1. Bacterial agents of bioterroism

2. Laboratory network for biological terrorism

3. Bacillus anthracis Anthrax

4. Primarily disease of herbivores Humans usually infected by contact with infected animals or contaminated animal products Soil reservoir Woolsorter’s disease (inhalation anthrax) No person-to-person transmission of inhalational anthrax Anthrax: Overview CDC

5. ANTHRAX Three forms of human anthrax occur: 1. Cutaneous 2. Gastrointestinal Oropharyngeal Abdominal 3. Inhalation (Woolsorter’s Disease)

6. Vesicle development, day 2Eschar formation, day 4 Cutaneous anthrax

7. Inhalation Anthrax Infective dose = 8,000 - 15,000 spores Incubation period = 1-6 days Duration of illness = 3-5 days Fever, malaise, and fatigue Short period of improvement = up to 2 days Abrupt respiratory distress…death <24hrs Person to person transmission = no

8. Anthrax: Specimen Selection Inhalation: Sputum and Blood Cutaneous: Vesicles and Eschar Gastrointestinal: Stool and Blood

9. Bacillus anthracis Key Sentinel Lab Tests Gram stain Growth characteristics on agar Sporulation, in air Motility Capsule by India Ink

10. Broad gram-positive rod: 1-1.5 X 3-5 µ Oval, central - subterminal spores: 1 X 1.5 µ with no significant swelling of cell Spores are NOT usually present in clinical specimens unless exposed to atmospheric O 2 Bacillus anthracis Gram Stain Morphology

11. B. anthracis, Gram stain demonstrating spores

12. Colonial morphology of 18-24hr @ 35 C: –Well isolated colonies are 2-5 mm in diameter –Flat or slightly convex, irregularly round –Edges: slightly undulate, often curly tailing edges –Ground glass appearance –“Sticky” consistency….stands up like beaten egg whites B. anthracis Colonial Morphology

13. B. anthracis, colony on SBA

14. “STICKY” consistency of B. anthracis’ colony on SBA

15. Gram-positive, broad rod, catalase- positive, spore-positive, aerobe: Bacillus sp. Spores are oval and nonswelling with ground glass colony appearance: Bacillus morphology group 1, includes B. anthracis, B. cereus, B cereus var mycoides, and B. thuringiensis Bacillus anthracis Presumptive Identification

16. Nonmotile: B anthracis and B cereus var mycoides (and B. megaterium) Nonhemolytic, forms capsule: Presumptive B. anthracis Refer to state lab for testing Bacillus anthracis Presumptive Identification, con’t

17. Yersinia pestis Plague

18. Natural vector - Rodent flea Mammalian hosts –rats, squirrels, chipmunks, rabbits, and carnivores Enzootic or Epizootic Plague: Overview

20. Plague Epidemiology U.S. averages 13 cases/yr 30% of cases are in Native Americans in the Southwest. 15% case fatality rate Most cases occur in summer and near the patient’s residence –bubonic (infected lymph nodes) –septicemic (blood-borne organisms) –pneumonic (transmissible by aerosol; deadliest)

21. Yersinia pestis Specimen Selection Specimen selection is important –Bubo - lymph node aspirate –Blood - organisms may be intermittent. Take three specimens 10-30 minutes apart –Pneumonic Sputum/throat - use Wayson stain Bronchial washings - Wayson stain Inoculate routine plating media

22. Sentinel Lab Procedures Yersinia pestis Gram stain Wayson stain Growth characteristics on agar Growth characteristics in broth

23. Yersinia pestis Gram stain Small, gram-negative coccobacilli

24. Yersinia pestis Wayson Stain Used for rapid assessment –when it is a part of the identification process Best with tissue, sputum, blood Stains of pure culture isolates tend to lose bipolarity Pink-blue cells with polar granules (safety pin appearance)

25. Yersinia pestis Wayson Stain Wayson stain alone is not diagnostic Pink-blue cells with a closed safety pin look

26. Y.pestis 48 h culture on SBA72 h culture on SBA

27. Small Gram-negative, poorly staining rods from blood, lymph node aspirate, or respiratory specimens Safety pin appearance in Gram, Wright, Giemsa, or Wayson stain More than one patient in a short, specified period with fever, lymphadenopathy Refer to state lab Yersinia pestis Technical Hints

28. Francisella tularensis Tularemia

29. Tularemia: Overview Disease of Northern Hemisphere In U.S., most cases associated with rabbits/hares and ticks About 200 cases/year in U.S. –most in South central and Western states –majority of cases in summer, some in winter

30. Reported Cases of Tularemia - 1990-1998

31. Low infectious dose –1 to 10 organisms by aerosol or intradermal route No person-to-person transmission Tularemia: Overview (cont’d) Several forms of human tularemia exist: - Ulceroglandular, glandular, oculoglandular, oropharyngeal, intestinal, pneumonic, and typhoidal

32. Tularemia: Specimen Selection Serum - acute and convalescent Blood cultures Sputum Swab – ulcer or eye

33. Sentinel Lab Procedures Francisella tularensis This is a dangerous, highly virulent organism and it should not be manipulated at the bench. Laboratory-acquired infections can occur easily. Gram stain Growth characteristics in broth Growth characteristics in agar

