Diagnostic potential of the peptide-mediated magnetic separation (PMS)-phage assay and PMS-culture to detect Mycobacterium avium subsp. paratuberculosis in bovine milk samples.

Controlling the spread of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), in domestic livestock is challenging. Current diagnostic methods lack sufficient sensitivity to detect subclinically infected animals, and thus, better diagnostic methods are needed. This study was carried out to investigate the diagnostic potential of two novel peptide-mediated magnetic separation (PMS)-based tests-a PMS-phage assay and PMS-culture-both of which have been developed and optimized to detect viable MAP cells in bovine milk. Individual milk samples (50 ml) were obtained from 105 "non-infected" and 40 "MAP-infected" animals (classified as such on the basis of prior faecal culture and serum-ELISA results) in three dairy herds and tested in parallel by the PMS-phage assay and PMS-culture. Diagnostic sensitivity (DSe) and specificity (DSp) of the PMS-phage and PMS-culture methods were determined relative to the MAP infection status of the animal contributing the milk sample. The PMS-based tests applied individually showed moderate DSe (PMS-culture 0.250 and PMS-phage assay 0.325) and high DSp (0.962 and 1.000, respectively). When results of the two PMS-based tests were combined, DSe increased substantially to 0.525, and the DSp was calculated to be 0.962. It was concluded that combined application of the PMS-phage assay and PMS-culture provided the most complete picture regarding the presence of viable MAP in bovine milk samples. A comprehensive validation of the PMS-based assays relative to currently used diagnostic methods (faecal culture and serum-ELISA) would be the next step in assessment of the diagnostic potential of these novel PMS-based methods.

Development of a novel phage-mediated immunoassay for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis.

AIMS: The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture. METHODS AND RESULTS: The new method couples Map-specific peptide-mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3 × 10(2) -6 × 10(8 ) phage ml(-1) . When low numbers of Map were present (10(2)  CFU ml(-1) ), the burst size of a single host Map cell was maximal (10(3) phage per cell) resulting in a highly sensitive screening assay. CONCLUSION: A rapid, sensitive immuno-based screening method suitable for the detection of viable Map in milk and faeces was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel PMS-phage-ELISA permits sensitive, qualitative detection of viable Map in milk or faeces samples within 48 h, representing a substantial decrease in time to detection compared with current culture methods for Map.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation.

AIMS: The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS). METHODS AND RESULTS: Four-phage display biopanning rounds were performed, and the peptides expressed by the two most Salmonella-specific (on the basis of phage-binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne™ tosylactivated Dynabeads(®). Peptide capture capability for whole Salmonella cells from nonenriched broth cultures was quantified by MS + plate counts and MS + Greenlight™ detection and compared to capture capability of anti-Salmonella (antibody-coated) Dynabeads(®). MS + Greenlight™ gave a more comprehensive picture of capture capability than MS + plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads(®) (mean capture values of 36·0 ± 18·2 and 31·2 ± 20·1%, respectively, over Salmonella spp. concentration range 3 × 10(1) -3 × 10(6) CFU ml(-1)) with cross-reactivity of ≤1·9% to three other foodborne pathogens: Escherichia coli, Listeria monocytogenes and Campylobacter jejuni. CONCLUSIONS: One of the phage display-derived peptide ligands was demonstrated by MS + Greenlight™ to be a viable antibody alternative for MS of Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates an antibody-free approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.

Immunological detection of Bacteroides fragilis in clinical samples.

A monospecific polyclonal antiserum, prepared against Bacteroides fragilis common polysaccharide antigen purified by polyacrylamide gel immunoblot detected B. fragilis, B. thetaiotaomicron, B. ovatus and Prevotella melaninogenica in pus samples from various anatomical sites by immunofluorescence microscopy of the pus. With standard clinical laboratory culture methods, 36% of 147 samples were positive for one or more of the above bacteria. Of these, B. fragilis accounted for 33%. By immunofluorescent labelling of pus with the common antigen antiserum the detection of these bacteria in the samples increased to 50%. All nine of the blood cultures in which B. fragilis was detected by culture contained bacteria positive for the common antigen. Immunofluorescent labelling of pus samples with a selection of monoclonal antibodies specific for surface polysaccharides which are known to be antigenically variable in culture in vitro and in an animal model of infection showed that these polysaccharides are also variable in natural infection. The results indicate that the common polysaccharide antigen, in contrast to the variable surface polysaccharides, is a suitable target for the immunodetection of B. fragilis in clinical samples from a range of anatomical sites.

