Isolation and Identification of Brucella species
International Brucellosis Epidemiology TrainingInternational Brucellosis Epidemiology TrainingDecember 7-9, 2004December 7-9, 2004
Prepared by Darla R. Ewalt
Presented by Barbara M. MartinPresented by Barbara M. Martin
Why Try to Isolate and Identify Brucella ?
Determine infection status Positive – confirms serology results Negative – does not mean animal is not
infected
Epidemiology Vaccine strain
Strain 19
RB51 Field strain
Arrival at the NVSL(Other Laboratories may differ)
Arrives in BMPS Given an accession number Logged into computer
Delivered to the Brucella Laboratory Given two unique numbers
MB number example 04-0231
Computer number example 983738 Processed usually within two days
Processing Tissue and Milk Samples
All identification written on containers or ear tags is transferred to worksheet Animal identification Type of sample
Supramammary lymph nodes
Milk from right front quarter (RF)
Specimens are not combined or pooled.
Processing Tissue Specimens
Lymph nodes Remove fat and other extraneous tissue Dip in 95% ethanol and burn off excess ethanol –
flame sterilizes surface
Udder, uterus, spleen, testicle, major organ Remove fat and other extraneous tissue Cut into 1-2 inch cube (Except do not cut into ovaries) Dip in 95% ethanol and burn off excess ethanol –
flame sterilizes surface
Processing Tissue SpecimensContinued
Tissue sample Cut into sample pieces Placed in a stomacher bag with equal volume
of PBS Stomacher bag placed in stomacher and
pulverized for a minimum of 2 minutes
Tissue suspension Swabbed onto a minimum of 5 media plates
Four different selective media
Processing Milk Samples
20 ml of milk (preferred) Centrifuge
15 minutes7000 X g8° C
CultureCreamSediment
Skim milkSerial BRT if requested
Incubation and Observation
Incubation period 10% CO2
37° C 5 to 10 days – isolation media observed twice
Observation Isolation media read approximately at 5 and
10 days Colonies with typical or suspicious colony
morphology are transferred
Identification of Brucella species
Identification requires a minimum of 5 days to complete.
In most cases identity is determine to biovar level.
Identification of Brucella species
Growth on dyes Basic fuchsin Thionin Thionin blue
Growth on additives Penicillin Erythritol Rifampicin
Growth in air
H2S production
Urease production
Catalase production
Lysis by phage Tbilisi Others available
Dominant antigen A M
Results
Basic FuchsinBasic Fuchsin
A = A = Brucella abortusBrucella abortus biovar 1biovar 1B = B = Brucella abortusBrucella abortus biovar 2biovar 2C = C = Brucella abortusBrucella abortus strain 19strain 19D = D = Brucella abortusBrucella abortus biovar 4biovar 4E = E = Brucella melitensis Brucella melitensis biovar 1biovar 1F = F = Brucella suis Brucella suis biovar 1biovar 1G = G = Brucella canisBrucella canisH = H = Brucella ovisBrucella ovis
ABCD
EFG
H
1:100,000
ABCD
EFG
H
1:50,000
ABCD
EFG
H
1:25,000
ResultsThionin
A = Brucella abortus biovar 1B = Brucella abortus biovar 2C = Brucella abortus strain 19D = Brucella abortus biovar 4
E = Brucella melitensis biovar 1F = Brucella suis biovar 1G = Brucella canisH = Brucella ovis
1:100,000
ABCD
EFG
H
1:50,000
ABCD
EFG
H
1:25,000
ABCD
EFG
H
Results
A = Brucella abortus biovar 1B = Brucella abortus biovar
2C = Brucella abortus strain 19D = Brucella abortus biovar 4E = Brucella melitensis biovar 1F = Brucella suis biovar 1G = Brucella canisH = Brucella ovis
A
B
C
D
EFG
H
Thionin Blue 1:500,000
Results
A = Brucella abortus biovar 1B = Brucella abortus biovar 2C = Brucella abortus strain 19D = Brucella abortus biovar 4E = Brucella melitensis biovar 1F = Brucella suis biovar 1G = Brucella canisH = Brucella ovis
ABCD
EFG
H
Penicillin 5 U/ml
ABCD
EFG
H
Erythritol 1 mg/ml
ABCD
EFG
H
Erythritol 2 mg/ml = 5% serum
Results
Growth in air
CO2 Required
Results
Lysis by phage
No lysis
Brucella species found in the United States
Brucella abortus Biovar 1
Strain 19Strain RB51
Biovar 2 Biovar 4
Brucella suis Biovar 1 Biovar 3 Biovar 4
Brucella ovisBrucella canisBrucella species found in marine mammals
Brucella neotomae
Animals infected with Brucella abortus
Animals infected with Brucella suis
Questions?
