Uros Glavinic

Clinical babesiosis and molecular identification of Babesia canis and Babesia gibsoni infections in dogs from Serbia

Canine babesiosis is a frequent and clinically significant tick-borne disease. Sixty symptomatic dogs with clinical findings compatible with babesiosis were included in this study conducted in Serbia. After clinical examination, blood samples were taken for microscopic examination, complete blood count (CBC), Canine SNAP 4Dx Test, DNA analyses and sequencing. The main clinical signs included apathy, anorexia, fever, brown/red discoloration of urine, pale mucous membranes, icterus, splenomegaly, and vomiting. The main clinicopathological findings in Babesia infections were a slight to severe thrombocytopenia and a mild to very severe normocytic normochromic anaemia. Microscopic evaluation revealed 58 positive samples with the presence of large and small intraerythrocytic piroplasms in 57 and 1 sample(s), respectively. No co-infections were found using SNAP test. Two Babesia species, B. canis (58/60) and B. gibsoni (2/60), were differentiated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Species identification was further confirmed by sequencing PCR products of B. gibsoni samples and six randomly selected B. canis samples. All dogs were treated with imidocarb dipropionate (6.6 mg/kg of body weight), given intramuscularly twice at an interval of 14 days. This report presents the first molecular evidence of the occurrence of B. gibsoni and B. canis, confirmed by DNA sequencing, in sick dogs from Serbia.

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.

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.

Efficacy of plant-derived formulation “Argus Ras” in Varroa destructor control

Abstract Varroa destructor is the most important honey bee parasite. There are various methods used in the control of this mite, but none of them meets all requested criteria, to be safe, effective and easy to apply. The objective of this study was to evaluate the varroacidal efficacy of newly created plant-derived formulation Argus Ras (mixture of extracts of Sophora flavescens, Ginkgo biloba, Gleditsia chinensis and Teucrium chamaedrys) in a field trial. The investigation was conducted on 240 Apis mellifera colonies equalized in respect of brood amount, adult bee population and food reserves. Efficiency was evaluated by applying Argus Ras consecutively with two other acaricides, amitraz and oxalic acid. Average acaricidal efficacy of Argus Ras was 80.89%, being higher of other previously tested essential oils. Besides, it showed a potential in knocking down the mites resistant to other acaricides. It should not be neglected that Argus Ras requires a smaller number of treatments and financial investments than other formulations used for the control of Varroa mites.

Dietary amino acid and vitamin complex protects honey bee from immunosuppression caused by Nosema ceranae

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.

Retrospective Analysis of the Bluetongue Outbreak in Serbia

Abstract Bluetongue, a vector-born disease caused by the Bluetongue virus (BTV) and transmitted by Culicoides biting midges, is considered to be one of the most important diseases of domestic ruminants. The first outbreak of bluetongue in Serbia was reported in 2001, when BTV serotype 9 was identified in sampled materials. In 2014, outbreak of BTV-4 in Serbia caused considerable economic losses affecting sheep, cattle and goats. During this outbreak, BTV-4 was recorded in 644 outbreaks within 49 municipalities, part of 17 administrative regions. From the total number of sheep kept in areas affected by bluetongue (n=1 748 110), 2 083 cases (0.2%) were proven to be BTV-4 infected. Total of 206 infected cattle and 24 infected goats were reported during this investigation period, which represents 0.06% and 0.03% of the total number of cattle and goats kept in affected areas, respectively. The highest incidence of infected sheep, cattle and goats was recorded on the territory covered by veterinary institute of Nis. Recorded lethality in cattle, sheep and goats was 18.45% (n=38), 48.10% (n=1002) and 54.17% (n=13), respectively. The peak of the outbreak was in September and October when 94.43% of the confirmed positive cases, regardless of the species, was recorded. Monitoring of bluetongue disease in Serbia relies on active surveillance programmes aimed at: (i) identification and tracing of susceptible and potentially infected animals and (ii) detection, distribution and prevalence of insect vectors. Vaccination of sheep is planned to be implemented as a control measure against bluetongue in Serbia.

Improved DNA-Based Identification of Cervidae Species in Forensic Investigations

Abstract The main reasons for wildlife forensic research are animal poaching, illegal trade, and falsified game meat products. Small trace amounts, old and degraded materials present the most common samples in revealing criminal activities in this field. This is the reason why it is crucial to use adequate and reliable methods and samples to identify animal species killed outside the hunting season or species protected by law. In this study, different endpoint PCR and real-time PCR protocols were compared in the identification of three Cervidae species (Capreolus capreolus, Cervus elaphus, Dama dama) from old and damaged material found in an enclosed area where the animals were kept. From a total of 129 samples, end point PCR provided results for 119 samples, while real-time PCR was successful in all cases. Also, we created and tested a protocol for simultaneous analyses of different types of samples, which is of great importance as when the amplification is carried out simultaneously it is more cost efficient and speeds up the process.

A molecular and haematological study of Theileria equi in Balkan donkeys

Equine piroplasmosis in donkeys has been recognised as a serious problem of major economic importance. The present molecular study is the first investigation of the presence of Theileria equi and Babesia caballi in Balkan donkeys and of the possible haematological alterations related to it. A total of 70 apparently healthy donkeys from Serbia were included in this study. The overall prevalence of T. equi infection in donkeys tested with multiplex PCR was 50%. There was no B. caballi-positive sample. Infections in donkeys included in this study seem to be associated with decreased red blood cell count, haemoglobin concentration, haematocrit and platelet count, and with increased white blood cell count, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. Altered haematological parameters in donkeys can lead to a decrease in working capacity and production performance. Further molecular research and long-term monitoring of equine piroplasmosis is needed in Serbia and throughout Europe.