BackgroundIn Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers.ResultsBetween 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2–100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5–100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5–100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes.ConclusionsA simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies.

To evaluate the prevalence rate of tick-borne bacterial pathogens, unfed adult Ixodes ricinus ticks were collected from vegetation in 2001, 2003, and 2004 at 18 localities throughout Serbia. A total of 287 ticks were examined by PCR technique for the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Francisella tularensis. The highest prevalence rate was that for B. burgdorferi sensu lato (42.5%), followed by A. phagocytophilum (13.9%) and F. tularensis (3.8%). The presence of five B. burgdorferi sensu lato genospecies, namely, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana was identified by restriction fragment length polymorphism (RFLP) analysis. The most frequent B. burgdorferi sensu lato genospecies was B. lusitaniae, followed by B. burgdorferi sensu stricto. Co-infection by B. burgdorferi sensu stricto and B. lusitaniae was frequently observed. Co-infection by B. burgdorferi sensu lato and A. phagocytophilum and co-infection by B. burgdorferi sensu lato and F. tularensis appeared in 24 ticks. Sequencing of p44/msp2 paralogs of Serbian A. phagocytophilum showed that they were unique and distinct from those of A. phagocytophilum in US and UK. This is the first report of B. garinii, B. lusitaniae, B. valaisiana, as well as A. phagocytophilum and F. tularensis infected ticks in Serbia. These findings indicate a public health threat in Serbia of tick-borne diseases caused by B. burgdorferi sensu lato, A. phagocytophilum and F. tularensis.

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The insecticidal and residual efficacy of three diatomaceous earth (DE) formulations, Insecto, PyriSec, and SilicoSec, against Sitophilus oryzae (L.) on barley and wheat was assessed. For this purpose, 4-kg lots of barley and wheat were treated with the above-mentioned DE formulations, in three dose rates (0.75, 1, and 1.5 g/kg grain) and stored at 26 degrees C. Samples of these lots were taken at the day of storage, and every 45 d, until the completion of a 450-d period of storage. Bioassays were conducted by exposing S. oryzae adults to these samples, at 26 degrees C and 57% RH. In these bioassays, the DE efficacy was evaluated by recording adult mortality after 24 h, 48 h, 7 d, and 14 d of exposure on the treated grains. After the 14-d count, all adults were removed, and the samples were left at the same conditions for an additional 45 d, to evaluate the capacity for progeny production in the treated grains. Adult mortality after 14 d of exposure was exponentially decreased with time. During the first 270 d of storage, mortality was > 90%, and progeny production was < 1 adult per sample, whereas after 270 d a gradual decrease in adult mortality occurred, with a resulting increase in progeny production. Generally, the three DE formulations tested were equally effective against S. onyzae adults. During the first 270 d of storage, the DE formulations were equally effective on both grains tested, but from 315 d of storage and on, S. oryzae mortality was higher on barley than on wheat. At this interval, progeny production was gradually increased, especially on grains treated with the lowest DE dose rate. However, even this rate caused a satisfactory level of mortality (> 90% after 14 d of exposure) during the first 270 d of storage.

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.