How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection?

Mycobacteria have thwarted detection by scientists for centuries. Mycobacterium paratuberculosis is one of the most fastidious of the Mycobacteriaceae, and has been implicated in both animal and human diseases. In domestic livestock, M. paratuberculosis has been associated with Johne's disease, which given its increasing incidence, is currently a cause for concern, due to the potential for M. paratuberculosis to enter our food chain. In addition, a tenuous link has been reported between M. paratuberculosis and Crohn's disease, however evidence to support this link is hampered by the lack of accurate methodologies for detection of M. paratuberculosis in humans. This review compares the sensitivity and specificity of traditional and more recent techniques to the culture and molecular detection of M. paratuberculosis. While serology and culture are popular choices for the livestock industry they have not produced useful data for human infection. Although the advent of molecular biology has enabled faster diagnosis of M. paratuberculosis in human infection, there is currently no gold standard such as culture on which to validate these findings. Even with DNA/RNA detection methods, there is the ever present issue of the genetic relatedness of M. paratuberculosis to other mycobacteria of the Mycobacterium avium complex, some of which also infect humans with very different pathological outcomes. Recent developments in this field include more rapid methods of M. paratuberculosis culture as well as the development of more accurate and sensitive PCR assays. The application of these techniques should offer a greater insight as to the role of M. paratuberculosis in human gastrointestinal diseases.     

European hares in Chile: a different lagomorph reservoir for Mycobacterium avium subsp. paratuberculosis?

Ruminants are the principal host for infection by Mycobacterium avium subsp. paratuberculosis (Map), the cause of Johne's disease. Based on studies of a Map-infected population of European rabbits (Oryctolagus cuniculus) in Scotland, lagomorphs as a broad taxonomic order were proposed as potential nonruminant reservoirs for Map. To determine whether a different lagomorph species may serve as a wildlife reservoir, we investigated Map infection in European hares (Lepus europaeus) sharing habitat with known Map-infected dairy cattle in southern Chile. Fecal, mesenteric lymph node, and ileal samples were aseptically collected from 385 wild hares for liquid culture and real-time polymerase chain reaction identification of acid-fast isolates. All tissue samples were also acid-fast stained and examined microscopically. We isolated Map from at least one tissue from 48 hares (12.6%) and fecal samples from 16 hares (4.2%). No Map was found in tissues of eight of the fecal-culture-positive hares. Histologically, all tissues from all hares were within normal limits, and no acid-fast organisms were observed in any sample. Active infection, implying amplification of the organism secondary to resultant disease, was not evident. With this report Map isolations on a population versus incidental detection have now been made from two lagomorph species. However, although the rabbit population studied in Scotland appears to function as a Map reservoir, the hares studied in Chile appear to be a dead-end host, serving only as potential mechanical vectors for the organism.     

Clustering of Mycobacterium avium subsp. paratuberculosis in Rabbits and the Environment: How Hot Is a Hot Spot?

Clustering of pathogens in the environment leads to hot spots of diseases at local, regional, national, and international levels. Scotland contains regional hot spots of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis) in rabbits, and there is increasing evidence of a link between paratuberculosis infections in rabbits and cattle. The spatial and temporal dynamics of paratuberculosis in rabbits within a hot spot region were studied with the overall aim of determining environmental patterns of infection and thus the risk of interspecies transmission to livestock. The specific aims were to determine if prevalence of paratuberculosis in rabbits varies temporally between seasons and whether the heterogeneous spatial environmental distribution of M. avium subsp. paratuberculosis on a large scale (i.e., regional hot spots) is replicated at finer resolutions within a hot spot. The overall prevalence of M. avium subsp. paratuberculosis in rabbits was 39.7%; the temporal distribution of infection in rabbits followed a cyclical pattern, with a peak in spring of 55.4% and a low in summer of 19.4%. Spatially, M. avium subsp. paratuberculosis-infected rabbits and, thus, the risk of interspecies transmission were highly clustered in the environment. However, this is mostly due to the clustered distribution of rabbits. The patterns of M. avium subsp. paratuberculosis infection in rabbits are discussed in relation to the host's socioecology and risk to livestock.     

Clustering of Mycobacterium avium subsp. paratuberculosis in rabbits and the environment: how hot is a hot spot?

