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.     

Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp. avium infections in a tule elk (Cervus elaphus nannodes) herd

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P) > or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P > or = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.     

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)     

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.     

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.     

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.     

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.     

Experimental infection of white-tailed deer (Odocoileus virginianus) with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis or Johne's disease, a chronic enteric disease of domestic ruminants as well as some nondomestic ruminants. Paratuberculosis is characterized by a protracted subclinical phase followed by clinical signs such as diarrhea, weight loss, and hypoproteinemia. Fecal shedding of Map is characteristic of both the subclinical and clinical phases, and it is important in disease transmission. Lesions of paratuberculosis are characterized by chronic granulomatous enteritis and mesenteric lymphadenitis. Animal models of paratuberculosis that simulate all aspects of the disease are rare. Oral inoculation of 9-day-old white-tailed deer (Odocoileus virginianus) on 3 June 2002 with 1.87 x 10(10) colony-forming units of Map strain K10 resulted in clinical disease (soft to diarrheic feces) as early as 146 days after inoculation; lesions consistent with paratuberculosis were observed in animals at the termination of the study. Intermittent fecal shedding of Map was seen between 28 and 595 days (4 March 2004) after inoculation. These findings suggest that experimental oral inoculation of white-tailed deer fawns may mimic all aspects of subclinical and clinical paratuberculosis.     

Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium are independently evolved pathogenic clones of a much broader group of M. avium organisms

Mycobacterium avium comprises organisms that share the same species designation despite considerable genomic and phenotypic variability. To determine the degree and nature of variability between subspecies and strains of M. avium, we used multilocus sequencing analysis, studying 56 genetically diverse strains of M. avium that included all described subspecies. In total, 8,064 bp of sequence from 10 gene loci were studied, with 205 (2.5%) representing variable positions. The majority (149/205) of these variations were found among M. avium subsp. hominissuis organisms. Recombination was also evident in this subspecies. In contrast, there was comparatively little variability and no evidence of recombination within the pathogenic subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. silvaticum. Phylogenetic analysis showed that M. avium subsp. avium and M. avium subsp. silvaticum strains clustered together on one branch, while a distinct branch defined M. avium subsp. paratuberculosis organisms. Despite the independent origin of these pathogenic subspecies, an analysis of their rates of nonsynonymous (dN) to synonymous (dS) substitutions showed increased dN/dS ratios for both: 0.67 for M. avium subsp. paratuberculosis and 0.50 for M. avium subsp. avium/M. avium subsp. silvaticum, while the value was 0.08 for M. avium subsp. hominissuis organisms. In conclusion, M. avium subsp. hominissuis represents a diverse group of organisms from which two pathogenic clones (M. avium subsp. paratuberculosis and M. avium subsp. avium/M. avium subsp. silvaticum) have evolved independently.     

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.     

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.     

The transport of Mycobacterium avium subsp. paratuberculosis through saturated aquifer materials

Aims:  To investigate the processes controlling the transport of Mycobacterium avium subsp. paratuberculosis ( Map ) through aquifer materials.
Methods and Results:  We measured two important surface characteristics known to affect bacterial attachment to sediment surfaces: surface charge and hydrophobicity. We then measured the transport of Map through laboratory columns packed with aquifer sand with varying ionic strength solutions and sediment surface charge. We found that Map has a strong negative charge and is highly hydrophobic and that the transport of Map through positively charged Fe-coated sands was reduced compared with transport through negatively charged clean quartz sand, although Map transport for all treatments was low compared with the transport behaviour reported in the literature for other bacteria.
Conclusions:  Our results suggest that the potential for groundwater contamination by Map is low; however, the organism may remain bound to the soil near the surface where it can be ingested by grazing animals or be released during run off events.
Significance and Impact of the Study:  This is the first study looking at the surface characteristics and transport behaviour of Map through aquifer materials and therefore provides important information for understanding the movement of Map in the environment.     

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.     

The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease

The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn’s disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn’s disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn’s disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC. We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC.