Zoonosis

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Zoonosis
Yersinia
Brucella

• Francisella

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

• Overview
• Morphology Physiology
• Epidemiology

• Symptoms
• Pathogenesis
• Diagnosis
• Treatment

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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

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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

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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.

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Microscopic Characteristics

• Brucella spp.
• poorly staining
• small gram-negative coccobacilli
• seen mostly as single cells
• appearing like fine sand

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

• Brucellosis caused by 1 of 4 Brucella species
• B. abortus
• Some strains
• require 5 CO2
• on initial
• isolation.

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2. B. melitenus
Sheep
Camels
Goats
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3. B. suis
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4. B. canis
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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

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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.

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Human - Brucellosis
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Human - Brucellosis
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Pathogenesis
Brucella
mucosal epithelium
Transported to lymph nodes, spleen, liver and
bone marrow.
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Pathogenesis
Lysozome
X
Phagosome
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Pathogenesis

• No exotoxin
• 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

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Diagnosis

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

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Treatment

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

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Tularemia (Francisella tularensis)
Gram stain
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Tularemia Overview

• Primary reservoir in US
• 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

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Tularemia Overview

• Gram-negative coccobacilli.
• Low infectious dose
• Two subspecies of F. tularensis
• subspecies tularensis (type A)
• subspecies holarctica (type B)

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Morphology Physiology

• Tiny gram-negative coccobacillus
• Nonmotile, encapsulated
• Aerobic slow growing (48 hours) 35-370 C
• Fastidious organism requires sulfhydryl
  (cysteine, IsoVitaleX) supplementation for growth
• Grows wells on
• Chocolate agar
• Buffered charcoal yeast extract agar

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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 on chocolate agar 48 hours growth.
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Microscopic Characteristics
Tiny, faintly staining, pleomorphic gram-negative
rods (0.2-0.5 mcm X 0.7-1.0 mcm) are noted cells
are smaller than those of Haemophilus species.
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Phenotypic Characteristics

• Grows slowly at 35-370 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)

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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)

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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

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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

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Tularemia Clinical Types

• Clinical presentation based on the route of
  infection

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Ulceroglandular Glandular tularemia

• Ulceroglandular accounts for 75-85 of naturally
  occurring cases.

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Typhoidal tularemia

• Bacteremia- Sepsis
• Fever, chills, headache, myalgias, malaise, sore
  throat, and anorexia.
• Likely bioterrorism presentation.

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Pneumonic tularemia

• Entry into lungs via
• Aerosols
• hematogenous
• Severe atypical pneumonia
• Likely BT presentation

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Oropharyngeal tularemia
Oculoglandular tularemia

• Inoculation of the conjunctivae
• Unilateral, purulent conjunctivitis
• preauricular, submandibular or cervical
  lymphadenopathy

• Primary disease is confined to the throat.
• Ingestion of infected meat or water can result in
  orpharyngeal or gastrointestinal tularemia

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Pathogenesis
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Macrophages engulf F. tularensis within a
pseudopod loop
Daniel L. Clemens, Bai-Yu Lee, and Marcus A.
Horwitz. INFECTION AND IMMUNITY, Sept. 2005, p.
58925902
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• 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

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Diagnosis

• Symptoms History
• Direct staining of clinical specimens with a
  fluorescein-labeled antibodies.
• Serum antibody titers of 1160 or greater
• Culture on cysteine-rich media
• Notify Laboratory personnel if you suspect
  Francisella since it is HIGLY INFECTIOUS

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Treatment of Tularemia

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

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Yersinia
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Overview 3 species cause human disease

• Yersinia pestis
• Yersinia enterocolytica
• Yersinia pseudotuberculosis

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Overview Plague

• Yersinia pestis a gram-negative bacterium.
• Three forms of clinical illness
• Bubonic
• Septicemic
• Pneumonic
• Pneumonic is the only one transmitted through
  aerosals.

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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.

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Plague Overview

• U.S. averages 13 cases/yr (17 in 2006)
• Plague is endemic in the desert southwest.
• Most cases occur in summer.

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Microscopic Characteristics

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

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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.
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Microscopic Characteristics

• Bipolar staining occurs when using Wayson, or
  Giemsa stain.

CDC
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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
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Y. pestis Physiology

• Non-motile
• Pleomorphic gram-negative bacillus
• Urease, and oxidase negative
• Facultative anaerobe
• Optimal growth at 28o C
• Facultative intracellular parasite

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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

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Yersinia pestis life-cycle
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Plague - Clinical types

• Bubonic
• infected lymph nodes.
• Pneumonic (most likely BT presentation)
• transmissible by aerosol deadliest.
• Septicemic
• blood-borne organisms.

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Bubonic Plague

• Regional lymphadenitis (Buboes)
• 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

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Bubo

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

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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.

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Septicemic Plague

• Primary or secondary
• 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)

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This patient presented with symptoms of plague
that included gangrene of the right hand causing
necrosis of the fingers. In this case, the
presence of systemically disseminated plague
bacteria Y. pestis, i.e. septicemia, predisposed
this patient to abnormal coagulation within the
blood vessels of his fingers.
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Y. pestis Virulence Determinants

• 3 virulence encoded Plasmids
• Virulence is up-regulated at 37C
• Capsule (F1 antigen)

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Yersinia Outer Proteins (Yops)

• 11 different proteins
• Antiphagocytic
• Inhibit production
• proinflammatory cytokines
• tumor necrosis factor
• Cytotoxin

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Yops

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

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F-1 Antigen

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

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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.

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Diagnosis

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

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Plague Treatment

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

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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.

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• 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.