L26 - Medical Microbiology Laboratory (Brucella spp.) by Hussein A. Abid

1. Medical Microbiology Laboratory
Gram Negative Coccobacilli
(Brucella spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. 2
TAXONOMY
Scientific name Rank
• Brucellaceae Family
• Brucella Genus
• B. melitensis
• B. suis
• B. abortus
• B. canis
Species
(medically important spp.)

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Brucella spp.
 Brucella are small, gram-negative, unencapsulated,
nonsporulating, nonmotile rods or coccobacilli that can
persist intracellularly.
 The genus Brucella includes four major clinically
relevant species:
1. B. melitensis (acquired by humans most commonly
from sheep, goats, and camels)
2. B. suis (from swine)
3. B. abortus (from cattle or buffalo)
4. B. canis (from dogs).

4. 4
Brucella spp.
The disease caused by them is called:
 Brucellosis
 Mediterranean fever
 Malta fever
 Undulant fever/remittent fever
 Gibraltar fever
 Cyprus fever

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TRANSMISSION
1. Ingesting infected food
2. Occupational exposure (e.g. exposure to cattle, sheep,..)
3. Consumption of unpasteurized milk products
4. Direct contact with an infected animal
5. Inhalation of aerosols
6. Transmission from human to human
7. Through sexual intercourse
8. From mother to child
NOTE: Incubation period (2 – 3 weeks)

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CLINICAL MANIFESTATIONS
 Regardless of the specific infecting species, brucellosis
often presents with one of three patterns:
1. A febrile illness similar to but less severe than typhoid
fever.
2. Fever and acute monarthritis, typically of the hip or
knee, in a young child (septic arthritis).
3. Long-lasting fever, misery, and low-back or hip pain in
an older man (vertebral osteomyelitis).

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CLINICAL MANIFESTATIONS
 High fever
 Malaise (discomfort)
 Anorexia (malnutrition)
 Arthralgia (pain in a joint)
 Fatigue
 Headache
 Sweating
 Weight loss
 Depression

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Brucella spp.
Clinical specimens:
 Blood for culture and serological testing
 Lymph nodes and liver biopsies
 Bonemarrow aspiration for culture
 Urine
 Sputum
 Breast milk
 Pleural, synovial and seminal fluid
 CSF and vaginal discharge
rare

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Brucella spp. (microscopy)
 Tiny, gram-negative coccobacilli

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Brucella (culture)
 The media employed currently are serum dextrose
agar, serum potato-infusion agar, trypticase soy agar
(Castaneda medium), or tryptose agar. (in 10% CO2)
Fastidious organism; growth on BA and CA, Punctate, grey, shiny, circular,
convex colonies at 48 - 72 h at 35/37 ºC, Non-hemolytic; non-pigmented

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BIOCHEMICAL TESTS
1. Catalase: positive (+ve)
2. Oxidase: positive (+ve)
3. Motility: non-motile
4. Indole, MR, VP, Citrate (IMViC): -, -, -, -
5. Urease test: positive (+ve)
6. Nitrate reduction test: positive (+ve)

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SEROLOGICAL TESTS
TIME NEEDED TEST NO.
2 min.
Rose Bengal test by Rapid Slide agglutination
(screening) test
1-
2 – 4 hrs. Rose Bengal test by Tube Agglutination test 2-
5 min.
Brucella IgG/IgM by Immunochromatographic
assay
3-
15 min. 2 Mercaptoethanol Test (ME) 4-
45 min. – 2 hrs. ELISA (IgG/ IgM) 5-
45 min. – 2 hrs. ECL (IgG/ IgM) 6-
- Complement fixation (CF) test 7-
15 – 20 min. Indirect Coombs test 8-

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ROSE BENGAL TEST (QUALITATIVE)
 It is one of the easiest methods to implement and the most
widely used for identifying Brucellosis antibodies in sera.
 Principle of the test:
o The RB is a rapid slide agglutination test.
o It is now often used widely for diagnosing human disease.
o The test uses a suspension of B. abortus smooth cells
stained with Rose Bengal dye (pink color) to detect Brucella
agglutinins.
o The stained bacterial suspension agglutinates when mixed
with samples containing specific IgG or IgM antibodies
present in the patient sample.

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ROSE BENGAL TEST (QUALITATIVE)
Procedure:
 Allow the reagents and sample to reach room temperature.
 Place 50 µL of the sample and one drop of each positive and
negative control into separate circles on the slide test.
 Shake the Rose Bengal reagent gently before using and add a
drop of this reagent next to the sample to be tested.
 Mix both drops with a stick, spreading them over the entire
surface of the circle. Use different stirrers for each sample.
 Rotate the slide with a mechanical rotator at 80-100 rpm for 2
minutes, and read the results (this is the optimum time
limited).

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ROSE BENGAL TEST (QUALITATIVE)
Results and interpretation
 No agglutination = absence of specific antibodies
 Agglutination (even slight) = presence of specific antibodies:
o If an agglutination appear after 15 seconds = (1:640)
o If agglutination appear after 30 seconds = (1:320)
o If agglutination appear after 1 min. = (1:160)
o If agglutination appear after 1.30 min. = (1:80)
 Patient history should be taken into account before giving the
result.
 This test is a screening test only for the detection of Brucella
agglutinins. If result is positive it must be confirmed by other
serological tests for Brucellosis.
Titration is
preferred

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ROSE BENGAL TEST (QUALITATIVE)
 Advantages:
o Rapid, inexpensive, sensitivity
and specificity.
 Limitations:
o Low sensitivity particularly in long chronic cases, and
relatively low specificity in endemic areas & Vaccinations
may produce agglutinins capable of reacting with the febrile
antigens.
Rose-Bengal kit

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2 MERCAPTOETHANOL TEST (ME)
 This test was designed to measure IgG in patients with chronic
brucellosis.
 Titers before and after treatment with 2-ME are compared.
 The test is available by only a few laboratories in Iraq.
Prophylaxis:
1. The disease could be prevented by pasteurization of milk which kill
the bacteria.
2. Affected animals are detected and eliminated from the herd.
3. General principles of hygiene are imposed to prevent spread or
reintroduction of infection.
4. In labs strict biosafety precautions.
5. Education campaign.

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OTHER METHODS
Description TEST NO.
Strip with colored brucella antigen. On
addition of patients serum, if antibodies
are present, prevents the flow of serum.
Castaneda strip test 1-
Delayed hypersensitivity Brucellin Skin test 2-
Molecular technique PCR 3-
Alterations in infected vertebrae; Pedro
Pons sign ( erosion of antero-superior
corner of lumbar vertebrae) and marked
osteophytosis are suspicious of
brucellic spondylitis.
Radiology 4-