Slide 1Slide 2Slide 3Slide 4Bordetella pertussisSlide 6DiagnosisEpidemiology of PertussisSlide 9Epidemiology of PertussisSlide 11Slide 12Virulence factors of Bordetella pertussisVirulence factors of Bordetella pertussisSlide 15Pertussis: Clinical ManifestationsSlide 17Questions?Slide 19Slide 20Haemophilus influenzaeVirulence Factors of H. influenzaeSlide 23Slide 24Slide 25Nontypeable H. influenzae (NTHI)NTHI –associated Otitis MediaSlide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Two serious Bacterial pathogensof theUpper Respiratory TractDr. Terry D. Connell: [email protected] influenzaeBordetella spp.Bordetella spp.Five pathogenic species of Bordetella B. avium Coryza (birds)B. bronchiseptica Kennel cough (dogs)Necrotic rhinitis (pigs)URT infection (human)B. parapertussis Mild whooping cough (human)B. homesii Pneumonia, bacteremia (newly emerging)Bordetella pertussis Whooping cough (human)DPT Vaccine !!Bordetella pertussisExtremely small, gram-negative coccobacilliStrictly aerobicObligate human pathogen. No animal reservoir (unlike Salmonella enteriditis)Fastidious, slow growing Three to six days for colonies on Bordet-Gengou agarInfects the upper respiratory tract (URT)T) - Ciliated respiratory epitheliumElaborates powerful toxins which elicit most of the symptoms of the disease“pertussis” - “violent cough”Diagnosis1. Culture bacteria from respiratory secretions (Not always successful!!)-Viable, but non-culturable bacteria - “VBNC” -Ex. - Cannot use cotton-swabs for sampling the throatFatty acids in cotton - kill the B. pertussis bacteria2. Immunofluorescence assay on secretions Antibodies that recognize pertussis proteins3. Agglutination reaction on secretions Antibodies that recognize pertussis proteins4. Diagnosis based on clinical diagnosis: Whoop-type coughing & lymphocytosisAgglutination reactionAbcamImmunofluorescenceEpidemiology of PertussisWorldwide problem - 60 million cases, 600,000 deaths/yrDeveloped countries - Dramatic recent increase in cases● Decreased vaccine use● Rise of strains resistant to the vaccineHighly contagious - attack rates of 50 -100%Transmission - aerosol droplets from coughingFemales - Higher attack rate, morbidity and mortality (???)Epidemiology of PertussisWorldwide problem - 60 million cases, 600,000 deaths/yrDeveloped countries - Dramatic recent increase in cases● Decreased vaccine use● Rise of strains resistant to the vaccineHighly contagious - Attack rates of 50 -100%Transmission - Aerosol droplets from coughingFemales (???) - Higher attack rate, morbidity, mortality Age distribution - Recent shifts in age groups19881998Shift in age distributionPre-vaccine - Predominately infected young children► Young children (<1 - 3 years of age)Post-vaccine - Increased rate of infection in two groups ► Older children (5 to 14) ► Young adults (18 to 25 yrs) A SHIFT in age-distribution of infectionVaccines exerts STRONG SELECTIVE PRESSURES on pathogensSelects for outgrowth of variants for which the vaccine doesn’t evoke immune protectionVaccination has “pushed” bacterium into other age groups. - Fewer maternal antibodies remaining in older children - Protection by vaccination in young children is not lifelongVirulence factors of Bordetella pertussisPili Attachment to host cellsCapsule Antiphagocytic, adherenceFilamentous hemagglutinin Adherence to glycolipidsPertactin Binds to host cellsBinds to: Ciliated epithelium of upper respiratory tract (URT)Multiple ADHESINS:Virulence factors of Bordetella pertussisPertussis toxin (Ptx) A1-B5 class of toxin (similar to CT) B pentamer – binding to receptors on cells A polypeptide – enzyme which increases cAMP cAMP = cyclic adenosine monophosphate Adenylate cyclase toxin Increases cAMP in infected URT cellsLymphocytosis-promoting toxin Increases lymphocyte numbers in URTTracheal cytotoxin Fragment of the peptidoglycan cell wall- destroys ciliated epithelial cellsDermonecrotic toxin Causes skin lesions and fatality in miceLPS (endotoxin) Activates alternative complement pathway FeverMultiple TOXINS:2. Catarrhal (or Prodromal) stage: Lasts from 7 to 14 days Nonspecific symptoms Malaise, rhinorrhea, lacrimation,low grade fever, anorexia (cold or flu-like!) Dry cough develops, worse at night1. Incubation stage:No overt symptomsLasts 7 to 10 daysInfected individual is already infectious!Pertussis: Clinical ManifestationsPertussis: Clinical Manifestations3. Paroxysmal stage:Paroxysmal coughing -Tenacious mucus-Series of repetitive coughs followed by a characteristic inspiratory “whoop” (cyanosis; convulsions; seizures)-Patient looks normal between paroxysms, with minimal fever Ciliostasis - Death of URT ciliated epithelial cells (trachael cytotoxin)- Failure of the respiratory escalator to move mucus from lungs to throatLymphocytosis Neutrophil count in the tissues - to 200,000 cells/mlPersistance Symptoms last 1-2 weeks !Until ciliated cells re-differentiate from basal cells Erythromycin Antibiotic treatment doesn’t ameliorate the symptoms. Why not?Convalescence 3 to 4 weeks; lymphocytes ↓ gradually; cough subsidesPertussis: Clinical ManifestationsQuestions?Next pathogen elicits infectionin the:Upper Respiratory TractLower Respiratory Tract Middle EarHaemophilus influenzaeUpper Respiratory TractLower Respiratory Tract Middle EarHaemophilus influenzaeAerobic, gram-negative bacteriumCoccobacillus or pleomorphic rodsObligate human pathogenMay be encapsulated or non-encapsulated (later!)Required for growth:- Fastidious bacterium to culture- Hemin- Nicotinamide adenine dinucleotide (NAD) Chocolate agar (Contains heat-treated, lysed erythrocytes)Virulence Factors of H. influenzaeCapsule Secreted polysaccharide “coat”ANTI-PHAGOCYTIC activityResists killing by macrophages andpolymorphonuclear neutrophilsPilus Rod-like appendagePromotes ATTACHMENT to target cells of the URT or middle earHAP protein Surface proteinPromotes more intimate ADHERENCEof bacterium to cellsEndotoxin (LPS) INFLAMMATORY, pyrogenic (fever) Impairs ciliary function of URT cellsIgA1 proteases DESTROYS IgAFacilitates colonization of themucosal surfaceIgAHow do we differentiate between strains of Haemophilus influenzae ?Six (6) serotypes ; based on antigenic differences