Yersinia
Brucella
Zoonosis
Francisella
ZOONOSIS
A disease, primarily of animals, which
is transmitted to humans as a result of
direct or indirect contact with the
infected animal population
Brucellosis
1. Overview
4. Symptoms
2. Morphology &
5. Pathogenesis
Physiology
6. Diagnosis
3. Epidemiology
7. Treatment
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
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
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.
Microscopic Characteristics
• Brucella spp.
– poorly staining
– small gram-negative
coccobacilli
– seen mostly as single cells
– appearing like “fine sand”
Brucella melitensis colonies
A. Grows slowly on
most standard
laboratory media.
Usually not visible
at 24h.
B. Pinpoint,
smooth,
translucent, nonhemolytic at 48h.
Public Health Aspects
Brucella: Sources
• Brucellosis caused by 1 of 4 Brucella species:
1. B. abortus
Some strains
require 5% CO2
on initial
isolation.
2. B. melitenus
Sheep
Camels
Goats
3. B. suis
4. B. canis
2 patient populations
1. Individuals who work
with unvaccinated
animals
•
B. abortus and B. suis
•
Infections result from:
• direct contact
• inhalation
2. Individuals who ingest
unpasteurized dairy
products
• B. melitensis is the most
common agent
Host Animal - Brucellosis
• Asymptomatic or mild disease.
• Predilection for organs rich in erythritol (breast,
uterus, placenta, epididymis).
• Causes sterility, abortions or carrier state in nonhuman animals.
Human - Brucellosis
Symptoms
Acute
Phase
Advanced
Disease
Chronic
Form
Pathogenesis
Brucella
mucosal epithelium
Transported to lymph nodes,
spleen, liver and bone marrow.
Pathogenesis
Lysozome
X
Phagosome
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
Diagnosis
• Symptoms and history
• Serological agglutination tests
• Culture
– Blood and bone marrow cultures
– Spleen, liver, joint fluid or abscesses
Treatment
Tetracycline, doxycycline, or
trimethoprimsulfamethoxazole in combination
and rifampin or gentamicin for 6 weeks to prevent
reoccurring infection.
Tularemia:
(Francisella tularensis)
Gram stain
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
Tularemia: Overview
• Gram-negative coccobacilli.
• Low infectious dose
• Two subspecies of F. tularensis:
– subspecies tularensis (type A)
– subspecies holarctica (type B)
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
Colony Characteristics
• After 48 hours incubation
• Colonies
– Very small
– white to gray to bluishgray
• Will not grow on
MacConkey or EMB plates.
F. tularensis on chocolate agar 48 hours growth.
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.
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)
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)
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
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 Clinical Types
Clinical presentation based on the
route of infection
Typhoidal tularemia
• Bacteremia- Sepsis
• Fever, chills, headache, myalgias, malaise,
sore throat, and anorexia.
• Likely bioterrorism presentation.
Pneumonic tularemia
• Entry into lungs via
– Aerosols
– hematogenous
• Severe atypical pneumonia
• Likely BT presentation
Pathogenesis
• 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
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
Treatment of Tularemia
• Prompt removal of ticks and insect repellent can
prevent disease.
• Antibiotics
– Streptomycin is the drug of choice
Yersinia
Overview: 3 species cause
human disease
• Yersinia pestis
• Yersinia enterocolytica
• Yersinia pseudotuberculosis
Overview: Plague
• Yersinia pestis; a gram-negative bacterium.
• Three forms of clinical illness;
– Bubonic
– Septicemic
– Pneumonic
• Pneumonic is the only one transmitted
through aerosals.
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.
Plague: Overview
• U.S. averages 13 cases/yr (17 in 2006)
• Plague is endemic in the desert
southwest.
• Most cases occur in summer.
Microscopic Characteristics
• Y. pestis appear as
single cells or short
chains of plump,
gram-negative rods.
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.
Microscopic Characteristics
• Bipolar staining
occurs when using
Wayson, or
Giemsa stain.
CDC
Colony Characteristics
• Grows well on most
standard laboratory
media.
• Sheep Blood Agar
– Gray-white
translucent colonies
– Pinpoint, graywhite, non-hemolytic
at 24 hours
Blood agar plate of Yersinia pestis at 48 hours.
CDC/Dr. Brodsky
Y. pestis: Physiology
• Non-motile
• Pleomorphic gram-negative bacillus
• Urease, and oxidase negative
• Facultative anaerobe
• Optimal growth at 28o C
• Facultative intracellular parasite
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
Yersinia pestis life-cycle
Plague - Clinical types
• Bubonic
• infected lymph nodes.
• Pneumonic (most likely BT presentation)
• transmissible by aerosol; deadliest.
• Septicemic
• blood-borne organisms.
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
Bubo
• swollen inguinal lymph node or bubo.
• After the incubation period of 2-7 days, symptoms
of the plague appear.
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.
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)
Y. pestis: Virulence Determinants
• 3 virulence encoded Plasmids
• Virulence is up-regulated at 37°C
• Capsule (F1 antigen)
Yersinia Outer Proteins
(Yops)
• 11 different proteins
• Antiphagocytic
• Inhibit production
– proinflammatory cytokines
– tumor necrosis factor
• Cytotoxin
Yops
• Targets
– dendritic cells
– macrophages
– Neutrophils
– does not target B and T lymphocytes
F-1 Antigen
• Glycoprotein capsule expressed at 370 C
• Not expressed in flea host
• Antiphagocytic
• Antibodies to F-1 are protective
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.
Diagnosis
• Examination of Bubo
aspirate, blood, sputum
• stained for bipolar staining
• Fluorescent-antibody
• Culture (hazardous)
Plague Treatment
• Y. pestis is susceptible to a variety of
antibiotics.
– streptomycin, tetracycline, and doxycycline
• Peumonic plague is contageous and
isolation is recommended.
Clinical Case
• 30 year old man from Colorado, went to a
hospital emergency department with a 3day 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.
• 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.