Uticaj metode uzorkovanja na detekciju Campylobacter coli iz pribora za uzorkovanje i mesa

A defined Campylobacter coli (C. coli) suspension was inoculated on sterile sampling materials (cotton bud, polyester bud, cellulose sponge) and pieces of lamb meat. Various combinations of diluents (phosphate buffer saline ± Tween®80) and sampling methods (direct homogenization, simulating the excision method for meat, and swabbing) were investigated for the recovery (detachment) of C. coli cells from the inoculated samples. The obtained C. coli bacteria, as quantified by real-time PCR with respect to the dilution factors and the initial inoculum, were used for the calculation of the recovery (%) per sampling material and method. Regarding artificially inoculated sampling materials, the lowest recovery was observed for cotton buds (2.8%) and the highest for cellulose sponge (28.9%), and the differences between the obtained results were statistically significant (P < 0.05). As regards lamb meat, the lowest recovery was observed for swabbing with cotton buds (3.2%) and the highest for direct homogenization (10.7%). The results indicate an overall low rate of bacterial recovery from contaminated samples, with cellulose sponges and polyester buds being significantly superior to cotton buds, and direct homogenization of meat with diluent better than swabbing. The type of sampling materials and methods applied for the quantification of C. coli entailsPrethodno određena suspenzija Campylobacter coli inokulisana je na sterilne materijale za uzorkovanje (pamučni štapić, poliesterski štapić, celulozni sunđer) i komade jagnjeć eg mesa. Različite kombinacije razređivača (fi ziološki rastvor fosfatnog pufera ± Tveen®80) i metode uzimanja uzoraka (direktna homogenizacija, simuliranje metode ekscizije mesa i uzimanje brisa) ispitivane su na uspešnost detekcije ć elija C. coli iz inokulisanih uzoraka. Kvantifi kacija je urađena PCR-om u realnom vremenu u odnosu na faktore razblaženja i početni inokulum i izračunat je nivo detekcije C. coli (%) prema materijalu i metodi uzimanja uzoraka. Iz veštački inokulisanih materijala za uzimanje uzoraka najniža detekcija ovih bakterija je zabeležena kod pamučnih štapića (2,8%), a najveća kod celuloznih sunđera (28,9%) i utvrdjena je statistički značajna razlika (

Zamjena masti u trajnim kobasicama emulzijom ekstra djevičanskog maslinovog ulja s dodatkom autohtonih bakterija mliječno-kiselog vrenja

