Emmanouil Liandris

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 microl. In order to obtain an indication of the methods 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.

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.

Avian mycobacteriosis in companion birds: 20-year survey.

The causative agents of avian mycobacteriosis in pet birds are rarely identified. The aim of this study is to add information about the etiology of avian mycobacteriosis. The identification of mycobacterium species in 27 cases of avian mycobacteriosis in pet birds was investigated by polymerase chain reaction (PCR) and sequencing of a rRNA hypervariable region. Avian mycobacteriosis appeared to be an infrequent diagnosis. Interestingly, a few cases of avian mycobacteriosis were recorded in very young birds. The most commonly affected species were the canary (Serinus canarius), the Eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus). All but one bird were infected with Mycobacterium genavense. Mycobacterium avium was identified only in one case.

Specific Detection of Unamplified Mycobacterial DNA by Use of Fluorescent Semiconductor Quantum Dots and Magnetic Beads

ABSTRACT 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. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a 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 to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 μl. In order to obtain an indication of the methods performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 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.

A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The methods minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

ESTIMATION OF THE SPREAD OF PATHOGENIC MYCOBACTERIA IN ORGANIC BROILER FARMS BY THE POLYMERASE CHAIN REACTION

Organic poultry breeding allows for increased exposure of birds to soil, faeces, and wildlife, which have been associated with the transmission of mycobacterial infections. Therefore the aim of this study was to investigate the spread of the major pathogenic mycobacteria in organically reared broilers in Greece using a diagnostic algorithm that relied on a combination of the polymerase chain reaction (PCR) and the restriction fragment length polymorphism analysis (RFLP). Liver, spleen and gonads from 81 to 150 days old broilers were aseptically collected post-mortem. 500 broilers from a population of 35,370, reared in the 25 registered as organic farms in Greece for the 2005 were used. DNA was isolated and incorporated to PCR targeted to 16S-rRNA gene (for Mycobacterium spp.), IS6110 (for Mycobacterium tuberculosis complex-MTBc), IS1245 (for Mycobacterium avium complex-MAC), IS901 (for M. avium subsp. avium-MAA) and hsp65 (for Mycobacterium genavense, by PCR-RFLP). The mean prevalence of mycobacteria detected by PCR with a 95% confidence interval was estimated to 4.4-8.8%. The relevant percentage with regard to the mycobacterial species that were included in this study was 0.17-2.03% for MAC, 2.11-3.39% for MTBc and 0.66-3.08% for mycobacteria not belonging to any of the above groups. None of the mycobacteria detected were identified as MAA or M. genavense. Considering that avian tuberculosis has been eradicated from conventional farms, the level and the pattern of positivity recorded here, indicates that our results may be associated with the specific conditions that apply to organic breeding.

Evaluation of the microbial safety of child food of animal origin in Greece.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

Associations between abortion-records in goats and test-positivity to Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis, is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects mainly ruminants. MAP can disseminate from the intestine to the genital tract. Therefore infection has been associated with decreased fertility but not directly with abortion in goats. The aim of this study was to obtain the evidence required to exploit the potential association between MAP-infection and abortion. For this purpose we have focused on three caprine herds geographically unrelated and keeping records of paratuberculosis and abortion of unspecified etiology. We collected in total 178 serum, milk, and fecal samples from 63, 2-4 years old, female goats. This material was processed for cultiva- tion, ELISA, and Real Time PCR (RT-PCR). No statistically significant association was recorded between abortion- record and RT-PCR positivity to MAP of fecal or milk samples. However ELISA - positivity was linked at a statistically significant level (p = 0.0275) with abortion. Goats that reacted positively by ELISA were 3.24 times more likely to abort than those with no detectable antibody titer. This study provides the first indications of the potential association of MAP- infection in goats and abortion-record, and suggests that a more extensive investigation should preferably rely on serol- ogy.