Bordetella pertussis: characteristics, virulence factors, pathogenesis, symptoms, treatment and vaccine

Bordetella pertussis

• Bordetella pertussis (Bordet-Gengou Bacillus; formally known as Hemophilus pertussis)

Morphology of Bordetella pertussis :

• The Bordetella spp are small, gram-negative coccobacilli with slight pleomorphism measuring 0.2-0.3 μm by 0.5 to 1.0 μm.
• They appear singly, in pairs, and in small clusters.
• On primary isolation, cells are uniform in size, but in subcultures they become quite pleomorphic and filamentous, and thick bacillary forms are common.
• It is nonmotile and non-sporing.
• Bipolar metachromatic staining may be demonstrated with toluidine blue.
• Capsules may be demonstrable in young, freshly isolated cultures only by special stains.
• In culture films, the bacilli tend to be arranged in loose clumps, with clear spaces in between giving a ‘thumb print’ appearance.
• Freshly isolated strains of B. pertussis have fimbriae.

Cultural Characteristics of Bordetella pertussis:

• It is an obligate aerobe.
• The optimum temperature for growth is 35-36°C.
• It does not require X and V factors for its growth.
• Complex media are necessary for primary isolation.
• The medium in common use is the Bordet-Gengou medium (potato-blood-glycerol agar).
• Primary isolation of B. pertussis requires the addition of charcoal, ion exchange resins, or 15-20 percent blood to neutralize growth-inhibiting effects.
• Potatoes impart a high starch content to the medium that neutralizes toxic materials.
• Glycerol acts as a stabilizing agent. Charcoal blood agar is a useful medium.
• The plates are incubated at 35-36°C in a moist environment (e.g., a sealed plastic bag).
• After incubation for 48-72 hours, colonies on Bordet-Gengou medium are small, dome shaped, smooth, opaque, viscid, greyish white, refractile and glistening, resembling ‘bisected pearls’ or ‘mercury drops’.
• Colonies are surrounded by a hazy zone of hemolysis. Confluent growth presents an ‘aluminium paint’ appearance.
• Subcultures of B. pertus­sis may be obtained on less exacting media, e.g. nutrient agar to which charcoal or starch has been added.

Biochemical Reactions of Bordetella pertussis:

• It is biochemically inactive.
• It does not ferment sugars, form indole, reduce nitrates, utilize citrate or split urea.
• It produces oxidase and usually catalase also.

Antigenic Constituents and Virulence Factors of Bordetella pertussis:

• B. pertussis is a delicate organism.
• It can be killed by heating at 55°C for 30 minutes, drying and disinfectants.
• Outside the body it can survive for five days on glass, three days on cloth and a few hours on paper.
• The organism is usually sensitive to ampicillin and erythromycin and these drugs have a reasonable therapeutic record.
• B. pertussis produces a number of factors that are involved in the pathogenesis of disease.
• 1. Agglutinogens
  • Bordetellae possess genus specific and species specific surface 14 agglutinogens associated with the capsular K antigens or fimbriae.
  • Factors 1 to 6 are found only in strains of B. pertussis, all of which carry Factor I and one or more of the other factors.
  • All three mammalian species of bordetellae has common Factor 7.
  • Factor 12 is specific’ for B. bronchiseptica and Factor 14 for B. parapertussis.
  • Agglutinogens promote virulence by help- ing bacteria to attach to respiratory epithelial cells.
  • They are useful in serotyping strains and in epidemiological studies.
  • On the basis of the agglutinogens Bordetellae carry they are classified into various types.
• 2. Pertussis toxin (PT)
  • PT, also known as lymphocytosis-promoting factor, pertussigen, histamine-sensitizing factor and islet-activating factor, has a wide spectrum of biologic activity.
  • Pertussis toxin promotes lymphocytosis, sensitization to histamine, and enhanced insulin secretion.
  • PT is expressed on the surface of the bacillus and secreted into the surrounding medium.
  • PT has a molecular weight of 117,000 and is a classic A-B toxin(dissociated into A and B subunits) made up of 6 polypeptide chains consisting of a A subunit, toxic subunit (SI) and five binding subunits (S2 to S5; two S4 subunits are present in each toxin molecule).
  • B unit consists of the remaining 5 polypeptide chains binds the toxin to the target cells and helps A unit to cross the membrane.
  • It can be toxoided. Pertussis toxoid is the major component of acellular pertussis vaccines.
  • It is apparently responsible for many of the clinical signs and symptoms of pertussis, as well as the relative and absolute lymphocytosis observed during the clinical illness.
  • Antibody against PT is also protective in animal models of pertussis. It can be toxoided.
  • The major component of acellular pertussis vaccines is PT toxoid.
  • Antibody to PT can protect mice against intranasal, intra- peritoneal or intracerebral challenge.
  • Outside the B. pertussis cells, the filamentous hemagglutinin and pertussis toxin are found which are secreted proteins .
• 3. Filamentous Hemagglutinin (FHA)
  • The filamentous hemagglutinin and pertussis toxin are secreted proteins and are found outside of the B. pertus­sis cells.
  • It is a protein that acquired its name through its ability to agglutinate erythrocytes.
  • It mediates adhesion to ciliated epithelial cells.
  • FHA is used in acellular pertussis vaccines along with PT toxoid.
  • Antibodies against filamentous hemagglutinin are protective.
  • FHA and PT hemagglutinins also promote secondary infection by coating other bacteria such as Haemo­philus influenzae and or Streptococcus pneumoniae and assisting their binding to respiratory epithelium besides facilitating adhesion of B. pertussis to respiratory epithelium.
  • This potential “piracy of adhesins” by other organisms may contribute to secondary bacterial invasion in pertussis .
• 4. Adenylate Cyclase (AC)
  • All mammalian Bordetellae but not B. avium produce adenylate cyclase .
  • At least two types of AC are known, only one of which has the ability to enter target cells and act as a toxin.
  • This is known as AC toxin (ACT).
  • It has the ability to enter target cells (leukocytes) and act as a toxin. It can be activated by eukaryotic calcium-dependent regulatory protein, calmodulin.
  • Inside the cell, after activation by calmodulin, this enzyme synthesizes cAMP(as pertussis toxin does) which is responsible for the biological effects such as interfering with leukocyte functions (inhibition of phagocytosis and chemotaxis).
• 5. Heat Labile Toxin (HLT) or Dermonecrotic Toxin
  • The heat–labile toxin (HLT) produced by all species of Bordetella appears to be a cytoplasmic protein it is a heat- labile toxin it is dermonecrotic and at high doses, this toxin causes fatal reactions in mice.
  • Role in disease is unknown.
• 6. Tracheal Cytotoxin (TCT)
  • Tracheal cytotoxin is a low-molecular-weight cell wall peptidoglycan monomer that has a specific affinity for ciliated epithelial cells.
  • It induces ciliary damage in hamster tracheal ring cultures and inhibition of DNA synthesis in the ciliated respiratory epithelial cells, resulting in accumulation in the lungs of mucus, bacteria and inflammatory debris leading to severe cough.
  • The disruption of ciliary function may also contribute to the secondary bacterial infections.
  • The toxin also stimulates the release of the cytokine interleukin-l, which leads to fever.
• 7. Lipopolysaccharide (Heat-Stable Toxin)
  • It is present in all bordetellae and exhibits features of gram-negative bacterial endotoxins.
  • Their role in the disease process is unknown.

Pathogenesis of Bordetella pertussis:

• Whooping cough is predominantly a pediatric disease.
• Whooping cough in 95 percent of cases is caused by B. pertussis.
• B. parapertussis causes about 5 percent of the cases and by B. bronchiseptica very infrequently (0.1%).

Stages of Whopping cough Disease:

• In human beings, after an incubation period of about 1-2 weeks, the disease takes a protracted course comprising three stages-the catarrhal, paroxysmal and convalescent -each lasting approximately two weeks.
• 1. Prodromal or Catarrhal Stage
  • The first stage, the catarrhal stage, resembles a com- mon cold, with serous rhinorrhea, sneezing, malaise, anorexia, and low grade fever.
  • Clinical diagnosis in the catarrhal stage is difficult.
  • During this stage, large numbers of organisms are sprayed in droplets, and the patient is highly infectious but not very ill.
  • This is unfortunate as this is the stage at which the disease can be arrested by antibiotic treatment.
• 2. Paroxysmal Stage
  • After 1 to 2 weeks, the paroxysmal stage begins.
  • As the catarrhal stage advances to the paroxysmal stage, the cough increases in intensity and comes on in distinctive bouts.
  • During the paroxysm, the patient is subjected to violent spasms of continuous coughing, followed by a long inrush of air into the almost empty lungs, with a characteristic whoop (hence the name).
  • The paroxysms of coughing may be so severe that cyanosis, vomiting and convulsions follow, completely exhausting the patient.
• 3. Convalescent Stage
  • After 2 to 4 weeks, the paroxysmal stage is followed by convalescence stage, during which the frequency and severity of coughing gradually decrease but secondary complications can occur.
  • The disease usually lasts 6-8 weeks though in some it may be very protracted.
• Complications:
  • Subconjunctival hemorrhage, subcutaneous emphysema, inguinal hernia or rectal prolapse due to pressure effects during the violent bouts of coughing.
  • Respiratory (bronchopneumonia, lung collapse)- Respiratory complications are self-limited, the atelectasis resolving spontaneously.
  • Neurological (convulsions, coma.)-neurological complications may result in permanent sequelae such as epilepsy, paralysis, retardation, blindness or deafness.

Epidemiology of Bordetella pertussis:

• Whooping cough is predominantly a pediatric disease, the incidence and mortality being highest in the first year of life.
• Maternal antibodies do not seem to give protection against the disease.
• Immunization should, therefore, be started early.
• The disease is commoner in the female than in the male at all ages.
• It is worldwide in distribution.
• It occurs in epidemic form periodically but the disease is never absent from any community.
• The source of infection is the patient in the early stage of the disease.
• Infection is transmitted by droplets and fomites contaminated with oropharyngeal secretions.
• Whooping cough is one of the most infectious of bacterial diseases and nonimmune contacts seldom escape the disease.
• The secondary attack rates are highest in close household contacts.
• The disease is often atypical.
• In adolescents and adults and may present as bronchitis.
• They may serve as a source of infection in infants and children.
• Natural infection confers protection though it may not be permanent, and second attacks have been reported.

Laboratory Diagnosis of whooping cough:

• Blood changes in the disease are distinctive and helpful in diagnosis.
• Pertussis typically causes an elevated white cell count, sometimes in excess of 50,000 cells/ μl (normal range=4500-11000 white blood cells/μl during the latter part of the catarrhal or early paroxysmal phase.
• A marked leukocytosis occurs, with relative lymphocytosis (total leukocytic counts 20,000-30,000 per/ μl with 60-80 percent lymphocytes).
• The erythrocyte sedimentation rate is not increased, except when secondary infection is present.

Three lab diagnosis methods are available:

1. Isolation of B. pertussis by culture from a pernasal swab;
2. Identification of the organism in a smear from a pernasal swab by immunofluorescence microscopy;
3. Serological demonstration of specific antibodies in the patient’s serum.

1. Microscopy:

• Microscopic diagnosis depends on demonstration of the bacilli in respiratory secretions by the fluorescent antibody technique.

2. Specimen Collection and Transport:

• Though the disease is mainly in the lower respiratory tract, the organism can be recovered readily from the nasopharynx.
• ‘Cough plates’ and postnasal swabs are unsatisfactory because of overgrowth by commensal bacteria.
• The optimal diagnostic specimen is a naso-pharyngeal aspirate.
• i. The Cough Plate Method
  • Here a culture plate is held about 10-15 cm in front of the patient’s mouth during about of spontaneous or induced coughing so that droplets of respiratory exudates impinge directly on the medium.
  • This has the advantage that specimen is directly inoculated at the bedside.
• ii. The Postnasal (Peroral) Swab
  • Secretions from the posterior pharyngeal wall are collected with a cotton swab on a bent wire passed through the mouth.
  • Salivary contamination should be avoided.
  • A West’s postnasal swab may be conveniently employed.
  • Cotton swabs should not be used because they contain fatty acids that are toxic to B. pertussis so it is preferable to use dacron or calcium alginate swabs for specimen collection.
• iii. The Pernasal Swab
  • A sterile swab on a flexible wire is passed gently along the floor of the nose until it meets resistance.
  • The swab, which will collect mucopus, is withdrawn and either plated immediately on charcoal blood agar, or placed in transport medium.
  • The use of transport medium reduces the isolation rate.
  • A single swab may yield a negative culture, but isolation rates of up to 80 percent may be achieved by taking specimens on several successive days.
  • The pernasal swab has generally replaced the cough plates or post- nasal swabs that were used in the past.

