Comparison of media for agar dilution susceptibility testing ofBordetella pertussis andBordetella parapertussis

Antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis andBordetella parapertussis is not standardized. In an attempt to find the optimal medium for agar dilution testing, the activity of erythromycin againstBordetella pertussis andBordetella parapertussis (34 isolates each) was assessed using homologous broth/agar combinations of Bordet-Gengou, charcoal, Iso-Sensitest (Oxoid) and Mueller-Hinton media. Each medium was supplemented with 5 % and 20 % whole defibrinated horse blood. Mueller-Hinton medium supplemented with 5 % horse blood performed best overall.     

Role of pertussis toxin in causing symptoms ofBordetella parapertussis infection

Whooping cough can be caused by eitherBordetella pertussis orBordetella parapertussis. Although the two species share an almost complete DNA identity,Bordetella parapertussis does not produce pertussis toxin, which is thought to be the main virulence factor ofBordetella pertussis. In order to elucidate the role of pertussis toxin in causing the typical symptoms of whooping cough, clinical information from 33 patients with culture-positiveBordetella parapertussis infection was collected and compared to that from 331 patients with infection caused byBordetella pertussis. Isolated strains ofBordetella parapertussis lacked pertussis toxin expression, as was demonstrated by negative tests for histamine sensitization. This was further substantiated in vivo by a significantly lower leukocyte count in the parapertussis group as compared to the pertussis group. Frequencies of typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting, were almost identical in the two groups. Nocturnal coughing and contact anamnesis were noted more often in theBordetella pertussis group. Children in the parapertussis group were significantly more often vaccinated with whole-cell pertussis vaccine than children infected withBordetella pertussis. The results indicate that pertussis toxin may not play a decisive role in causing the typical symptoms of whooping cough, such as paroxysmal coughing, whooping and vomiting.     

Activity of new macrolides againstBordetella pertussis andBordetella parapertussis

MICs and MBCs of four new macrolides (azithromycin, clarithromycin, dirithromycin and roxithromycin) and two older macrolides (erythromycin and josamycin) forBordetella pertussis andBordetella parapertussis were determined. The activity of the new macrolides was as good as that of erythromycin, while josamycin was slightly less active.Bordetella parapertussis was more resistant thanBordetella pertussis.     

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.     

Recovery ofBordetella pertussis from four kinds of swabs

Calcium alginate, dacron, rayon and cotton wool tipped swabs, in combination with charcoal horse blood transport medium with cephalexin, were compared with regard to their ability to maintain viability of Bordetella pertussis,Calcium alginate proved to be the most suitable swab material. A field trial confirmed the results of the laboratory study.     

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.     

Detection ofBordetella pertussis by determination of adenylate cyclase activity

The adenylate cyclase activity ofBordetella pertussis in clinical isolates was measured in calmodulin-supplemented Stainer-Scholte broth by the rate of conversion of ATP to cyclic AMP. Analysis of 250 stock strains ofBordetella pertussis showed that measurable adenylate cyclase activity was produced by all strains. In clinical testsBordetella pertussis was isolated from 135 (22 %) of 605 swab samples. Increased adenylate cyclase activity was detected in 124 (92 %) Stainer-Scholte broth cultures of these samples. A total of 475 swabs contained other bacteria or had no growth; only one of the Stainer-Scholte broth cultures of these swab samples contained measurable adenylate cyclase activity. The results indicate that testing for adenylate cyclase activity provides a specific and sensitive means for detectingBordetella pertussis in clinical specimens.     

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.     

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.     

Alterations in serum proteins of rats after immunization with Freund's adjuvant andBordetella pertussis vaccine

Immunization is known to modify the level of certain circulating serum proteins. In this study we describe changes in the electrophoretic pattern of serum proteins after immunization with two different antigens: RaTE and BSA incorporated with three different adjuvants: complete Freund's adjuvant, incomplete Freund's adjuvant, andBordetella pertussis vaccine. Serum albumin and globulins were decreased while ₁, ₂, and globulins had a tendency to increase. The serum protein modifications were observed as early as 24 h after immunization and were not reversed by histamine or serotonin antagonists. Some physiopathological hypotheses of their possible effects in immunization are discussed.This study was supported by PHS Research Grant CA 02357 from the National Cancer Institute. It is publication no 75 from the Center for Immunology.     

Evaluation of Pooled and individual components ofBordetella pertussis as antigens in an enzyme immunoassay for diagnosis of pertussis

Six different antigen preparations for use in an enzyme immunoassay (EIA) to detect IgM, IgA and IgG antibodies toBordetella pertussis were evaluated using sera from 13 randomly selected culture-positive patients and from 87 patients with suspected pertussis during a pertussis outbreak. Based on results in 80 healthy control sera a specificity limit of 99.9 % was selected. Sera from all culture-positive patients reacted with at least one of the antigens. The sensitivity of the EIA using the individual antigen preparations was 85 % for filamentous hemagglutinin, 92 % for pertussis toxin, 62 % for 69 kDa outer membrane protein, 85 % for a pool of these three antigens, 54 % for sonicated whole bacteria and 69 % for 21 kDa pertussis toxin subunit S1. In the outbreak patient group 49 (56 %) of the initial sera reacted with at least one of five antigen preparations. The EIA using sonicated bacteria detected only 41 % of all seropositive cases compared with 51 % using filamentous hemagglutinin, 61 % using pertussis toxin, 65 % using 69 kDa OMP and 65 % using pooled antigen. It is concluded that either the pooled antigen or pertussis toxin antigen are suitable antigen preparations for use in the EIA for diagnosis of pertussis.     

Shelf life of prepared Bordet-Gengou and Regan-Lowe agar plates for isolation ofBordetella pertussis

The shelf life of prepared agar plates used for the isolation ofBordetella pertussis was studied. They contained Bordet-Gengou agar, Bordet-Gengou agar with cephalexin, Regan-Lowe agar, Regan-Lowe agar with cephalexin, or Regan-Lowe agar with both cephalexin and amphotericin B. Plates stored were compared to freshly prepared control plates for up to a maximum of 18 weeks. They were inoculated with clinical isolates ofBordetella pertussis, either in pure culture, or mixed with a defined oropharyngeal flora. Bordet-Gengou agar plates may be used, with proper storage at 4 °C in airtight-sealed plastic bags, for up to 10 weeks, Regan-Lowe agar plates for up to 14 weeks. Field studies are needed to substantiate our findings.     

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.     

Comparison of three media for agar dilution susceptibility testing ofBordetella pertussis using six antibiotics

Since antimicrobial susceptibility testing of the fastidious speciesBordetella pertussis is not standardized, the most suitable medium for agar dilution testing of this species has not yet been determined. In the present study, Mueller-Hinton, Bordet-Gengou, and Oxoid charcoal agars (each supplemented with 5% horse blood) were evaluated for agar dilution susceptibility testing ofBordetella pertussis against ampicillin, chloramphenicol, ciprofloxacin, doxycycline, erythromycin, and trimethoprim-sulfamethoxazole. Mueller-Hinton agar was the most suitable medium.