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Background and Aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and Methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy–Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

The honey bee is of fundamental significance for the ecological balance of the planet and in effect, global crop and animal production. In recent years, the Deformed wing virus (DWV) has been recog...

Within the context of an investigation focused at improving the effectiveness of test-and-removal for the control of ovine paratuberculosis, we recently conducted a preliminary study using ELISA and real time PCR to assess whether parturition affects test-positivity, in animals with subclinical paratuberculosis. Samples of faeces and blood were collected from 42 adult female animals, before (PP1) and after parturition (PP2), and before mating (PP3). In the preliminary stage of the analysis, only one of the animals tested reacted positively to ELISA (2.38%, 1 of 42), which corresponds to 2.8% of PCR-reactors (1 of 36). Therefore, the final stage the investigation was conducted using only real time PCR, which was applied to test samples of faeces collected from 85 animals, in 5 periods of sampling: 4–15/1–3 days (FP1/FP2) before and after parturition (FP3/FP4), and before mating (FP5). The result of the preliminary analysis indicated that PCR-positivity in terms of the number of shedders and the amount of MAP, is statistically significantly lower before parturition (PP1), whereas that of the final, higher in FP4 compared to FP5. Significantly higher levels of positivity in PP2 and FP4 were also recorded in connection with animals reacting positively to PCR more than once. In conclusion, in sheep with subclinical paratuberculosis, the period of 4–15 days postpartum is more suitable for the application of test-and-removal aiming to the control of the disease, using real time PCR. The use of ELISA for the same purpose is not recommended in the specific category of animals, due to low sensitivity.

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.

Background and Aim: Canine degenerative myelopathy (CDM) is an adult-onset fatal disorder associated with a point mutation of the superoxide dismutase 1 (SOD1) gene (SOD1:c.118G>A). This study aimed to determine the allele and genotype frequencies of this mutation in a group of Belgian Malinois dogs in Greece. Materials and Methods: Samples (n=72) of whole blood were collected from 72 purebred dogs of the Hellenic Armed Forces; these samples were processed for DNA isolation, polymerase chain reaction, and digestion with the restriction endonuclease AcuI. Sample testing was conducted in compliance with ISO17025 accreditation requirements. Results: The observed relative genotype frequencies were 71% for the homozygous (GG), 25% for the heterozygous (AG), and 4% for the homozygous mutant (AA) alleles. These frequencies were close to those expected, indicating no significant departure from Hardy–Weinberg equilibrium (HWE, p=0.395). The frequency of heterozygous animals indicates that a high risk of developing CDM in forthcoming generations exists in the tested population because mating among carriers would result in 25% AA progeny. The medical record of the group of study animals indicated selection against leishmaniosis, as applied throughout generations by owners and breeders. The potential association of this selection with the HWE status of the study population was discussed. Conclusion: The SOD1:c.118G>A mutation was common in the tested group of dogs; thus, they are suitable for a follow-up investigation on the development and progression of CDM. A case-control study on animals with evidence of sensitivity to infectious myelopathy could provide new insights into disease pathogenesis.

We report on a case of a sea bass and sea bream aquaculture farm in Greece, with many incidents of severe dermal and visceral granulomatous lesions detected during routine inspection and post-mortem examination. The investigation was conducted on samples of fish feeds and fish feed ingredients available commercially, and tissue samples collected from the affected population, and consisted of molecular analysis and histopathology. The former was performed using five PCR assays targeting the main mycobacterial pathogens. Histopathology examination relied on staining of tissue sections of various visceral organs with hematoxylin-eosin and Ziehl-Neelsen. Presence of mycobacteria was demonstrated in the fish feeds (100% and 46.2% positive for Mycobacterium spp. and Mycobacterium avium, respectively), in the fish feed ingredients (73.3% and 33.3% positive for Mycobacterium spp. and Mycobacterium avium, respectively) and the tissue sections (76.5% and 3.9% positive for Mycobacterium marinum and Mycobacterium avium, respectively) that were examined. Sequence analysis of the PCR products was confirmatory of the specificity of the amplification process, at a 98% minimum level of identification. Histopathology revealed in all cases evidence consistent with mycobacterial infection. The result of the analysis indicates that fish are exposed to different mycobacterial species; this probably accumulates in large numbers over the years in the organic sediment collected on the seabed of the aquaculture, causing severe infections.

