CMR Matoo and Cherry 2005

Molecular Pathogenesis, Epidemiology, and
Clinical Manifestations of Respiratory

Infections Due to Bordetella pertussis and
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Other Bordetella Subspecies
Seema Mattoo and James D. Cherry
Clin. Microbiol. Rev. 2005, 18(2):326. DOI:
10.1128/CMR.18.2.326-382.2005.
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CLINICAL MICROBIOLOGY REVIEWS, Apr. 2005, p. 326–382 Vol. 18, No. 2
0893-8512/05/$08.00⫹0 doi:10.1128/CMR.18.2.326–382.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of

Respiratory Infections Due to Bordetella pertussis and
Other Bordetella Subspecies
Seema Mattoo1† and James D. Cherry2*
Department of Microbiology, Immunology, and Molecular Genetics,1 and Department of Pediatrics,2
David Geffen School of Medicine, University of California, Los Angeles, California
INTRODUCTION .......................................................................................................................................................327
HISTORY.....................................................................................................................................................................328
PHYLOGENETIC RELATIONSHIPS BETWEEN BORDETELLA SUBSPECIES...........................................328
VIRULENCE DETERMINANTS AND MOLECULAR PATHOGENESIS ........................................................329
Animal Models ........................................................................................................................................................329
Bordetella Virulence Regulon .................................................................................................................................330
Commonly Expressed Loci ....................................................................................................................................332
Differentially Expressed and Differentially Regulated Loci..............................................................................332
Virulence Determinants .........................................................................................................................................334
Filamentous hemagglutinin ...............................................................................................................................334
Agglutinogens ......................................................................................................................................................335
Fimbriae ...............................................................................................................................................................336
Pertactin and other autotransporters ..............................................................................................................337
Adenylate cyclase ................................................................................................................................................338
Dermonecrotic toxin ...........................................................................................................................................339
Lipopolysaccharides............................................................................................................................................339
Type III secretion system...................................................................................................................................340
Tracheal cytotoxin...............................................................................................................................................341
Pertussis toxin.....................................................................................................................................................341
PATHOLOGY..............................................................................................................................................................343
B. pertussis Infection ...............................................................................................................................................343
B. bronchiseptica Infection......................................................................................................................................344
Dogs ......................................................................................................................................................................344
Swine.....................................................................................................................................................................344
Laboratory animals ............................................................................................................................................344
(i) Guinea pigs ................................................................................................................................................344
(ii) Rabbits.......................................................................................................................................................344
PATHOGENESIS AND IMMUNITY.......................................................................................................................344
CLINICAL MANIFESTATIONS ..............................................................................................................................346
B. pertussis ................................................................................................................................................................346
Classic illness ......................................................................................................................................................346
Mild illness and asymptomatic infection ........................................................................................................346
Infants ..................................................................................................................................................................347
Adults....................................................................................................................................................................347
B. parapertussishu .....................................................................................................................................................348
B. bronchiseptica.......................................................................................................................................................348
Swine.....................................................................................................................................................................348
Dogs ......................................................................................................................................................................348
Laboratory animals ............................................................................................................................................348
Humans ................................................................................................................................................................348
B. holmesii ................................................................................................................................................................349
DIAGNOSIS ................................................................................................................................................................349
Differential Diagnosis of Bordetella Infections ....................................................................................................349
Infections in humans..........................................................................................................................................349
Infection in animals............................................................................................................................................349
Specific Diagnosis of B. pertussis Infections........................................................................................................349
* Corresponding author. Mailing address: 10833 Le Conte Ave.

MDCC 22-442, Los Angeles, CA 90095-1752. Phone: (310) 825-5226.
Fax: (310) 206-4764. E-mail: [email protected].
† Present address: Leichtag Biomedical Research Building, Univer-
sity of California at San Diego, La Jolla, CA 92093-0721.
326
VOL. 18, 2005 BORDETELLA RESPIRATORY INFECTIONS 327
Culture of B. pertussis.........................................................................................................................................349
(i) Specimen collection...................................................................................................................................349
(ii) Specimen transport..................................................................................................................................350
(iii) Culture......................................................................................................................................................350
(iv) DFA testing of nasopharyngeal secretions...........................................................................................350
(v) Detection of B. pertussis by PCR.............................................................................................................350
(vi) Serologic diagnosis of B. pertussis infection.........................................................................................351
Specific Diagnosis of Other Bordetella Infections...........................................................................................351
TREATMENT..............................................................................................................................................................351
VACCINATION AND PREVENTION......................................................................................................................352
B. pertussis Vaccines................................................................................................................................................352
Whole-cell DTP vaccines....................................................................................................................................353
(i) Reactogenicity ............................................................................................................................................353
(ii) Vaccine efficacy .........................................................................................................................................354
Acellular pertussis component DTP vaccines .................................................................................................355
(i) Reactogenicity ............................................................................................................................................355
(ii) Vaccine efficacy .........................................................................................................................................357
B. bronchiseptica Vaccines ......................................................................................................................................359
Dogs (kennel cough)...........................................................................................................................................359
Swine (atrophic rhinitis)....................................................................................................................................359
EPIDEMIOLOGY: IMPLICATIONS FOR THE CONTROL OF HUMAN INFECTIONS.............................359
B. pertussis Epidemiology .......................................................................................................................................359
Observed (reported pertussis) ..........................................................................................................................359
(i) Incidence.....................................................................................................................................................359
(ii) Mortality....................................................................................................................................................360
(iii) Sex and race ............................................................................................................................................360
(iv) Season and geographic areas.................................................................................................................360
B. pertussis infection............................................................................................................................................360
(i) Percentage of cough illnesses due to B. pertussis in adolescents and adults ....................................361
(ii) Rate of B. pertussis infections .................................................................................................................362
(iii) Rate of cough illnesses due to B. pertussis infections ........................................................................362
B. parapertussishu Epidemiology ............................................................................................................................362
Incidence and mortality .....................................................................................................................................362
Sex, race, season, and geographic areas..........................................................................................................363
Interrelationship between B. parapertussishu and B. pertussis infections.....................................................363
B. bronchiseptica Epidemiology..............................................................................................................................363
B. holmesii Epidemiology........................................................................................................................................363
LONG-TERM GOALS OF PERTUSSIS PREVENTION......................................................................................363
Eradication of B. pertussis Circulation.................................................................................................................363
Lesser Goals ............................................................................................................................................................364
Infant and Childhood DTaP Immunization Schedules .....................................................................................364
FUTURE RESEARCH................................................................................................................................................365
Pathogenesis of Disease .........................................................................................................................................365
Better Vaccines........................................................................................................................................................365
Role of B. holmesii in Human Disease .................................................................................................................366
More Complete Epidemiologic Study...................................................................................................................366
ACKNOWLEDGMENTS ...........................................................................................................................................366
REFERENCES ............................................................................................................................................................366
INTRODUCTION (149, 831). Human-adapted B. parapertussis (B. parapertussishu)

causes a milder pertussis-like disease and, like B. pertussis, lacks
Whooping cough (pertussis) is a highly contagious, acute
an environmental reservoir (149). B. holmesii, the most recent
respiratory illness of humans that is caused by the gram-neg-
of the Bordetella species associated with human respiratory
ative bacterial pathogen Bordetella pertussis (149). B. pertussis
tract infection, has been found in the blood of young adults and
is a strict human pathogen with no known animal or environ-
occasionally in the sputum (752, 814, 839). Little is known
mental reservoir (174). As such, transmission of disease is
postulated to occur via respiratory droplets. While nine species about the biology, virulence mechanisms, and pathogenic sig-
of Bordetella have been identified to date, only three additional nificance of B. holmesii; in contrast, B. pertussis, B. bronchisep-
members, B. bronchiseptica, B. parapertussis, and B. holmesii, tica, and B. parapertussis have been extensively studied.
have been associated with respiratory infections in humans and Although pertussis is relatively well controlled at present by
other mammals (174, 504). B. bronchiseptica infects a wide extensive vaccination programs, it is evident that the circula-
range of hosts and occasionally causes cough illnesses in hu- tion of B. pertussis throughout the world continues largely
mans; in particular, severe infections have been noted in per- unabated (149). Whooping cough is still common in areas of
sons who are immunocompromised such as patients with AIDS the world where vaccine use is low. Recent studies suggest that
328 MATTOO AND CHERRY CLIN. MICROBIOL. REV.
there are presently ⬃48.5 million yearly cases of pertussis

worldwide, with as many as 295,000 deaths (187). One effect of
vaccination has been a shift in the incidence of reported per-
tussis from children aged 1–9 years in unvaccinated popula-
tions to infants, adolescents, and adults in vaccinated popula-
tions (149). Reasons for this shift include incomplete immunity
in infants who have received fewer than three doses of vaccine,
the relatively short-lived immunity that results from vaccina-
tion, and the recent greater awareness of pertussis in adoles-
cents and adults. Although adolescent and adult pertussis is
significant in terms of medical costs and lost work, the most
worrisome consequence is epidemiological (149, 645). Numer-
ous studies have shown that adults and adolescents provide a
reservoir of B. pertussis and are the major source of transmis-
sion to partially immunized infants and children (35, 56, 135,
149, 186, 583).
During the last 20 years, many good reviews have been
written relating to the microbiology of Bordetella species and
the clinical and epidemiologic aspects of pertussis (136, 147, FIG. 1. Phylogenetic relationships among the nine known Borde-
149, 174, 418, 504, 802). The purpose of the present review is tella species based on a combination of multilocus enzyme electro-
to consolidate data from the previous literature and new in- phoresis, IS element, and sequence analysis. These species appear to
have descended from a common B. petrii ancestor. Further, B. bron-
formation, as well as to correlate clinical events with the latest
chiseptica appears to be the evolutionary progenitor of B. pertussis,
molecular evidence. B. parapertussishu, and B. parapertussisov; as such, these species have
been reclassified as subspecies of the “B. bronchiseptica cluster.”
HISTORY
B. pertussis and was not associated with lymphocytosis, a hall-
In contrast to other severe epidemic infectious diseases of
mark of B. pertussis infection in children.
humans (i.e., smallpox, polio, and measles), pertussis lacks an
B. holmesii was presented as a new gram-negative species
ancient history (356). Lapin stated that the first mentioning of
associated with septicemia in 1995 (814). This organism was
the disease was found in Moulton’s The Mirror of Health, in
first isolated in 1983 but was not associated with respiratory
1540 but he also refers to a paper by Nils Rosen von Rosen-
illness until 1998. During the period from 1995 through 1998,
stein which suggested that the illness began in France in 1414
B. holmesii was recovered from nasopharyngeal specimens of
(442). The first epidemic was noted in Paris, France, in 1578
33 patients in Massachusetts with pertussis-like symptoms
(162). In 1679, Sydenham named the illness pertussis (meaning
(508, 839).
violent cough).
Bordet and Gengou reported the isolation of B. pertussis in
1906, although they had observed the organism microscopically PHYLOGENETIC RELATIONSHIPS BETWEEN
in the sputum of a patient with pertussis in 1900 (63, 356). BORDETELLA SUBSPECIES
Since pertussis was such a severe disease in infants, vaccine Figure 1 depicts the phylogeny of all nine known Bordetella
development began soon after the growth of the organism in species, namely, B. pertussis, B. bronchiseptica, B. parapertussishu,
the laboratory (136, 141, 147). Initially, experimental vaccines B. parapertussisov (ovine-adapted B. parapertussis), B. avium,
were used to treat and prevent pertussis. Epidemic pertussis B. hinzii, B. holmesii, B. trematum, and B. petrii. B. avium is a
was brought under control in the United States with the wide- bird pathogen causing coryza and rhinotracheitis in poultry
spread use of whole-cell pertussis vaccines in the 1940s and (263, 718). B. hinzii is found mainly as a commensal of the
1950s. Control of the disease has continued in the United respiratory tracts of fowls but has pathogenic potential in im-
States over the last decade with the use of acellular pertussis munocompromised humans (171, 404). B. trematum has been
component DTP vaccines (diphtheria-tetanus toxoids, acellu- isolated from ear infections and skin wounds in humans but
lar pertussis vaccine, adsorbed vaccines) (referred to as DTaP has never been associated with respiratory tract infections
vaccines) (149). (777). B. parapertussisov causes a chronic infection of the sheep
B. bronchiseptica was first isolated during the first decade of respiratory tract (631). B. petrii, the most recently identified
the 20th century by Ferry, McGowan and perhaps others in Bordetella strain, was isolated from the environment and is
studies of dogs suffering from distemper (240, 242, 243, 283, capable of anaerobic growth (791, 792). It was assigned to the
511, 658, 761, 831). Further studies in the early 20th century Bordetella genus based on comparative 16S rDNA sequence
demonstrated B. bronchiseptica infections in many animals and analysis, DNA base composition, isoprenoid quinone content,
also humans (79, 241, 283, 511, 658, 689, 761, 831). DNA-DNA hybridization experiments, and several metabolic
B. parapertussis was first isolated from children with pertussis properties and may represent the only Bordetella strain not
in the 1930s by Eldering and Kendrick (220, 221) and Bradford known to occur in a close pathogenic, opportunistic, or com-
and Slavin (73). Pertussis associated with B. parapertussis in- mensal relationship with an animal or human host.
fections was in general somewhat less severe than that due to The dendrogram in Fig. 1 is based on a combination of mul-
tilocus enzyme electrophoresis, insertion sequence (IS) poly- genes known or suspected to be involved in pathogenicity are
morphisms, and sequence data (including comparative 16S missing in the genomes of human-adapted bordetellae. It is
rDNA sequence analysis and microarray based comparative interesting that while Bordetella subspecies have been studied
genome hybridization) analyses (189, 264, 608, 783). It con- extensively for years, full functional data are available for only
firms the close genetic relationship of all known bordetellae, a small portion of the Bordetella genomes. For instance, ge-
with the B. pertii facultative anaerobe as the proposed environ- nome sequence analysis predicts that at least 30 genes are in-
mental progenitor of pathogenic bordetellae. It further dem- volved in biosynthesis of lipopolysaccharides (LPS) for B. bron-
onstrates remarkably limited genetic diversity among B. per- chiseptica, but functional data are available for only 13 of these
tussis, B. parapertussis, and B. bronchiseptica strains; as such, genes. A detailed list of the functional annotation for predicted
these strains have been reclassified as “subspecies” of a single proteins from the sequenced Bordetella strains is presented in
species with different host adaptations. For these subspecies, Table 1.
B. bronchiseptica is the likely evolutionary progenitor and
B. pertussis and B. parapertussishu are considered two separate
VIRULENCE DETERMINANTS AND
human-adapted lineages of B. bronchiseptica. B. pertussis,
MOLECULAR PATHOGENESIS
B. parapertussis (human and ovine), and B. bronchiseptica
strains are collectively referred to as the “B. bronchiseptica Animal Models
cluster” (264). It must be noted that although 16S rDNA anal-
ysis and IS element polymorphisms place B. holmesii as part of B. pertussis pathogenesis has been studied mainly by using
the B. bronchiseptica cluster, B. holmesii does not share any the mouse model of respiratory tract infection (90, 91, 265,
characteristics of virulence protein expression with the mem- 514, 778, 805). Intranasal and aerosol challenge experiments
bers of the B. bronchiseptica cluster based on immunological using B. pertussis and B. parapertussishu in mice have yielded
detection with specific antisera and DNA hybridization exper- important insights into the roles of specific virulence factors in
iments (264). determining colonization. Mouse respiratory as well as intra-
B. parapertussishu strains are particularly interesting. They cerebral challenge experiments have been used to determine
comprise a single electrophoretic type and, based on PCR- immunity generated in response to B. pertussis infection (548,
based RAPD fingerprinting and IS element analyses, are near- 549, 687). However, since B. pertussis and B. parapertussishu are
ly identical regardless of their geographic origin or year of restricted to humans, often large infectious doses are required
isolation (783, 842). A plausible hypothesis is that B. para- to colonize the animals. This suggests that the above animal
pertussishu evolved relatively recently from a closely related model systems are limited in their degree of sensitivity to
B. bronchiseptica strain (Fig. 1). Given its long-standing posi- accurately reflect events occurring during infection of the hu-
tion as a host-restricted human pathogen, the isolation of man host. In contrast, animal models have been developed for
strains identified as B. parapertussis from asymptomatic and B. bronchiseptica that reflect both the natural course of infec-
pneumonic sheep came as a considerable surprise and prompt- tion and infections that are skewed towards disease (175–177,
ed speculation that cross-species transmission may occur. Sub- 324, 325, 506, 841). Specific-pathogen-free rabbits, rats, and
sequent studies, however, clearly demonstrated that human mice inoculated intranasally by delivery of a 5-␮l droplet of a
and ovine strains of B. parapertussis represent distinct clonal B. bronchiseptica culture to the nares become persistently col-
lineages that diverged independently from B. bronchiseptica onized in the nasal cavity, larynx, trachea, and lungs without
(782). B. parapertussisov isolates are genetically diverse, and showing any signs of clinical disease. Larynx, trachea, and lung
there appears to be little or no transmission between the sheep specimens show no gross pathology, and histological examina-
and human reservoirs. tions of tissue sections rarely show inflammation or abnormal
Investigators at the Sanger Center recently sequenced the tissue structure. A B. bronchiseptica strain, RB50, was isolated
genomes of three Bordetella subspecies (B. pertussis strain To- from the nose of a naturally infected New Zealand White
hama 1, B. parapertussishu strain 12822, and B. bronchiseptica rabbit and has been used extensively to understand mecha-
strain RB50) (608). The genome of RB50 is 5.34 Mb, while nisms of Bordetella pathogenesis in animal models (175). Its
those of Tohama 1 and 12822 are 4.09 and 4.77 Mb, respec- intranasal 50% infective dose for rabbits, rats, and mice is less
tively. The differences in genome sizes and sequence compar- than 200, 25, and 5 CFU, respectively, indicating the ability of
ison of the three genomes support the hypothesis that B. per- these model systems to accurately reflect the characteristics of
tussis and B. parapertussis recently and independently evolved naturally occurring infection. The availability of mice with
from B. bronchiseptica-like ancestors. Interestingly, this restric- knockout mutations in genes required for immune effector
tion to the human host included significant loss of DNA, per- functions has allowed an investigation of interactions between
haps corresponding to a more “streamlined” genome. In com- Bordetella virulence factors and host defense (324, 424, 491,
parison with Tohama 1 and 12822, a large portion of the extra 621). These models are appropriate for probing mechanisms of
DNA in RB50 is attributed to prophage and prophage rem- colonization and signal transduction, since the balance is
nants (608). Other genes lost by B. pertussis and B. parapertussis tipped towards disease in immunocompromised animals (324).
include loci involved in small-molecule metabolism, membrane Such model systems also provide an excellent opportunity to
transport, and biosynthesis of surface structures. In addition to understand how bacteria establish persistent infections without
this substantial gene deletion, B. pertussis and B. parapertussis causing damage to their hosts. As a result of the extremely high
contain 358 and 200 pseudogenes, respectively, many of which degree of genetic relatedness of members for the B. bronchi-
have been inactivated by insertion of IS elements, in-frame septica cluster, a comparative analysis of the similarities and
stop codons, or frameshift mutations. Interestingly, very few differences in the infectious cycles of Bordetella subspecies
TABLE 1. Functional annotation of predicted proteins based on genome sequence analysis of B. pertussis strain Tohama I,
B. parapertussishu strain 12822 and B. bronchiseptica strain RB50
No. of proteins with assigned COGs inb:
Functional annotationa
B. pertussis (3,436) B. parapertussishu (4,185) B. bronchiseptica (4,994)
Information storage and processing
Translation, ribosomal structure, and biogenesis 162 191 201
Transcription 269 362 442
DNA replication, recombination, and repair 337 143 133
RNA processing 1 1
Chromatin structure and dynamics 4 4 5
Cellular processes
Cell division and chromosome partitioning 30 30 35
Posttranslational modification, protein turnover, chaperones 100 132 140
Cell envelope biogenesis, outer membrane 175 208 226
Cell motility and secretion 57 64 82
Inorganic ion transport and metabolism 176 203 249
Signal transduction mechanisms 73 102 116
Intracellular trafficking and secretion 52 59 64
Defense mechanisms 25 31 41
Metabolism
Energy production and conversion 212 284 352
Carbohydrate transport and metabolism 139 197 237
Amino acid transport and metabolism 352 475 556
Nucleotide transport and metabolism 50 59 60
Coenzyme metabolism 107 114 121
Lipid metabolism 141 200 253
Secondary metabolites biosynthesis, transport and catabolism 98 143 172
Poorly characterized
General function prediction only 275 350 402
Unknown function 286 432 516
Undetermined COGs 315 401 591
Total no. of proteins with assigned COGs 3,121 3,784 4,403

