A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 ± 36.8 min at 37°C compared to 63 ± 17.5 min for M. smegmatis mc2 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37°C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc2 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.
Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7×) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.
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.
The etiological agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. Controlling the spread of this disease is hindered by the lack of sensitive, selective, and rapid detection methods for M. avium subsp. paratuberculosis. By using a recently optimized sandwich immunoassay (B. J. Yakes, R. J. Lipert, J. P. Bannantine, and M. D. Porter, Clin. Vaccine Immunol. 15:227-234, 2008), which incorporates a new monoclonal antibody for the selective capture and labeling of M. avium subsp. paratuberculosis and surface-enhanced Raman scattering for sensitive readout, detection limits of ∼630 and ∼740 M. avium subsp. paratuberculosis cells/ml are achieved in phosphate-buffered saline and whole milk samples, respectively, after spiking with heat-treated M. avium subsp. paratuberculosis. Surprisingly, these detection limits are 3 orders of magnitude lower than expected based on theoretical predictions. Experiments designed to determine the origin of the improvement revealed that the major membrane protein targeted by the monoclonal antibody was present in the sample suspensions as shed protein. This finding indicates that the capture and labeling of shed protein function as a facile amplification strategy for lowering the limit of detection for M. avium subsp. paratuberculosis that may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.
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.
Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSPAs) and those only present in the latter (LSPPs). We examined the distribution of 3 LSPAs and 17 LSPPs across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSPA8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSPPs, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.
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 D68°C, mean D63°C, and D72°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.
Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.
In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.
We have developed and optimized a 96-well microtiter plate assay, based on the reduction of alamarBlue, to assess the efficacies of much needed new antimicrobials against Acanthamoeba species. This assay has been optimized for determination of drug efficacy against two potentially pathogenic species, Acanthamoeba castellanii and Acanthamoeba polyphaga, and has been validated by comparison of their relative susceptibilities to chlorhexidine, a drug widely used to treat Acanthamoeba keratitis. The results demonstrate that the assay is comparable to a manual counting assay and that A. polyphaga is more resistant to chlorhexidine than A. castellanii. Thus, by use of the manual counting assay, 3.125 μM chlorohexidine was almost completely effective against A. castellanii, whereas this concentration was less than 20% effective against A. polyphaga. Similar results were obtained by the alamarBlue assay. The new assay was used to determine the relative susceptibilities of A. castellanii and A. polyphaga to the alkylphosphocholines (APCs) hexadecylphosphocholine (hexadecyl-PC; miltefosine) and octadecylphosphocholine (octadecyl-PC) as well as an alkylgycerolphosphocholine, edelfosine. Both APCs studied were equally effective against A. castellanii, but octadecyl-PC was less effective than hexadecyl-PC against A. polyphaga. Both APCs were more effective than edelfosine against both Acanthamoeba species. A. polyphaga was found to be significantly less susceptible to each of the phosphocholine analogues. The newly described assay offers a number of advantages over those described previously. It is less labor-intensive than previously described assays and is sensitive and rapid, and the results can be read in a nonsubjective manner. As it is based on a standard 96-well, microtiter plate, it is amenable to automation and high throughput.
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.
Sera from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (n = 56) and naturally (n = 4) and experimentally (n = 8) infected with Mycobacterium bovis were tested for the presence of antibodies against paratuberculosis antigens. An enzyme-linked immunosorbent assay (ELISA) was established based on absorption of M. avium subsp. paratuberculosis antigens on a hyperimmune antiserum against M. avium subsp. avium proteins in order to remove cross-reacting antigens. This absorbed-antigen ELISA recognized 66% of animals with paratuberculosis (37 of 56), while none of the animals with naturally occurring bovine tuberculosis (TB) had detectable antibodies. However, the animals with experimental bovine TB also responded in this ELISA. Similar results were found in a commercial ELISA, showing that neither of these tests was able to distinguish between paratuberculosis and bovine TB. The sera were further tested for antibody activities against purified AhpC and AhpD, which are proteins constitutively expressed by M. avium subsp. paratuberculosis, and against a secreted 14-kDa protein present in culture filtrates from the M. avium complex. Elevated antibody levels to AhpC, AhpD, and the 14-kDa antigen were found in 27% (13 of 48), 15% (7 of 48), and 27% (13 of 48), respectively, of the cattle with paratuberculosis. Together these ELISAs were positive with 35% (17 of 48) of the animals. None of the animals with bovine TB had detectable antibodies against any of the purified proteins despite their high levels of cross-reacting antibodies. These results show that purified specific antigens are needed to differentiate between paratuberculosis and bovine TB in ELISA.
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.
Specific antibodies, available in unlimited quantities, have not been produced against Mycobacterium avium subsp. paratuberculosis, the bacterium that causes Johne's disease (JD). To fill this gap in JD research, monoclonal antibodies (MAbs) against M. avium subsp. paratuberculosis were produced from BALB/c mice immunized with a whole-cell extract of M. avium subsp. paratuberculosis. A total of 10 hybridomas producing MAbs to proteins ranging from 25 to 85 kDa were obtained. All MAbs showed some degree of cross-reactivity when they were analyzed against a panel of whole-cell protein lysates comprising seven different mycobacterial species. The MAbs were characterized by several methods, which included isotype analysis, specificity analysis, epitope analysis, reactivity in immunoblot assays, and electron microscopy. The identities of the antigens that bound to two selected MAbs were determined by screening an M. avium subsp. paratuberculosis lambda phage expression library. This approach revealed that MAb 9G10 detects MAP1643 (isocitrate lyase) and that MAb 11G4 detects MAP3840 (a 70-kDa heat shock protein), two proteins present in high relative abundance in M. avium subsp. paratuberculosis. The epitopes for MAb 11G4 were mapped to the N-terminal half of MAP3840, whereas MAb 9G10 bound to the C-terminal half of MAP1643. Aptamers, nucleic acids that bind to specific protein sequences, against the hypothetical protein encoded by MAP0105c were also generated and tested for their binding to M. avium subsp. paratuberculosis as well as other mycobacteria. These detection reagents may be beneficial in many JD research applications.
This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102 CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 102 CFU/g for the IS900 element and 103 CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.
Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6.3%) (P < 0.05). Positive IS900 PCR results occurred more often in U.S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U.S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-γ) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-γ tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.
A sandwich immunoassay for the rapid, low-level detection of Mycobacterium avium subsp. paratuberculosis has been developed. M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle, and one of the major obstacles in controlling the spread of this disease is the inability to rapidly detect small amounts of bacteria or other diagnostic markers shed during the subclinical stage of infection. This paper details the development and performance of an assay for sonicated M. avium subsp. paratuberculosis lysate that is based on surface-enhanced Raman scattering (SERS). There are two key components of the assay: (i) an immobilized layer of monoclonal antibodies that target a surface protein on the microorganism; and (ii) extrinsic Raman labels (ERLs) that are designed to selectively bind to captured proteins and produce large SERS signals. By correlating the number of M. avium subsp. paratuberculosis bacilli present prior to sonication to the amount of total protein in the resulting sonicate, the detection limit determined for total protein can be translated to the microorganism concentration. These findings yield detection limits of 100 and 200 ng/ml (estimated to be 500 and 1,000 M. avium subsp. paratuberculosis bacilli/ml) for sonicate spiked in phosphate buffer and sonicate spiked in whole milk, respectively. Moreover, the time required to complete the assay, which includes sample preparation, antigen extraction, ERL incubation, and readout, is less than 24 h. The potential for incorporation of this novel assay into diagnostic laboratories is also briefly discussed.
Nisin A is the most widely characterized lantibiotic investigated to date. It represents one of the many antimicrobial peptides which have been the focus of much interest as potential therapeutic agents. This has resulted in the search for novel lantibiotics and more commonly, the engineering of novel variants from existing peptides with a view to increasing their activity, stability and solubility.
The aim of this study was to compare the activities of nisin A and novel bioengineered hinge derivatives, nisin S, nisin T and nisin V. The microtitre alamar blue assay (MABA) was employed to identify the enhanced activity of these novel variants against M. tuberculosis (H37Ra), M. kansasii (CIT11/06), M. avium subsp. hominissuis (CIT05/03) and M. avium subsp. paratuberculosis (MAP) (ATCC 19698). All variants displayed greater anti-mycobacterial activity than nisin A. Nisin S was the most potent variant against M. tuberculosis, M. kansasii and M. avium subsp. hominissuis, retarding growth by a maximum of 29% when compared with nisin A. Sub-species variations of inhibition were also observed with nisin S reducing growth of Mycobacterium avium subsp. hominissuis by 28% and Mycobacterium avium subsp. paratuberculosis by 19% and nisin T contrastingly reducing growth of MAP by 27% and MAC by 16%.
Nisin S, nisin T and nisin V are potent novel anti-mycobacterial compounds, which have the capacity to be further modified, potentially generating compounds with additional beneficial characteristics. This is the first report to demonstrate an enhancement of efficacy by any bioengineered bacteriocin against mycobacteria.
mycobacteria; nisin variants; alamar blue; peptide engineering; lantibiotic; bacteriocin
With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against M. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis.
