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Microsporidium Nosema ceranae is well known for exerting a negative impact on honey bee health, including down-regulation of immunoregulatory genes. Protein nutrition has been proven to have beneficial effects on bee immunity and other aspects of bee health. Bearing this in mind, the aim of our study was to evaluate the potential of a dietary amino acid and vitamin complex \"BEEWELL AminoPlus\" to protect honey bees from immunosuppression induced by N. ceranae. In a laboratory experiment bees were infected with N. ceranae and treated with supplement on first, third, sixth and ninth day after emergence. The expression of genes for immune-related peptides (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) was compared between groups. The results revealed significantly lower (p<0.01 or p<0.001) numbers of Nosema spores in supplemented groups than in the control especially on day 12 post infection. With the exception of abacein, the expression levels of immune-related peptides were significantly suppressed (p<0.01 or p<0.001) in control group on the 12th day post infection, compared to bees that received the supplement. It was supposed that N. ceranae had a negative impact on bee immunity and that the tested amino acid and vitamin complex modified the expression of immune-related genes in honey bees compromised by infection, suggesting immune-stimulation that reflects in the increase in resistance to diseases and reduced bee mortality. The supplement exerted best efficacy when applied simultaneously with Nosema infection, which can help us to assume the most suitable period for its application in the hive.

The most common types of beta-casein in cow’s milk are A1 and A2, which differ in one amino acid. During the gastrointestinal proteolysis of A1 beta-casein in humans this difference results in the release of beta-casomorphin-7, an opioid which may lead to severe effects on human health, causing various ailments (type-1 diabetes mellitus, ischemic heart disease, arteriosclerosis, sudden infant death syndrome, autism, schizophrenia, gastrointestinal digestive discomfort, as well as increased gastrointestinal transit time). By contrast, A2 beta-casein cannot exert these effects owing to its different composition and metabolism. Furthermore, studies have shown that it can influence milk productivity traits. Our research aimed to screen the frequency of A1 and A2 alleles of beta-casein gene in a population of Serbian Holstein-Friesian cows and to detect how the genotypes influence milk production, and milk protein and fat yields. Out of 106 animals, 13 (12.26%) were of A1A1 genotype, 58 (54.72%) of A1A2, and 35 (33.02%) of A2A2 genotype. Milk yield was significantly (P<0.01) higher in A2A2 compared to both A1A1 and A1A2 genotypes. Milk protein concentrations were significantly (P<0.01) higher in A2A2 compared to A1A2 genotype, while milk fat concentrations were significantly (P<0.01) higher in A2A2 compared to both A1A1 and A1A2 genotypes.

Agaricus bisporus water crude extract was tested on honey bees for the first time. The first part of the cage experiment was set for selecting one concentration of the A. bisporus extract. Concentration of 200 µg/g was further tested in the second part of the experiment where bee survival and food consumption were monitored together with Nosema infection level and expression of five genes (abaecin, hymenoptaecin, defensin, apidaecin, and vitellogenin) that were evaluated in bees sampled on days 7 and 15. Survival rate of Nosema-infected bees was significantly greater in groups fed with A. bisporus-enriched syrup compared to those fed with a pure sucrose syrup. Besides, the anti-Nosema effect of A. bisporus extract was greatest when applied from the third day which coincides with the time of infection with N. ceranae. Daily food consumption did not differ between the groups indicating good acceptability and palatability of the extract. A. bisporus extract showed a stimulative effect on four out of five monitored genes. Both anti-Nosema and nutrigenomic effects of A. bisporus extract were observed when supplementation started at the moment of N. ceranae infection or preventively (before or simultaneously with the infection).

Colony losses, including those induced by the colony collapse disorder, are an urgent problem of contemporary apiculture which has been capturing the attention of both apiculturists and the research community. CCD is characterized by the absence of adult dead bees in the hive in which few workers and a queen remain, the ratio between the brood quantity and the number of workers is heavily disturbed in favor of the former, and more than enough food is present. Robbing behavior and pests usually attacking the weakened colony do not occur. In the present paper, the causes of the emergence of this problem are discussed, as well as the measures of its prevention. The following factors, which lead to colony losses, are analyzed: shortage of high-quality food (pollen and honey); infestation with parasites, primarily with , and mixed virus infections; bacterial infections (American and European foulbrood), fungal infections (nosemosis and ascosphaerosis) and trypanosomal infections (lotmariosis); and, finally, general management of the apiary. Certain preventive measures are proposed: (1) providing ample high-quality forage and clean water, (2) avoiding sugarisation, i.e. superfluous use of sugar syrup, (3) meeting the nutritional needs of the colony, (4) when feeding bees, taking care of the timing and the composition of diet, avoiding pure sugar syrup which in excessive quantities may induce energetic and oxidative stress, (5) when there is a shortage of natural feed – honey in the brood chamber – use sugar syrup with natural/artificial supplements to avoid protein starvation, (6) organized control of in the colonies is obligatory due to its vector role, and (7) compliance with hygienic and sanitary measures and principles of good apiculture practice and management in apiaries. To conclude, all preventive measures are feasible in compliance with rules and regulations concerning regular spring and autumn bee health monitoring by licensed veterinarians, who can propose adequate treatments if necessary.

Among numerous factors that contribute to honey bee colony losses and problems in beekeeping, pesticides and Nosema ceranae have been often reported. In contrast to insecticides, whose effects on bees have been widely studied, fungicides did not attract considerable attention. Prochloraz, an imidazole fungicide widely used in agriculture, was detected in honey and pollen stored inside hives and has been already proven to alter immune gene expression of honey bees at different developmental stages. The aim of this study was to simulate the realistic conditions of migratory beekeeping, where colonies, both uninfected and infected with N. ceranae , are frequently transported to the vicinity of crop fields treated with prochloraz. We investigated the combined effect of prochloraz and N. ceranae on honey bees that faced fungicide during the larval stage through food consumption and microsporidium infection afterwards. The most pronounced changes in gene expression were observed in newly emerged Nosema -free bees originating from colonies previously contaminated with prochloraz. As exclusively upregulation was registered, prochloraz alone most likely acts as a challenge that induces activation of immune pathways in newly emerged bees. The combination of both stressors (prochloraz and Nosema infection) exerted the greatest effect on six-day-old honey bees. Among ten genes with significantly altered expression, half were upregulated and half downregulated. N. ceranae as a sole stressor had the weakest effects on immune gene expression modulation with only three genes significantly dysregulated. In conclusion, food contaminated with prochloraz consumed in larval stage could present a threat to the development of immunity and detoxification mechanisms in honey bees.

There is a growing interest in using agri-food by-products and a demand for natural substances that might help maintain healthy honey bee colonies. We investigated a by-product of the wine industry, a grape pomace (GP) of the autochthonous Prokupac grape cultivar from Serbia. A hydroethanolic extract (50% (w/v) ethanol) of GP (Prokupac GP extract) obtained by the pressurized liquid extraction (PLE) method was subjected to qualitative profiling of phenolic composition by liquid chromatography with OrbiTrap Exploris 120 mass spectrometer. Then, the extracts’ antioxidant and redox-modulatory activities were evaluated through Electron Paramagnetic Resonance (EPR) spectroscopy. Finally, the extract’s potential to modulate cellular redox status was evaluated using cultured AmE-711 honey bee cells. The results show that the Prokupac GP extract contains a wide array of flavonoids, anthocyanins, stilbenes, and their various conjugated derivatives and that anthocyanins, particularly malvidin-based compounds, dominate. EPR measurements showed strong scavenging activity against superoxide anion (O2•−) and hydroxyl radicals (•OH), with inhibition efficiencies of 84.37% and 81.81%, respectively, while activity against the DPPH radical was lower (17.75%). In the cell-based assay, the Prokupac GP extract consistently provided strong antioxidant protection and modulated the cellular response to oxidative stress by over 14%. In conclusion, while the Prokupac GP extract demonstrated antioxidant properties and the ability to modulate cellular responses to oxidative stress, in vivo studies on honey bees are required to confirm its efficacy and safety for potential use in beekeeping practice.

Nosema ceranae is the most widespread microsporidian species which infects the honey bees of Apis mellifera by causing the weakening of their colonies and a decline in their productive and reproductive capacities. The only registered product for its control is the antibiotic fumagillin; however, in the European Union, there is no formulation registered for use in beekeeping. Thymol (3-hydroxy-p-cymene) is a natural essential-oil ingredient derived from Thymus vulgaris, which has been used in Varroa control for decades. The aim of this study was to investigate the effect of thymol supplementation on the expression of immune-related genes and the parameters of oxidative stress and bee survival, as well as spore loads in bees infected with the microsporidian parasite N. ceranae. The results reveal mostly positive effects of thymol on health (increasing levels of immune-related genes and values of oxidative stress parameters, and decreasing Nosema spore loads) when applied to Nosema-infected bees. Moreover, supplementation with thymol did not induce negative effects in Nosema-infected bees. However, our results indicate that in Nosema-free bees, thymol itself could cause certain disorders (affecting bee survival, decreasing oxidative capacity, and downregulation of some immune-related gene expressions), showing that one should be careful with preventive, uncontrolled, and excessive use of thymol. Thus, further research is needed to reveal the effect of this phytogenic supplement on the immunity of uninfected bees.

To improve the genomic evaluation of milk-related traits in Holstein-Friesian (HF) cattle it is essential to identify the associated candidate genes. Novel SNP-based analyses, such as the genetic mapping of inherited diseases, GWAS, and genomic selection, have led to a new era of research. The aim of this study was to analyze the association of each individual SNP in Serbian HF cattle with milk production traits and inbreeding levels. The SNP 60 K chip Axiom Bovine BovMDv3 was deployed for the genotyping of 334 HF cows. The obtained genomic results, together with the collected phenotypic data, were used for a GWAS. Moreover, the identification of ROH segments was performed and served for inbreeding coefficient evaluation and ROH island detection. Using a GWAS, a polymorphism, rs110619097 (located in the intron of the CTNNA3 gene), was detected to be significantly (p < 0.01) associated with the milk protein concentration in the first lactation (adjusted to 305 days). The average genomic inbreeding value (FROH) was 0.079. ROH islands were discovered in proximity to genes associated with milk production traits and genomic regions under selection pressure for other economically important traits of dairy cattle. The findings of this pilot study provide useful information for a better understanding of the genetic architecture of milk production traits in Serbian HF dairy cows and can be used to improve lactation performances in Serbian HF cattle breeding programs.

The aim of the study was to investigate the effect of the immunostimulant Mycobacterium Cell Wall Fraction (MCWF) on the treatment of S. aureus SCM by intravenous application. The study included 45 HF dairy cows in 2nd and 3rd month after parturition divided into three groups (n = 15 per group): the MC + group – cows with S. aureus SCM treated with MCWF; the MC- group – cows with S. aureus SCM, with no treatment; and the C group – the control group of healthy cow with no treatment. Samples were collected 0th (I sample), 7th (II), and 14th day (III) from the day of SCM diagnosis and on day 21st (IV). A greater influx of leukocytes was confirmed into milk after 7 days after MCWF treatment in MC + group, which was followed by increase of WBC and LYM in blood. These results support the hypothesis of effective action of MCWF, and in quarters with lower-grade infection, bacteriological cure was achieved. The MC- group had a statistically higher concentration of TBARS and CAT activity in milk, while MC + group had lower blood serum LDH activity, which indicates a positive effect of the MCWF application and a lower exposure of the tissue to lipide peroxidation and inflammation caused by S. aureus. The application of MCWF would give new possibilities in the prevention and therapy of mammary gland diseases without fear of the presence of residues and the emergence of bacterial resistance. In future studies, the effects of local and systemic application of MCWF in the treatment of S. aureus SCM should be compared.

One of the most important approaches in the prevention and treatment of nosemosis is the use of herbal preparations as food supplements for bees. Therefore, the aim of this study was to investigate the effects of a plant-based supplement branded as “B+” on honeybees in a laboratory experiment. Four experimental groups were established: treated group (T), N. ceranae-infected and treated group (IT), N. ceranae-infected group (I) and non-infected group (NI). Survival, N. ceranae spore load and oxidative stress parameters together with expression levels of antioxidant enzyme genes and vitellogenin gene were monitored. The mortality in the T, IT and NI groups was significantly (p < 0.001) lower than in than in the I group. Within Nosema-infected groups, the IT group had a significantly lower (p < 0.001) number of N. ceranae spores than the I group. In addition, expression levels of genes for antioxidant enzymes were lower (p < 0.001) in the IT group compared to the I group. The concentration of malondialdehyde and the activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione S-transferase) were significantly lower (p < 0.001) in the IT group compared to the I group. No negative effects of the tested supplement were observed. All these findings indicate that the tested supplement exerted beneficial effects manifested in better bee survival, reduced N. ceranae spore number and reduced oxidative stress of bees (lower expression of genes for antioxidant enzymes and oxidative stress parameters).