Effects of Plant-Based Supplement on Oxidative Stress of Honey Bees (Apis mellifera) Infected with Nosema ceranae.

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).

Serum oxidant-antioxidant status and butyrylcholinesterase activity in dogs with idiopathic epilepsy - A pilot study.

Oxidative stress plays an important role in pathogenesis of idiopathic epilepsy (IE). Although IE is the most common neurological condition, oxidant-antioxidant status in epileptic dogs is still unknown. The aim of this study is to evaluate the serum oxidant-antioxidant status in dogs with newly diagnosed IE. The status in 15 dogs with IE and 15 healthy dogs is estimated through spectrophotometric determination of two oxidant markers: advanced oxidation protein products-albumin index (AOPP) and thiobarbituric acid reactive substances (TBARS); and three antioxidant markers: total thiols (R-SH) level, glutathione (GSH) level, and paraoxonase-1 (PON-1) activity. Also, butyrylcholinesterase (BChE) activity is assessed in both groups of dogs. Higher AOPP is observed in the dogs with newly diagnosed IE, while TBARS level shows no difference when compared to the healthy dogs. In contrast, lower levels of antioxidants (R-SH, GSH, and PON-1) and BChE activity are found in the dogs with IE. No significant differences are observed in the oxidant and antioxidant markers and BChE activity across the investigated IE cases with focal and generalized seizures. Our findings provide evidence that dogs with IE are characterized by an impaired serum oxidant-antioxidant balance and lower BChE activity, which may contribute to a better understanding of IE pathogenesis.

Genotypes and virulence factors in Staphylococcus aureus isolated from dairy cows with subclinical mastitis in Serbia.

This study aimed to determine the prevalence of Staphylococcus aureus subclinical mastitis and to genotype the S. aureus isolates using the 16S-23S rRNA intergenic spacer (RS-PCR) method. In addition, the genes responsible for adherence, biofilm formation, host evasion, tissue necrosis, methicillin resistance, and enterotoxin production of S. aureus were investigated. The overall prevalence of S. aureus subclinical mastitis in lactating cows was 5.4% (95% confidence interval, CI=4.7-6.1%). An increased risk of S. aureus intramammary infection was observed on small family farms (odds ratio, OR=4.2, 95% CI=2.6-6.6, P < 0.001) and medium-sized farms (OR=3.5, 95% CI=2.2-5.7, P < 0.001). The RS-PCR analysis revealed 44 genotypes and genotype variants, of which 15 new genotypes and five new variants were detected within small and medium-sized farms. S. aureus isolates of new genotypes and genotype variants carried the clfA gene responsible for adherence at a lower frequency (64.8%) and enterotoxin-producing genes sea (20.4%), seb (14.8%) and sec (14.8%) at a higher frequency than the other known genotypes (P < 0.001), and were confirmed to carry the sej and sep genes. The spa gene was detected in all S. aureus isolates, whereas none harbored bap, ser, or tsst-1 genes. Methicillin-resistant strains of S. aureus (MRSA) were also detected, with a higher prevalence (19.2%) on large farms with more than 50 cows (P < 0.001). Using molecular techniques as diagnostic tools provides a better understanding of intramammary staphylococcal infections' occurrence, spread, and eradication.

"Slow kill" treatment reduces DNA damage in leukocytes of dogs naturally infected with Dirofilaria immitis.

