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Clinical Veterinary Microbiology

Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium aviumsubsp. paratuberculosis from Sheep

R. J. Whittington, I. Marsh, S. McAllister, M. J. Turner, D. J. Marshall, C. A. Fraser
R. J. Whittington
NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570, and
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I. Marsh
NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570, and
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S. McAllister
NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570, and
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M. J. Turner
NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570, and
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D. J. Marshall
Orange Agricultural Institute, Orange, New South Wales 2500,Australia
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C. A. Fraser
Orange Agricultural Institute, Orange, New South Wales 2500,Australia
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DOI: 10.1128/JCM.37.4.1077-1083.1999
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Tables

  • Table 1.

    Studies in which culture of M. avium subsp.paratuberculosis from sheep has been attempted

    Facility and country of cultureaSample(s)Medium(a)SensitivityTimeReference
    Difficulty
     United KingdomLymph nodesModified Dubos50 CFU/g NAb6
     United KingdomGut mucosaModified Dubos-actidione-chloramphenicol75 CFU/ft of intestineNA6
     New ZealandFecesHerrold’s5 of 47 infected sheep; 1 to 15 colonies 6 mo9
     United StatesTissues, fecesHerrold’s, Lowenstein-Jensen3 of 20 infected sheep 6 mo32
     United KingdomTissuesEgg basedTiny colonies after 7 mo in 3 of 12 tubes 7 mo15
     AustraliacMucosaHerrold’s, Lowenstein-Jensen1 of 3 infected ewes after 12 wk 5 mo31
     AustraliacMucosa, lymph node, fecesEgg based4 of 50 infected sheep 5 mo7
     AustraliadMucosaHerrold’s0 of 1 infected sheep 9 mo25
     MoroccoFecesLowenstein-Jensen–mycobactin2 of 10 infected ewes16 wk5
     South AfricaFeces, tissuesHerrold’s2% of 59 infected sheepNA18
     IcelandMucosa, lymph nodesFinlayson’s, modified Dubos, Herrold’s9 to 54% of 22 infected samples 8 mo17
     SpainFeces, tissuesHerrold’s, Lowenstein-Jensen7 and 93% of 41 infected samples, respectively but colonies minute4 to 8 mo20
    Ease
     IndiaFecesSerum agar57% of 100 infected sheepNA22
     IranFeces, tissuesFinlayson-Taylor, Lowenstein-Jensen75% (sheep and goats not differentiated)NA3
     ChinaNAPotato broth60% of 38 infected sheepNA14
     SpainFeces, tissuesLowenstein-Jensen, Middlebrook 7H11-OADC with or without mycobactin J86% of 25 infected sheep 4 mo1
    • ↵a Countries are divided with regard to whether difficulty or ease was noted or implied in obtaining results.

    • ↵b NA, not available.

    • ↵c New South Wales.

    • ↵d Victoria.

  • Table 2.

    Composition of Middlebrook media used to culture strains of M. avium subsp. paratuberculosis from sheep in this study

    IngredientAmt/liter in:
    Modified BACTEC 12BaModified 7H10 agarbModified 7H11 agarc
    For base medium
     Casein digest667 mge800 mgd800 mg
     Ammonium sulfate333 mg400 mg400 mg
     Monopotassium phosphate667 mg1.2 g1.2 g
     Disodium phosphate1.7 g1.2 g1.2 g
     Sodium citrate67 mg320 mg320 mg
     Magnesium sulfate33 mg20 mg40 mg
     Calcium chloride0.33 mg0.4 mg
     Zinc sulfate0.67 mg0.8 mg
     Copper sulfate0.67 mg0.8 mg
     l-Glutamic acid333 mg400 mg400 mg
     Ferric ammonium citrate27 mg32 mg32 mg
     Pyridoxine0.67 mg0.8 mg0.8 mg
     Biotin0.33 mg0.4 mg0.4 mg
     Malachite green0.2 mg0.8 mg
     Bacto Agar12 g12 g
    For enrichment
     Oleic acid40 mg
     Albumin fraction V, bovine3.3 ge4.0 g4.0 g
     Dextrose1.6 g1.6 g
     Catalase32,000 Ue2.4 mg3.2 mg
     Sodium chloride680 mg
     C14 palmitic acid667 μCie
    As additive
     Egg yolk167 ml200 ml200 ml
     Mycobactin J0.83 mg1.0 mg1.0 mg
     PANTA PLUSf33.3 ml40 ml40 ml
    • ↵a Additives (and 700 μl of water) were added to the enriched BACTEC media at the following rates per 4-ml vial, resulting in a final volume of 6 ml/vial: 1 ml of egg yolk, 5 μg (100 μl of mycobactin J, and 200 μl of PANTA PLUS (4a, 13a).

    • ↵b ADC was used for enrichment.

    • ↵c OADC was used for enrichment.

    • ↵d As added Casitone.

    • ↵e Added by manufacturer to standard Middlebrook 7H9 broth to form BACTEC 12B medium.

    • ↵f Consists of polymyxin B (1,000 U/ml), amphotericin B (100 μg/ml), nalidixic acid (400 μg/ml), trimethoprim (100 μg/ml), azlocillin (100 μg/ml), and polyoxyethylene stearate (4 mg/ml).

  • Table 3.

    Culture of ovine tissues and feces in modified BACTEC 12B radiometric mediuma

    Histological gradeTissue samplesFecal samples
    nNo. positivenNo. positive
    No lesions14530
    Paucibacillary4473
    Multibacillary18181919
     Total36272922
    • ↵a Group A. Sheep were selected from numerous farms based on clinical, pathological, and serological criteria. Tissues and feces were not from the same sheep. Data are the number of sheep with a positive radiometric GI and IS900 PCR result.

  • Table 4.

    Culture of ovine tissues and feces in modified BACTEC 12B radiometric medium and on modified Middlebrook agar slopesa

    Histological gradenModified BACTEC 12BModified Middlebrook agar
    No. of positive tissue samplesNo. of positive fecal samplesNo. of tissue samples:No. of fecal samples:
    Positive on 7H10Positive on 7H11ContaminatedbPositive on 7H10Positive on 7H11Contaminatedb
    Paucibacillary1616915161851
    Multibacillary141413141212110
     Total3030222928120161
    • ↵a Group B. The sheep came from nine farms and were selected for culture based on histological findings alone. Tissues and feces were matched from the same sheep. Data are the number of sheep with a positive radiometric GI and IS900 PCR result or the number of sheep with growth on solid medium and a positive IS900 PCR result.

    • ↵b Cultures on 7H11 that were overgrown by contaminants.

  • Table 5.

    Culture of ovine feces in modified BACTEC 12B radiometric mediuma

    Histological gradenNo. of positive fecal samples
    No lesions113
    Paucibacillary62
    Multibacillary1414
     Total3119
    • ↵a Group C. Sheep were selected based on clinical signs and came from a single farm. Data are the number of sheep with a positive radiometric GI and IS900 PCR result.

  • Table 6.

    Culture of ovine tissues and feces in modified BACTEC 12B radiometric mediuma

    Histological gradenNo. of positive tissue samplesNo. of positive fecal samples
    No lesions2784
    Paucibacillary221
    Multibacillary111111
     Total402116
    • ↵a Group D. Twenty sheep were selected based on serology from each of two farms. Tissues and feces were matched from the same sheep. Data are the number of sheep with a positive radiometric GI and IS900 PCR result.

  • Table 7.

    Combined results of all trials with modified BACTEC 12B radiometric mediuma

    Histological gradeTissue samplesFecal samples
    nNo. (%) positivenNo. (%) positive
    No lesions4113 (31.7)417 (17.1)
    Paucibacillary2222 (100)3115 (48.4)
    Multibacillary4343 (100)5857 (98.3)
    • ↵a Tissues and feces are not necessarily matched from the same sheep. Data are the number and percentage of sheep with a positive radiometric GI and IS900 PCR result.

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Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium aviumsubsp. paratuberculosis from Sheep
R. J. Whittington, I. Marsh, S. McAllister, M. J. Turner, D. J. Marshall, C. A. Fraser
Journal of Clinical Microbiology Apr 1999, 37 (4) 1077-1083; DOI: 10.1128/JCM.37.4.1077-1083.1999

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Evaluation of Modified BACTEC 12B Radiometric Medium and Solid Media for Culture of Mycobacterium aviumsubsp. paratuberculosis from Sheep
R. J. Whittington, I. Marsh, S. McAllister, M. J. Turner, D. J. Marshall, C. A. Fraser
Journal of Clinical Microbiology Apr 1999, 37 (4) 1077-1083; DOI: 10.1128/JCM.37.4.1077-1083.1999
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KEYWORDS

bacteriological techniques
culture media
Mycobacterium avium subsp. paratuberculosis
sheep

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