3. Culture:

• The swab is inoculated immediately on charcoalhorse blood agar and Bordet-Gengou medium both with and without methicillin or cephalexin and incubated for at least seven days before being discarded as negative.
• The specimen may be transported in Regan-Lowe semi-solid medium if delay in transport is unavoidable.
• Plates are incubated in high humidity at 35-36°C and colonies appear in 48-72 hours.
• Typical ‘bisected pearl’ colonies appearing after 3-5 days must be investigated further.

4. Identification:

• Identification is confirmed by microscopy and slide agglutination with specific antisera.
• Immunofluorescence is useful in identifying the bacillus in direct smears of clinical specimens and of cultures.

5. Detection of Bacterial Antigens:

• Bordetella antigens may be detected in serum and urine in tests with specific antiserum.
• Alternatively, bacteria in nasopharyngeal secretions are labelled with fluorescein-conjugated antiserum and examined by ultraviolet microscopy.
• This method has the theoretical advantage, compared with culture, of detecting dead bordetellae.

6. Polymerase Chain Reaction (PCR):

• Polymerase chain reaction (PCR) is used for the detection of bordetella DNA in nasopharyngeal specimens, by the use of various primers with a sensitivity of 80 percent to 100 percent.

7. Serology:

• Rise in antibody titer may be demonstrated in paired serum samples by ELISA, agglutination.
• Complement fixation, immunoblotting, indirect hemagglutination, and toxin neutralization.
• Demonstration of specific secretory IgA antibody in nasopharyngeal secretions by ELISA has been proposed as a diagnostic method in culture negative cases.

Treatment of whooping cough:

• B. pertussis is susceptible to several antibiotics (except penicillin).
• The drug of choice is erythromycin (or one of the newer macrolides such as clarithromycin), which may reduce the severity of the illness if given before the paroxysmal stage.
• Chloramphenicol and cotrimoxazole are also useful.
• Treatment with pertussis immunoglobulin has been tried, but with limited success.
• Prophylaxis:
  • Preventing the spread of infection by isolation of cases is seldom practicable as infectivity is highest in the earliest stage of the disease when clinical diagnosis is not easy.
• Treatment and Quarantine:
  • Antibiotics and immunoglobulins currently available are not very effective for the treatment of patients or the protection of contacts.
  • Control of the disease by quarantine is unrealistic.

Vaccination for whooping cough :

• Specific immunization with killed B. pertussis vaccine has been found very effective.
• It is of utmost importance to use a smooth phase I strain for vaccine production.
• The vaccines in general use are suspensions of whole bacterial cells, killed by heat or chemicals.
• Adsorption of the bacteria on to an adjuvant, such as aluminium hydroxide, enhances the immune response (particularly important with factor3) and also causes fewer adverse reactions.
• B. pertussis acts as an adjuvant for the toxoids (diphtheria and tetanus toxoid) producing better antibody response.
• Infants and young children should be kept away from cases.
• Those known to have been in contact with whooping cough may be given prophylactic antibiotic (erythromycin or ampicillin) treatment for 10 days to prevent the infecting bacteria to become established.
• The best protection that can be given to an infant is to administer a booster dose of DPT/DT to his siblings before he is born.
• Adverse Reactions:
  • Pertussis vaccination may induce reactions ranging from local soreness and fever to shock and neurological complications like convulsions and encephalopathy.
  • Provocation poliomyelitis is a rare complication.
• Contraindications:
  • If severe complications such as encephalopathy, seizures. ‘shock or hyperpyrexia develop following the vaccine, subsequent doses of the vaccine are contraindicated.
  • Routine pertussis vaccination is not advisable after the age of seven years as adverse reactions are likely and the risk of severe disease is low.
• Acellular Pertussis Vaccine:
  • Acellular vaccines containing the protective components of the pertussis bacillus (PT. FHA. agglutinogens 1, 2. 3) first developed in Japan, cause far fewer reactions, particularly in older children.
  • Both whole cell and acellular vaccines have a protection rate of about 90 percent.