## 4 Discussion
As regards the others apiaries, B, E and H, they halved the prevalence of _N. ceranae_ from 2020 to 2021, while the apiaries D, F and G showed an increase in the number of positive colonies. Only apiary I kept at 100% _N. ceranae_ prevalence in both years. Moreover, the mean value of the number of _N. ceranae_ spores per bee per apiary was higher in 2020 than in 2021. Given that _N. ceranae_ could negatively affect honey bee health causing weakening and depression of the immune system [94] of insects, favouring viral infections or under certain conditions bring the colony to collapse [95], the presence of a significant association between _N. ceranae_ and other pathogens was analysed in this study. The data obtained from statistical analysis of the observed infections highlighted particular significant associations between _N. ceranae_ with ABPV and the DWV-A variant. In addition, _N. ceranae_ infected colonies tested positive to SBV and/or CBPV and/or BQCV, supporting the fact that _N. ceranae_ occurrence could increase honey bees susceptibility to other pathogens [65]. Besides _N. ceranae_, the prevalence of trypanosomatid _L. passim_ was very high in both years: indeed more than half of the analysed hives tested positive to _L. passim_ (51.3% in 2020 and 62.2% in 2021, respectively) and in 2021 its occurrence increased in all apiaries, except for apiaries D, E and H, confirming its greater diffusion in the world [48]. Although many researches have demonstrated that _L. passim_ can be present with other pathogens too and especially a relationship between _N. ceranae_ and _L. passim_ have been reported [96] as well as between _L. passim_ and viruses, making plausible synergistic effects [51], the results here reported did not show a significant association of _L. passim_ with _N. ceranae_, but it was significantly associated with ABPV. How _L. passim_ could influence the occurrence and virulence of the other pathogens in the host is unknown and requires further studies. The other protozoan _C. mellifera_, recently correlated with colony losses in the USA and Belgium, was often detected in association with _N. ceranae_[51; 53; 97]. However, in the present study it was found only in one hive of the apiary B in 2020. Several studies reported interactions, sometimes controversial, between _N. ceranae_ and DWV virus [27; 96; 98]. Our statistical analysis confirms a significant association between _N. ceranae_ and DWV-A variant in both years of sampling. DWV was described as the most prevalent virus in Europe [2] and it has been suggested that its widespread was most probably due to the _Varroa_ mites infestation [99], which could be a new viral transmission route, in particular in summer and autumn than in spring [78; 79], causing elevated viral loads [45; 100; 101] and selecting for DWV variants [102]. About these variants, three were mainly reported, each one with a different prevalence [42]. DWV-A was initially the dominant genotype, but over the years, DWV-B prevalence has increased and numerous studies reported now DWV-B as the dominant variant in Europe [103]. DWV-B has been considered a more virulent variant than the DWV-A genotype [41] and mostly associated to _Varroa_ mite infestation, whose transmission could exert strong selection on DWV genomes [104], favouring DWV-B. However, little is still known about the prevalence of DWV variants in Italy. The data provided by this study reported overall prevalence values of DWV-A variant ranging between 50.6% (2020) and 32.2% (2021) of hives, while for DWV-B between 17.8% (2020) and 12.2% (2021). In particular, apiary I kept high levels of DWV infection in both years, and this apiary was the one in which most of all investigated pathogens were found. Except for the apiary A, DWV-A variant was found in 2020 in all the apiaries, while for DWV-B variant apiaries A, C and H resulted negative.