Development and validation of a luminescence-based, medium-throughput assay for drug screening in Schistosoma mansoni

Schistosomiasis, one of the world's greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs. METHODOLOGY/PRINCIPAL FINDINGS: The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery. CONCLUSIONS: The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies

Valutazione della tossicità acuta sul tessuto retinico dopo somministrazione di aminoglicosidi e fluorochinoloni in un modello sperimentale animale su ratto albino.

Titolo della tesi: Valutazione della tossicità acuta sul tessuto retinico dopo somministrazione di aminoglicosidi e fluorochinoloni in un modello sperimentale animale su ratto albino. Introduzione – L'occhio è un organo sferoidale, posto nella cavità orbitaria, direttamente a contatto con l’ambiente esterno e pur essendo protetto dalle palpebre e da altri annessi oculari è soggetto all’attacco di numerosi batteri gram-positivi e gram-negativi. Le infezioni oculari più frequenti sono la congiuntivite, la cheratite e l’endoftalmite. La terapia ad oggi più utilizzata nelle patologie oftalmiche batteriche è costituita da due classi di antibiotici: aminoglicosidi e fluorochinoloni, in particolare da Tobramicina, Netilmicina (aminoglicosidi) e Norfloxacina (fluorochinolone). In letteratura sono noti alcuni aspetti circa la tossicità oculare a carico di alcuni aminoglicosidi mentre mancano studi dettagliati circa la possibile tossicità oculare di fluorochinoloni. Scopo della ricerca – Nel nostro studio abbiamo voluto valutare le eventuali alterazioni retiniche causate dalla Tobramicina, dalla Netilmicina e dalla Norfloxacina dopo somministrazione topica in acuto, in un modello sperimentale su ratto. Abbiamo quindi valutato l'attività elettrica della retina in risposta a particolari stimoli luminosi, il numero delle file di fotorecettori presenti nello strato nucleare esterno della retina e l’intera morfologia retinica. Materiali e metodi – Sono stati utilizzati 18 ratti maschi Wistar (Harlan, Italy) adulti sani, con un peso di circa 250g ± 25g che sono stati randomizzati nei seguenti gruppi sperimentali:1.Gruppo trattato con Norfloxacina; 2.Gruppo trattato con Tobramicina; 3.Gruppo trattato con Netilmicina. Gli animali di ciascun gruppo hanno ricevuto il rispettivo farmaco topicamente nell’occhio destro, 2 gocce applicate 6 volte al giorno per 5 giorni, mentre l’occhio sinistro, usato come controllo, è stato trattato con soluzione fisiologica con identica modalità di somministrazione. Prima dell’inizio del trattamento, dopo la prima applicazione e a fine trattamento è stato effettuato l’ERG da flash per andare a valutare l’attività elettrica della retina. Subito dopo l’ultima registrazione elettroretinografica gli animali sono stati anestetizzati, operati per l’asportazione di entrambi gli occhi e sacrificati. Abbiamo poi isolato il tessuto retinico per l’allestimento dei preparati istologici. Le retine sono state fissate, incluse e criosezionate seguendo procedure standard. Parte delle sezioni sono state colorate con ioduro di propidio per valutare la morfologia dello strato esterno plessiforme di cellule nervose della retina, andando a contare il numero di file di nuclei presenti in tale strato. Le sezioni rimanenti sono state colorate con ematossilina-eosina in modo da valutare la morfologia dell’intero tessuto retinico. Risultati – Le registrazioni elettroretinografiche prima del trattamento, dopo 2h, e dopo 5gg di somministrazione topica di Norfloxacina, di Netilmicina e di Tobramicina, non evidenziano alterazioni significative dell’ampiezza dell’onda b, se confrontati con il controllo. Limitando l’osservazione al tracciato dell’occhio che ha ricevuto Tobramicina si osserva una riduzione dell’ampiezza dell’onda b. Tale riduzione è tuttavia non significativa e costituisce, al momento, solo un trend. Dalla valutazione isto-patologica dello strato nucleare esterno della retina non si osservano diminuzioni, in numero, delle file di cellule nervose successivamente a somministrazione dei tre antibiotici. La morfologia retinica non evidenzia alterazioni patologiche successivamente a somministrazione dei tre antibiotici. Conclusioni – Dai risultati sperimentali ottenuti possiamo concludere che la somministrazione topica di Netilmicina e Norfloxacina risulta priva di effetti collaterali sulla visione, infatti non si evidenziano variazioni delle ampiezze dell’onda b dopo 5gg; mentre è necessario utilizzare con cautela la Tobramicina; dal momento che è stato rilevato un trend negativo nell’andamento delle ampiezze dell’onda b dopo 5gg, Tuttavia dato che da un punto di vista morfologico, per nessuno dei tre farmaci, non sono state rilevate alterazioni, per escludere il verificarsi di degenerazione del tessuto retinico si rendono necessarie ulteriori future analisi con tempi di somministrazione più prolungati