Parasitic diseases are considered to be a cause of oxidative stress which leads to oxidative damage of various molecules including DNA. This can result in mutations, replication errors, and genome instability. Therefore, aim of this study was to measure DNA damage induced by Dirofilaria immitis in the single cells such as dogs' leukocytes using the comet assay. Also, we monitored the effects of antiparasitic treatment on mitigation of sensitivity to DNA damage in leukocytes treated with H2O2 using the in vivo and ex vivo comet assay. The whole blood samples from 34 dogs from Serbia were used, both males and females, from one to 13 years old, both pure and mixed-breeds. A rapid immunochromatographic test (Antigen Rapid Heartworm Ag 2.0 Test Kit, Bionote, Minnesota, USA) was used for the detection of D. immitis antigens. The modified Knott's test and PCR were used in the aim of detecting D. immitis microfilariae in dogs' blood, and evaluating the number of circulating microfilariae during the treatment. The genotoxicity evaluation showed that D. immitis infection resulted in DNA damage in naturally infected dogs, with the highest DNA damage occurring in the group of dogs with severe clinical signs. Treatment with ivermectin and doxycycline decreased DNA damage in leukocytes of dogs in all groups, as the intensity of infection decreased due to applied therapy. Ex vivo comet assay results showed that leukocytes exhibited decreased sensitivity to H2O2-induced DNA damage during treatment. The results of the modified Knott's test and PCR in our study showed that treatment with ivermectin and doxycycline was successful in decreasing the average number of microfilariae during the time and at the end eliminating them from the dogs' blood.

Genotoxic Potential of Thymol on Honey Bee DNA in the Comet Assay.

Thymol is a natural essential oil derived from the plant Thymus vulgaris L. It is known to be beneficial for human and animal health and has been used in beekeeping practice against Varroa mite for years. In this study, the genotoxic and antigenotoxic potential of thymol were evaluated on the honey bee (Apis mellifera L.) continuous cell line AmE-711 for the first time. Using the Comet assay, three increasing concentrations (10, 100, and 1000 µg/mL) of thymol were tested. Negative control (non-treated cells) and positive control (cells treated with 100 µM H2O2) were also included. The absence of thymol cytotoxicity was confirmed with the Trypan blue exclusion test. Thymol in the concentration of 10 µg/mL did not increase DNA damage in AmE-711 honey bee cells, while 100 and 1000 µg/mL concentrations showed genotoxic effects. For testing the antigenotoxic effect, all concentrations of thymol were mixed and incubated with H2O2. The antigenotoxic effect against was absent at all concentrations (10, 100, 1000 µg/mL) tested. Moreover, thymol enhanced the H2O2-induced DNA migration in the Comet assay. The obtained results indicate genotoxic effects of thymol on cultured honey bee cells suggesting its careful application in beekeeping practice to avoid possible negative effects on honey bees.

First Report of Sarconema eurycerca Heart Infection in Mute Swans in Serbia.

BACKGROUND: The Sarconema eurycerca heart infection may be one of the main causes of death in swans and geese due to severe heart lesions. To date, there have been no reports of Sarconema eurycerca in mute swans (Cygnus olor) in Serbia and this parasite has never been morphologically characterized in wild birds from this region. METHODS: In spring 2021, eight dead mute swans (Cygnus olor) were collected during avian influenza surveillance on the territory of the Province of Vojvodina and submitted to Scientific Veterinary Institute "Novi Sad" to determine the cause of death. The presence of highly pathogenic avian influenza subtype H5N1 was determined in all swans using molecular methods. RESULTS: Necropsy of the swans revealed changes characteristic for highly pathogenic avian influenza infection, such as severe haemorrhages and necrosis in different organs. Three of the eight swans showed massive subepicardial haemorrhages and myocarditis with intralesional 2-4 cm long whitish roundworms. Histopathologically, haemorrhages and many adult heart worms were observed in the myocardium. Based on morphological features of parasites, infection by Sarconema eurycerca was concluded. CONCLUSION: According to the available scientific literature, the presence of heart roundworms in swans has not yet been confirmed in our country. This report is the first published case of cardiac filariasis associated with S. eurycerca infection in mute swans in the Republic of Serbia.

Contact varroacidal efficacy of lithium citrate and its influence on viral loads, immune parameters and oxidative stress of honey bees in a field experiment.

With an almost global distribution, Varroa destuctor is the leading cause of weakening and loss of honey bee colonies. New substances are constantly being tested in order to find those that will exhibit high anti-Varroa efficacy at low doses/concentrations, without unwanted effects on bees. Lithium (Li) salts stood out as candidates based on previous research. The aims of this study were to evaluate Li citrate hydrate (Li-cit) for its contact efficacy against Varroa, but also the effect of Li-cit on honey bees by estimating loads of honey bee viruses, expression levels of immune-related genes and genes for antioxidative enzymes and oxidative stress parameters on two sampling occasions, before the treatment and after the treatment. Our experiment was performed on four groups, each consisting of seven colonies. Two groups were treated with the test compound, one receiving 5 mM and the other 10 mM of Li-cit; the third received oxalic acid treatment (OA group) and served as positive control, and the fourth was negative control (C group), treated with 50% w/v pure sucrose-water syrup. Single trickling treatment was applied in all groups. Both tested concentrations of Li-cit, 5 and 10 mM, expressed high varroacidal efficacy, 96.85% and 96.80%, respectively. Load of Chronic Bee Paralysis Virus significantly decreased (p < 0.01) after the treatment in group treated with 5 mM of Li-cit. In OA group, loads of Acute Bee Paralysis Virus and Deformed Wing Virus significantly (p < 0.05) increased, and in C group, loads of all viruses significantly (p < 0.01 or p < 0.001) increased. Transcript levels of genes for abaecin, apidaecin, defensin and vitellogenin were significantly higher (p < 0.05-p < 0.001), while all oxidative stress parameters were significantly lower (p < 0.05-p < 0.001) after the treatment in both groups treated with Li-cit. All presented results along with easy application indicate benefits of topical Li-cit treatment and complete the mosaic of evidence on the advantages of this salt in the control of Varroa.

Analysis of the concentrations of some haematological parameters, C-reactive protein and anti-Müllerian hormone in bitches affected by pyometra.

The work was aimed to research into the vital signs, haematological and biochemical parameters, acute-phase protein concentrations (CRP, albumin), progesterone (PG) and anti-Müllerian hormone (AMH) concentrations in bitches with pyometra and their comparison with those in healthy bitches. In addition, the goal of this work was to assess if some of the tested parameters may be used as a biomarker in the diagnostics of pyometra and the monitoring of the postoperative recovery. Forty 3-6-year-old bitches of various breeds were included in the research and allotted to two groups: the control (C, n = 20) and the diseased - bitches diagnosed with pyometra (P, n = 20). Blood samples for the analyses were taken immediately before surgery (0 h), and 12 h, 24 h, 48 h and 72 h after, excepting for AMH and PG concentrations, which were determined only at 0 h. Clinical examination detected significantly higher respiration rates in bitches with pyometra in comparison to healthy ones at all time points, higher heart rates before and 12 h after surgery, and higher body temperature before and 12 h, 48 h and 72 h post operation (p < .01). Significantly higher concentrations of CRP (p < .01) and lower concentrations of albumin were detected in bitches with pyometra in comparison to their healthy counterparts at all time points. Progesterone and AMH concentration analysis immediately before surgery detected significantly higher levels of the hormone in bitches with pyometra than in the control (p < .01). The inflammatory reaction of the endometrium resulted in a significant AMH concentration increase, whilst increased CRP concentrations and lower albumin concentrations throughout the research may be used as biomarkers of proinflammatory activities, that is of the acute-phase response.

Effects of Agaricus bisporus Mushroom Extract on Honey Bees Infected with Nosema ceranae.

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).

Plant-based supplement containing B-complex vitamins can improve bee health and increase colony performance.

