Search

Bovine tuberculosis (bTB) is an ongoing issue in several countries within the European Union. Microbiological culture is the official confirmation technique for the presence of Mycobacterium tuberculosis complex (MTBC) members in bovine tissues, but several methodological issues, such as moderate sensitivity and long incubation times, require the development of more sensitive and rapid techniques. This study evaluates the analytical and diagnostic performance, comparative to culture, of a real-time PCR targeting the MTBC-specific IS6110 transposon using a panel of bovine tissue samples sourced from the Spanish bTB eradication campaign. Robustness and repeatability were evaluated in an interlaboratory trial between European Union National Reference Laboratories. The limit of detection with 95% confidence was established at 65 fg/reaction of purified genomic equivalents. Diagnostic sensitivity (Se) and specificity (Sp) were, respectively, 96.45% and 93.66%, and the overall agreement (κ) was 0.88. Cross-reactivity was detected against two mycobacterial isolates identified as Mycobacterium marinum and "Mycobacterium avium subsp. hominissuis," and whole-genome sequencing (WGS) analysis of the latter isolate revealed an IS6110-like sequence with 83% identity. An identical IS-like element was found in other Mycobacterium avium complex species in the NCBI nucleotide and WGS databases. Despite this finding, this methodology is considered a valuable alternative to culture, and the strategy of use should be defined depending on the control or eradication programs.

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.

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.

Although the post-mortem diagnosis of bovine tuberculosis is mainly achieved through microbiological culture, the development of other techniques to detect Mycobacterium tuberculosis complex (MTBC) members directly from tissue samples has been pursued. The present study describes the development, optimization and validation of a Real-Time PCR based on the mpb70 gene to detect MTBC members in clinical tissue samples from cattle. Specific primers and a hybridization probe were used to amplify MTBC-specific sequences in order to avoid cross-reaction with non-MTBC species. An Internal Amplification Control (IAC) was included in order to assess the presence of PCR inhibitors in the samples. The PCR was optimized to achieve maximum efficiency, and the limit of detection, limit of quantification and dynamic range of the reaction were determined. The specificity of the reaction was tested against 34 mycobacterial and non-mycobacterial species. The diagnostic sensitivity, specificity and positive and negative predictive values (PPV and NPV) of the method were assessed on 200 bovine tissue samples in relation to bacteriological culture. The dynamic range of the reaction spanned from 5 ng/reaction (106 genome equivalents) to 50 fg/reaction (10 genome equivalents). The efficiency of the reaction was 102.6% and the achieved R2 was 0.999. The limit of detection with 95% confidence was 10 genome equivalents/reaction. No cross-reactions with non-MTBC species were observed. The diagnostic sensitivity and specificity values of the mpb70 specific Real-Time PCR respect to culture were 94.59% (95% CI: 86.73–98.51%) and 96.03% (95% CI: 90.98–98.70%), respectively, with a PPV of 93.33% (95% CI: 85.55–97.07%) and a NPV of 96.80% (95% CI: 92.10–98.74%). The concordance of the Real-Time PCR based on mpb70 is comparable to that of culture (K = 0.904) showing a great potential for the detection of members of the MTBC in animal tissues.

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.

The official technique for diagnosis of bovine tuberculosis (bTB) worldwide is the tuberculin skin test, based on the evaluation of the skin thickness increase after the intradermal inoculation of a purified protein derivative (PPD) in cattle. A number of studies performed on experimentally infected or sensitized cattle have suggested that the relative sensitivity of the cervical test (performed in the neck) may vary depending on the exact location in which the PPD is injected. However, quantitative evidence on the variation of the test accuracy associated to changes in the site of inoculation in naturally infected animals (the population in which performance of the test is most critical for disease eradication) is lacking. Here, the probability of obtaining a positive reaction (>2 or 4 millimeters and/or presence of local clinical signs) after multiple inoculations of bovine PPD in different cervical and scapular locations was assessed in animals from five bTB-infected herds (818 cattle receiving eight inoculations) using a hierarchical Bayesian logistic regression model and adjusting for the potential effect of age and sex. The effect of the inoculation site was also assessed qualitatively in animals from four officially tuberculosis free (OTF) herds (two inoculations in 210 animals and eight inoculations in 38 cattle). Although no differences in the qualitative outcome of the test were observed in cattle from OTF herds, a statistically important association between the test outcome and the inoculation site in animals from infected herds was observed, with higher probabilities of positive results when the test was performed in the neck anterior area. Our results suggest that test sensitivity may be maximized by considering the area of the neck in which the test is applied, although lack of effect of the inoculation site in the specificity of the test should be confirmed in a larger sample.

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.