34. Francisella tularensis Poorly staining, tiny Gram-negative coccobacilli

35. Francisella tularensis Growth Characteristics Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal –Enriched chocolate agar + 9% sheep blood + cysteine –Not part of Sentinel Lab routine procedures –BCYE (for Legionella) also works Will grow initially on sheep and chocolate blood agar and Thayer-Martin agar, but poorly or not at all on passage Grows slowly at 35 o C, poorly at 28 o C

36. Francisella tularensis Growth Characteristics 24 hours –gray-white, translucent colonies –usually too small to be seen individually 48 hours –Sheep Blood Agar - <1 mm, gray-white, opaque, no hemolysis

37. Francisella tularensis Sheep blood agarChocolate agarCysteine heart agar

38. Francisella tularensis Technical Hints Tiny, Gram-negative coccobacilli from blood, lymph node aspirate, or respiratory specimens Blood isolates that will grow slowly on chocolate agar but poorly or not at all on blood agar in 24 hours Faint growth in thio; requires cysteine in other broth Refer to state lab If you see:

39. Brucella spp. Brucellosis

40. BRUCELLOSIS A zoonotic disease caused by any of 4 Brucella sp.: abortus, melitensis, suis, and canis A systemic infection characterized by an undulant fever pattern But relatively rare in the U.S. with approximately 100 cases/yr

42. BRUCELLOSIS: TRANSMISSION Unpasteurized dairy products –The most common mode of transmission Direct skin contact –Occupational hazard for farmers, butchers, veterinarians, and laboratory personnel Aerosols –Highly infectious

43. Infective dose = 10 -100 organisms Incubation period = 5 days - > 6 months Duration of illness = weeks to months Fever, profuse sweating, malaise, headache and muscle/back pain. Person to person transmission = no Mortality = <5% Persistence of organism = very stable BRUCELLOSIS

44. Brucella spp. Specimen Selection Serum –The diagnosis of brucellosis is frequently achieved by serology. An acute & convalescent phase specimen should be collected (21d apart) Blood or bone marrow –Sources from which Brucellae are most often isolated Tissue (spleen, liver) –Brucellae occasionally isolated

45. Brucellosis is THE most commonly reported laboratory-associated bacterial infection. Cases have occurred in clinical laboratory settings by “sniffing” cultures, direct skin contact with cultures, and aerosol generating procedures Brucella spp. Biosafety Alert

46. Colonial morphology on SBA Gram stain morphology Oxidase positive Urea hydrolysis positive Sentinel Lab Tests Brucella spp.

47. Colonial morphology on SBA –Fastidious –Visible growth may take 48 - 72 hrs –Small (0.5-1.0mm), convex, glistening – Non-hemolytic and non-pigmented Brucella spp. Key Sentinel Lab Tests

48. B. melitensis on sheep blood agar

49. Gram Stain Morphology –Tiny (very) –Faintly staining –Gram-negative coccobacilli –0.5 - 0.7  x 0.6 - 1.5  Brucella spp. Key Sentinel Lab Tests

50. Tiny, faintly staining, gram-negative coccobacilli from blood or bone marrow Slow growth on Sheep Blood Agar, 2-3 days for colony appearance Oxidase + Urease + Handle plates with care Refer to state lab Brucella spp. Review of Key Tests

51. Clostridium botulinum Botulism

52. Caused by toxin from Clostridium botulinum –toxin types A, B, E, most commonly associated with human disease –most potent lethal substance known to man (lethal dose 1ng/kg) C. botulinum spores found in soil worldwide Approximately 100 reported cases/year in the U.S. –infant most common (72%) –food borne not common No person-to-person transmission Botulism: Overview

53. Infective dose: 0.001  g/kg Incubation period: 18 - 36 hr (6hr to 10 d) Dry mouth, double vision, droopy eyelids, dilated pupils Generalized, progressive descending bilateral muscle weakness & paralysis Respiratory failure and death Mortality usually 5 – 10% FOODBORNE BOTULISM

54. Among 309 persons with clinically diagnosed botulism reported to CDC from 1975 to 1988: –Stool cultures for C. botulinum: 51% + –Serum botulinum toxin testing: 37% + –Stool botulinum toxin testing: 23% + Overall, at least one of the above tests was positive for 65% of all patients Diagnosis is primarily clinical

55. Sentinel Lab Procedures for Botulism Event Properly collected specimens are to be referred to designated testing laboratories Prior to the shipment of any botulism- associated specimen, testing must be arranged with MDCH laboratory

56. Clinical specimens to be collected: 1. Serum 2. Feces 3. Food samples Autopsy specimens: 1. Serum 2. Gastric and intestinal contents Sentinel Lab Procedures for Botulism Event

57. Materials suspected of containing botulism toxin must be handled: –Biological Safety Cabinet (Class II) –Laboratory Coats –Disposable surgical gloves –Face shield (as needed) Botulism Biosafety Alert

58. BOTULISM The diagnosis of botulism is made clinically, i.e., based on the patient’s case history and physical findings Health care providers suspecting botulism should contact the Michigan Department of Community Health

59. Botulism Referral Lab Procedures Mouse bioassay Isolation of C. botulinum