General Concepts

Introduction

Bordetella pertussis was first isolated in pure culture in 1906 by Bordet and Gengou. Today, B pertussis belongs to the genus Bordetella in the family Alcaligenaceae, which contains several species of closely related bacteria with similar morphology. B pertussis and B parapertussis cause whooping cough (pertussis) in humans. Other members of the genus are B bronchiseptica, which causes respiratory disease in various animals and is only occasionally found in humans. Recent additions to the genus are B avium and B hinzii, which both cause respiratory disease in poultry and are very rarely found in humans.

Clinical Manifestations

After an incubation period of 1 to 2 weeks, whooping cough begins with the catarrhal phase (Fig. 31-1). This phase lasts 1 to 2 weeks and is usually characterized by low-grade fever, rhinorrhea, and progressive cough; the patient is highly infectious. The subsequent paroxysmal phase, lasting 2 to 4 weeks, is characterized by severe and spasmodic cough episodes. At the end of the catarrhal phase, a leukocytosis with an absolute and relative lymphocytosis frequently begins, reaching its peak at the height of the paroxysmal stage. At this time, the total blood leukocyte levels may resemble those of leukemia (≥ 100,000/mm³), with 60 to 80 percent being lymphocytes. The convalescent phase, lasting 1 to 3 weeks, is characterized by a continuous decline of the cough before the patient returns to normal. Serious complications, sometimes fatal, are bronchopneumonia and acute encephalopathy, the latter being characterized primarily by convulsions and frequently resulting in death or lifelong brain damage.

Figure 31-1

Pathogenesis of whooping cough.

Structure

Bordetella pertussis is a small (approximately 0.8 μm by 0.4 μm), rod-shaped, coccoid, or ovoid Gram-negative bacterium that is encapsulated and does not produce spores. It is a strict aerobe. It is arranged singly or in small groups and is not easily distinguished from Haemophilus species. B pertussis and B parapertussis are nonmotile. Numerous antigens and biologically active structural components have been demonstrated in B pertussis (Fig. 31-2), although their exact chemical structure and location in the bacterial cell are known only in part.

Figure 31-2

Virulence factors of B pertussis.

Pertussis Toxin

Various immunologic, physiologic, and pharmacologic effects are induced by killed B pertussis cells in experimental animals (e.g., increased sensitivity to histamine and serotonin and active and passive anaphylaxis). Adjuvant activity, leukocytosis, splenomegaly, cell proliferation, hypoglycemia, and hypoproteinemia also occur. Many additional features have been described, including increased sensitivity to factors such as endotoxins, X irradiation, infection, cold stress, pollen extracts, peptone shock, and methacholine; increased resistance to infection; increased capillary permeability; and accelerated production of experimental “allergic” encephalomyelitis.

It is now generally accepted that all those biologic activities are caused by a single active protein produced by B pertussis. To avoid confusion caused by the many different names for this protein, the uniform term pertussis toxin was proposed by Pittman. Pertussis toxin is a protein exotoxin, secreted during in vivo and in vitro growth; it consists of five different subunits, designated S1, S2, S3, S4, and S5. Since the toxin molecule contains two S4 subunits, it is a hexamer. Like many other protein toxins, pertussis toxin consists of an A subunit that carries the biologic activity and a B subunit that binds the complex to the cell membrane. In pertussis toxin, the S 1 subunit constitutes the A protomer and the B oligomer is formed by the remaining five subunits (Fig. 31-3). The toxin binds to cell receptors by two dimers, one consisting of S2 and S4 and the other of S3 and S4. Since glutaraldehyde-inactivated pertussis toxin is capable of adherence, this binding activity evidently has little to do with the various toxic activities of pertussis toxin. The toxin reacts with different cell types, including T lymphocytes, and acts on different cellular regulatory processes. Pertussis toxin is a member of the family of ADP-ribosylating bacterial toxins. The S1 subunit of pertussis toxin ADP-ribosylates the Cys³⁵² of protein G_i (GTP-binding protein), as well as the corresponding cysteine of protein G_α and of transducin. Although pertussis toxin is synthesized solely by B pertussis, both B parapertussis and B bronchiseptica possess genes for pertussis toxin without expressing them. Bordetella parapertussis expresses pertussis toxin when the toxin gene from the B pertussis chromosome is introduced into B parapertussis.

