MICR 454L

Emerging and Re-EmergingInfectious Diseases

Lecture 5: Bordetella pertussis

Dr. Nancy McQueen & Dr. Edith Porter

Overview History Morphology Growth and metabolic characteristics Virulence factors Diseases Diagnosis

Latex agglutination Spot test Culture PCR Immune response

Therapy Threats

Bordetella pertussis

Bordetella pertussis

Respiratory epithelial cell

Cilia

History of Pertussis

Lacks ancient history unlike small pox and measles

Illness probably began first in France in 1414 First epidemic noted in Paris, France, 1578 Named pertussis (violent cough) in 1679 1900 first microscopic observation 1906 first isolation by Bordet and Gengou Soon thereafter vaccine development

Bordetella pertussis

Gram-negative rods beta-proteobacterium Fastidious

Bordet-Gengou medium Potato Sheep blood glycerol

Regan-Lowe Charcoal 10 % horse blood Cephalexin http://medinfo.ufl.edu/year2/mmid/bms5300/images/d7053.jpg

B. pertussis: Numerous Virulence Factors

Mattoo and Cherry (2005) Clin. Microbiol. Rev. 18 (2): 326.

B. pertussis: Studies on Pathogenicity

Mouse animal model of respiratory tract infection Intranasal or aerosol application Large doses required as not a mouse pathogen

B. bronchiseptica often used as model organisms in pathogen-free rabbits, rats and mice Colonization studies

In vitro tissue culture

B. pertussis: Virulence Regulon

Virulence control system regulated by the bvgAS locus

Two component signal transduction system that uses a four step His-Asp-His-Asp phosphotransfer signaling system BvgA: DNA binding

response regulator BvgS: transmembrane

sensor kinase

B. pertussis: Selected Virulence Factors Adhesins

filamentous hemagglutinin (FHA) fimbriae (FIM)

Toxins Pertussis toxin (PT)

AB toxin ADP-ribosylates G proteins

Adenylate cyclase-hemolysin (AC-Hly) Bi-functional Anti-inflammatory/antiphagocytic

Tracheal cytotoxin (TCT) Type III secretion

ActSynergistically

Pertussis Toxin 1 A subunit for action, 4 B

subunits for binding Once intracellular, the A

subunit ADP ribosylates a critical cysteine residue on the Gi regulatory proteins involved in control of host cell adenylate cyclase resulting in increased intracellular cAMP.

This causes cellular dysfunction.

Activation of Pertussis Toxin

Adenylate Cyclase-Hemolysin (AC-Hly)

Produced as pretoxin Activated by palmitoylation Mainly bound to surface of BP Binds to CD11b expressed on many cells, in

particular leukocytes

400 aa 1300 aa

N C

Adenylate Cyclase

Delivery of adenylate cyclaseWeak hemolysin

Increases in cAMP

Consequences of Increased Intracellular cAMP

Inhibition of Chemotaxis Oxidative burst Phagocytosis Co-stimulatory molecule

expression IL12 production

Increased apoptosis

Anti-phagocyticAnti-Inflammatory

Tracheal Cytotoxin (TCT)

Small glycopeptide toxin Monomeric subunit of bacterial peptidoglycan

N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-(L)-alanyl-g-(D)-glutamyl-meso-diaminopimelyl-(D)-alanine

Secreted in large amounts Selectively damages ciliated respiratory epithelial

cells Induces nitric oxide synthase in conjunction with

LPS

B. pertussis: Whooping Cough Incubation time

7 – 10 days Catarrhal period (1 – 2 weeks)

Symptoms of common cold Paroxysmal period

Obstruction by mucus Paroxysmal cough Inspiratory “whoop” Posttussive vomiting

Convalescent period (> 2 weeks) Paroxysms gradually decrease

Apnea often only symptom in neonates and

unvaccinated infants!

