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.     

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity

Background

A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen.

Results

This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes.

Conclusions

Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1889-2) contains supplementary material, which is available to authorized users.     

Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.

Results

Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.

Conclusions

This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.     

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.     

Biofilm formation by Mycobacterium avium isolates originating from humans, swine and birds

Background  

Mycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.     

Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

Background  

Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects.     

Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis

Background

Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis.

Results

In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria.

Conclusions

Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1076) contains supplementary material, which is available to authorized users.     

Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms by Quantitative PCR

It has been suggested that Mycobacterium avium subspecies paratuberculosis has a role in Crohn''s disease. The organism may be acquired but is difficult to culture from the environment. We describe a quantitative PCR (qPCR) method to detect M. avium subsp. paratuberculosis in drinking water and the results of its application to drinking water and faucet biofilm samples collected in the United States.Mycobacterium avium subspecies paratuberculosis is a member of the Mycobacterium avium complex. M. avium subsp. paratuberculosis causes Johne''s disease in bovine and ovine animals and has been hypothetically linked to Crohn''s disease in humans. Several review articles have been written describing the association between M. avium subsp. paratuberculosis and Crohn''s disease (1, 2, 10, 11, 16, 23). Most mycobacterial infections are acquired from the environment; however, M. avium subsp. paratuberculosis can elude laboratory culture from environmental samples (28). M. avium subsp. paratuberculosis has been cultured only once from drinking water in the United States; therefore, its occurrence in drinking water is unknown (17). There are several reasons one could expect to find M. avium subsp. paratuberculosis in drinking water. The bacterium has been isolated from surface water used as a source of drinking water (19, 20, 24, 26). It is resistant to chlorine disinfection (25). Also, other subspecies of M. avium have been detected in biofilms obtained from drinking water pipes in the United States (8, 22, 27).Due to the potential for waterborne transmission of mycobacteria and the association of M. avium subsp. paratuberculosis with human illness, the focus of this study was to estimate the organism''s occurrence in drinking water in the United States using quantitative PCR (qPCR) (15). A comprehensive method was developed for detection of M. avium subsp. paratuberculosis in drinking water and biofilms that includes the concentration of microorganisms from samples using membrane filtration, total DNA extraction and purification, and detection of two targets unique to this bacterium: IS900 and target 251. IS900 is a common target used to identify M. avium subsp. paratuberculosis, and the average number of copies per genome is 14 to 18 (13). Target 251 qPCR analysis, which corresponds to the M. avium subsp. paratuberculosis gene 2765c (David Alexander, personal communication), was developed by Rajeev et al. (21). Samples positive for both targets are considered positive for M. avium subsp. paratuberculosis. TaqMan primer and probe sequences and qPCR assay characteristics are described in Table ​Table1.1. The complete method is described in Fig. S1 in the supplemental material.

TABLE 1.

qPCR assay primers, probes, DNA targets, and assay characteristics^a

Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 10³ to 10⁴ CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD₅₀) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.The prospect of being able to detect viable Mycobacterium avium subsp. paratuberculosis organisms in food or veterinary samples within 48 h using a commercially available phage amplification assay (FASTPlaqueTB assay; Biotec Laboratories Limited, Ipswich, United Kingdom), rather than waiting weeks for conventional culture results, is an exciting recent development (7, 8, 26). However, the mycobacteriophage used in the phage amplification assay has a broader mycobacterial host range than M. avium subsp. paratuberculosis alone (23). Consequently, plaques obtained when naturally infected, rather than artificially spiked, samples are tested may not necessarily emanate from M. avium subsp. paratuberculosis alone if other Mycobacterium spp. are also present in the sample. Some additional selective step prior to phage infection, such as magnetic separation (12), is needed to introduce selectivity for M. avium subsp. paratuberculosis.Magnetic separation (MS) has become a routine method in food and veterinary microbiology laboratories and is commonly used in combination with culture or molecular methods for the detection and isolation of pathogenic bacteria such as Listeria monocytogenes (13, 31), Salmonella spp. (22, 25), and Escherichia coli O157:H7 in both the food (15) and veterinary (20) clinical sample testing context. Magnetic-separation methods selectively separate the target bacterium from other, nontarget microorganisms and inhibitory sample components while concentrating the target bacterial cells into a smaller volume. Collectively, these properties of magnetic separation enhance the analytical specificity and sensitivity of the subsequent detection method, which can be culture, PCR, microscopy, an antigen detection immunoassay, or a phage assay. The latter is our proposed endpoint detection method. The combination of phage amplification and MS is not a new concept. Immunomagnetic (IMS)-phage assays for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 have been described previously (5, 6).The original IMS approach for M. avium subsp. paratuberculosis, employing a polyclonal anti-M. avium subsp. paratuberculosis antibody, was described by Grant et al. (9). This IMS approach showed good detection specificity for M. avium subsp. paratuberculosis as well as high detection sensitivity, because it was able to recover ≤10 CFU/ml directly from both spiked broth and milk. Its subsequent use in combination with IS900 PCR enhanced the speed of detection of M. avium subsp. paratuberculosis (10), and IMS-PCR was able to detect as few as 10³ CFU/50 ml, 1 to 2 log₁₀ units lower than the number detected by IS900 PCR applied directly to milk. However, our experience of using this and another polyclonal-antibody-based IMS method (Pathatrix PM-50 beads; Matrix Microscience, Newmarket, England) in conjunction with culture on Herrold''s egg yolk medium for the isolation of M. avium subsp. paratuberculosis from mixed-broth cultures from milk (unpublished data) and from raw-milk cheeses (27) has been that these polyclonal-antibody-based IMS methods lack sufficient specificity for M. avium subsp. paratuberculosis, and that consequently, nontarget bacteria, which bind nonspecifically to the beads, often overgrow this bacterium in culture. With other food-borne pathogens, an appropriate selective culture medium can be employed after IMS to prevent the outgrowth of any nontarget bacteria. Unfortunately, no truly selective culture medium exists for M. avium subsp. paratuberculosis at present, so specificity for this bacterium via magnetic separation must be achieved by optimizing the types of bead and capture ligands used.A monoclonal-antibody-based IMS method for M. avium subsp. paratuberculosis was reported by Metzger-Boddien et al. (17). Other groups have been attempting to produce monoclonal antibodies for application in IMS (3, 4). However, as an alternative to either polyclonal or monoclonal antibodies for the capture of M. avium subsp. paratuberculosis, new magnetic-separation approaches involving an M. avium subsp. paratuberculosis-specific peptide, aMp3 (30) or aMptD (28), have been described (i.e., peptide-mediated magnetic separation [PMS]). The first peptide (aMp3) was screened from nine recombinant bacteriophages specifically binding M. avium subsp. paratuberculosis that were produced using a commercially available phage-peptide display library (30). The second peptide, aMptD, was identified by biopanning of the M. avium subsp. paratuberculosis-specific ABC transponder operon (mpt) (29). The two chemically synthesized peptides, aMp3 and aMptD, were linked via carbodiimide to paramagnetic beads and were used in peptide-based capture PCR. Both PMS methods were reported to have high selectivity for M. avium subsp. paratuberculosis (i.e., no cross-reaction with other Mycobacterium spp.), and the analytical detection sensitivity, 5 ×10² CFU per ml (28), was comparable to the results previously reported by Grant et al. (10).As with other pathogenic bacteria that are likely to be present in raw milk, low numbers of viable M. avium subsp. paratuberculosis organisms are expected to be encountered in milk and dairy products (2, 11, 24). For other food-borne pathogens, such as Listeria monocytogenes (31), Salmonella spp. (22), and Escherichia coli O157:H7 (15), magnetic separation is generally applied after an enrichment culture step. This enrichment culture step aims to dilute food components known to be growth/PCR inhibitors, revive stressed or injured cells, and boost the numbers of the target bacterium (18, 21), so that magnetic separation and subsequent detection are likely to be more successful. Unfortunately, a prior enrichment culture step is impractical for M. avium subsp. paratuberculosis, since it would take too long, due to the slow-growing nature of this bacterium; thus, MS really needs to be applied directly to the sample. Consequently, any IMS or PMS method for M. avium subsp. paratuberculosis must achieve close to 100% capture efficiency, with minimal nonspecific binding by other mycobacteria, to limit false-negative or false-positive results. Capture efficiency is a measure of the completeness of capture of the original population of target cells present in the sample. Analytical specificity refers to the ability of an assay to measure one particular organism or substance, rather than others, in a sample (19). Therefore, the objectives of this study were (i) to identify the best magnetic-separation approach for the isolation of M. avium subsp. paratuberculosis from milk, in terms of capture efficiency and the percentage of nonspecific binding, by comparing as many paramagnetic-bead-coating antigen combinations as possible and (ii) to evaluate the potential use of the best magnetic-separation approach in conjunction with the previously optimized phage assay (7) as a novel IMS- or PMS-phage assay for the detection of M. avium subsp. paratuberculosis in milk.     