a
Functional classifications are based on COG (Clusters of Orthologous Groups) categories. Additional annotated sequence information can be obtained from the
Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes/) and from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genomes
/MICROBES/Complete.html).
b
Numbers of predicted proteins are given in parentheses.
serves as a guide to understanding fundamental features of and IV were relatively harmless. Based on further extensive
bacterium-host interactions. analyses, Lacey pioneered a hypothesis that Bordetella could
exist in three distinct phenotypic modes, designated X, I, and
C, in response to environmental signals (437). Several subse-
Bordetella Virulence Regulon
quent studies have demonstrated that in the laboratory, bvgAS
B. pertussis, B. parapertussis (human and ovine), and B. bron- expression can be activated by growth at 37°C in the relative
chiseptica share a nearly identical virulence control system absence of MgSO4 or nicotinic acid (527, 528). Bordetellae
encoded by the bvgAS locus. BvgA and BvgS are members of a grown under such “nonmodulating” conditions are referred to
two-component signal transduction system that uses a four- as Bvg⫹-phase-specific bacteria and correspond to Lacey’s X
step His-Asp-His-Asp phosphotransfer signaling mechanism mode (Fig. 2A). Signal inputs detected by the periplasmic
(Fig. 2A) (773–775). BvgA is a 23-kDa DNA-binding response domain of BvgS are relayed through the membrane to the
regulator (70). BvgS is a 135-kDa transmembrane sensor ki- transmitter domain, which autophosphorylates at His-729 by a
nase containing a periplasmic domain, a linker region (L), a reaction that is reversible in vitro (544, 773–775). His-729 then
transmitter (T), a receiver (R), and a histidine phosphotransfer donates the phosphoryl group to Asp-1023 of the receiver
domain (HPD) (730). BvgA and BvgS from B. pertussis and domain. Asp-1023 can donate the phosphoryl group to His-
B. bronchiseptica have 100 and 96% amino acid sequence iden- 1172 of the HPD or to water to form inorganic phosphate. The
tity, respectively, and the loci are functionally interchangeable HPD can then transfer the phosphate back to BvgS or, alter-
(496). natively, can phosphorylate (and thus activate) BvgA at Asp-
BvgAS is environmentally responsive, although the relevant 54. On phosphorylation by BvgS, BvgA promotes the transcrip-
signals for regulating the bvgAS locus in vivo are yet to be tion of Bvg⫹-phase-specific genes called vag genes (for “vir-
determined. Over 70 years ago, Leslie and Gardner studied activated genes” [bvgAS was originally termed vir]) by binding
agglutinogenic properties of B. pertussis and described four to cis-acting sequences in their promoter regions. An addi-
phases (phases I, II, III, and IV) of the organism in response to tional class of genes, termed vrg (for “vir-repressed genes), is
varied environmental conditions (442, 454). Phases I and II repressed by the products of the bvgAS locus (7, 8, 425). The
were highly toxic for guinea pigs and mice, whereas phases III repression of these genes is mediated via a 32-kDa cytoplasmic
FIG. 2. (A) The BvgAS phosphorelay. BvgS is a transmembrane sensor protein consisting of a periplasmic domain (P), a linker region (L), and
histidine kinase (HPK), receiver (R), and histidine phosphotransfer domains (HPD). BvgA is a response regulator that contains receiver (R) and
helix-turn-helix (HTH) domains. Under inducing signals, BvgS autophosphorylates and initiates a phosphorelay that eventually leads to the
phosphorylation and activation of BvgA. The sequential steps in the phosphorelay and the amino acid residues involved are shown. The bvgS-C3
allele confers constitutive activity. BvgAS controls as least three distinct phenotypic phases in response to environmental conditions. The Bvg⫹
phase or X mode is necessary and sufficient for respiratory tract colonization and is associated with the expression of virulence factors. The Bvgi
phase is hypothesized to be important for respiratory transmission and is characterized by the expression of a subset of Bvg⫹ phase-specific factors
as well as factors expressed maximally in the Bvgi phase. B. pertussis and B. bronchiseptica express a significantly different array of proteins in their
Bvg⫺ phase. The Bvg⫺ phase of B. bronchiseptica is necessary and sufficient for growth under nutrient-limiting conditions and is predicted to play
a role in survival in the environment. Other abbreviations: om, outer membrane; cm, cell membrane. (B) Expression curves for the four classes
of genes regulated by BvgAS. Genes expressed maximally in the Bvg⫹ phase (such as cyaA) are referred to as “late” Bvg-activated genes and are
represented by the black curve (curve 1). Genes that are expressed maximally under both Bvg⫹ and Bvgi phase conditions (such as fhaB) are
referred to as “early” Bvg-activated genes and are represented by the green curve (curve 2). Genes expressed maximally only under Bvgi phase
conditions (such as bipA) are represented by the gold curve (curve 3). Finally, genes that are repressed by BvgAS and expressed maximally only
under Bvg⫺ phase conditions are represented by the red curve (curve 4). Abbreviation: nic, nicotinic acid.
repressor protein called BvgR (533). The gene encoding BvgR of vag genes and repression of vrg genes. The Bvg⫺ phase
is located immediately downstream of the bvgAS locus and is corresponds to Lacey’s C mode (Fig. 2A).
also activated by BvgA (531, 532). The BvgAS phosphorelay The BvgS receiver is a pivotal component of the phosphore-
can be inactivated by growing bordetellae under “modulating” lay, acting as a biochemical checkpoint by mediating phosphor-
conditions, such as at 25 or 37°C in the presence of ⱖ10 mM ylation and dephosphorylation of the HPD and BvgA, as well
nicotinic acid or ⱖ40 mM MgSO4 (527). Under these Bvg⫺ as dephosphorylation of the transmitter. Mutational analyses
phase conditions, BvgAS is unable to activate the transcription of bvgAS have provided a number of tools for deciphering the
structure of the virulence regulon and for investigating the role growing within biofilms appear to be more resistant to antibi-
of Bvg-mediated signal transduction in vivo. Mutations that otics and host immune defenses than are their planktonic
alter as well as those that completely abrogate signal transduc- counterparts (457). While the physiological relevance of Bvg-
tion have been identified. The bvgS-C3 allele locks BvgS into dependent biofilm formation in B. bronchiseptica remains to be
an active form, rendering it insensitive to modulating signal determined, studying biofilm formation has potential implica-
(175, 544). Strains containing this mutation constitutively ex- tions in understanding the life-style of B. bronchiseptica (versus
press all known Bvg-activated virulence factors. A deletion in B. pertussis) as a chronically colonizing pathogen. Systematic
bvgS, on the other hand, locks the bacteria in the Bvg⫺ phase analysis of gene expression in the Bvg⫹, Bvgi, and Bvg⫺ phases
and renders them avirulent (544). The Bvg⫺ phase of B. bron- of Bordetella reveals the existence of at least four classes of
chiseptica is characterized by expression of motility and several Bvg-regulated genes: (i) those that are expressed maximally
metabolic processes involved in redox reactions and amino only in the Bvg⫹ phase, (ii) those that are expressed maximally
acid transport (8, 230, 268, 517). In contrast, B. pertussis and in both the Bvg⫹ and Bvgi phases, (iii) those that are expressed
B. parapertussishu are nonmotile due to multiple frameshifted exclusively in the Bvgi phase, and (iv) those that are expressed
and transposon-disrupted genes in their flagellar loci (608). only in the Bvg⫺ phase (Fig. 2B). From a phylogenetic per-
The Bvg⫺ phase of B. pertussis is characterized by the expres- spective, however, Bvg-regulated genes fall into two categories.
sion of several outer membrane proteins of unknown function Some loci are commonly expressed by B. pertussis, B. paraper-
(287). Experiments with phase-locked and ectopic expression tussis (human and ovine), and B. bronchiseptica. Their products
mutants have demonstrated that the Bvg⫹ phase is necessary are highly similar and in some cases interchangeable between
and sufficient for respiratory tract colonization by B. pertussis different subspecies. In contrast, other loci which are present in
and B. bronchiseptica (175, 496). These experiments also dem- the genomes of all four subspecies appear to be differentially
onstrated that the Bvg⫺ phase of B. bronchiseptica was neces- expressed. These genes provide important clues for under-
sary and sufficient for survival under nutrient-limiting condi- standing fundamental differences between Bordetella-host in-
tions, suggesting the existence of an environmental reservoir teractions.
(175). An environmental reservoir for B. pertussis and B. para-
pertussishu seems less plausible, as these strains are more fas- Commonly Expressed Loci
tidious and appear to be confined to transmission by the re-
spiratory droplet route. A role for the Bvg⫺ phase of these Based on in vitro attachment assays and in vivo colonization
human-adapted bordetellae remains to be identified. experiments, several surface-exposed and secreted factors have
So, why is this BvgAS phosphorelay so complex? One pos- been proposed to play a role in Bordetella pathogenesis (Table
sibility is that multiple steps allow multiple levels of control. 2). Putative adhesins commonly expressed in the Bvg⫹ phase
The complexity of the system may also reflect the ability to of all four subpecies of the B. bronchiseptica cluster include
respond to signal intensity in a graded manner. Indeed, it was filamentous hemagglutinin (FHA), fimbriae (FIM), and per-
recently demonstrated that instead of controlling a biphasic tactin (PRN), 1 of the 13 autotransporter proteins encoded in
transition between the Bvg⫹ and Bvg⫺ states, BvgAS controls the Bordetella genomes: Additional autotransporters expressed
expression of a spectrum of phenotypic phases in response to by members of the B. bronchiseptica cluster include BrkA,
quantitative differences in environmental cues (176, 203, 204, SphB1, and Vag8. Commonly expressed Bvg⫹ phase toxins
732). Wild-type bordetellae grown in the presence of sub- include a bifunctional adenylate cyclase/hemolysin (CyaA) and
modulating conditions, such as concentrations of 0.4 to 2 mM dermonecrotic toxin (DNT). The first identified Bvgi-phase-
nicotinic acid for B. bronchiseptica, express a phenotypic phase specific factor, BipA, also seems to be commonly expressed in
distinct from those described above. This phase is character- B. pertussis and B. bronchiseptica and at significantly reduced
ized by the absence of Bvg-repressed phenotypes, the presence levels in B. parapertussisov. Bvg⫺-phase-specific loci expressed
of a subset of Bvg-activated virulence factors and the expres- in both B. pertussis and B. bronchiseptica include wlb, which is
sion of several polypeptides that are expressed maximally or involved in LPS synthesis. In addition, commonly expressed
exclusively in this phase. Bordetellae growing in this phase Bvg-independent factors such as tracheal cytotoxin (TCT) play
display phenotypes intermediate between the Bvg⫹ and Bvg⫺ an important role in pathogenesis. Orthologous gene products
phases; as such, this phase has been designated the Bvg-inter- display high levels of amino acid sequence identity when com-
mediate (Bvgi) phase and corresponds to Lacey’s I mode (Fig. pared between Bordetella subspecies. For instance, the B. per-
2A) (176). A single nucleotide change in bvgS at position 733 tussis and B. bronchiseptica FHA, PRN, CyaA, and BipA pro-
resulting in a Thr-to-Met substitution mimics a Bvgi-phase teins have 92, 91, 97, and 95% amino acid sequence identity,
phenotype. Bvgi phase bordetellae containing this mutation respectively (608). These factors are likely to perform similar,
(designated bvgS-II) display increased resistance to nutrient if not identical, functions during respiratory tract infection and
limitation and a decreased ability to colonize the respiratory polymorphisms may in some cases reflect specific host adapta-
tract compared to wild-type Bvg⫹-phase bacteria (176). The tions.
Bvgi phase appears to be conserved between B. pertussis and
B. bronchiseptica, and is predicted to play a role in the respi- Differentially Expressed and Differentially
ratory transmission of these strains (257). Recently, the Bvgi Regulated Loci
phase of B. bronchiseptica was shown to be associated with
biofilm formation (383). Biofilms are bacterial communities From an evolutionary viewpoint, differentially expressed loci
that are attached to a solid surface and have characteristics are an informative class of Bvg-regulated genes. The ptx-ptl
different from free-living planktonic bacteria (173). Bacteria operon, which encodes the structural subunits of pertussis
TABLE 2. Expression and function information for various virulence determinants for B. pertussis and B. bronchiseptica
Gene expression Protein expressiona
Virulence determinant Description
B. pertussis B. bronchiseptica B. pertussis B. bronchiseptica
Adhesins
Filamentous hemagglutinin 220-kDa surface-associated and secreted protein; dominant ⫹ ⫹ ⫹ ⫹
(FHA) adhesin; required for tracheal colonization; highly immu-
nogenic; primary component of acellular pertussis vaccines
Fimbriae (FIM) Filamentous cell surface structures; required for persistent tra- ⫹ ⫹ ⫹ ⫹
cheal colonization; component of some acellular pertussis
vaccines: required for protective immunity to infection
Autotransporters
Pertactin (PRN) 68–70-kDa surface protein; mediates eukaryotic cell binding ⫹ ⫹ ⫹ ⫹
in vitro; enhances protective immunity
Vag8 95-kDa outer membrane protein ⫹ ⫹ ⫹ ⫹
BrkA 73-kDa surface-associated N-terminal passenger domain ⫹ ⫹ ⫹ ⫹
with 30-kDa outer membrane C-terminal protein; putative
adhesin; confers serum resistance and protection against
antimicrobial peptides in B. pertussis
SphB1 Subtilisin-like Ser protease/lipoprotein required for FHA ⫹ ⫹ ⫹ ⫹
maturation in B. pertussis
Tracheal colonization factor 60-kDa secreted protein; role in tracheal colonization in ⫹ ⫺ ⫹ ⫺
(TcfA) murine model
Toxins
Pertussis toxin (PT) A-B-toxin; ADP-ribosylates G proteins; responsible for per- ⫹ ⫺ ⫹ ⫺
tussis-associated lymphocytosis; strong adjuvant and pri-
mary component of pertussis vaccines
Adenylate cyclase (CyaA) Calmodulin-activated RTX family toxin with dual adenylate ⫹ ⫹ ⫹ ⫹
cyclase/hemolysin activity; acts as anti-inflammatory and
antiphagocytic factor during infection
Type III secretion Allows Bordetella to translocate effector proteins directly into ⫹ ⫹ ⫺ ⫹
host cells; required for persistent tracheal colonization; in-
hibits host immune response; activates ERK1/2; mislocalizes
NF-␬B; causes caspase-independent cell death
Dermonectrotic toxin (DNT) 160-kDa heat-labile secreted toxin; activates Rho; induces ⫹ ⫹ ⫹ ⫹
necrosis in vitro
Tracheal cytotoxin (TCT) Disaccharide-tetrapeptide monomeric by-product of pepti- ⫹ ⫹ ⫹ ⫹
doglycan synthesis; causes mitochondrial bloating, disrup-
tion of tight junctions, damage to cilia, IL-1␣ and NO˙
production
LPS
wlb locus Consists of 12 genes required for LPS (band A) biosynthesis ⫹ ⫹ ⫹ ⫹
wbm locus Encodes O antigen; may be important for confering serum ⫺ ⫹ ⫺ ⫹
resistance
PagP Mediates palmitoylation modification of lipid A; may be ⫺ ⫹ ⫺ ⫹
important for persistence and resistance to serum killing
Additional loci
Flagella Peritrichous cell surface appendages required for motility; ⫺ ⫹ ⫺ ⫹
highly antigenic; ectopic expression of flagella in the Bvg⫹
phase is detrimental to the infection cycle
Type IV pili Polar pili usually with an N-methylated phenylalanine as the ⌬ ND NA ND
N-terminal residue; possible functions include adherence,
twitching motility, and DNA uptake
Capsule A type II polysaccharide coat predicted to be comprised of ⌬ ND ND ND
an N-acetylgalactosaminuronic acid Vi antigen-like poly-
mer; possible role in protection against host defense
mechanisms or survival in the environment
Alcaligin A siderophore for complexing iron, which is internalized ⫹ ⫹ ⫹ ⫹
through outer membrane receptors (B. bronchiseptica
encodes 16 such receptors while B. pertussis encodes 12);
iron uptake may be important for survival within
mammalian hosts
Vrg loci Several loci of uncharacterized function ⫹ ⫺ ⫹ ⫺
a
⫹, positive for expression; ⫺, no expression; ⌬, genome contains deletion mutations in these genes; ND, not determined; NA, not applicable.
toxin (PT) and its export apparatus, falls into this category. The exist in the LPS structures of all four subspecies (discussed in
ptx-ptl locus is present in all four subspecies of the B. bronchi- detail later in this review). A type III secretion system (TTSS),
septica cluster, but expression and toxin production are ob- which allows gram-negative pathogens to deliver bacterial ef-
served only in the Bvg⫹ phase of B. pertussis. Differences also fector proteins directly into eukaryotic cells and alter host cell
signaling functions, has been identified and characterized in centered at position ⫺88.5 relative to the start of transcrip-
Bordetella subspecies. Type III genes are intact and highly tion (677). Binding of a phosphorylated BvgA dimer to this
conserved in members of the B. bronchiseptica cluster; how- site, followed by cooperative binding of two additional Bvg⬃P
ever, only B. bronchiseptica and B. parapertussisov readily dimers 3⬘ to the high-affinity site, leads to the activation of
display TTSS-associated phenotypes in vitro. Comparative fhaB transcription. Binding of the first BvgA⬃P dimer to the
sequence analysis of B. pertussis, B. parapertussishu, and B. primary high-affinity binding site seems to be the critical first
bronchiseptica has also revealed the existence of a type IV pilin step for fhaB transcription, since binding of the second and
biogenesis cluster present only in B. bronchiseptica; further third dimers was found to be entirely cooperative and in-
analysis of this recently discovered locus is pending (638). Like- dependent of nucleotide sequence (69, 71).
wise, a locus comprising three regions predicted to be involved FHA is synthesized as a 367-kDa precursor, FhaB, which
in export/modification, biosynthesis, and transport of a type II undergoes extensive N- and C-terminal modifications to form
capsule has been identified in the B. bronchiseptica genome the mature 220-kDa FHA protein. It is exported across the
(608). Capsules are often key contributors for enabling patho- cytoplasmic membrane by a Sec signal peptide-dependent
gens to survive host defense mechanisms or harsh ex vivo en- pathway. Its translocation and secretion across the outer mem-
vironments. While the central part of the capsular locus is brane requires a specific accessory protein, FhaC. FhaC folds
mostly intact in B. pertussis, its 5⬘ end appears to have under- into a transmembrane ␤-barrel that facilitates secretion by
gone massive IS element-mediated rearrangements and dele- serving as an FHA-specific pore in the outer membrane (304,
tions (608). In B. parapertussishu, two genes have undergone 392). FHA most probably crosses the outer membrane in an
nonsense and frameshift mutations (608). The above listed dif- extended conformation and acquires its tertiary structure at
ferences may contribute to determining host specificity or the the cell surface, following extensive N- and C-terminal proteo-
nature of infection. lytic modifications which have recently been characterized in a
Although differences in Bvg⫹-phase phenotypes expressed series of elegant experiments (180, 181, 303, 304, 391–393). On
by Bordetella subspecies are apparent, they exist in a back- translocation across the cytoplasmic membrane, the N termi-
ground of overall similarity. In contrast, the Bvg⫺ phases of nus of FhaB undergoes cleavage of an additional 8 to 9 kDa at
these organisms are remarkably different. To date, there are a site that corresponds to a Lep signal peptidase recognition
few loci that are coexpressed in the Bvg⫺ phases of all four sequence. This portion of the N terminus is predicted to be
subspecies (174). An interesting example involves the motility important for interacting with FhaC. Once at the cell surface,
phenotype of B. bronchiseptica. Although B. pertussis and approximately 130 kDa of the C terminus of FhaB is proteo-
B. parapertussis contain the entire complement of motility lytically removed by a newly identified subtilisin-like autotrans-
genes, they are not expressed and these subspecies are there- porter/protease, SphB1 (180, 181). FHA release depends on
fore nonmotile (7, 8). In a similar vein, the B. pertussis vrg SphB1-mediated maturation. The C terminus of the FhaB pre-
loci encode several outer membrane proteins that are specif- cursor is predicted to serve as an intramolecular chaperone,
ically expressed in the Bvg⫺ phase. The vrg genes in the preventing premature folding of the protein. Together, FHA
B. bronchiseptica genome appear to be transcriptionally silent. and FhaC serve as prototypes for members of the two-partner
Understanding the role of Bvg-mediated signal transduction secretion (TPS) system, which typically include secreted pro-
in the Bordetella life cycle is crucial in determining the patho- teins with their cognate accessory proteins from several gram-
genic mechanisms and evolutionary trends involved in Borde- negative bacteria. Although efficiently secreted via this process,
tella-host interactions. It provides insightful details into the a significant amount of FHA remains associated with the cell
dynamics of virulence gene regulation and its implications for surface by an unknown mechanism.
host adaptations. In vitro studies using a variety of mammalian cell types
suggest that FHA contains at least four separate binding do-
mains that are involved in attachment. The Arg-Gly-Asp
Virulence Determinants
(RGD) triplet, situated in the middle of FHA and localized to
The virulence determinants of B. pertussis and B. bronchi- one end of the proposed hairpin structure, stimulates adher-
septica are discussed in Table 2. ence to monocytes/macrophages and possibly other leukocytes
Filamentous hemagglutinin. The virulence determinants of via the leukocyte response integrin/integrin-associated protein
B. pertussis and B. bronchiseptica are discussed in Table 2. FHA (LRI/IAP) complex and complement receptor type 3 (CR3)
is a highly immunogenic, hairpin-shaped molecule which serves (384, 654, 690). Specifically, the RGD motif of FHA has been
as the dominant attachment factor for Bordetella in animal implicated in binding to very late antigen 5 (VLA-5; an ␣5␤1-
model systems (174, 655). It has been included as a component integrin) of bronchial epithelial cells (387). Ligation of VLA-5
in most acellular pertussis vaccines (149). Protein structure and by the FHA RGD domain induces activation of NF-␬B, which
immunological analyses suggest that the FHA proteins from then causes the up-regulation of epithelial intercellular adhe-
B. pertussis and B. bronchiseptica are similar in their molecular sion molecule 1 (ICAM-1) (385, 386). ICAM-1 up-regulation is
mass, structure dimensions, and hemagglutination properties involved in leukocyte accumulation and activation at the site of
and have a common set of immunogenic epitopes (529, 594, 683). bacterial infection (59, 593, 762). Interestingly, purified PT can
FHA is encoded by fhaB, one of the strongest BvgAS-acti- abrogate NF-␬B activation by this mechanism, suggesting the
vated genes. It is maximally expressed under both Bvg⫹- and involvement of a PT-sensitive G protein in the signaling pro-
Bvgi-phase conditions. The fhaB promoter contains a primary cess (the role of PT is discussed in detail later in this review)
high-affinity BvgA-binding site consisting of two nearly perfect (386). The CR3 recognition domain in FHA has yet to be
inverted heptanucleotide repeats [TTTC(C/G)TA] that are identified. FHA also possesses a carbohydrate recognition do-
main (CRD), which mediates attachment to ciliated respira- McGuirk and Mills have demonstrated that interaction of
tory epithelial cells as well as to macrophages in vitro (636). In FHA with receptors on macrophages results in suppression of
addition, FHA displays a lectin-like activity for heparin and the proinflammatory cytokine, interleukin-12 (IL-12), via an
other sulfated carbohydrates, which can mediate adherence to IL-10 dependent mechanism (513, 515). These data reveal a
nonciliated epithelial cell lines. This heparin-binding site is role for FHA in facilitating persistence by curbing protective
distinct from the CRD and RGD sites and is required for Th1 immune responses. In contrast, a subsequent study sug-
FHA-mediated hemagglutination (530). FHA is also required gests that FHA can elicit proinflammatory and proapoptotic
for biofilm formation in B. bronchiseptica (383). responses in human monocyte-like cells and bronchial epithe-
A role for FHA in vivo has been more difficult to discern lial cells (2). As mentioned earlier, binding of FHA to the
mainly due to the lack of a natural animal host (other than VLA-5 integrin induces the expression of proinflammatory
humans) for B. pertussis, as well as the complexity of this mol- genes, such as ICAM-1, in an NF-␬B-dependent manner in
ecule and its associated biological activities. In a rabbit model human bronchial epithelial cells (386). FHA-specific antibod-
of respiratory tract infection, fewer FHA mutants compared to ies are also necessary to protect against reinfection by B. bron-
wild-type B. pertussis were recovered from the lungs at 24 hs chiseptica in the rat model (503). Specifically, animals were
after intratracheal inoculation (690). A comparison of in vivo challenged with marked B. bronchiseptica strains 30 days after
results with in vitro binding characteristics of the various mu- receiving a primary inoculation of wild-type or mutant B. bron-
tant strains used in the above study suggested that wild-type chiseptica. The animals developed a robust anti-Bordetella se-
B. bronchiseptica was capable of adhering to both ciliated ep- rum antibody response by the 30-day time point, which was
ithelial cells and macrophages. Further, competition experi- monitored both qualitatively and quantitatively by enzyme-
ments with lactose and anti-CR3 antibody suggested that both linked immunosorbent assay (ELISA). The presence of anti-
CRD- and RGD-dependent binding was involved (690). Using FHA serum titers was correlated with the ability of the animal
mouse models, however, others have found FHA mutants to be to resist further infection with the marked B. bronchiseptica
indistinguishable from wild-type B. pertussis in their ability to challenge strains. However, antibodies to FHA also inhibit the
persist in the lungs but defective for tracheal colonization (421, phagocytosis of B. pertussis by neutrophils (554). Taken to-
557). Still others, also using mouse models, have observed no gether, these data suggest FHA performs several immuno-
difference between FHA mutants and wild-type B. pertussis modulatory functions in vivo.
(284, 419, 663, 810). Data regarding the role played by FHA in the pathogenesis
Construction and analysis of two types of FHA mutant de- of B. pertussis infections in humans can be gleaned from recent
rivatives of B. bronchiseptica, one containing an in-frame de- pertussis vaccine studies. Vaccinees who received FHA con-
letion in the structural gene fhaB and one in which FHA is taining pertussis vaccines mounted a substantial antibody re-
expressed ectopically in the Bvg⫺ phase, in the absence of the sponse to this protein (217, 218, 332). In general, acellular
array of Bvg⫹-phase virulence factors with which it is normally vaccines which contain FHA as well as PT toxoid have slightly
expressed, proved invaluable in determining a role for FHA (7, greater efficacy than monocomponent PT toxoids (3, 131, 149,
177). Comparison of these mutants with wild-type B. bronchi- 734, 735, 737). However, one whole-cell component DTP vac-
septica showed that FHA is both necessary and sufficient to cine in which vaccinees did not mount an antibody response to
mediate adherence to rat lung epithelial cells in vitro. Using a FHA was nevertheless highly efficacious (218, 332, 334, 719).
rat model of respiratory infection, FHA was shown to be ab- Most importantly, in two studies in which serologic correlates
solutely required, but not sufficient, for tracheal colonization in of immunity were determined, it was found that FHA made no
healthy, unanesthetized animals (177). FHA was not required contribution to protection (148, 736).
for initial tracheal colonization in anesthetized animals, how- Analysis of the B. pertussis, B. bronchispetica, and B. para-
ever, suggesting that its role in establishment may be dedicated pertussishu genomes revealed the existence of two additional
to overcoming the clearance activity of the mucociliary esca- genes, fhaS and fhaL, which encode FHA-like proteins (608).
lator (177). While all the in vitro and in vivo studies so far While orthologs of these genes are conserved among the Bor-
demonstrate a predominant role for FHA as an adhesin, the detella subspecies, differences exist in their internal sequences.
release of copious amounts of FHA from the cell surface seems Bordetella subspecies display differential binding to ciliated
counterintuitive since adhesins typically remain associated with cells derived from different hosts, suggesting that host speci-
the bacterial surface to promote maximum attachment. The ficity may in part be dependent on the interaction of bacterial
significance of FHA release during bacterial infection was in- adhesins to their host receptors (770). Analysis of fhaS and
vestigated using a B. pertussis SphB1-deficient mutant in a fhaL gene products may be of interest in this regard. It may
mouse model of respiratory tract infection (179). SphB1 mu- also explain the exact contribution of FHA in modulating the
tants are incapable of secreting FHA, and mature FHA re- host immune response.
mains surface associated in these strains. These mutants were Agglutinogens. Agglutinogens (AGGs) are surface proteins
found to be defective in their ability to multiply and persist in that, with infection, elicit the production of antibodies that
the lungs of mice, despite their increased adhesiveness in vitro. cause the agglutination of Bordetella organisms in vitro (10,
Since surface-associated FHA also causes autoagglutination of 219, 489, 666, 667, 803). Early studies determined 14 antigenic
bordetellae, a secondary role for FHA may be to facilitate the types of AGGs, 6 of which were specific for B. pertussis (666).
dispersal of bacteria from microcolonies and their detachment A serotyping scheme was developed from the results of the
from epithelial surfaces to promote bacterial spread. agglutination studies using antisera raised against Bordetella in
B. pertussis inhibits T-cell proliferation to exogenous anti- rabbits following multiple injections of killed organisms. The
gens in vitro in an FHA-dependent manner (67). Further, antisera were made “monospecific” by adsorption with heter-
ologous strains. within a stretch of cytosine residues located between the ⫺10
Of the six AGGs specific for B. pertussis, AGG1 was com- and ⫺35 elements of the fim2, fim3, fimX, and fimN promoters
mon to all strains while AGG2 to AGG6 were found in various (401, 817). The putative promoter region of fimA does not
combinations in different isolated strains (666). Three AGGs contain a “C stretch” and therefore is predicted not to undergo
(AGG1, AGG2, and AGG3) have subsequently been deter- phase variation where expressed. Since slip-strand mispairing
mined to be the main agglutinating antigens, while AGG4, affects transcription of the individual fimbrial genes indepen-
AGG5, and AGG6 have been classified as minor antigens that dently of each other, bacteria may express Fim2, Fim3, FimX,
apparently associate with either AGG2 or AGG3. AGG2 and FimN, FimA, or any combination at any given time. However,
AGG3 have since been determined to be fimbrial in nature all fimbrial serotypes have a common minor fimbrial subunit,
(fimbriae are discussed in detail later in this review). FimD, which forms the tip adhesin (265). The fimD gene is
The nature of AGG1 is not known (666). Since both PRN located within the fimbrial biogenesis operon downstream of
and LPS can function as AGGs, either could be AGG1 (75, fimB and fimC (472, 819). Interestingly, this operon is posi-
462, 551). It must be noted, however, that the original sero- tioned between fhaB and fhaC, genes required for synthesis
typing scheme was based on heat labile antigens, thereby dis- and processing of FHA. Based on the predicted amino acid
counting LPS as AGG1. sequence similarity to the E. coli PapD and PapC proteins,
Studies done over 50 years ago found that protection from FimB and FimC have been proposed to function as a chaper-
pertussis in exposed children correlated with high titers of one and usher, respectively (471, 819). Mutation of any one of
serum agglutinating antibody (agglutinins) (546, 682). In the the genes in the fimBCD locus results in a complete lack of
early 1960s it was noted that the apparent efficacy of British fimbriae on the bacterial cell surface, suggesting that fimBCD
pertussis vaccines had decreased. Preston suggested that this is the only functional fimbrial biogenesis locus on the Borde-
decline in vaccine efficacy was because the vaccine used in tella chromosome (818).
England at the time did not contain AGG3 and the most Attachment to host epithelia is often a crtical, early step in
prevalent circulating B. pertussis strains were serotype 1.3 (640, bacterial pathogenesis. Although fimbriae are implicated in
643). Efficacy in England apparently increased following the this process, it has been difficult to establish a definitive role for
addition of serotype 3-containing strains to the vaccine. This Bordetella fimbriae as adhesins for several reasons. First, the
seemed to support Preston’s opinion (641, 642, 644). However, multiple, unlinked major fimbrial subunit genes, as well as the
the protective unitage of the British vaccine was also increased transcriptional and translational coupling of the fimbrial bio-
at the time, so that the increased efficacy may not have been genesis operon with the fha operon, have impeded the ability
due to the inclusion of serotype 3 strains in the vaccine (667). to construct strains completely devoid of fimbriae. Second, the
Since pertussis seems to have been well controlled in Japan presence of several other putative adhesins with potentially
since 1981 and since none of the DTaP vaccines used in Japan redundant functions has obscured the detection of clear phe-
contain AGG3, it seems that antibody to this antigen is of notypes for Fim⫺ mutants. Finally, since the interactions be-
minor importance in protection against disease (422). How- tween bacterial adhesins and host receptor molecules are ex-
ever, in a small study of B. pertussis isolates in Japan it was pected to be highly specific, the use of heterologous hosts for
found that six of seven collected between 1992 and 1996 were studies with B. pertussis has severely limited the ability to detect
serotype AGG 3 (306). in vivo roles for putative adhesins. Nonetheless, several studies
Fimbriae. Like many gram-negative pathogenic bacteria, suggest that fimbriae may mediate the binding of Bordetella to
bordetellae express filamentous, polymeric protein cell surface the respiratory epithelium via the major fimbrial subunits and
structures called fimbriae (FIM). The major fimbrial subunits to monocytes via FimD (328, 329, 557). Geuijen et al. have
that form the two predominant Bordetella fimbrial serotypes, shown that purified B. pertussis fimbriae, with or without FimD,
Fim2 and Fim3 (AGG2 and AGG3), are encoded by unlinked were able to bind to heparan sulfate, chondroitin sulfate, and
chromosomal loci fim2 and fim3, respectively (470, 558). A dextran sulfate, sugars that are ubiquitously present in the
third unlinked locus, fimX, is expressed only at very low levels mammalian respiratory tract (266). Heparin-binding domains
if at all (660), and recently a fourth fimbrial locus, fimN, was within the Fim2 subunit were identified and found to be similar
identified in B. bronchiseptica (401). B. bronchiseptica and to those of the eukaryotic extracellular matrix protein, fibro-
B. parapertussis contain a fifth gene, fimA, located immediately nectin. Studies by Hazenbos et al. suggest that FimD mediates
upstream of the fimbrial biogenesis operon fimBCD and 3⬘ of the binding of nonopsonized B. pertussis to VLA-5 on the sur-
fhaB, which is expressed and capable of encoding a fimbrial face of monocytes, which then causes activation of CR3,
subunit type, FimA (68). Genome sequence analysis of B. per- thereby enhancing its ability to bind FHA (328, 329). Fimbriae
tussis, B. parapertussishu and B. bronchiseptica reveals that all have also been suggested to play a minor role in biofilm for-
three subspecies contain fim2 and fim3, although the predicted mation (383).
C terminus of Fim2 is variable in B. pertussis (638). FimX is In vivo studies have shown that Fim⫺ B. pertussis strains are
intact in B. pertussis and B. bronchiseptica but frameshifted in defective in their ability to multiply in the nasopharynx and
B. parapertussishu. While fimA is truncated, fimN is deleted in trachea of mice (265, 557). By using a B. bronchiseptica strain
B. pertussis. Further, variations are seen in the FimN C termini devoid of fimbriae but unaltered in its expression of FHA and
of B. bronchiseptica and B. parapertussishu. There is also a novel other putative adhesins, fimbriae have been shown to contrib-
putative fimbrial subunit upstream of fimN in B. bronchiseptica ute to the efficiency of establishment of tracheal colonization
and B. parapertussishu that is missing in B. pertussis (638). and are absolutely required for persistence in the trachea using
In addition to positive regulation by BvgAS, the fim genes both rat and mouse models (506). Moreover, the serum anti-
are subject to fimbrial phase variation by slip-strand mispairing body profiles of animals infected with Fim⫺ bacteria differ
qualitatively and quantitatively from those of animals infected known to date (225, 226). In support of the autotransporter
with wild-type B. bronchiseptica (506). Specifically, fimbriae secretion model, Charles et al. have shown that deletion of the
play an immunomodulatory role by (i) acting as T-independent 3⬘ region of prnBp prevents surface exposure of the molecule
antigens for an early immunoglobulin M IgM response and (ii) (127).
inducing a Th2-mediated component of the host immune re- Other Bordetella proteins with predicted autotransport abil-
sponse to Bordetella infection (503). Challenge experiments ity include TcfA (originally classified as a tracheal coloniza-
suggest that the presence of fimbriae is important for eliciting tions factor) (248), BrkA (238), SphB1 (180), and Vag8 (247).
an immune response that is protective against superinfection All of these proteins show significant amino acid sequence
(S. Mattoo et al., unpublished data). Fimbriae are also impor- similarity in their C termini and contain one or more RGD
tant for exerting an anti-inflammatory function and inhibiting tripeptide motifs. Unlike PRN, BrkA, SphB1, and Vag8, TcfA
killing by lung macrophages in mice (784). is expressed exclusively in B. pertussis. Based on predicted
Data from the two trials in which serologic correlates of amino acid sequence similarity to all of these proteins, the
immunity in children were determined also suggest that anti- B. pertussis genome appears to encode at least three additional
body to FIM contributes to protection (148, 736). In addition, members of this autotransporter family. A lot has been learned
when a vaccine containing PT, FHA, and PRN was compared about Bordetella autotransporters in recent years. As men-
to one which contained FIM 2/3 as well as PT, FHA, and PRN, tioned earlier, SphB1 has been characterized as a subtilisin-like
the latter vaccine displayed significantly greater efficacy (597) Ser protease/lipoprotein that is essential for cleavage and C-
(see the section on DTaP vaccine efficacy, below). terminal maturation of FHA (180). SphB1 is the first reported
Taken together, all the above results suggest FIM-mediated autotransporter whose passenger protein serves as a matura-
interactions with epithelial cells and/or monocytes/macro- tion factor for another protein secreted by the same organism.
phages may play important roles not only in adherence but also BrkA is expressed as a 103-kDa preproprotein that is pro-
in the nature and magnitude of the host immune response to cessed to yield a 73-kDa ␣ (passenger)-domain and a 30-kDa
Bordetella infection. ␤-domain that facilitates transport by functioning dually as a
Pertactin and other autotransporters. Bordetella strains ex- secretion pore and an intramolecular chaperone that effects
press a number of related surface-associated proteins belong- folding of the passenger concurrent with or following translo-
ing to the autotransporter secretion system, that are positively cation across the outer membrane (598, 599). Like PRN and
regulated by BvgAS. The autotransporter family includes func- SphB1, BrkA remains tightly associated with the bacterial sur-
tionally diverse proteins, such as proteases, adhesins, toxins, face. Vag8 is a 95-kDa outer membrane protein that is ex-
invasins, and lipases, that appear to direct their own export to pressed in B. pertussis, B. bronchiseptica, and B. parapertussishu
the outer membrane (344). Autotransporters typically consist (247). The B. pertussis and B. bronchiseptica Vag8 homologs
of an N-terminal region called the passenger domain, which are highly similar, and their C termini show significant homol-
confers the effector functions, and a conserved C-terminal re- ogy to the C termini of PRN, BrkA, and TcfA, suggesting that
gion called the ␤-barrel, which is required for the secretion of Vag8, too, may function as an autotransporter. However,
the passenger proteins across the membrane. The N-terminal cleavage of the ␣-domain from the C terminus may not occur
signal sequence facilitates translocation of the preproprotein in Vag8, since the predicted size of the entire protein encoded
across the inner membrane via the Sec pathway. On cleavage by vag8 corresponds to the size seen by sodium dodecyl sulfate-
of the N-terminal signal in the periplasm, the C terminus folds polyacrylamide gel electrophoresis (247). In contrast, TcfA is
into a ␤-barrel in the outer membrane, forming an aqueous produced as a 90-kDa cell-associated precursor form that is
channel. The linker region between the N and C termini directs processed to release a mature 60-kDa protein (248). It is in-
the translocation of the passenger through the ␤-barrel chan- teresting that TcfA, the only known B. pertussis-specific auto-
nel. On the surface, passenger domains may be cleaved from transporter, is also the only Bordetella autotransporter that is
the translocation unit and remain noncovalently associated not surface associated.
with the bacterial surface or may be released into the extra- The ability of PRN and the other autotransporters to func-
cellular milieu following an autoproteolytic event (for example, tion as adhesins has been tested both in vitro and in vivo. In
when the passenger domain is a protease) or cleavage by an vitro studies demonstrated that purified PRN could promote
endogenous outer membrane protease. the binding of CHO cells to tissue culture wells and that ex-
The first member of autotransporter family to be identified pression of prn in Salmonella or E. coli could increase the
and characterized in Bordetella is PRN. Mature PRN is a 68- adherence and/or invasiveness of these bacteria to various
kDa protein in B. bronchiseptica (556), a 69-kDa protein in mammalian cell lines (228). In contrast, a PRN⫺ strain of
B. pertussis (128), and a 70-kDa protein in B. parapertussis B. pertussis did not differ from its wild-type parent in its ability
(human) (461). It has been proposed to play a role in attach- to adhere to or invade HEp2 cells in vitro or to colonize the
ment since all three PRN proteins contain an Arg-Gly-Asp respiratory tracts of mice in vivo (664). Similarly, a B. bronchi-
(RGD) tripeptide motif as well as several proline-rich regions septica strain with an in-frame deletion mutation in prn was
and leucine-rich repeats, motifs commonly present in mole- indistinguishable from wild-type B. bronchiseptica in its ability
cules that form protein-protein interactions involved in eukary- to establish a persistent respiratory tract infection in rats (P. A.
otic cell binding (226). The B. pertussis, B. bronchiseptica, and Cotter and J. F. Miller, unpublished data). In contrast to the
B. parapertussis PRNs differ primarily in the number of proline- animal model studies discussed above, several pieces of data
rich regions they contain (460). The X-ray crystal structure of derived from vaccine trials and household contact study sug-
B. pertussis PRN suggests that it consists of a 16-strand parallel gest that PRN may be the most important adhesin of B. per-
␤-helix with a V-shaped cross section and is the largest ␤-helix tussis (148, 203, 736). Of the seven vaccine efficacy trials con-