Clustering of pathogens in the environment leads to hot spots of diseases at local, regional, national, and international levels. Scotland contains regional hot spots of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis) in rabbits, and there is increasing evidence of a link between paratuberculosis infections in rabbits and cattle. The spatial and temporal dynamics of paratuberculosis in rabbits within a hot spot region were studied with the overall aim of determining environmental patterns of infection and thus the risk of interspecies transmission to livestock. The specific aims were to determine if prevalence of paratuberculosis in rabbits varies temporally between seasons and whether the heterogeneous spatial environmental distribution of M. avium subsp. paratuberculosis on a large scale (i.e., regional hot spots) is replicated at finer resolutions within a hot spot. The overall prevalence of M. avium subsp. paratuberculosis in rabbits was 39.7%; the temporal distribution of infection in rabbits followed a cyclical pattern, with a peak in spring of 55.4% and a low in summer of 19.4%. Spatially, M. avium subsp. paratuberculosis-infected rabbits and, thus, the risk of interspecies transmission were highly clustered in the environment. However, this is mostly due to the clustered distribution of rabbits. The patterns of M. avium subsp. paratuberculosis infection in rabbits are discussed in relation to the host's socioecology and risk to livestock.     

Improved Short-Sequence-Repeat Genotyping of Mycobacterium avium subsp. paratuberculosis by Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Accurate sequence analysis of mononucleotide repeat regions is difficult, complicating the use of short sequence repeats (SSRs) as a tool for bacterial strain discrimination. Although multiple SSR loci in the genome of Mycobacterium avium subsp. paratuberculosis allow genotyping of M. avium subsp. paratuberculosis isolates with high discriminatory power, further characterization of the most discriminatory loci is limited due to inherent difficulties in sequencing mononucleotide repeats. Here, a method was evaluated using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as an alternative to Sanger sequencing to further differentiate the dominant mycobacterial interspersed repetitive-unit (MIRU)–variable-number tandem-repeat (VNTR) M. avium subsp. paratuberculosis type (n = 37) in Canadian dairy herds by targeting a highly discriminatory mononucleotide SSR locus. First, PCR-amplified DNA was digested with two restriction enzymes to yield a sufficiently small fragment containing the SSR locus. Second, MALDI-TOF MS was performed to identify the mass, and thus repeat length, of the target. Sufficiently intense, discriminating spectra were obtained to determine repeat lengths up to 15, an improvement over the limit of 11 using traditional sequencing techniques. Comparison to synthetic oligonucleotides and Sanger sequencing results confirmed a valid and reproducible assay that increased discrimination of the dominant M. avium subsp. paratuberculosis MIRU-VNTR type. Thus, MALDI-TOF MS was a reliable, fast, and automatable technique to accurately resolve M. avium subsp. paratuberculosis genotypes based on SSRs.     

How does a Mycobacterium change its spots? Applying molecular tools to track diverse strains of Mycobacterium avium subspecies paratuberculosis

Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne's disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed‐field gel electrophoresis, single sequence repeats, variable‐number tandem repeats, genome rearrangements, single nucleotide polymorphisms and genomewide comparisons to identify insertions and deletions. Combinations of these methods can now provide discrimination sufficient for dependable strain tracking. These molecular epidemiology techniques are being applied to understand transmission of Johne's disease within dairy cattle herds as well as identify which strains predominate in wildlife.     

Can Immune Response Mechanisms Explain the Fecal Shedding Patterns of Cattle Infected with Mycobacterium avium Subspecies paratuberculosis?

Johne’s disease (JD) is a chronic disease in ruminants and is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). At late stages of the disease, MAP bacilli are shed via feces excretion and in turn create the potential for oral-fecal transmission. The role of the host immune response in MAP bacteria shedding patterns at different stages of JD is still unclear. We employed mathematical modeling to predict if the variation in MAP shedding could be correlated to the immune response in infected animals. We used a novel inverse modeling approach that assumed biological interactions among the antigen-specific lymphocyte proliferation response (cell-mediated response), antibody/humoral immune responses, and MAP bacteria. The modeling framework was used to predict and test possible biological interactions between the measured variables and returns only the essential interactions that are relevant in explaining the observed cattle MAP experimental infection data. Through confronting the models with data, we predicted observed effects (enhancement or suppression) and extents of interactions among the three variables. This analysis enabled classification of the infected cattle into three different groups that correspond to the unique predicted immune responses that are essential to explain the data from cattle within these groups. Our analysis highlights the strong and weak points of the modeling approach, as well as the key immune mechanisms predicted to be expressed in all animals and those that were different between animals, hence giving insight into how animals exhibit different disease dynamics and bacteria shedding patterns.     

