Culture of Mycobacterium avium subsp. paratuberculosis: challenges, limitations and future prospects.

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants and is suspected to be involved in the development of Crohn's disease and several autoimmune disorders. As such, sensitive and specific MAP detection methods are required to confirm infection in animals and identify potential sources of animal and human exposure. Despite recent developments in immunological and nucleic acid-based detection methods, culture-based detection of MAP remains the 'gold standard' against which the sensitivity and specificity of other detection methods are measured. However, not all culture-based approaches are equivalent in terms of detection capability, which can lead to errors in the evaluation of other detection methods. This review will provide an overview of the chronological development of culture methods for MAP, and will consider the unique growth requirements of MAP, the merits of solid versus liquid culture media, the relative performance of the commonly used MAP culture media, and sample preparation/decontamination protocols for different sample types. The limitations of current MAP culture methods and prospects for improvements are discussed.

The potential role of molecular mimicry by the anaerobic microbiota in the aetiology of autoimmune disease.

Autoimmune diseases are thought to develop as a consequence of various environmental and genetic factors, each of which contributes to dysfunctional immune responses and/or a breakdown in immunological tolerance towards native structures. Molecular mimicry by microbial components is among the environmental factors thought to promote a breakdown in immune tolerance, particularly through the presence of cross-reactive epitopes shared with the human host. While resident members of the microbiota are essential promoters of human health through immunomodulation, defence against pathogenic colonisation and conversion of dietary fibre into nutritional resources for host tissues, there may be an underappreciated role of these microbes in the aetiology and/or progression of autoimmune disease. An increasing number of molecular mimics are being identified amongst the anaerobic microbiota which structurally resemble endogenous components and, in some cases, for example the human ubiquitin mimic of Bacteroides fragilis and DNA methyltransferase of Roseburia intestinalis, have been associated with promoting antibody profiles characteristic of autoimmune diseases. The persistent exposure of molecular mimics from the microbiota to the human immune system is likely to be involved in autoantibody production that contributes to the pathologies associated with immune-mediated inflammatory disorders. Here-in, examples of molecular mimics that have been identified among resident members of the human microbiota and their ability to induce autoimmune disease through cross-reactive autoantibody production are discussed. Improved awareness of the molecular mimics that exist among human colonisers will help elucidate the mechanisms involved in the breakdown of immune tolerance that ultimately lead to chronic inflammation and downstream disease.

Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology.

AIM: To develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP). METHODS AND RESULTS: Seven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM). CONCLUSIONS: MAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 µg ml-1 Mycobactin J. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.

Multilaboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M.caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture.

Development of a novel immunochromatographic lateral flow assay specific for Mycobacterium bovis cells and its application in combination with immunomagnetic separation to test badger faeces.

BACKGROUND: The European badger is an important wildlife reservoir of Mycobacterium bovis implicated in the spread of bovine tuberculosis in the United Kingdom and Ireland. Infected badgers are known to shed M. bovis in their urine and faeces, which may contaminate the environment. To aid bovine tuberculosis control efforts novel diagnostic tests for detecting infected and shedding badgers are needed. We proposed development of a novel, rapid immunochromatographic lateral flow device (LFD) as a non-invasive test to detect M. bovis cells in badger faeces. Its application in combination with immunomagnetic separation (IMS) to detect Mycobacterium bovis cells in badger faeces is reported here. RESULTS: A novel prototype LFD for M. bovis cells was successfully developed, with unique specificity for M. bovis and a limit of detection 50% (LOD50%) of 1.7 × 104 M. bovis cells/ml. When IMS was employed to selectively capture and concentrate M. bovis cells from badger faeces prior to LFD testing, the LOD50% of the IMS-LFD assay was 2.8 × 105 M. bovis cells/ml faecal homogenate. Faeces samples collected from latrines at badger setts in a region of endemic bovine tuberculosis infection were tested; 78 (18%) of 441 samples tested IMS-LFD assay positive, whereas 140 (32%) tested IMS-qPCR positive (Kappa agreement -0.009 ± 0.044, p = 0.838). Subsequently, when 130 faeces samples from live captured, or captive, badgers of known infection status (on the basis of StatPak, interferon-γ and/or culture results) were tested, the IMS-LFD assay had higher relative diagnostic specificity (Sp 0.926), but poorer relative diagnostic sensitivity (Se 0.081), than IMS-qPCR (Sp 0.706, Se 0.581) and IMS-culture (Sp 0.794, Se 0.436). CONCLUSIONS: The novel IMS-LFD assay, although very specific for M. bovis, has low analytical sensitivity (indicated by the LOD50%) and would only detect badgers shedding high numbers of M. bovis (>104-5 cells/g) in their faeces. The novel LFD would, therefore, have limited value as a non-invasive test for badger TB surveillance purposes but it may have value for alternative veterinary diagnostic applications.

Variations in the Physical Properties and Microbial Community of Dairy Cow Manure-Implications for Testing and Efficacy of Footbathing Products.

Footbaths containing disinfectants are used on dairy farms to reduce the spread of digital dermatitis; however, they commonly become contaminated with manure. This trial investigated the physical properties and microbial composition of dairy cow manure from two production systems and examined whether the source of manure impacted the efficacy of footbathing disinfectants. Manure was collected from eighteen dairy cows, nine housed and fed grass silage (HOUSED) and nine at pasture (PASTURE). The pH and dry matter content was determined, total DNA was extracted and the region v3-v4 of the 16s rRNA gene sequenced. The efficacy of formalin and two trial products (TP1: peracetic acid and hydrogen peroxide; TP2: chlorocresol and triamine) was evaluated when mixed with manure from the two production systems. Production system differences were found in manure dry matter content, bacterial microbiome and the efficacy of both trial footbathing products but not formalin. The properties of manure affected the results of laboratory testing and therefore have the potential to influence footbathing disinfectant efficacy when footbaths are contaminated with manure. Further research into the impact of organic contaminants on the efficacy of disinfectants could facilitate the development of improved testing programmes and disinfectant products.

Production and evaluation of antibodies and phage display-derived peptide ligands for immunomagnetic separation of Mycobacterium bovis.

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10(5) CFU/ml) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

Peroxidase-Mimicking Activity of Biogenic Gold Nanoparticles Produced from Prunus nepalensis Fruit Extract: Characterizations and Application for the Detection of Mycobacterium bovis.

In the present study, a facile, eco-friendly, and controlled synthesis of gold nanoparticles (Au NPs) using Prunus nepalensis fruit extract is reported. The biogenically synthesized Au NPs possess ultra-active intrinsic peroxidase-like activity for the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Chemical analysis of the fruit extract demonstrated the presence of various bioactive molecules such as amino acids (l-alanine and aspartic acids), organic acids (benzoic acid and citric acid), sugars (arabinose and glucose), phenolic acid, and bioflavonoids (niacin and myo-inositol), which likely attributed to the formation of stable biogenic Au NPs with excellent peroxidase-mimicking activity. In comparison with the natural horseradish peroxidase (HRP) enzyme, the biogenic Au NPs displayed a 9.64 times higher activity with regard to the reaction velocity at 6% (v/v) H2O2, presenting a higher affinity toward the TMB substrate. The Michaelis-Menten constant (KM) values for the biogenic Au NPs and HRP were found to be 6.9 × 10-2 and 7.9 × 10-2 mM, respectively, at the same concentration of 100 pM. To investigate its applicability for biosensing, a monoclonal antibody specific for Mycobacterium bovis (QUBMA-Bov) was directly conjugated to the surface of the biogenic Au NPs. The obtained results indicate that the biogenic Au NPs-QUBMA-Bov conjugates are capable of detecting M. bovis based on a colorimetric immunosensing method within a lower range of 100 to 102 cfu mL-1 with limits of detection of ∼53 and ∼71 cfu mL-1 in an artificial buffer solution and in a soft cheese spiked sample, respectively. This strategy demonstrates decent specificity in comparison with those of other bacterial and mycobacterial species. Considering these findings together, this study indicates the potential for the development of a cost-effective biosensing platform with high sensitivity and specificity for the detection of M. bovis using antibody-conjugated Au nanozymes.

Development and single-laboratory validation of a pseudofunctional biosensor immunoassay for the detection of the okadaic acid group of toxins.

A rapid analytical optical biosensor-based immunoassay was developed and validated for the detection of okadaic acid (OA) and its structurally related toxins from shellfish matrix. The assay utilizes a monoclonal antibody which binds to the OA group of toxins in order of their toxicities, resulting in a pseudofunctional assay. Single-laboratory validation of the assay for quantitative detection of OA determined that it has an action limit of 120 microg/kg, a limit of detection of 31 microg/kg, and a working range of 31-174 microg/kg. The midpoint on the standard matrix calibration curve is 80 microg/kg, half the current regulatory limit. Inter- and intra-assay studies of negative mussel samples spiked with various OA concentrations produced average coefficient of variation (CV) and standard deviation (SD) values of 7.9 and 10.1, respectively. The assay was also validated to confirm the ability to accurately codetect and quantify dinophysistoxin-1 (DTX-1), DTX-2, and DTX-3 from shellfish matrix. Alkaline hydrolysis was not required for the detection of DTX-3 from matrix. Excellent correlations with the data generated by the biosensor method and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were obtained using a certified reference material (R(2) = 0.99), laboratory reference material, and naturally contaminated mussel samples (R(2) = 0.97). This new procedure could be used as a rapid screening procedure replacing animal-based tests for DSP toxins.

Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation.

The generation of novel Mycobacterium avium subsp. paratuberculosis (MAP)-specific monoclonal antibodies and phage-display derived peptide binders, along with their application for the magnetic separation (MS) of MAP cells, is described. Our aim was to achieve even greater MAP capture capability than is possible with peptide-mediated magnetic separation (PMS) using a 50:50 mix of biotinylated-aMp3 and biotinylated-aMptD peptide-coated beads. Gamma-irradiated whole MAP cells and ethanol extracted antigens (EEA) from these cells were used to elicit an immune response and as phage-display biopanning targets. A range of novel binders was obtained and coated onto paramagnetic beads, both individually and in various combinations, for MS evaluation. IS900 PCR was employed after MS to provide quick results. Capture sensitivity was assessed using a range of MAP concentrations after which the most promising beads were tested for their specificity for MAP, by performing MS followed by culture using 10 other Mycobacterium species. Magnetic beads coated with the biotinylated EEA402 peptide demonstrated a greater level of MAP capture than the current PMS method, even when low numbers of MAP (<10 cfu/ml) were present; however these beads also captured a range of other mycobacteria and so lacked capture specificity. Magnetic beads coated with monoclonal antibodies 6G11 and 15D10 (used as a 50:50 mix or as dually coated beads) also demonstrated improved MAP capture relative to the current PMS method, but with little cross-reactivity to other Mycobacterium spp. Therefore, two new MS protocols are suggested, the application of which would be dependent upon the required endpoint. Biotinylated EEA402-coated beads could potentially be used with a MAP-specific PCR to ensure detection specificity, while beads coated with 6G11 and 15D10 monoclonal antibodies could be used with culture or the phage amplification assay.

Improved detection of Mycobacterium bovis in bovine tissues using immunomagnetic separation approaches.

Immunomagnetic separation (IMS) represents a simple but effective method of selectively capturing and concentrating Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), from tissue samples. It is a physical cell separation technique that does not impact cell viability, unlike traditional chemical decontamination prior to culture. IMS is performed with paramagnetic beads coated with M. bovis-specific antibody and peptide binders. Once captured by IMS, M. bovis cells can be detected by either PCR or cultural detection methods. Increased detection rates of M. bovis, particularly from non-visibly lesioned lymph node tissues from bTB reactor animals, have recently been reported when IMS-based methods were employed.

Improved detection of Mycobacterium bovis infection in bovine lymph node tissue using immunomagnetic separation (IMS)-based methods.

Immunomagnetic separation (IMS) can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR) or culture (IMS-MGIT). This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL), 74 non-visibly lesioned (NVL)) collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1%) lymph node samples tested positive by culture, 162 (57.8%) by IMS-PCR (targeting IS6110), and 191 (68.2%) by IMS-MGIT culture. Twelve (6.9%) of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 79 M. bovis positive lymph node samples (27 (13.1%) VL and 52 (70.3%) NVL) were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle) decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant) which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 70.3% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

Phage display-derived binders able to distinguish Listeria monocytogenes from other Listeria species.

The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors.

Okadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology.

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0-1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.