ducted in the early 1990s, two were performed in a manner in catalyzing the palmitoylation of an internal lysine residue
which antibody values at the time of exposure were known (Lys-983) (37, 311). The E. coli HlyA protoxin is also activat-
(148, 736). In both of these trials it was noted that antibody to ed by fatty acyl group modification (322, 375, 388). Whereas
PRN was most important in protection. In addition to these E. coli hemloysin is released in the extracellular medium, the
observations, it is clear that DTaP vaccines which contain PRN majority of the Bordetella CyaA remains surface associated,
in addition to PT and FHA are significantly more effective in
with only a small portion being released in the supernatant.
preventing B. pertussis illness (131, 149, 310) (this is discussed It was recently suggested that FHA may play a role in retain-
in detail in the section on DTaP vaccine efficacy studies below). ing CyaA toxin on the bacterial cell surface; B. pertussis mu-
With regard to the above studies, we predict that protection tants lacking FHA released significantly more CyaA into the
may be afforded by blocking PRN-mediated attachment of medium, and CyaA toxin association with the bacterial sur-
B. pertussis to host cells. More recently, Hellwig et al. have pre- face could be restored by expressing FHA from a plasmid in
sented evidence that anti-pertactin antibodies are required for trans (844). CyaA also inhibits biofilm formation in B. bronchi-
efficient phagocytosis of B. pertussis by the host immune cells septica, possibly via its interaction with FHA and subsequent
(343). interference with FHA function (383).
Potential adhesive functions for TcfA, BrkA, and Vag8 have The eukaryotic surface glycoprotein CD11b serves as the
not been investigated directly, although TcfA⫺ B. pertussis receptor for mature CyaA toxin. Interestingly, surface-bound
strains show a decreased ability to colonize the murine trachea CyaA does not appear to be responsible for host cell intoxica-
compared to wild-type B. pertussis (248). BrkA has been pro- tion; a recent report demonstrates that intoxication requires
posed to play a role in serum resistance and contribute to the close contact of live bacteria with target cells and active secre-
adherence of B. pertussis to host cells in vitro and in vivo. It also tion of CyaA (292). CyaA can enter a variety of eukaryotic cell
protects against lysis by certain classes of antimicrobial pep- types (350). Once inside, CyaA is activated by calmodulin (830)
tides (239). Interestingly, BrkA does not appear to be required and catalyzes the production of supraphysiologic amounts of
for serum resistance of B. bronchiseptica (647). Most recently, cyclic AMP (cAMP) from ATP (89, 163, 164, 321). Purified
Vag8 has been proposed to facilitate the secretion of type III CyaA inhibits chemiluminescence, chemotaxis and superoxide
proteins in B. bronchiseptica (507). This is the first reported
anion generation by peripheral blood monocytes and polymor-
example of an autotransporter involved in regulating type III
phonuclear neutrophils in vitro (611). CyaA also induces apo-
secretion.
ptosis in cultured murine macrophages (419) and inhibits the
Adenylate cyclase. All of the Bordetella species that infect
phagocytosis of B. pertussis by human neutrophils (808, 809).
mammals secrete CyaA, a bifunctional calmodulin-sensitive
Recently, CyaA was shown to inhibit the surface expression of
adenylate cyclase/hemolysin. CyaA is expressed maximally in
costimulatory molecule CD40 and IL-12 production in bone
the Bvg⫹ phase. Unlike the promoter for fhaB, cyaA does not
marrow-derived dendritic cells from C57BL/6 mice infected
contain any high-affinity BvgA-binding sites in its promoter
with B. bronchiseptica (709). It was further shown to be re-
region. Instead, it contains several heptameric variants of the
quired for p38 phosphorylation, suggesting that it plays a role
BvgA-binding consensus 5⬘-TTTCCTA-3⬘ which extend be-
in inhibiting the p38 mitogen-activated protein kinase pathway
tween nucleotides ⫺137 and ⫺51 from the transcriptional
start site. Phosphorylation of BvgA is absolutely required for (709). In vivo studies have shown that, compared to wild-type
binding at these sites. The main target sequence for the B. pertussis, CyaA-deficient mutants are defective in their abil-
BvgA⬃P and DNA interaction is located between positions ity to cause lethal infections in infant mice (305, 810) and to
⫺100 and ⫺80; binding to this centrally located site is pre- grow in the lungs of older mice (284, 305). Taken together,
dicted to trigger cooperative interactions of BvgA⬃P with these results suggest that CyaA functions primarily as an anti-
the neighboring low-affinity sites. inflammatory and antiphagocytic factor during infection.
CyaA is synthesized as a protoxin monomer of 1,706 amino The importance of CyaA in resisting constitutive host de-
acids. Its adenylate cyclase catalytic activity is located within fense mechanisms was further demonstrated by using mice that
the N-terminal 400 amino acids (277, 349). The 1,300-amino- lack the ability to mount an adaptive immune response. SCID,
acid C-terminal domain mediates delivery of the catalytic do- SCID-beige, and Rag-1⫺/⫺ mice, which are deficient in T and
main into the cytoplasm of eukaryotic cells and possesses low B cells and NK cell activities, are dependent on constitutive,
but detectable hemolytic activity for sheep red blood cells (46, innate defense mechanisms for protection against microbial
349, 668). Amino acid sequence similarity between the C- pathogens. When these mice were inoculated with wild-type B.
terminal domain of CyaA, the hemolysins of E. coli (HlyA) and bronchiseptica, they died within 50 days, while those inoculated
Actinobacillus pleuropneumoniae (HppA), and the leukotoxins with the CyaA-deficient strain remained healthy (324). Con-
of Pasteurella hemolytica (LktA) and Actinobacillus actinomy- versely, neutropenic mice, made so by treatment with cyclo-
cetemcomitans (AaLtA) places CyaA within a family of calci- phosphamide or by a homozygous null mutation in the gran-
um-dependent, pore-forming cytotoxins known as RTX (re- ulocyte colony-stimulating factor gene, were killed by both
peats-in-toxin) toxins (659, 672, 676, 813). Each of these toxins wild-type and CyaA-deficient strains of B. bronchiseptica, indi-
contains a tandem array of a nine amino acid repeat [LXGG cating that in the absence of neutrophils, CyaA is not required
XG(N/D)DX] that is thought to be involved in calcium bind- to cause a lethal infection (324). These data indicate that T and
ing (813). Before the CyaA protoxin can intoxicate host cells, B cells are required to prevent killing by wild-type B. bronchi-
it must be activated by the product of the cyaC gene, which septica but innate defenses alone are adequate to control in-
is located adjacent to, and transcribed divergently from, the fection by a CyaA-deficient mutant. It also suggests that phago-
cyaABDE operon (36). CyaC activates the CyaA protoxin by cytic cells, particularly polymorphonuclear neutrophils, are a
primary in vivo target of the adenylate cyclase toxin. creased turbinate atrophy in infected pigs (483, 671), transpo-
Primary infections of children with either B. pertussis or son mutants of B. pertussis lacking dermonecrotic toxin are no
B. parapertussishu stimulate a vigorous serum antibody re- less virulent than wild-type bacteria in mice (810).
sponse to CyaA (153). In contrast, children immunized with Lipopolysaccharides. Like endotoxins from other gram-neg-
DTP or DTaP vaccines who later became vaccine failures ative bacteria, the LPS of Bordetella species are pyrogenic,
and developed pertussis had only minimal serum antibody mitogenic, and toxic and can activate and induce tumor necro-
responses to CyaA. This apparent induced tolerance is of sis factor production in macrophages (19, 582, 806). Bordetella
interest, and it may be evidence of the phenomenon called LPS molecules differ in chemical structure from the well-
“original antigenic sin” (395). With this phenomenon, a known smooth-type LPS expressed by members of the family
child’s serum immunologic response at initial exposure is to Enterobacteriaceae. Specifically, B. pertussis LPS lacks a repet-
all presenting epitopes of the infecting agent or vaccine. On itive O-antigenic structure and is therefore more similar to
subsequent exposure to the pathogen, the child responds rough-type LPS. It resolves as two distinct bands (A and B)
preferentially to the epitopes shared with the original in- on silver-stained sodium dodecyl sulfate-polyacrylamide gels
fecting agent or vaccine and the response to new epitopes of (612). The faster-migrating moiety, band B, consists of a lipid
the infecting agent are blunted. In the present scenario, A molecule linked via a single ketodeoxyoctulosonic acid res-
both vaccines contained multiple antigens and the vacci- idue to a branched oligosaccharide core structure containing
nated children responded to the antigens with which they heptose, glucose, glucuronic acid, glucosamine, and galactos-
had been primed but had only a minimal response to the aminuronic acid (GalNAcA) (92, 443, 447). The charged sug-
new antigen (CyaA) following infection. CyaA is not present ars, GalNAcA, glucuronic acid, and glucosamine, are not
in DTaP vaccines, but very small amounts might be present commonly found as core constituents in other LPS mole-
in DTP vaccines. cules. The slower-migrating moiety (band A) consists of band
Dermonecrotic toxin. Although initially misidentified as an B plus a trisaccharide consisting of N-acetyl-N-methylfucos-
endotoxin, DNT was one of the first B. pertussis virulence amine (FucNAcMe), 2,3-deoxy-di-N-acetylmannosaminuronic
factors to be described (62). This heat-labile toxin induces acid (2,3-diNAcManA), and N-acetylglucosamine (GlcNAc)
localized necrotic lesions in mice and other laboratory animals (92, 443, 447). B. bronchiseptica LPS is composed of band A
when injected intradermally and is lethal for mice at low doses and band B plus an O-antigen structure consisting of a single
when administered intravenously (62, 377, 470, 609). The sugar polymer of 2,3-dideoxy-di-N-acetylgalactosaminuronic
DNTs of B. pertussis, B. bronchiseptica, and B. parapertussishu acid (208). B. parapertussishu isolates contain LPS that lacks
are nearly identical (⬃99% amino acid identity) cytoplasmic, band A, has a truncated band B, and contains an O antigen
single polypeptide chains of about 160 kDa (183, 370, 580, 846). that, like B. bronchiseptica, consists of 2,3-dideoxy-di-N-acetyl-
Bordetella DNT is a typical A-B toxin, composed of a 54-amino- galactosaminuronic acid. B. parapertussisov isolates lack O an-
acid N-terminal receptor-binding domain and a 300-amino-acid tigen and contain band A- and B-like moieties that appear to
C-terminal enzymatic domain. While the receptor for DNT has be distinct from those of the other Bordetella species (780).
not yet been identified, in vitro assays using fibroblast and The wlb locus, which is well conserved among the Bordetella
osteoblast-like cell lines determined that on receptor binding, subspecies, is required for the biosynthesis and assembly of the
DNT is internalized via a dynamin-dependent endocytosis. band A LPS trisaccharide (639). It is composed of 12 genes,
Translocation is independent of acidification of endosomes wlbA to wlbL. Based on mutational analyses, certain putative
and retrograde vesicular transport and requires the N-terminal functions have been assigned for these genes (639). WlbA to
region of the DNT enzymatic domain, which includes a puta- WlbD are involved in the biosynthesis of 2,3-diNAcManA,
tive transmembrane domain. On endocytosis, DNT undergoes the second sugar of the band A trisaccharide. WlbE encodes
proteolytic nicking by mammalian proteases such as furin, a transferase that adds 2,3-diNAcManA to the growing tri-
which is necessary for the cellular activity of DNT (502). saccharide chain. WlbF is a putative enzyme involved in
In vitro studies have shown that purified DNT from B. bron- FucNAcMe biosynthesis, and WlbG is a transferase that adds
chiseptica induces dramatic morphological changes, stimulates this sugar to an acyl carrier lipid on which the trisaccharide
DNA replication, and impairs differentiation and proliferation unit is synthesized prior to transfer en bloc to band B. WlbH is
in osteoblastic clone MC 3T3 cells (369, 372). Recent evidence a GlcNAc transferase that adds the third and final sugar of the
indicates that these effects are due to DNT-mediated activa- trisaccharide. WlbI is a predicted integral membrane protein
tion of the small GTP-binding protein Rho (371), which results which is likely to be involved in the transfer of band A and/or
in tyrosine phosphorylation of focal adhesion kinase (p125fak) assembly of the final full-length LPS molecule rather than its
and paxillin (436). p125fak and paxillin are involved in embry- biosynthesis. Interestingly, while wlbJ and wlbK are two appar-
onic development and cell locomotion (378), and their activa- ently separate genes in B. pertussis, they are fused into a single
tion leads to profound alterations in the actin cytoskeleton and open reading frame in B. bronchiseptica and B. parapertussishu.
the assembly of focal adhesions (648, 703–705). Lacerda et al. Mutations in wlbJK do not affect LPS biosynthesis or alter any
also showed that DNT stimulates DNA synthesis without ac- phenotypes compared to the wild-type strains, and their func-
tivation of p42mapk and p44mapk, providing evidence for a novel tion(s) remains unclear. WlbL is a putative dehydratase in-
p21rho-dependent signaling pathway that leads to entry into the volved in the biosynthesis of the band A sugar FucNAcMc.
S phase of the cell cycle in Swiss 3T3 cells (436). If and how Finally, the wbm locus, which lies adjacent to the wlb locus
these effects of DNT contribute to Bordetella pathogenesis is in B. bronchiseptica and B. parapertussishu, is required for
not known. Although B. bronchiseptica strains with decreased the assembly of O antigen (85). Deletion of wbm in B. bron-
dermonecrotic toxin activity have been associated with de- chiseptica and B. parapertussishu leads to loss of O-antigen
expression, thus removing the major structural differences membrane or cytoplasm of eukaryotic cells through a needle-
between the LPS molecules of these bacteria and B. pertussis like injection apparatus (433). These bacterial effector proteins
(85). then alter normal host cell-signaling cascades and other pro-
Although a distinct role(s) for LPS in Bordetella pathogen- cesses to promote the pathogenic strategies of the bacteria
esis has not yet been demonstrated, its importance is suggested (450). Type III secretion has been identified in a variety of
by the observation that changes in LPS structure in B. bron- pathogens including those infecting humans, such as Yersinia,
chiseptica are controlled by the BvgAS virulence regulatory Shigella, Salmonella, and enteropathogenic E. coli, as well as
system (780). Recently, pagP was shown to encode a BvgAS- the plant pathogens Pseudomonas syringae and Erwinia (for
regulated lipid A palmitoyl transferase that mediates a palmi- reviews, see references 172 and 374). Most recently, TTSSs
toylation modification of B. bronchiseptica lipid A. Identical have also been identified in endosymbionts and invertebrate
pagP open reading frames have been identified in the B. per- pathogens, such as Rhizobium spp., Sodalis glossinidius, and
tussis and B. parapertussishu genomes. Preliminary evidence Photorhabdus luminescens (192, 254, 789). Type III secretion
suggests that the B. parapertussishu lipid A also undergoes represents one of the most complex mechanisms of protein
BvgAS-regulated, PagP-mediated palmitoylation. However, translocation in biology. The complete TTSS often requires
pagP is not expressed in B. pertussis due to a disruption of the over 20 genes, which encode a secretion apparatus that spans
putative promoter region by an IS element. These differences the bacterial cytoplasmic and outer membranes, as well as
may contribute to determining host specificity or the nature of translocator proteins that form pores in the eukaryotic cell
infection. Analysis of B. bronchiseptica pagP mutants showed membrane and a type III secretion-specific ATPase required
that pagP is not required for the initial colonization of the for apparatus assembly and secretion (420). While the genes
mouse respiratory tract but may be important for persistence. encoding the secretion apparatus and translocators are rela-
Likewise, compared with their wild-type parental strains, B. tively highly conserved among different genera, the effector
pertussis, B. parapertussishu, and B. bronchiseptica strains which proteins secreted by these systems are quite diverse.
synthesize only band B LPS show decreased colonization in a The Bordetella TTSS was first identified in B. bronchiseptica
mouse model of respiratory infection (325). For B. bronchisep- as a BvgAS-activated virulence factor. The Bordetella type III
tica and B. parapertussishu, this difference may be attributed to secretion locus, termed the bsc locus, includes 22 genes that
differences in sensitivity to antibody-dependent serum killing, encode components of the type III secretion apparatus, se-
for which palmitoylation by PagP may also be important (325). creted proteins, and putative chaperones. Interestingly, while
Analysis of B. bronchiseptica and B. parapertussishu wbm mu- type III genes are intact and highly conserved in members of
tants, which lack the O antigen, showed that these strains the B. bronchiseptica cluster, only B. bronchiseptica and B. para-
activated complement and were highly susceptible to comple- pertussisov readily display type III secretion-associated pheno-
ment-mediated killing in vitro. Interestingly, while the B. para- types in vitro. These include induction of cytotoxicity in several
pertussishu ⌬wbm mutant was severely defective in colonization cultured cell lines (779, 841), dephosphorylation of specific
of the tracheas and lungs of mice, the B. bronchiseptica wbm host cell proteins (841), and activation of the mitogen-acti-
mutant showed no defect. Perhaps the most interesting obser- vated protein kinases, ERK1 and ERK2 (840). Bone mar-
vation, however, is that B. pertussis strains which did not display row-derived dendritic cells derived from mice infected with
significant resistance to killing by serum containing excess B. bronchiseptica display a TTSS-mediated increase in sur-
complement in vitro rapidly acquired resistance to comple- face expression of major histocompatibility complex class II
ment-mediated killing in vivo in a BrkA-independent manner and of CD86 and CD80 costimulatory molecules (709). In
(620). Since B. pertussis does not express O antigen, this ob- B. bronchiseptica, the TTSS also prevents translocation of the
servation came as a considerable surprise, leading to the spec- transcription factor NF-␬B to the nucleus even on stimulation
ulation that B. pertussis may have developed another means of of the cells with tumor necrosis factor alpha; instead it causes
resisting antibody-independent complement-mediated killing aberrant aggregation of NF-␬B within the host cell cytoplasm
in vivo. A possible solution to this mystery seems to have been (840). B. bronchiseptica also causes very rapid cell death in
provided by the recent observation that LPS protects B. per- macrophage and epithelial cell lines in a type III secretion-
tussis from surfactant protein A (SP-A)-mediated clearance, dependent manner (840). Cell death does not require cas-
presumably by sterically limiting access of SP-A to the lipid A pase-1, is not blocked by the pancaspase inhibitor zVAD, and
region, the target for SP-A binding (691). Thus, B. pertussis and does not involve cleavage of procaspase-3, procaspase-7, and
B. bronchiseptica seem to have utilized their individual re- poly(ADP-ribose) polymerase, suggesting that the cell death
sources to acquire resistance to complement-mediated killing pathway induced by the B. bronchiseptica TTSS is distinct from
in vivo—B. bronchiseptica deploys serum resistance via its O- the death pathways induced by the Yersinia, Shigella, and Sal-
antigen-containing LPS and therefore does not requires BrkA monella TTSSs (731). The Bordetella TTSS most probably in-
for this purpose; B. pertussis lacks O antigen and therefore uses duces a necrotic form of cell death, since dying cells morpho-
a combination of its O-antigen-deficient LPS and BrkA to logically resemble necrotic rather than apoptotic cells and
acquire serum resistance. Further in vivo characterization of since death is efficiently blocked by the addition of nonspecific
mutants with defined mutations affecting LPS structure will cytoprotectants such as glycine. Despite extensive efforts, the
greatly facilitate deciphering the precise role(s) of LPS in Bor- Bordetella type III effector proteins and their cellular targets
detella pathogenesis. for the above phenotypes are presently unknown.
Type III secretion system. A TTSS has been identified in In vivo, the B. bronchiseptica TTSS contributes to persistent
Bordetella subspecies (841). TTSSs allow gram-negative bacte- colonization of the trachea in both rat and mouse models of
ria to translocate effector proteins directly into the plasma respiratory infection (840, 841). The inflammatory cells that
infiltrate the lungs during infection undergo apoptosis in mice evolutionary pressure to maintain the TTSS in B. pertussis: in
infected with a wild-type strain but not in those infected with a comparing B. pertussis and B. bronchiseptica, BtrS, BtrW, and
mutant strain deficient in type III secretion (840). Additionally, BtrV are identical, BtrU differs at six amino acid residues, and
mice infected with the type III secretion-deficient strain elicit of the nucleotide differences observed in the genes encoding
higher titers of anti-Bordetella antibodies (specifically serum TTSS proteins, substitutions are dramatically skewed toward
IgA) than do animals infected with wild-type Bordetella (840). those that are silent or conservative (507). It is therefore con-
Consistent with this, rats infected with the type III secretion- ceivable that B. pertussis does express a functional TTSS but in
deficient strain are completely protected against superinfection a manner where signal inputs are recognized, integrated, and
with wild-type B. bronchiseptica (Mattoo et al., unpublished regulated differently from in B. bronchiseptica. If so, then taken
data) and adoptive transfer of anti-Bordetella antibodies pro- together with the immunomodulatory role suggested for the
tects SCID-beige mice from tracheal and lung colonization by TTSS, this hypothesis provides new insight into how we view
B. bronchiseptica (324). Interestingly, infection of immunocom- and vaccinate against B. pertussis infection. It further raises the
promised SCID and SCID-beige mice with type III secretion possibility that TTSS-deficient Bordetella strains could serve as
mutants resulted in a hypervirulent phenotype (840). Taken live vaccine delivery vehicles. Additional phylogenetic analyses
together, these data suggest the B. bronchiseptica. TTSS may of type III-related loci will help clarify the relationship be-
be involved in modulating the host immune response and could tween expression of the bsc and btr loci, host range, and course
contribute to the typically chronic rather than pathological of disease.
nature of B. bronchiseptica infections. Tracheal cytotoxin. TCT corresponds to a disaccharide-tet-
Recently, an additional locus consisting of four genes, btrS, rapeptide monomer of peptidoglycan that is produced by all
btrU, btrW, and btrV, situated 3⬘ to the bsc locus was identified gram-negative bacteria as they break down and rebuild their
and shown to encode regulatory proteins for the Bordetella cell wall during growth. Its structure is N-acetylglucosaminyl-
TTSS (507). Like the bsc genes, this locus, called btr (for 1,6-anhydro-N-acetylmuramyl-(L)-alanyl-␥-(D)-glutamyl-meso-
“Bordetella type III regulation”), is also transcriptionally acti- diaminopimelyl-(D)-alanine (170). While other bacteria, such
vated by BvgAS. btrS encodes an extracytoplasmic function as E. coli, recycle this peptidoglycan fragment by transporting
sigma factor, homologous to the HrpL protein which activates it back into the cytoplasm via an integral cytoplasmic mem-
type III secretion in P. syringae (507). btrU, btrW, and btrV brane protein called AmpG, Bordetella spp. release it into the
encode proteins predicted to contain an array of domains that environment due to the lack of a functional AmpG (170, 394,
define “partner-switching” complexes that were traditionally 608, 674). As such, TCT is constitutively expressed and is
thought to function only in gram-positive bacteria (507). Anal- independent of BvgAS control.
ysis of the btr locus in B. bronchiseptica revealed an intricate The activities of TCT have been studied in vitro using ham-
level of regulation of type III secretion, which falls downstream ster tracheal organ culture and cultured hamster tracheal ep-
of the role of BvgA. BtrS was found to be necessary and ithelial (HTE) cells (169, 280). TCT causes mitochondrial
sufficient for transcription of all bsc type III genes. Deletions in bloating, disruption of tight junctions, and extrusion of ciliated
btrU and btrW revealed an uncoupling of protein expression cells, with little or no damage to nonciliated cells, in hamster
from secretion, since these mutants expressed type III proteins tracheal ring cultures and a dose-dependent inhibition of DNA
at normal levels but failed to secrete them. Finally, while tran- synthesis in HTE cells. TCT also causes loss of ciliated cells,
scription of type III loci was unaffected, type III secretion- cell blebbing, and mitochondrial damage, as is evident in hu-
specific proteins could not be detected in a btrV deletion mu- man nasal epithelial biopsy specimens (820). TCT alone is
tant. Thus, the btr locus encodes a novel regulatory cascade necessary and sufficient to reproduce the specific ciliated- cell
where BtrS is required for the expression of type III loci, BtrU cytopathology characteristic of B. pertussis infection in ex-
and BtrW are required for secretion, BtrV is essential for planted tracheal tissue (280). TCT-dependent increase in nitric
translation and/or protein stability, and BvgAS exerts control oxide (NO˙) is proposed to mediate this severe destruction of
over the entire TTSS by regulating btrS (507). ciliated cells. TCT triggers IL-1␣ production in HTE cells, and
Like the bsc genes, the btr genes are highly conserved (97 to both TCT and IL-1␣ result in increased NOF production when
100% amino acid sequence identity) between B. bronchiseptica added to HTE cells (341, 342). It is hypothesized that, in vivo,
and B. pertussis (507, 608). The bsc and btr genes of B. pertussis TCT stimulates IL-1␣ production in nonciliated mucus-secret-
are predicted to be transcribed and translated to yield full- ing cells, which positively controls the expression of inducible
length polypeptides. However, unlike B. bronchiseptica and B. nitric oxide synthase, leading to high levels of NOF production.
parapertussov, B. pertussis and B. parapertussishu failed to confer NOF then diffuses to neighboring ciliated cells, which are
cytotoxicity to mammalian cell lines in vitro, consistent with the much more susceptible to its damaging effects (250). TCT also
lack of expression of type III proteins, as determined by im- functions synergistically with Bordetella LPS to induce the pro-
munoblot analysis (507). Surprisingly, reverse transcription- duction of NOF within the airway epithelium (251). The ability
PCR analysis of B. pertussis and B. parapertussishu showed that to construct TCT-deficient mutants by expressing a heterolo-
all bsc and btr loci were actively transcribed and BvgAS acti- gous ampG gene in Bordetella will allow this hypothesis to be
vated. Further, the BtrS regulon appears to be intact and tested by using in vivo models.
functional in B. pertussis and B. parapertussishu. Thus, the block Pertussis toxin. PT is an ADP-ribosylating toxin synthesized
in type III secretion observed in human-adapted Bordetella and secreted exclusively by B. pertussis. It is an A-B toxin
strains under standard in vitro laboratory conditions appears to composed of six polypeptides, designated S1 to S5, which are
be post-transcriptional, similar to the phenotype observed in a encoded by the ptxA to ptxE genes, respectively. The S1 poly-
B. bronchiseptica btrV deletion mutant. There appears to be an peptide comprises the A subunit of the toxin, while the pen-
tameric B subunit consists of polypeptides S2, S3, S4, and S5 ulatory immunologic effects (577, 623). The various effects of
assembled in a 1:1:2:1 ratio (472, 590, 750). Each subunit is PT in pertussis vaccines, in purified form and released during
synthesized with an N-terminal signal sequence, suggesting infection, have been studied in various model systems and in
that transport into the periplasmic space occurs via a general humans and have been reviewed in depth previously (147). To
export pathway analogous to the sec system of E. coli. Secre- summarize, histamine sensitization in mice in response to B.
tion across the outer membrane requires a specialized trans- pertussis infection was first described over 50 years ago (606).
port apparatus composed of nine Ptl (for “pertussis toxin lib- Subsequently, histamine sensitization was studied by many in-
eration”) proteins (231, 812). The ptl locus bears extensive vestigators, but only five studies involving humans were carried
similarity to the prototype type IV secretion system involved in out (269, 486, 501, 561, 574, 576, 618, 622, 625, 686, 701, 801).
exporting single-stranded “T-DNA” encoded by the Agrobac- In a 1948 study, Parfentjev and Goodline found that mice
terium tumefaciens virB operon, suggesting that both these sys- injected intraperitoneally with a pertussis vaccine subsequently
tems function by a common mechanism to transport large became hypersensitive to histamine challenge; 2 mg of hista-
protein complexes (182, 434, 799). Furthermore, there is evi- mine had similar lethality in sensitized (previously vaccinated)
dence that only the fully assembled PT holotoxin is efficiently mice as did 50 mg in unvaccinated mice (606). In these animals,
secreted (585, 626). histamine sensitivity following intraperitoneal injection of per-
The ptl genes are located directly 3⬘ to, and within the same tussis vaccine increased over 4 to 5 days, plateaued, and then
transcriptional unit as, the ptxA to ptxE genes (430, 812). While diminished over 3 to 4 weeks (801). Following intravenous
the chromosomes of B. parapertussis and B. bronchiseptica also pertussis vaccine administration, however, sensitization oc-
contain ptx-ptl loci that encode functional polypeptides, these curred within 90 min and peaked in 1 day. Histamine sensiti-
genes are transcriptionally silent due to mutations in the pro- zation also occurs in mice following B. pertussis respiratory
moter regions (13, 302, 494, 589). In both B. parapertussis and infection (625).
B. bronchiseptica, replacement of native ptx-ptl promoter se- In three controlled studies in children, histamine sensitiza-
quences with those from B. pertussis results in the secretion of tion could not be demonstrated in association with B. pertussis
biologically active Ptx (327). The biological relevance of dif- infection or following vaccination (269, 501, 618). In the most
ferential PT expression among bordetellae is not known. recent study, Gifford et al. compared skin test sensitivity to
The A component of PT, consisting of the enzymatically histamine in DT- and DTP-immunized children and found no
active S1 subunit, sits atop the B oligomer, a ringlike structure significant difference in wheal size between the two groups (269).
formed by the remaining S2 to S5 subunits (403, 700, 749). The In 1949 it was reported that mice that received pertussis
subunits are held together by noncovalent interactions. The B vaccine intraperitoneally had a marked decrease in their blood
oligomer binds to eukaryotic cell membranes and dramatically glucose concentrations and that this hypoglycemia persisted
increases the efficiency with which the S1 subunit gains entry for at least a week (607). This hypoglycemia was due to hyper-
into host cells (749). It has been proposed that PT traverses the insulinemia. Peak levels of insulin following ip injection oc-
membrane directly without the need for endocytosis, since it curred 7 days after exposure in the mouse, and elevated levels
does not require an acidic environment for entry into eukary- persisted for 17 days (307). It was also noted that mice immu-
otic cells (396). Subsequent reports, however, have proposed nized with pertussis vaccine did not experience the usual hy-
that PT binds to cell surface receptors and undergoes endocy- perglycemic effect following histamine administration (743).
tosis via a cytochalasin D-independent pathway. Early and late There is considerable evidence indicating that PT causes an
endosmes, as well as the Golgi apparatus, have been implicated increase in serum insulin levels in humans (21, 147). In the
in the PT trafficking process (507, 836, 837). Once within the most definitive study, Toyota et al. administered PT intrave-
host cell cytosol, the B oligomer intercalates into the cytoplas- nously to six volunteers and observed increased plasma insulin
mic membrane and binds ATP, causing the release of the S1 levels at the time when glucose tolerance tests were performed
subunit, which then becomes active on reduction of its disulfide (763). Specifically, the ratio of the increment of the plasma
bond (402). While the exact mechanism of how and when PT insulin level to that of blood glucose at 30 min after the glucose
assembles and interacts with the Ptl transporter in vivo is challenge remained significantly greater 4, 30, and 60 days after
currently unknown, a recent report suggests that subassemblies the initial intravenous PT injection compared with the baseline
of PT consisting of the S1 subunit and a partial B oligomer can glucose tolerance test response. Infants vaccinated with DTP
interact with the Ptl system (84). vaccines have also been noted to have an increase in serum
Based on extensive in vitro characterization of PT, the S1 insulin levels (319, 552). Neonates with severe pertussis are
subunit in its reduced form has been shown to catalyze the occasionally found to be markedly hypoglycemic, suggesting
transfer of ADP-ribose from NAD to the ␣ subunit of guanine hyperinsulinemia (156, 165, 360).
nucleotide-binding proteins (G proteins) in eukaryotic cells Almost 100 years ago, it was observed that children with
(61, 403, 749). PT can ADP-ribosylate and thus inactivate G pertussis often had marked leukocytosis in association with
proteins such as Gi, Gt (transducin), and Go. When active, Gi significant lymphocytosis (256). Early studies of mice found a
inhibits adenylyl cyclase and activates K⫹ channels, Gt acti- similar leukocytosis (562–565, 575, 600). This occurred follow-
vates cyclic GMP phosphodiesterase in specific photorecep- ing the injection of either living B. pertussis organisms, killed B.
tors, and Go activates K⫹ channels, inactivates Ca2⫹ channels, pertussis cells, or B. pertussis culture supernatants. PT was later
and activates phospholipase C-␤ (776). Biological effects at- found to cause absolute lymphocytosis in many diverse species
tributed to the disruption of these signaling pathways include such as lampreys, swine, guinea pigs, rabbits, monkeys, calves,
histamine sensitization, enhancement of insulin secretion in sheep, and mice (600). In the mouse model, PT administered
response to regulatory signals, and both suppressive and stim- intravenously elicits a leukocytosis which peaks in about 4 days
and then decreases to normal values over a 2- to 3-week period clear that PT does not play a role in causing the paroxysmal
(565). Both neutrophil and lymphocyte numbers increase, but coughing, whooping, and vomiting characteristic of pertussis.
the lymphocytic response is generally more pronounced. The The exact role of PT in the establishment of infection, disease,
leukocytosis is not due to increased cell production (562). and/or transmission of pertussis remains to be determined.
Instead, there is an increase in the release of cells into the
blood from extravascular sites and these cells continue to re-
circulate rather than emigrate from the blood, as would occur PATHOLOGY
normally. Of the lymphocytes, both B and T cells are increased B. pertussis Infection
in the peripheral blood (53, 565). In most primary infections in
children, there is a distinct leukocytosis with an absolute lym- In 1943 Lapin wrote a 14-page chapter on the pathology of
phocytosis (149). In contrast, infections in adults (which are pertussis, and there has been little new information added
always reinfections) are not associated with an absolute lym- during the ensuing 60 years (442). In his review Lapin cited 35
phocytosis (149, 550). Occasional primary infections in young papers relating to postmortem data on children dying of per-
infants also do not elicit a lymphocytosis. This is probably due tussis. In spite of such a complete data set, there are no data
to the presence of transplacentally acquired antibody to PT. available on the pathology of mild or typical pertussis in pa-
PT is a strong adjuvant in several immunologic systems in tients that survived.
several animals and humans (14, 15, 190, 235, 246, 452, 453, In 1912 Mallory and Horner reported the findings of three
456, 553, 561, 573, 575, 578, 600, 610, 649, 699, 745, 746, 801, children with illnesses of relatively short durations prior to
804). This adjuvancy in the experimental-animal model is as- death (487). However, all three of these children had high
sociated with enhancement of serum antibody responses to fevers prior to death, suggesting a secondary problem. Micro-
other antigens, increased cellular immune responses to various scopic study in these cases showed large numbers of bacteria
protein antigens, contribution to hyperacute experimental au- between the cilia of the cells lining the trachea. The microor-
toallergic encephalomyelitis, and increased anaphylactic sensi- ganisms usually extended to the base of the cilia, and the long
tivity. Of these adjuvant activities demonstrated in animal axis of the organisms tended to coincide with the direction of
model systems, only the enhancement of serum antibody re- the cilia. Frequently there was a lateral spreading of the cilia
sponses to other vaccine antigens has been demonstrated to (mushrooming) of individual cells, and in many places the cilia
occur in vaccinated children. were reduced to stubs or were entirely gone. In two of the
Although, on the one hand, PT displays adjuvant properties, cases, no bacteria were found between the cilia of the cells
it has also been shown to inhibit chemotaxis, oxidative re- lining the bronchi or bronchioles. In the third case, masses of
sponses, and lysosomal enzyme release in neutrophils and mac- bacteria were found between the cilia of the cells lining the
rophages (61, 72, 74, 438, 521, 555, 595, 717, 785). This phe- bronchi and bronchioles. There was an unevenness in the dis-
notype has been confirmed using mouse and rat models, where tribution with some bronchi free of bacteria.
PT was shown to inhibit chemotaxis and migration of neutro- In experiments using rhesus and ringtail monkeys, Sauer and
phils, monocytes/macrophages, and lymphocytes (76, 479, 522). Hambrecht found pure cultures of B. pertussis between the cilia
Most recently, PT was shown to display an immunosuppressive of the smaller bronchi and bronchioli during uncomplicated
activity, since mice infected with a PT⫺ mutant elicited much pertussis (442, 689). They also noticed endobronchitis and
higher anti-Bordetella serum antibody titers than did mice in- peribronchitis with an abundance of B. pertussis on and be-
fected with wild-type B. pertussis (90). PT has also been sug- tween the cilia of the finer bronchi and bronchioli at 11 days
gested to function as an adhesin involved in the adherence of postinoculation in one monkey. The upper respiratory tract
B. pertussis to human macrophages and ciliated respiratory was normal macroscopically. The bronchi were found to con-
epithelial cells (654, 771). tain leukocytes, mucus, and debris.
PT is commonly cited as the major virulence factor ex- Most patients with fatal cases of pertussis have broncho-
pressed by B. pertussis, responsible for many, if not all, of the pneumonia; this may be due to B. pertussis or to secondary in-
symptoms typically associated with pertussis (623). However, fection with other respiratory bacteria (149). The initial stages
despite a plethora of experimental evidence demonstrating PT of infection entail congestion and infiltration of the mucosa by
function in vitro, in animal models, and in human studies, clear lymphocytes and polymorphonuclear leukocytes. The lumens
evidence for a substantial in vivo role for PT in human disease of the bronchi contain inflammatory debris. Lapin, in review-
is lacking. Since B. pertussis and B. parapertussishu differ pri- ing the data of several reports, suggests that the initial pulmo-
marily in the absence of PT expression by B. parapertussishu, a nary lesion in pertussis is a lymphoid hyperplasia of peribron-
comparative analysis of the symptoms in children infected with chial and tracheobronchial lymph nodes (442). A necrotizing
either of these organisms has been adopted as a means of process that affects the midzonal and basilar layers of the
isolating the effects of PT. Such studies have indicated that the bronchial epithelim follows (149, 469). Necrosis and desqua-
most notable difference between the two is leukocytosis with mation of the superficial epithelial layers of the small bronchi
lymphocytosis in B. pertussis- but not B. parapertussis-infected may subsequently occur. Numerous small areas of atelectasis
children (340, 825). However, PT does appear to contribute to occur, and there is an increase in fibrous tissue around the
morbidity in B. pertussis infections because the duration and bronchi.
severity of illness tend to be greater in B. pertussis infections Of the more recent cases, postmortem data are available in
than in B. parapertussis infections. In this regard, the frequent the deaths of five infants (156, 712). A 27-day-old infant was
finding of extreme leukocytosis in neonates and young infants found to have extensive hemorrhage in the apex of the left lung
with fatal B. pertussis infections is noteworthy. However, it seems (156). Bilateral pulmonary edema and focal hemorrhages were
noted microscopically. There was also a diffused infiltration by purative bronchopneumonia with the loss of the pulmonary
macrophages and moderate focal infiltrations with inflamma- histologic architecture. Variable amounts of fibrinous exudate
tory cells, accompanied by extensive necrotizing bronchopneu- fill the terminal air passages.
monia and thromboemboli. Smith and Vyas noted similar post- (ii) Rabbits. Suppurative bronchopneumonia with intersti-
mortem findings in four infants (712). Widespread mucus tial pneumonitis occurs. Histologically there may be peribron-
plugging and extensive mucosal damage were reported. They chial lymphocytic cuffing (23).
also noted a severe depletion of lymphocytes from the thymus,
lymph nodes, and spleen. PATHOGENESIS AND IMMUNITY
In fatal cases there are often pathologic changes in the brain.
There may be microscopic or gross cerebral hemorrhage and There are four important steps relating to infection and
cortical atrophy. Although it has been suggested that these disease due to bacterial pathogens in general and specifically to
findings may be the direct effect of bacterial toxins (624), it is B. pertussis: (i) attachment, (ii) evasion of host defenses, (iii)
most likely that they are the result of hypoxia and anoxic brain local damage, and (iv) systemic manifestations (147, 174, 279,
damage. 346, 571, 577, 581, 641, 811). The Bordetella virulence deter-
minants were discussed earlier in this review, and a summary of
B. bronchiseptica Infection these factors is presented in Table 2.
Dogs. B. bronchiseptica infection is a major cause of kennel Infection is initiated by the attachment of B. pertussis organ-
cough in dogs (49, 509). This disease is a tracheobronchitis isms to the cilia of epithelial cells of the upper respiratory tract
characterized by congestion of the mucosal lining of the tra- (811). Various factors (FHA, FIM, PT, LPS, TcfA, BrkA,
chea and bronchi and a mucoid or mucopurulent exulate. In Vag8, and PRN) have been implicated in facilitating attach-
addition, patchy areas of exudative pneumonia as well as pe- ment (174, 418, 577, 641, 769, 811). Of note is the redundancy
techiae and hemorrhages over the pleural surface may be of protein adhesins that may contribute to the attachment
noted. Often, no macroscopic abnormalities are seen in the process. As a consequence of this redundancy, individual ad-
respiratory tract. However, in most dogs, histologic examina- hesive functions are often masked, and it has been difficult to
