Tick-borne pathogens remain insufficiently investigated in donkeys, particularly in southeastern Europe. This study investigated the occurrence of piroplasms, Anaplasmataceae, and hemotropic Mycoplasma spp. in donkeys from Croatia and assessed the genetic diversity of detected Theileria equi. Blood samples from 135 apparently healthy donkeys originating from 15 herds in six Croatian counties were examined using PCR. Positive T. equi samples were further characterized by sequencing, haplotype determination, and phylogenetic analysis. Theileria equi DNA was detected in 31/135 animals (22.96%; 95% CI: 15.99–29.94%), whereas no Anaplasmataceae or hemotropic Mycoplasma spp. DNA was detected. Infection showed marked geographical and herd-level variation. The highest prevalence was recorded in Šibensko-Kninska County (17/34; 50.0%), while all positive animals originated from three herds in the Croatian littoral region. Prevalence differed significantly among herd-size categories (p = 0.012), with no positive animals detected in small herds. Sequence analysis identified three T. equi haplotypes: D was predominant (22/31; 70.97%), followed by A (8/31; 25.81%) and E (1/31; 3.23%). Phylogenetic analysis confirmed all Croatian sequences as T. equi and indicated the presence of multiple genetic lineages within the sampled donkey population. These findings provide new molecular epidemiological data on T. equi in Croatian donkeys and demonstrate a heterogeneous distribution of infection among the investigated regions and herds. Among the selected tick-borne pathogens investigated, T. equi was the only pathogen detected. Further studies integrating larger and longitudinal datasets with detailed management, environmental, and tick-vector data are needed to better understand the factors underlying the observed epidemiological patterns.

Nutritional supplementation with pollen and protein-based patties is widely used in apiculture to support honey bees (Apis mellifera carnica, Pollmann 1879) colonies during periods of limited natural forage, particularly in early spring and late summer. This study evaluated the effects of different supplemental diets on the survival, food intake, and gross energy content of caged adult worker bees of mixed ages under controlled laboratory conditions. Worker bees were fed four different diets: sucrose syrup (control), sugar patty, protein patty, and pollen patty. Survival, food intake, and gross energy content were assessed over an 18-day experimental period. Calorimetric analyses demonstrated that diet composition significantly influenced the gross energy content of adult bee tissues. Bees fed pollen patty exhibited the highest energy values (22.05 ± 0.38 MJ/kg), particularly among young (22.28 ± 0.18 MJ/kg) and middle-aged workers (22.26 ± 0.23 MJ/kg), indicating enhanced energy accumulation during early adult development. In contrast, although sucrose syrup had the highest intrinsic caloric value among the tested diets (16.56 ± 0.01 MJ/kg), it resulted in the lowest gross energy accumulation in bee tissues (20.92 ± 0.40 MJ/kg), suggesting that nutrient composition, rather than caloric density alone, determines physiological outcomes. These findings highlight the importance of nutritionally complex pollen‑ and protein‑based supplements for supporting worker bee physiological conditions during periods of food scarcity and provide new insights into the relationship between dietary energy content and energy reserves accumulated in bee tissues.

Nutritional supplementation with pollen and protein-based patties is widely used in apiculture to support honey bee (Apis mellifera carnica) colonies during periods of limited natural forage, particularly in early spring and late summer. This study evaluated the effects of different supplemental diets on the survival, food intake, and gross energy content of caged adult worker bees of mixed ages under controlled laboratory conditions. Worker bees were fed four different diets: sucrose syrup (control), sugar patty, protein patty, and pollen patty. Survival, food intake, and gross energy content were assessed over an 18-day experimental period. Survival differed significantly among dietary treatments in both spring and summer experiments, with the highest final survival observed in bees receiving sugar patty (HBP). Aged-related differences in survival were treatment-dependent. Descriptive calorimetric data showed that bees receiving the pollen-enriched patty (HBP-PO) had the highest mean GE of bee body (22.05 ± 0.38 MJ/kg), with similarly high values observed in the 18-day old bees (22.28 ± 0.18 MJ/kg) and 20-day old bee workers (22.26 ± 0.23 MJ/kg). Notably, the GE content of the experimental diets did not correspond directly to the GE measured in bee bodies: the sucrose syrup had the highest dietary GE (16.56 ± 0.01 MJ/kg), whereas bees fed pollen-enriched patty showed the highest descriptive mean GE despite this diet having the lowest GE among the tested patties. These findings indicate that dietary caloric density alone may not predict the energetic status or survival of worker bees and suggest that diet composition and worker age contribute to different physiological responses to supplemental feeding. The experiment was conducted under controlled cage conditions; further studies at the colony level under apiary conditions are needed to determine whether these individual-level responses are maintained within functioning honey bee colonies.