Paratuberculosis is a disease affecting mainly ruminants, and it is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Control of the disease relies primarily on test-and-removal that is not easy to apply effectively for reasons associated with its pathogenesis. Therefore the aim of this preliminary study was to investigate whether positivity of sheep to MAP-specific antibody and DNA, detected by ELISA and faecal real time polymerase chain reaction (PCR) respectively, varies between specific stages of breeding i.e. before and after parturition, and before the mating season. Rectal faeces (n = 120) and whole blood (n = 120) samples were collected from a flock of sheep with no record of clinical paratuberculosis. The sheep under study are maintained throughout the year, in isolated confinement with no access to pasture or other animals, and a continuously monitored diet. The proportion of positive animals to real time PCR before parturition (13.9%) was found to be significantly lower (χ 2 = 10.67, P = 0.0015 ) than the proportion of positive animals after parturition (59.5%), and before the mating season (47.6%). The proportion of positive animals after parturition and before the mating season did not differ significantly. PCR analysis of one sample collected from each animal after parturition, allowed detection of more than 71% of the reactors. In conclusion, faecal shedding of MAP detected by real time PCR seems to be decreased before parturition, which probably makes the specific period less suitable for detection of shedders in sheep with no clinical signs of paratuberculosis.

Background: Zoonotic tuberculosis has been mainly associated with the consumption of bovine dairy products and its control has been prioritized by the Food and Agriculture Organisation. Objective: As such, the aim of this study is to assess whether the polymerase chain reaction (PCR) can be used as an early diagnostic indicator of Mycobacterium bovis infection in bovine dairy farms. Methods: Milk samples (n=78) were collected from all the animals older than 8 years of age, in all (n=4) bovine dairy farms located in a specific agricultural region of Greece, with high prevalence in bovine tuberculosis. Detection of DNA belonging to slow growing members of the genus Mycobacterium was conducted in pooled samples using two PCR assays targeting 16S-rRNA and 65-kDa heat shock protein (hsp65). Randomly selected PCR products were submitted to sequence analysis for confirmation of the specificity of the amplification process. DNA isolation and PCR testing were conducted in compliance with ISO17025 accreditation requirements. Results: The overall percentage of positivity was 47.7%, and ranged among farms from 0% to 76.9%. PCRpositive animals were detected in both farms that were at the time of investigation positive with the tuberculin skin test (TST), whereas the only farm with a negative TST record tested also negatively with PCR. Interestingly, one farm that was negative with TST since 2012 but had a long prior record of high level TST-positivity, tested positively with PCR. Conclusion: In conclusion it can be stated that PCR can be used for the detection of mycobacteria in pooled samples of milk collected from the older animals of a dairy farm, as an early and sensitive diagnostic indicator. This can support TST monitoring for the control of bovine tuberculosis, and improve detection of farms, in which routine monitoring should be intensified. The specific approach offers significant practical benefits that compensate for the additional cost of PCR.

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.

Leishmaniosis is a zoonotic disease that affects millions of people especially in resource-poor settings. The development of reliable diagnostic assays that do not require dedicated equipment or highly trained personnel would improve early diagnosis and effective control. For this purpose, a combination of magnetic bead and cadmium selenite quantum dot probes was applied for the detection of Leishmania-specific surface antigens (proteins) and DNA. Both analytes are isolated from the solution using magnetic bead capture probes whereas the presence of the targeted molecules is demonstrated by quantum dot detection probes. The sensitivity and specificity of this method reached 100% based on an assessment performed on 55 cultured isolates of various microbial pathogens. The low limit of detection was 3125 ng/μl and 10(3)cells/ml for Leishmania DNA and protein, respectively. The method shows considerable potential for clinical application in human and veterinary medicine, especially in resource-poor settings.

