Detection of Mycobacterium avium subsp. paratuberculosis in cheeses from small ruminants in Tuscany

Paratuberculosis is an infectious disease which affects mainly domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). Map has been associated with human diseases like Crohn disease, type-1 diabetes, sarcoidosis, multiple sclerosis and Hashimoto's thyroiditis. The aim of this study was to determine the level of Map positivity of cheeses produced in Tuscany (Italy) as an indication of human exposure to the specific pathogen. Sampling was focused on artisanal cheeses produced without commercial starter culture from raw sheep or goat milk, on small-scale farms.Samples were tested by quantitative PCR (qPCR) and culture. Map DNA was detected in 4/7 (57.14%) goat, and in 14/25 (56%) sheep cheeses by qPCR, whereas cultivation produced a positive result in only one case. This corresponded to a goat cheese that had also reacted positively by qPCR and yielded a viable Type S (sheep) strain of Map. The Map load of the tested samples based on qPCR ranged from 6×10 to 1.8×104Map cells/g of cheese. The results indicate on average 56.57% and 66.6% positivity of cheese samples and farms, respectively. Hence, the type of cheeses that were analyzed within the context of this study seem to constitute a considerable source of human exposure to Map; although the question remains of whether the Map cells were present in a viable form, since positive results were almost exclusively recorded by qPCR

Driving laboratory standardisation of bacterial culture and antimicrobial susceptibility testing in veterinary clinical microbiology in Europe and beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for standardized training of clinical microbiologists and harmonisation of diagnostic procedures. This is particularly true for veterinary clinical microbiology where additional challenges exist when microbiologists are trying to fulfil a professional role very similar to their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include 1) harmonisation of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); 2) specific guidelines for interpretation and reporting of AST results for animal pathogens; 3) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; 4) standardisation of diagnostic procedures for animal clinical specimens and 5) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues led by the European Network for Optimisation of Veterinary Antimicrobial Treatment (ENOVAT) which is bringing together experts in veterinary microbiology, pharmacology, epidemiology and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners towards standardisation and harmonisation of laboratory methodologies and optimisation of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardisation and harmonisation of veterinary clinical microbiology methodologies, which could then be used as a template for implementation at a wider international level

Detection of Pathogenic Mycobacteria Based on Functionalized Quantum Dots Coupled with Immunomagnetic Separation

Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 104 bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples

Linking Chronic Infection and Autoimmune Diseases: Mycobacterium avium Subspecies paratuberculosis, SLC11A1 Polymorphisms and Type-1 Diabetes Mellitus

(MAP) has been reported to be a possible trigger in the development of T1DM. gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls. alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients

Specific detection of unamplified mycobacterial DNA using fluorescent semiconductor quantum dots and magnetic beads

Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp. dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmim selenite QDs conjugated with streptavidin and species specific probes were used to produce fluorescent signal. MBs conjugated with streptavidin and a genus specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method on isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined at 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage of patients with tuberculosis, and Mycobacterium avium subsp. paratuberculosis in faeces and paraffin embedded tissues, in comparison with culture, Ziehl-Neelsen stain and Real Time PCR. The concordance of these methods compared to the proposed with connection to positive and negative samples varied between 53.84% - 87.23% and 84.61%-100% respectively. The overall accuracy of the QD assay compared to Real Time PCR was 70-90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples

Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles

Mycobacterial infections have a high economic, human and animal health impact. Herein, we present the development of a colorimetric method that relies on the use of gold nanoparticles for fast and specific detection of Mycobacterium spp. dispensing with the need for DNA amplification. The result can be recorded by visual and/or spectrophotometric comparison of solutions before and after acid induced AuNP-probe aggregation. The presence of a complementary target prevents aggregation and the solution remains pink, whereas in the opposite event it turns to purple. The application of the proposed method on isolated bacteria produced positive results with the mycobacterial isolates and negative with the controls. The minimum detection limit of the assay was defined at 18.75 ng of mycobacterial DNA diluted in a sample-volume of 10 μl. In order to obtain an indication of the method's performance on clinical samples we applied the optimized assay to the detection of Mycobacterium avium subsp. paratuberculosis DNA in faeces, in comparison with real-time PCR. The concordance of the two methods with connection to real-time PCR positive and negative sample was defined respectively as 87.5% and 100%. The proposed method could be used as a highly specific and sensitive screening tool for the detection of mycobacteria directly from clinical samples in a very simple manner, without the need of high-cost dedicated equipment. The technology described here, may develop into a platform that could accommodate detection of many bacterial species and could be easily adapted for high throughput and expedite screening of samples

The Infectivity of sarcoid clinical material and its bacterial content inoculated in CBA mice

Background: Sarcoidosis is a multisystemic disorder that is currently viewed as the consequence of chronic immunological response associating genetic susceptibility and specific environmental or transmissible agents. Relevant evidence, although conflicting, justifies a concern about the involvement of specific pathogens to disease causation. In this study we assessed the infectivity of sarcoid clinical material, and of the pathogens found in it, to normal CBA mice used as a model of an immuno-competent host. Materials and Methods: One hundred and eleven mice were inoculated into their footpads with fresh, filtered, and autoclaved, sputum and bronchoalveolar lavage homogenates, collected from patients with sarcoidosis, and with the mycobacterial and propionibacterial pathogens isolated from this material. Results: The total number of positive reactors of the animals that received raw clinical material and the pathogens it contained was statistically significant compared to those of the control groups. However, the number of affected mice per group was in most cases less than 50% and inflammation was almost always mild and local. Conclusion: Based on the evidence provided by inoculation of normal CBA mice, some of the material under study, although of mild potency, can be infectious to an immuno-competent host

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

Driving Laboratory Standardization of Bacterial Culture and Antimicrobial Susceptibility Testing in Veterinary Clinical Microbiology in Europe and Beyond

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level