**2. Results**

#### *2.1. N. ceranae Infection and Spore Counts*

The amplification of the part of the *16S rDNA* gene showed the presence of *N. ceranae* in both experimental groups (NH and NHP) and the control group (C) during the two pre-treatment sessions (August 2019 and April 2020) and about two months after the two post-treatment sessions (October 2019 and June 2020). There were no PCR products in the negative controls. The results from the obtained sequences have shown the presence of *N. ceranae* species after standard nucleotide BLAST (BLASTN programs) in the GenBank database.

The numbers of *N. ceranae* spores per bee for the pre- and post-treatment periods each year are shown in Figure 1. Even after the first treatment, a significant reduction in the number of *N. ceranae* spores per bee (*p* < 0.01) was observed (Figure 1a). Compared to the pre-treatment period (August 2019) (NH: 3.8 ± 0.61 × 106; NHP: 3.5 ± 0.78 × 106), in October 2019 the two supplement-treated groups (NH and NHP) showed a significant decrement in *N. ceranae* spore counts: about 68% in the NH group and about 60% in the NHP group (Figure 1a). In contrast to the two experimental groups, the control C group showed a reduction by only 19% of the number of *N. ceranae* spores (pre- treatment 2.7 ± 0.68 × <sup>10</sup>6; post-treatment 2.2 ± 0.59 × <sup>10</sup>6). During the next year, at the beginning of the second pre-treatment session (April 2020), in contrast to October 2019, a much more pronounced augmentation of *N. ceranae* spore counts was observed in the control C group—about 33% (ANOVA with Tukey's HSD post hoc test, *p* = 0.6047; *df* = 8) and less in the experimental groups NH and NHP—18% and 22% (ANOVA with Tukey's HSD post hoc test, *p* = 0.6104, *df* = 8; *p* = 0.5406, *df* = 8, respectively) (Figure 1b). At the end of the second experimental period in June 2020, compared to the pre-treatment period (April 2020), the NH and NHP groups treated with herbal supplements showed a significant reduction in *N. ceranae* spores (*<sup>p</sup>* < 0.01)—about 67% (0.47 ± 0.58 × 106) and 60% (0.68 ± 0.64 × <sup>10</sup>6), respectively (Figure 1b). The reduction in the number of *N. ceranae* spores in the control C group at the end of the second experimental period (June 2020) was only 11% (2.61 ± 0.72 × <sup>10</sup>6).

The treatment with the herbal supplements NH and NHP showed a significant reduction in the *N. ceranae* spore levels compared to the control C group (post-hoc Tukey HSD Test: *F* = 24.199, *p* = 0.0004; *F* = 18.919, *p* = 0.0009, respectively).
