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Yersinia Brucella Zoonosis Francisella.

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Presentation on theme: "Yersinia Brucella Zoonosis Francisella."— Presentation transcript:

1 Yersinia Brucella Zoonosis Francisella

2 ZOONOSIS A disease, primarily of animals, which is transmitted to humans as a result of direct or indirect contact with the infected animal population

3 Brucellosis Overview Morphology & Physiology Epidemiology Symptoms
Pathogenesis Diagnosis Treatment

4 Brucella: Overview Primarily a disease of animals.
Common where significant disease among domestic animals. Common names- Undulant fever, Malta fever, Mediterranean remittent fever. Brucella can go through intact skin. Facultative intracellular bacteria

5 Morphology & Physiology
Small gram-negative coccobacillus Grows slowly (7 days), at 370 C. On subculture, a minimum of 48 h growth Aerobic growth on Chocolate agar and Sheep blood agar Will not grow on MacConkey or Eosin methylene blue (EMB) agar

6 Morphology & Physiology
Non-pigmented and non-hemolytic Non-motile Oxidase: positive Catalase: positive Urease: strongly positive, less than 2 hours. Some species within 5 minutes.

7 Microscopic Characteristics
Brucella spp. poorly staining small gram-negative coccobacilli seen mostly as single cells appearing like “fine sand”

8 Brucella melitensis colonies
A. Grows slowly on most standard laboratory media. Usually not visible at 24h. B. Pinpoint, smooth, translucent, non-hemolytic at 48h.

9 Public Health Aspects Brucella: Sources
Brucellosis caused by 1 of 4 Brucella species: B. abortus Some strains require 5% CO2 on initial isolation. Brucella abortus is enzootic in elk and bison in the greater Yellowstone National Park area

10 2. B. melitenus Sheep Camels Goats

11 3. B. suis Brucella suis is enzootic in feral swine in the southeast

12 4. B. canis

13 2 patient populations Individuals who work with unvaccinated animals
B. abortus and B. suis Infections result from: direct contact inhalation Individuals who ingest unpasteurized dairy products B. melitensis is the most common agent Farmers, veterinarians, slaughterhouse workers, hunters and laboratory workers. People who travel to or migrate from rural areas of Latin America and the Middle East. Endemic in dairy animals, particularly goats and camels.

14 Host Animal - Brucellosis
Asymptomatic or mild disease. Predilection for organs rich in erythritol (breast, uterus, placenta, epididymis). Causes sterility, abortions or carrier state in non-human animals.

15 Human - Brucellosis Symptoms Acute Phase Advanced Disease Chronic Form Symptoms may not develop for up to 2 months from time of exposure Acute phase is < 8 weeks from onset with non-specific “flu like” symptoms and intermittent fever Advanced disease is the undulant form, <1 year from onset. Symptoms are undulant fevers, arthritis and epididymo-orchitis in males. Chronic disease (>1 year from onset) can mimic miliary tuberculosis with suppurative lesions in the liver, spleen, and bone. The patient may have recurrent fevers, arthritis, depression, and chronic fatigue syndrome. Brucellosis may lead to granulomatous hepatitis, peripheral arthritis, leucopenia, thrombocytopenia, meningitis, and endocarditis.

16 Pathogenesis Brucella mucosal epithelium
Following penetration of the mucosal epithelium (ingestion/inhalation routes, abraded skin, or the conjunctiva) the bacteria are transported via macrophages to the regional lymph Transported to lymph nodes, spleen, liver and bone marrow.

17 Pathogenesis X Phagosome
Lysozome X Phagosome Inside the macrophage the brucella causes an inhibition of phagosome-lysosome fusion. Survival in macrophages establishes chronic infections

18 Pathogenesis No exotoxins
LPS does not activate the alternative complement pathway Acute lymphadenitis Granulocyte production in lymphatic tissue, spleen, liver, bone marrow, lymph nodes and kidneys. A potential bioterrorist agent

19 Diagnosis Symptoms and history Serological agglutination tests Culture
Blood and bone marrow cultures Spleen, liver, joint fluid or abscesses

20 Treatment Tetracycline, doxycycline, or trimethoprimsulfamethoxazole in combination and rifampin or gentamicin for 6 weeks to prevent reoccurring infection.

