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Isolation and Identification of Brucella

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  • Isolation and Identification of Brucella species

    International Brucellosis Epidemiology TrainingDecember 7-9, 2004

    Prepared by Darla R. EwaltPresented by Barbara M. Martin

  • Why Try to Isolate and Identify Brucella ?Determine infection statusPositive confirms serology resultsNegative does not mean animal is not infectedEpidemiologyVaccine strainStrain 19RB51Field strain

  • Arrival at the NVSL(Other Laboratories may differ)Arrives in BMPSGiven an accession numberLogged into computerDelivered to the Brucella LaboratoryGiven two unique numbersMB number example 04-0231Computer number example 983738Processed usually within two days

  • Processing Tissue and Milk SamplesAll identification written on containers or ear tags is transferred to worksheetAnimal identificationType of sample Supramammary lymph nodesMilk from right front quarter (RF)Specimens are not combined or pooled.

  • Processing Tissue SpecimensLymph nodesRemove fat and other extraneous tissueDip in 95% ethanol and burn off excess ethanol flame sterilizes surfaceUdder, uterus, spleen, testicle, major organRemove fat and other extraneous tissueCut 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 SpecimensContinuedTissue sampleCut into sample piecesPlaced in a stomacher bag with equal volume of PBSStomacher bag placed in stomacher and pulverized for a minimum of 2 minutesTissue suspensionSwabbed onto a minimum of 5 media platesFour different selective media

  • Processing Milk Samples20 ml of milk (preferred)Centrifuge15 minutes7000 X g8 CCultureCreamSedimentSkim milkSerial BRT if requested

  • Incubation and ObservationIncubation period10% CO237 C5 to 10 days isolation media observed twiceObservationIsolation media read approximately at 5 and 10 daysColonies with typical or suspicious colony morphology are transferred

  • Identification of Brucella speciesIdentification requires a minimum of 5 days to complete.

    In most cases identity is determine to biovar level.

  • Identification of Brucella speciesGrowth on dyesBasic fuchsinThioninThionin blue

    Growth on additivesPenicillinErythritolRifampicinGrowth in airH2S productionUrease productionCatalase productionLysis by phageTbilisiOthers availableDominant antigenAM

  • ResultsBasic FuchsinA = 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

  • ResultsThionin

  • Results

  • Results

  • ResultsGrowth in airCO2 Required

  • ResultsLysis by phageNo lysis

  • Brucella species found in the United StatesBrucella abortusBiovar 1Strain 19Strain RB51Biovar 2Biovar 4

    Brucella suisBiovar 1Biovar 3Biovar 4

    Brucella ovisBrucella canisBrucella species found in marine mammals

    Brucella neotomae

  • Animals infected with Brucella abortus

  • Animals infected with Brucella suis

  • Questions?

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