tion reveals tracheobronchitis. designate one protein as the primary adhesin. Various animal
There are two patterns of microscopic findings. One pattern model systems and tissue culture systems give different results
consists of focal, occasionally coalescing, areas of epithelial in regard to the importance of the various proteins in the
degeneration and necrosis. The cells are disorganized, with attachment process. In vitro attachment assays with a variety of
vacuolation and pyknosis. The lamina propria is congested and cell lines, however, establish FHA as a potent adhesin, at least
infiltrated with only a few macrophages and lymphocytes. In under laboratory conditions.
the other pattern, a mucopurulent exudate is present in the Information from vaccine efficacy studies can also provide
lumen of the airway and there is edema of the lamina propria some indirect evidence for the role of a particular protein as an
with a marked infiltration of polymorphonuclear leukocytes. adhesin. For instance, a vaccine containing FIM2/3, PT, FHA,
Clumps of gram-negative bacteria are seen among the cilia and PRN, which elicited a strong antibody response to FIM2/3
of the tracheobronchial epithelium. Infection complicated antigens, had significantly greater efficacy than a vaccine con-
by pneumonia is accompanied by alveolar capillary congestion taining just PT, FHA, and PRN (597) (see the sections on DTP
and exudation of fluid with polymorphonuclear leukocytes and and DTaP vaccine efficacy, below). This observation suggests
macrophages into the air spaces. The lymph nodes and pala- that FIM (either FIM2, FIM3, or both) may serve as adhesins
tine tonsils frequently appear immunologically reactive, and and that antibodies directed against these antigens may block
lymphadenitis and tonsillitis are occasionally present. attachment of Bordetella to host cells via FIM. However, as
Swine. B. bronchiseptica causes upper respiratory illness in presented in the section on fimbriae (see above), the beneficial
young piglets, which leads to atrophic rhinitis (201, 274, 483). effect of antibody to FIM2/3 could be due to the inhibition of
Very young piglets (3 to 4 days old) may experience broncho- another antigenic function.
pneumonia. Hypoplasia occurs within the snout, with the ven- While it has been suggested in several in vitro and in vivo
tral scroll of the ventral turbinate most often affected. It may studies that FHA is the major Bordetella adhesin, data relating
be a slightly shrunken and distorted scroll, or, at the extreme, to two recent vaccine efficacy trials suggest that in the presence
there may be a complete absence of the scroll. The dorsal of other adhesins FHA may not be necessary for attachment.
scrolls of the ventral turbinate may also be involved in more For example, the former Lederle DTP vaccine (Tri-Immunol)
severe cases. contained a minimal amount of FHA and generated a mini-
Histologically there is hyperplasia of the epithelium with mal anti-FHA response, but it was more efficacious than the
some metaplasia. The epithelium is more stratified with poly- Lederle-Takeda DTaP vaccine (ACEL-IMMUNE), which
hedral cells which are devoid of cilia. There is some infiltration contained a large amount of FHA and generated a vigorous
with neutrophils and mononuclear cells and fibroblastic pro- antibody response to FHA (332, 334, 719). The most definitive
liferation in the lamina propria. The osseous core may be re- data relating to the importance of PT, FHA, PRN, and FIM
duced in size and replaced with fibrous tissue. There is an in- are presented in two studies of serologic correlates of immu-
crease in the osteoblasts number of around the trabeculae, and nity (148, 736). These two investigations were nested house-
osteoclasts are rare. hold contact studies within cohort vaccine efficacy trials in
Laboratory animals. (i) Guinea pigs. Lesions include muco- which vaccinees had received two-component (PT and FHA),
purulent or catarrhal exudates in the upper respiratory tract four-component (PT, FHA, PRN, and FIM2), or five-compo-
and the tympanic bullae (23). Microscopically there is a sup- nent (PT, FHA, PRN, and FIM2/3) DTaP vaccines and two
different DTP vaccines with markedly different immunogenic 12, 19, 45, 326, 423, 440, 575, 605, 823). Two investigators
profiles. Both of these studies indicated that PRN was the most found that mice were more susceptible to fatal infections with
important vaccine antigen, and in one of the studies (148) the Proteus vulgaris, Pasteurella multocida, Pseudomonas fluore-
data suggested a synergistic relationship between PRN and PT. scens, and Escherichia coli following immunization with per-
Interestingly, in both studies antibody to FHA did not contrib- tussis vaccines than were unimmunized mice (12, 423). In an-
ute to protection. Supporting these observations is the fact that other study, it was found that vaccinated mice had an increased
vaccines that contain PRN as well as PT and FHA have sig- susceptibility to influenza virus infection (605). In contrast,
nificantly greater efficacy than do vaccines containing PT alone Landy found that B. pertussis LPS increased the resistance of
or PT plus FHA (131, 149). However, in a study in which a PT mice to Salmonella enterica serovar Typhi (440). Bell and Mu-
toxoid vaccine was compared with a vaccine containing both noz found that B. pertussis extracts increased the resistance of
PT and FHA, it was found that the two-component vaccine had mice to rabies virus infection, and Winters et al. noted that
significantly greater efficacy (3, 734, 735, 737). These findings pertussis vaccine rendered normally sensitive mice resistant to
suggest that in the absence of antibody to PRN (and perhaps fatal adenovirus infections (575, 823). Pertussis vaccine was
FIM), antibody to FHA contributes to protection. also found to increase the resistance in animals to Crytococcus
Collectively, the above data suggest that pertactin may serve and Candida infections, and B. pertussis LPS fragments were
as an important adhesin but that, in its absence, other proteins found to offer protection against encephalomyocarditis virus,
can carry out this function. In addition, there may be a syner- Semliki Forest virus, and influenza A and B viral infections in
gistic relationship between PRN and high concentrations of PT mice (1, 19, 326).
(148). In the first blinded controlled study in Sweden with acellular
Evasion of host defenses is facilitated by adenylate cyclase pertussis vaccines, four deaths were associated with invasive
toxin (CyaA) and PT (147, 348, 577). Specifically, CyaA enters bacterial disease in the vaccine groups (3, 738). Both vaccines
neutrophils and catalyzes the excessive production of cAMP, in this trial contained residual amounts of active PT. Because
which intoxicates the cells such that phagocytosis is compro- of the possibility of increased susceptibility to invasive bacterial
mised. Like CyaA, PT also adversely affects phagocytosis and infections relating to DTP immunization, one of us (J.D.C.)
killing of organisms by inhibiting migration of lymphocytes and participated in two large case-control studies of two different
macrophages to areas of infection. populations (57, 194). In neither study was there evidence of
Local tissue damage of the ciliated epithelial cells may be an increased risk of invasive bacterial disease following DTP
due to TCT, DNT, and perhaps CyaA (147, 280, 581). Of these immunization (DTP vaccines contain both active PT and LPS),
toxins, it is likely that TCT is most potent in this regard. The and in one of the studies there was an apparent decreased risk
hallmark of B. pertussis infection is paroxysmal cough, and it of invasive disease following immunization (57). In the other
seems likely that local tissue damage is responsible for this study, a second analysis looked at the occurrence of any illness
cough. However, the total duration of the cough in typical in the period, before and after immunization. In this analysis
pertussis is longer than the period during which local damage there was no increase in the occurrence of infectious illnesses
would be expected to last. Therefore, it seems possible that in the postimmunization period compared with the preimmu-
there is another, unidentified toxin that contributes to the nization period (194).
continued paroxysmal cough. Following infection, antibodies develop to many B. pertussis
In contrast to many other severe bacterial diseases in which antigens including PT, FHA, PRN, FIM2/3, CyaA, and LPS
systemic manifestations are most important, B. pertussis infec- (149, 153, 346, 347, 490, 666, 757, 758, 767). Agglutinating
tion is unique in that there are no direct physically evident antibodies (agglutinins) develop specifically to FIM2/3, PRN,
systemic events. Encephalopathy occurs, but this most proba- and LPS. Neutralizing antibody to PT also develops. Using
bly is a secondary event due to anoxia associated with coughing ELISA, the qualitative and quantitative differences between
paroxysms (149). The most pronounced systemic laboratory class-specific antibodies (IgA, IgE, IgG, and IgM) can be de-
manifestation is leukocytosis with lymphocytosis, which is termined. In general, IgG and IgM antibodies, but not IgA
due to PT. PT may also cause hyperinsulinemia, and this may antibodies, develop against vaccine antigens following primary
manifest in some young infants as hypoglycemia (156, 712). immunization (146, 332, 579, 757, 821). Interestingly, persons
Some researchers have suggested that clinical pertussis is a who have been primed by infection respond to immunization
PT-mediated disease (623, 624, 661, 662). Although this idea is with not just an IgG response but also an IgA response (446).
still entertained by some, there is little evidence to support it. Specific secretory IgA antibodies to the various B. pertussis
Contradicting this notion is the observation that paroxysmal antigens can be detected in nasopharyngeal secretions and
cough, the main manifestation of pertussis, is clearly not due to saliva of previously infected persons (288, 843).
PT. This opinon is based on the fact that human infections with Infection with B. pertussis and immunization with DTP and
B. parapertussishu, which does not express PT, also elicit an DTaP vaccines clearly elicit protection of various degrees and
identical proxosymal cough (143, 149). durations against pertussis. The past literature suggested that
Another area of interest has been the effect of pertussis immunity after B. pertussis infection was lifelong whereas vac-
vaccines with active PT in the susceptibility of animals to other cine-induced immunity was relatively short lived (137, 141, 146,
infections (575). These effects are discussed here even though 244, 439, 695). Extensive studies during the last 30 years doc-
they may be due to other biologically active components of ument the limited duration of immunity following immuniza-
whole-cell vaccines (LPS or CyaA) or to combinations of fac- tion, and additional careful review of old literature and the
tors. While increased susceptibility has been observed in some more recent study of pertussis in adults indicate that immunity
model systems, increased resistance has occured in others (1, following disease is also relatively short-lived. An analysis of
surveillance data from the prevaccine era of reported pertussis illness lasts 6 to 12 weeks or longer and has three stages:
would suggest that pertussis was a rarity in adults (141). How- catarrhal, paroxysmal, and convalescent. Initially, in the ca-
ever, the experts at the time clearly recognized that atypical tarrhal stage, there is rhinorrhea, lacrimation, and mild cough,
pertussis was not uncommon in adults and that these illnesses similar to events which occur with rhinovirus infections. Over
were reinfections (141, 442, 478, 492). a 7- to 14-day period, the cough worsens in both frequency and
More recently, studies in Germany (where pertussis was degree. The temperature is normal or occasionally mildly ele-
epidemic because pertussis immunization was not routinely vated. The paroxysmal stage, which has its onset during the
carried out) found numerous cases of pertussis in adults. In- second week of illness, is characterized by repeated coughing
terestingly, many of these adults had had pertussis during fits with 5 to 10 or more forceful coughs during a single expi-
childhood (695, 828). One of us (J.D.C.) had the opportunity ration (a paroxysm). At the end of a paroxysm, there is a
to observe and study pertussis in adults in the United States massive inspiratory effort during which the classic whoop oc-
and also in Germany. In general, pertussis in German adults curs. In conjunction with a paroxysm, cyanosis, bulging eyes,
tended to be more typical and severe than cases observed in protrusion of the tongue, salivation, lacrimation, and disten-
the United States. This suggested that vaccine-induced immu- tion of neck veins may occur. The paroxysms are associated
nity was actually better than disease-induced immunity. Studies with tenacious mucus, but the production of purulent sputum
of IgG antibody to PT, FHA, PRN, FIM2, and agglutinins in does not occur. Posttussive vomiting is common.
similarly aged young adults in the United States and Germany The paroxysmal episodes usually occur in groups, with mul-
found that the geometric mean titers (GMTs) in the Ameri- tiple group episodes occurring every hour both night and day.
cans were two- to four-fold higher than those noted in the Following a group of paroxysmal episodes, the children are
Germans (146). This suggested that priming by vaccine was exhausted and may appear apathetic. Weight loss may occur
better that priming by infection. because of frequent vomiting and because of the refusal to eat
In addition to serum and secretory antibody responses fol- by the child because he or she recognizes that eating often
lowing infection or immunization, cell-mediated immune re- triggers paroxysms. Interestingly, between paroxysms the af-
sponses to the various B. pertussis antigens occur regularly fected children may appear normal without any respiratory
(16–18, 34, 60, 94, 451, 547–549, 679–681, 766, 845). Studies distress.
with the mouse respiratory infection model indicate that cel- Common complications of classic pertussis include pneumo-
lular immunity plays a major role in bacterial clearance and nia, otitis media, seizures, and encephalopathy. The pneumo-
augments the effects of antibody by predominantly Th1 cell nia may be a primary event in response to B. pertussis infection
stimulation (547, 548). In humans a cellular immune response or may be due to a secondary infection with other pathogens.
occurs shortly after the onset of a natural infection with Seizures and encephalopathy are most probably due to cere-
B. pertussis (816). A Th1 response to PT, FHA, and PRN is bral hypoxia related to severe paroxysms. In association with
preferentially induced. Immunization with a whole-cell pertus- paroxysms, interstitial or subcutaneous emphysema may result
sis vaccine also resulted in a Th1 response whereas the re- from rupture of alveoli. Other complications include subarach-
sponse to acellular pertussis vaccines was more heterogeneous, noid and intraventricular hemorrhage, subdural and spinal epi-
with the stimulation of both Th1 and Th2 cells. Several studies dural hematoma, ulcer or laceration of the frenulum of the
of murine and other rodent animal models also suggest a tongue, epistaxis, melena, subconjuctival hemorrhage, rupture
complementary role for humoral and cell-mediated immunity of the diaphragm, umbilical and inguinal hernia, rectal pro-
in protection against Bordetella infection (503, 547–549). Per- lapse, apnea, rib fracture, severe alkalosis with associated te-
sistent memory T and B cells lead to anamnestic antibody tanic seizures, and dehydration.
responses, which are important in long-term immunity (485). The paroxysmal stage lasts for 2 to 8 weeks and sometimes
longer. The transition to the convalescent stage is gradual and
is associated with an initial decrease in the frequency of the
CLINICAL MANIFESTATIONS paroxysms and subsequently a decrease in the severity of the
events as well. The convalescent stage usually lasts for 1 to 2
B. pertussis
weeks but is occasionally prolonged.
Several factors known to affect the clinical manifestations of It is important to note that children who have had classic
B. pertussis include patient age, previous immunization or in- pertussis often have a reccurrence of typical coughing parox-
fection, presence of passively acquired antibody, and antibiotic ysms when they have respiratory viral infections. Most children
treatment (149). Additional factors which may also play a role with classic pertussis which resulted from a primary B. pertussis
in clinical manifestations include the number of organisms at infection have an elevated white blood cell count with an ab-
exposure, host genetic and acquired factors, and the genotype solute lymphocytosis. Unless there are complications as noted
of the organism. above, the physical examination in pertussis is normal except
The incubation period is most commonly 7 to 10 days. How- for the coughing episodes. Neither fever nor pharyngitis is
ever, it was noted that in the household setting, 22% of sec- typical of pertussis.
ondary cases occurred more than 28 days after the onset of Mild illness and asymptomatic infection. In household con-
illness in the primary case (334). This suggests either a more tact studies, asymptomatic infections in family members are
prolonged incubation time or a delay in exposure within the common (203, 474, 475). In one study, 52 (46%) of 114 house-
household. hold contacts who remained well and 23 (43%) of 53 of those
Classic illness. Classic illness most often occurs as a primary with mild respiratory illnesses (rhinorrhea, tearing, sneezing,
infection in unimmunized children (141, 147, 149, 155). The conjunctivitis, fever, hoarseness, sore throat, or cough of ⬍2
weeks’ duration) had laboratory evidence of B. pertussis infec- the same population it was observed that many deaths attrib-
tion (203). Most of the silent infections or mild respiratory uted to SIDS were in fact related to B. pertussis infection (588).
illnesses noted above occurred in previously vaccinated chil- In a more recent study, Heininger et al. found evidence of
dren and adults or in adults who had previously had B. pertussis B. pertussis DNA in nasopharyngeal swabs from 9 (18%) of 51
infections. In another recent study, 21 (5.3%) of 399 appar- infants who had experienced sudden unexpected deaths (339).
ently healthy infants who were controls in the study had evi- As a follow-up to this study, a carefully controlled study was
dence of B. pertussis infection by PCR assay (335). Follow-up performed in which specimens for PCR assay were collected
information was available for 15 of these subjects; 4 had cough from 254 infants who had experienced sudden unexpected
illnesses, and 11 remained asymptomatic. deaths and from 441 matched controls (335). The rate of PCR
In addition to asymptomatic infections and trivial respira- positivity was 5.1% in the sudden-death cases and 5.3% in the
tory infections as noted above, a substantial number of both controls. The high rate of infection in the controls was a surprise
unvaccinated and previously vaccinated children have mild but does not negate the possibility that the B. pertussis infections
cases of pertussis (333, 336, 337, 693, 719). In one study it was in the SIDS cases may have contributed to the fatalities.
noted that 47% of 247 subjects had cough illnesses which Adults. During the last two decades, a number of studies
lasted ⱕ28 days (333). A continuation of the above study have indicated that B. pertussis infections in adolescents and
involved 2592 culture-positive, previously unvaccinated chil- adults are common (55, 200, 271, 354, 390, 397, 534, 550, 586,
dren; of these children, 38% had cough illnesses of ⱕ28 days 634, 665, 675, 695, 739, 759, 788, 828, 835). There has also been
and in 17% had cough illnesses of ⱕ21 days (336). In another an increase in the number reported cases of pertussis in ado-
study, in which PCR as well as culture was used diagnostically, lescents and adults in concert with these studies (112, 135, 199,
it was found that 32% of the subjects had cough illnesses which 232, 309, 702, 710, 838). All adults and most adolescents who
lasted ⱕ4 weeks (693). Of this group, however, the majority have B. pertussis infections have had a previous B. pertussis
(57%) had paroxysmal coughing; 32% also had whoops. infection, have been vaccinated, or both (146, 200, 206). Pre-
Infants. Most deaths due to B. pertussis infection occur in vious infection or previous immunization tends to modify ill-
infants, and severe morbidity is most common in this age group ness in adults. Nevertheless, adolescents and adults can have
(35, 44, 112, 136, 147, 156, 185, 186, 270, 335, 338, 339, 360, symptomatic infection, mild disease, or classic pertussis. The
510, 512, 537, 583, 588, 619, 630, 669, 670, 712, 751, 790). In the experience of one of us (J.D.C.) and the results of several
United States from 1997 through 2000, there were 7,203 cases studies suggest that adults who were primed by previous infec-
of reported pertussis in infants younger than 6 months of age. tion (as opposed to priming by immunization) are more likely
Of this group, 63.1% were hospitalized, 11.8% had pneumonia, to have typical pertussis.
1.4% had seizures, 0.2% had encephalopathy, and 0.8% died. In a U.S. study of 27 adolescents and adults, the following
In the 6- to 11-month age group, 28.1% were hospitalized, clinical findings were noted: (i) the median duration of cough
8.6% had pneumonia, 0.7% had seizures, 0.1% had encepha- was 42 days, with a range from 27 to 66 days; (ii) all subjects
lopathy, and ⬍0.1% died (112). had paroxysmal cough; (iii) 26% of the subjects had whooping;
The source of infection in infants is frequently an adolescent (iv) 56% had post-tussive vomiting; and (v) 100% had post-
or adult family member (35, 56, 186, 200). In a recent study of tussive gagging (739). In another study, the attending doctors
616 infants with pertussis, the source was identified in 264 cases failed to diagnose pertussis in 31 university students who
(43%) (56). Of this group, mothers were the source of 32% of showed laboratory evidence of Bordetella infection (550). In-
infections and another family member was the source in 43%. stead, their diagnoses included upper respiratory tract infec-
Of the source persons, 20% were 10 to 19 years of age and 56% tion (39%), bronchitis (48%), and others (16%). In this group
were adults. of patients with pertussis, the median duration of cough illness
Neonatal infection is particularly severe, with up to a 3% risk prior to being seen was 21 days; 94% of patients had one or
of death (44, 156, 165, 360, 512). The initial finding is fre- more coughing episodes per hour, and 90% of the coughing fits
quently apnea, and although the babies cough, their cough is so had a staccato or paroxysmal quality. Only two findings in this
faint that it often goes unrecognized. Seizures due to hypoxia study differentiated patients with Bordetella infection from
resulting from apnea are common. Severe pulmonary hyper- those with cough illnesses without evidence of Bordetella in-
tension, the cause of which is unknown, is a frequent cause of fection. Patients with pertussis were less likely to have a pro-
death (510, 630, 790). In young infants, the severity of disease ductive cough (3% versus 21%) and were less likely to have an
and risk of death correlates directly with the white blood cell antibiotic prescribed at the time of the visit (39% versus 64%).
count and in particular the number of lymphocytes (44, 156, In contrast to the two U.S. studies, the findings in two German
338, 512, 537). White blood cell counts in the range of 30,000 studies noted more typical illness (634, 695). It should be noted
to ⬎100,000 cells/ml are common. Coinfections with adenovi- that most of the German adults had not been immunized
ruses or respiratory syncytial virus are relatively frequent (44, during childhood whereas most of the adolescents and adults
186, 459, 712). Other risk factors for fatal disease in young in the U.S. studies had.
infants are premature birth and the presence of pneumonia In a household contact study in Germany relating to a pe-
(537, 790). diatric vaccine efficiency trial, there were 79 adults with labo-
The relationship between B. pertussis infection and sudden ratory-confirmed B. pertussis infections (634). Of this group,
infant death syndrome (SIDS) is interesting but far from clear. 80% coughed for ⱖ3 weeks and in 63% the cough was parox-
In a study done more than 20 years ago, it was found that infant ysmal, 53% had post-tussive choking or vomiting. Complica-
deaths due to B. pertussis infection were often misdiagnosed as tions included pneumonia, rib fracture, inguinal hernia, and
respiratory viral infections (136). Similarly, in another study of severe weight loss. Unique sweating episodes were noted in 5%
of the ill subjects (634). 93, 199, 201, 224, 275, 283, 409, 427, 509, 511, 646, 671, 713,
In another adult study in conjunction with a cohort vac- 794, 834).
cine efficiency trial in children, 64 laboratory-confirmed cases Swine. In pigs, infection with B. bronchiseptica may be
were noted (695). Of this group, 70% had paroxysms, 38% had asymptomatic or may be associated with upper respiratory
whooping, 66% had post-tussive phlegm, and 17% had post- tract disease characterized by sneezing and coughing (275).
tussive vomiting. In adults, fainting may occur in association
This is followed by a deformity of the bony structures of the
with coughing fits (205). nose (atrophic rhinitis). Disease in pigs usually occurs during
infancy. Most cases occur in animals that are 3 to 8 weeks of
B. parapertussishu age, but animals that are slightly older (11 to 13 weeks of age)
may also develop turbinate atrophy. Pigs with atrophic rhinitis
B. parapertussis infection in humans can cause unrecognized have a shortening of the upper jaw and a twisting of the snout
infection, mild pertussis, or classic pertussis (51, 222, 314, 330, to one side. In some very young pigs the turbinate damage may
340, 363, 444, 465, 468, 498, 499, 545). B. parapertussishu was not persist due to regeneration of the bone.
first isolated in the United States and during the first half of the Dogs. Infectious tracheobronchitis of dogs (kennel cough) is
20th century was studied in Michigan, New York, and Califor- a highly contagious respiratory disease (427). The illness may
nia, as well as in Denmark (222, 445, 545). After that, little be mild with only a dry hacking cough or severe with a parox-
attention was paid to B. parapertussishu in the United States ysmal dry or mucoid cough, accompanied by ocular and nasal
and Europe until the end of the century. At this time, in con- discharge. Dogs with severe disease frequently have pneumonia.
junction with vaccine efficacy trials, extensive studies compar- Post-tussive retching and vomiting occur (283). The illness lasts 1
ing illness due to B. pertussis with that due to B. parapertussishu to 3 weeks, and deaths due to pneumonia are not uncommon.
have been performed in Sweden, Italy, and Germany (51, 340, Laboratory animals. Outbreaks of respiratory disease have
465, 498, 499). In addition, a similar non-vaccine-related study occurred in animals in commercial facilities which produce
was carried out in Finland (330). animals for research (283). Illness can vary from nasal dis-
In the first of these studies, illnesses in 38 children with charge, sneezing, loss of appetite, and weight loss (rabbit ca-
B. parapertussishu infections were compared with B. pertussis
tarrh) to bronchopneumonia and septicemia (93, 646, 713).
illnesses in 76 children matched by sex, season, and age (340).
Humans. In 1991, Woolfrey and Moody reviewed human
Comparative findings (B. pertussis versus B. parapertussishu)
illnesses associated with B. bronchiseptica infections (831).
showed the following: cough for ⬎4 weeks, 57% versus 37%
Subsequently there have been many additional case reports,
(P ⫽ 0.06); whooping, 80% versus 59% (P ⫽ 0.07); whooping
and these have most recently been summarized by Ner et al.
for ⬎2 weeks, 26% versus 18% (P ⫽ 0.05); paroxysms, 90%
(587). In 1910 McGowan studied 13 laboratory workers ex-
versus 83% (P ⫽ 0.5); fever (temperature ⱖ 38°C), 9% versus
posed to various animals with B. bronchiseptica infections
0%; post-tussive vomiting, 47% versus 42%; and mean leuko-
(511). While all of these workers had nasal symptoms, B. bron-
cyte and lymphocyte counts, 12,500/mm3 and 7,600/mm3 versus
chiseptica was isolated from only one of them. This worker
7,800/mm3 and 3,500/mm3 (P ⬍ 0.0001), respectively.
constantly handled rabbits and guinea pigs and had severe
In a study in Italy, Mastrantonia et al. identified 76 children
with B. parapertussishu infections and found that in this group, chronic nasal symptoms (catarrh) over an 18-month period.
100% had cough, 76% had paroxysms, 33% had whooping, The illness was intractable and had acute exacerbations. Dur-
42% had post-tussive vomiting, 29% had apnea, and 12% had ing an exacerbation, a pure culture of B. bronchiseptica was
cyanosis (499). With the exception of the frequency of parox- grown from “a mass of muco-pus hanging down over the soft
ysms, the frequency of the other manifestations were all less palate.” In 1926 Brown described a mild pertussis-like illness in
common than those noted in children with B. pertussis infec- a 5-year-old girl whose illness commenced about 10 to 12 days
tions. Similar findings were noted in a study in Munich, Ger- after she had been given a rabbit with mild “snuffles” (79). B.
many, where illnesses in 64 children with B. parapertussishu bronchiseptica was isolated from this child as well as from the
infections were compared with B. pertussis illnesses in 116 rabbit.
children (465). In the review by Woolfrey and Moody (831), they described
The results in some older studies have been less conclusive. an analysis of the results in 23 papers as well as two of their
This is probably because concomitant infections with B. per- own experiences (77, 79, 82, 88, 125, 126, 191, 241, 262, 267,
tussis and B. parapertussishu are fairly common and difficult to 400, 405, 431, 432, 604, 714, 733, 822). Of this group of 25
interpret (363, 389, 535). patients, 56% had a compromising factor and all but 3 (12%)
had a known exposure to animals. A recent paper by Ner et al.
(587) notes 20 additional reports and presents a detailed de-
B. bronchiseptica
scription of six patients (43, 65, 129, 154, 188, 202, 212, 227,
B. bronchiseptica causes respiratory infections in many dif- 261, 282, 476, 477, 613, 614, 650, 748, 786, 797, 824, 847). Over
ferent mammals including mice, rats, guinea pigs, skunks, half of the recent cases of B. bronchiseptica illness have in-
opossums, rabbits, raccoons, cats, dogs, ferrets, foxes, pigs, volved human immunodeficiency virus-infected patients. In
hedgehogs, sheep, koalas, leopards, horses, lesser bushbabies, one report of nine patients, all had had at least one AIDS-
and occasionally humans (283). The most important and best defining condition before the B. bronchiseptica infection (212).
described natural infections are in dogs and pigs. Infections in Pneumonia usually occurs in AIDS patients as well as in other
laboratory animals have also provided a wealth of information immunocompromised patients and is frequently cavitary. Dis-
for understanding B. bronchiseptica pathogenesis (23, 48, 83, seminated infections are known to occur. Other respiratory
manifestations include sinusitis and bronchitis. ELISA to measure increases in IgG and IgA antibody titers to
Normal children exposed to farm animals or pets usually PT in paired serum samples.
have pertussis-like illnesses. Access to diagnostic or laboratory methods is a major prob-
lem in both developed and developing countries. In developed
countries, in which pertussis is apparently well controlled by
B. holmesii
immunization, few laboratories are equipped for the routine
B. holmesii was identified as a Bordetella sp. in 1995 and was diagnosis of B. pertussis infection. Few laboratories maintain
initially noted as a cause of septicemia (814). Subsequent stud- fresh culture media for diagnosis, and kits for specimen col-
ies noted further septicemic illnesses as well as respiratory lection and transport are not routinely available. B. pertussis
infections (294, 467, 508, 560, 591, 678, 752, 839). Of particular serologic testing using acceptable techniques is rarely avail-
interest were the results of studies of persons with pertussis- able. Commercial laboratories provide an array of B. pertussis
like illnesses in Massachusetts from 1995 through 1998 (839). ELISA techniques, but, in general, there is no evidence of
The investigators isolated B. holmesii from nasopharyngeal sensitivity or specificity related to serum specimens from pa-
specimens from 33 patients suspected of having pertussis. Of tients. Furthermore, PCR is not routinely available.
the 23 patients with available clinical data, 100% had cough, In developing countries, laboratory services and resources
61% had paroxysms, 26% had post-tussive vomiting, and 9% vary markedly from country to country. However, the facilities
had whooping. for the routine culture of B. pertussis are frequently more
readily available and laboratory workers are more knowledge-
DIAGNOSIS able than in many developed countries, in which pertussis is
Differential Diagnosis of Bordetella Infections controlled. Routine serologic diagnostic services and PCR are
generally not available.
Infections in humans. In classic pertussis, the clinical There is currently no evidence of failure to detect new
diagnosis should be made without difficulty based on the par- B. pertussis variants by culture, PCR, or serologic testing. The
oxysomal cough with post-tussive vomiting and whooping, ab- experience throughout the vaccine era suggests that pertussis
solute lymphocytosis, and lack of significant fever (149). How- variants are unlikely to be a future problem. Surveillance stud-
ever, the etiologic diagnosis of illness due to B. pertussis versus ies using pulsed-field gel electrophoresis are likely to be useful
B. parapertussishu requires definitive laboratory study. Many in identifying variant strains that would affect PCR and/or
other infectious agents cause illnesses with cough that can be serologic diagnoses (306).
confused with Bordetella infections. Most important in this re- Laboratory techniques for the determination of B. pertussis
gard are Mycoplasma pneumoniae, Chlamydia pneumoniae, ad- infections have existed for over 90 years. A specific medium for
enoviruses, and other respiratory viruses (33, 136, 147, 161, the culture of B. pertussis was described in 1906 (63), and the
166, 196, 314, 584, 829). In addition, spasmodic attacks of demonstration of serum agglutinating antibody for diagnosis
coughing may be observed in children with bronchiolitis, bac- was noted in 1916 (635). In the present era, the extensive
terial pneumonia, cystic fibrosis, or tuberculosis. The cough acellular pertussis vaccine efficacy trials and other epidemio-
associated with sinusitis can also be confused with that caused logical studies have made it possible to evaluate multiple meth-
by Bordetella spp., as can the cough associated with an airway ods for the laboratory diagnosis of B. pertussis infections (229,
foreign body. 293, 310, 315, 317, 337, 493, 536, 693, 719, 722, 740, 781).
Infection in animals. In dogs and laboratory animals, the Common laboratory diagnostic methods currently include cul-
clinical manifestations of B. bronchiseptica are the same as ture, direct antigen detection (direct fluorescent-antibody
those associated with tracheobronchitis. There are many viral [DFA] test) PCR, and serologic demonstration (ELISA with
and bacterial causes of tracheobronchitis, so that in isolated many B. pertussis antigens and agglutination) by measuring
cases, laboratory study is necessary to diagnose B. bronchisep- rises in titer or high single serum values. Factors such as past
tica infection. In general, the same is true for the early stages exposure to the bacterium, age, antibiotic administration, im-
of illness in pigs. munization, timing of specimens, and laboratory sophistication
can affect the sensitivity and specificity of the individual tests.
Specific Diagnosis of B. pertussis Infections
The greatest sensitivity is obtained when culture is supple-
An excellent, detailed account of the laboratory diagnosis of mented by PCR and serologic testing.
Bordetella infections is presented by Loeffelhalz in the eighth Culture of B. pertussis. B. pertussis is a fastidious small
edition of the Manual of Clinical Microbiology (473). (0.2- by 0.7-␮m), faintly staining, gram-negative coccobacillus.
Many practical problems markedly affect the sensitivity and Suboptimal culture results can occur because of inadequate
specificity of the laboratory diagnosis of pertussis. These in- specimen collection and transport, as well as poor laboratory
clude delay in specimen collection, poor specimen collection methods. Methods for specimen collection, transport and cul-
technique, specimen transport problems, laboratory medium, ture have been well described (272, 273, 276, 359, 418, 473, 572,
problems, laboratory inexperience, laboratory contamination, 827).
equipment expense, and the conventional need for two serum (i) Specimen collection. B. pertussis colonizes the ciliated
samples for serologic diagnosis. In addition, there may also be epithelial cells in the upper and lower respiratory tracts. There-
confusion in the interpretation of serologic results. fore, a specimen for culture should be obtained from the sur-
The sensitivity and specificity of the laboratory diagnosis of face of respiratory ciliated epithelial cells of the upper respi-
B. pertussis infection can equal those for many other bacterial ratory tract. Specimens obtained from the throat, sputum, or
infections, with the proper performance of culture, PCR, and anterior nose, which are not lined with ciliated epithelium, are
not adequate (495). from correctly collected and transported specimens are bacte-
Two methods have been used successfully in the collection rial and fungal contamination and the lack of fresh media.
of specimens; these are nasopharyngeal swab and nasopharyn- (iv) DFA testing of nasopharyngeal secretions. The use of
geal aspiration. In young children, nasal wash specimens may DFA for the rapid diagnosis of B. pertussis and B. parapertussishu
also be satisfactory; however, dilution may be a problem, and infections has been recently reviewed (572). This method has
the method has not been adequately evaluated in the diagnosis been used for 40 years because it is rapid and inexpensive and
of B. pertussis infection (313). For nasopharyngeal swabs, cot- can provide a positive result when cultures are negative due to
ton and rayon should not be used because they contain fatty antibiotic use. However, DFA is an insensitive method since it
acids that are toxic to B. pertussis. Calcium alginate is the does not employ amplification and lacks specificity because of
preferred swab material, although Dacron is acceptable. The cross-reactions with members of the normal nasopharyngeal
swab shaft should be a fine, flexible wire. flora (229). As noted by Loeffelholz, commercially available
The correct use of a nasopharyngeal swab is an unpleasant antibodies are polyclonal (Becton Dickinson) or monoclo-
experience for the patient, and this unpleasantness can lead to nal, identifying a lipooligosaccharide eptiope (Accu-Mab; Al-
suboptimal specimen collection. For optimal results, the tip of tachem Pharma, Edmonton, Alberta, Canada) (473). Both
the swab must come into contact with the ciliated cells of the products have similar low sensitivity compared with culture,
respiratory epithelium. Failure to achieve this is a major cause but the Canadian product appears to have greater specificity
of negative cultures. Although there are data suggesting that (518, 572). False-positive results with polyclonal antibodies are
nasopharyngeal aspiration leads to a higher isolation rate of due to several organisms of the oral and nasopharyngeal flora
B. pertussis, this method is generally less practical in the clinical including unencapsulated Haemophilus influenzae, diphthe-
setting. rioids, and anaerobic or facultatively anaerobic bacterial spe-
(ii) Specimen transport. Immediate culture of a specimen is cies (229).
preferred but is not always practical in the clinical setting (v) Detection of B. pertussis by PCR. The use of PCR has
(367). For successful transport, the transport medium must made the rapid diagnosis of many infectious diseases possible,
prevent the loss of B. pertussis and inhibit the growth of other and its use in the diagnosis of pertussis infection is rapidly
organisms that obscure the identification of B. pertussis. The evolving (6, 81, 229, 337, 463, 520, 572, 652, 653, 693, 781). Key
transport medium of choice is Regan-Lowe agar (half-strength factors for the successful application of PCR in the diagnosis of
charcoal agar supplemented with horse blood and cephalexin), infection by Bordetella spp. involve sample collection and prep-
a nutritive medium that inhibits the growth of the normal aration, primer selection and amplification conditions, detec-
nasopharyngeal flora. Preincubation of this medium at 36°C tion, and internal and external positive and negative controls.
overnight before shipment of the specimen may increase the Aspects of these factors have been summarized by Müller et al.
yield of B. pertussis but may also result in a greater growth rate (572).
of other bacterial or fungal contaminants. Nonnutritive bacte- Calcium alginate swabs should not be used for PCR speci-
riological transport media, such as Ames medium, can be used mens because of inhibitory factors present in the fiber, and
if they contain charcoal and the period between specimen aspirate specimens need to be treated with a mucolytic agent
collection and culture is less than 24 h. to remove PCR-inhibiting substances. The use of a Dacron
(iii) Culture. Methods for culture are well described (272, swab with a fine, flexible wire shaft is recommended. Following
273, 359, 362, 418, 473, 572). Although many types of culture swabbing, the swab should be shaken vigorously in 0.4 ml of
medium have allowed the successful isolation of B. pertussis sterile 0.9% saline solution in a vial, the swab should be dis-
from clinical specimens, charcoal agar (Regan-Lowe agar) sup- carded, and the vial should be sealed (693). Primers have been
plemented with 10% horse blood and cephalexin (40 mg/liter) derived from four chromosomal regions: (i) the promoter re-
is currently the medium of choice (572). In situations when gion of the genes encoding PT, (ii) a DNA region upstream of
culture plates are inoculated directly without transport, it is the poron gene, (iii) repeated insertion sequences, and (iv) the
also advisable to inoculate an enrichment medium and then adenylate cyclase toxin gene, cyaA. All except cyaA primers are
replate following 48 h of incubation. Satisfactory enrichment specific for B. pertussis. Common primers include IS481, which
media include Regan-Lowe transport medium and Stainer- detects both B. pertussis and B. holmesii; IS1001, which detects
Scholte broth. both B. parapertussis and B. holmesii; and PTp1 and PTp2,
Although Bordet-Gengou agar is frequently used for cul- which amplify a 191-bp DNA fragment from the PT promoter
ture, the need for this medium to be freshly made makes it less region and is B. pertussis specific (158, 210, 229, 234, 373, 572,
practical than charcoal agar (which has an 8-week shelf life) in 627, 651–653, 693, 756).
most laboratories. Since cephalexin inhibits some B. pertussis A number of detection systems have been used that differ
strains, some laboratories inoculate specimens onto charcoal considerably in expense and sensitivity. During the last 15
agar both with and without this antibiotic. Agar plates are years, a number of PCR assays have been evaluated by com-
incubated at 35 to 36°C in high-humidity ambient air. B. per- paring results with culture and clinically typical pertussis. Over-
tussis is catalase and oxidase positive and urease negative and all, this diagnostic tool has the advantage of a much higher
is identified by specific antiserum via agglutination or fluores- sensitivity compared with conventional culture. In a prospec-
cence. Cultures are usually examined daily for 7 days. How- tive study, in which swabs for PCR and culture were obtained
ever, Katzko et al. found that an increased yield of positive simultaneously from 555 individuals with cough illness, the use
results occurred if they held the cultures for an additional of PCR increased the identification of B. pertussis infections by
5 days (406). almost fourfold, from 28 to 111 (693). In a similar but larger
The main reasons for failure of bacterial growth in culture study, Schmidt-Schläpfer et al. (694) found a 2.6-fold increase
in the detection of B. pertussis by PCR compared with culture. 550).

