Effect of short- and long-term antibiotic exposure on the viability of Mycobacterium avium subsp. paratuberculosis as measured by propidium monoazide F57 real time quantitative PCR and culture.

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis in ruminants, has a lipid-rich cell wall which facilitates its survival and persistence in the environment. This property of the organism is exploited when it is cultured as decontaminating agents and antibiotics are used to suppress the growth of contaminating microflora, but such treatments can also negatively affect the isolation of MAP itself. The objective of this study was to assess the effect of the 'VAN' antibiotics (vancomycin, amphotericin B and nalidixic acid) on the viability of MAP using a propidium monoazide real time quantitative PCR (PMA qPCR) and culture. Long-term (5 week) treatment with VAN antibiotics resulted in a larger decrease in bacterial numbers compared to short-term (3 day) exposure. The PMA qPCR assay indicated that 50 µg/mL of vancomycin, 50 µg/mL of nalidixic acid, and 200 µg/mL of amphotericin B were 'threshold' concentrations, respectively, above which the decline in the viability of MAP was statistically significant. Using culture, these threshold concentrations were 100 µg/mL of vancomycin, 50-100 µg/mL of nalidixic acid, and 100 µg/mL of amphotericin B, respectively. Given that the two methods were found to be comparable, the PMA qPCR is a potentially more convenient and effective alternative to culture in detecting MAP.

Correlation of Mycobacterium avium subsp. paratuberculosis counts in gastrointestinal tract, muscles of the diaphragm and the masseter of dairy cattle and potential risk for consumers.

Tissues of cattle intended for human consumption can be contaminated by Mycobacterium avium subsp. paratuberculosis (MAP). Although different studies attribute varying roles of MAP in Crohn's disease progression it is thought that the exposure of humans to this bacterium should in any case be minimised. In this study, we have collected samples of intestine, mesenteric lymph nodes, muscles of diaphragm (musculus diaphragma) and masseter muscles (musculus masseter) from twenty-five cows in a slaughterhouse. The infectious status of all animals was confirmed by culture of faeces. MAP was found in almost all the intestines and mesenteric lymph nodes examined, including three faecal culture-negative animals indicating intermittent shedding. As intestine is used for the traditional production of sausages, it is alarming that 84.2% of intestine samples were positive for MAP. F57 and IS900 real time PCR revealed MAP in 40 to 68% of diaphragms and 11.1 to 38.9% of masseters. A noticeable dependence of the probability of MAP positivity of faeces versus gastrointestinal tract (GIT) and of GIT and muscles was observed. Due to the changing behaviour of consumers, both of these muscles have started to be widely used in cuisine. Therefore, the results of this paper imply that the processing of cows with paratuberculosis in abattoirs without any precautions (restrictions) and the usage of meat for human consumption should be rethought.

Mycobacterial screening of Czech red deer (Cervus elaphus) populations in overwintering sites, 2004-2006.

One isolate of Mycobacterium avium subsp. paratuberculosis was detected in 2,212 fecal samples of wild deer assembled in overwintering sites (OwS). Neither M. bovis nor M. a. subsp. avium was found. Therefore, congregating deer in OwSs does not automatically lead to the amplification of these pathogens among animals in OWSs.

Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6°c, 65°c and ph 2.0

The aim of this work was to study the expression of selected Mycobacterium avium subsp. paratuberculosis (MAP) genes connected with MAP virulence, adhesion and stress response. The temperature of 6°C and 65°C were chosen with regard to the food industry, storage conditions (refrigerator) and low-temperature pasteurization. A pH of 2.0, using lactic acid, was selected to mimic the natural environment of the stomach. Expression of selected genes was studied using real time reverse transcription PCR on three different MAP isolates. MAP isolates were chosen according to the number of their preceding cultivations. While isolates 8672 and 8819 were previously cultivated only once, MAP isolate 12146 went through four passages. Different expression profiles were observed in each of the three MAP isolates. However, particular similar patterns were observed. SigE, sigF and ahpC were up-regulated, while sigL was down-regulated under temperature stress. Mmp gene was found to be down-regulated under acidic conditions. Low passage isolates (8672 and 8819) showed certain level of acid resistance.

Soil and plant contamination with Mycobacterium avium subsp. paratuberculosis after exposure to naturally contaminated mouflon feces.

The aim of this study was to demonstrate the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil and colonization of different plant parts after deliberate exposure to mouflon feces naturally contaminated with different amounts of MAP. Samples of aerial parts of plants, their roots, and the soil below the roots were collected after 15 weeks and examined using IS900 real-time quantitative PCR (qPCR) and cultivation. Although the presence of viable MAP cells was not demonstrated, almost all samples were found to be positive using qPCR. MAP IS900 was not only found in the upper green parts, but also in the roots and soil samples (from 1.00 × 10(0) to 6.43 × 10(3)). The level of soil and plant contamination was influenced mainly by moisture, clay content, and the depth from which the samples were collected, rather than by the initial concentration of MAP in the feces at the beginning of the experiment.

Mycobacterium avium subsp. Paratuberculosis and the expression of selected virulence and pathogenesis genes in response to 6°C, 65°c and ph 2.0.

The aim of this work was to study the expression of selected Mycobacterium avium subsp. paratuberculosis (MAP) genes connected with MAP virulence, adhesion and stress response. The temperature of 6°C and 65°C were chosen with regard to the food industry, storage conditions (refrigerator) and low-temperature pasteurization. A pH of 2.0, using lactic acid, was selected to mimic the natural environment of the stomach. Expression of selected genes was studied using real time reverse transcription PCR on three different MAP isolates. MAP isolates were chosen according to the number of their preceding cultivations. While isolates 8672 and 8819 were previously cultivated only once, MAP isolate 12146 went through four passages. Different expression profiles were observed in each of the three MAP isolates. However, particular similar patterns were observed. SigE, sigF and ahpC were up-regulated, while sigL was down-regulated under temperature stress. Mmp gene was found to be down-regulated under acidic conditions. Low passage isolates (8672 and 8819) showed certain level of acid resistance.

Oligonucleotide microarray technology and its application to Mycobacterium avium subsp. paratuberculosis research: a review.

Microarrays represent a modern powerful technology, which have potential applications in many areas of biological research and provide new insights into the genomics and transcriptomics of living systems. The aim of this review is to describe the application of microarray technology for Mycobacterium avium subsp. paratuberculosis (MAP) research. The main focus points include a summary of results obtained for MAP using microarrays, examination of the fields of MAP research which are currently being investigated and possible areas of future research. This article is divided into two parts according to the type of nucleic acid used for array hybridisation. Articles related to MAP research using microarray technology are then divided according to the field of study, such as comparative genome analysis, diagnostics, expression or environmental studies.