Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and assessment of entomological risk index at localities in Belgrade.

Background/Aim: The first case of human Lyme borreliosis (LB) in Serbia was recorded in 1987. The number of reported LB cases has increased in the past decade. The aim of this study was to estimate the density of Ixodes ricinus (I. ricinus) ticks, the prevalence of Borrelia burgdorferi sensu lato (B. burgdorferi) in them, and entomological risk index (ERI) at 19 Belgrade localities which were grouped into three categories (forests, parkforests, parks). The values of ERI were compared with the number of tick bites in humans. Methods: Ticks were collected monthly by using the flag hours method and the infection rate was determined by using dark field microscopy. The ERI value was calculated for each locality where the ticks were collected. The related data about tick bites was obtained from the patient protocol of the Institute of Epidemiology, Military Medical Academy, Belgrade. Results: The total number of collected ticks, the number of nymphs and the infection rates of the nymphs were significantly higher in forests (p < 0.05) than park-forests and parks. Statistically, the ERI value was significantly higher in forests than parks of Belgrade (χ2 = 7.78, p < 0.01). In March and July, the ERI value was also significantly higher in forests, than park-forests (p < 0.01) and parks (p < 0.01). May was the month with the highest ERI value in each ecological category (forests p < 0.05; park-forests p < 0.01; parks p < 0.001). However, the number of tick bites in humans did not correlate with ERI values. Conclusion: The obtained results indicate that the risk of tick bite and human exposure to B. burgdorferi sensu lato is present at all selected localities in Belgrade. For a more comprehensive Lyme disease risk assessment the method of entomological risk index assessment should be combined with other methods, taking into consideration all tick stages and the behaviour and habits of people who may get infected B. burgdorferi sensu lato.

Evidence of recombination in Tula virus strains from Serbia.

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

[Risk of Lyme disease development after a tick bite].

BACKGROUND/AIM: Despite numerous research of Lyme disease (LD), there are still many concerns about environmental of infectious agent of LD, as well as its prophylaxis, diagnosis and treatment. The aim of this work was to determine the risk of LD in relation to the way of removing ticks and duration of tick attachment. METHODS: In the period from 2000 to 2007 a prospective study was conducted including persons with tick bite referred to the Institute of Epidemiology, Military Medical Academy, and followed for the occurrence of early Lyme disease up to six months after a tick bite. Epidemiological questionnaire was used to collect relevant information about the place and time of tick bites, the way of a removing tick, duration of tick attachment, remnants of a tick left in the skin (parts of the mouth device) and the signs of clinical manifestations of LD. Duration of tick attachment was determined on the basis of size of engorged tick and epidemiological data. Removed ticks were determined by the key of Pomerancev. Professional removing of attached tick was considered to be removing of tick with mechanical means by healthcare personnel. Fisher's exact test, Chi squares test and calculation of the relative risk (RR) were used for data analysis. RESULTS: Of 3 126 patients with tick bite, clinical manifestations of LD were demonstrated in 19 (0.61%). In the group of subjects (n = 829) in which a tick was not removed professionally there were 17 (2.05%) cases with LD, while in the group of respondents (n = 2 297) in who a tick was removed professionally there were 2 (0.09%) cases with LD after tick bite (RR, 23.55; p < 0.0001). The disease was most frequent in the group of respondents with incompletely and unprofessionally removed ticks (2.46%). In the groups of patients with unprofessionally but completely removed ticks LD occurred in 0.89%, while in the group of subjects with a tick removed by an expert, but incompletely in 0.78% cases. The disease occurred rarely in the group with a tick removed completely and professionally (0.05%). There was no case of LD in the group of patients with a tick removed within 24 hours. The longer time of exposure after 24 hours, the higher absolute risk of disease was reported. CONCLUSION: In prevention of Lyme disease it is important to urgent remove a tick, to use a correct procedure of removing and to remove the whole tick without any remnants.

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in Belgrade area.

OBJECTIVE: Lyme borreliosis is vector-borne zoonosis. The causative agent of Lyme borreliosis is a spirochete of Borrelia burgdorferi (Bb) sensu lato complex, which is transmitted by ticks of the Ixodes ricinus complex. The aim of our paper is to estimate the prevalence of I. ricinus ticks, the level of their infectivity by Bb, and the prevalence of certain genospecies of Bb sensu lato in ixodide ticks inhabiting Belgrade. MATERIALS AND METHODS: An estimate of the tick population density was expressed by the value of flag/hour. For isolation and cultivation of Borrelia, selective Barbour-Stonner-Kelly II media was used. Typization of Borrelia was made by applying the species-specific polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism analysis. In statistical analysis, Chi(2) test was used. RESULTS: Values of flag/hour have varied in relation to year observed and type of habitat: The lowest values were recorded in the city parks (17.9). The values were higher in parks-woods (19.7 and 33.4, respectively). The highest values were detected in localities similar to wooded areas (48.0). The estimated average infestation of ticks with Bb was 21.9%, excluding statistically significant differences by years of investigation. We found the dominance of Borrelia afzelii (75%). Bb sensu stricto (22.2%) as well as Borrelia garinii (2.8%) was much less present. Statistically significant difference was established in the prevalence of the above-mentioned genospecies in relation to the examined localities. CONCLUSIONS: We have established the prevalence of all three genospecies in the city of Belgrade. Bb sensu lato was found, with the dominance of B. afzelii.

[Risk for infection by lyme disease cause in green surfaces maintenance workers in Belgrade].

BACKGROUND/AIM: Lyme disease (LD) is a cosmopolitan disease from the group of zoonoses that in humans can affect skins, joints, heart and/or nervous system. Research conducted in endemic areas, shows not only that the population, which lives or works in tick habitats, is in a high risk for getting infected by LD cause, but also that every enviroment itself is specific in regard to both vectors and B. burgdoferi species. In our enviroment, research of seropositivity to B. bugdorferi in persons exposed to vectors of LD have not been conducted. The aim of this study was to determine whether there was a higher risk for getting B. burgdofei infection in the fieldworks of JKP "Zelenilo Beograd" than in other persons. METHODS: Risk for getting LD in the fieldworkers of JKP "Zelenilo Beograd" (park-maintanance company) was determined according to seropositivity, the number and infection rate of LD vectors, and entomological risk index (ERI) value. Serum samples of 34 fieldworkers ofJKP "Zelenilo Beograd", and 35 persons of the control group, were tested for the presence of specific IgM and IgG anti-B. burgdofedi antibodies by using ELISA test In the research localities, the number of ticks was estimated by using the flag hours method, and tick infection rate was determined by dark field microscopy, with magnification rate of 400x. The data were analyzed statistically using chi(2) test, and Pearson's correlation analysis. RESULTS: Out of 34 fieldworkes serum samples, 8 (23.5%) were positive on anti-B. buagdorfei antibody presence, while in the control group only one (2.9%/0) sample was positive (p < 0,05). Seropositivity rate varied from 0-66.6% among localities. Flag hours value among the localities ranged from 8.2-29.9, and tick infection rate ranged from 10.8-22.3%. ERI value was 0.03-0.15. Correlation between flag hour rate and seropositivity, tick infection rate and seropositivity, and also ERI and seropositivity was established. CONCLUSION: Frequency of B. burgdorferi seropositivity was significantly higher in green-surface maintenance workers, than in the control group not exposed to these habitats. A significant correlation was found between anti-B. burgdofedi antibodies findings and the number of ticks, tick infection rate, and ERI. The results obtained in our study indicate that there was a significantly higher risk for getting LD cause in the fieldworkers than in the control group.

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.

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain.

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.