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.

Equine piroplasmosis is significant tick-borne disease with wide distribution. The prevalence of equine piroplasmosis in Serbia, Montenegro and Bosnia and Herzegovina is unknown. In aim to obtain a first insight into the prevalence we performed molecular epidemiological study which included 142 horses, on seven locations in these three countries. We first performed PCR for the detection of a 450bp long section of the 18S rRNA of piroplasma-specific region. For all positive samples we have done multiplex PCR for the species detection. Species determination was further confirmed by sequencing PCR products of 10 randomly selected Theileria equi and all Babesia caballi samples. The overall prevalence rates in analysed region for T. equi and B. caballi were 22.5% and 2.1%, respectively. Possible risk factors (such as location, age, sex and activity) associated with PCR positivity were evaluated. Marked differences were found in prevalence between geographic areas. There was no significant association between positivity and age group. T. equi was more prevalent in females and farming horses. This is the first report on the molecular survey of T. equi and B. caballi in central Balkan. Further prevalence studies on definitive host and vectors in this region are necessary.

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.

The aim of this study was to determine the concentrations of oxidative stress parameters and DNA damage in horses infected by Theileria equi. Initial screening of 110 horses with duplex PCR enabled the selection of 30 infected horses with T. equi and 30 free of infection (control). Specimens from the 60 horses were further analysed by determining the following oxidative stress parameters: extent of haemolysis (EH), plasma free haemoglobin (PHb), catalase (CAT), Cu,Zn superoxide dismutase (SOD1), paraoxonase (PON1), nitrite (NO), total nitrate and nitrite (NOx), malondialdehyde (MDA) and free thiol groups (-SH). In addition, relative distribution of lactate dehydrogenase (LDH-LDH) activity and the DNA-damaging effects of T. equi infection were evaluated. Compared to control horses, horses infected with T. equi had significantly higher SOD1 activities (P <0.05) and PHb (P <0.01), NO (P <0.001), NOx (P <0.05) and MDA concentrations (P <0.001), and significantly lower EH (P <0.001), CAT (P <0.01) and PON1 (P <0.001) activities, and thiol group concentrations (P <0.05). The comet assay demonstrated significantly increased DNA damage in T. equi infected cells compared to non-infected cells (P <0.001). Infected horses had significantly increased LDH isoenzyme activities (P <0.05). There was higher production of ROS/RNS in T. equi-infected horses, which resulted in changes in osmotic fragility, damage to lipids, proteins and DNA, haemolysis and hepatocellular damage. Oxidative stress in horses naturally infected with T. equi could contribute to the pathogenesis of the infection.

The main reasons for wildlife forensic research are animal poaching, illegal trade, and falsifi ed game meat products. Small trace amounts, old and degraded materials present the most common samples in revealing criminal activities in this fi eld. 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 identifi cation 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 amplifi cation is carried out simultaneously it is more cost effi cient and speeds up the process.