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

1. Overview
2. Morphology Physiology
3. Epidemiology

1. Symptoms
2. Pathogenesis
3. Diagnosis
4. 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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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 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

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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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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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Pathogenesis
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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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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.