Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 × 10² CFU ml⁻¹ for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 × 10² CFU ml⁻¹) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 × 10³ and an association constant of 1.33 × 10⁹. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Contrasting Results of Culture-Dependent and Molecular Analyses of Mycobacterium avium subsp. paratuberculosis from Wood Bison

Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.     

Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 × 10¹ to 2 ×10⁶ copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M.avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.     

Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.     

Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

Relationship between Presence of Cows with Milk Positive for Mycobacterium avium subsp. paratuberculosis-Specific Antibody by Enzyme-Linked Immunosorbent Assay and Viable M. avium subsp. paratuberculosis in Dust in Cattle Barns

Paratuberculosis, or Johne''s disease, in cattle is caused by Mycobacterium avium subsp. paratuberculosis, which has recently been suspected to be transmitted through dust. This longitudinal study on eight commercial M. avium subsp. paratuberculosis-positive dairy farms studied the relationship between the number of cows with M. avium subsp. paratuberculosis antibody-positive milk and the presence of viable M. avium subsp. paratuberculosis in settled-dust samples, including their temporal relationship. Milk and dust samples were collected in parallel monthly for 2 years. M. avium subsp. paratuberculosis antibodies in milk were measured by enzyme-linked immunosorbent assay (ELISA) and used as a proxy for M. avium subsp. paratuberculosis shedding. Settled-dust samples were collected by using electrostatic dust collectors (EDCs) at six locations in housing for dairy cattle and young stock. The presence of viable M. avium subsp. paratuberculosis was identified by liquid culture and PCR. The results showed a positive relationship (odds ratio [OR], 1.2) between the number of cows with ELISA-positive milk and the odds of having positive EDCs in the same airspace as the adult dairy cattle. Moreover, the total number of lactating cows also showed an OR slightly above 1. This relationship remained the same for settled-dust samples collected up to 2 months before or after the time of milk sampling. The results suggest that removal of adult cows with milk positive for M. avium subsp. paratuberculosis-specific antibody by ELISA might result in a decrease in the presence of viable M. avium subsp. paratuberculosis in dust and therefore in the environment. However, this decrease is likely delayed by several weeks at least. In addition, the data support the notion that M. avium subsp. paratuberculosis exposure of young stock is reduced by separate housing.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.     

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg²⁺ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 10⁶ to 3 × 10¹ copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

Efficacy of Various Pasteurization Time-Temperature Conditions in Combination with Homogenization on Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effect of various pasteurization time-temperature conditions with and without homogenization on the viability of Mycobacterium avium subsp. paratuberculosis was investigated using a pilot-scale commercial high-temperature, short-time (HTST) pasteurizer and raw milk spiked with 10¹ to 10⁵ M. avium subsp. paratuberculosis cells/ml. Viable M. avium subsp. paratuberculosis was cultured from 27 (3.3%) of 816 pasteurized milk samples overall, 5 on Herrold's egg yolk medium and 22 by BACTEC culture. Therefore, in 96.7% of samples, M. avium subsp. paratuberculosis had been completely inactivated by HTST pasteurization, alone or in combination with homogenization. Heat treatments incorporating homogenization at 2,500 lb/in², applied upstream (as a separate process) or in hold (at the start of a holding section), resulted in significantly fewer culture-positive samples than pasteurization treatments without homogenization (P < 0.001 for those in hold and P < 0.05 for those upstream). Where colony counts were obtained, the number of surviving M. avium subsp. paratuberculosis cells was estimated to be 10 to 20 CFU/150 ml, and the reduction in numbers achieved by HTST pasteurization with or without homogenization was estimated to be 4.0 to 5.2 log₁₀. The impact of homogenization on clump size distribution in M. avium subsp. paratuberculosis broth suspensions was subsequently assessed using a Mastersizer X spectrometer. These experiments demonstrated that large clumps of M. avium subsp. paratuberculosis cells were reduced to single-cell or “miniclump” status by homogenization at 2,500 lb/in². Consequently, when HTST pasteurization was being applied to homogenized milk, the M. avium subsp. paratuberculosis cells would have been present as predominantly declumped cells, which may possibly explain the greater inactivation achieved by the combination of pasteurization and homogenization.     

Isolation of Mycobacterium avium subsp. paratuberculosis from Free-Ranging Birds and Mammals on Livestock Premises

Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants (“7,5”) appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock.     

Fate of Mycobacterium avium subsp. paratuberculosis in Swiss Hard and Semihard Cheese Manufactured from Raw Milk

Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 10⁴ to 10⁵ CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 10³ to 10⁴ cells of M. avium subsp. paratuberculosis per g will be inactivated.     

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.     

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.     

Discovery of Stable and Variable Differences in the Mycobacterium avium subsp. paratuberculosis Type I, II, and III Genomes by Pan-Genome Microarray Analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.     

Novel Feature of Mycobacterium avium subsp. paratuberculosis,Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.     

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne''s disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne''s disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.     

Detection of Mycobacterium avium subsp. paratuberculosis in Retail Cheeses from Greece and the Czech Republic

We investigated the presence of Mycobacterium avium subsp. paratuberculosis in retail cheeses from Greece and the Czech Republic. We found that 31.7% and 3.6% of our samples reacted positive by PCR and culture, respectively. Consumption of these cheeses is likely to result in human exposure to M. avium subsp. paratuberculosis, albeit at a low level for viable cells.