The study aimed to investigate whether the genetic polymorphisms in the 3 ′ UTR of the caprine SLC11A1 gene are functional, and to assess the role of MAP as a regulatory parameter in gene expression. To this goal we constructed plasmids expressing the Luciferase reporter gene in transient transfections of a mouse (Balb/c) macrophage cell line (RAW264.7), incorporating those polymorphisms that our previous work indicated as more prominent in terms of SLC11A1 expression and responsiveness to MAP infection. Gene expression variation was recorded on the average of the respective measurements after exposure to Mycobacterium avium subsp. paratuberculosis (MAP) combined with microbial antigens and cytokines. In silico analysis of the region under study allowed identification of one cis-acting RNA element, five putative transcriptional regulatory elements and 85 3 ’ end microRNA binding sites. The two polymorphic regions (regions A and B) of the 3 ’ UTR of the caprine SLC11A1 gene were recognized as regulators of its activity, at transcriptional and post-transcriptional level. The GT16 polymorphism at region A, combined with the GT8 polymorphism at region B, results in up-regulation of the SLC11A1 gene. The specific genotype was also found to be more responsive to MAP exposure at a statistically significant level.

Natural infection of domestic cats by Leishmania infantum (synonym: L. chagasi) has been demonstrated in several European, Latin American, and Asian countries, and the estimated prevalence of infection, based mainly on blood PCR, ranges from 0.3% up to 60.6%. In this study we aimed to: (a) estimate the prevalence of the infection by L. infantum in clinically normal cats (group A; n = 50) and in cats with various clinical signs (group B; n = 50), living in an endemic region, by both cytological examination of four different tissues (lymph node, skin, bone marrow, and conjunctiva) and by PCR in four different tissues (blood, skin biopsies, bone marrow, and conjunctiva); (b) compare the diagnostic sensitivity of the above methods and evaluate for possible associations between their results; and (c) investigate the possible associations between infection by L. infantum and signalment, living conditions, season of sampling, and health status of the cats. The prevalence of the infection in the study population was 41% and did not differ (P = 0.839) between group A (42%) and B (40%) cats. Lymph node, skin, bone marrow and conjunctiva cytology was always negative. Therefore, the diagnosis of the infection was based only on PCR in blood, skin biopsy, bone marrow and conjunctiva, which was positive in 13%, 18.2%, 16% and 3.1% of the cats, respectively. PCR was positive in only one of the four tissues in 80.5% of the infected cats. The results differed (P = 0.014) among the four tissues and were less frequently positive in conjunctiva compared to skin biopsies and bone marrow (P = 0.007 for both comparisons), thus highlighting the need for multiple tissue PCR testing in order to minimize false-negative results. More PCR-positive cats were found when sampling was performed during the period of sandfly activity (odds ratio: 2.44; P = 0.022). Also, in group B cats, the likelihood of PCR-positivity was higher (odds ratio: 3.93; P = 0.042) among those presenting at least one systemic clinical sign that had been previously reported in cats with leishmaniosis.

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.

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.

SLC11A1 (solute carrier family 11 member A1) protein is located on the phagolysosome membrane of macrophages and participates in bacterial killing. Here we have extended our previous work on the investigation of the potential association of polymorphisms of the 3′untranslated region (UTR) of SLC11A1 gene with test-positivity of goats to Mycobacterium avium subsp. paratuberculosis (MAP). Blood, serum and faeces were collected from 223 adult goats, from nine goat farms from Greece with a long-term record of paratuberculosis but no vaccination or tuberculin testing. The samples were subjected to sequence and structure analysis of the SLC11A1 gene and were evaluated by ELISA, culture and real time polymerase chain reaction. The 3′UTR region of the targeted gene revealed 2 microsatellites consisting of a variable number of guanine-thymine repeats named regions A and B. Statistically significant association was recorded between genotypes of region B and ELISA results, whereas the presence of B 7 allele was found to contribute to ELISA negativity. The comparison of the SLC11A1 mRNA level pre- and post-exposure to MAP shows elevated gene expression especially at the 3-h time point, in all macrophages tested regardless of their genotype. Unfortunately the latter could not be linked at a statistically significant level with any of the targeted genetic polymorphisms separately. In conclusion it can be stated that the evidence reported here provide the first indications on the association of B genotypes of the SLC11A1 gene and the detection of MAP-specific antibody by ELISA in goats.

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 mu l. 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. (C) 2009 Elsevier B.V. All rights reserved.

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.

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 1.8×10(4)Map 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.

Spoligotyping was undertaken with 38 Mycobacterium tuberculosis isolates from Greek sarcoidosis patients and 31 isolates from patients with tuberculosis. Fifty percent of the isolates from sarcoidosis patients and 16.13% of the isolates from patients with tuberculosis were represented by a unique pattern, whereas the remaining isolates belonged to seven shared types. Interestingly, half of the isolates from sarcoidosis patients did not resemble the spoligotypes of the isolates from patients with tuberculosis, most of which pertained to shared spoligotypes.

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150–200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 103M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 104M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.

Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania . The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis. In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use. The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis. The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum , Leishmania donovani and Leishmania chagasi , showed optimum performance on our test samples, and detected 41.9% Leishmania -positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre ≥200), and 19% by microscopic examination of lymph node aspirates.

H βρουκέλλωση των αιγοπροβάτων eίναι ένα νόσημα eυρέως διαδeδομένο στις παραμeσόγeιeς χώρeς. Το νόσημα eίναι ιδιαίτeρα σημαντικό για την προστασία της Δημόσιας Υγeίας δeδομένου ότι μeταδίδeται στον άνθρωπο προκαλώντας σοβαρή νόσο. Τον Απρίλιο του 2005 έγινe η διeρeύνηση της παρουσίας του νοσήματος eιδικά μeταξύ των αρσeνικών ζώων μίας πιστοποιημένης βιολογικής eκτροφής αιγών στο νομό Ηλeίας. Τα κοπάδι αριθμούσe 250 θηλυκά και 13 αρσeνικά ζώα και παρουσίαζe σποραδικά πeριστατικά αποβολών που eκδηλώνονταν συνήθως κατά το τeλeυταίο τρίτο της κυοφορίας. Τα ζώα κατά τη διάρκeια της δeιγματοληψίας ήταν κλινικά υγιή, eνώ δeν ήταν δυνατό βάσeι του ιστορικού να προσδιοριστeί μe ακρίβeια eάν eίχαν παρουσιάσeι στο παρeλθόν συμπτώματα προσβολής των όρχeων ή της eπιδιδυμίδας.Η eργαστηριακή διαγνωστική διeρeύνηση για τα συγκeκριμένα ζώα αποφασίστηκe να γίνeι μe την eφαρμογή της αλυσιδωτής αντίδρασης της πολυμeράσης (Polymerase Chain Reaction - PCR) για την ανίχνeυση DNA της Brucella melitensis σe δeίγματα αίματος. Δυο από τα 13 δeίγματα αίματος που συλλέχθηκαν από το κοπάδι αιγών αντέδρασαν θeτικά μe την PCR eπιτρέποντας την eνίσχυσητ ου eιδικού για την Brucella melitensis τeμαχίου DNA μeγέθους 844 ζeυγών βάσeων. Στην παρούσα μeλέτη, η PCR eπέτρeψeτην ανίχνeυση δυο ζώων που αν και κλινικά υγιή ήταν φορeίς Brucella μe μικροβιακό φορτίο αρκeτά υψηλό ώστe να γίνeι ταυτοποίηση του βακτηρίου μe μία μόνο δeιγματοληψία αίματος. Το eύρημα αυτό φαίνeται να συμφωνeί μe την αντίληψη ότι δeξαμeνή της βρουκέλλωσης στην Eλλάδα eίναι οι αίγeς που νοσούν πιο eλαφρά από τα πρόβατα και συντηρούν τη λοίμωξη για μακρόχρονικό διάστημα. Το ένα από τα θeτικά δeίγματα που ανιχνeύθηκαν ταυτοποιήθηκe μe χαρτογράφηση ως Brucella suis, κάτι που αναφέρeται για πρώτη φορά σe σχέση μe τις αίγeς. Το eύρημα αυτό και η eυπάθeια του ανθρώπου στην Brucella suis καθιστούν την έκθeση του στο συγκeκριμένο παθογόνο μέσω των αιγών έναν eπιδημιολογικό παράγοντα που αξίζeι πιθανώς να μeλeτηθeί eνδeλeχώς. Η αναγκαιότητα γι' αυτό καταδeικνύeται και από το γeγονός ότι η κατανάλωση αιγοπρόβeιων γαλακτοκομικών προϊόντων στην Eλλάδα γίνeται σe ορισμένeς πeριπτώσeις χωρίς να έχeι προηγηθeί ή απαραίτητη θeρμική τους eπeξeργασία, κάτι που δeν ισχύeι γιατην κατανάλωση χοίρeιου κρέατος.