Research background. Formulations based on vegetable or fish oil and modifications in the production technology of dry fermented sausages have emerged in recent years aiming to achieve the desirable target of reducing the fat content of these meat products. However, previous efforts have confronted many difficulties, such as high mass loss and unacceptable appearance due to intensely wrinkled surfaces and case hardening. The objective of this study is to produce and evaluate dry fermented sausages by utilising a meat protein-olive oil emulsion as fat substitute and indigenous lactic acid bacteria (LAB) with probiotic properties isolated from traditional Greek meat products. Experimental approach. A novel formulation with extra virgin olive oil and turkey protein was developed to totally replace the conventionally added pork fat. Probiotic and safety characteristics of autochthonous LAB isolates from spontaneously fermented sausages were evaluated and three LAB isolates were finally selected as starter cultures. Physicochemical, microbiological and sensory analyses were carried out in all treatments (control, Lactobacillus acidophilus, L. casei, L. sakei and Pediococcus pentosaceus) during fermentation. Results and conclusions. Ready-to-eat sausages were found to be microbiologically stable. The olive oil-based formulation produced in this study generated a mosaic pattern visible in the sliced product simulating the fat in conventional fermented sausages and was regarded as an ideal fat substitute for the production of fermented sausages. An autochthonous isolate of Lactobacillus casei adapted the best to the final products as it was molecularly identified to be present in the highest counts among the LAB isolates used as starter cultures. Novelty and scientific contribution. Α novel and high-quality dry fermented meat product was produced by replacing the added pork fat with a fat substitute based on a meat protein-olive oil emulsion. Autochthonous LAB with in vitro probiotic properties could have a potential use in large-scale novel dry fermented sausage production. Such isolates could be used as starters in an effort to standardise the production process and retain the typical organoleptic and sensory characteristics. Moreover, isolates like L. casei 62 that survived in high counts in the final products can increase the safety of fermented sausages by competing not only with pathogens but also with the indigenous microbiota and could have a potential functional value for the consumer.Pozadina istraživanja. Posljednjih godina se u proizvodnji trajnih kobasica koriste novi pripravci na bazi biljnog ili ribljeg ulja koji smanjuju udjel masti u tim mesnim proizvodima. Međutim, dosadašnji napori u dobivanju takvih proizvoda nailazili su na mnoge prepreke, kao što su veliki gubitak mase i neprihvatljiv izgled proizvoda zbog prevelike naboranosti površine i tvrdoće crijeva. Svrha je ovoga rada bila proizvesti trajne kobasice s emulzijom maslinovog ulja i proteina mesa kao zamjenom za masti, uz dodatak autohtonih bakterija mliječno-kiselog vrenja s probiotičkim svojstvima izoliranih iz tradicionalnih grčkih mesnih proizvoda, te ispitati svojstva dobivenih proizvoda. Eksperimentalni pristup. Razvijen je novi pripravak s ekstra djevičanskim maslinovim uljem i proteinima iz puretine koji bi u potpunosti zamijenio tradicionalno dodavanu svinjsku mast. Ispitana su probiotička svojstva i sigurnost primjene autohtonih bakterija mliječno-kiselog vrenja izoliranih iz tradicionalnih fermentiranih kobasica, i izdvojena su tri izolata koja su zatim upotrijebljena kao starter kulture. Tijekom fermentacije provedene su fizikalno-kemijske, mikrobiološke i senzorske analize svih ispitanih uzoraka (kontrolnog uzorka, te kobasica proizvedenih s pomoću Lactobacillus acidophilus, L. casei, L. sakei i Pediococcus pentosaceus). Rezultati i zaključci. Gotovi proizvodi bili su mikrobiološki stabilni. Dodatkom pripravka na bazi maslinovog ulja dobivena je kobasica koja je na presjeku imala mozaičan izgled sličan onom tradicionalne kobasice, pa se pripravak smatra idealnom zamjenom za mast u proizvodnji trajnih kobasica. Molekularnom je analizom utvrđeno da je među bakterijama mliječno-kiselog vrenja koje su korištene kao starter kulture u gotovom proizvodu bilo najviše bakterija Lactobacillus casei, što potvrđuje da se ta vrsta najbolje prilagođava uvjetima proizvodnje. Novina i znanstveni doprinos. Proizveden je novi visoko kvalitetni sušeni fermentirani mesni proizvod zamjenom svinjske masti emulzijom maslinovog ulja i proteina iz puretine. Autohtone bakterije mliječno-kiselog vrenja s potvrđenim probiotičkim svojstvima in vitro mogle bi se primijeniti u proizvodnji trajnih kobasica na veliko. Dobiveni bi se izolati mogli primijeniti kao starter kulture za standardizaciju postupka i očuvanje tipičnih organoleptičkih i senzorskih svojstava kobasica. Osim toga, izolati poput L. casei 62, koji su pronađeni u velikom broju u gotovom proizvodu, svojom kompetitivnošću ne samo s patogenim sojevima, već i s autohtonom mikrobiotom, povećavaju sigurnost fermentiranih kobasica te imaju potencijalnu funkcionalnu vrijednost za potrošače

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

BACKGROUND: Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. METHODS: The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. CONCLUSIONS: The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. SIGNIFICANCE: The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food

Comparison of 2016–17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016–17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016–17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005–06 and 2016–17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016–17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control

Comparison of 2016–17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016–17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016–17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005–06 and 2016–17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016–17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control

Evolution and Phylogenetic Analysis of Full-Length VP3 Genes of Eastern Mediterranean Bluetongue Virus Isolates

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979–2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an ‘eastern’ (BTV-9, -16 and -1) and a ‘western’ (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe

Characterization and development of detection methods of Closteroviruses, Foveaviruses and Vitiviruses in grapevine

