Mixed outbreak ofBordetella pertussis andBordetella parapertussis infection in Finland

The epidemiology of whooping cough in a vaccinated population was studied during an outbreak of paroxysmal cough in an elementary school with 258 pupils in Turku, Finland. Nasopharyngeal specimens for isolation ofBordetella pertussis and/or sera for ELISA detection of antipertussis immunoglobulin M, A and G antibodies were taken from 94 % of children who were prospectively followed for two months.Bordetella pertussis was isolated in six patients, and 17 culture-positive cases withBordetella parapertussis were identified. Patients withBordetella pertussis orBordetella parapertussis were found simultaneously in the same classrooms. Comparison of immunoglobulin M responses toBordetella pertussis andBordetella parapertussis was used for differential diagnosis of these two infections. Twenty-six cases with pertussis and 27 cases with parapertussis were diagnosed. The results of this prospective study suggest thatBordetella parapertussis is a more common etiologic agent of mild respiratory tract infection among vaccinated school-aged children than is generally recognised. The possibility thatBordetella pertussis was converted toBordetella parapertussis during this outbreak is discussed.     

Protective role of immunoglobulin G antibodies to filamentous hemagglutinin and pertactin ofBordetella pertussis inBordetella parapertussis infection

An outbreak of parapertussis was studied prospectively in 38 first and second grade pupils of an elementary school. Eleven (29%) children were confirmed to be culture positive forBordetella parapertussis. Serum samples were collected from 31 children for assay of antibodies to filamentous hemagglutinin (FHA), pertactin (PRN), and pertussis toxin ofBordetella pertussis. At the first sampling, ten children were found to have a cough and 21 were asymptomatic. Of the latter, 12 remained asymptomatic and eight developed cough within 11 to 53 days (mean ± standard deviation, 31±12 days) after sampling. One child was identified as culture positive forBordetella pertussis and, thus, not included in the analysis ofBordetella parapertussis infection. The mean levels of IgC antibodies to FHA and PRN were significantly higher in the 12 asymptomatic children than in the eight children who later developed cough or in 20 healthy control children of the same age (for FHA, p=0.009 and < 0.001, respectively; for PRN, p=0.002 and 0.002, respectively). These preliminary data suggest thatBordetella parapertussis infection is more prevalent than documented, and that children with high levels of IgG antibodies to FHA and PRN can remain asymptomatic.     

Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.     

Serum Immunoglobulin G Antibody Responses to Bordetella pertussis Lipooligosaccharide and B. parapertussis Lipopolysaccharide in Children with Pertussis and Parapertussis

Serum immunoglobulin G (IgG) antibodies against the lipooligosaccharide (LOS) of Bordetella pertussis and the lipopolysaccharide (LPS) of Bordetella parapertussis were measured by enzyme-linked immunosorbent assay in paired sera from 40 children with pertussis and 14 with parapertussis. Wide differences in the individual responses were noted. Both anti-LOS and -LPS IgG levels increased significantly in the children with pertussis, as did anti-LPS but not anti-LOS in those with parapertussis.     

Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis

Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685–91. A triplex real‐time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single‐target real‐time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony‐forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra‐ and inter‐assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture‐positive samples were also PCR positive. Our single‐tube triplex real‐time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis.     

Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen

Bordetella pertussis and Bordetella parapertussis are both causative agents of whooping cough outbreaks. Although not expressing the pertussis toxin, B. parapertussis induces, in a murine model, an acute hemorrhagic edematous alveolitis, similar to that observed with B. pertussis. These data suggest that the pertussis toxin may only play an accessory role in the acute pulmonary syndrome observed during Bordetella infection. Both with B. pertussis and B. parapertussis, the ability to induce lethal pulmonary lesions is associated with enhanced in vitro adenylate cyclase expression and activity. We also demonstrate that passive immunization with specific anti-B. pertussis adenylate cyclase antibodies or active immunization with purified B. pertussis secreted adenylate cyclase protect mice against a lethal respiratory challenge with B. pertussis or B. parapertussis. Our results suggest that adenylate cyclase might be the primary cytotoxin responsible for mouse pulmonary lesions during respiratory tract infection with B. pertussis or with the related species B. parapertussis and is a protective antigen of B. pertussis.     

Rapid molecular genetic assay for direct identification of Bordetella from patients specimens

Biological diagnosis of whooping cough is increasingly necessary to confirm respiratory tract infection. Indeed, clinical symptoms are variable especially in adolescents and adults who contaminate newborns too young to be vaccinated. The PCR assay was proven highly sensitive for the diagnosis of pertussis. In this study, we reported the use of a new test (GenoQuick^® Bordetella [GQB], Hain Life Science, Germany) which permits the fast molecular genetic identification of Bordetella pertussis and parapertussis directly from patients specimens, i.e. swabs from nose or throat. The test was performed over a three months period on 40 specimens from patients (1 month to 65 years old), most of them were young children admitted in paediatric emergency with paroxysmal cough or prolonged cough.     

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.     

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.     

