Analysis of the distribution of the tick Ixodes ricinus L. (Acari: Ixodidae) in a nature reserve of western Germany using Geographic Information Systems.

From May to November 2003, at five selected sites of the 'Siebengebirge', a nature reserve near Bonn, Germany, data on microclimate, pedology, plant sociology and tick abundance were collected weekly and correlated. A total of 2832 host-seeking Ixodes ricinus (2660 nymphs and 172 adults) were collected, with maximal abundance in June and a minor second increase in abundance in August. The spatial and temporal variation in tick abundance was closely related to the air temperature, relative humidity, soil water content and specific vegetation type. Increases in tick abundances in individual habitats were most likely caused by climate change and/or anthropogenic interventions such as increased numbers of wild boar, a host of I. ricinus. When combined with data from previous investigations a correlation of plant communities with six semi-qualitative tick abundance categories (ranging from 'no ticks' to 'very high tick abundance') was revealed, and GIS analysis indicated that 56.5% of the 'Siebengebirge' is made up of plant communities belonging to the three highest tick abundance categories. Therefore, the risk of visitors in the 'Siebengebirge' of being exposed to tick bites and being infected by Borrelia burgdorferi sensu lato had increased.

Multiple environmental factor analysis in habitats of the harvest mite Neotrombicula autumnalis (Acari: Trombiculidae) suggests extraordinarily high euryoecious biology.

The harvest mite Neotrombicula autumnalis (Trombiculidae) has become a great nuisance in various vegetated areas in Germany over the last 15 years. According to reports of dermatologists, this species appears to have propagated and spread significantly. Moreover, cases of severe trombidiosis or trombidiosis-like skin reactions have been noticed at unusually early times of the year. Due to the lack of scientific studies, little is known about the ecology of N. autumnalis and its distribution, and preferred habitats cannot be predicted. A four-year study was conducted to identify trombiculid foci in different areas of Bonn in order (1) to determine the timing of larvae appearance in different years, (2) to identify the factors that lead to high larvae abundances at the mite foci ('multiple factor analysis'), and (3) to develop an ecological control strategy. By means of the 'tile catch method' (TCM) which turned out to be most appropriate to collect data on the distribution and abundances of trombiculid mites, larvae of N. autumnalis were caught from mid July until the end of October/beginning of November. The distribution of the mites was patchy, supporting the hypothesis that certain factors cause a concentration in foci. Most of the mite foci had a fixed location for at least three years. Efforts to isolate nymphs and adults in larger quantities to gain knowledge about their preferred soil areas and soil depths failed. Only some nymphs of N. autumnalis could be found living 10-40 cm deep in the soil. Due to the restriction that the nymphs and adults can only rarely be isolated in the ground, the analysis of environmental factors was executed based on abundances of questing larvae on the soil surface. The detailed analysis of soil-physical, soil-chemical and meso-faunistic factors could not finally explain the unequal distribution of the mites, although the porosity of the soil had a statistically significant slight influence on the abundance of larvae, and soil pH bordered significance, also suggesting a slight influence. Furthermore, soil temperatures during the winter seasons in three subsequent years appeared too high to affect the harvest mite. The field experiments suggest that N. autumnalis and particularly its larval stages are extremely euryoecious (meaning tolerating very different environmental conditions). Further studies are necessary: additional investigations on the influence of certain abiotic environmental factors on N. autumnalis, the search for factors underlying the rhythmicity of its life cycle ('zeitgeber'), and the reasons and mechanisms for heterogeneous distribution of soil fauna in general. Ecological control of the mite is, in principle, possible but only after identifying the foci and ascertaining their approximate dimensions with the TCM. This control strategy is the most promising one, albeit very laborious, emphasising the need of further research on the ecology of the harvest mite.

Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany.