Flow cytometric analysis of within-strain variation in polysaccharide expression by Bacteroides fragilis by use of murine monoclonal antibodies.

The reactivity of four different monoclonal antibodies (MAbs) with populations of Bacteroides fragilis NCTC 9343, enriched by density gradient centrifugation for a large capsule, small capsule and electron-dense layer (EDL) only visible by electronmicroscopy, was examined. The MAbs reacted strongly with polysaccharides present in both the large capsule- and EDL-enriched populations but not in the small capsule-enriched populations. The pattern of labelling was determined by immunoblotting, immunofluorescence and immuno-electronmicroscopy, and flow cytometry. The MAbs labelled cell membrane-associated epitopes in the large capsule- and EDL-enriched populations and cell-free material in the EDL population. By immunoblotting, ladders of repeating polysaccharide subunits were evident in the EDL population but not in the large capsule population. The proportion of cells labelled within each population was determined by flow cytometry. The reactivity of another MAb with the small capsule population was confirmed by flow cytometry. A qualitative indication of epitope expression was obtained by examination of the flow cytometric profiles. Differential expression of the same saccharide epitope was observed both between and within structurally distinct B. fragilis populations. The MAbs were species-specific and cross-reacted with several recent clinical isolates. These polysaccharides may be relevant to the virulence of B. fragilis.

Prospective study of natural cytotoxicity in peripheral blood of patients with nonlymphoid solid malignancies.

Natural killer (NK) cells have been implicated as an initial immunosurveillance mechanism for carcinogenesis in humans. Work in the murine system as well as the findings of depressed NK activity in patients with advanced malignancies and the discovery of increased incidences of cancer in humans congenitally deficient in NK ability have supported this. Few prospective studies have demonstrated a prognostic change in NK activity with respect to malignant disease course. In 32 healthy donors, NK activity against K562 was determined. No race or sex difference existed with respect to NK cell function. Esophageal (5), bronchogenic (3), breast (3), cervical (3), and endometrial (1) cancer patients who had received no prior chemotherapy were compared to controls. All patients subsequently received radiotherapy. Prior to such treatment NK activity could not be associated with stage of malignancy. Of the 15 patients studied, 11 were sequentially followed. Five of eight patients with stable or improving clinical courses as assessed by weight and Karnofsky scores were found to have increasing NK activity. Two of three patients with poor clinical courses presented with subnormal killing which never rose to normal while the third declined to subnormal before expiring. Esophageal, cervical, and endometrial carcinoma patients all presented with low or subnormal NK activity. Of these, only cervical and endometrial cancer patients exhibited an increase to normal levels.

Human natural killer cells: correlation of a lytic assay with a visual binding assay.

Different populations of human effector cells were examined for their ability to bind and to lyse K562 target cells as monitored by a fluorochrome-labeled batch system binding assay and a 6-hour chromium release assay respectively. Binding and cytolysis were found to increase, or decrease, concomitantly with null cell purification, and both were abrogated by trypsin treatment of effectors. At 4 degrees C, cytolysis was abolished whereas binding was only decreased. When binding and cytolysis data were correlated, the nylon wool nonadherent cells (i.e., T and null cells) were found to be the most efficient (i.e., fewer bound effectors per target cell lysed) while mononuclear cells were the least efficient with respect to natural killing. These findings support the current hypotheses of spontaneous killer ontogeny and peripheral blood compartmentalization. Furthermore, this study confirms observations made for mononuclear and enriched T plus null cell populations in single conjugate in agarose assays and extends those to the null cell population--a population relatively enriched for large granular lymphocytes.