New provisions for the labelling of fishery and aquaculture products: Difficulties in the implementation of Regulation (EU) n. 1379/2013

The European Union (EU), within the renewal plan of the Common Fisheries Policy and the Common Market Organization, with the Cape IV of Reg. (EU) n. 1379/2013 have introduced new requirements for the labelling of fisheries and aquaculture products. These, as well as providing consumers with more complete information, integrate the provisions of Reg. (EU) n. 1169/2011 and acts as a tool to prevent frauds and illegal fishing. In this work the new seafood labelling provisions were evaluated, starting from the analysis of the art. 35 of the Chapter IV and comparing it with the previous EU dispositions (Reg. (EC) no. 104/2000 and no. 2065/2001). The exclusion of prepared and processed products and aquatic invertebrates from the application of the mandatory seafood labelling provisions and the role of the mass caterer operators with respect to the labelling requirements were identified as the two major shortcomings that still need to be better addressed by the legislator. Overall, what emerged from this work is that, if on the one hand the European legislation on seafood labelling has achieved important goals, evolving and improving itself, on the other it is still controversial and plagued by the same problems as 15 years ago. Therefore, the authors suggest that the regulation is modified at least extending its scope to all products and to at all stages of the fishery logistic chai

Occurrence and viability of Anisakis spp. larvae in ready to eat products made of herring (Clupea harengus) sold in Italy

The study aimed to assess the occurrence and viability of Anisakis spp. larvae in ready to eat products made of herring, a common host of Anisakis spp. and the third most commercialized fish species in the EU. A total of 135 products consisting of 50 smoked whole specimens and 85 filleted products (25 smoked, 30 marinated, 30 canned) were sampled from 2016 to 2018. Viscera and muscle of whole herrings were visually inspected and separately digested. Filleted products were also visually inspected and digested. Larvae viability was assessed, then they were counted and microscopically identified to genus level. A subsample was molecularly identified. At least one Anisakis spp. larva was found in 56 products (41.5%), for a total of 1715 larvae collected (0-172 larvae/product). Most of the larvae (91%) were found in the viscera of 49 of the 50 whole herrings (98%). A highly significant difference was observed between the positivity rate and larval density at muscle level, as 149 larvae were found in the muscle of 31 whole herrings (positivity rate 62%, 0.022 larval density/g), while only 7 larvae were found in the 85 filleted products (positivity rate 7%, 0.001 larval density/g). Larvae were molecularly identified as A. simplex. The study showed that obviously contaminated products are commercialized. All the larvae were dead, proving a negligible risk of developing anisakiasis. However, their allergenic potential is debated and the significant difference between muscle infection levels in whole and filleted herrings may result in a different risk of exposure to antigens

Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)

Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products

A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method to Combat Health Frauds

The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds

Development of a Simple and Cost-Effective Bead-Milling Method for DNA Extraction from Fish Muscles

In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs

The uncertainty of seafood labeling in China: A case study on Cod, Salmon and Tuna

Exotic marine fish products are increasingly appreciated in China. In this study, 100 samples of Cod, Salmon and Tuna products were collected from supermarkets in Shanghai, Nanjing and Hangzhou. First the information reported on the label were assessed in the light of the Chinese legislation, paying particular attention to the fish names and the geographical origin. Then, a comparative analysis of the official trade denominations adopted by five European countries (Italy, France, Germany, Spain and United Kingdom) for Cod, Salmon and Tuna was performed. Finally, the Chinese names of the species considered in the EU list were verified consulting the available international lists. Overall, 95% of the samples employed just generic names. In particular, 98% of Salmon and 100% of Tuna products were generically labeled while the labeling of Cod products was more diversified, even though 80% reported misleading or fake denominations. The results of this work highlighted the lack of a mandatory legislation on seafood traceability and of an official naming system. In particular, this study propose the introduction of a detailed Chinese naming system based on the Chinese Latin Dictionary for Seafood Names, following the EU approach. In fact, inaccurate labeling can have both economic and health implications for consumers as well as it may distort the true abundance of fish stocks. These drawbacks can be particularly serious considering the pivotal role of China in the global fishery industry

Evolution of the Anisakis risk management in the European and Italian context

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy