Diagnostic microbiology
lecture: 15
Bordetella pertussis
Abed ElKader Elottol
MSc. Microbiology
2010
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Organism:
Short, gram negative bacilli (coccobacilli).
 Aerobic, non spore forming
Non motile,.
Specific to humans.
Complex, enriched media is required for isolation (BordetGengou agar )or ( Regan-Lowe Charcoal Agar) (charcoal act as
adsorbents of toxic fatty acids, radicals and peroxides) .
urease negative, nitrate negative.
 Pertussis is highly contagious
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Bordetella pertussis growing in
Regan-Lowe Agar Incubated in CO2
for 72 hours
mercury drop" colonies on Bordet' Gengou
virulence factors
 adhesions such as filamentous hemagglutinin, peractin,
and fimbriae.
 a number of toxins including pertussis toxin, adenylate
cyclase toxin, trachael cytotoxin, dermonecrotic toxin .
 lipopolysaccharide endotoxin .
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Disease: whooping cough (Pertussis)
•Pertussis = Transmitted by the respiratory tract rout from
early case and possibly via carriers.
•The organism adheres to and multiply rapidly on the surface
of the epithelium in the trachea and bronchi and interfere with
ciliary action .
• Disintegrating organisms liberate a toxin that irritate surface
cells, giving rise to catarrhal symptoms.
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An acute respiratory disease with three stages:
•
•
A catarrhal stage with an irritating cough, lasts 1 to 2 weeks.
A paroxysmal stage characterized by violent coughs followed by a high
respiratory whoop, lasts 2 to 6 weeks.
• A convalescent stage where the cough gradually decreases in
frequency and severity, lasts several weeks.
Note : 75% of deaths are among infants.
Signs & Symptoms of Pertussis
The first symptoms of pertussis are similar to those of a
common cold:
 runny nose
 sneezing
 mild cough
 low-grade fever
• Most B.pertussis strains contains peptide that promotes
marked lymophocytosis in the host
• Elevated white blood cell count with a lymphocytosis,
Confirmed with laboratory testing.

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Laboratory Diagnosis:
Nasopharyngeal swabs or cough droplets, expelled onto "Cough
plates" held in front of the mouth of the patient during a
paroxysms.
Incubate (Bordet-gengou plates) at 35 oC for 2-5 days.
Typical colonies resemble a 1mm droplets of mercury
surrounded by a zone of hemolysis.
Colonies are confirmed with specific antiserum
 Direct immunofluorescent staining of smears made from
nasopharyngeal swabs may give a rapid positive test.
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Human Disease & Associated Pathogens
Genus
Species
Bordetella
pertussis
Pertussis
parapertussis
Pertussis (milder form)
bronchiseptica
Bronchopulmonary disease
Disease
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Legionella pneumophila
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•Legionellosis was first recognized in association with the
epidemic of legionnaires disease which occurred in
Philadelphia in July 1976.
• 182 person contracted pneumonia, 34 died.
•Shortly following the epidemic, the causative agent was
isolated from lung tissues of patient who died of this disease.
•The organism was named Legionella pneumophila. Slow
grower (2-5 days in enriched media containing L-cystein and
supplemented source of ferric iron).
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General characteristics
• Motile Gram-negative, aerobic, non-spore-forming bacteria .
• Stain poorly with gram stain unless safranin is applied for long
periods.
• Non capsulated.
• In tissue and clinical specimens the organisms are
coccobacillary, measuring 1-2 µm.
• Elongated filamentous forms may be seen after growth on
some culture media.
Clinical Manifestation:
Transmission: Via respiratory tract.
Acute pneumonia begins 2-10 days after exposure with a
brief malaise, myalgia, and headache, followed rapidly by
prostration ‫انهيار‬, high fever.
an rigors ‫ شديد‬Cough, dyspnea, pleuritic and abdominal pain,
vomiting, diarrhea, and unexplained encephalopathy are often
seen.
Leukocytosis, elevated ESR, proteinurea, hematuria and
abnormal serum enzyme determination are common.
 The common causes of death due to respiratory failure and
shock.
 Gram stain of sputum is not diagnostic.
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Laboratory Diagnosis
• Culture gold standard
• Require L-cysteine and iron salts
• Use BCYE (buffered charcoal yeast extract agar)
• Antibiotics might be used to help inhibit the growth
of fastidious bacteria.
• Grown aerobically using 3-5% CO2 at 35 c° for 2 to 5
days
• pH 6.9 for optimum growth
• Colonies are small with a crystalline-like, ground glass
appearance.
Florescence stain of Legionella
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Biochemical Characteristics:
Weak oxidase positive
Strong catalase positive
Liquefy gelatine
Hydrolysis of hippurate Positive (This differentiate it from other
legionella species)
Virulence factors:
1. Proteolytic enzymes
2. Exotoxin with lysed laboratory animal RBCs
3. Cytotoxin which caused inhibition of cell growth in laboratory
animals.
4.endotoxin.
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Collection & Processing of Speciemn:
Caution: Specimen must be handled in a biological safety
cabinet.
•Lung tissue obtained at autopsy or biopsy is optimally
selected from areas of necrosis.
• A representative portion of specimen should be placed in
10% neutral formalin for Direct Fluorescent Antibody and
histopathological examination.
• Pleural fluid, transtracheal aspirate.
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Diagnosis:
1. Isolation and identification of Legionella from clinical
specimens either on artificial media or guinea pig inoculation
followed by embryonated hen`s egg.
2. Demonstration of a four-fold or greater rise in antibody titer
from acute phase to convalescent phase by the IFA test.
3. Demonstration of the organism in clinical specimen by DFA.
Treatment:
Erythromycin is the drug of choice.
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BRUCELLA
Undulating fever
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Medically Important Species
• B. abortus
Cattles
• B. melitensis Goats & sheeps
• B. canis
Dogs
• B.suis
swine
The Many Names of Brucellosis
Human Disease
Animal Disease
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Malta Fever
Undulant Fever
Mediterranean Fever
Rock Fever of Gibraltar
Gastric Fever
Bang’s Disease
Enzootic Abortion
Epizootic Abortion
Slinking of Calves
Ram Epididymitis
Contagious Abortion
• Gram-negative bacilli (coccobacilli )
• Chronically infected domestic animals.
•non-motile, non-spore forming.
•Some spp. Require supplemental carbon dioxide for
primary isolation.
•Isolation form clinical specimens require prolonged
incubation.
•Brucella strains always catalse -positive
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.)Tiny, faintly staining coccobacillus (0.5 to 0.7 by 6.0 to 1.5 microns
TRANSMISSION
• Primarily animal disease : Causes abortion & sterility.
Transmission
From animals to animals & humans by:
 Abrasion in skin
Inhalation
Ingestion of contaminated milk & cheese
Persons at High Risk
 Dairy workers & farmers
 Live stock handlers & veternarians
°Slaughterhouse employees
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CLINICAL FEATURES OF BRUCELLOSIS (Undulating fever)
• Incubation period: 1-3 weeks
• Initially influenza-like.
Headache, weakness, weight lost, arthralgia, liver
dysfunction.
• Undulating (rising & falling) fever for weeks & months.
• A chronic illness
• Splenomegaly
• Hepatomegaly
• Enlarged lymph nodes
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LAB DIAGNOSIS
• Specimens
Blood, LN & BM biopsy
• Culture
Require 5-10% CO2
• Serology
° Antibody titre of 1:160 or more (significant)
° Titre returns to normal within a year of successful treatment
TREATMENT
• Ciprofloxacin
May be up to 6 weeks to prevent relapses
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PREVENTION
• Pasteurization of milk
• Immunization of animals
• Eradication of infected livestock
• Minimize occupational exposure
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End of Lecture
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