Author(s)

Roberto Zoccola^1*, Alessandra Pautasso¹, Carla Grattarola¹, Milena Monnier¹, Ilaria Miceli¹, Francesco Ingravalle¹, Maria Ines Crescio¹, Federica Giorda¹, Loretta Masoero¹, Guendalina Zaccaria², Giuseppa Purpari³, Antonella Cersini⁴, Maurizio Viscardi⁵, Laura Serracca⁶, Iolanda Padalino⁷, Giantonella Puggioni⁸, Cristina Canonico⁹, Anna Toffan¹⁰, Cinzia Centelleghe¹¹, Cristina Esmeralda Di Francesco¹², Sandro Mazzariol¹¹, Giovanni Di Guardo¹², Walter Mignone¹³, Cristina Casalone¹, Maria Goria¹

¹Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Torino, Italy.
²Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy.
³Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy.
⁴Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Roma, Italy.
⁵Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici (NA), Italy.
⁶Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, La Spezia, Italy.
⁷Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, Foggia, Italy.
⁸Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy.
⁹Istituto Zooprofilattico Sperimentale dell’ Umbria e delle Marche, Ancona, Italy.
¹⁰Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy.
¹¹University of Padova, Department of Comparative Biomedicine and Food Science (BCA), Legnaro (PD), Italy.
¹²Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy.
¹³Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d’Aosta, Imperia, Liguria, Italy.

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% - 99.3%), mean Sp = 98.1% (95% CI 72.5% - 99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 - 0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.

Dolphin Morbillivirus, RT-PCR, Interlab Ring Test, Method Comparison, Stranded Cetaceans, Mediterranean Sea

Zoccola, R. , Pautasso, A., Grattarola, C., Monnier, M., Miceli, I., Ingravalle, F., Crescio, I., Giorda, F., Masoero, L., Zaccaria, G., Purpari, G., Cersini, A., Viscardi, M., Serracca, L., Padalino, I., Puggioni, G., Canonico, C., Toffan, A., Centelleghe, C., Di Francesco, C.E., Mazzariol, S., Di Guardo, G., Mignone, W., Casalone, C. Goria, M. and Goria, M. (2019) Inter-Laboratory Ring Trial to Evaluate Reverse Transcription Polymerase Chain Reaction Methods Used for Dolphin Morbillivirus Detection in Italy. Advances in Microbiology, 9, 423-437. doi: 10.4236/aim.2019.95025.