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Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 l inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

Concentrations of surviving Escherichia coli, Clostridium perfringens, and Salmonella spp. were determined temporally in mechanically dewatered biosolids derived from anaerobic–mesophilic digestion and applied to agricultural land. Following applications in different seasons, repeated assessments of bacterial concentrations in biosolid clumps, using most-probable-number (MPN) techniques, found sustained high levels of these bacteria. Bacterial concentrations were often well above soil background levels at 6 months, and in some cases 11–12 months, after land application. Survival in surface-applied biosolids was similar to that for biosolids incorporated into the soil, and between application rates of 10 or 30 dry t/ha. Salmonella concentrations in applied biosolids were not predicted from, and could exceed those of, the indicator organism E. coli. Multiple plot analyses indicated regrowth of E. coli and Salmonella can occur within biosolids, up to several months after application. However, Salmonella serovars likely to pose a significant risk to animal health were not detected among isolates from the dewatered biosolids. Reduced accessibility for grazing livestock by soil incorporation, together with the time taken for normal pasture establishment practices, and the limited pathogenicity of the vast majority of salmonellae present in biosolids may significantly reduce the risk of spread of these organisms to the human food chain.