Η σημασία των μυκοβακτηριακών λοιμώξeων των πτηνών συστηματικής eκμeτάλλeυσης μπορeί σήμeρα να eίναι μeιωμένη λόγω της eυρeίας eφαρμογής συγχρόνων μeθόδων eκτροφής, η μeλέτη τους όμως συνeχίζeι να eίναι eπίκαιρη αναφορικά μe την προστασία της υγeίας των πτηνών, των ζώων και του ανθρώπου. Στις μυκοβακτηριακές λοιμώξeις των πτηνών eμπλέκονται κυρίως μέλη του Mycobacterium avium complex (MAC), το οποίο διακρίνeται σe 28 ορότυπους που ανά ομάδeς συνιστούν eίδη: οι ορότυποι 1 έως 6, 8 έως 11 και ο ορότυπος 21 αποτeλούν το eίδος Μ. avium subsp. avium (ΜΑ), οι ορότυποι 4 και 8 αναφέρονται και ως Μ. avium subsp. hominisuis, eνώ οι ορότυποι 7, 12 έως 20 και 22 έως 28 συνιστούν το Mycobacterium intracellulare. Από τα μυκοβακτήρια που συνιστούν το MAC, eκeίνα που προκαλούν γeνικeυμένη φυματίωση στις όρνιθeς eίναι μόνο οι ορότυποι 1 έως 3 του ΜΑ. Λοίμωξη από τους υπόλοιπους ορότυπους του ΜΑ και από το Μ. intracellulare προκαλeί, συνήθως, μόνο eστιακές φυματιώδeις αλλοιώσeις. Στα παραπάνω έχeι προστeθeί τeλeυταία και ένα ακόμη eίδος, το Mycobacterium genavense, που αναγνωρίζeται ως παθογόνο για τα πτηνά. Πeριστατικά μυκοβακτηριακών λοιμώξeων στα πτηνά έχουν καταγραφeί παγκοσμίως. Η eισαγωγή του νοσήματος σe μια eκτροφή γίνeται κυρίως μe την αγορά μολυσμένων πτηνών που αποβάλλουν τα μυκοβακτήρια μe τα κόπρανα τους. Η eίσοδος του μικροβίου στον οργανισμό γίνeται στις πeρισσότeρeς πeριπτώσeις μέσω της στοματικής, και σπανιότeρα, μέσω της άνω αναπνeυστικής οδού. Η φυματίωση των πτηνών σe μία eκτροφή eκδηλώνeται συνήθως μe σποραδικά κρούσματα αιφνίδιων θανάτων, απώλeια βάρους και μeίωση της ωοπαραγωγής. Τα κλινικά συμπτώματα μπορeί eπίσης να προέρχονται από την προσβολή του eντέρου, των οστών, των πνeυμόνων ή του δέρματος. Τα νeκροτομικά eυρήματα eίναι παθογνωμονικά μόνο eφόσον ανeυρeθουν φυμάτια στα παρeγχυματικά όργανα και κυρίως στο μυeλό των οστών. Για την eργαστηριακή διάγνωση των μυκοβακτηριακών λοιμώξeων των πτηνών χρησιμοποιούνται ορολογικές eξeτάσeις, η μικροσκοπική eξέταση eπιχρισμάτων κοπράνων και η απομόνωση του μυκοβακτηρίου μe καλλιέργeια από παθολογικά υλικά, που αποτeλeί και σήμeρα τη μέθοδο αναφοράς. Τα τeλeυταία χρόνια eφαρμόζονται και κάποιeς μέθοδοι που βασίζονται σe τeχνικές της Μοριακής Βιολογίας, από τις οποίeς πιο eυρέως διαδeδομένη eίναι η αλυσιδωτή αντίδραση της πολυμeράσης (ΑΑΠ). Η καλλιέργeια παθολογικών υλικών γιατην απομόνωση μυκοβακχηρίων ξeκινά μe την απομόλυνση (decontamination) του δeίγματος από άλλα συνυπάρχοντα βακτήρια, των οποίων η ανάπτυξη δeν θα eπέτρeπe την ανάδeιξη της παρουσίας των μυκοβακχηρίων. Στη συνέχeια ακολουθeί eνοφθαλμισμός σe eιδικά θρeπτικά υποστρώματα που για το MAC eίναι συνήθως το Lowenstein-Jensen και το Middlebrook 7Η11. Η eπώαση γίνeται σe ατμόσφαιρα 5-10% διοξeιδίου του άνθρακα για τουλάχιστον 4-6 eβδομάδeς. Η ταυτοποίηση των αποικιών που αναπτύσσονται γίνeται μe eιδικές βιοχημικές δοκιμές ή μe την ΑΑΠ που μeιώνeι κατά πολύ το χρόνο που απαιτeίται για την τeλική γνωμάτeυση. Οι γeνωμικές πeριοχές, που χρησιμοποιούνται συνήθως γιατην ανίχνeυση και ταυτοποίηση του MAC μe ΑΑΠ, eίναι οι ακόλουθeς: 16 S rRNA , hsp 65, IS 1245 και η IS 901. Eιδικά για το Μ genavense, που eίναι από γeνeτικής απόψeως πολύ συγγeνές μe το ΜΑ, eφαρμόζeται ΑΑΠ για την eνίσχυση μίας eπιλeγμένης πeριοχής του γονιδίου της πρωτeίνης θeρμικού shock 65kDa και στη συνέχeια η πέψη του προϊόντος eνίσχυσης μe το ένζυμο πeριορισμού SaR.

The basic role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab for first-line treatment of cancer patients. Recent anticancer therapeutics based on active tumor targeting by conjugating tumor-specific antibodies has become of great interest in oncology. Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers able to deliver specific molecule payloads in a selective manner to improve the efficacy and safety of cancer imaging and therapy. We herein aimed to determine the targeting ability of bevacizumab-conjugated quantum dots (QDs) in vitro and in vivo.We used QDs labeled with bevacizumab, in various in vitro experiments using cell lines derived from colorectal cancer (CRC) and breast cancer (BC). For a competition study of QD-bevacizumab complex and bevacizumab, the cells were pre-treated with bevacizumab (100 nmol/L) for 24 h before exposure to the QD-bevacizumab complex. The breast cancer cells (MDA-MB-231) were injected to 9 nude mice to make the xenograft tumor model. The QD-bevacizumab complex was injected into the tumor model and fluorescence measurements were performed at 1, 12, and 24 h post-injection.Immunocytochemical data confirmed strong and specific binding of the QD-bevacizumab complex to the cell lines. The cells pre-treated with an excess of bevacizumab showed absence of QD binding. The in vivo fluorescence image disclosed that there was an increased signal of tumor after the injection of QDs. Ex vivo analysis showed 3.1 ± 0.8%, 28.6 ± 5.4% and 30.8 ± 4.2% injected dose/g accumulated in the tumors at 1, 12 and 24 h respectively. Tumor uptake was significantly decreased in the animals pretreated with excess of bevacizumab (p=0.001).In conclusion, we could successfully detect the VEGF-expressing tumors using QDs-bevacizumab nanoprobes in vitro and in vivo, opening new perspectives for VEGF-targeted non-invasive imaging in clinical practice.

Highlights • IFAT for anti-Leishmania IgG was positive in 10/100 cats. • ELISA for anti-Leishmania IgG was positive in 1/100 cats. • IFAT for anti-Leishmania IgM was positive in 1/100 cats. • The results of IFAT and ELISA for anti-Leishmania IgG disagreed (P = 0.039). • IFAT or ELISA for anti-Leishmania IgG disagreed with the results of PCR., Graphical Abstract, Cats that live in areas where canine and human leishmaniosis due to Leishmania infantum is endemic may become infected and may develop anti-Leishmania antibodies. In this study 50 clinically normal and 50 cats with cutaneous and/or systemic signs that lived in an endemic area and had been previously examined for infection by L. infantum using PCR in four different tissues were serologically tested for the presence of anti-Leishmania IgG (IFAT and ELISA) and IgM (IFAT). The aim was to compare the results of IFAT, ELISA and PCR and to investigate the possible associations between seropositivity to Leishmania spp and signalment, living conditions, season of sampling, health status of the cats, and seropositivity to other infectious agents. Low concentrations of anti-Leishmania IgG were detected by IFAT in 10% of the cats and by ELISA in 1%, whereas anti-Leishmania IgM were detected by IFAT in 1%. There was disagreement between the results of IFAT and ELISA for anti-Leishmania IgG (P = 0.039) and between all serological tests and PCR (P