Difference in virulence of Mycobacterium avium isolates sharing indistinguishable DNA fingerprint determined in murine model of lung infection

Background

Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence.

Methods and Findings

In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild- type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner.

Conclusions/Significance

The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.     

The Thioamides Methimazole and Thiourea Inhibit Growth of M. avium Subspecies paratuberculosis in Culture

Background

Thyrotoxicosis is conceptualized as an “autoimmune” disease with no accepted infectious etiology. There are increasingly compelling data that another “autoimmune” affliction, Crohn disease, may be caused by Mycobacterium avium subspecies paratuberculosis (MAP). Like M. tb, MAP is systemic. We hypothesized that some cases of thyrotoxicosis may be initiated by a MAP infection. Because other thioamides treat tuberculosis, leprosy and M. avium complex, we hypothesized that a mode of action of some thioamide anti-thyrotoxicosis medications may include MAP growth inhibition.

Methods

The effect of the thioamides, thiourea, methimazole and 6-propo-2-thiouracil (6-PTU) were studied in radiometric Bactec® culture, on ten strains of three mycobacterial species (six of MAP, two of M. avium and two of M. tb. complex). Data are presented as “cumulative growth index,” (cGI) or “percent decrease in cumulative GI” (%-ΔcGI).

Principal Findings

Methimazole was the most effective thioamide at inhibiting MAP growth. At 128µg/ml: MAP UCF-4; 65%-ΔcGI & MAP ATCC 19698; 90%-ΔcGI. Thiourea inhibited MAP “Ben” maximally; 70%-ΔcGI. Neither methimazole nor thiourea inhibited M. avium or M. tb. at the doses tested. 6-PTU has no inhibition on any strain studied, although a structurally analogous control, 5-PTU, was the most inhibitory thioamide tested.

Significance

We show inhibition of MAP growth by the thioamides, thiourea and methimazole in culture. These data are compatible with the hypothesis that these thioamides may have anti-prokaryotic in addition to their well-established eukaryotic actions in thyrotoxic individuals.     

Rapid Assessment of the Viability of Mycobacterium avium subsp. paratuberculosis Cells after Heat Treatment,Using an Optimized Phage Amplification Assay

Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10⁶ to 10⁷ CFU/ml) and dispensed in 100-μl aliquots in thin-walled 200-μl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63°C for 3, 6, and 9 min; (ii) 68°C for 20, 40, and 60 s; and (iii) 72°C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold''s egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r² = 0.943) and heated (r² = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D_68°C, mean D_63°C, and D_72°C for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9°C. Complete inactivation of 10⁶ to 10⁷ CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log₁₀ reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.Due to the possible association of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne''s disease in cattle, with Crohn''s disease in humans, the consumption of milk and dairy products contaminated with this pathogenic bacterium has been suggested as a possible source of infection for humans (18). So far, the presence of viable M. avium subsp. paratuberculosis cells has been reported for pasteurized cows'' milk (6, 14, 23) and various cheeses (1, 4, 19). However, the rapid detection of viable M. avium subsp. paratuberculosis cells in food remains problematic. Culture is considered the gold standard method of demonstrating the viability of M. avium subsp. paratuberculosis cells, yet this approach is far from perfect and is not really appropriate for risk assessment purposes. First, M. avium subsp. paratuberculosis is a fastidious, slow-growing bacterium requiring a long incubation period before producing visible colonies (4 to 6 weeks minimum). Second, there is no selective growth medium for M. avium subsp. paratuberculosis, and chemical decontamination is required before plating samples on solid Herrold''s egg yolk medium (HEYM). This decontamination step, which aims to inactivate the competitive microflora, is often not totally effective, and cultures can be overgrown quickly by non-acid-fast bacteria during incubation. Third, the decontamination step has been demonstrated to have adverse effects on M. avium subsp. paratuberculosis viability (5). This extends the time required for primary isolation (to up to 20 weeks) and undoubtedly underestimates the number of cells originally present in the sample.Recently, we reported an optimization of the conditions of a commercially available phage amplification assay involving D29 mycobacteriophage (FASTPlaqueTB assay; Biotec Laboratories, Ipswich, United Kingdom) to permit accurate enumeration of M. avium subsp. paratuberculosis cells in milk (7). The main advantage of using phage amplification to detect M. avium subsp. paratuberculosis is that the number of viable cells can be estimated quickly, within 24 to 48 h, based on the count of plaques produced when D29-infected cells burst on a lawn of M. smegmatis indicator cells in an agar plate. Moreover, there is no need to carry out chemical decontamination of the sample before the phage assay because the D29 phage will infect only viable mycobacterial cells, and thus the detection sensitivity of the test is enhanced. For these reasons, the optimized phage amplification method may be used to speed up the acquisition of results during inactivation experiments involving samples artificially spiked with M. avium subsp. paratuberculosis.So far, the optimized phage amplification assay has been applied for the detection of viable M. avium subsp. paratuberculosis cells in spiked broth and milk samples. However, the performance of the test in assessing the viability of M. avium subsp. paratuberculosis cells subjected to physical or chemical treatments, which are likely to comprise mixtures of viable cells, injured/stressed cells, and dead cells, still needed to be investigated. For this reason, thermal inactivation experiments were carried out in order to assess the utility of this optimized phage assay for use instead of conventional culture for research involving artificially spiked milk samples. The main objectives of this study were to evaluate the correlation between colony and plaque counts for heat-treated M. avium subsp. paratuberculosis and to demonstrate a quicker acquisition of accurate results than that obtainable by culture.     

Isolation of Mycobacterium avium Subspecies paratuberculosis Reactive CD4 T Cells from Intestinal Biopsies of Crohn's Disease Patients

Background

Crohn''s disease (CD) is a chronic granulomatous inflammation of the intestine. The etiology is unknown, but an excessive immune response to bacteria in genetically susceptible individuals is probably involved. The response is characterized by a strong Th1/Th17 response, but the relative importance of the various bacteria is not known.

Methodology/Principal Findings

In an attempt to address this issue, we made T-cell lines from intestinal biopsies of patients with CD (n = 11), ulcerative colitis (UC) (n = 13) and controls (n = 10). The T-cell lines were tested for responses to various bacteria. A majority of the CD patients with active disease had a dominant response to Mycobacterium avium subspecies paratuberculosis (MAP). The T cells from CD patients also showed higher proliferation in response to MAP compared to UC patients (p<0.025). MAP reactive CD4 T-cell clones (n = 28) were isolated from four CD patients. The T-cell clones produced IL-17 and/or IFN-γ, while minimal amounts of IL-4 were detected. To further characterize the specificity, the responses to antigen preparations from different mycobacterial species were tested. One T-cell clone responded only to MAP and the very closely related M. avium subspecies avium (MAA) while another responded to MAP, MAA and Mycobacterium intracellulare. A more broadly reactive T-cell clone reacted to MAP1508 which belongs to the esx protein family.

Conclusions/Significance

The presence of MAP reactive T cells with a Th1 or Th1/Th17 phenotype may suggest a possible role of mycobacteria in the inflammation seen in CD. The isolation of intestinal T cells followed by characterization of their specificity is a valuable tool to study the relative importance of different bacteria in CD.     

Resequencing the Mycobacterium avium subsp. paratuberculosis K10 Genome: Improved Annotation and Revised Genome Sequence

We report the resequencing and revised annotation of the Mycobacterium avium subsp. paratuberculosis K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.The American bovine isolate K10 remains the only Mycobacterium avium subsp. paratuberculosis genome to be fully sequenced and published to date (1). Although this 4.8-Mbp genome likely contains some assembly errors (3), it has provided, and will continue to provide, an invaluable resource for Mycobacterium research. The assembly errors were identified through optical mapping of related M. avium subsp. paratuberculosis strain ATCC 19698, which revealed a 648-kb inversion around the origin of replication and two additional copies of the insertion sequences IS1311 and IS_MAP03 (3). These findings were subsequently validated via PCR, Southern blotting, and (Sanger) sequence analysis in ATCC 19698 and were also confirmed to be present in K10 (3). We designate this interim corrected genome M. avium subsp. paratuberculosis K10′. To further improve this resource, we undertook a resequencing project of the original M. avium subsp. paratuberculosis K10 genome.Whole-genome sequencing was performed on the Illumina GAIIx platform using one flow cell lane with 36-cycle paired-end chemistry. Reads were variably trimmed at the 3′ end based on the Illumina Read Segment Quality Indicator (Illumina manual), and read pairs containing ambiguous bases were removed. Read mapping onto the K10′ genome sequence was performed using SHRiMP (ver. 1.3.2) (2), and single-nucleotide polymorphisms and indels (deletion and insertion polymorphisms [DIPs]) were called using Nesoni (ver. 0.29; Monash University Victorian Bioinformatics Consortium) with default parameters. Read mapping determined that the data set comprised an average sequence coverage of 72.6 across the K10′ genome. This high sequence coverage allowed differences between K10\K10′ and the resequenced version of the genome, designated K10", to be identified with high confidence.Ninety single-nucleotide differences and one 51-bp indel were identified in the K10" genome. As confirmation that these differences are likely to represent errors in the original genome sequence, we have also detected these polymorphisms in two additional bovine M. avium subsp. paratuberculosis genomes recently sequenced and assembled within our laboratory (data not shown). Seven of the 90 differences and the 51-bp indel were subjected to PCR and Sanger sequencing for verification. All of the polymorphisms were confirmed to be present in K10" compared to the original genome sequence.Thirty-six single-nucleotide deletions and four nucleotide insertions were identified in K10" compared to the reference. These DIPs resulted in 27 frameshift mutations of protein coding loci. As a consequence of these frameshifts, one complete coding sequence (CDS) feature was removed (MAPK_3751), one novel CDS was created (MAPK_2081b), and one pseudogene was repaired (MAPK_4158-4159). In almost all of the other cases, the frameshifts resulted in proteins which more closely resembled their orthologs in M. avium subsp. hominissuis and M. intracellulare. Other frameshifts of biological interest include the truncation of a PPE family protein (MAPK_1173) and the extension of an MCE (mammalian cell entry) family protein (MAPK_4086). Compared to the reference, K10" also had a 51-bp indel within a possible MCE family protein (MAPK_1575). This indel consisted of an 11-bp deletion (bases 2436510 to 2436520 in the original K10 sequence) and an insertion of 51 bp. The resulting protein sequence now more closely resembles orthologs of the MCE family in other Mycobacterium spp. In conclusion, the fact that so many of the amended bases have resulted in revised coding regions indicates the underlying importance of this exercise.     

