Analysis of pathogen co-occurrence in host-seeking adult hard ticks from Serbia.

Past studies in Serbia have reported concurrent infections of Ixodes ricinus ticks with Borrelia burgdorferi sensu lato genospecies, Anaplasma phagocytophilum and Francisella tularensis. As a step forward, this investigation included a broader range of microorganisms and five most common and abundant tick species in Serbia. Five tick species were identified (Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. concinna and I. ricinus) and analyzed for the presence of seven pathogens. Anaplasma ovis, A. phagocytophilum, Babesia canis, B. burgdorferi s.l., Coxiella burnetii, Rickettsia helvetica and R. monacensis were detected. Sequencing of samples positive for F. tularensis revealed the presence of Francisella-like endosymbionts. No Bartonella spp. DNA was amplified. Concurrent infections were present in three tick species (D. reticulatus, H. concinna and I. ricinus). The rate of co-infections was highest in I. ricinus (20/27), while this tick species harbored the broadest range of co-infection combinations, with dual, triple and a quadruple infection(s) being detected.

First detection of spotted fever group Rickettsiae in ticks in Serbia.

Ticks can transmit multiple pathogenic bacteria responsible for diseases in animals and humans such as Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and spotted fever group Rickettsia sp. The current study aimed to investigate the presence of Rickettsiae in ticks collected from seven localities in Serbia. One hundred thirty-one (131) questing ticks belonging to 5 tick species (Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Haemaphysalis Concinna, and Ixodes ricinus) were collected in 2007 and 2009. Ticks were tested by polymerase chain reaction, amplifying gltA, ompA, and 17-kd genes, and sequencing analysis, revealing the presence of Rickettsia helvetica and Rickettsia monacensis in I. ricinus ticks only (infection rates 7.7% and 15.4% for R. helvetica and R. monacensis, respectively). R. helvetica has been isolated from I. ricinus ticks and has been implicated in fatal perimyocarditis. R. monacensis was first identified in I. ricinus samples collected in Germany and has recently been implicated in human infection. The results of the current study make fundamental the need to evaluate the incidence of infection with R. helvetica and R. monacensis among the resident population.

Potential infectivity of Anaplasma phagocytophilum strains in Ixodes ricinus ticks from Serbia.

Anaplasma phagocytophilum strains from different geographical regions are characterised by diverse potential infectivity for humans and domesticated animals. We investigated the potential pathogenicity of A. phagocytophilum strains in Ixodes ricinus ticks from 11 geographically different localities in Serbia. Sequences obtained in this study showed a high variability of p44 paralogues. Some of them, however, formed groups with similarities greater than 86% ('similarity groups'). Previous studies showed that 'similarity groups' were nearly always country specific. Our results correlated with this observation, and we also observed significant clustering of paralogues according to vector and reservoir origin of A. phagocytophilum strains. According to the high genetic similarity of sequences isolated from ticks collected in four localities, namely Avala, Batrovci, Hajducka cesma and Ljubovija, with paralogues with proven pathogenicity isolated from human granulocytic anaplasmosis (HGA) patients and A. phagocytophilum infected sheep, we could assume that strains with potential infectivity for humans and domestic animals were present in Serbia.

Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Francisella tularensis and their co-infections in host-seeking Ixodes ricinus ticks collected in Serbia.

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.

Molecular characterization of Borrelia burgdorferi sensu lato strains isolated in the area of Belgrade, Serbia

This is the first report of the molecular characterization and identification of Borrelia burgdorferi sensu lato strains isolated in Serbia. Isolates A1, A2 and M1, from Ixodes ricinus, belong to Borrelia burgdorferi sensu stricto, while isolate K1 from Apodemus flavicollis is a mixture of Borrelia afzelii and B. burgdorferi s.s.

Trata-se do primeiro relato de identificação e caracterização molecular de cepas de Borrelia burgdorferi sensu lato isoladas na região de Belgrado, Sérvia. As cepas A1, A2 e M1, isoladas de Ixodes ricinus, pertencem à Borrelia burgdorferi sensu stricto, enquanto a cepa K1, isolada de Apodemus flavocollis é uma mistura de Borrelia afzelii e B. burgdorferi s.s.

Allozyme polymorphism of Mdh and alpha-Gpdh in Ixodes ricinus populations: comparison of borreliae-infected and uninfected ticks.

Ixodes ricinus Linnaeus (Acari: Ixodidae) ticks are vectors of numerous infectious diseases in humans and animals. The allozyme variability of MDH and alpha-Gpdh was detected by native polyacrylamide gel electrophoresis in I. ricinus natural populations in three localities in Serbia. Four alleles of Mdh locus (MDH 1, MDH 2, MDH 3 and MDH X) and four alleles of alpha-Gpdh locus (VS, S, F and VF) were detected. Interpopulation differences in Mdh and alpha-Gpdh allele frequencies were statistically insignificant. Significant difference in alpha-Gpdh allele frequencies between males and females was recorded in the largest sample only. Differences in allele frequencies, detected between borreliae-infected and uninfected I. ricinus ticks, were close to the level of statistical significance, especially for alpha-Gpdh locus. Clear significant difference appeared in females when sexes were tested separatelly (P = 0.037). It is interesting that genotypes containing rarer alleles (MDH 1 and S) were infected in higher proportion in comparison to other genotypes. Our results point towards a possible role of Mdh and alpha-Gpdh loci in I. ricinus ticks in the determination of energy requirements for host seeking. Sex differences in alpha-Gpdh allele frequencies suggest that selective pressure, concerning efficiency of reserve materials utilisation, points to alpha-Gpdh rather than to Mdh locus.

Persistence and efficacy of three diatomaceous earth formulations against Sitophilus oryzae (Coleoptera: Curculionidae) on wheat and barley.

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.