Irene R. Grant

Zoonotic potential of Mycobacterium avium ssp. paratuberculosis : the current position

A possible association between Mycobacterium avium ssp. paratuberculosis (Map) and Crohns disease was first suggested in 1913, when similarities between the gross pathology and symptoms of Johnes disease in cattle and those of Crohns disease in humans were first noted. Crohns disease is now generally believed to have a multifactorial aetiology with genetic predisposition, environmental factors (infectious agent, diet or smoking), and abnormal inflammatory response all playing a part. Evidence supporting a link between Map and Crohns disease includes: higher detection rates of Map by PCR and culture in gut samples from Crohns patients compared with controls; demonstration of a serological response to Map antigens in Crohns patients; and anti-Map antibiotic therapy resulting in remission, or substantial improvement in disease condition, in many patients. The available scientific evidence has been reviewed by a number of expert groups in recent years. The consensus opinion, at present, is that the available information is insufficient to prove or disprove that Map is a cause of Crohns disease, but the hypothesis is still plausible. The recent discovery of a susceptibility gene in Crohns patients, NOD2/CARD15, does not preclude a role for Map in the pathogenesis of at least some cases of Crohns disease, as the function of this gene is bacterial sensing in the gut. If Map does contribute to the causation of Crohns disease then it may not be acting as a conventional infectious agent.

Comparative evaluation of four decontamination protocols for the isolation of Mycobacterium avium subsp. paratuberculosis from milk

Aims: Four chemical decontamination protocols for milk were compared with respect to mean percentage recovery of spiked Mycobacterium avium subsp. paratuberculosis, minimum detection limit and ease of application.

Rapid and Sensitive Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

ABSTRACT Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection of Mycobacterium avium subsp. paratuberculosis from milk and/or fecal samples from cattle and American bison. Samples were spiked with M. avium subsp. paratuberculosis organisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detect M. avium subsp. paratuberculosis in feces and milk. By use of this simple and rapid technique, 10 or fewer M. avium subsp. paratuberculosis organisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johnes disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.

Effect of higher pasteurization temperatures, and longer holding times at 72°C, on the inactivation of Mycobacterium paratuberculosis in milk

Raw cow’s milk spiked with 106 cfu ml−1 of Mycobacterium paratuberculosis was subjected to heat treatments of 72, 75, 78, 80, 85 or 90 °C for 15 s, and 72 °C for 20 and 25 s, using laboratory pasteurizing units. Three bovine strains of Myco. paratuberculosis were studied (NCTC 8578, B2 and DVL 943). Each strain was subjected to all the heat treatments indicated on three separate occasions. Although each of the heat treatments achieved a substantial (5–6 log10) reduction in numbers of viable Myco. paratuberculosis, small numbers of the organism (4–16 cfu 10 ml−1) survived in a proportion of the milk samples at each of the higher temperatures investigated, right up to 90 °C for 15 s. A longer holding time of 25 s at 72 °C was found to be more effective at inactivating Myco. paratuberculosis. Only one of the three strains studied, B2, yielded small numbers of survivors after heating at 72 °C for 20 s, but it was completely inactivated by extending the holding time at 72 °C by a further 5 s to 25 s. It was concluded that a longer holding time is more likely to achieve the complete inactivation of Myco. paratuberculosis in milk than a higher pasteurization temperature.

Efficacy of Various Pasteurization Time-Temperature Conditions in Combination with Homogenization on Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

ABSTRACT The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 101 to 105M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrolds egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in2, applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log10. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in2. Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 aw). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 aw for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

Sensitivity of foodborne pathogens to irradiation in the components of a chilled ready meal

The sensitivity of five foodborne pathogens to irradiation in the components of a roast beef meal (beef, gravy, cauliflower, roast potato and mashed potato) was investigated. Bacillus cereus (vegetative cells) was the most radiation-sensitive (D 10 = 0·126–0·288 kGy) of the pathogens studied. Clostridium perfringens (vegetative cells), Salmonella typhimurium and Listeria monocytogenes had similar D 10 ranges (0·342–0·586, 0·371–0·697 and 0·301–0·648 kGy, respectively). Staphylococcus aureus had D 10 values ranging from 0·252–0·427 kGy. When irradiated in gravy the pathogens generally had lower D 10 values than in any of the other four components.

Phylogenetic characterization of a novel radiation-resistant bacterium from irradiated pork: description of Hymenobacter actinosclerus sp. nov.

A phylogenetic analysis was performed on a red-pigmented, radiation-resistant, Gram-negative, rod-shaped organism originating from irradiated pork. Comparative 16S rRNA gene sequencing showed the bacterium was a member of the Cytophaga-Flavobacterium-Bacteroides line of descent and represents a new subline within the genus Hymenobacter. A new species, Hymenobacter actinosclerus, is described for this novel radiation-resistant bacterium. The type strain of Hymenobacter actinosclerus is CCUG 39621T.

Mycobacterium avium ssp. paratuberculosis and its potential survival tactics.

Mycobacterium avium ssp. paratuberculosis (Map) is an important animal pathogen with a potential, but as yet unproven, role in human disease. This review briefly describes the characteristics of Map that distinguish it from other Mycobacterium spp., presenting new information arising from completion of the sequencing of the Map genome. It then focuses on the potential mechanisms Map might employ to survive and disseminate in the environment, including interaction with protozoa and insects, dormancy, biofilm formation and aerosolization.

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture.

ABSTRACT UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log10 reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log10 reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrolds egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log10 lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).