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle. The complex, multifaceted interaction of M. avium subsp. paratuberculosis with its host includes dampening the ability of infected cells to respond to stimuli that promote M. avium subsp. paratuberculosis clearance. By disrupting host defenses, M. avium subsp. paratuberculosis creates an intracellular environment that favors the establishment and maintenance of infection. Toll-like receptors (TLRs) are important sensors that initiate innate immune responses to microbial challenge and are also immunotherapeutic targets. For example, TLR9 contributes to host defense against M. avium subsp. paratuberculosis, and its agonists (CpG oligodeoxynucleotides [ODNs]) are under investigation for treatment of Johne's disease and other infections. Here we demonstrate that M. avium subsp. paratuberculosis infection changes the responsiveness of bovine monocytes to TLR9 stimulation. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite a 10-fold increase in TLR9 expression and maintained uptake of CpG ODNs. Other TLR9-mediated responses, such as oxidative burst, which occur through noncanonical signaling, remain functional. Kinome analysis verifies that classic TLR9 signaling is blocked by M. avium subsp. paratuberculosis infection and that signaling instead proceeds through a Pyk2-mediated mechanism. Pyk2-mediated signaling does not hinder infection, as CpG ODNs fail to promote M. avium subsp. paratuberculosis clearance. Indeed, Pyk2 signaling appears to be an important aspect of M. avium subsp. paratuberculosis infection, as Pyk2 inhibitors significantly reduce the number of intracellular M. avium subsp. paratuberculosis bacteria. The actions of M. avium subsp. paratuberculosis on TLR9 signaling may represent a strategy to generate a host environment which is better suited for infection, revealing potential new targets for therapeutic intervention.
The cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study, Mycobacterium avium subsp. paratuberculosis recombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity to M. avium subsp. paratuberculosis antigens were compared in calves inoculated with live M. avium subsp. paratuberculosis, M. avium subsp. avium (M. avium), Mycobacterium kansasii, or Mycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinant M. avium subsp. paratuberculosis proteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected with M. bovis. M. avium subsp. paratuberculosis proteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with either M. avium subsp. paratuberculosis or M. avium after antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+ CD25+ T cells from calves inoculated with M. avium subsp. paratuberculosis and M. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated with M. avium subsp. paratuberculosis, the differences in immune responses to M. avium subsp. paratuberculosis antigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated with M. avium subsp. paratuberculosis and those inoculated with M. avium that were somewhat disparate from the responses in calves infected with M. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.
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.
Nine isolates from swine produced biofilm. There was a significant higher frequency of porcine isolates forming biofilm compared to human isolates. All isolates were previously characterised by IS1311- and IS1245-RFLP typing. The ability to form biofilm did not correlate with the RFLP-type, hsp65 sequevar, colony morphology or the presence of gene sequences related to GPL synthesis.
The observed differences in biofilm forming abilities between porcine and human isolates raises questions regarding the importance of biofilm formation for infectious potential. The optimised method worked well for screening of multiple isolates.
Johne's disease (JD) is one of the most costly bacterial diseases in cattle. In the U.S., economic losses from the disease have been estimated to exceed $1,500,000,000 per year, mainly from the effects of reduced milk production. Current diagnostic tests for JD are laboratory based and many of those tests require specialized equipment and training. Development of rapid and inexpensive diagnostic assays, which are adapted for point-of-care applications, would aid in the control of JD. In this study, a polyaniline (Pani)-based conductometric biosensor, in an immunomigration format, was fabricated for the detection of serum antibody (IgG) against the causal organism of JD, Mycobacterium avium subsp. paratuberculosis (MAP). Immobilized Mycobacterium avium purified proteins in the capture membrane were used to detect MAP IgG, previously bound with Pani/anti-bovine IgG* conjugate in the conjugate membrane. After detection, the Pani in the sandwiched captured complex bridges an electrical circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by Pani, was measured as drop in electrical resistance. Testing of the biosensor with known JD positive and negative serum samples demonstrated a significant difference in the mean resistance observed between the groups. This proof-of-concept study demonstrated that a conductometric biosensor could detect MAP IgG in 2 minutes. The biosensor's speed of detection and the equipment involved would, among other things, support its application towards the various point-of-care opportunities aimed at JD management and control.
Conductometric; biosensor; immunoassay; electrochemical immunosensor; polyaniline; paratuberculosis; Johne's disease; on-site diagnosis
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.
Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (109 CFU/animal) or medium (107 CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (103 CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines.