A validated molecular protocol to differentiate pure wolves, dogs and wolf x dog hybrids through a panel of multiplexed canine {STR} markers

The conservation of pure wolf populations in Europe is currently threatened by anthropogenic hybridization with dogs, through potential extinction of local gene pools due to replacement with domestic gene variants. Distinction of wolf–dog hybrids from wild ancestors is essential for development and implementation of management and conservation plans. Morphological traits are rarely helpful, and a genetic approach is the most effective to distinguish admixed individuals back to old backcrosses. To provide a molecular tool to address the issue, we optimized and validated a cost-effective protocol in multiplexed PCR format based on 22 STR canine loci, which allows us to distinguish genetically pure wolves from dogs, and, when used in association with a Bayesian assignment approach, is capable of statistically assigning admixed individuals to classes of hybrids with different levels of dog ancestry. Our method demonstrated high reliability, showing full repeatability and reproducibility of data with as little as 0.125 ng of genomic DNA, and was therefore suitable for the analysis of non-invasively collected samples and degraded DNA. The application of our STR panel to the appropriate assignment procedure unambiguously defined two genetically separated clusters for wolves and dogs, and successfully identified known hybrids as admixed individuals, which eventually were classified as recent hybrids and older backcrosses. The protocol, which is described here in detail, can be adopted by various laboratories that need this kind of diagnosis; furthermore, it would be capable of producing concordant results through inter-laboratory comparisons with wolf and dog control DNAs

Oceanic giants in the Mediterranean: first mitochondrial analysis of leatherback turtles (Dermochelys coriacea) in the Adriatic and Tyrrhenian seas

The leatherback turtle Dermochelys coriacea (Vandelli, 1761) is an occasional trophic visitor of the Mediterranean basin. So far, very few individuals have been genetically analysed from this sea and none from Italy. We sequenced a mitochondrial DNA fragment of three specimens of D. coriacea found in recent years along the North-Adriatic and Tyrrhenian shores. They were all females approaching adult stage. Causes of death were attributable to the main threats for sea turtles in Mediterranean waters, all related to human activities (collisions with boats, entanglement in fishing nets and plastic debris ingestion). Two different mitochondrial haplotypes were observed, with the two North-Adriatic turtles sharing the same one. Compared to known Dermochelys sequences and previous genetic characterization of rookeries, these results suggest that the most probable origin of at least two of the three leatherbacks was the western Atlantic

Cardiovascular flukes (Trematoda: Spirorchiidae) in Caretta caretta Linnaeus, 1758 from the Mediterranean Sea

Background: The northern Adriatic Sea represents one of the most important neritic foraging grounds for the loggerhead sea turtle Caretta caretta L. in the Mediterranean Sea. Four genera of blood flukes with variable prevalence and pathogenic impact have been reported worldwide in this species. Hapalotrema Looss, 1899 and Amphiorchis Price, 1934 are the only two genera reported in Mediterranean waters; however, updated data describing spirorchiidiasis in the central and eastern Mediterranean and infection prevalence are still lacking. This work aimed to investigate the presence and pathology of spirorchiidiasis in C. caretta in the Mediterranean Sea. Methods: One hundred sixty-eight animals stranded along the northwestern Adriatic coast between 2009 and 2015 were submitted to necropsy and subsequent analyses for the detection of adult flukes, detection of eggs in the faeces and spleen and histopathology. Molecular analyses were carried out on hosts (mitochondrial D-loop) and parasites (28S gene and ITS2 spacer) to trace the turtle origins and identify the fluke phylogenetic relationships. Results: Spirorchiidiasis was detected in 16.7% of the animals. Hapalotrema mistroides (Monticelli, 1899) and Neospirorchis sp. were found in twenty-six and ten cases, respectively. Adult flukes were found in six cases, while eggs were detectable through copromicroscopic examination for all infected turtles, and the results for the detection of eggs in the spleen agreed with the copromicroscopic analysis. Only mild lesions were observed. Eggs of types 1 and 3 were grossly visible in the gastrointestinal mucosa, vasculitis was rarely observed in the heart and great vessels, and multifocal granulomas were widespread in the tissues. Molecular identification unambiguously assigned the spirorchiid samples to H. mistroides and Neospirorchis sp. Genetic characterization of loggerhead mtDNA pointed to a Mediterranean origin of the turtle hosts. Conclusion: This survey provides new data on the spread of spirorchiidiasis in the Mediterranean loggerhead sea turtle population and reports for the first time the presence of Neospirorchis spp. in this basin. The infections did not have a causal effect on the death nor a strong impact on the general health status of the animals

Oceanic giants in the Mediterranean: first mitochondrial analysis of leatherback turtles (Dermochelys coriacea) in the Adriatic and Tyrrhenian seas

The leatherback turtle Dermochelys coriacea (Vandelli, 1761) is an occasional trophic visitor of the Mediterranean basin. So far, very few individuals have been genetically analysed from this sea and none from Italy. We sequenced a mitochondrial DNA fragment of three specimens of D. coriacea found in recent years along the North-Adriatic and Tyrrhenian shores. They were all females approaching adult stage. Causes of death were attributable to the main threats for sea turtles in Mediterranean waters, all related to human activities (collisions with boats, entanglement in fishing nets and plastic debris ingestion). Two different mitochondrial haplotypes were observed, with the two North-Adriatic turtles sharing the same one. Compared to known Dermochelys sequences and previous genetic characterization of rookeries, these results suggest that the most probable origin of at least two of the three leatherbacks was the western Atlantic

The position of nonsense mutations can predict the phenotype severity : A survey on the DMD gene

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used theDMDgene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, theDMDgene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, becauseDMDmutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.Peer reviewe

Polyphasic characterization of microbiota of “Mastredda”, a traditional wooden tool used during the production of PDO Provola dei Nebrodi cheese

11openInternationalItalian coauthor/editorThe biofilms of the wooden tables used for the acidification of the curd were investigated for PDO Provola dei Nebrodi cheese, a traditional stretched cheese made in eastern Sicily (southern Italy) from raw cows’ milk. To this purpose the wooden tables of four dairy facilities were analysed for their microbiota by scanning electron microscopy (SEM) analysis and a combined culture-independent and -dependent microbiological approach. SEM inspection showed an almost continuous biofilm formation. MiSeq Illumina analysis identified 8 phyla, 16 classes, 25 orders, 47 families and 50 genera. Corynebacterium, Bifidobacterium and lactic acid bacteria (LAB) were detected in all samples. In particular, the LAB genera detected on all wooden tables were Lactobacillus, Streptococcus and Lactococcus. LAB dominated the surfaces of all wooden tables with levels higher than 7.0 Log CFU/cm2. In particular, the LAB found at the highest levels were mesophilic cocci. Coagulase positive staphylococci, Salmonella spp., Listeria monocytogenes and Shiga-toxigenic Escherichia coli were never detected. Twenty-seven dominating LAB strains were identified within the genera Enterococcus, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. This work showed that the wooden table used during the production of PDO Provola dei Nebrodi cheese is a safe system and a microbiologically active toolopenBusetta, Gabriele; Garofalo, Giuliana; Mangione, Guido; Botta, Luigi; Franciosi, Elena; Di Gerlando, Rosalia; Todaro, Massimo; Licitra, Giuseppe; Scatassa, Maria Luisa; Gaglio, Raimondo; Settanni, LucaBusetta, G.; Garofalo, G.; Mangione, G.; Botta, L.; Franciosi, E.; Di Gerlando, R.; Todaro, M.; Licitra, G.; Scatassa, M.L.; Gaglio, R.; Settanni, L

Pyogenic Granuloma of the Sigmoid Colon causing Intussusception in an Infant

Pyogenic granuloma is a benign vascular tumor that may affect the gastrointestinal tract. This report describes a rare case of sigmoid-colon pyogenic granuloma in a 4-month-old boy causing intussusception. Resection and anastomosis were curative. The mother had history of high dose of progesterone exposure during initial weeks of conception for vaginal bleeding. This may point towards etiology of the lesion

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads b 5.0 Log cfu g−1 . Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy produc

Reduction of PDO Pecorino Siciliano cheese making duration: microbial dynamics and quality attributes deriving from replacing whey permeate with hot water during cooking

This work was carried out with the aim to reduce the transformation duration of Protected Designation of Origin (PDO) Pecorino Siciliano cheese. To this purpose, the cooking in hot water (experimental production, EXP) was compared to the traditional cheese cooking under whey permeate (control production, CTR). The microbiological composition of under rind (UR) and core (Co) section of CTR and EXP cheeses was determined by a combined culture-dependent and -independent approach. Total mesophilic microorganisms and lactic acid bacteria (LAB) present in raw ewes' milk (5.0 log CFU/mL) increased during cheese making and reached values of about 8.0 log CFU/g in both sections (UR and Co) of 5-month ripened cheeses of both productions (CTR and EXP) monitored. The identification of the viable LAB populations in ripened cheeses showed that Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus and Streptococcus dominated UR and Co sections of all cheeses. MiSeq Illumina analysis demonstrated that LAB populations (lactobacilli, lactococci and streptococci) dominated the bacterial community of cheeses at 95.63-98.41 % of relative abundance. The two different cooking operations did not influence the physicochemical characteristics of PDO Pecorino Siciliano cheeses. Sensory evaluation performed by artificial senses analysis and trained panelists confirmed that the modification of PDO Pecorino Siciliano cheese production protocol did not significantly affect product characteristics and overall acceptance. Thus, data of this work confirmed that cooking under hot water allowed to reduce transformation duration and safeguard typicality of PDO Pecorino Siciliano cheese

Targeted gene panel screening is an effective tool to identify undiagnosed late onset Pompe disease

Mutations in the GAA gene may cause a late onset Pompe disease presenting with proximal weakness without the characteristic muscle pathology, and therefore a test for GAA activity is the first tier analysis in all undiagnosed patients with hyperCKemia and/or limb-girdle muscular weakness. By using MotorPlex, a targeted gene panel for next generation sequencing, we analyzed GAA and other muscle diseasegenes in a large cohort of undiagnosed patients with suspected inherited skeletal muscle disorders (n = 504). In this cohort, 275 patients presented with limb-girdle phenotype and/or an isolated hyperCKemia. Mutational analysis identified GAA mutations in ten patients. Further seven affected relatives were identified by segregation studies. All the patients carried the common GAA mutation c.-32-13T > G and a second, previously reported mutation. In the subcohort of 275 patients with proximal muscle weakness and/or hyperCKemia, we identified late-onset Pompe disease in 10 patients. The clinical overlap between Pompe disease and LGMDs or other skeletal muscle disorders suggests that GAA and the genes causing a metabolic myopathy should be analyzed in all the gene panels used for testing neuromuscular patients. However, enzymatic tests are essential for the interpretation and validation of genetic results. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe