Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

“…The PMS-culture and confirmation of suspected positive cultures was carried out as follows: 500 µL of each bead suspension after PMS was inoculated into screwcapped glass test tubes containing 5 mL of modified Middlebrook 7H9 liquid medium (Pozzato et al, 2011), consisting of (per 900 mL) 4.7 g of Middlebrook 7H9 powder, 1.0 g of casitone, 5 mL of glycerol supplemented with 10% vol/vol OADC supplement and PANTA plus antibiotic supplement (all Becton Dickinson) and mycobactin J (2 mg/L; Synbiotics Europe SAS, Lyon, France), but without the addition of 16% egg yolk. The broth cultures were incubated at 37°C and absorbance at optical density at 600 nm (OD 600nm ) was measured at time 0 and then from 4 wk onward every 2 wk up to 16 wk using a Biowave CO8000 Density meter (Biochrom Ltd., Cambridge, UK).…”

“…Only a milk sample preparation and culture approach, optimized over recent years by researchers at QUB, was successful in isolating viable MAP from 12 CMR samples during this study. This approach included (1) allowing time (overnight at 4°C following reconstitution of CMR) for complete rehydration of MAP cells present in CMR before testing commenced, (2) PMS to selectively capture MAP cells from CMR rather than exposing potentially injured MAP cells to a chemical decontamination treatment, (3) primary culture for up to 16 weeks in a modified Middlebrook 7H9 liquid medium (first described by Pozzato et al, 2011), which had casitone added but no egg yolk, to permit resuscitation of sub-lethally injured MAP cells, and (4) sub-culture of any primary cultures once evidence of MAP growth was observed into richer Dubos liquid medium (without egg yolk) and Herrold's egg yolk medium to stimulate more copious growth of MAP. It is difficult to explain the discrepant culture results at UW and QUB; we can only speculate on possible reasons.…”

“…The question arises, are sub-lethally injured MAP cells likely to benefit from, or be adversely affected by, such a rich source of nutrients when trying to repair heat or dessication damage? Third, the primary culture medium adopted by QUB contained added casitone (0.1%, 1 g/L) and a higher than normal amount of 9733 glycerol (0.5%, as per the Pozzato et al (2011) recipe, rather than 0.2%, which is the amount indicated by the manufacturer (Difco) of Middlebrook 7H9 broth). The additional ingredient, casitone, is a pancreatic enzymatic digest of casein (milk protein) that contains a particularly high content of AA and peptides of varying sizes, making it a nutritious hydrolysate (BD Biosciences, 2017).…”

“…Glycerol would also represent a carbon source; a very old publication (Sattler and Youmans, 1948) relating to the effects of glycerol on tubercle bacilli (M. tuberculosis) reported that glucose and glycerol increased their total amount of growth but not their initial rate of multiplication. In light of the MAP isolation success at QUB during this study, we would like to suggest that the optimal culture approach for isolation of MAP from powdered milk products could be liquid culture in the modified 7H9 medium of Pozzato et al (2011), with PANTA but without egg yolk, following PMS to capture MAP cells from reconstituted CMR that has had time to completely rehydrate before testing commences. Chemical decontamination with HPC should definitely be avoided because of its known deleterious effects on the viability of MAP cells (Dundee et al, 2001, Gao et al, 2005, and therefore, the distinct possibility that false-negative results will be obtained when testing dairy products containing low numbers of MAP.…”

Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

“…In 2012, BD discontinued the supply of Bactec 12B medium, an antibiotic additive (PANTA Plus; BD) containing polyoxyethylene stearate (POES), and support for the associated Bactec 460 instrumentation, as the radiometric technology had become redundant in nonveterinary applications, such as for Mycobacterium tuberculosis diagnosis. Therefore, researchers in Europe, New Zealand, and Australia commenced research to find a suitable replacement medium, and the results of these studies are beginning to be published (16).…”