Intron 1 of the human H-ras gene possesses a polymorphism consisting of repetitions of the GGGCCT consensus. Three alleles have been reported at this locus. We confirmed that two, P1 and P2, display four and two repeats, respectively, with their internal sequence structure similar to that previously described. The third, P3, previously assigned as a three-unit repetition allele according to its electrophoretic mobility and with no other information regarding its internal structure, was also found. Sequence analysis of the P3 allele revealed that it consists of three perfect repeats of the GGGCCT consensus. This polymorphism is present only in human c-H-ras gene, although single hexanucleotide repeats are found scattered within intron 1 of this gene in rodents. Analysis of this locus in matched tumor/distant normal samples from: (i) 38 patients with non-small-cell lung carcinoma (NSCLC), and (ii) 35 patients with sporadic invasive breast carcinoma, revealed: (1) 6.6% and 19% loss of heterozygosity (LOH) respectively, and (2) 10.5% and 2.9% hexanucleotide instability (HI) respectively, detected by the presence of shifted in length alleles. Shifted alleles exhibited altered internal sequence structure in comparison to normal ones, suggesting complex mutational events. The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias adjacent to squamous cell carcinomas (7.7% LOH, 5.9% HI), implying that abnormalities at this locus may be early events in lung carcinogenesis. The frequency of alterations (LOH vs. HI) was significantly different among NSCLC and breast cancer (P=.005), probably due to the different tumor biology of each system. Finally, altered mRNA expression of H-ras gene was detected in all cases with HI, but this finding was also observed in samples without HI. In view of reports showing that elements in intron 1 of H-ras gene potentially influence its transcriptional regulation, from our results we cannot exclude that the hexanucleotide locus could be an element with possible involvement in expressional regulation of this gene.

Data on human papilloma virus (HPV) involvement in preneoplastic and neoplastic lesions of the larynx and lung are limited and conflicting. The presence of HPV was investigated in a series of laryngeal specimens and non-small cell lung carcinomas (NSCLCs). The laryngeal samples (154) comprised 14 cases with hyperplasia without dysplasia, 49 with dysplasia, and 91 squamous cell carcinomas (SqCCs). The NSCLCs included 31 SqCCs, 32 adenocarcinomas, and 5 undifferentiated large cell carcinomas. Furthermore, we examined, for HPV DNA sequences, 14 bronchial metaplastic squamous lesions located next to cancerous areas. We used a sensitive nested polymerase chain reaction assay (NPCR), dot blotting, and in situ hybridization. The findings were correlated with clinicopathologic features of the patients. In the laryngeal specimens, NPCR analysis showed HPV DNA in 20 (13%) of the 154 specimens. Notably, 19 of 20 HPV-positive cases were carcinomas and only one was a mild dysplastic lesion. Typing of the carcinomas showed single HPV 6, 16, 18, and 33 infection in 1 (1.1%), 12 (13.2%), 2 (2.2%), and 1 (1.1%) samples, respectively, and HPV 6/33, 16/33, and 6/18 coinfection in three carcinomas. In situ hybridization findings were in agreement with PCR results, with the exception of two cases in which HPV 18 DNA was detected only by PCR. HPV was more frequently observed in heavy smokers than in patients with low daily cigarette consumption and nonsmokers (P = .03). There was no correlation between virus infection and gender, grade, and lymph node status of the carcinomas. None of the NSCLCs or adjacent metaplastic squamous epithelium contained HPV DNA sequences. The presented data suggest a contributory role of HPV in late stages of laryngeal carcinogenesis, because all premalignant lesions were negative but one. This study does not support a potential role of HPV in the development of NSCLCs.

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 method's 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.

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.

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.

Johne's disease or paratuberculosis is a chronic, progressive intestinal disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). One of the genes that have been targeted with regard to resistance or sensitivity to paratuberculosis is the SLC11A1 (solute carrier family 11 member A1). Here we extend our previous work to the sequence and structure analysis of the caprine SLC11A1 gene and we assess the functional impact of the most frequent polymorphisms of the 3' UTR region of the SLC11A1 gene to its expression in goat macrophages exposed in vitro to MAP. The role of these polymorphisms in primary immune response is also investigated with connection to gene expression of two interleukins (IL), one of which pro (IL-1a), and the other anti-inflammatory (IL-10). In order to assess gene response, quantitative detection of the SLC11A1, IL-10 and IL1a mRNA was performed by real time PCR before, and at 1, 3 and 24h after exposure of primary cultures of peripheral blood monocyte-derived macrophages to MAP, collected from 54 goats of the Greek native goat breed. Sequence analysis of the 3' UTR end of the caprine SLC11A1 gene determined its full length to be 522 bases. Structure analysis confirmed the presence of two microsatellites consisted of a variable number of guanine-thymine repeats (regions A and B). The homozygous B7 genotype [B(GTn)7/7] was associated at a statistically significant level with increased expression of the SLC11A1 and IL-1α genes indicating increased in vitro responsiveness and therefore resistance of mononuclear derived macrophages to MAP infection.

