Survival of Mycobacterium avium subsp. paratuberculosis in Dam Water and Sediment

In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.     

Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment

In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohn's disease.     

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Environmental Survival of Mycobacterium avium subsp. paratuberculosis in Different Climatic Zones of Eastern Australia

The duration of survival of both the S and C strains of Mycobacterium avium subsp. paratuberculosis in feces was quantified in contrasting climatic zones of New South Wales, Australia, and detailed environmental temperature data were collected. Known concentrations of S and C strains in feces placed on soil in polystyrene boxes were exposed to the environment with or without the provision of shade (70%) at Bathurst, Armidale, Condobolin, and Broken Hill, and subsamples taken every 2 weeks were cultured for the presence of M. avium subsp. paratuberculosis. The duration of survival ranged from a minimum of 1 week to a maximum of 16 weeks, and the provision of 70% shade was the most important factor in extending the survival time. The hazard of death for exposed compared to shaded samples was 20 and 9 times higher for the S and C strains, respectively. Site did not affect the survival of the C strain, but for the S strain, the hazard of death was 2.3 times higher at the two arid zone sites (Broken Hill and Condobolin) than at the two temperate zone sites (Bathurst and Armidale). Temperature measurements revealed maximum temperatures exceeding 60°C and large daily temperature ranges at the soil surface, particularly in exposed boxes.     

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants

Aims

To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis‐infected dairy herds.

Methods and Results

Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one‐stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two‐stage plant.

Conclusions

Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two‐stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit.

Significance and Impact of the Study

Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.     

Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10³ to 10⁴ organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented >6-log₁₀ reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72°C for 15 s, 75°C for 25 s, and 78°C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of >6 log₁₀ in 85% of runs and >4 log₁₀ in 14% of runs.     

Heat inactivation of Mycobacterium avium subsp. paratuberculosis in milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10(3) to 10(4) organisms/ml were processed with combinations of three temperatures of 72, 75, and 78 degrees C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented > 6-log10 reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72 degrees C for 15 s, 75 degrees C for 25 s, and 78 degrees C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of > 6 log10 in 85% of runs and > 4 log10 in 14% of runs.     

Mycobacterium avium subsp. paratuberculosis in scavenging mammals in Wisconsin

The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-ruminant wildlife has raised questions regarding the role of these species in Johne's disease transmission. In this study we tested 472 tissues from 212 animals of six different species of scavenging mammals. All animals were taken from within a 210-square-mile area in Dane and Iowa counties of south central Wisconsin from September to May in 2003-04 and tested for the presence of MAP. We detected MAP-specific DNA in 81 of 212 (38%) scavenging mammals, in 98 of the 472 (21%) tissues; viable MAP was cultured from one coyote's ileum and lymph node tissue. Despite the low numbers of viable MAP isolated in this study, our data adds to the increasing evidence demonstrating the potential for transmission and infection of MAP in nonruminant species and provides possible evidence of interspecies transmission. The apparently high exposure of nonruminant wildlife provides potential evidence of a spill-over of MAP to wildlife species and raises the question of spillback to domestic and wild ruminants. These results demonstrate the importance of understanding the role of wildlife species in developing management strategies for Johne's disease in domestic livestock.     

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

Background  

Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated.     

Adsorption of Mycobacterium avium subsp. paratuberculosis to Soil Particles

Attachment of Mycobacterium avium subsp. paratuberculosis to soil particles could increase their availability to farm animals, as well as influence the transportation of M. avium subsp. paratuberculosis to water sources. To investigate the possibility of such attachment, we passed a known quantity of M. avium subsp. paratuberculosis through chromatography columns packed with clay soil, sandy soil, pure silica, clay-silica mixture, or clay-silica complexes and measured the organisms recovered in the eluent using culture or quantitative PCR. Experiments were repeated using buffer at a range of pH levels with pure silica to investigate the effect of pH on M. avium subsp. paratuberculosis attachment. Linear mixed-model analyses were conducted to compare the proportional recovery of M. avium subsp. paratuberculosis in the eluent between different substrates and pH levels. Of the organisms added to the columns, 83 to 100% were estimated to be retained in the columns after adjustment for those retained in empty control columns. The proportions recovered were significantly different across different substrates, with the retention being significantly greater (P < 0.05) in pure substrates (silica and clay-silica complexes) than in soil substrates (clay soil and sandy soil). However, there were no significant differences in the retention of M. avium subsp. paratuberculosis between silica and clay-silica complexes or between clay soil and sandy soil. The proportion retained decreased with increasing pH in one of the experiments, indicating greater adsorption of M. avium subsp. paratuberculosis to soil particles at an acidic pH (P < 0.05). The results suggest that under experimental conditions M. avium subsp. paratuberculosis adsorbs to a range of soil particles, and this attachment is influenced by soil pH.Mycobacterium avium subsp. paratuberculosis is a pathogen of great significance for livestock since it causes a fatal and economically important disease called paratuberculosis or Johne''s disease (JD). The significance of M. avium subsp. paratuberculosis has further increased due to speculation over its role in the causation of Crohn''s disease in humans (10). Although reports trying to establish a causative association between M. avium subsp. paratuberculosis and Crohn''s disease are conflicting and inconclusive, they have aroused concerns among public health authorities (13); therefore, greater attention is now being paid to understand the natural ecology of M. avium subsp. paratuberculosis (32, 34). We investigated a largely unexplored aspect of the natural ecology of M. avium subsp. paratuberculosis: its attachment to soil particles, which could influence its availability to farm animals and humans (see below).Bacteria can become loosely associated with clay or soil particles through reversible adsorption mediated by electrostatic and van der Waals'' forces or by cell surface hydrophobicity (20). An irreversible firm attachment may later occur usually mediated by extracellular bridging polymers (8). The attachment of microbiota such as Escherichia coli, Arthrobacter spp., and poliovirus to soil or clay particles has been reported previously (2, 3, 11, 22, 26), but there is only indirect evidence of the association of mycobacteria with soil particles. A study reported the recovery of only 3.5% of nontuberculous mycobacteria inoculated into soil samples and attributed this to their adsorption to clay particles (5). Later, a similar phenomenon was inferred for M. avium subsp. paratuberculosis because 99% of these organisms in feces could not be detected upon culture of feces mixed with soil, suggesting the binding of M. avium subsp. paratuberculosis to soil particles (33). An association between M. avium subsp. paratuberculosis and clay particles was also suggested by an epidemiological study conducted to investigate the risk factors for ovine JD, indicating the possibility of bacterial attachment to clay particles (6).M. avium subsp. paratuberculosis is transmitted primarily by the feco-oral route. Infected animals shed huge numbers of M. avium subsp. paratuberculosis in their feces (29, 35), thus contaminating soil and the farm environment. The ability of M. avium subsp. paratuberculosis to survive for extended periods in an external environment, in spite of it being an obligate parasite (32, 34), facilitates the build-up of soil and pasture contamination levels over time. The attachment of M. avium subsp. paratuberculosis to soil particles could help retain the bacteria in the upper layers of the soil, thus further enhancing contamination levels. The contaminated farm environment thus becomes a potential source of infection for farm animals because grazing ruminants normally consume soil with pasture, and the amounts can be substantial, up to 300 or more grams per day for sheep (9, 21).In addition, runoff from contaminated farm soils can contaminate water bodies (23), which can be a potential health hazard for humans because the routine chlorine disinfection of water is not able to eliminate M. avium subsp. paratuberculosis completely (28). The transportation of bacteria from the farm environment to water sources is influenced by their attachment to soil or clay particles (11, 12). Generally, bacterial adsorption to soil particles decreases the rate of transportation through soil (3), but it also helps retain bacteria in the top surface layers of the soil, thus increasing the possibility of the contamination of runoff water (24). Note that soil particles can be dislodged and moved by wind, water, and mechanical factors.The aim of the present study was to verify whether M. avium subsp. paratuberculosis attaches to clay and other soil particles and whether this attachment is influenced by soil pH. The study findings improve our knowledge and understanding of the natural ecology of M. avium subsp. paratuberculosis.     

Role of the mitogen-activated protein kinase pathway in the differential response of bovine monocytes to Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium

We compared the kinetics of activation and antimicrobial activities of MAPK-p38 and MAPK-ERK in bovine monocytes infected with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. avium (Maa). Monocytes were incubated with MAP or Maa organisms with or without a specific inhibitor of the MAPK-p38 pathway (SB203580), and MAPK phosphorylation and antimicrobial functions of monocytes were evaluated. At early time points MAPK-p38 phosphorylation was greater in MAP-infected bovine monocytes than in Maa-infected monocytes. At later time points MAPK-p38 phosphorylation by both organisms was similar. MAPKp38 phosphorylation in MAP-infected monocytes was similar to negative control cells, whereas in Maa-infected this activation remained greater than negative control cells. Increase phosphorylation MAPK-ERK was similar at all time points for both organisms. Bovine monocytes had minimal capacity to kill MAP organisms, to acidify MAP-containing phagosomes, or to form phagolysosome. Alternatively, bovine monocytes were able to kill Maa organisms. Addition of SB203580 to monocyte cultures increased phagosome acidification, phagolysosome formation, and killing of MAP and Maa organisms. Taken together these data indicate that early transient activation of MAPK-p38 in bovine mononuclear phagocytes by MAP organisms may be a key mechanism involved in the capacity of MAP to survive in bovine monocytes.     

Effect of Turbulent-Flow Pasteurization on Survival of Mycobacterium avium subsp. paratuberculosis Added to Raw Milk

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooled D values (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolated D_72°C was <2.03 s. This was equivalent to a >7 log₁₀ kill at 72°C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log₁₀. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C.     

Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (or paratuberculosis). Paratuberculosis is a chronic gastroenteritis mainly affecting cattle, sheep and other ruminants. MAP is also of concern due to the heretofore unresolved issue of its possible role in Crohn's disease in humans. We present here a review of MAP (i) mobile genetic elements; (ii) repetitive elements; (iii) single nucleotide polymorphisms; and (iv) whole-genome comparisons to study the molecular epidemiology of MAP. A summary of the findings to date is presented, and the discriminatory power, advantage and disadvantages of each of the methods are compared and discussed.     

Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.     

Insertion and Deletion Events That Define the Pathogen Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium comprises genetically related yet phenotypically distinct subspecies. Consistent with their common origin, whole-genome sequence comparisons have revealed extensive synteny among M. avium organisms. However, the sequenced strains also display numerous regions of heterogeneity that likely contribute to the diversity of the individual subspecies. Starting from a phylogenetic framework derived by multilocus sequence analysis, we examined the distribution of 25 large sequence polymorphisms across a panel of genetically defined M. avium strains. This distribution was most variable among M. avium subsp. hominissuis isolates. In contrast, M. avium subsp. paratuberculosis strains exhibited a characteristic profile, with all isolates containing a set of genomic insertions absent from other M. avium strains. The emergence of the pathogen from its putative M. avium subsp. hominissuis ancestor entailed the acquisition of approximately 125 kb of novel genetic material, followed by a second phase, characterized by reductive genomics. One genomic deletion is common to all isolates while additional deletions distinguish two major lineages of M. avium subsp. paratuberculosis. For the average strain, these losses total at least 38 kb (sheep lineage) to 90 kb (cattle lineage). This biphasic pattern of evolution, characterized by chromosomal gene acquisition with subsequent gene loss, describes the emergence of M. avium subsp. paratuberculosis and may serve as a general model for the origin of pathogenic mycobacteria.Mycobacterium avium organisms are nontuberculous mycobacteria prevalent in environmental and clinical settings. M. avium infections result in diverse diseases, including avian tuberculosis, Johne''s disease, and Lady Windermere''s syndrome. Isolates are phenotypically different and were historically classified as separate species. However, current taxonomy, based on molecular analyses, recognizes a single species, M. avium, which is divided into distinct subgroups (21, 22).At present, M. avium subsp. hominissuis denotes environmental organisms associated with opportunistic infections in humans and swine (13, 23). M. avium subsp. avium is the classical agent of tuberculosis in birds and, along with M. avium subsp. silvaticum, represents a distinct lineage of bird pathogens (22). M. avium subsp. paratuberculosis causes Johne''s disease (Paratuberculosis), a chronic granulomatous intestinal disease (5). Although primarily associated with livestock, the bacterium may infect a wide range of mammalian hosts. A number of studies, using molecular testing for the M. avium subsp. paratuberculosis-specific insertion element IS900, have found an association between the presence of M. avium subsp. paratuberculosis and Crohn''s disease in humans (1, 9).Previous studies, including bigenomic comparisons of the sequenced strains M. avium subsp. hominissuis 104 and M. avium subsp. paratuberculosis K-10 (11), have revealed inter- and intrasubspecies differences (6, 12, 15, 16, 18, 19, 26). The phenotypic heterogeneity of M. avium strains may stem from genomic differences, but in the absence of a phylogenetic framework it has been difficult to define the key variations associated with the emergence of an individual subspecies. Recently, we proposed a phylogeny for M. avium based on multilocus sequence analysis (MLSA) of 10 genes and 56 M. avium isolates. This phylogeny is consistent with the current taxonomy and indicates that M. avium subsp. paratuberculosis is a distinct, clonal lineage of M. avium (22). To better understand the evolution of this subspecies, we have now examined the distribution of large sequence polymorphisms among a genetically defined panel of M. avium strains. Our findings reveal a characteristic genomic profile for M. avium subsp. paratuberculosis and provide insight into the biphasic evolution of this successful pathogen.