Only a few studies have been reported in which the sensitivity The greatest sensitivity and specificity for the serological
and specificity of PCR in the diagnosis of B. pertussis infection diagnosis of B. pertussis infection is achieved by ELISA and
were determined by comparison with serologically identified measurement of IgG and IgA antibodies to PT. A significant
cases (337, 463, 466, 652, 781). In one recent study, PCR rise in titer (greater than or equal to twofold) between acute-
results were compared with serologic diagnoses, and PCR had phase and convalescent-phase sera needs to be demonstrated.
a sensitivity of 61% and a specificity of 88% (337). Similar In adolescents and adults, single high values of IgG or IgA
findings have been observed in other studies. False-positive antibodies to PT also indicate infection. Although IgA anti-
results are a potential problem associated with PCR in the body to PT is more indicative of a recent antibody response, it
diagnosis of pertussis and other respiratory illnesses (6, 520, is less consistent than a PT IgG response (781). The younger
572). False-positive results can occur if specimens for PCR are the child, the less consistent the IgA antibody response. Simo-
opened in the pertussis laboratory before going to the PCR ndon et al. (707) noted that a significant fall in titer is also
laboratory (J. Cherry, unpublished data). In addition, they can diagnostic of infection.
occur as a result of contamination of the air in a room where
DTP immunization is carried out (754). Specific Diagnosis of Other Bordetella Infections
(vi) Serologic diagnosis of B. pertussis infection. Natural
The same transport and culture methods used for B. pertussis
infection with B. pertussis is followed by an increase in the
can be used for other Bordetella subspecies. In addition, the
concentrations in serum of IgA, IgG, and IgM antibodies to
other subspecies are generally less fastidious and grow on
specific antigens as well as to preparations of the whole organ-
blood and MacConkey agars (473). B. holmesii and B. bronchi-
ism (255, 289, 293, 310, 490, 519, 536, 572, 601, 719, 826). Prior
septica are inhibited by cephalexin, and so the isolation of these
to the development of ELISA techniques, the main serologic
organisms will be missed if Regan-Lowe transport medium is
test for the diagnosis of pertussis was the demonstration of a
used and if cephalexin is used in the culture media. The use of
fourfold increase in agglutinating-antibody titer. The antibod-
methicillin or oxacillin in place of cephalexin should allow the
ies measured in this test are directed against FIM2/3, PRN,
growth of B. holmesii and B. bronchiseptica (508, 831).
and LPS. Experience suggests that this test had reasonably
B. parapertussis, B. bronchiseptica, and B. holmesii can all be
good specificity but lacked sensitivity. The mainstay of the
identified in respiratory secretions by PCR (158, 210, 234, 627,
serologic diagnosis of pertussis during the last 15 years has
651, 652, 756, 787). Several methods, involving different prim-
been ELISA, using specific B. pertussis proteins as antigens.
ers and organism-specific oligonucleotide probes or restriction
Standardized techniques have been developed, and antibodies
enzyme cleavage patterns, have been developed for routine
are quantitated by the reference line computer programme
laboratory testing.
and reference sera from the Center for Biologics Evaluation
and Research, U.S. Food and Drug Administration. The pre-
TREATMENT
cision of the test is such that intra-assay coefficients of variation
have been reduced to ⬍10%, which makes a twofold increase A number of antibiotics have in vitro activity against B. per-
in titer significant. tussis (33, 39–41, 52, 361, 364–366). The mainstay of treatment
In contrast to natural infection, the primary immunization of over the last 30 years has been oral erythromycin. Erythromy-
children induces mainly IgM and IgG antibodies. Serologic cin administration during the catarrhal stage of illness shortens
diagnosis of pertussis may be suspected by the demonstration the duration of symptoms and eliminates the organism from
of an increase in agglutinin titer or the use of ELISA, showing the upper respiratory tract within 5 days of initiation of therapy
an increase in IgA or IgG antibody titer to PT, FHA, PRN, in most instances (41, 52, 308). Since untreated patients with
FIM, or to sonicated whole organisms in two serum samples pertussis are contagious for 2 to 4 weeks or more, erythromycin
collected 2 to 4 weeks apart. It is now clear that antibody treatment significantly shortens the period of individual con-
responses to FHA and PRN also occur following other Borde- tagiousness. Initiation of erythromycin treatment after the par-
tella infections, so that isolated increases in titers of antibody oxysmal stage has begun is generally thought not to benefit the
against these antigens are not specific for B. pertussis infection. patient in regard to either duration or severity of illness. How-
In addition, high titers of antibody to FHA may be the result of ever, in one small open randomized study, the children who
cross-reacting epitopes of nonencapsulated H. influenzae, were treated with erythromycin developed significantly fewer
M. pneumoniae, C. pneumoniae, and perhaps other bacteria. whoops than did children in the control group, even though
Some culture-positive patients, particularly children younger most had reached the paroxysmal state at the time of onset of
than 3 months, fail to develop measurable antibodies. treatment (52). In addition, it was the feeling of one of us
The primary problem in the serologic diagnosis of B. pertus- (J.D.C.), based on studies of German children, that treatment
sis infection by ELISA is the delay in obtaining the acute-phase early in the paroxysmal phase of illness was also beneficial
specimen. In patients with reinfections, a rapid increase in titer (333). The dose for pediatric patients is 40 to 50 mg/kg/day
occurs, so that with a delayed acute-phase sample, the titer is given every 6 h for 14 days. In 1997 the findings in a relatively
likely to have already peaked and further titer increases be- large study suggested that a 7-day course of treatment with
tween the acute-phase and convalescent-phase sera may not be erythromycin was as effective as a 14-day course (316). Al-
observed. However, for those not recently immunized, this though uncommon, the use of erythromycin in neonates has
problem can be circumvented by using single-serum ELISA. been associated with the occurrence of hypertrophic pyloric
Specifically, patients with illness will have ELISA titers that are stenosis (357). To our knowledge, the dose for adults has never
significantly higher than GMT, of sera from well controls (493, been adequately studied, but 1 to 2 g/day given every 6 h has
been used. It is the opinion of one of us (J.D.C.) that the lower that contained culture media, whole cells that were washed,
dose (1 g/day) is not optimal; the gastrointestinal side effects of mixed vaccines that contained other bacteria from the upper
erythromycin in adults, even with the lower dose, make it a less respiratory tract flora as well as B. pertussis cells, fractionated
than an optimal choice (139). vaccines (extracted vaccines), “detoxified vaccines,” and vac-
During the last decade, three B. pertussis isolates have been cine enriched with “toxic factors.” In his book, Lapin reviewed
found to be resistant to erythromycin (429, 449, 459). Of these 29 studies of a number of candidate vaccines which were per-
three erythromycin-resistant B. pertussis strains, two were stud- formed between 1933 and 1942 (442). It was recognized during
ied for resistance to trimethoprim-sulfamethoxazole and found early vaccine development that alterations in the conditions of
to be sensitive. It is likely that erythromycin-resistant strains culture, such as the use of human blood rather than horse
are also resistant to other macrolides, but this was demon- blood, resulted in products that were less reactogenic but con-
strated for only one of the isolates (459). At present, there is tained larger numbers of bacterial cells.
no evidence of an emerging erythromycin resistance pattern in In the mid-1940s, Kendrick et al. (416) developed a labora-
B. pertussis isolates (361, 429) and there is no evidence to tory test which could be used to predict vaccine efficacy. Prior
suggest that resistant strains are contributing to the increase in to this time, efficacy could be determined only in human trials.
reported pertussis in recent years. However, since PCR is now This test (the mouse potency test) employed intraperitoneal
being used in many laboratories for the diagnosis of B. pertussis vaccination of young mice with dilutions of test vaccine and a
illness, the possibility of missing resistant strains is a possible standard vaccine. The animals were challenged 14 to 17 days
problem. Since B. pertussis is likely to be sensitive to tri- later by the intracerebral inoculation of 100,000 live bacteria.
methoprim-sulfamethoxazole, this agent can be used in chil- Potency was determined by comparing survival in the two vac-
dren who cannot tolerate erythromycin or in situations of dem- cine groups at 14 days. A World Health Organization potency
onstrated resistance (365). unit was established such that vaccines were required to con-
The newer macrolides (azithromycin and clarithromycin) tain greater than 4 international units (IU) per dose and the
are also effective for the treatment of pertussis (11, 20, 441, immunization dose was three doses (12 IU) (833). In the
448, 497, 617). Although the dose and duration of treatment United States, the potency of DTP vaccines is based on the
with these two macrolides have not been well studied, we number of protective units in 1.5 ml of a specific vaccine lot
suggest the following based on the available data: azithromycin (159). The vaccine must have an approximate minimum po-
at 10 mg/kg on day 1 and 5 mg/kg on days 2 to 5 as a single dose tency of 12 units per 1.5 ml. This is based on either a single test
for 5 days for children and 500 mg on day 1 and 250 mg on days estimate of no less than 8 units or a 2, 3, 4 test geometric mean
2 to 5 for adults; clarithromycin at 15 to 20 mg/kg/day in two estimate of no less than 9.6, 10.8, or 12 units, respectively. The
divided doses for 7 days for children and 1 g/day in two doses maximum allowed potency is 36 units per 1.5 ml.
for 7 days for adults. Vaccines standardized by the mouse potency test were ex-
Humans infected with B. parapertussishu or B. holmesii tensively evaluated in trials conducted by the British Medical
should also respond to the macrolide therapy indicated above. Research Council after World War II, and the results of these
In contrast, however, B. bronchiseptica is usually resistant to studies validated the mouse potency test as a surrogate for
erythromycin (831). Most B. bronchiseptica strains are sensitive vaccine efficacy (523–525). The toxicity of whole-cell vaccines
to aminoglycosides, extended-spectrum third-generation peni- was evaluated using the mouse weight gain test.
cillins, tetracyclines, quinolones, and trimethoprim-sulfameth- Beginning in the mid-1940s, routine immunizations of chil-
oxazole. Therefore, for outpatient management, children can dren with pertussis vaccines was started in the United States,
be treated orally with trimethoprim-sulfamethoxazole and and this continued until the replacement of whole-cell vaccines
adults can be treated with the same drug or a tetracycline or with acellular pertussis vaccine in the late 1990s (136, 147, 149,
quinolone. 410–417). Initial vaccines were monocomponent whole-cell
pertussis vaccines, but by 1947 combination vaccines with diph-
theria and tetanus toxoids (DTP) were available and recom-
VACCINATION AND PREVENTION mended. During the 1950s, other countries also started per-
B. pertussis Vaccines forming routine pertussis immunization. In the early years,
reactogenicity of pertussis vaccines and the temporal associa-
Since pertussis was such a severe disease with high mortality, tion of immunization with catastrophic neurologic events and
attempts to create vaccines for both treatment and prevention deaths created major problems for many national programs.
were made soon after B. pertussis was first isolated (136, 147, As a result, vaccine manufacturers tried to make their vaccines
216, 442, 481, 482). The initial vaccines consisted of killed “safer.” These attempts in the United States had some suc-
whole B. pertussis organisms. Early on, it was recognized that cesses, but one vaccine was later shown to have poor efficacy
the production of serum antibodies and clinical protection in (293, 310). In Sweden, where there was great concern about
vaccine recipients correlated directly with the number of or- “vaccine encephalopathy,” the vaccine was changed and by
ganisms in the vaccine. It was also realized at this time that 1979 was found to be ineffective; its use was therefore discon-
clinical toxicity also correlated directly with the number of tinued (670).
organisms in the vaccine. From 1962 until 1977, a DTP vaccine with an extracted
In the 1930s, many candidate pertussis vaccines were devel- pertussis component (TriSlogan; Eli Lilly Co.) was available in
oped and used for the treatment and prevention of pertussis in the United States (147, 167, 807). This crude DTaP vaccine
limited studies. These initial vaccines were prepared by many was prepared by chemical extraction from whole bacteria, and
different methods; the products included whole-cell vaccines the cellular debris was removed by centrifugation. This vaccine
was extensively used because it was thought to be less reacto- were controlled, however, so that all rate estimates included
genic, but few data support this assumption. children with temporally related neurologic disease that may
At present, DTP vaccines are still routinely used in the have been caused by other factors. The first well done study,
developing world and many developed countries, whereas DTP and the most definitive to date, was a prospective case-control
vaccines with acellular pertussis components (DTaP vaccines) study (National Childhood Encephalopathy Study [NCES])
have replaced DTP vaccines in many developed countries. that evaluated all hospital admissions of children from 2 to 36
Whole-cell DTP vaccines. (i) Reactogenicity. The reactoge- months of age with acute serious neurologic illnesses (9). This
nicity of DTP vaccines has been extensively evaluated by one of study occurred in England, Wales, and Scotland between 1976
us (J.D.C.) in numerous research papers, chapters, and reviews and 1979. The results of this study have been analyzed and
(26–28, 30, 130, 133, 136, 137, 140, 142, 143, 145, 147, 149, 160, reanalyzed on multiple occasions and, in our opinion, present
236, 346, 458). Only a brief summary of these analyses and conclusive evidence against a causal relationship between DTP
related studies is presented here. immunization and brain damage.
From January 1978 to December 1979, a study involving The initial results of the NCES noted a statistically signifi-
15,752 doses of DTP and 784 doses of DT was carried out in cant association between pertussis immunization and neuro-
the Los Angeles area (26–32, 160). Children in this study were logic illness. Hospitalized patients with acute neurologic illness
evaluated for reactions that occurred within 48 h of vaccina- were about twice as likely to have been vaccinated with a
tion. In general, all common local and systemic reactions were pertussis vaccine during the week before the event than were
more common in DTP recipients that in DT recipients. Red- the controls for the matching period. However, the causal
ness, swelling, and pain at the injection occurred in 37.4, 40.7, inference based on this study must be questioned because
and 50.9%, respectively, of DTP recipients but in only 7.6, 7.6, when the period is increased from 7 days to 1 month preceding
and 9.9%, respectively, of DT recipients. The percentage of the event onset or the comparable period in the controls, the
these reactions in DTP vaccinees increased from the first dose frequency of pertussis immunization is the same (140, 142).
to the fifth dose. Fever (ⱖ38°C) occurred in 46.5% of DTP This indicates that the DTP immunization called attention to
recipients and in only 9.3% of DT recipients. Over the first or brought out an event that was to occur anyway but was
four doses, the rate of fever increased from 39.6 to 54.2%. moved forward in time.
Drowsiness, fretfulness, vomiting, anorexia, and persistent cry- A further analysis of the NCES which was restricted to
ing were all more common in DTP vaccinees than in DT infants with infantile spasms (an identifiable seizure disorder
recipients. With the exception of anorexia, these systemic re- of infancy) failed to find a statistical association between per-
actions decreased in frequency from the first to the fifth DTP tussis immunization and the event (47). Finally, an analysis of
doses. The overall rates for these systemic events in DTP vac- the NCES data with the exclusion of infantile spasm cases was
cinees were as follows: drowsiness, 31.5%; fretfulness, 53.4%; interpreted by the investigators to indicate a cause-and-effect
vomiting, 6.2%; anorexia, 20.9%; and persistent crying, 3.1%. relationship. Specifically, it was suggested that the risk of per-
Other more notable events in DTP vaccinees in this study, manent brain damage following pertussis immunization was 1
which were not frequent enough to compare statistically with per 330,000 vaccine doses and the risk of any encephalopathy
similar events in DT recipients, were the following: high- following immunizations was 1 per 140,000 vaccinations (539–
pitched, unusual cry, 0.1%; convulsions, 0.06%; and hypotonic- 541). However, as demonstrated by Stephenson (723) and
hyporesponsive episodes (shock, collapse), 0.06%. However, MacRae (480), both of these estimates are incorrect. In regard
none of the DT vaccinees had similar events. to any encephalopathy, the risk estimate is an artifact due to
Throughout the entire pertussis vaccine era, there has been the inclusion of nine children with febrile convulsions. In re-
concern about the temporally related occurrence of severe gard to the increase risk of brain damage, it is noted that
neurologic disease and death and DTP vaccination (4, 5, 9, 22, vaccinees and controls had the same rate of pertussis immu-
25, 42, 47, 50, 54, 80, 86, 87, 95, 106, 108, 110, 130, 133, 136, nization during the 1-month period, indicating a redistribution
140, 142, 143, 145, 147, 178, 195, 207, 209, 213, 214, 223, 233, of events over time and not a cause-and-effect relationship.
236, 237, 258, 259, 281, 295, 297–301, 312, 318, 320, 323, 345, During a period of extensive study of neurologic events
351–353, 355, 358, 368, 376, 379, 382, 398, 399, 408, 428, 435, temporally related to pertussis immunization, it became appar-
480, 488, 526, 538–543, 559, 566, 568, 570, 628, 629, 637, 706, ent that what was being called pertussis vaccine encephalopa-
712, 716, 720, 721, 723–729, 741, 742, 744, 755, 760, 795, 796). thy was not an encephalitis-like event but instead was the first
Over 70 years ago, Madsen (481) noted the deaths of two seizure or seizures of infantile epilepsy. During the late 1980s,
newborns shortly after receiving pertussis immunization, and four carefully performed studies were carried out in the United
in 1948, Byers and Moll reported a series of 15 patients in States and Denmark (142, 259, 296, 706, 795). Collectively in
whom severe neurologic disease had its onset after immuniza- these studies about 1 million pertussis immunizations were
tion (87). By 1979, more than 1,000 patients with neurologic evaluated to see if they were a causative factor in epilepsy. No
damage in association with pertussis immunization had been evidence of a causative relationship was found. Finally, a 10-
reported (160). In these initial case reports and small studies, year active surveillance study (1993 to 2002) in Canada found
no data were available to allow rate calculations and compar- no evidence of encephalopathy following ⬎6.5 million doses of
ison with unvaccinated subjects. In addition, alternative causes pertussis vaccines (559). To summarize, it is our opinion that
for the neurologic events were rarely studied. there is no such entity as “pertussis vaccine encephalopathy.”
During the period from 1967 to 1980, population studies SIDS has its peak age of occurrence at about 10 weeks of
were undertaken to determine the frequency of neurologic age, and since the first dose of DTP in the United States is
events following pertussis vaccination. None of these studies administered to infants at 2 months of age the temporal asso-
ciation of immunization and the occurrence of SIDS was to be These studies were carried out mainly in the United Kingdom
expected. However, in the late 1970s and 1980s there was and the United States, but data from Denmark, Sweden, Hun-
considerable media coverage which inferred a cause-and-effect gary, New Zealand, and Japan were also noted. In the United
relationship (178). The impetus for this view followed the States, four household contact studies and one case-control
occurrence of a time cluster of four deaths due to SIDS in study were reported for the periods between 1979 and 1983
Tennessee in 1974 which occurred within 24 h after DTP vac- (78, 109, 111). The reported efficacy in these investigations
cination (107). This was extensively studied by the Centers for varied from 64 to 96%. The highest efficacy estimate was noted
Disease Control and Prevention (CDC), and a weak statistical in the case-control study. These were all CDC studies, and dur-
association with one lot of a DTP vaccine was noted. However, ing the periods studied, vaccines from four different manufac-
the data from this study were carefully evaluated by the inves- turers (Wyeth, Lederle, Connaught, and Parke-Davis) were in
tigators and a panel of outside consultants, who concluded that use.
there was no causal relationship between the specific vaccine From mid-1982 to mid-1983, Blennow et al. carried out a
lot and the SIDS cases. nonblinded but prospective cohort study involving 1,140 in-
During the same period, two retrospective studies of limited fants in Sweden (58). In this trial, 525 infants received three
populations suggested that DTP immunization may have been doses of a plain whole-cell vaccine (Pertussis Vaccine Well-
causally related to SIDS (25, 760). However, it was noted that come; Wellcome) licensed in the United Kingdom and 615
there was considerable bias in these investigations (568). At unvaccinated infants served as controls. The subsequent attack
the same time, a case-control study in Sheffield, England, rate was 1.5% (8 of 525) among vaccinated children and 7.6%
found no association between DTP immunization and SIDS (47 of 615) among the controls (P ⬍ 0.001). The vaccine
(755). One further relatively small study in the state of Wash- efficacy was 80% (95% confidence interval [CI] ⫽ 58 to 90).
ington was thought by the authors to indicate a causal rela- The vaccine efficacy was 93% (95% CI ⫽ 72 to 98) in prevent-
tionship between DTP and SIDS (796). However, the sample ing pertussis with a cough duration of 4 weeks or more.
was small, there were only four cases within 3 days of immu- A more definitive CDC study of DTP vaccine efficacy was
nization, and the authors deleted nonimmunized SIDS victims carried out from 1984 through 1986 in three urban areas (Bal-
and their controls in the analysis process. timore, Md.; Denver, Colo.; and Milwaukee, Wis.) of the
Three large controlled studies present convincing data United States (602). A total of 347 children aged 1 through 4
against a cause-and-effect relationship between DTP immuni- years were exposed in the household to a primary case. To be
zation and SIDS (295, 355, 716). The first of these investiga- included in the study, a household had to have a culture or
tions was conducted in Norway over an 8-year period (716). serologically confirmed case or an illness that met the Council
The investigators compared the observed dates of SIDS occur- of State and Territorial Epidemiologists case definition for
rence in 222 infants with the expected frequency distribution of endemic pertussis (ⱖ14 days of cough and either vomiting,
SIDS and found no evidence indicating that DTP immuniza- whooping, or paroxysms). In this study, efficacy was calculated
tion was a causative factor. The second study was a large by using many different case definitions and by using all house-
case-control study in the United States, involving 800 cases holds and households with laboratory-confirmed cases. In ad-
(355). In this study the SIDS victims were less likely to have dition, efficacy rates were examined by the number of vaccine
received DTP immunization than the controls in the compar- doses and severity of disease in primary cases and in household
ative periods. This negative association between DTP immu- contacts. The efficacy varied from a low of 59% (95% CI ⫽ 24
nization and SIDS was noted in the Washington state study, to 78) to a high of 97% (95% CI ⫽ 86 to 99). In general, and
the study in Norway, and a recent analysis from a German as expected, the calculated efficacy increased directly with in-
investigation (335). The third large study which failed to show creased duration of cough and the addition of paroxysms and
a cause-and-effect relationship between DTP immunization whooping to the case definition. When any cough illness was
and SIDS was carried out in Tennessee and involved a cohort considered, the efficacy was 64% (95% CI ⫽ 49 to 75).
of 129,834 children who were born in four urban Tennessee Vaccine efficacy correlated directly with the number of vac-
counties during the period from 1974 through 1984 (295). In cine doses the vaccinee received. It was 18% for one DTP
this large cohort there was no increased risk of SIDS after DTP dose, 48% for two DTP doses, 58% for three DTP doses, and
immunization compared with a period distant from immuni- 68% for four or more DTP doses.
zation. In summary, it is clear to us that DTP immunization is A Los Angeles household contact study was carried out
not a causative factor in SIDS. between July 1987 and October 1990 (200). In this study, im-
(ii) Vaccine efficacy. The use of whole-cell pertussis vaccines munization data were available for 103 household contacts
and whole-cell pertussis component DTP vaccines has clearly who were 15 years of age or younger. Efficacy values were 37%
been highly efficacious (136, 147, 149). In the prevaccine era, (95% CI ⫽ 21 to 50) for any respiratory illness; 66% (95% CI
the average yearly rate of reported pertussis in the United ⫽ 49 to 77) for clinical pertussis (a cough illness lasting for at
States was 157 per 100,000 population. Following the routine least 2 weeks with paroxysms, whooping, or post-tussive vom-
immunization of children, this rate had fallen to ⬍1 per iting and without another apparent cause); and 89% (95%
100,000 population in the 1970s. Of interest, however, is that CI ⫽ 73 to 95) for laboratory-confirmed pertussis. At the time
no formal efficacy trials were ever carried out in the United of this study, the only DTP vaccines available were those man-
States on any individual DTP vaccines that were used during ufactured by Lederle and Connaught.
the last 50 years. The most definitive studies of vaccine efficacy occurred be-
In 1987, Fine and Clarkson reviewed the literature for effi- tween 1990 and 1995 in six vaccine efficacy trials in four coun-
cacy data and found 43 papers dating from 1923 to 1983 (245). tries in which various DTaP vaccines were evaluated and DTP
TABLE 3. Efficacy of DTP vaccines in six trials conducted in four countries from 1990 to 1995
Study location % Efficacy againsta:

Vaccine manufacturer
(reference) Mild and typical pertussis Typical pertussis
Senegal (708) Pasteur Mérieux Sérums et Vaccins 92 (86–99)

Sweden (310) Connaught USA 41 (30–51) 48 (37–58)
Italy (293) Connaught USA 23 (1–40) 36 (14–52)
Germany (719) Lederle 83 (76–88) 93 (89–96)
Germany (697) Behringwerke AG or SmithKline Beecham Biologicals 81 (68–89) 89 (77–95)
Germany (464) Behringwerke AG 96 (71–100)
a
95% CI is given in parentheses.
vaccines were used as comparative controls (293, 310, 334, 464, known components in identifiable quantities (688). Following
697, 708, 719). These trials (which are discussed more com- Sato’s lead, DTaP vaccines were developed by six manufactur-
pletely below in the section on DTaP vaccine efficacy) included ers in Japan and, because of epidemic pertussis, were put into
three cohort trials, two household contact studies, and one immediate routine use in Japan in 1981 (151, 422, 592). Use of
case-control study. The DTP vaccines were all licensed vac- these vaccines during the last 23 years has significantly con-
cines produced by Connaught and; Lederle (United States), trolled epidemic pertussis in Japan.
Pasteur Mérieux Sérums and Vaccines (France), SmithKline- The use of first-generation Japanese-made DTaP vaccines
Beecham Biologicals (Belgium), and Behringwerke (Germany). was limited to Japan because adequate efficacy, immunogenic-
The subjects in all studies received three doses of vaccine at ity, and reactogenicity data were not available on any single
about 2, 4 and 6 months of age, and in two studies a fourth dose vaccine or on vaccine use in early infancy. However, the suc-
was administered in the second year of life. cesses in Japan stimulated extensive studies and trials in Japan,
As noted in Table 3, all vaccines except the Connaught United States, Europe, and Africa, which were carried out over
vaccine had excellent efficacy values. The Connaught values a 15-year period from 1980 to 1995. Immunogenicity and re-
are surprisingly low and hard to reconcile with the values in the actogenicity studies were carried out on all candidate acellular
U.S. studies presented above, since the Connaught vaccine pertussis vaccines and DTaP vaccines. Subsequently, nine de-
enjoyed widespread use in the United States during these study finitive efficacy trials were carried out with 11 different vac-
periods. However, in the 1980s because of concerns relating to cines.
vaccine reactions, manufacturers tried variations in production (i) Reactogenicity. To meet regulatory requirements, numer-
techniques to make their whole-cell vaccines less reactogenic. ous phase II and III studies with candidate DTaP vaccines have
In this venture, Lederle was able to reduce the cell count in been carried out. Since all DTaP vaccines do not contain LPS,
their vaccine but nevertheless retain potency. We assume, or contain only trace amounts, it is not surprising that all DTaP
without any proof, that Connaught also made attempts to pro- vaccines are, in general, less reactogenic than whole-cell DTP
duce a less reactogenic vaccine. We do know, however, that the vaccines. The most definitive study of common vaccine reac-
Connaught DTP vaccine used in a Tennessee study in 1988 to tions was a National Institute of Allergy and Infectious Dis-
1989 elicited good antibody responses to PT, FHA, PRN, and eases (NIAID)-sponsored study which was carried out in the
FIM in 16 vaccinated infants whereas the Connaught DTP six NIAID-supported Vaccine Treatment Evaluation Units
vaccine used in the vaccine efficacy trials in Sweden and Italy in (VTEU) located in five geographic regions of the United
1992 produced only minimal responses to PT, FHA, and PRN States (198). In this study, the reactogenicity of 13 candidate
(24, 293, 310). DTaP vaccines given as the primary series at 2, 4, and 6 months
Acellular pertussis component DTP vaccines. Some of the of age was evaluated. There were 2,200 subjects who received
candidate vaccines reviewed by Lapin in 1943 were crude one of the 13 candidate DTaP vaccines or a DTP control
DTaP vaccines (442). In addition, a DTP vaccine with an ex- vaccine following a double-blind protocol. Between 113 and
tracted (fluid) pertussis component (TriSlogen) was available 217 infants received three doses of one of the 13 DTaP prod-
in the United States from 1962 to 1977 (147). This vaccine ucts, and 370 infants received the comparative DTP vaccine.
enjoyed considerable use even though its content was never The results of this study were presented in detail in a supple-
determined and its efficacy was never formally studied. As ment to Pediatrics (197). Of the 13 candidate vaccines, 7 were
technology became available in the 1970s, it was noted that PT, later evaluated in phase III efficacy trials. Two additional vac-
FHA, and LPS were liberated into liquid medium during cul- cines which were not evaluated in the NIAID study were also
ture (147, 149, 346, 688). These antigens could be concentrated evaluated in efficacy trials. The composition of the seven vac-
and separated by density gradient centrifugation. Although cines noted above as well as the two additional vaccines not
there was limited knowledge at the time about the function and studied are presented in Table 4. As can be seen, there is
importance of various B. pertussis antigens, it was thought early considerable variation among the vaccines in the number of B.
on that toxoided PT was the most important antigen to be pertussis antigens and their amounts, the amount of adjuvant
included in an acellular vaccine and that LPS was a major (aluminum), and the amounts of diphtheria and tetanus
cause of vaccine reactions and therefore should be removed toxoids.
(623, 688). In the overall study of the 13 candidate vaccines, local reac-
Sato et al. in Japan were the first to describe the production tions (redness, swelling, and pain), fever, fussiness, drowsiness,
of aceilular pertussis component vaccines which contained anorexia, and the use of antipyretics were all more frequently
TABLE 4. Composition of nine DTaP vaccines evaluated in efficacy trialsa