Bordetella parapertussis Infection in Children: Epidemiology, Clinical Symptoms, and Molecular Characteristics of Isolates

The clinical trial conducted in Italy to evaluate the efficacy of acellular pertussis vaccines provided an opportunity to estimate the frequency of clinical infections with Bordetella parapertussis and to compare the clinical characteristics of children suffering from Bordetella pertussis illness with those of children with B. parapertussis illness. This study dealt with 76 B. parapertussis infections diagnosed from a population of 15,601 children participating in the follow-up of suspected cases of pertussis. An overall incidence of 2.1 cases of laboratory-confirmed parapertussis per 1,000 person-years was observed. Children affected by B. parapertussis infections showed a less severe clinical picture both in the duration of symptoms and in the percentage of patients affected, even when compared with vaccinated children with pertussis. To characterize the isolated strains, we performed assays for susceptibility to erythromycin and sulfamethoxazole-trimethoprim, and we examined the genomic DNAs by pulsed-field gel electrophoresis. The results showed a high degree of genetic stability among B. parapertussis strains regardless of time of collection and geographical distribution.     

The O antigen enables Bordetella parapertussis to avoid Bordetella pertussis-induced immunity

Bordetella pertussis and Bordetella parapertussis are closely related endemic human pathogens which cause whooping cough, a disease that is reemerging in human populations. Despite how closely related these pathogens are, their coexistence and the limited efficacy of B. pertussis vaccines against B. parapertussis suggest a lack of cross-protective immunity between the two. We sought to address the ability of infection-induced immunity against one of these pathogens to protect against subsequent infection by the other using a mouse model of infection. Immunity induced by B. parapertussis infection protected against subsequent infections by either species. However, immunity induced by B. pertussis infection prevented subsequent B. pertussis infections but did not protect against B. parapertussis infections. The O antigen of B. parapertussis inhibited binding of antibodies to the bacterial surface and was required for B. parapertussis to colonize mice convalescent from B. pertussis infection. Thus, the O antigen of B. parapertussis confers asymmetrical cross-immunity between the causative agents of whooping cough. We propose that these findings warrant investigation of the relative role of B. parapertussis in the resurgence of whooping cough.     

A search for subclinical infection during a small outbreak of whooping cough: implications for clinical diagnosis

The transmission of whooping cough in a general practice community was followed after the identification of the first case for nearly three years. Intensive case-finding was undertaken to detect contacts of known cases of whooping cough and to take pernasal swabs from those with any cough; 102 swabs were taken. In three months 39 cases of whooping cough were clinically diagnosed, 17 (44%) of which were confirmed bacteriologically. All had a prolonged paroxysmal cough, one-third reported a catarrhal phase, 18 (46%) vomited with paroxysms and nine (23%) whooped. No isolations of Bordetella pertussis were obtained from the 84 contacts with non-paroxysmal coughs. There was no evidence that subclinical bordetella infection (showing none of the signs of whooping cough) is a common occurrence.

It is probable that many recognizable cases of whooping cough are missed because it can be a milder illness than is often realized and commonly exhibits neither whooping, vomiting nor a catarrhal phase. Paroxysms may be infrequent. The diagnosis of whooping cough should be suspected from a prolonged paroxysmal cough alone.

     

Bordetella parapertussis Survives the Innate Interaction with Human Neutrophils by Impairing Bactericidal Trafficking inside the Cell through a Lipid Raft-Dependent Mechanism Mediated by the Lipopolysaccharide O Antigen

Whooping cough is a reemerging disease caused by two closely related pathogens, Bordetella pertussis and Bordetella parapertussis. The incidence of B. parapertussis in whooping cough cases has been increasing since the introduction of acellular pertussis vaccines containing purified antigens that are common to both strains. Recently published results demonstrated that these vaccines do not protect against B. parapertussis due to the presence of the O antigen on the bacterial surface that impairs antibody access to shared antigens. We have investigated the effect of the lack of opsonization of B. parapertussis on the outcome of its interaction with human neutrophils (polymorphonuclear leukocytes [PMNs]). In the absence of opsonic antibodies, PMN interaction with B. parapertussis resulted in nonbactericidal trafficking upon phagocytosis. A high percentage of nonopsonized B. parapertussis was found in nonacidic lysosome marker (lysosome-associated membrane protein [LAMP])-negative phagosomes with access to the host cell-recycling pathway of external nutrients, allowing bacterial survival as determined by intracellular CFU counts. The lipopolysaccharide (LPS) O antigen was found to be involved in directing B. parapertussis to PMN lipid rafts, eventually determining the nonbactericidal fate inside the PMN. IgG opsonization of B. parapertussis drastically changed this interaction by not only inducing efficient PMN phagocytosis but also promoting PMN bacterial killing. These data provide new insights into the immune mechanisms of hosts against B. parapertussis and document the crucial importance of opsonic antibodies in immunity to this pathogen.     

Importance of culture in laboratory diagnosis of Bordetella pertussis infections.

