micr 454l emerging and re-emerging infectious diseases lecture 5: bordetella pertussis dr. nancy...
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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.