The aim of this study was to characterise the effects of maternal undernutrition during gestation on hepatic gluconeogenic enzyme gene expression and to determine whether such effects are mediated through epigenetic changes in the glucocorticoid receptor (GR). Pregnant ewes were fed a 50% nutrient-restricted diet from Day 0 to 30 (R1) or from Day 31 to 100 of gestation (R2) or a 100% diet throughout gestation (Control). After parturition lambs were fed to appetite. At 10 months of age offspring were euthanised and livers were removed. Maternal undernutrition did not affect offspring bodyweight at birth or at 10 months of age. However, liver weight of males of the R2 group was lower (P

The aim of the present study was to assess the presence of single nucleotide polymorphisms (SNPs) in the Growth Differentiating Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) genes, in two Greek sheep breeds i.e. Chios and Karagouniki, with high and moderate litter size (LS) i.e. number of lambs born, respectively. An examination of association(s) between the polymorphisms and LS, within breeds, was also conducted. Blood samples from 92 Chios and 96 Karagouniki ewes were collected, while repeated records on LS, were also available ( n = 239 and 259, respectively). Detection of SNPs was performed on the DNA products by PCR-RLFP analysis. Data analysis included examination of allelic and genotypic differentiation between the breeds, testing for Hardy–Weinberg Equilibrium (HWE) and association analysis between polymorphisms and LS. Genetic analysis showed that the G1 and G8 mutations of the GDF9 gene were significantly over-presented only in the highly prolific breed (Chios). The B4 mutation of the BMP15 gene was significantly over-presented only in the low prolific breed (Karagouniki). Both breeds were in Hardy–Weinberg disequilibrium (HWD), possibly due to selection pressure, population sub-structure and genetic drift. Certain polymorphisms of the G1, G4 and G8 sites of the GDF9 gene in the Chios breed were found to show heterozygote advantage with no evidence of infertility for the homozygous females. A similar trend was observed in the BMP15 gene although at no statistically significant level. In the Karagouniki breed, no significant associations between the studied polymorphisms and LS were detected.

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.

It has been suggested that infection with Corynebacterium pseudotuberculosis, the pathogen responsible for caseous lymphadenitis (CLA), might interfere with diagnostic testing for tuberculosis (TB), due to antigenic similarities between this particular type of bacterium and those expressed by mycobacteria. The aim of the present study was to investigate whether experimental infection with C. pseudotuberculosis in goats impacted on TB testing, using single and comparative intradermal tuberculin (SIT and SCIT respectively) tests and interferon (IFN)-γ assay. No positive reactors were detected among the CLA-affected goats using the SIT/SCIT tests or the interferon IFN-γ assay. A proportion of goats showed inconclusive results to the SIT test and reactions to Mycobacterium avium. There was no evidence that infection with C. pseudotuberculosis interferes with diagnostic testing for TB using standard interpretation of the SIT, SCIT and IFN-γ tests.

Tuberculosis (TB) in goats (. Capra hircus) is due to infection with members of the Mycobacterium tuberculosis complex (MTC), mainly Mycobacterium bovis and Mycobacterium caprae. We report a comparative experimental infection of goats with M. bovis, M. caprae and M. tuberculosis strains. We hypothesized that goats experimentally infected with different members of the MTC would display different clinical pictures. Three groups of goats were challenged with either M. bovis SB0134 (group 1, n=. 5), M. caprae SB0157 (group 2, n=. 5) and M. tuberculosis SIT58 (group 3, n= 4). The highest mean total lesion score was observed in M. bovis challenged goats (mean 15.2, range 9-19), followed by those challenged with M. caprae (10.8, 2-23). The lowest score was recorded in goats challenged with M. tuberculosis (3, 1-6). Culture results coincided with the lesion scores in yielding more positive pools (7/15) in M. bovis challenged goats. By contrast, only three pools were positive from goats challenged M. tuberculosis (3/12) and with M. caprae (3/15), respectively. Differences in the performance of the intradermal and gamma-interferon (IFN-γ) tests depending of the group were observed since all goats from group 1 were diagnosed using intradermal test and these goats reacted earlier to the IFN-γ assay in comparison to the other groups. This study confirmed that goats experimentally infected with different members of the MTC display different clinical pictures and this fact may have implications for MTC maintenance and bacterial shedding., TB research funding is acknowledged to AGL2012-36171 and AGL2014-56305 (MINECO, Spain and FEDER) and EU FP7 grants WildTBvac #613779 and ANTIGONE #278976. Javier Bezos is recipient of a Torres-Quevedo contract (PTQ-12-05812) funded by the Ministry of Economy and Competitiveness and the European Social Fund.

Staphylococcus aureus causes many serious diseases in humans and animals, and it is the most common aetiologic agent of contagious bovine mastitis. The bacteria produce several virulence factors and the importance of evaluating the combination of these virulence factors has been recently emphasized. In study, the combination of several virulence factors: coagulase gene (coa), protein A gene (spa), collagen-binding protein gene (cna), fibrinogen-binding protein gene (efb), Panton-Valentin leukocydin gene (pvl) and enterotoxins (sea,seb, sec, sed, see, seg, seh, sei, sej) was considered. The analysis of the relationship between presence/absence of the different genes and the udder inflammatory response measured by milk somatic cell counts was performed by general linear models and logistic regression. The classification of isolates in clusters by virulence genes combinations showed that at least one cluster induced a higher inflammatory response. Moreover, the analysis of the association between virulence genes and the presence of a subclinical mastitis showed the role of spa and sej gene as risk factors. These results support that the development of subclinical mastitis could be related to strains characteristics and to the expression of specific combinations of the virulence factors.

