MICR 454L Emerging and Re-Emerging Infectious 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 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 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

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 Act Synergistically

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 G i 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 aa1300 aa N C Adenylate Cyclase Delivery of adenylate cyclase Weak hemolysin

Increases in cAMP

Consequences of Increased Intracellular cAMP Inhibition of Chemotaxis Oxidative burst Phagocytosis Co-stimulatory molecule expression IL12 production Increased apoptosis Anti-phagocytic Anti-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 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: 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 ects2004/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, Brock Biology of Microorganisms, by Madigan and Martinko, Pearson Prentice Hall, Upper Saddle River, NJ, 11 th ed, Microbiology: An Introduction, by Tortora, Funke and Case; Pearson Prentice Hall; 9 th ed, Immunobiology, by Janeway,, Travers, Walport, and Shlomchik, Garland Science, 6 th ed, Nicole Guiso (2005) Is Bordetella pertussis Changing. Microbe Malak Kotb Genetics of Susceptibility to Infectious Diseases Volume 70, Number 10, 2004 / ASM News Y Bernard Dixon MicrobeLibrary Article: Microbe 2005 J Clin Microbiol Oct;43(10): Nucleic Acid amplification tests for diagnosis of Bordetella infections. Riffelmann M, Wirsing von König CH, Caro V, Guiso N; Pertussis PCR Consesus Group.