNA containing the genes responsible for pertussis toxin expression. All six B. parapertussis strains tested had nucleic acid sequences that hybridized with the cloned 4.5-kb fragment in Southern blot analyses. When the B. parapertussis DNA was digested with restriction endonuclease PstI, the pattern of hybridization was identical to that obtained with B. pertussis. Only five of the seven B. bronchiseptica strains tested had sequences that hybridized with the 4.5-kb fragment. Three of these B. bronchiseptica strains had a hybridization pattern identical to B. pertussis upon PstI digestion and Southern blot analyses. Two B. bronchiseptica strains were shown to lack a PstI cleavage site downstream from the region analogous to that coding for the pertussis toxin structural genes. Monoclonal antibody analyses were unable to detect pertussis toxin subunits S1 and S2 in Western blots with cellular material or culture supernatant from several B. bronchiseptica and B. parapertussis strains that possessed the DNA homologies. In addition, preliminary Northern hybridizations with RNA isolated from B. bronchiseptica and B. parapertussis strains suggested that the homologous regions were not transcribed. The data show that the gene coding for the toxic component of B. pertussis is common in other Bordetella species, though the gene probably is not expressed.     

Development of a real-time PCR for the identification of Bordetella pertussis and Bordetella parapertussis

This study describes a real-time PCR assay for the detection and identification of Bordetella pertussis and Bordetella parapertussis. The assay is based on amplification of a fragment from the repeat sequence regions IS481 and IS1001 found in B. pertussis and B. parapertussis, respectively, with subsequent species identification by melting curve analysis using SYBR Green chemistry. Discrimination between the two species was straightforward, as the corresponding melting points showed a significant difference of 7 degrees C. The assay was evaluated first with reference strains and retrospective human clinical samples, and then prospectively with 132 human clinical specimens received between March 2003 and December 2005. The assay allowed the rapid detection of 22 positive clinical samples, of which 15, including one fatal case, were not identified by standard culture techniques. The new assay was sensitive and specific, and can be implemented easily using any real-time PCR apparatus.     

Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children,Including Newly Identified Serotypes 6C and 6D

Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.Infection with Streptococcus pneumoniae is a leading cause of death in children worldwide (15, 19, 25). S. pneumoniae comprises 48 capsular serogroups containing more than 90 serotypes. Serogroup 6 classically consisted of serotypes 6A and 6B. The 7-valent conjugate vaccine (Prevnar; PCV7) includes the 6B antigen and confers some cross-protection against serotype 6A but not 6C (20, 22). Serotype 6C is important in carriage and invasive disease, and its prevalence has increased following widespread use of PCV7 (4, 5, 9, 10, 14, 16, 24).The existence of serotype 6D has been postulated (8) and was created experimentally (2), but naturally occurring isolates have not been identified (2, 8, 17) until recently, when we identified 14 naturally occurring serotype 6D isolates from nasopharyngeal swabs from Fijian children (11). Recently, two serotype 6D isolates have been found in Korean children (1).Initially, serotype 6C was postulated to have arisen from a single, possibly recent, evolutionary event in which serotype 6A wciN was replaced by serotype C wciN (21). Subsequent analyses, predominantly by multilocus sequence typing (MLST), have shown that 6C is genetically diverse, believed to be a consequence of multiple separate conversion events or a single event occurring sufficiently early in pneumococcal evolution (3, 4, 8, 9, 17).Serotype 6C is usually associated with clonal complexes (CCs) containing predominantly serotype 6A, and less commonly, 6B or non-serogroup-6 serotypes (3, 4, 9, 17). To date, MLST has been conducted on two serotype 6D isolates from Korea (ST282) (1), two from China (ST982 and ST4190), and one from Australia (ST4241) (http://spneumoniae.mlst.net). However, the molecular epidemiology of serotype 6D isolates is otherwise uncharacterized.In this study, we conducted MLST of serogroup 6 isolates to determine the genetic diversity and likely evolutionary origin of serotype 6C and 6D isolates.S. pneumoniae was isolated and identified as described elsewhere (18) from children aged 6 to 18 months participating in the Fiji Pneumococcal Project (FiPP) who had received 0, 1, 2, or 3 doses of PCV7 and 0 or 1 dose of 23-valent pneumococcal polysaccharide vaccine (PPV23) (23). Isolates were serotyped by a multiplex PCR-based reverse line blot (mPCR/RLB) assay and/or quellung reaction (before factor serum 6d was available), plus serogroup 6 serotype-specific PCR as previously described (11, 12).MLST was applied to all unique serotype 6C and 6D strains from the FiPP study (n = 52 and 24, respectively), of which 24 and 14 isolates, respectively, were included in our previous report (without ST results) (11). For comparison, we performed MLST on a subset of randomly selected serotype 6A (n = 16) and 6B (n = 17) isolates from the FiPP study.Fresh 18- to 24-h subcultures of S. pneumoniae isolates were suspended in nuclease-free water (Ambion), and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) per the manufacturer''s instructions. MLST was performed using primer pairs described by the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm) (aroE, recP, spi, xpt and ddl) or Enright and Spratt (6) (gdh and gki), except as described below.PCRs were conducted with 25-μl volumes containing approximately 5 ng of genomic DNA, 1 U of AmpliTaq DNA polymerase (Applied Biosystems), 1 × PCR buffer II (50 mM KCl, 10 mM Tris-HCl [pH 8.3]), 3.0 mM MgCl₂, 250 μM each deoxynucleoside triphosphate (dNTP), 0.5 μM forward primer, and 0.5 μM reverse primer (Sigma-Aldrich). PCR cycling conditions were a 5-min hold at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. Some isolates which produced no or small amounts of PCR product from spi (seven 6C and three 6D isolates) and/or recP (three 6C and one 6A isolate) were successfully amplified with primers described by Enright and Spratt (6) at an annealing temperature of 52°C in 4.5 mM MgCl₂.Amplicons were sequenced in both directions using capillary separation on the ABI 3730xl DNA analyzer with ABI BigDye Terminator labeling (version 3.1) (Australian Genome Research Facility) using the same primers as for amplification. Contiguous sequences were formed and edited using Sequencher 4.9 (Gene Codes Corporation).Allelic profiles and sequence types (STs) were obtained and compared to those of other isolates in the MLST database (http://spneumoniae.mlst.net). Relationships between STs were explored using eBURST version 3 software (Imperial College, London), which is available at the MLST website. For this study, a group was defined as two or more isolates which shared alleles at six of seven loci. Using this stringent definition, a group also defined a clonal complex (CC). Bootstrap analyses of the CC founder are also presented where appropriate.Generally, Fijian serogroup 6 isolates were highly clonal, with the predominant clone in each serotype representing >44% of the isolates. This is not surprising, given that all strains were isolated from children of similar ages over a relatively short period of time in a small, geographically isolated area.Serotype 6A isolates included STs 490 (7/16), 4778, 4779, 499, and 460 (Table ​(Table1).1). ST490 is predicted to be the CC founder (bootstrap value, 96%) and contains predominantly serotype 6A isolates in the MLST database. ST4778 and ST4779 are newly identified in this study and were not assigned to a CC. The only other ST499 isolate in the database (serotype 6A isolate from Finland) was also not assigned to a CC. ST460 is predicted to be a CC founder (bootstrap value, 88%). Most serotype 6A isolates (10/16) belonged to STs which contain only serotype 6A in the database (i.e., ST490, ST499, and ST460).

