DEVELOPMENT OF INNOVATIVE DIAGNOSTIC PROTOCOLS TO BE APPLIED TO THE STUDY OF THE EPIDEMIOLOGY AND GENETICS OF AVIAN ROTAVIRUSES

Rotaviruses (RVs) are the major cause of acute gastroenteritis in infants and young animals of mammalian and avian species. In avian species, group A and D are represented with high frequency, while group F and G are sporadic. Enteric syndrome in young birds is a major concern to the poultry industry, causing severe economic losses. Monitoring Rotavirus distribution in different avian species is crucial to uncover diversity among strains and to better understand RV ecology in the field. The aims of this project were: i) to provide information on distribution of the different RV groups in avian species suffering enteritis in Italy; genetic diversity of RVs in these species; dynamics and timing of RV infection within flocks; and ii) to develop new and updated diagnostic protocols to be applied in the diagnostic routine and for research use. The data collected during a period of nine years, indicate that the infection is widespread in the Italian production, with an average prevalence of 20% in commercial flocks and 15% in game-hunting species. All the 117 samples analysed in the present study originated from poultry flocks that presented one or more clinical signs and lesions associated with enteric diseases. Clinical manifestations consisted mostly of diarrhoea, dehydration, anorexia, weight loss and increased mortality. By statistical analysis it was not observed any significant correlation between the general clinical signs/lesions or gastro-enteric lesions observed in the field and at post mortem examination with the presence of different AvRV groups or different combinations of groups, even with regard to the different species involved (p>0.05). From the 117 samples tested by group-specific RT-PCRs, AvRV-D was detected in 107 (91.5%), AvRV-A in 70 (59.9%), AvRV-F in 61 (52.1%) and AvRV-G in 31 (26.5%). Only 17% of samples showed the presence of a single rotavirus group (AvRV-A or AvRV-D), but dual or multiple presence of rotaviruses of different AvRV groups was observed in the majority of samples. Group D is the predominant RV in Italy, representing the most commonly found RV in all the species investigated. The identification of several avian rotaviruses (AvRVs) belonging to group F and group G in partridge, pheasant and guinea fowl, suggests that the lack of specific detection methods could have underestimated the real diffusion of these two groups in the past. The use of new group-specific RT-PCRs developed and performed on a set of clinical specimens tested positive for RVs by Electron Microscopy (EM), allowed the rapid genetic screening of AvRVs circulating within the avian rotavirus main groups. In this study, 175 complete sequences were obtained (17 of NSP4A, 20 of VP6A, 5 of VP4A, 35 of NSP4D, 34 of VP6D, 9 of VP4D, 17 of VP7D, 21 of VP6F and 13 of VP6G). No correlation between year of isolation nor avian species and the different RV-groups was observed. Regarding results obtained from longitudinal studies in turkeys, the infection was already present in the first weeks of life; after 2/3 weeks a second infection outbreak happened in the same herds. Phylogenetic analysis of samples collected during the longitudinal study in turkey flocks showed that different RV-groups and different strains from the same group were present in the same flock, evidencing complex RV groups/strains patterns that modified in time. Results of chicken longitudinal study reinforced the hypothesis of a primary pathogenetic role of astrovirus-rotavirus infections in enteric syndrome during first weeks of life.