1. Diagnostic microbiology lecture: 15 Bordetella pertussis Abed ElKader Elottol MSc. Microbiology 2010 1Abed ElKader ElOttol

2. 2 Organism: Short, gram negative bacilli ( coccobacilli). Aerobic, non spore forming Non motile,. Specific to humans. Complex, enriched media is required for isolation (Bordet- Gengou agar )or ( Regan-Lowe Charcoal Agar) (charcoal act as adsorbents of toxic fatty acids, radicals and peroxides). urease negative, nitrate negative. Pertussis is highly contagious

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4. mercury drop" colonies on Bordet' Gengou Bordetella pertussis growing in Regan-Lowe Agar Incubated in CO2 for 72 hours

6. virulence factors adhesions such as filamentous hemagglutinin, peractin, and fimbriae. a number of toxins including pertussis toxin, adenylate cyclase toxin, trachael cytotoxin, dermonecrotic toxin. lipopolysaccharide endotoxin.

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8. Disease: whooping cough (Pertussis) Pertussis = Transmitted by the respiratory tract rout from early case and possibly via carriers. The organism adheres to and multiply rapidly on the surface of the epithelium in the trachea and bronchi and interfere with ciliary action. Disintegrating organisms liberate a toxin that irritate surface cells, giving rise to catarrhal symptoms. 8Abed ElKader ElOttol

9. An acute respiratory disease with three stages: A catarrhal stage with an irritating cough, lasts 1 to 2 weeks. A paroxysmal stage characterized by violent coughs followed by a high respiratory whoop, lasts 2 to 6 weeks. A convalescent stage where the cough gradually decreases in frequency and severity, lasts several weeks. Note: 75% of deaths are among infants. Note : 75% of deaths are among infants.

10. Signs & Symptoms of Pertussis  The first symptoms of pertussis are similar to those of a common cold:  runny nose  sneezing  mild cough  low-grade fever Most B.pertussis strains contains peptide that promotes marked lymophocytosis in the host Elevated white blood cell count with a lymphocytosis, Confirmed with laboratory testing.

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13. Laboratory Diagnosis: Nasopharyngeal swabs or cough droplets, expelled onto "Cough plates" held in front of the mouth of the patient during a paroxysms. Incubate (Bordet-gengou plates) at 35 o C for 2-5 days. Typical colonies resemble a 1mm droplets of mercury surrounded by a zone of hemolysis. Colonies are confirmed with specific antiserum Direct immunofluorescent staining of smears made from nasopharyngeal swabs may give a rapid positive test. 13Abed ElKader ElOttol

14. Genus Species Disease Bordetella pertussis Pertussis parapertussis Pertussis (milder form) bronchiseptica Bronchopulmonary disease Human Disease & Associated Pathogens

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17. Legionella pneumophila 17Abed ElKader ElOttol

18. Legionellosis was first recognized in association with the epidemic of legionnaires disease which occurred in Philadelphia in July 1976. 182 person contracted pneumonia, 34 died. Shortly following the epidemic, the causative agent was isolated from lung tissues of patient who died of this disease. The organism was named Legionella pneumophila. Slow grower (2-5 days in enriched media containing L-cystein and supplemented source of ferric iron). 18Abed ElKader ElOttol

19. General characteristics Motile Gram-negative, aerobic, non-spore-forming bacteria. Stain poorly with gram stain unless safranin is applied for long periods. Non capsulated. In tissue and clinical specimens the organisms are coccobacillary, measuring 1-2 µm. Elongated filamentous forms may be seen after growth on some culture media.

20. Transmission: Via respiratory tract. Acute pneumonia begins 2-10 days after exposure with a brief malaise, myalgia, and headache, followed rapidly by prostration انهيار, high fever. an rigors شديد Cough, dyspnea, pleuritic and abdominal pain, vomiting, diarrhea, and unexplained encephalopathy are often seen. Leukocytosis, elevated ESR, proteinurea, hematuria and abnormal serum enzyme determination are common. The common causes of death due to respiratory failure and shock. Gram stain of sputum is not diagnostic. 20Abed ElKader ElOttol Clinical Manifestation:

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24. Laboratory Diagnosis Culture gold standard Require L-cysteine and iron salts Use BCYE (buffered charcoal yeast extract agar) Antibiotics might be used to help inhibit the growth of fastidious bacteria. Grown aerobically using 3-5% CO2 at 35 c° for 2 to 5 days pH 6.9 for optimum growth Colonies are small with a crystalline-like, ground glass appearance.

25. Abed ElKader ElOttol25 Florescence stain of Legionella

26. Biochemical Characteristics: Weak oxidase positive Strong catalase positive Liquefy gelatine Hydrolysis of hippurate Positive (This differentiate it from other legionella species) Virulence factors: 1. Proteolytic enzymes 2. Exotoxin with lysed laboratory animal RBCs 3. Cytotoxin which caused inhibition of cell growth in laboratory animals. 4.endotoxin. 26Abed ElKader ElOttol

27. Collection & Processing of Speciemn: Caution: Specimen must be handled in a biological safety cabinet. Lung tissue obtained at autopsy or biopsy is optimally selected from areas of necrosis. A representative portion of specimen should be placed in 10% neutral formalin for Direct Fluorescent Antibody and histopathological examination. Pleural fluid, transtracheal aspirate. 27Abed ElKader ElOttol

28. Diagnosis: 1. Isolation and identification of Legionella from clinical specimens either on artificial media or guinea pig inoculation followed by embryonated hen`s egg. 2. Demonstration of a four-fold or greater rise in antibody titer from acute phase to convalescent phase by the IFA test. 3. Demonstration of the organism in clinical specimen by DFA. Treatment: Erythromycin is the drug of choice. 28Abed ElKader ElOttol

29. BRUCELLA Undulating fever 29Abed ElKader ElOttol

30. Medically Important Species B. abortus Cattles B. melitensis Goats & sheeps B. canis Dogs B.suis swine

31. The Many Names of Brucellosis Human Disease Malta Fever Undulant Fever Mediterranean Fever Rock Fever of Gibraltar Gastric Fever Animal Disease Bang’s Disease Enzootic Abortion Epizootic Abortion Slinking of Calves Ram Epididymitis Contagious Abortion

32. Gram-negative bacilli (coccobacilli ) Chronically infected domestic animals. non-motile, non-spore forming. Some spp. Require supplemental carbon dioxide for primary isolation. Isolation form clinical specimens require prolonged incubation. Brucella strains always catalse -positive 32Abed ElKader ElOttol

33. Tiny, faintly staining coccobacillus (0.5 to 0.7 by 6.0 to 1.5 microns).

34. TRANSMISSION Primarily animal disease : Causes abortion & sterility. Transmission From animals to animals & humans by: Abrasion in skin Inhalation Ingestion of contaminated milk & cheese Persons at High Risk Dairy workers & farmers Live stock handlers & veternarians °Slaughterhouse employees 34Abed ElKader ElOttol

36. CLINICAL FEATURES OF BRUCELLOSIS (Undulating fever) Incubation period: 1-3 weeks Initially influenza-like. Headache, weakness, weight lost, arthralgia, liver dysfunction. Undulating (rising & falling) fever for weeks & months. A chronic illness Splenomegaly Hepatomegaly Enlarged lymph nodes 36Abed ElKader ElOttol

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39. LAB DIAGNOSIS Specimens Blood, LN & BM biopsy Culture Require 5-10% CO2 Serology ° Antibody titre of 1:160 or more (significant) ° Titre returns to normal within a year of successful treatment TREATMENT Ciprofloxacin May be up to 6 weeks to prevent relapses 39Abed ElKader ElOttol

42. PREVENTION Pasteurization of milk Immunization of animals Eradication of infected livestock Minimize occupational exposure 42Abed ElKader ElOttol

43. End of Lecture 43Abed ElKader ElOttol