Η σπουδαιότητα της αμπελοκαλλιέργειας στην Ε.Ε. και την Ελλάδα ειδικότερα, σε συνδυασμό με τις επερχόμενες αλλαγές και αυξημένες απαιτήσεις στο χώρο της πιστοποίησης πολλαπλασιαστικού υλικού καθιστούν αναγκαία την ύπαρξη αξιόπιστων τεχνικών για την ανίχνευση αλλά και τον χαρακτηρισμό των ιών της αμπέλου. Παρά την εντυπωσιακή αύξηση των γνώσεων τα τελευταία χρόνια σε ότι αφορά την αιτιολογία της συστροφής των φύλλων και της βοθρίωσης του ξύλου της αμπέλου, ο μεγάλος αριθμός των ιών που σχετίζονται με τις ασθένειες αυτές και τα μειονεκτήματα των διάφορων δοκιμών ανίχνευσης/ταυτοποίησης, καθιστούν τον έλεγχο του πολλαπλασιαστικού υλικού μια διαδικασία σύνθετη, συχνά με μικρή αξιοπιστία, επίπονη, χρονοβόρα, και με υψηλό κόστος. Επίσης, η συνεχιζόμενη ανακάλυψη νέων clostero-και viti-ιών δείχνει ότι στην αιτιολογία των ασθενειών εμπλέκονται και άλλοι άγνωστοι ιοί που ανήκουν στα γένη αυτά και για τα οποία προς το παρόν δεν υπάρχει δυνατότητα ανίχνευσης. Αντικείμενο της διδακτορικής διατριβής ήταν η ανάπτυξη αξιόπιστων μεθόδων ανίχνευσης καθώς και ο χαρακτηρισμός νέων άγνωστων ιών που σχετίζονται με τη συστροφή των φύλλων και τη βοθρίωση του ξύλου της Αμπέλου. Αρχικά, έγινε έλεγχος πρέμνων αμπέλου προερχόμενων από κλωνική επιλογή για την παρουσία ιών που σχετίζονται με τις δύο αυτές ασθένειες, με τις διαθέσιμες ορολογικές και μοριακές και τεχνικές, με στόχο τη συλλογή απομονώσεων ιών των γενών Closterovirus, Foveavirus και Vitivirus. Στη συνέχεια, αναπτύχθηκε μια μέθοδος πολλαπλής εστιασμένης RT-PCR η οποία χρησιμοποιεί εκκινητές με εκφυλισμό, για την ταυτόχρονη ανίχνευση ιών που ανήκουν στα γένη Foveavirus και Vitivirus. Η μέθοδος αυτή συνδυάσθηκε με μια απλουστευμένη και αξιόπιστη μέθοδο επεξεργασίας των δειγμάτων (αποτύπωση εκχυλίσματος φυτικού χυμού σε νάιλον μεμβράνη) και με μια δοκιμή, ανιχνεύει ταυτόχρονα τους RSPaV-1, GVA, GVB, και GVD στο αμπέλι, ενώ θεωρητικά είναι δυνατή η ανίχνευση και άλλων μελών που ανήκουν στα γένη Foveavirus και Vitivirus και προσβάλλουν άλλους ξενιστές. Επίσης, αναπτύχθηκε μια ακόμη μέθοδος πολλαπλής εστιασμένης RT-PCR για την ταυτόχρονη ανίχνευση ιών που ανήκουν στα γένη Closterovirus, Foveavirus και Vitivirus όπου μαζί με τους εκφυλισμένους εκκινητές που περιέχουν δεοξυινοσίνη, χρησιμοποιούνται αντίστοιχο

Incidence of viruses infecting spinach in Greece, highlighting the importance of weeds as reservoir hosts

The aim of this survey was to identify viruses infecting spinach (Spinacia oleracea L.) in the most important spinach-producing areas in Greece. A total of 1074 spinach samples were collected from eleven districts belonging to the prefectures of Thessaloniki, Chalkidiki and Imathia in northern Greece, and Evia in central Greece. Samples were tested by ELISA, mechanical inoculation onto indicator plants and immunoelectron microscopy. Beet western yellows virus (BWYV), Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) were identified in 13.5%, 7% and 5.4% of samples, respectively, infected samples being detected in all regions examined. This is the first record of CMV and TuMV infecting spinach in Greece, and the first report of BWYV occurrence in any crop nationwide. Surveys were also conducted to assess the potential reservoir hosts of BWYV, CMV and TuMV in weeds collected from spinach fields. All three viruses were detected among 125 samples tested by ELISA. TuMV prevailed as it occurred in 14.4% of all weed sample