Figure 31-3

Binding of pertussis toxin to cell membranes.

Like many other bacteria, B pertussis possesses hemagglutinating activity, expressed as its capacity to agglutinate red cells from geese, chickens, and other animals. Pertussis toxin is one of the hemagglutinins, whereas another component with hemagglutinating activity is called filamentous hemagglutinin. This component appears as fine filaments, about 2 nm in diameter and 40 to 100 nm in length. Like pertussis toxin, it has hemagglutinating activity as well as the ability to effect the adherence of B pertussis to cilia by its lectin-like binding to lactose-containing moieties.

Classification and Antigenic Types

The genus Bordetella contains species of serologically related bacteria with similar morphology, size, and staining reactions. B pertussis and B parapertussis are genomically extremely closely related. Other members of the genus are B bronchiseptica, which by DNA-DNA and DNA-rRNA hybridization is also closely related. Recent additions to the genus are B avium (formerly designated Alcaligenes faecalis) and B hinzii (formerly designated A faecalis type II), which cause respiratory disease in poultry and are very rarely found in humans.

Bordetella pertussis was first isolated in pure culture in 1906 and was long considered the sole agent of whooping cough. Later studies revealed that this disease also can be caused in a mild form by B parapertussis and occasionally by B bronchiseptica. A phenomenon of B pertussis organisms is their variation during growth on agar plates: the antigenically competent, smooth, virulent form (phase I) can mutate to the antigenically incomplete, nonvirulent, rough form (phase IV). This change is associated with a loss of capacity to synthesize pertussis toxin, filamentous hemagglutinin, heat-labile toxin, adenylate cyclase toxin, agglutinogens, and certain outer membrane proteins. There are also two intermediate forms, called phases II and III.

In addition to this spontaneous phase variation, B pertussis undergoes antigenic modulation in response to changes in environmental conditions, such as growth at low temperatures or on agar plates with high concentrations of MgSO₄ or nicotinic acid. Bordetella pertussis organisms grown under such conditions are avirulent and are characterized by the loss of the capacity to synthesize the numerous toxic factors and other structural components. Both phase variation and antigenic modulation are reversible and also occur in B parapertussis and B bronchiseptica. Both phenomena are under the control of a single genetic locus. The virulent strains are therefore designated Bvg⁺, and the avirulent strains Bvg^–. Phase variation has been observed in vivo. Another type of serotype variation in B pertussis—the loss of one or more agglutinogens—occurs independently of phase variation.

Pathogenesis

The agent of whooping cough is transmitted primarily via droplets. Infection results in colonization and rapid multiplication of the bacteria on the mucous membranes of the respiratory tract. Bacteremia does not occur. Electron microscopic studies have demonstrated that phase I strains of B pertussis adhere only to the tuft of ciliated cells in the mucosa of the human respiratory tract; no attachment to nonciliated cells was observed. Convincing experimental data indicate that the adherence of B pertussis to human cilia is effected by a synergistic action of pertussis toxin and filamentous hemagglutinin, each acting as a bivalent bridge between the bacterium and the ciliary receptor (Fig. 31-4).

Figure 31-4

Synergy between pertussis toxin and the filamentous hemagglutinin in binding to ciliated respiratory epithelial cells.

Studies of numerous B pertussis toxins and their corresponding biologic activities have yielded plausible explanations for many of the symptoms of whooping cough. These include, for example, the frequent occurrence of absolute lymphocytosis (an unusual phenomenon in bacterial infections), hypoglycemia, and the adjuvant effect of pertussis toxin on the immune response to unrelated antigens. The finding that phase I isolates of B bronchiseptica produce almost complete ciliostasis within 3 hours in ciliated epithelial cell outgrowths from canine tracheal explants may be explained by the action of adenylate cyclase toxin and tracheal cytotoxin. The same toxins evidently inhibit the phagocytic activities of the host. In humans, an initial local peribronchial lymphoid hyperplasia occurs, accompanied or followed by necrotizing inflammation and leukocyte infiltration in parts of the larynx, trachea, and bronchi. Usually, peribronchiolitis and variable patterns of atelectasis and emphysema also develop.

To date, there is no plausible explanation for the development of the paroxysmal coughing syndrome characteristic of pertussis. According to Pittman, pertussis is mediated by pertussis toxin and is characterized by a two-stage process—infection (colonization) and disease—thus resembling other bacterial toxicoses such as diphtheria, tetanus, and cholera. This fascinating idea can be accepted only if it is clearly demonstrated that pertussis toxin causes paroxysmal coughing. Such a demonstration is lacking. Moreover, paroxysmal coughing occurs in infections with B parapertussis, which does not synthesize pertussis toxin. On the other hand, an additional infection with B pertussis cannot be excluded in such cases. There is no convincing explanation for the acute encephalopathy sometimes observed in pertussis. Research into the pathogenetic mechanisms of pertussis are hampered by the lack of an adequate animal model showing the characteristic paroxysmal coughing syndrome and by the limited opportunity to perform direct studies of the respiratory tract of babies and children.

Host Defenses

A case of whooping cough confers substantial immunity, which usually lasts for many years. Second infections of adults, usually with atypical symptoms and thus not regularly diagnosed as pertussis, may be more frequent than previously assumed. Immunity acquired after infection with B pertussis does not protect against the other Bordetella species. Pertussis toxin is assumed to be one essential protective immunogen, but numerous findings indicate that other components, such as filamentous hemagglutinin, heat-labile toxin, agglutinogens, outer membrane proteins, and adenylate cyclase toxin, may also contribute to immunity after infection or vaccination. The immunogenicity of the substances may be significantly increased by the presence of pertussis toxin. This synergism indicates that pertussis toxin could function as an adjuvant to a variety of protective antigens of B pertussis. The defense mechanisms are both nonspecific (local inflammation, increase in macrophage activity, and production of interferon) and specific (proliferation of B and T cells). The basis of immunity in whooping cough is, however, incompletely understood. A role of circulating antibody in immunity is indicated by the correlation between protection of human vaccinees and their serum agglutinin titers. However, effective immunity does not necessarily depend on the presence of serum agglutinins, and immunity to whooping cough may therefore be mediated essentially by cellular mechanisms. This cell-mediated immunity may be considered the crucial carrier of long-term immunity, and titers of specific humoral antibodies may diminish over the years. This may be the reason why infants usually do not benefit significantly from maternal antibody.

Epidemiology

The mucous membranes of the human respiratory tract are the natural habitat for B pertussis and B parapertussis. Although B pertussis can survive outside the body for a few days and so may be transmitted by contaminated objects, most infections occur after direct contact with diseased persons—specifically, by inhalation of bacteria-bearing droplets expelled in cough spray. The patient is most infectious during the early catarrhal phase, when clinical symptoms are relatively mild and noncharacteristic (Fig. 31-5). Subclinical cases may have similar epidemiologic significance. Healthy carriers of B pertussis or B parapertussis are assumed to play no significant epidemiologic role. The natural habitat of B bronchiseptica is the respiratory tract of smaller animals such as rabbits, cats, and dogs. Therefore, human infections with B bronchiseptica are extremely rare and occur only after close contact with carrier animals.

Figure 31-5

Relationship of B pertussis to the developing antibody response during whooping cough.

Whooping cough, a highly communicable, worldwide infection, was once common and dangerous, killing many thousands of children per year. Widespread vaccination has caused a continuous decrease in incidence and mortality over the years, but large numbers of patients still die in countries where vaccination is inadequate. Whooping cough is mainly an infection of infants and children, although susceptibility is general. The disease is especially dangerous in the first 6 months of life. Neither season nor climate seems to affect the morbidity rate.

Diagnosis

Bordetellae can be cultured from nasopharyngeal swabs or nasopharyngeal secretions. The sensitivity of the method depends mainly on the technique of taking the swabs or secretions. Swabs (one for each nostril) should be introduced deeply into the nose as to reach the nasopharynx. Swabs should be made of dacron or calcium alginate, and they should be transported in half strength charcoal blood agar. Secretions should be from the nasopharynx using a suction device with a mucus trap. Nasopharyngeal secretions should be immediately plated onto Regan-Lowe medium, which has replaced Bordet-Gengou medium as the medium of choice. The transportation time for both materials should be kept as short as possible. For culture isolation, Bordet-Gengou agar containing blood, potato extract, and glycerol remains one of the effective means, although minor modifications regarding blood concentrations and addition of penicillin and nicotinamide have been recommended. For routine use, charcoal-blood agar (REGAN-LOWE medium) is most widely used. A (2,6-O-dimethyl)-b-cyclodextrin supplemented STAINER-SCHOLTE broth can be used as an enrichment medium. The Bordetella species do not need factors X and V (NAD⁺ and hemin).

Bordetella pertussis usually grows after 3 to 4 days of incubation at 37° C. The small, transparent colonies are indistinguishable from those of B bronchiseptica, but usually are smaller than those of B parapertussis. All three species produce hemolysis. Biochemically they are relatively inert and do not ferment carbohydrates or produce H₂S and indole. An important characteristic of B parapertussis is its capacity to produce brown pigmentation on blood-free peptone agar. B pertussis and B parapertussis can be distinguished by certain biochemical and culture characteristics (Table 31-1) in addition to slide agglutination with specific antisera. B bronchiseptica as well as B avium and B hinzii can be differentiated by conventional methods for typing gram-negative nonfermenting rods (such as API-NE).

Table 31-1

Differential Characteristics of B pertussis and B parapertussis.

Detection of B pertussis and B parapertussis DNA by PCR has been described, and various primers and detection methods were applied. Ongoing studies will define the role of a standardized PCR method in pertussis diagnostics.

Circulating antibodies, appearing as late as week 3 of illness and reaching their maximum at weeks 8 to 10, have been demonstrated by agglutination and complement fixation tests. The agglutination test is applied mainly in epidemiologic studies. Although no direct relationship has been shown between the agglutinin concentration and the degree of protection, high agglutinin titers (>1:320) are assumed to correlate with protection from disease. Modern serologic techniques, such as enzyme-linked immunosorbent assay (ELISA), have been used to detect IgG, IgM, IgA, and IgE antibodies to both whole B pertussis cells and certain isolated components. In accordance with other serologic methods, seroconversion could be observed only 2 to 4 weeks after the onset of the disease (Fig. 31-5). The detection of specific IgA and IgM antibodies, however, is indicative of recent infection and is useful for the differential diagnosis of pertussiform syndromes of longer duration. IgA antibodies to pertussis toxin and FHA (filamentous hemagglutinin are found mainly after natural infection; the same is true of secretory IgA in nasopharyngeal secretions, which usually appears during week 2 or 3 of illness. Unfortunately, infants do not regularly produce IgA antibody before 6 months of age. Specific IgM antibodies may be used in infants as an indicator of acute infection.

Control

Although B pertussis is susceptible in vitro to several antibiotics, such as tetracycline, erythromycin, and chloramphenicol, the efficacy of these drugs in patients during the paroxysmal phase is not convincing. Treatment with erythromycin, which is usually considered the antibiotic of choice, will eliminate viable B pertussis organisms from the respiratory tract within a few days. The treatment, however, has no influence on the course of the disease. Human hyperimmune pertussis globulin is still used occasionally, but no reliable data support its efficacy. Further treatment is symptomatic.

Susceptible children (unimmunized children without a history of whooping cough) should have no contact with pertussis patients during the first 4 weeks of illness, although such isolation is often difficult. A patient treated with erythromycin may be contagious for only 5 to 10 days. Exposed immunized children younger than 4 years are given booster doses of pertussis vaccine. Exposed unimmunized children are given erythromycin for 10 days after contact is discontinued or after the patient ceases to be contagious.

Pertussis vaccine is produced from smooth forms (phase I) of the bacteria as a killed whole-cell vaccine. In the United States, pertussis vaccination of infants and children is recommended. Owing to a relatively mild course of disease and to occasional neurologic complications after vaccination, it has been argued by numerous pediatricians that general vaccination with the whole-cell vaccine is no longer justified.

Acellular pertussis vaccines have been developed, and were licensed in Japan since 1981 for children (older than two years), and also have been used in infants since 1990. These vaccines are composed very differently and contain various amounts of structural components from the bacteria. Components available for vaccine production include pertussis toxin (which is detoxified), filamentous hemagglutinin, a 69 kDa outer-membrane protein called pertactin, and fimbrial antigens 2 and 3. Some of these vaccines have been licensed in the U.S. for booster vaccinations since 1991. Recent data suggest that after primary vaccinations of infants these vaccines can convey similar levels of protection as the whole-cell vaccine. Thus, acellular vaccines have also been licensed in some European countries for primary vaccination.

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