Nasopharyngeal secretions from 223 patients were examined for the presence of Bordetella pertussis and B. parapertussis by culturing on Regan-Lowe agar (J. Regan and F. Lowe, J. Clin. Microbiol. 6:303-309, 1977) and by direct fluorescent-antibody testing. B. pertussis was found in 38 patients; B. parapertussis was recovered from 2. Culturing was more sensitive (38 of 38 patients) than direct fluorescent-antibody testing (26 of 38 patients) for the detection of B. pertussis. Overgrowth by other organisms (7 of 223 patients) was uncommon. The patients with B. pertussis infections were generally less than 1 year old, had received no or one immunization, and had coughing spells but infrequently had whooping cough. Accurate diagnosis of B. pertussis infections should include culturing.     

Western blot analysis of antibody responses of young infants to pertussis infection

Western blot and agglutination techniques were used to analyze the antibody responses toBordetella pertussis in 27 infants less than six month of age with presumed pertussis infection. The antibody response to theBordetella pertussis adhesins filamentous hemagglutinin, pertactin and agglutinogens, and to theBordetella pertussis toxins pertussis toxin and adenylate cyclase-hemolysin were compared. Infection induced intense antibody responses to filamentous hemagglutinin, pertussis toxin and adenylate cyclase-hemolysin. Antibodies to agglutinogens were never detected, and antibodies to pertactin were rarely detected in infected infants' sera. Therefore, determination of anti-agglutinogens levels only is not suitable for the serological diagnosis of pertussis in young infants. Use of purified filamentous hemagglutinin, pertussis toxin and adenylate cyclase-hemolysin in Western blot analysis may improve the serodiagnosis ofBordetella infections. However, care must be exercised in distinguishing between the antibody response in young infants and maternally derived antibodies.     

O antigen protects Bordetella parapertussis from complement

Bordetella pertussis, a causative agent of whooping cough, expresses BrkA, which confers serum resistance, but the closely related human pathogen that also causes whooping cough, Bordetella parapertussis, does not. Interestingly, B. parapertussis, but not B. pertussis, produces an O antigen, a factor shown in other models to confer serum resistance. Using a murine model of infection, we determined that O antigen contributes to the ability of B. parapertussis to colonize the respiratory tract during the first week of infection, but not thereafter. Interestingly, an O antigen-deficient strain of B. parapertussis was not defective in colonizing mice lacking the complement cascade. O antigen prevented both complement component C3 deposition on the surface and complement-mediated killing of B. parapertussis. In addition, O antigen was required for B. parapertussis to systemically spread in complement-sufficient mice, but not complement-deficient mice. These data indicate that O antigen enables B. parapertussis to efficiently colonize the lower respiratory tract by protecting against complement-mediated control and clearance.     

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.     

Interactions between Bordetella pertussis and the complement inhibitor factor H

Bordetella pertussis causes whooping cough in humans, a highly contagious disease of the upper respiratory tract. An increase in cases of whooping cough in adolescents and adults in many countries has been reported, despite high immunization rates in children. To efficiently colonize the host the bacteria have to resist complement, the first defence line of innate immunity. B. pertussis has previously been shown to bind the classical pathway inhibitors C4b-binding protein and C1-inhibitor being thereby able to escape the classical pathway of complement. In this study recent clinical isolates of B. pertussis and B. parapertussis were found to survive alternative pathway attack in fresh non-immune serum better than the reference B. pertussis strain, Tohama I. By using adsorption assays, flow cytometry and a radioligand binding assay we observed that both B. pertussis and B. parapertussis bound the alternative pathway inhibitor factor H (FH) from normal human serum. The surface attached FH maintained its complement regulatory activity and promoted factor I-mediated cleavage of C3b. The main binding region was located to the C-terminal part of FH, into short consensus repeat domains 19-20. In contrast, the avian pathogen B. avium did not bind FH and was sensitive to the alternative pathway of human complement. In conclusion, the human pathogens B. pertussis and B. parapertussis are able to evade the alternative complement pathway by surface acquisition of the host complement regulator FH.     

Update on epidemiology,diagnosis, and treatment of pertussis

AlthoughBordetella pertussis was identified as the causative organism of whooping cough in 1906 (1), numerous questions regarding epidemiology, diagnosis, treatment, and prevention of this disease remain unanswered. Widespread immunization has drastically reduced the prevalence of pertussis in many countries, but epidemics still occur, even in well-immunized communities (2, 3), which explains the continued interest in this old disease.This editorial will focus on some recent developments in the areas of epidemiology, diagnosis, and treatment of pertussis; it is not, however, intended as a comprehensive review of these fields.     

Methods for isolation ofBordetella pertussis from patients with whooping cough

Culture remains the standard method for diagnosis of whooping cough. While in the past attempts at isolatingBordetella pertussis from patients with suspected whooping cough were often unsuccessful, new methods have recently been developed which are suitable for use in routine microbiology laboratories. Recent advances include the use of calcium alginate tipped swabs for taking nasopharyngeal swabs, use of charcoal horse blood agar for transport and culture, and the inclusion of cephalexin as a selective agent in agar media. Experience shows that careful application of these new methods often enablesBordetella pertussis to be isolated from clinical specimens, thereby permitting a diagnosis to be made at an early stage of the disease.