From March to October 2003, a total of 2,518 host-seeking Ixodes ricinus ticks (1,944 nymphs, 264 females, 310 males) were collected by blanket dragging at 45 sites all over the city area of Bonn, western Germany, to be checked for Borrelia burgdorferi infection. The collection sites included 20 private gardens, nine public recreational parks, the boundaries of 14 sylvatic suburban areas and two footpaths between suburban farmed fields. Generally, numbers of specimens collected along sylvatic suburban areas and at urban sites with dense tree populations were significantly higher than at the other collection sites. Out of 1,394 specimens (865 nymphs, 241 females, 288 males) that were randomly chosen for Borrelia analysis by a simple PCR, 250 (17.9 %) were found to be infected with B. burgdorferi sensu lato. While the infection prevalences varied significantly between females (26.6%), males (12.5%) and nymphs (17.3%), there were no striking differences between sylvatic and unwooded sites. A total of 92.8% of the ticks Borrelia-positive by the simple PCR were also positive in a diagnostic nested PCR. Using genospecies-specific oligonucleotide probes, single Borrelia genospecies infections (91.4%) could be assigned to B. afzelii (39.5%), B. garinii (27.9%), B. burgdorferi sensu stricto (15.6%) and B. valaisiana (8.6%) by DNA hybridization. Various combinations of double infections were observed in 4.3% of the infected ticks. Another 4.3% of the Borrelia infections were untypeable. The B. burgdorferi genospecies distribution in the city area was shown to be variable from site to site and, even more, it was distinct from rural collection sites near Bonn. This is ascribed to a different spectrum of reservoir hosts. Taking into account the infection prevalences of host-seeking ticks in the forested surroundings of Bonn, our study demonstrates that the risk of acquiring Lyme disease after a tick bite in urban/suburban areas is comparably as high as in woodlands outside of the city.

Substantial rise in the prevalence of Lyme borreliosis spirochetes in a region of western Germany over a 10-year period.

More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.

Polymerase chain reaction-based differentiation of the mosquito sibling species Anopheles claviger s.s. and Anopheles petragnani (Diptera: Culicidae).

A polymerase chain reaction (PCR)-based diagnostic assay was developed that rapidly and reliably differentiates the sibling species of the Anopheles claviger complex, An. claviger s.s. and An. petragnani. The assay makes use of nucleotide differences in the internal transcribed spacer 2 ribosomal DNA sequences to generate PCR products of specific length for each of the two species. In evaluating the test, 580 of 592 field-collected An. claviger s.l. specimens were unambiguously identified as one of the two sibling species. Due to poor DNA quality, the remaining 12 specimens yielded no PCR product. Of the 592 mosquitoes, 407 larval specimens had been identified morphologically prior to species-specific DNA amplification, and in all instances PCR identification corroborated with morphologic identification. Mosquitoes identified as An. claviger s.s. came from various localities all over Europe and from Israel. Those identified as An. petragnani were collected in southern France and Spain. The species-diagnostic PCR assay would facilitate data collection on the temporal and spatial distribution of the two An. claviger sibling species because they represent possible vectors of disease in Europe, the Near and Middle East, and north Africa.

Spermidine metabolism in parasitic protozoa--a comparison to the situation in prokaryotes, viruses, plants and fungi.