Amt of B. pertussis antigens (␮g/dose) Amt. of Flocculationc
Vaccineb aluminum Preservative
PT FHA PRN FIM (mg/dose) Diphtheria toxoid Tetanus toxoid
d
Certiva 40 0.50 Thimerosal 25 7
Tripediad
23.4 23.4 0.17 Thimerosal 6.7 5
PM-2e 25 25 0.30 Thimerosal 15 5
SKB-2e 25 25 0.50 Phenoxyethanol 25 10
Acelluvax 5 2.5 2.5 0.35 Thimerosal 25 10
Infanrixd 25 25 8 0.50 Phenoxyethanol 25 10
ACEL-IMMUNEd 3.5 35 2 0.8 f 0.23 Thimerosal 9 5
DAPTACELd 10 5 3 5g 0.33 Phenoxyethanol 15 5
Daptacel-like (enhanced) 20 20 3 5g 0.33 Phenoxyethanol 15 5
a
Data from references 149 and 216.
b
Trade names.
c
Limit of flocculation per dose.
d
Licensed in the United States.
e
No product name.
f
FIM2.
g
FIM2/3.
detected in DTP recipients than in DTaP vaccinees (Table 5)

TABLE 5. Percentage of DTaP- and DTP-vaccinated infants
(198). The rate of vomiting was similar in DTaP and DTP reported to have local or systemic reactions
vaccine recipients. Over the course of the three-dose series, the within 3 days of immunizationa
frequency of fever, redness, and swelling increased whereas
% of infants with reaction to:
that of drowsiness decreased in DTaP vaccine recipients. Category
There were only minor differences between some of the DTaPb DTPc
reaction categories among the seven DTaP vaccines that were Systemic
later evaluated in efficacy trials. In general, the two vaccines Fever (temp ⬎ 100°F)
with the smallest amounts of diphtheria toxoid (ACEL- First dose 4.2 27.3
IMMUNE and Tripedia) were less likely to be associated with Second dose 11.3 34.1
Third dose 15.8 37.7
fever, redness, or swelling after the third dose than were the Antipyretic use
other five vaccines with greater amounts of this toxoid. First dose 39.3 60.5
A subsequent study evaluated reaction rates for the same 13 Second dose 36.7 59.8
vaccines following a fourth dose at 15 to 20 months of age Third dose 36.3 61.4
Fussiness
(615). Overall, when rates in DTP recipients were compared First dose 6.6 20.6
with the collective rates in DTaP vaccinees, it was found that Second dose 7.7 23.5
DTP recipients had a greater frequency of irritability and in- Third dose 6.7 17.3
jection site redness, swelling, and pain. When the reaction Drowsy
rates in recipients of the seven DTaP vaccines that were used First dose 29.9 43.5
Second dose 17.6 31.0
in efficacy trials were compared, there were considerable Third dose 12.9 24.6
differences. Similar to the findings after the third dose, the Anorexia
two vaccines with the smaller amounts of diphtheria toxoid First dose 9.3 19.5
(ACEL-IMMUNE and Tripedia) were also less likely to be Second dose 8.9 16.5
Third dose 8.9 14.3
associated with fever, irritability, and redness, swelling, and Vomiting
pain at the injection site after the fourth dose. They were also First dose 6.3 7.0
less likely to be associated with severe irritability or marked Second dose 4.5 4.5
(⬎50-mm diameter area) redness at the injection site. Third dose 4.2 5.3
In the NIAID study, it was noted that 20 children had entire Local (injection site)
thigh swelling in the leg where the fourth dose of a DTaP Redness
vaccine was injected (656). This reaction was noted to have a First dose 13.5 49.4
significant linear association with the amount of diphtheria Second dose 17.1 47.7
toxoid in the DTaP vaccine, and it was not observed after Third dose 21.5 47.6
Swelling
receipt of either ACEL-IMMUNE or Tripedia, the two vac- First dose 8.7 39.7
cines with the smaller amounts of diphtheria toxoid. Also, Second dose 12.1 34.1
lesser degrees of swelling (⬎50 mm but less than the entire Third dose 13.3 35.7
limb) correlated with the vaccine PT content after dose 4 and Pain
First dose 3.8 27.3
the aluminum content after dose 5. Second dose 2.0 18.7
In several additional studies, a high frequency of relatively Third dose 2.1 15.8
large reactions (redness and swelling) at the injection site were a
Data from reference 198.
noted following four and five doses of DTaP vaccines (278, b
Thirteen candidate DTaP vaccines.
616, 692, 696, 711, 764). In all these studies except one, the c
Tri-Immunol (Lederle DTP).
DTaP vaccines contained either 15 or 25 limits of flocculation (6 ␮g of protein nitrogen per dose), and the other vaccine
(Lf) of diphtheria toxoid. Interestingly, the NIAID VTEU contained toxoided PT and FHA (3.75 ␮g of protein nitrogen
study results indicate that vaccines with high concentrations of per dose for each antigen). Depending on the case definition,
both PT and diphtheria toxoid were the most likely to cause the efficacy of the PT toxoid varied from a high of 54% (95%
redness and swelling of ⬎50 mm in diameter. The three vac- CI ⫽ 26 to 72) to a low of 32% (95% CI ⫽ 6 to 51). The efficacy
cines with the lowest rates of redness and swelling of ⬎50 mm
of the two-component PT toxoid/FHA vaccine varied from a
in diameter were Tripedia, Acelluvax, and ACEL-IMMUNE. high of 69% (95% CI ⫽ 47 to 82) and a low of 45% (95% CI ⫽
The efficacy trials carried out in the early 1990s allowed the 22 to 62).
gathering of data relating to less common and more clinically Three further analyses of data from this trial were subse-
significant vaccine-associated events (157, 293, 310, 331, 464, quently published (735–737). These included household con-
768, 772). Overall, persistent crying, temperature of ⱖ40.5°C, tact substudy data, cases diagnosed serologically, and analyses
hypotonic-hyporesponsive episodes, and convulsions were all using many different clinical case definitions. As might be ex-
less frequent during the primary series following DTaP immu- pected, the calculated efficacy increased when the case defini-
nization than following DTP immunization. For example, the tion required paroxysmal cough of a long duration as well as
likelihood of persistent crying for ⱖ3 h, temperature of laboratory confirmation.
ⱖ40.5°C, hypotonic- hyporesponsive episodes, and seizures in Since (i) no whole-cell pertussis vaccine had been included
the Italian trial was, respectively, 7, 7, 17, and 6 times more in the initial trial in Sweden, (ii) the efficacies of the PT toxoid
likely following DTP immunization than following one of the and the PT toxoid/FHA vaccines were lower than expected,
two DTaP vaccines (157). Similar findings were noted in the and (iii) there were still no efficacy data relating to the first year
Stockholm (Sweden) trial, except that the occurrence of con- of life, a number of trials with seven of the vaccines evaluated
vulsions was similar in DTP and DTaP recipients. In the Er- for immunogenicity and reactogenicity in the NIAID VTEU
langen (Germany) trial, the likelihood of persistent inconsol- study as well as two additional candidate vaccines were planned
able crying, temperature of ⱖ40.5°C, and convulsions were and carried out in the early 1990s. These vaccines and their
four, three, and four times more common, respectively, in DTP contents are listed in Table 4. Because of the confusion related
recipients than in DTaP vaccinees (772). Only one child in this
to the variation of efficacy depending on the case definition, it
trial, a DTP recipient, had a hypotonic-hyporesponsive epi-
was felt by leading investigators that a universal case definition
sode. It is clear from the above that true but infrequent severe
should be developed. With this goal, a World Health Organi-
reactions following DTP immunization (persistent crying, tem-
zation (WHO) committee met in January 1991 in Geneva
perature of ⱖ40.5°C, hypotonic-hyporesponsive episodes, and
(832). Resulting from this meeting was the WHO primary case
the induction of febrile convulsions in children prone to febrile
definition. This definition or a slight variation of it was used in
convulsions) are significantly reduced in infants following im-
six of the eight subsequent efficacy trials which were carried
munization with DTaP vaccines.
out. The WHO case definition required an illness with ⱖ21
(ii) Vaccine efficacy. As noted above, the first DTaP vaccines
days of spasmodic cough and either culture-confirmed B. per-
were developed at the Japanese National Institutes of Health
tussis infection or serologic evidence of infection as indicated
in the late 1970s (151, 592, 688). First-generation DTaP vac-
cines produced by six companies were put into routine use in by a significant rise in IgA or IgG ELISA antibody against PT
Japan in 1981 despite the lack of proof of efficacy of any of the or FHA in paired sera or, as an alternative, contact with a case
six vaccines (149). Subsequently, six small household contact of culture-confirmed pertussis in the vaccinee’s household with
studies were published, and the calculated efficacy varied from onset within 28 days before or after the onset of the cough
78 to 92% (592). However, in none of the studies were vaccines illness in the study subject.
from a single manufacturer evaluated. Later, one household Not all members of the WHO committee, including one of
contact study carried out by multiple investigators evaluated a us (J.D.C.), agreed with this primary case definition (131, 149).
single vaccine (the Takeda vaccine) (569). In this study, efficacy A definition which discarded bona fide cases seemed illogical
against all infections was 81% and efficacy against typical in- at the time, and this is even more apparent today. With this
fections was 98%. In retrospect, all of these studies had signif- definition, vaccines that lessen the severity of disease but do
icant problems relating to the method of diagnosis, the method not prevent disease will have calculated efficacies similar to
of case ascertainment, and observer bias. However, the biggest those of much better vaccines that prevent mild disease as well
problem was that no vaccinated infants were studied; immuni- as typical disease.
zation was instituted after the second birthday. As an example of how illogical the WHO case definition was,
Since adequate data were not available on any single vaccine one need only look at data from the original Swedish trial (3,
or on vaccine use in infancy, a prospective double-blind study 735–737). In recipients of JNIH-7 vaccine (the PT toxoid),
was carried out in Sweden in the mid-1980s (3). In this trial, there were 23 culture-confirmed cases with 1 day or more of
which was supported by NIAID, two acellular pertussis vac- coughing spasms. When ⱖ21 days of coughing spasms is re-
cines were studied. A control group received a placebo, but no quired for the definition of a case, there were only 12 cases;
whole-cell pertussis vaccine control was used in the trial. For 48% of the cases have been removed from the data set. Sim-
reasons which are not entirely clear today, the 2-, 4-, 6-month ilarly with JNIH-6 PT/FHA vaccine recipients, 4 (29%) of 14
primary immunization schedule was not followed. Rather, two cases are removed from the data set by the more rigorous
doses of vaccine were administered. The first dose was given to definition. There were 39 placebo recipients with spasmodic
infants 5 to 11 months of age, and the second dose followed the cough for ⱖ1 day with culture confirmation. When ⱖ21 days of
first by 7 to 13 weeks. One vaccine (JNIH-7) was a PT toxoid spasmodic cough is required, 4 cases (10%) are removed from
TABLE 6. Vaccine efficacy data for eight acellular pertussis vaccines evaluated in seven trials carried out in the 1990s
% Efficacy
Location (reference) Design Vaccine used Schedule Typical Mild and typical
pertussis pertussis
Sweden (Göteborg)a (768) Double-blind prospective cohort Certiva 3 doses (3, 5, 12 mo) 71 54
Sweden (Stockholm)a (310) Double-blind prospective cohort SKB-2 3 doses (2, 4, 6 mo) 59 42
Daptacel 85 78
Italy (Rome) (293) Double-blind prospective cohort Acelluvax 3 doses (2, 4, 6 mo) 84 71
Infanrix 84 71
Germany (Erlangen) (719) Prospective cohort ACEL-IMMUNE 4 doses (3, 4, 5, 6, 15–18 mo) 83 72
Germany (Mainz) (697) Household contact Infanrix 3 doses (3, 4, 5 mo) 89 81
Germany (Munich)a,b (464) Case control Tripedia 4 doses (2, 4, 6, 15–25 mo) 80, 93
Senegalc (708) Household contact Triavax 3 doses 74 31
a
Significant observer bias occurred in this trial.
b
Laboratory diagnosis based on culture only; 80% efficacy was against cough illness of 21 or more days, and 93% efficacy was against the WHO case definition.
c
The 31% efficacy was based on 21 days or more of cough illness; 74% efficacy was against the WHO case definition.
the data set. cases in vaccinees.

Prior to the onset of the different efficacy trials, there was In the study in which one of us (J.D.C.) was the co-principal
considerable concern relating to the study methodology. It was investigator in Erlangen, Germany, Wyeth-Lederle made a
felt by many that reliable data could be obtained only in trials considerable effort in correcting for the biases of the unblinded
which were cohort in nature and completely double-blinded DT group. However, there was no evidence that use of the
and that both placebo and DTP controls were included. This of unblinded DT group led to an artificial increase in efficacy. In
course was a major ethical problem in countries where DTP fact, as noted in the substudy household contact analysis, the
vaccines were available and their use was recommended. In the attack rate in DT subjects was lower than expected due to the
end, only two trials (Stockholm no. 1 and Italy) followed this use of prophylactic macrolides (334).
format (293, 310). In Sweden the Göteborg trial was double- In the course of the Erlangen study, it became apparent to
blinded but there was no DTP vaccine control and the schedule one of us (J.D.C.) that the most significant bias in all efficacy
of immunization was 3, 5, and 12 months rather than 2, 4, and trials, including double-blind placebo-controlled trials, was ob-
6 months (preferred for comparing efficacy with other studies) server bias (150). Specifically, if vaccines make disease less
(768). The second, later Stockholm study was double-blinded typical but do not prevent illness, then if the observers (such as
but did not contain a placebo arm (597). In addition, in this parents and physicians) “know pertussis” and initiate study
trial two different vaccination schedules were employed (2, 4, only if they think the illness is pertussis, atypical cases will be
and 6 months and 3, 5, and 12 months). missed and efficacy will be inflated. This type of bias can be
In Germany, three trials were carried out and all were faced reduced if the vaccinees are monitored regularly by scheduled
with the problem that a double-blinded control group (given phone calls and all children with cough illnesses lasting ⬎7
DT vaccine) was not ethical (334, 464, 697, 719). However, days are evaluated. In this regard, the Erlangen protocol was
since at the time the majority of children were not being vac- the most satisfactory of all the cohort trials since telephone
cinated, it was relatively easy to obtain DT controls. Because of calls were made every 2 weeks. In Italy, scheduled telephone
the fear of DTP vaccines in Germany, it was difficult to per- calls were made monthly, and in Stocklholm no. 1, scheduled
form a cohort study with double-blind administration of DTP calls were made every 6 to 8 weeks (293, 310). Apparently,
or DTaP. Therefore, only one of the three German studies scheduled telephone calls were not made in the Götenburg
(Erlangen) was a cohort study with a double-blinded compar- trial (719, 768).
ison of DTP and DTaP and an unblinded DT group (719). The A measure of this type of observed bias can be made in the
Mainz study was a household contact study in which primary trials in which efficacy data for mild as well as typical disease
cases in households in six areas of Germany were identified were presented. Specifically, for DT recipients about 18 to 25%
and prospective follow-up of young children in the families was more cases are expected if mild and typical cases are compared
carried out (697). with just typical cases (333, 336). In Italy, Erlangen, Stockholm
The Munich study was a case-control study in which controls no. 1, and Götenburg, differences of 20, 18, 12, and 4%, re-
were selected randomly from records in the offices of partici- spectively, were noted (149, 293, 310, 768). This indicates that
pating physicians (464). Major weaknesses of this study were observer bias was greatest in the Götenburg trial and least in
that it was a case-control evaluation (which inflates efficacy) Erlangen and Italy.
and that laboratory diagnosis did not include serologic testing A summary of the efficacy data from the seven trials con-
(245). In both the Munich and Erlangen trials, the vaccinees ducted in the early 1990s is presented in Table 6. In general,
received a fourth dose of vaccine in the second year of life. three- and four- or five-component vaccines (vaccines that
Since the use of a DT control group was clearly not ethical, contain PRN and FIM as well as PT and FHA) have greater
the Senegal trial initially was a double-blind study in which a efficacy against both mild and typical pertussis than do PT or
DTP vaccine was compared with the candidate DTaP vaccine PT-plus-FHA vaccines.
(708). Subsequently, a household contact analysis was added so The final study, done in Sweden, was a comparative but
that cases in unvaccinated children could be compared with blinded study without a DT control group (597). In a head-to-
different epidemiologies. The first is the epidemiology of re-

ported clinical pertussis and, the second is the epidemiology of
B. pertussis infection. These are considered separately below.
Observed (reported pertussis). (i) Incidence. In the United
States in the prevaccine era, the average yearly incidence of
reported pertussis was 157 cases per 100,000 population (136).
In England and Wales in the prevaccine era, the average yearly
incidence of reported pertussis was 230 per 100,000 popula-
tion. It was realized at the time that only 18% of cases were
reported in the United States, suggesting that the actual rate
was in the range of 872 cases per 100,000 population. In the
prevaccine era, pertussis was an ever-present disease and in
addition there were epidemic cycles every 2 to 5 years (on
average about every 3 years). In the United States, the average
interval between epidemic peaks in the prevaccine era (1922 to
FIG. 3. Number of cases of pertussis in the United States from 1942) was similar to that in the early vaccine era (1962 to 1982)
1940 to 2002 (http://www.cdc.gov/nip/ed/slides/pertussis8p.ppt). and the present vaccine era (1983 to 2004) (136, 144). Fine and
Clarkson carefully studied the interepidemic interval in En-
gland and Wales for the 32-year period from 1950 to 1982 and
head comparison, the enhanced Daptacel-like vaccine had sim- found that it did not change significantly (244).
ilar efficacy to that of the effective DTP vaccine used in the With the introduction and widespread use of pertussis vac-
United Kingdom and was significantly more efficacious than cines in the 1940s and early 1950s, the rate of reported per-
the three-component Acelluvax vaccine. In the same trial, tussis fell approximately 150-fold (Fig. 3). For the 7-year pe-
Acelluvax was more efficacious than the two-component SKB-2 riod between 1976 and 1982, the rate of reported pertussis in
vaccine. the United States remained between 0.5 and 1.0 case per
100,000 population (136). From 1982 to the present, there has
B. bronchiseptica Vaccines
been a modest linear increase in the rate of reported pertussis,
Dogs (kennel cough). A number of kennel cough vaccines which reached 3.1 cases per 100,000 population in 2002 (144)
are available in the United States and recently the American (Fig. 4). The reason for this upward trend is unknown, but in
Animal Hospital Association Canine Vaccine Task Force 2003 our opinion it is most probably due to a greater awareness of
presented recommendations (23, 89a; Intra-Trac II Product pertussis by physicians, public health professionals, and the
Insert [http://medi-vet.com/IntraTracII.aspx]). The following public. Another contributing factor may be the decreased ef-
vaccines are among those available: killed whole-cell ficacy of one of the two DTP vaccines in use from around 1988
B. bronchiseptica (KWC Bb), live avirulent Bb (LABb), LABb/ until 1995 and more recently due to the use of DTaP vaccines
live attenuated parainfluenza virus (LAPV), and LABb/LAPV/ which, with one exception, are less efficacious than most DTP
live attenuated adenovirus type 2 (CAV-2). The KWC Bb vaccines (24, 131, 152, 290, 293, 310).
vaccine is given to puppies at 6 to 8 and 10 to 12 weeks of age Throughout history, reported pertussis was a disease of chil-
and then annually. Initial immunization of older animals is two dren. In the prevaccine era, ⬃95% of cases were noted in
doses 4 weeks apart. The LABb/LAPV can be given at 3 weeks children younger than 10 years (136). Data from Massachu-
of age with a second dose 3 weeks later and then administered setts from the period from 1933 to 1939 revealed the following
annually. The LAPV/LABb/CAV-2 vaccine is given at ⬎8 percent distribution by age group: ⬍1 year, 7.5%; 1 to 4 years,
weeks of age and is followed by annual single doses. Controlled
data for vaccine efficacies are not available.
Swine (atrophic rhinitis). A number of atrophic rhinitis vac-
cines are available (23, 201, 484). These include killed or non-
pathogenic strains of B. bronchiseptica with various prepara-
tions of Pasteurella multocida. No efficacy data are available.
Recommendations stress vaccination of sows prior to delivery,
and of piglets shortly after birth and again at 3 to 4 months of
age.
EPIDEMIOLOGY: IMPLICATIONS FOR THE

CONTROL OF HUMAN INFECTIONS
B. pertussis Epidemiology
There is much confusion today relating to the epidemiology
of pertussis. The main cause of this confusion is the failure to
recognize that there are major differences in the dynamics of
reported pertussis compared to the dynamics of B. pertussis FIG. 4. Number of cases of pertussis in the United States from
infection (132). For practical purposes, there are two vastly 1980 to 2002 (http://www.cdc.gov/nip/ed/slides/pertussis8p.ppt).
were noted in children 5 to 9 years of age, and two deaths

(3.6%) were reported in adults.
(iii) Sex and race. Historically, one of the most remarkable
features of reported pertussis was that the attack rate in fe-
males was higher than in males (136). The male/female ratio of
reported pertussis cases in children aged 0 to 9 years in En-
gland and Wales from 1945 to 1982 was 0.88 (95% CI ⫽ 0.84
to 0.92). Interestingly, the reported attack rate in infant boys
and girls during this period was almost equal. Data obtained in
the United States during the early vaccine era was similar to
the data noted above for England and Wales. Recent data
from 1999 to 2002 in the United States also noted more cases
in females than in males (113–116). The male/female ratio of
reported pertussis cases for this 4-year period is 0.85.
In the prevaccine era, reported pertussis attack rates in the
FIG. 5. Age distribution and incidence of reported cases of pertus- United States did not vary appreciably by race (285). Interest-
sis in the United States from 1997 to 2000 (http://www.cdc.gov/nip/ed
/slides/pertussis8p.ppt). ingly, recent data from the United States suggest considerable
differences in attack rates by race. During the period from 1999
to 2002, the highest attack rates occurred in American Indians
41.1%; 5 to 9 years, 46%; 10 to 14 years, 4.1%; and ⱖ15 years, or Alaska Natives and the lowest occurred in Asian or Pacific
0.9%. Similar prevaccine era data from England and Wales for Islanders (113–116). The reported attack rates in whites were
the period from 1945 to 1949 revealed the following percent consistently higher than those in blacks. For example, in 2002
distribution by age group: ⬍1 year, 10.4%; 1 to 4 years, 56.9%; the following rates per 100,000 were reported: American In-
5 to 9 years, 29.2%; 10 to 14 years, 2.0%; and ⱖ15 years, 1.4%. dian or Alaska Natives, 4.17; Asian or Pacific Islanders, 1.00;
With the marked reduction in reported pertussis resulting blacks, 1.55; and whites, 3.71 (116).
from universal pediatric immunization, there was a dramatic (iv) Season and geographic areas. Reported pertussis is
shift in the percent age distribution. For the period from 1978 noted in all populated areas of the world. However, reporting
to 1981 in the United States, when the rate was ⬍1 case per varies considerably. In the prevaccine era and early vaccine
100,000 population, the following percent distribution by age era, no seasonal pattern was observed for epidemic pertussis
group was noted: ⬍1 year, 53.5%; 1 to 4 years, 26.5%; 5 to 9 (136). For the 23-year period from 1980 to 2003, pertussis has
years, 8.2%; 10 to 14 years, 5.4%; and ⱖ15 years, 6.5% (136). been reported in every month of every year (96–105, 113–124).
Over the last 20 years, in conjunction with the modest in- In general, however, more cases are reported during the sec-
crease in reported pertussis, there has been an increase in ond half of each year. Between 1980 and 1986, the peak
reported pertussis in adolescents and adults (144). During the months of reported pertussis were August, September, and
period from 1997 to 2000, the following percent distribution by October. Since 1987, the peak month of reported pertussis has
age group was observed: ⬍1 year, 29.4%; 1 to 4 years, 11.1%; been December. However, surprisingly, the smallest number of
5 to 9 years, 9.8%; 10 to 19 years, 29.4%; and ⱖ20 years, 20.4% reported cases occurred in January. During the 4-year period
(109) (Fig. 5). from 1999 to 2002, the number of reported pertussis cases in
(ii) Mortality. Pertussis was a major cause of death in the December was 5.8 times the number of subsequent cases in
prevaccine era (136). In the period from 1926 to 1930, there January. This large difference seems biologically unlikely, and
were 36,013 reported deaths from pertussis in the United therefore it seems that the large number of cases in December
States. Mortality due to pertussis is markedly age related; the may reflect a reporting bias rather than a true increase in the
vast majority of deaths occur in infants. However in the pre- number of cases. Taking this into consideration, it appears that
vaccine and early vaccine era, some deaths were noted in older the true pattern from 1987 to 2002 is similar to that noted for
children. For example, the mean annual pertussis death rate in the period 1980 to 1986: most cases are observed in August,
the United States from 1940 to 1948 was 64 per 100,000 for September, and October.
infants, 6.4 per 100,000 for children 1 to 4 years of age, and 0.2 At the Hospital for Sick Children, Toronto, Canada, be-
per 100,000 for children 5 to 14 years of age. During this tween 1980 and 1990 the peak incidence of cases occurred in
period, more reported deaths in the first year of life resulted August, September, and October (286). In Britian the peak
from pertussis than from measles, scarlet fever, diphtheria, months of reported pertussis have been August and September
poliomyelitis, and meningitis combined. Recent studies suggest (710). In the United States from 1990 through 1999, reported
that pertussis is an occasional factor in deaths in older adults pertussis in infants peaked each year in the period from July to
(112, 124a, 534). September (790).
In the vaccine era, pertussis mortality declined following the B. pertussis infection. During the last 20 years, the study of
curve of the decline in reported pertussis (136). In contrast, in adolescent and adult B. pertussis infections has shed consider-
the prevaccine era, pertussis mortality was falling whereas the able light on the epidemiology of B. pertussis infections. As
incidence of reported pertussis was constant (567). noted above, the cyclic pattern of epidemic pertussis is similar
During the period from 1997 to 2000, there were 62 reported today to what it was in the prevaccine era. This pattern is
pertussis deaths in the United States (112). Of these, 56 (90%) different from that of other diseases which have been con-
occurred during the first 6 months of life. Two deaths (3.6%) trolled by immunization (135, 149, 550). For example, with the
TABLE 7. Percentage of prolonged cough illnesses in adolescents ence of a significantly high single serum titer as determined by
and adults as a result of infections with serologica PCR, comparison of titers in well controls. In addition to serologic
or culture evidence of Bordetella infections
study, diagnosis was attempted by culture, PCR, and DFA. The
% of individual studies were done under variable conditions, which
Author (reference) Location Yr illness due affected the results. Some studies were done by following a
to Bordetella
formal protocol over a relatively long period, whereas others
Jackson et al. (390) Seattle 1983–1987 15 were done during pertussis outbreaks. The latter, of course, is
Robertson et al. (665) New South Wales 1985–1986 26
Mink et al. (550) Los Angeles 1986–1989 26
a possible major bias in that the investigators might study only
Schmitt-Grohé et al. (695) Germany 1992–1994 32 subjects they thought had pertussis and might not study pro-
Wright et al. (835) Nashville 1992–1994 21 longed cough illnesses that they did not think were pertussis.
Wirsing von König et al. Germany 1992–1994 31 As noted in Table 7, the percentage of cough illnesses with
(828)
Rosenthal et al. (675) Chicago 1993–1994 26 laboratory evidence of Bordetella infection varied between a
Jansen et al. (397) San Diego 1993–1994 17 low of 12% and a high of 52%. The median value was 26%. A
Nennig et al. (586) San Francisco 1994–1995 12 major factor which affected the results of these studies was the
Strebel et al. (739) Minneapolis/St. Paul 1995–1996 13
Birbebaek et al. (55) Denmark 1995–1997 17
number of antigens used in the ELISAs. From several recent
Vincent et al. (788) Korea 1997–1998 50 studies, it has become clear that infections with other Borde-
Gilberg et al. (271) Paris 1999 52 tella subspecies can cause IgG and IgA antibody responses to
a
Significant rise in ELISA titer or high titer to PT, FHA, PRN, or FIM, or
FHA and PRN and to a lesser extent FIM (149, 719). Other
significant agglutinin titer or titer rise. studies suggest that there may be cross-reacting antibody re-
sponses to FHA and perhaps PRN due to M. pneumoniae and
C. pneumoniae infections (390, 550, 788). In addition, other
control of measles, the interepidemic cycle lengthened. This infectious agents such as H. influenzae may also have FHA-like
was because the circulation of measles virus had been reduced antigens that can result in high titers due to cross-reactivity
in addition to the control of the disease. With pertussis, how- (788).
ever, it was pointed out by Fine and Clackson 22 years ago that Due to the above, it may be more appropriate to evaluate
immunization controlled disease but did not decrease the cir- only PT antibody to determine B. pertussis infections in studies
culation of B. pertussis (244). It was also noted in the 1970s that of prolonged cough illnesses. Table 8 lists 11 studies for which
the source of infection in hospitalized infants was most often specific PT ELISA data were available (55, 271, 397, 550, 586,
an adult (583). The above observations led one of us (J.D.C.) 675, 695, 739, 788, 828, 835). Using only PT, the variation is
as well as others to conclude that B. pertussis infection was between 1 and 52%. In evaluating these data, it is important to
endemic in adolescents and adults (135, 149, 550). This ado- know which studies were carried out during pertussis out-
lescent and adult reservoir of sporadic infections is the source breaks and which were not related to outbreaks. All studies
of B. pertussis infections in nonimmune children. The tradi- with the higher percentages (17 to 52%) were done during
tional cyclic pattern occurs because it takes a few years for a pertussis outbreaks, whereas all the others except one (695)
significant number of susceptables in the population to develop were performed in nonoutbreak situations. The median value
so that continued transmission will occur. is 13%, and it appears that the one very low value (1%) may
The development of the ability to measure IgG and IgA have been the result of a laboratory assay problem (397). The
antibodies to B. pertussis antigens by ELISA made it possible to data in the studies by Mink et al., Wright et al., Nennig et al.,
accurately study adolescent and adult B. pertussis infections. and Strebel et al. strongly support a frequency of 12 to 16% of
During the last 20 years, three types of studies of adolescent prolonged cough illnesses as being due to B. pertussis infection
and adult populations have been carried out. These studies (550, 586, 739, 835). Since some persons with pertussis due to
have determined: (i) the percentage of prolonged cough ill-
nesses that are due to B. pertussis infections, (ii) the rate of
infections, and (iii) the rate of infections with cough illness. In TABLE 8. Percentage of prolonged cough illnesses in adolescents
addition, surveys of IgA antibody in various populations has and adults as a result of B. pertussisa infections
indicated the age-related occurrence of unrecognized B. per-
% of
tussis infections. IgA antibody studies can be used for this Author (reference) Location Yr illness due
purpose in both vaccinated and nonvaccinated populations to B. pertussis
because IgA antibody to B. pertussis antigens is not stimulated
Mink et al. (550) Los Angeles 1986–1989 13
by primary immunization. Schmitt-Grohé et al. (695) Germany 1991–1994 ⬃11
(i) Percentage of cough illnesses due to B. pertussis in ado- Wright et al. (835) Nashville 1992–1994 16
lescents and adults. Prolonged cough illness was evaluated in Wirsing von König et al. Germany 1992–1994 26
(828)
13 studies published between 1987 and 2002 (Table 7) (55, 271, Rosenthal et al. (675) Chicago 1993–1994 26
390, 397, 550, 586, 665, 675, 695, 739, 788, 828, 835). These Jansen et al. (397) San Diego 1993–1994 1
studies, in six countries, involved subjects with cough illnesses Nennig et al. (586) San Francisco 1994–1995 12
Strebel et al. (739) Minneapolis/St. Paul 1995–1996 13
seen between 1983 and 1999. The primary mode of diagnosis Birbebaek et al. (55) Denmark 1995–1997 17
was the measurement of IgG or IgA antibodies to one or more Vincent et al. (788) Korea 1997–1998 7
B. pertussis antigens. Diagnosis was made by the demonstration Gilberg et al. (271) Paris 1999 52
of a significant antibody titer rise between acute-phase and a
Significant rise in IgA or IgG antibody titer or high titer to PT, or culture or
convalescent-phase sera to one or more antigens or the pres- PCR positive.
TABLE 9. Rate of B. pertussis infections in adolescents and adultsa However, this high value assumes that the respiratory illnesses
Author Annual
during the periods of seroconversion were due to B. pertussis.
Location Yr
(reference) rate (%)
Deville et al. (206) Los Angeles, Calif. 1984–1989 8 B. parapertussishu Epidemiology

Cromer et al. (184) Columbus, Ohio 1985–1990 ⬃1
Incidence and mortality. In the prevaccine and early vaccine
Hodder et al. (354) Cleveland, Ohio 1989–1992 3
Ward et al., submitted Eight U.S. cities 1997–1999 1.3 eras, B. parapertussishu infections were noted in three areas of
a
Infections were determined by the demonstration of a significant rise in the United States (California, Michigan, and New York) and in
serum antibody titer to PT in successive serum samples. England, France, Czechoslovakia, Bulgaria, Denmark, Mexico,
Chile, Australia, Japan, Spain, Portugal, Hungary; Yugoslavia,
and Finland (51, 222, 314, 330, 340, 363, 444, 445, 465, 468, 498,
B. pertussis infection fail to mount a serum antibody response 499, 545). However, most studies compared the percentage of
to PT but do have a response to FHA, the use of only PT may cases of pertussis due to either B. pertussis or B. parapertussishu,
underestimate the true rate of prolonged cough illnesses due and population rates were not determined. Nonetheless, from
to B. pertussis (719). available data in Denmark, an average yearly rate can be cal-
(ii) Rate of B. pertussis infections. There are limited data culated for the period from 1950 to 1957 for children aged 0 to
available from which to access the rate of infections. There are 9 years (444, 445). The average yearly rate for pertussis due to
four studies in which sera from a number of subjects were B. parapertussishu was 73 per 100,000. For the same period it
available over time (184, 206, 354; b J. Ward, J. Cherry, S. was 1,569 per 100,000 for pertussis due to B. pertussis infection.
Chang, S. Partridge, W. Keitel, K. Edwards, M. Lee, J. Eldering and Kendrick (222) studied pertussis in the Grand
Treanor, D. Greenberg, S. Barenkamp, D. Bernstein, R. Edel- Rapids area of Michigan for a 16-year period (1935 to 1950).
man, and R. Rabinovich, submitted for publication). In two Of 4,483 Bordetella isolates, 106 (2.4%) were B. parapertussishu.
studies two sera per subject were available, and in the other In Czechoslovakia during a 3-year period (1967 to 1969), Bor-
two studies multiple sera over 3- and 5-year intervals could be ska and Simkovicova noted rates of infection of 2.66 to 2.69%
comparatively evaluated. In these studies, the same problem among preschool children and 0.33 to 1.11% among school
existed in regard to false-positives due to the use of FHA, children (66). In this study specimens were collected every
PRN, and FIM. In Table 9, specific data for ELISA-measured three months from all children. Of the children with positive
increases in the titer of antibody to PT over time are presented. cultures, 70.5% had clinical symptoms; of this group, the ma-
The rates varied from ⬃1 to 8%. The two studies with the jority (64.7%) had only very mild symptoms.
lower rates involved only two sera per subject, whereas the 3% In the present era, the DTaP vaccine efficacy trials in the
rate involved three sera per year over a 3-year period and the early 1990s revealed considerable evidence of the frequency of
8% rate involved two sera per year over a 5-year period. This B. parapertussishu infections in young children. In the Erlangen
later study was reevaluated by one of us (J.D.C.) because of trial, there was laboratory evidence of B. pertussis infections in
concerns that some of the seroconversions may have been due 103 control subjects (DT recipients) and B. parapertussishu
to nonspecific PT polyclonal responses rather than true rises in infections in 27 control subjects (719). The B. parapertussishu
antibody titers (206). Removal of low values resulted in a rate illness rate was 0.9 case per 100 person years. A total of 21%
of 6.7 per 100 person-years. of all cases were due to B. parapertussishu.
(iii) Rate of cough illnesses due to B. pertussis infections. In Of the Bordetella isolates from study subjects in the other
several of the prolonged cough illness studies, attempts were trials, 20, 12.2, 8, 2.1, and 2.1% were found to be B. paraper-
made to determine population rates of cough illnesses due to tussishu in Munich, Italy, Stockholm, Mainz, and Gothenburg,
B. pertussis infection. In three initial studies the rates were respectively (499).
calculated to be 64, 133, and 176 per 100,000 population (550, Agglutinating-antibody studies using B. parapertussishu as
586, 695). All three investigations were hampered by imprecise the antigen suggested that infections are common in all age
denominators. Subsequently, two prospective studies were car- groups and that the majority of these infections are unrecog-
ried out (739, 800) (Table 10). The first study revealed a rate nized clinically (252, 545, 793). Miller et al. found B. paraper-
of 507 per 100,000 population, and the second had a rate of 370 tussishu agglutinins in the sera of 40% of children who had not
per 100,000 population. In addition, the Hodder et al. study in had clinical pertussis and who had not received pertussis vac-
Cleveland included infection data and respiratory illness data cine (545). Vysoka-Burianova found agglutinating antibodies
over a 3-year period (354). The data in this study suggest that to B. parapertussishu in the sera of 59% of 6- to 9-year-old
the rate could be as high as 1,500 per 100,000 population. children (793).
In contrast to the findings in Europe and the prevaccine era
in the United States, B. parapertussishu isolates in the United
TABLE 10. Rate of B. pertussis infections in States during the last 30 years have been uncommon. En-
adolescents and adultsa
hanced surveillance activities by the CDC have processed over
Author
Location Yr
Annual 4,000 B. pertussis isolates during the last 8 years. During the
(reference) rate (%) same time period there were only 10 B. parapertussishu isolates,
Strebel et al. (739) Minneapolis/St. Paul 1995–1996 0.5 and, interestingly, all of these were from Ohio (G. Sanden,
Ward et al. (800) Eight U.S. cities 1997–1999 0.37 personal communication).
Hodder et al. (354) Cleveland, Ohio 1989–1992 1.5
Similar to B. pertussis, there appear to be epidemic cycles of
a
B. pertussis infection diagnosed by culture, PCR, or serologic study. B. parapertussishu infections. In Denmark during the period
between 1950 and 1957 there were three peaks, suggesting a B. bronchiseptica Epidemiology
3-year cycle (445). Further surveillance by Lautrop over a 20-
Although infection with B. bronchiseptica has been associ-
year period suggested peaks every fourth year (444). He also
ated with respiratory disease in at least 18 mammals, little is
noted that the B. pertussis and B. parapertussishu peaks did not
known about its epidemiology in nature (38, 64, 79, 211, 215,
occur concomitantly. In the study in Erlangen, there were no
240, 243, 249, 260, 267, 283, 291, 407, 426, 455, 500, 511, 516,
identified cases of pertussis due to B. parapertussishu between
673, 698, 715, 747). The primary host of B. bronchiseptica is not
April 1991 and April 1992. Following this, there were B. para-
known, and little is known about intra- and interspecies trans-
pertussishu-caused illnesses each month through March 1993
mission in the wild. Most experience over the last 90 years has
(340).
been gained by the study of domesticated animals living in
Clinical disease due to B. parapertussishu infection is most
closed quarters. Transmission from animal to animal is by
common during the first 5 years of life. In Denmark, 65% of direct contact with respiratory secretions, fomites, and perhaps
the cases occurred in children younger than 5 years and 96.1% by aerosol (23, 765). Porter et al. have shown that the organism
occurred in children younger than 10 years (444). In other can grow in lakewater, which suggests that B. bronchiseptica
studies, the vast majority of B. parapertussishu symptomatic in- may occur as a free-living organism (632, 633). If this is the
fections have been noted in children (51, 66, 330, 340, 363, 465, case, then transmission to multiple animal species could occur
498). In a study in Finland, 12 cases were noted in persons aged without direct contact.
16 years or older (330). In contrast to B. pertussis infection, death A major difficulty in the study of the epidemiology of
due to B. parapertussishu infection is rare. Zwelzer and Wheeler B. bronchiseptica in laboratory animal research colonies, ken-
noted two deaths in infants from whom B. parapertussishu was nels, and pens is the high rate of asymptomatic infection with
isolated at autopsy (848). Both of these very young infants had prolonged shedding of the organism. In some guinea pig col-
bronchopneumonia (848). onies, the incidence of asymptomatic infection can be as high
Sex, race, season, and geographic areas. Unfortunately as 20% (23). High asymptomatic infection rates also occur in
there are no data available from the larger B. parapertussishu rabbit colonies, and most conventionally managed swine herds
studies on the incidence of clinical disease by sex (66, 444, 445). are infected with B. bronchiseptica (23, 201).
In four relatively recent studies, more girls than boys had
pertussis due to B. parapertussishu infection (51, 340, 498, 825).
B. holmesii Epidemiology
Overall in these four studies there were 228 cases, and 122
(54%) were noted in girls (male/female ratio ⫽ 0.87). These B. holmesii appears to be a rare human pathogen, whose
findings are similar to those noted above for pertussis due to epidemiology is presently not known (508, 814, 839).
B. pertussis infection.
No data are available in regard to B. parapertussishu illness by LONG-TERM GOALS OF PERTUSSIS PREVENTION
race. In a 3-year study in Italy in conjunction with a DTaP
vaccine efficacy trial, the seasonality of B. parapertussishu ill- Eradication of B. pertussis Circulation
nesses was similar to that of B. pertussis illnesses (499). An
The ultimate long-term goal of the prevention of a disease
increase in the number of cases was noted from April through by immunization should always be the elimination of the in-
July. In examining the epidemic curves in Denmark for the fectious agent. This is theoretically possible with all human
period 1950 through 1957, there were four peak periods, all of infections in which there is no animal reservoir (e.g., rabies) or
which were winter peaks (445). B. parapertussis infection ap- environmental reservoir (e.g., tetanus). This goal was achieved
pears to occur worldwide, but in recent years it has been noted with smallpox in 1978 and may soon be achieved with all three
most often in Europe (51, 330, 340, 465, 498, 499, 825). types of polio (134, 138). In addition, toxigenic strains of
Interrelationship between B. parapertussishu and B. pertussis Corynebacterium diphtheriae have been eliminated from large
infections. As noted above, during the early studies in Den- geographic regions of the world (815).
mark it was found that epidemics of illness due to B. pertussis The ultimate long-term goal for the prevention of pertussis
and B. parapertussis were not concomitant (445). However, due to B. pertussis infection should also be the elimination of
during Lautrop’s investigation he noted 79 persons who had the circulation of the organism. However, it is readily apparent
bacteriologically confirmed infections with both B. pertussis that this goal is not realistic in the foreseeable future because
and B. parapertussishu. In about one-third of the studied sub- of numerous obstacles such as lack of funding, understanding,
jects, the infections with the two organisms occurred simulta- and commitment. Similar to the experience with diphtheria,
neously. In sequential infections, the first infection was with however, it does seem possible to us that the circulation of
B. parapertussishu in all but four instances. During the 7-year B. pertussis could be stopped in countries with aggressive, com-
observation period, two successive infections with B. paraper- prehensive immunization programs.
tussis were not encountered. More recent studies have noted As noted above, the universal immunization of infants and
both mixed infections and sequential infections with B. pertus- children such as in the United States with pertussis vaccines
sis and B. parapertussishu (51, 330, 389, 535, 719, 753). From the prevents more than 95% of all reported illnesses. However,
available data, there is no evidence of more or less severe dis- with the present increased awareness of pertussis, the absolute
ease in children with concommitant infections with both or- number of reported cases of pertussis has increased substan-
ganisms than would occur with B. pertussis infection. In addi- tially during the last two decades (144). It is apparent today
tion, in sequential infections there is no evidence that the first that the reservoir of B. pertussis in the population is symptom-
infection affected the manifestations of the second infection. atic infections in adolescents and adults of all ages. This is not
TABLE 11. Contents of two DTaP vaccines formulated measured following adolescent and adult immunization may
for adolescents and adults not be as functionally specific against B. pertussis as that which
Aventis (Adacel)a GSK (Boostrix)b followed primary immunization in young children. If this were
Antigen
amt or dose amt or dose the case, the protection following adolescent and adult booster
Diphtheria toxoid 2 Lf 2.5 Lf immunizations would probably be of shorter duration than the
10-year projected duration.
Tetanus toxoid 5 Lf 5 Lf
PT toxoid 2.5 ␮g 8 ␮g Since adults should receive dT boosters every 10 years, it is
FHA 5 ␮g 8 ␮g reasonable to assume that such a schedule with a DTaP vaccine
PRN 3 ␮g 2.5 ␮g
FIM2/3 5 ␮g
could be effective. As noted above, the demonstrated projected
2-Phenoxyethanol 0.6% 2.5 mg decay pattern of IgG antibody to PRN following adult booster
a
immunization supports a 10-year booster schedule. To be ef-
Aventis: Product information, September 2002.
b
GlaxoSmithKline (GSK): Product information, November 2003. fective, a universal pertussis immunization program (infants,
children, adolescents, and adults) would need to elicit both
individual protection and herd immunity. Experience indicates
a new phenomenon (it was present in the prevaccine era), but that the pediatric aspects of this proposed program are pres-
it is more apparent today with the control of disease in vacci- ently in place. The adolescent portion of this program also
nated children (141). Clinical studies of prolonged cough ill- seems attainable through the development of school-based
nesses and serologic surveillance studies indicate that all age programs. Immunization of adults with boosters every 10 years
groups and all geographic areas are involved. is a difficult goal and presently impossible. However, innova-
The availability of DTaP vaccines leads to the possibility of tive ways can be developed to reach this goal. Of necessity in
adolescent and adult booster immunizations. In addition, two this regard will be active committees (similar to the Committee
specific adolescent and adult DTaP vaccines have been devel- of Infectious Diseases of the American Academy of Pediatrics)
oped and are licensed in several countries. The compositions involving internists, family practitioners, and obstetricians.
of these two vaccines are presented in Table 11. Work-related immunization programs could capture a substan-
With the goal of the elimination of the circulation of tial number of adults, and if these programs could include
B. pertussis, an immunization program must include both ad- spouses they may well reach enough of the population to in-
olescents and all ages of adults in addition to universal child- duce herd immunity. Clearly for success a new public health
hood immunization. The duration of protection and of the ex- commitment will be required.
pected decrease in circulation of B. pertussis following booster
immunizations in adults is not known. It is clear, however, that Lesser Goals
the decay in the level of antibodies following a booster dose in
adults varies by antigen (446). Specifically, antibody to PT falls At present, multiple committees, working groups, and task
substantially during an 18-month period postvaccination where- forces are addressing the future control of pertussis with ado-
as titers to PRN and FHA have considerably lengthened decay lescent and adult immunization. In general, these groups have
curves. Using the serum antibody decay curves in adolescents shied away from universal adult immunization, presumably
and adults who received one dose of an adult-formulated aP because it is presently logistically impossible. In general, most
vaccine, Le et al. predicted the duration of the time that the groups favor a universal adolescent booster dose and various
IgG GMT of antibody to the various antigens would remain attempts to reach adults who, if infected, would spread infec-
above the limits of quantitation of their ELISA (446). This tion to infants too young to be adequately immunized. Two
analysis suggested that the GMT for PT would fall below 6 recent modeling studies support a booster program involving
ELISA units (Eu)/ml in 2.3 years, whereas the GMT for PRN adolescents (645, 784). This approach could be expected to
would stay above 8 Eu/ml for 9.1 years. Since there is evidence reduce the number of B. pertussis illnesses in infants but not
that a low level (ⱖ7 Eu/ml) of antibody to PRN is highly have a major effect on the circulation of B. pertussis. B. pertussis
protective, it seems probable that an interval between booster would continue to circulate in persons aged 25 to over 75 years.
doses of 10 years would be effective (148, 736). In this regard, With school-based immunization programs, a high proportion
one concern relates to what is being measured with respect to of adolescents could be immunized. However, selective immu-
antibody to PRN. In the serologic correlates of immunity stud- nization of adults for other diseases (e.g., influenza) has not
ies, the protected subjects were young children who had re- had high compliance rates, and there is no reason to expect
cently been immunized with PRN-containing vaccines. The better results with DTaP vaccines.
antibodies to PRN in these subjects were specifically due to the
direct immunologic response to B. pertussis PRN. Infant and Childhood DTaP Immunization Schedules
For the adults, however, the delayed antibody decay pattern
suggests that the measured antibody may be the result of ex- The present infant and childhood immunization schedule
posure to cross-reacting antigens and not just the result of the evolved using DTP vaccines and has been effective. The same
specific immunization. For example, the PRN of other Borde- schedule is now used with DTaP vaccines, and it is not optimal.
tella species is slightly different from that of B. pertussis. In ad- In general, good DTP vaccines contained more antigens than
dition, it seems possible that cross-reacting antibodies to PRN the present DTaP vaccines but the concentrations of the anti-
may also be stimulated by infections with M. pneumoniae and gens in the DTP vaccines were modest. In contrast, the specific
perhaps C. pneumoniae (550, 788). With these possibilities in antigens (PT, FHA, PRN, and FIM) in DTaP vaccines are
mind, it seems possible that the antibody to PRN that was present in higher concentrations than their counterparts in
DTP vaccines. Furthermore, the two good DTaP vaccines Better Vaccines
available in the United States contain significantly larger
amounts of diphtheria toxoid than did the former U.S. DTP Basic research on previously known virulence factors has
vaccines (759a, 759b). The excessive amounts of diphtheroid greatly enhanced our understanding of Bordetella pathogenesis
toxoid (i.e., 25 Lf and 15 Lf) in the DTaP vaccines are respon- and the host response to infection. Sequence information ob-
tained from the Bordetella Genome Project has further accel-
sible for marked local reactions in children who received
erated the discovery of novel virulence factors. Above all, com-
booster doses (656). In addition, one of the two vaccines has a
parative analyses of the genomes as well as the pathogenic and
large amount of PT, which contributes to local reactions with
environmental life cycles of various Bordetella subspecies have
booster doses (759b).
provided crucial insights into the infection mechanisms em-
The vaccine efficacy trials in Sweden and Italy demonstrate
ployed by Bordetella. As with most fields, information obtained
good antibody responses and protection for more than 5 years
from such research has facilitated the development of new
following three doses of vaccine at 2, 4, and 6 months of age
vaccines. To avoid the side effects associated with DTP vac-
and no booster dose during the second year of life (596, 684, cines, scientists and clinicians have developed two generations
685). These data indicate that with these two vaccines, a of efficacious DTaP vaccines generally free of such alarming
booster dose in the second year of life may be unnecessary and side effects. However, pertussis vaccine efficacy trials con-
is the cause of excessive local reactions with the fourth and fifth ducted throughout the 1990s clearly demonstrated the gener-
doses. Although further study may be necessary, it is clear that ally greater efficacy of DTP control vaccines over their coun-
we do not need five doses of the present DTaP vaccines in terpart first- and second-generation DTaP vaccines (464, 697,
childhood. The simplest change is to omit the dose in the 708, 719). These observations suggest that the inclusion of
second year of life. Another approach which was demonstrated additional antigens in the next generation of DTaP vaccines
to give a good primary response was a schedule of 3, 5, and 12 could improve vaccine efficacy.
months of age (597). Current DTaP vaccines are single-component (PT toxoid),
Following either schedule, a booster dose at 5 years of age two-component (PT and FHA), three-component (PT, FHA,
should provide good protection until the time of an adolescent and PRN), four-component (PT, FHA, PRN, and FIM2), and
booster at 13 to 15 years of age. five-component (PT, FHA, PRN, and FIM2/3) vaccines. Of
these vaccines, only a five-component vaccine has been shown
to be as efficacious as one highly immungenic DTP vaccine
(597). FIM2/3 serve as important adjuvants for inducing a
FUTURE RESEARCH protective immune response (148, 503, 506, 597, 736). The role
Pathogenesis of Disease of FHA in generating anti-Bordetella immunity has come into
question; however, the discovery of fhaS and fhaL suggests that
Although much has been accomplished relating to Bordetella their gene products could overcome the action of anti-FHA
spp. and their illnesses during the last two decades, there is still antibodies.
much that is not known. A fundamental question is why people Inclusion of CyaA in an aP vaccine concoction has also been
and animals infected with the four Bordetella subspecies dis- suggested; however, anti-CyaA antibodies could cross-react
cussed in this review have such isolated prolonged cough ill- with RTX-family hemolysins generated by other members of
nesses. The cough in pertussis is truly unique. It is difficult to the microflora. For this very reason, tracheal cytotoxin, a by-
believe that this cough is solely due to local damage to ciliated product of bacterial cell wall synthesis, would also not make a
epithelial cells, because it continues over an extensive period good vaccine candidate. In contrast, additional surface-associ-
ated proteins (e.g., autotranporters such as BrkA, Vag8, and
with the appearance of being completely normal between par-
SphB1) serve as promising vaccine components. Antibodies
oxysms. There are clinical similarities to tetanus, suggesting
generated against SphB1, for instance, could target agglutina-
that there may well be an as yet undiscovered toxin which is
tion via SphB1-FHA interactions. Alternatively, antibody-me-
common to respiratory Bordetella spp. Careful investigation of
diated blocking of SphB1 function could impede proper mat-
the commonly expressed unknown proteins of Bordetella may
uration and secretion of FHA.
shed light on this issue.
The suitability of adding purified LPS to an aP vaccine
Present animal model systems are not adequate for the study concoction has also been a subject of discussion. While active
of coughs. Since other primates have pertussis-like illnesses, B. pertussis LPS is present in all DTP vaccines in different
studies of these animals by using various B. pertussis strains concentrations and is a major contributor to local and systemic
with various deletions might be useful. In addition, human reactions with these vaccines (147, 149), B. pertussis infection,
volunteer studies are being planned. interestingly, is not accompanied by the usual LPS toxicity
Another area of interest is the possible relationship between (fever and shock). Thus, genetically modified LPS may be used
B. pertussis strain differences and clinical manifestations of as a putative vaccine component, serving as an antigen and/or
disease. Careful analysis of different protein variations can be an adjuvant.
analyzed by disease severity. Most recently, the intriguing possibility of type III secretion
Further study of Bvg⫺ and Bvgi phase organisms in more serving as a potent virulence factor in human disease was pre-
natural circumstances such as primate and human volunteer sented (507). Until this report, type III secretion was consid-
studies with B. pertussis and studies of more natural swine ered to be B. bronchiseptica specific. Since B. pertussis ex-
populations with B. bronchiseptica should be useful. presses PT, which, like the TTSS, displays immunosuppressive
phenotypes, it was assumed not to require a functional TTSS Role of B. holmesii in Human Disease
for pathogenesis. Mattoo et al. showed that although type
The findings in Massachusetts in 1995 through 1998 of per-
III-specific proteins were not expressed by B. pertussis in vitro,
tussis-like illness due to B. holmesii is of interest and suggests
transcription of all TTSS genes was surprisingly intact and
that illness due to this agent may be overlooked. Increased
regulated in a manner similar to that seen for B. bronchiseptica. awareness that this organism could be responsible for culture-
It seems unlikely that B. pertussis would expend the energy negative cases of pertussis should be encouraged. Culture-
required to transcribe the entire bsc and btr loci (which consist negative samples should be studied by PCR and also recul-
of over 26 genes), unless the products of these genes were tured in media without cephalexin.
required for pathogenesis. It was therefore proposed that B.
pertussis may express type III secretion in vivo (507). If this is
More Complete Epidemiologic Study
so, proteins secreted by this system would serve as good vac-
cine candidates, provided that they are also antigenic. A type Presently, there is much misunderstanding relating to B. per-
III-secreted effector for B. bronchiseptica has recently been tussis epidemiology because of the failure to recognize spo-
identified (603). Preliminary results suggest that this effector is radic, endemic cases of pertussis in adolescents and adults. The
functionally conserved in B. pertussis as well, thus validating the use of single-serum ELISA on sera from persons with pro-
inclusion of this effector in the vaccine concoctions for Borde- longed cough illnesses should be systematically studied in all
tella-mediated human and animal infections. Another good, age groups throughout the world.
and perhaps better, type III-secreted vaccine candidate is Bsp22. ACKNOWLEDGMENTS
This 22-kDa protein is unique to Bordetella, highly conserved
throughout the sequenced Bordetella strains, secreted in copi- We appreciate the veterinary research input provided by Marcelo A.
Couto and the encouragement and assistance provided by Jeffery F.
ous amounts by the Bordetella TTSS, and highly antigenic Miller. We are also grateful to Laura Wennstrom for her invaluable
based on experiments conducted with mouse, rat and rabbit assistance.
models (507, 840, 841). While Bsp22 is secreted by the TTSS, REFERENCES
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Molecular Pathogenesis, Epidemiology, and

Clinical Manifestations of Respiratory

Updated information and services can be found at:

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml

Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of

* Corresponding author. Mailing address: 10833 Le Conte Ave.

INTRODUCTION (149, 831). Human-adapted B. parapertussis (B. parapertussishu)

there are presently ⬃48.5 million yearly cases of pertussis

Information storage and processing

Total no. of proteins with assigned COGs 3,121 3,784 4,403

in the detection of B. pertussis by PCR compared with culture. 550).

Study location % Efficacy againsta:

Senegal (708) Pasteur Mérieux Sérums et Vaccins 92 (86–99)

TABLE 4. Composition of nine DTaP vaccines evaluated in efficacy trialsa

detected in DTP recipients than in DTaP vaccinees (Table 5)

the data set. cases in vaccinees.

different epidemiologies. The first is the epidemiology of re-

EPIDEMIOLOGY: IMPLICATIONS FOR THE

were noted in children 5 to 9 years of age, and two deaths

Deville et al. (206) Los Angeles, Calif. 1984–1989 8 B. parapertussishu Epidemiology

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