Pertussis Complications

Major complications most common among infants and young children

Include hypoxia, apnea, pneumonia, seizures, encephalopathy, and malnutrition

Young children can die from pertussis 13 children died in the United States in 2003. Most deaths occur among unvaccinated

children or children too young to be vaccinated

Copyright ©2003 American Academy of Pediatrics

Halasa, N. B. et al. Pediatrics 2003;112:1274-1278

Fig 1. A bronchus contains sloughed debris (B); its accompanying artery (A) is occluded by a fresh thrombus

B. pertussis: Diagnosis Culture

Thought to be almost 100% specific

Nasopharyngeal aspirates or swabs

Direct plating and preincubation before transport

Incubate at least for 1 week DFA (direct fluorescence

assay) Lacks sensitivity and specificity Not accepted as proof in

notifying countries Serology

ELISA detecting pertussis toxin IgG detection not useful

Real time PCR From nasopharyngeal

aspirates or swabs More sensitive than culture Accepted in notifying countries

B. pertussis: Therapy

Macrolide antibiotics Azithromycin Clarithromycin Erythromycin

Post exposure prophylaxis

B. pertussis: Prevention Vaccination Whole cell preparation

Side effects Brain damage?

Acellular or subunit protein vaccine 1 – 5 purified Bordetella

virulence factors detoxified PT—either alone or

combined either with adhesin(s)

Part of DTP : dipohteria-tetanus toxoid, acellular pertussis vaccine, also referred to DTaP

Requires boosters

http://www.brown.edu/Courses/Bio_160/Projects2004/pertussis/sepsis1.jpg

B. pertussis : Epidemiology Highly contagious preventable disease

Person-person Aerosolized droplets Direct contact

Endemic illness with epidemics every 3-5 years in the US

Overall increase in cases since 1990, with disproportionate increase in adolescents and adults 60% of all cases in adolescents and adults 80% secondary attack rates in susceptible persons

25,827 cases in 2004 in the US Highest number since 1959

Age Distribution and Incidence of Reported Cases of Pertussis

United States from 1997 to 2000

Threats by B. pertussis

Case numbers rise Improved awareness of pertussis Waning of immunity from childhood vaccination

Pertussis boosters Decreases in vaccine efficacy over time

Filamentous hemagglutin inhibits immune response Virulence or antigenic changes in the circulating

bacteria ? Compare strains collected before and after implementation of

vaccination Mismatch between vaccine and circulating strains High number of insertion sequences in the genome) over 250 IS!)

Take Home Message

Bordetella pertussis exhibits numerous pathogenic factors representing adhesins, cytotoxins and Type III secretion apparatus.

There is a re-emergence of the formerly early childhood disease among adolescents.

Active Learning Exercise

Assume you have developed a new vaccine candidate.

Explain how you would test the efficacy of this vaccine in an animal model. Which animal model? How to administer the vaccine? How to survey whether immune response has

taken place? How to test whether the immune response is

protective?

Resources The Microbial Challenge, by Krasner, ASM Press, Washington DC, 2002. Brock Biology of Microorganisms, by Madigan and Martinko, Pearson

Prentice Hall, Upper Saddle River, NJ, 11th ed, 2006. Microbiology: An Introduction, by Tortora, Funke and Case; Pearson

Prentice Hall; 9th ed, 2007. Immunobiology, by Janeway,, Travers, Walport, and Shlomchik, Garland

Science, 6th ed, 2005. Nicole Guiso (2005) Is Bordetella pertussis Changing . Microbe 71 231-234. Malak Kotb Genetics of Susceptibility to Infectious Diseases Volume 70,

Number 10, 2004 / ASM News Y 457-463 Bernard Dixon MicrobeLibrary Article: Microbe

2005 J Clin Microbiol. 2005 Oct;43(10):4925-9. Nucleic Acid amplification

tests for diagnosis of Bordetella infections. Riffelmann M, Wirsing von König CH, Caro V, Guiso N; Pertussis PCR Consesus Group.