Targeting polyamines of parasitic protozoa in chemotherapy has attracted attention because polyamines might reveal novel drug targets for antiparasite therapies (Müller et al. 2001). The biological function of the triamine spermidine in parasitic protozoa has not been studied in great detail although the results obtained mainly imply three different functions, i.e., cell proliferation, cell differentiation, and biosynthesis of macromolecules. Sequence information from the malaria genome project databases and inhibitor studies provide evidence that the current status of spermidine research has to be extended since enzymes of spermidine metabolism are present in the parasite (Kaiser et al. 2001). Isolation and characterisation of these enzymes, i.e., deoxyhypusine synthase (EC 1.1.1.249) (DHS) and homospermidine synthase (EC 2.5.1.44) (HSS) might lead to valuable new targets in drug therapy. Currently research on spermidine metabolism is based on the deposition of the deoxyhypusine synthase nucleic acid sequence in GenBank while the activity of homospermidine synthase was deduced from inhibitor studies. Spermidine biosynthesis is catalyzed by spermidine synthase (EC 2.5.1.16) which transfers an aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine. Spermidine is also an important precursor in the biosynthesis of the unusual amino acid hypusine (Wolff et al. 1995) and the uncommon triamine homospermidine in eukaryotes, in particular in pyrrolizidine alkaloid-producing plants (Ober and Hartmann 2000). Hypusine is formed by a two-step enzymatic mechanism starting with the transfer of an aminobutyl moiety from spermidine to the epsilon-amino group of one of the lysine residues in the precursor protein of eukaryotic initiation factor eIF5A by DHS (Lee and Park 2000). The second step of hypusinylation is completed by deoxyhypusine hydroxylase (EC 1.14.9929) (Abbruzzese et al. 1985). Homospermidine formation in eukaryotes parallels deoxyhypusine formation in the way that in an NAD(+)-dependent reaction an aminobutyl moiety is transferred from spermidine. In the case of homospermidine synthase, however the acceptor is putrescine. Thus the triamine homospermidine consists of two symmetric aminobutyl moieties while there is one aminobutyl and one aminopropyl moiety present in spermidine. Here, we review the metabolism of the triamine spermidine with particular focus on the biosynthesis of hypusine and homospermidine in parasitic protozoa, i.e., Plasmodium, Trypanosoma and Leishmania, compared to that in prokaryotes i.e., Escherichia coli, a phytopathogenic virus and pyrrolizidine alkaloid-producing plants (Asteraceae) and fungi.

Individual cases of autochthonous malaria in Evros Province, northern Greece: entomological aspects.

Prompted by four autochthonous cases of malaria in 1994 and 1995 in Evros Province, northern Greece, we conducted an entomological study between 1997 and 1999 in Nipsa and Chandras, rural locations where two of the four cases had occurred, and in Feres where two additional autochthonous malaria cases had been diagnosed in 1998. In Nipsa and Chandras, we identified 29 Anopheles breeding sites and characterized them by physicochemical parameters. Larvae were collected both at these sites and in a brackish water breeding site near Feres in the Evros River delta. Adults were caught in sheds at all three locations. Morphology was used to classify larvae and adults as A. superpictusor as species belonging to the A. claviger or A. maculipennis species complexes. The latter were further identified by PCR as being A. maculipennis s.s., A. melanoon and A. sacharovi. Of the A. maculipennis complex larvae collected inland, approximately 94% were A. maculipennis s.s. and 6% A. melanoon, whereas all larvae collected in the coastal region were A. sacharovi. In contrast, the A. maculipennis adults were A. maculipennis s.s. and A. melanoon (both 47%), and A. sacharovi (6%). In the coastal region, no A. maculipennis s.s. adults were caught. The ratio of A. melanoon adults collected to A. sacharovi was about 3:1. As shown by a bloodmeal ELISA, only 5 of 266 fed females (1.9%) had ingested human blood, whereas 232 (87%) had fed on goats. Of the mosquitoes containing human blood, two were A. melanoon, one A. sacharovi and one A. maculipennis s.s. One human blood specimen could no longer be assigned to a particular mosquito.

Individual cases of autochthonous malaria in Evros Province, northern Greece: serological aspects.

From 1994 to 1995 four presumably autochthonous malaria cases were diagnosed by blood smear microscopy in Evros Province, northern Greece. Alarmed by these unexpected infections a serological survey was performed from 1997 to 1999 in ten rural villages, including those where the malaria cases had occurred. Among the 1,102 blood samples examined, nine turned out to contain specific antibodies against plasmodial parasites as detected by indirect fluorescent antibody test, including two of the former patients. The remaining seven samples were taken from healthy individuals with no history of recent infection or of having travelled to endemic areas. A further 21 sera showed borderline reactivity with Plasmodium falciparum antigen. Although no retrospective examination of the blood specimens could be performed to confirm the serological results by direct parasite detection, we can conclude that at least the seropositive persons have actually undergone infection with malaria parasites but developed no or only mild clinical symptoms which went unnoticed. It is becoming obvious that even in European countries where climatic and vector conditions are favourable for the development of the parasite there is a potential risk of incidental malaria transmission by indigenous Anopheles mosquitoes.