To the Editor: Rabies is caused by 12 recognized virus species within the Lyssavirus genus (family Rhabdoviridae) (1) and each year causes 55,000 deaths worldwide among humans. In Europe, the main reservoir and vector of rabies is the red fox (Vulpes vulpes), followed by the raccoon dog (Nyctereutes procyonoides) in central and Baltic Europe (2). Among these virus species, the rabies virus (former genotype 1, classical rabies virus) is maintained in reservoir mammals, mainly carnivores. Despite the successful oral vaccination campaigns of wildlife that eliminated rabies in large parts of Europe (2), the disease still occurs in Europe; 4,884 cases in animals were recorded in 2012. Until 2012, Greece had been free of rabies since 1987; the last case occurred in a dog. During 1971–1987, a total of 248 cases were recorded in domestic animals, of which only 6 occurred during 1981–1987 (3). The wide compulsory vaccination of dogs, along with control of stray dogs, were the main measures leading to the elimination of rabies in Greece. Greece shares land borders with Turkey, Former Yugoslav Republic of Macedonia (FYROM), and Bulgaria, where rabies is reported in wild and domestic animals (4; www.who-rabies-bulletin.org/). After the fox rabies case reported in FYROM in 2011, ≈0.3 km from Greece (4), rabies surveillance was improved along the northern and eastern land borders of Greece, and wild and domestic animals found dead or suspected of having rabies were collected. This program was approved by the European Commission. The first rabies case was diagnosed on October 19, 2012, in a red fox in Palaiokastro, 60 km from the Albanian border. The animal was wandering in the village during daytime and attacked a dog before being killed by local hunters and sent to the National Reference Laboratory for Animal Rabies (Athens, Greece) for rabies testing. The second case, isolated in Ieropigi, 4 km from the Albanian border, was a shepherd dog demonstrating aggressive behavior against dogs and sheep of the flock. Seven additional cases (6 foxes, 1 dog) were reported in December 2012 in 2 prefectures of northern Greece that share borders with FYROM (5). In 2012, a total of 237 domestic and wild animals were submitted to the National Reference Laboratory for Animal Rabies for rabies testing. Samples were tested by the fluorescent antibody test, rabies tissue culture infection test, real-time reverse transcription PCR, and heminested reverse transcription PCR, as described (6,7). In brief, viral RNA was extracted (Viral RNA Mini Kit, QIAGEN, Hilden, Germany) from 140 μL of homogenized brain suspension supernatant and subjected to the partial nucleoprotein (N) gene amplification (positions 71–644 compared with PV strain genome). The PCR products were bidirectionally sequenced by using the same primers in a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The phylogenetic tree was constructed by using the neighbor-joining method with 1,000 replicates using MEGA5 (8). Of 237 animals tested, 9 (7 foxes, 2 dogs) were rabies positive by fluorescent antibody test, rabies tissue culture infection test, and PCR. Positive samples were subjected to sequencing analysis of the N gene. The sequence analysis of the first 567 nt of the N gene of the 3 isolates (GR64C/12, GR112C/12, GR238C/12) showed 99.8% nt identity with the N gene of a rabies virus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JQ973884","term_id":"409191659","term_text":"JQ973884"}}JQ973884) isolated from a red fox in FYROM in 2011. Nucleotide identity was 100% between the 6 isolates from Greece (GR177C/12, GR187C/12, GR217C/12, GR231C/12, GR236C/12, GR242C/12) and isolate {"type":"entrez-nucleotide","attrs":{"text":"JQ973884","term_id":"409191659","term_text":"JQ973884"}}JQ973884, as well as a Serbia strain (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JF973785","term_id":"337752481","term_text":"JF973785"}}JF973785). This perfect nucleotide identity shown with the Serbia isolate collected >15 years ago suggests the persistence of some viral strains over time in the Balkans, in accordance with previous studies (4,9). Amino acid identity was perfect among all 9 isolates from Greece. Phylogenetic analysis of the partial sequences of the N gene of isolates from Greece compared with representative sequences from the Balkans (Figure) showed that isolates from Greece resolved within the East Europe (EE) group of the cosmopolitan lineage. The EE group encompasses the 9 Greek isolates with referenced viral sequences from FYROM, Bulgaria, Serbia, Bosnia Herzegovina, and Montenegro. Within the EE group

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.

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.

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.

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.

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.

International audience; Systemic mycobacteriosis associated with avian polyomavirus infection was diagnosed histologically in an 8-year-old, captive European goldfinch with a history of nervous signs. Severe mycobacterial lesions were observed in central nervous system, lungs, cervical air sacs and adrenal glands, without involvement of the gastrointestinal tract. In addition to mycobacteriosis, intranuclear inclusions, typical of polyomavirus, were identified in the adrenal glands. Polymerase chain reaction assays were used to identify Mycobacterium genavense and finch polyomavirus as the causative agents. The absence of involvement of the gastrointestinal tract and the severity of the lesions in the respiratory tract suggested that inhalation may have been the primary route of infection with M. genavense.