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.

Results

Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.

Conclusions

The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.     

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.     

Presence of Mycobacterium avium subsp. paratuberculosis in Environmental Samples Collected on Commercial Dutch Dairy Farms

Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne''s disease in cattle, was identified in settled-dust samples of Dutch commercial dairy farms, both in the dairy barn and in the young stock housing. Bioaerosols may play a role in within-farm M. avium subsp. paratuberculosis transmission.Paratuberculosis is an infectious enteric disease caused by Mycobacterium avium subsp. paratuberculosis leading to economic losses in dairy cattle globally (2, 10). The main transmission route is the fecal/oral route from infectious adult cattle to susceptible calves (12).Preventive calf management was a key point in model studies (7), but 20-year implementation did not lead to farm-level eradication, suggesting uncontrolled routes of transmission (1, 7).Environmental samples were used to classify commercial dairy herds (3, 9, 11), based on long-term survival of M. avium subsp. paratuberculosis in the environment (16). Recently, bioaerosols containing viable M. avium subsp. paratuberculosis were identified in an experimental setting with 100% M. avium subsp. paratuberculosis prevalence (6) and may thus be a mode of transmission. Dust containing M. avium subsp. paratuberculosis might be ingested or inhaled by calves (4). Experimental M. avium subsp. paratuberculosis challenge studies in sheep successfully used inhalation (8). These transmission routes could hamper current control programs. Our objective was to study whether M. avium subsp. paratuberculosis could be detected in bioaerosols on commercial Dutch dairy farms.Dairy herds in three Dutch veterinary practices were sampled in 2009. All farms participated in a Dutch M. avium subsp. paratuberculosis monitoring program in 2008, either the Dutch Paratuberculosis Program (PPN; n = 2) or the Dutch Bulk Milk Quality Assurance Program (BMQAP; n = 22) (15). Both PPN herds were certified M. avium subsp. paratuberculosis-free. Herds corresponding to the BMQAP had at least one positive animal identified by enzyme-linked immunosorbent assay (ELISA) (Pourquier ELISA; Institut Pourquier, France). Farms were grouped into three M. avium subsp. paratuberculosis test prevalence levels (control, zero positive animals; group A, one positive animal; group B, two or more positive animals; Table ​Table11).

TABLE 1.

Overview of the results of the questionnaire about relevant M. avium subsp. paratuberculosis management practices^a

Association of ISMav6 with the Pattern of Antibiotic Resistance in Korean Mycobacterium avium Clinical Isolates but No Relevance between Their Genotypes and Clinical Features

The aim of this study was to genetically characterize clinical isolates from patients diagnosed with Mycobacterium avium lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of M. avium isolates from 92 Korean patients revealed that all isolates were M. avium subspecies hominissuis. In hsp65 sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS1311 positive and IS900 negative. Four of the M. avium subsp. hominissuis isolates did not harbor IS1245 and 1 of the M. avium isolates intriguingly harbored DT1, which is thought to be a M. intracellulare-specific element. M. avium subsp. hominissuis harboring ISMav6 is prevalent in Korea. No significant association between clinical manifestation and treatment response has been found in patients with the hsp65 code type and ISMav6, indicating that no specific strain/genotype among M. avium subsp. hominissuis organisms was a major source of M. avium lung disease. Interestingly, the presence of ISMav6 was correlated with greater resistance to moxifloxacin. Conclusively, the genotype of Korean M. avium subsp. hominissuis isolates is not a disease determinant responsible for lung disease and specific virulent factors of M. avium subsp. hominissuis need to be investigated further.