TABLE 1.

Distribution of STs and eBURST analysis for 109 serotype 6 isolates from Fijian children

Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis.

A polymerase chain reaction (PCR) assay which allows the simultaneous detection and discrimination of the two causative agents of pertussis, Bordetella pertussis and Bordetella parapertussis, was developed. Primer pairs were based on insertion sequence elements IS481 and IS1001. IS481 is specific for B. pertussis and is present in about 80 copies per cell, while IS1001 is specific for B. parapertussis and is found in 20 copies per cell. An internal control was included in the PCR assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. Discrimination of amplified DNA derived from the internal control, B. pertussis, or B. parapertussis was accomplished by differential spacing of the primers. The sensitivity of the combined PCR method was found to be very high and allowed the detection of one cell of either pathogen. The usefulness of the method was investigated by using a limited number of clinical samples derived from patients with serologically proven pertussis.     

Reciprocal protective immunity against Bordetella pertussis and Bordetella parapertussis in a murine model of respiratory infection

The protective immunity induced by infection with Bordetella pertussis and with Bordetella parapertussis was examined in a murine model of respiratory infection. Convalescent mice that had been infected by aerosol with B. pertussis or with B. parapertussis exhibited a protective immune response against B. pertussis and also against B. parapertussis. Anti-filamentous hemagglutinin (anti-FHA) serum immunoglobulin G (IgG) and anti-FHA lung IgA antibodies were detected in both mice infected with B. pertussis and those infected with B. parapertussis. Antibodies against pertussis toxin (anti-PT) and against killed B. pertussis cells were detected in mice infected with B. pertussis. Pertactin-specific antibodies and antibodies against killed B. parapertussis cells were detected in mice infected with B. parapertussis. Spleen cells from mice infected with B. pertussis secreted interferon-gamma (IFN-gamma) in response to stimulation by FHA or PT. Spleen cells from mice infected with B. parapertussis also secreted IFN-gamma in response to FHA. Interleukin-4 was not produced in response to any of the antigens tested. The profiles of cytokine secretion in vitro revealed induction of a Th1-biased immune response during convalescence from infection by B. pertussis and by B. parapertussis. It is possible that Th1 and Th2 responses against FHA might be related to the reciprocal protection achieved in our murine model.