It is common knowledge that nutritive stress resulting from decreased diversity and quality of food, pollution of food sources and beekeeping errors may lead to increased susceptibility of bees to pathogens and pesticides. The dearth of adequate food is frequently compensated with supplements. Thus, this research was aimed to study the effects of the plant-based supplement B + on colony strength (assessed according to open and sealed brood area, honey and pollen/bee bread reserves, and the number of adult bees). In addition, Nosema ceranae spores and viruses were quantified and the level of infestation with Varroa destructor assessed. The experiment was conducted in late summer and early spring. In colonies which were given B + in feed a significant increase (p < 0.05) in the parameters of colony strength were noticed in comparison to the control (colonies fed on sugar syrup). Moreover, it was proven that the bees from these colonies had significantly lower (p < 0.05) N. ceranae spore counts, and acute bee paralysis, deformed wing and sacbrood virus loads. Our results suggest that the addition of B + supplement to the colonies provide them with nutrients, contribute to their strengthening, might prevent nutritive stress and increase the success of bees in combating pathogens.

Evaluation of the effects of two anaesthetic protocols on oxidative status and DNA damage in red-eared sliders (Trachemys scripta elegans) undergoing endoscopic coeliotomy.

The aim of this study was to assess how red-eared sliders (Trachemys scripta elegans) respond to anaesthesia itself and coelioscopy. For that purpose, the turtles were anaesthetised with ketamine-medetomidine or propofol, and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and the level of malondialdehyde (MDA) were determined by spectrophotometry. The possible genotoxic effects of the anaesthetic agents were estimated by comet assay. A total of 24 turtles were included in this study. The animals were divided into four groups according to the anaesthetic protocol and according to whether endoscopy would be performed. Significantly decreased activities of CAT were found only in the propofol group and in turtles undergoing coelioscopy. Both anaesthetic protocols induced significantly increased MDA levels, while no differences were observed after the intervention. A significant increase in GST activity was detected in turtles after both anaesthetic protocols, but after coelioscopy significant changes in GST activity were found only in the propofol group. However, no differences in SOD activity and no DNA damages were detected in either group. These findings suggest that ketamine-medetomidine may be more suitable anaesthetic agents in red-eared sliders than propofol.

Effects of oral supplementation with organically modified clinoptilolite during prepartum period on colostrum quality in primiparous dairy cows.

This research paper addresses the hypothesis that an oral supplementation with organically modified clinoptilolite will improve colostrum quality in primiparous dairy cows whilst having no adverse effects on the cows' health. A total of 36 pregnant Holstein primiparous dairy cattle were randomly assigned to receive daily oral drenching, two hours following morning feeding, with 1 l of water containing either 0 g/l (n = 16) or 150 g/l (n = 20) of clinoptilolite. Treatment lasted from 24 ± 4 d prior to expected parturition until two days postpartum (pp). Colostrum was collected at 2 to 3 h, 12, 24 and 36 h pp and blood samples were collected at 24 ± 4 and 4 ± 2 d prior to parturition and 1, 2 and 7 d pp. Overall mean dry matter, fat and total protein percentage as well as IgG concentration and mass were significantly greater in colostrum collected from cattle drenched with clinoptilolite (total protein increased by 15% and IgG concentration and mass by 21 and 38% respectively at first sampling and further at second sampling). Total γ globulin and most other blood serum biochemistry parameters did not differ between cattle treated and not treated with clinoptilolite, the only exception being the fast anionic γ globulin fraction that was 17% greater at 4 ± 2 d prior to parturition and 10% lower on the 1st day pp in treated cattle. These results showed that organically modified oral clinoptilolite supplementation at 150 g/d significantly increases the IgG concentration in colostrum and has no adverse effects on the energy status, protein, lipid, and mineral metabolism in primiparous dairy cattle during prepartum period.

Oxidative Stress, Endoparasite Prevalence and Social Immunity in Bee Colonies Kept Traditionally vs. Those Kept for Commercial Purposes.

Commercially and traditionally managed bees were compared for oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and malondialdehyde (MDA)), the prevalence of parasites (Lotmaria passim, Crithidia mellificae and Nosema ceranae/apis) and social immunity (glucose oxidase gene expression). The research was conducted on Pester plateau (Serbia-the Balkan Peninsula), on seemingly healthy colonies. Significant differences in CAT, GST and SOD activities (p < 0.01), and MDA concentrations (p < 0.002) were detected between commercial and traditional colonies. In the former, the prevalence of both L. passim and N. ceranae was significantly (p < 0.05 and p < 0.01, respectively) higher. For the first time, L. passim was detected in honey bee brood. In commercial colonies, the prevalence of L. passim was significantly (p < 0.01) lower in brood than in adult bees, whilst in traditionally kept colonies the prevalence in adult bees and brood did not differ significantly. In commercially kept colonies, the GOX gene expression level was significantly (p < 0.01) higher, which probably results from their increased need to strengthen their social immunity. Commercially kept colonies were under higher oxidative stress, had higher parasite burdens and higher GOX gene transcript levels. It may be assumed that anthropogenic influence contributed to these differences, but further investigations are necessary to confirm that.

Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to adrenaline in vitro.

Diabetes represents one of the major health concerns, especially in developed countries. Some hormones such as the stress hormone adrenaline can induce reactive oxygen species (ROS) and may worsen the diabetes. Therefore, the main aim of the investigation was to find out whether peripheral blood mononuclear cells (PBMCs) from normal persons have less DNA damage induced by adrenaline (0.1, 1 and 10 µM) in comparison to PBMCs from obese, prediabetic and diabetic patients. Also, the biochemical parameters of oxidative stress (TBARS, catalase) and lactate dehydrogenase were monitored. It was observed that higher concentrations of adrenaline (1 and 10 µM) induced DNA damage in the obese, prediabetic and diabetic groups. In healthy individuals only the highest concentration of adrenaline caused significant increase in the DNA damage. In summary, total comet score (TCS) comparison has shown significant differences between groups, and DNA damaging effects of adrenaline were most evident in diabetic patients. The results of the biochemical analysis also demonstrate that adrenaline exerts most obvious effects in diabetic individuals which is manifested as significant change of parameters of oxidative stress. In summary, the obtained results demonstrated that diabetics are more sensitive to genotoxic effects of adrenaline and this effect probably resulted from decreased antioxidative defence mechanisms in various stages of progression through diabetes. Therefore, these results could contribute to a better understanding of a role of endocrine factors to damage of cellular biomolecules which could be useful in finding novel therapeutic approaches and lifestyle changes with an aim to lower the possibility of diabetes complications.

Efficacy of oral fluralaner for the treatment of canine generalized demodicosis: a molecular-level confirmation.

BACKGROUND: Canine generalized demodicosis is a common parasitic disease caused by the proliferation of Demodex mites. The introduction of isoxazoline class treatments in veterinary dermatology has resulted in apparently effective treatment of generalized demodicosis. The objective of this study was to evaluate the effectiveness of fluralaner for the treatment of canine generalized demodicosis using real-time PCR for the detection and quantification of Demodex DNA. METHODS: Twenty privately owned dogs with clinical symptoms of generalized demodicosis and deep skin scrapings positive for Demodex canis mites were enrolled in the study. Following diagnosis (day 0) each dog was treated with fluralaner at the recommended commercial dose for tick and flea treatment (25-56 mg/kg) based on body weight. Clinical and mite count assessments, and hair sampling for molecular analyses were performed on days 0, 28, 56, 84 and 112. Demodex DNA was detected and quantified using real-time PCR. RESULTS: A single oral dose of fluralaner reduced Demodex mite counts in skin scrapings by an average of 98.9% in all dogs by day 28. No mites were recovered from skin scrapings from any treated dog by day 56, at which time the dog was considered to be clinically cured, with total hair regrowth. There were significant differences among examined dogs in qPCR cycle threshold (Ct) values on days 0, 28, 56, 84 and 112. Demodex DNA levels decreased (increasing Ct values) throughout the study. Mite DNA was present on day 112, possibly from dead mites, at values significantly lower than in samples taken on days 0, 28 and 56. Based on qPCR testing of diluted samples, the Demodex mite population was reduced by approximately 1000-fold on day 112. CONCLUSIONS: Oral administration of fluralaner at the recommended dose to dogs with generalized demodicosis is highly effective for reducing Demodex mite populations and resolving clinical signs of generalized demodicosis. The presence of mite DNA may indicate that treatment did not kill all Demodex mites.

Quantitative PCR assessment of Lotmaria passim in Apis mellifera colonies co-infected naturally with Nosema ceranae.

A recently described trypanosomatid species Lotmaria passim and the microsporidium Nosema ceranae infect the honey bee (Apis mellifera), but the interspecific dynamic of these two common gut parasites is unknown. In this study, a real-time qPCR assay was developed to enable the specific detection and quantification of L. passim. The annual dynamics of N. ceranae and L. passim infections were evaluated in ten A. mellifera colonies naturally infected with both parasites at one apiary in Serbia from March 2016 to March 2017. Ten samples (60 bees abdomens) were taken from each colony on 8 sampling occasions. L. passim infection level was evaluated with qPCR, while N. ceranae infection was measured by spore counts. N. ceranae infection level was significantly higher in comparison with that of L. passim (spore or cell equivalents/bee, respectively). Significant positive correlation between infection levels of the parasite species indicates their similar annual dynamics, whilst the differences in the levels of infection between particular months point to a seasonal pattern in the incidence of both parasites. The assay which has been developed and validated creates opportunity for detailed study of L. passim infection kinetics and the improvement in the management practices in beekeeping related to these two parasites.

Haplotype identification and detection of mitochondrial DNA heteroplasmy in Varroa destructor mites using ARMS and PCR-RFLP methods.

In the present study, amplification refractory mutation system (ARMS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used for identification of recently described Serbia 1 (S1) and Peshter 1 (P1) mitochondrial haplotypes of Varroa destructor. Based on single nucleotide polymorphisms (SNPs) within cytochrome oxidase 1 (cox1) and cytochrome b (cytb) gene sequences, a total of 64 adult V. destructor females were analyzed from locations where the S1 and P1 haplotypes had been detected previously. Results of haplotype identification obtained by ARMS and PCR-RFLP methods were completely consistent with the sequencing data. Furthermore, in some analyzed samples the occurrence of site heteroplasmy at haplotype-defining sites was detected, as it was confirmed by double peaks in the sequence chromatograms. Neither mites with simultaneous nucleotide variability, nor those with combined SNP and heteroplasmy in cox1 and cytb were found. Given that this is the first occurrence of site heteroplasmy in V. destructor, the origin of this phenomenon and possible specific traits of heteroplasmic mites have yet to be determined.

Species-specific diagnostics of Apis mellifera trypanosomatids: A nine-year survey (2007-2015) for trypanosomatids and microsporidians in Serbian honey bees.

In this study, honey bees collected in Serbia over 9 consecutive years (2007-2015) were retrospectively surveyed to determine the prevalence of eukaryotic gut parasites by molecular screening of archival DNA samples. We developed species-specific primers for PCR to detect the two known honey bee trypanosomatid species, Crithidia mellificae and the recently described Lotmaria passim. These primers were validated for target specificity under single and mixed-species conditions as well as against the bumblebee trypanosomatid Crithidia bombi. Infections by Nosema apis and Nosema ceranae (Microsporidia) were also determined using PCR. Samples from 162 colonies (18 from each year) originating from 57 different localities were surveyed. L. passim was detected in every year with an overall frequency of 62.3% and annual frequencies ranging from 38.9% to 83.3%. This provides the earliest confirmed record to date for L. passim and the first report of this species in Serbia. N. ceranae was ubiquitous, occurring in every year and at 95.7% overall frequency, ranging annually from 83.3% to 100%. The majority of colonies (60.5%) were co-infected with L. passim and N. ceranae, but colony infections by each species were statistically independent of one another over the nine years. Although C. mellificae and N. apis have both been reported recently at low frequency in Europe, neither of these species was detected in Serbia. These results support the hypothesis that L. passim has predominated over C. mellificae in A. mellifera during the past decade.