Microbial culture is exemplified by the Petri dish, a tool that (one century after its invention) still remains one of the “gold standards” for microbiological analysis. However, current trends towards automation, massively paralleled assays, and miniaturization (as well as the observation that we still cannot culture most microorganisms), suggest that new ideas in microbial culture are required. In the Petri dish, nutrient containing agar is typically used as the matrix on which microorganisms are cultured. However, new materials such as nanofibres and nanoporous materials may be better choices as supporting matrixes. Further, emerging techniques in microengineering and the fabrication of low cost materials are helping to create new porous disposables that are of sufficiently low cost that they may be used in the routine microbiology laboratory. These disposables are in turn allowing the development of novel miniature culture methods to take place, methods such as microchemostats, cages for growing microorganisms, and “habitats on a chip”. One particularly useful porous ceramic is Porous Aluminium Oxide (PAO), which can be utilized to generate highly subdivided culture chips that possess up to one million separate, miniaturized, growth areas. Indeed, this material has applications in microbiological diagnostics, microbiological research and industrial microbiology. In this chapter, the applications, advantages, and limitations of porous matrixes and accompanying culture chips will be examined. It is expected that these advances will yield significant improvements in microbial culture when compared to the classical Petri dish.

Liposomes applications in health care include meanly their ability to carry drugs and genes inside the human body for therapeutic purposes. Nevertheless their applicability can extend far beyond and could be used as analytical tools in order to perform rapid, low-cost, sensitive and specific analyses. Their physical characteristics, such as large internal volume and extended surface area, render them ideal for these applications and specifically for improving the specificity and sensitivity of the analytical assay. The purpose of this study was to develop a simple, stable and low-cost oligonucleotide-tagged liposomal formulation consisting of EggPC and DPPG with a simple to synthesize thiol-reactive conjugate (Mal-SA) incorporated into the lipid bilayer of liposomes. The prepared liposomes, having also the water soluble dye Sulforhodamine B encapsulated in their inner cavity, were characterized in terms of their physicochemical (size, size distribution, zeta-potential, lipid content) and mechanical (morphology, rigidity) properties. The results showed that the final liposomal formulation could be used in the future as analytical tool for detecting pathogen strains of microorganism in biological milieu.

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 method's 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.

BackgroundThe etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.Methodology/principal findingsFifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 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.Conclusions/significanceThe 274C/T SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.

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.

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.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.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.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.

Sarcoidosis, similarly to Crohn's disease (CD), is a complex inflammatory disease of unknown etiology. The belief that a genetic susceptibility to the development of sarcoidosis exists was derived from observations of familial clustering of sarcoidosis cases and racial differences in disease prevalence. Taking into account the remarkable similarity in the immunopathophysiology of sarcoidosis and CD, and in further exploring the genetic background of sarcoidosis, we study gene polymorphisms known for their implication in CD. These polymorphisms are in the CARD15/NOD2 gene (R702W, G908R and 3020insC), as well as mutations in the promoter of the CD14 gene (T/C at position -159) and in the TLR4 gene (Asp299Gly and Thr399Ile).DNA was obtained from 100 sarcoidosis patients and 150 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.Although CARD 15/NOD2 mutations were more frequent in cases than in controls, the difference was significant only for the G908R polymorphism (p = 0.024). Interestingly, the same was recorded with reference to the T allele (p = 0.002) and TT genotype (p = 0.017) frequencies of the CD14 promoter. No differences were observed in the 299Gly and 399Ile allele frequencies between patients and controls. Finally, the co-existence of a mutation in the CARD15/NOD2 and the CD14 genes was associated with sarcoidosis at a higher level of significance than any of these mutations separately.Our results suggest that the G908R mutation of the CARD15/NOD2 gene, as well as the T allele and TT genotype of the CD14 promoter are associated with increased susceptibility for developing sarcoidosis.