On the evolution of ‘Indian Bison type’ strains of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease (JD) in animals, has also been linked with Crohn's disease in human beings. Lack of indigenous diagnostics and vaccine hampered control of JD in India. Designing effective control strategies require thorough understanding of the etiological agent at phenotypic and molecular levels. On the basis of cultural phenotypes and IS1311 PCR-REA typing, MAP strains have been genotyped as ‘Cattle type’, ‘Sheep type’ and ‘Bison type’. Information exists on genetic differences and comparative evolution of ‘Cattle type’ and ‘Sheep type’ strains after divergence from M. avium; however, emphasis has been little on ‘Bison type’ strains. Recently, a new ‘Indian Bison type’ genotype has been reported as principal strain infecting different animal species and human beings in India. The study analyzed few genetic markers to have inferences on the molecular evolution of native MAP isolates belonging to ‘Bison type’ genotype. Results pointed towards recent evolution of ‘Bison type’ genotype.     

Antibodies recognizing specific Mycobacterium avium subsp. paratuberculosis’s MAP3738c protein in type 1 diabetes mellitus children are associated with serum Th1 (CXCL10) chemokine

Recently Mycobacterium avium subsp. paratuberculosis (Map) was associated to type 1 diabetes mellitus (T1DM). In this study we investigated for Map presence in children affected by T1DM compared to healthy children. A pool of 212 sera from T1DM children at onset was compared to sera from 57 healthy children for humoral immune response towards the Map specific protein MAP3738c by ELISA. Serum concentrations of CXCL10 (pro-Th1) and CCL2 (pro-Th2) chemokines were also measured in both sera pool. Results showed that T1DM children had a stronger seropositivity towards MAP3738c protein compared to healthy children. Data highlighted also the correlation between serum activity of T1DM patients towards the specific protein of Map and the increase of CXCL10 concentration if compared to non-diabetic subjects. In conclusion, an immune response to Map in T1DM patients at onset was observed and this may indicate a role of the bacterium in triggering or precipitating the disease.     

Mycobacterium avium–intracellulare contamination of mammalian cell cultures

Mycobacterium avium contamination has been described as a putative contaminant of nonphagocytic mammalian cells. Screening of numerous cultured nonphagocytic mammalian cell lines revealed the presence of intracellular bacteria that were identified as M. avium-intracellulare. An extensive and critical analysis of the origin of infection, of cure protocols, and of biological manifestations in M. avium-infected cells is presented. As no tremendous visible alteration of turbidity or pH of cell culture media, and no morphological change occurred in most M. avium-infected cell cultures, detection of an infection by these bacteria is rather difficult. Recommendations are given for treatment of irreplaceable cultures and prevention of mycobacterial contamination in a tissue culture facility.     

What Is a Story?

This paper attempts to discover the criteria by which a listener accepts or rejects an item of oral narrative as being a story. The hypotheses are: (1) such criteria relate to structure rather than content, although the listener does not consciously distinguish structure from content; (2) the structure must have a certain minimal and maximal degree of complexity and be of a certain kind; (3) the criteria will hold cross-culturally. Information gathered from ethnographies and folklore literature is inadequate to confirm the hypotheses, but it does not contradict them. An experiment to test hypotheses (1) and (2) produced a certain degree of negative confirmation: listeners rejected as a story any narrative item which did not conform to the structural criteria regarded as minimal by myself; however, they also rejected items which I regarded as well structured but which were bizarre or nonsensical in content. The confusion of structure and content tended to confirm hypothesis (1).     

What Is Phloem Unloading?

Several studies of phloem unloading have failed to distinguish between transport events occurring at the sieve element/companion cell boundary and subsequent short-distance transport through parenchyma cells. Indirect evidence has been obtained for symplastic unloading in storage and utilization sinks. In other sinks transfer to the apoplast may occur, but not necessarily at the sieve element/companion cell complex, and the evidence for apoplastic phloem unloading is equivocal, as is the role of apoplastic acid invertase in this process. The ability of several types of sink cells to accumulate sugars from the apoplast is discussed in the conflicting light of functional symplastic continuity between sink cells. Attention is drawn to the complexity of the postunloading pathway in many sinks and the difficulty of determining the exact sites of symplast/apoplast solute exchange. Potential future areas for study in the field are highlighted.     

Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective?

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.