21 Tularemia: (Francisella tularensis)
Gram stain

22 Tularemia: Overview Primary reservoir in US Insect vectors
Rabbits and muskrats Insect vectors Ticks Infection via Insect bites Handling contaminated animal tissues Inhalation of aerosols Ingestion of contaminated food or water Exposure in a laboratory setting Rabbit fever

23 Tularemia: Overview Gram-negative coccobacilli. Low infectious dose
Two subspecies of F. tularensis: subspecies tularensis (type A) subspecies holarctica (type B) 10 to 50 organisms by aerosol or intradermal route. Subspecies tularensis Highly virulent Most common biovar in North America Typically associated with rabbits Subspecies holarctica Less virulent Most common biovar in Europe and Asia

24 Morphology & Physiology
Tiny gram-negative coccobacillus Nonmotile, encapsulated Aerobic slow growing (48 hours) C Fastidious organism requires sulfhydryl (cysteine, IsoVitaleX) supplementation for growth Grows wells on Chocolate agar Buffered charcoal yeast extract agar fastidious organism and requires cysteine supplementation for good growth on general laboratory media- Cysteine heart agar Enriched chocolate agar Buffered charcoal yeast extract agar

25 Colony Characteristics
After 48 hours incubation Colonies Very small white to gray to bluish-gray Will not grow on MacConkey or EMB plates. F. tularensis may at first grow on SBA, but upon subsequent passage will fail to grow on standard SBA. On cysteine supplemented agar plates, it is a gray-white, opaque colony, usually too small to be seen at 24 h. After incubation for 48 h or more, colonies are very small, white to grey to bluish-grey. F. tularensis will not grow on MacConkey or EMB plates. F. tularensis on chocolate agar 48 hours growth.

26 Microscopic Characteristics
Gram Stain shows Tiny Gram negative coccobacillus Pleomorphic Pale staining Seen mostly as individual cells Difficult to interpret due to minute size and poor staining. Tiny, faintly staining, pleomorphic gram-negative rods ( mcm X mcm) are noted; cells are smaller than those of Haemophilus species.

27 Phenotypic Characteristics
Grows slowly at C Oxidase-negative Weakly catalase-positive (may be negative) Urea-negative Nitrate-negative Non-motile Beta-lactamase-positive Satellite or XV test-negative (unlike Haemophilus)

28 Tularemia: Public Health
Modes of humans infection Bite of infected flies, or ticks Handling contaminated animal tissues or fluids Direct contact with or ingestion of contaminated water, food, or soil Inhalation of infective aerosols (most likely BT route) Occupational exposure risk for laboratory workers, landscapers, farmers, veterinarians, hunters, trappers, cooks, and meat handlers

29 Tularemia: Public Health
Endemic in US Majority of cases occur May – September (tick exposure) or winter (hunters). Most in rural areas. Arkansas, Missouri and Oklahoma

30 Symptoms Incubation period: 3-5 days (range 1-21 days)
Clinical presentation can be divided into groups Ulceroglandular (45-85%) /glandular (10% to 25%) Typhoidal Pneumonic Oculoglandular Oropharyngeal/Gastrointestinal Prominent lymphadenopathy Recovery followed by permanent immunity Tularemia can range from a mild infection to a severe life-threatening illness.

31 Tularemia Clinical Types
Clinical presentation based on the route of infection F. tularensis, able to pass through unbroken skin.  Incubation period = 1-21 days

32 Typhoidal tularemia Bacteremia- Sepsis
Fever, chills, headache, myalgias, malaise, sore throat, and anorexia. Likely bioterrorism presentation. Typhoidal tularemia involves a systemic illness without anatomic localization of infection. Organisms enter the bloodstream through breaks in the skin or through mucous membranes and may affect the lungs and reticuloendothelial organs Necrotic foci can occur in any involved organ, and caseating granulomas may develop. Sepsis may occur, leading to shock, organ system failure, adult respiratory distress syndrome, and disseminated intravascular coagulation.

33 Pneumonic tularemia Entry into lungs via Severe atypical pneumonia
Aerosols hematogenous Severe atypical pneumonia Likely BT presentation Organisms enter the lungs either through inhalation of infectious aerosols or through hematogenous spread. The infectious dose by the respiratory route is 10 to 50 organisms Once in the lungs, the organisms rapidly enter pulmonary macrophages (within minutes) and begin replicating. An accumulation of neutrophils and macrophages, can be seen at sites of bacterial replication. The neutrophils appears to play more of a destructive than protective role in the host response.

34 Pathogenesis Pathogenesis
Virulence factors for F tularensis have not been well defined. Tularensis is a facultative intracellular pathogen that multiplies predominantly within macrophages.

35 Facultative intracellular pathogen
Capsule protects against complement killing Macrophage uptake bacterial surface polysaccharides serum complement complement C3 receptors LPS - O antigen prevents maturation of the phagosome multiply to high levels in cytosol Bacterial release via apoptosis

36 Diagnosis Symptoms & History
Direct staining of clinical specimens with a fluorescein-labeled antibodies. Serum antibody titers of 1:160 or greater Culture on cysteine-rich media Notify Laboratory personnel if you suspect Francisella since it is HIGLY INFECTIOUS

37 Treatment of Tularemia
Prompt removal of ticks and insect repellent can prevent disease. Antibiotics Streptomycin is the drug of choice

38 Yersinia

39 Overview: 3 species cause human disease
Yersinia pestis Yersinia enterocolytica Yersinia pseudotuberculosis

40 Overview: Plague Yersinia pestis; a gram-negative bacterium.
Three forms of clinical illness; Bubonic Septicemic Pneumonic Pneumonic is the only one transmitted through aerosals.

41 Plague: Overview Natural disease of rodents
Fleas that live on rodents transmit the bacteria to humans, in the bubonic form. This disease occurs in many areas of the world, including the United States.

42 Plague: Overview U.S. averages 13 cases/yr (17 in 2006)
Plague is endemic in the desert southwest. Most cases occur in summer. New Mexico and Colorado had the majority of plague cases in 2006

43 Microscopic Characteristics
Y. pestis appear as single cells or short chains of plump, gram-negative rods.

44 Microscopic Characteristics
Gram stain: In direct smears, bacterial cells may be inside or outside of leukocytes. The Gram smear morphology is suggestive but not specific for Y. pestis. Bipolar staining of a plague smear prepared from lymph aspirated from a bubo of plague patient.

45 Microscopic Characteristics
Bipolar staining occurs when using Wayson, or Giemsa stain. CDC

46 Colony Characteristics
Grows well on most standard laboratory media. Sheep Blood Agar Gray-white translucent colonies Pinpoint, gray-white, non-hemolytic at 24 hours Blood agar plate of Yersinia pestis at 48 hours. CDC/Dr. Brodsky

47 Y. pestis: Physiology Non-motile Pleomorphic gram-negative bacillus
Urease, and oxidase negative Facultative anaerobe Optimal growth at 28o C Facultative intracellular parasite

48 Public Health Aspects of Plague
Fleas carry Y. pestis in their intestinal tract. When feeding the fleas regurgitate uncapsulated organisms. Bacteria re-encapsulate and grow. Progeny are resistant to intracellular killing

49 Yersinia pestis life-cycle

50 Plague - Clinical types
Bubonic infected lymph nodes. Pneumonic (most likely BT presentation) transmissible by aerosol; deadliest. Septicemic blood-borne organisms.

51 Bubonic Plague Regional lymphadenitis (Buboes) Cutaneous findings
Inguinal, axillary, or cervical lymph nodes most common 80% can become septic 60% mortality if untreated Cutaneous findings Possible papule, vesicle, or pustule at inoculation site Purpuric lesions - late

52 Bubo swollen inguinal lymph node or bubo.
After the incubation period of 2-7 days, symptoms of the plague appear.

53 Pneumonic Plague Pneumonic
From aerosol or septicemic spread to lungs. Person-to-person transmission by respiratory droplet. 100% mortality untreated. Pneumonia progresses rapidly to dyspnea, cyanosis. Death from respiratory collapse/sepsis.

54 Septicemic Plague Primary or secondary Severe endotoxemia
Secondary from bubonic or pneumonic forms 100% mortality if untreated Severe endotoxemia Systemic inflammatory response syndrome Shock, Disseminated intravascular coagulopathy (DIC) Adult Respiratory Distress Syndrome (ARDS)

55 Y. pestis: Virulence Determinants
3 virulence encoded Plasmids Virulence is up-regulated at 37°C Capsule (F1 antigen)

56 Yersinia Outer Proteins (Yops)
11 different proteins Antiphagocytic Inhibit production proinflammatory cytokines tumor necrosis factor Cytotoxin 2 are antiphagocytic Others inhibit the proinflammatory cytokine, tumor necrosis factor production and induces macrophage apoptosis another Yops is a cytotoxin that causes actin filament disruption

57 Yops Targets dendritic cells macrophages Neutrophils
does not target B and T lymphocytes

58 F-1 Antigen Glycoprotein capsule expressed at 370 C
Not expressed in flea host Antiphagocytic Antibodies to F-1 are protective

59 Plasminogen activator (fibrinolysin) and Coagulase
Plasmid encoded proteins Promote dissemination of organisms from the clot at the bite site Coagulase is produced at 280 C but not at 320 C.

60 Diagnosis Examination of Bubo aspirate, blood, sputum
stained for bipolar staining Fluorescent-antibody Culture (hazardous)

61 Plague Treatment Y. pestis is susceptible to a variety of antibiotics.
streptomycin, tetracycline, and doxycycline Peumonic plague is contageous and isolation is recommended.

62 Clinical Case 30 year old man from Colorado, went to a hospital emergency department with a 3-day history of fever, nausea, vomiting, and right inguinal lymphadenopathy. Patient was not a hunter nor had he been in the woods recently but he did have dogs. He was discharged home without treatment. Lymphadenopath could be from Francisella tularensis (Ulceroglandular or Glandular) or Yersinia pestis (bubonic). What should you look for? Answer: Look for papules or pustules. No papules or pustules were seen on right leg.

63 Cultures of blood and a lymph node aspirate.
Three days later, the man returned and was hospitalized with sepsis and bilateral pulmonary infiltrates. One of the patient's dogs had serologic evidence of past Y. pestis infection. Cultures of blood and a lymph node aspirate. What should have been asked the first day? Answer: Any recent tick or flea bites.


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