Single nucleotide polymorphism analysis of the ITS2 region of two sympatric malaria mosquito species in Sweden: Anopheles daciae and Anopheles messeae.

Four species of the Anopheles maculipennis complex have previously been recorded in Sweden. A recent addition to the complex is Anopheles daciae, which is considered to be closely related to, but distinct from Anopheles messeae. The original designation of An. daciae was based on five genetic differences (161, 165, 167, 362 and 382) in the second internal transcribed spacer (ITS) 2 of the ribosomal RNA. Further studies have shown that only two nucleotide differences (362 and 382) robustly separate the species. Thirty-three An. maculipennis complex mosquitoes were collected in the province of Uppland, Sweden. All were An. daciae but showed double peaks for three variable positions (161, 165 and 167). When cloned, the intra-individual nucleotide variation was almost exclusively fixed with either TTC or AAT, originally diagnostic for An. messae and An. daciae, respectively. To further investigate the intra-individual variation, nine An. daciae and 11 An. messeae were collected in southern Sweden and their ITS2 fragments were amplified and sequenced using Illumina MiSeq sequencing (Illumina, Inc., San Diego, CA, USA). For the diagnostic nucleotide 382 no intra-individual variation could be detected. However, although each An. daciae specimen carried several ITS2 sequence variants for the four other nucleotides, there was no intra-individual variation in the An. messeae specimens.

Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden.

The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick-borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly-central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia-infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia-infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia-infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the only mammalian tick host.

High levels of hybridization between molecular forms of Anopheles gambiae from Guinea Bissau.

In the malaria vector Anopheles gambiae Giles sensu stricto, two molecular forms denoted M and S are considered units of incipient speciation within this species. Very low hybrid frequencies and significant genetic differentiation have been found in sympatric M- and S-form populations. We studied the molecular form composition and the degree of genetic differentiation at 15 microsatellites in two samples of An. gambiae collected in two consecutive years from Bissau, Guinea Bissau. High frequencies of M/S hybrids (19-24%) were found in this area. Coincidently, very low levels of genetic differentiation were detected between forms when analysis involved microsatellites mapped at chromosome-3 (mean Fst, 0.000-0.002). The single exception was the X-linked AGXH678, for which high differentiation was measured (Fst, 0.158-0.301). This locus maps near the centromere of chromosome X, a low recombination region in which selection is likely to promote divergence between M and S forms. These results strongly suggest that the degree of isolation between M and S forms, considered the units of incipient speciation within An. gambiae, is not homogenous throughout the species distribution range.

Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus.

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

Evaluation of extracts and oils of tick-repellent plants from Sweden.

Abstract. Leaves of Myrica gale Linnaeus (Myricaceae), Rhododendron tomentosum (Stokes) H. Harmaja (formerly Ledum palustre Linnaeus: Ericaceae) and Artemisia absinthium Linnaeus (Asteraceae) were extracted with organic solvents of different polarities and the essential oils of leaves were obtained by steam distillation. The extracts or oils were tested in the laboratory for repellency against host-seeking nymphs of Ixodes ricinus Linnaeus (Acari: Ixodidae). Rhododendron tomentosum oil, 10%, diluted in acetone, exhibited 95% repellency; R. tomentosum and A. absinthium extracts in ethyl acetate, > 70% repellency; A. absinthium extract in hexane, approximately 62% repellency; and M. gale oil, 10%, approximately 50% repellency on I. ricinus nymphs. Compounds in the leaf extracts or in the oils were collected by solid phase microextraction (SPME) and identified by gas chromatography-mass spectrometry (GC-MS) and/or MS. Characteristic volatiles detected from oil or extract of M. gale were the monoterpenes 1,8-cineole, alpha-terpineol, 4-terpineol and thujenol; and of R. tomentosum myrcene and palustrol. Characteristic volatiles from leaf extracts of A. absinthium were sabinene, oxygenated monoterpenes, e.g. thujenol and linalool, and geranyl acetate. Each plant species synthesized numerous volatiles known to exhibit acaricidal, insecticidal, 'pesticidal' and/or arthropod repellent properties. These plants may be useful sources of chemicals for the control of arthropods of medical, veterinary or agricultural importance.

New and earlier records of ticks (Acari: Ixodidae, Argasidae) from Guinea-Bissau.

Ticks were collected from pastures and domestic and wild vertebrates during the rainy seasons of 1994 and 1996 in Guinea-Bissau. We collected the following species: from pastures Rhipicephalus lunulatus Neumann, R. muhsamae Morel & Vassiliades, R. senegalensis Koch, and R. sulcatus Neumann; from reptiles Amblyomma nuttalli Dönitz and Aponomma flavomaculatum (Lucas); and from mammals Amblyomma paulopunctatum Neumann, Amblyomma splendidum Giebel, Amblyomma variegatum (F.), Hyalomma truncatum Koch, R. muhsamae, R. sanguineus (Latreille), R. senegalensis, and R. sulcatus. New host records are as follows: Amblyomma nuttalli on Senegal flap shelled turtle (Cyclanorbis senegalensis Dumeril & Bibron), Aponomma flavomaculatum on West African dwarf crocodile (Osteolaemus tetraspis Cope), and R. sulcatus on bushbuck (Tragelaphus scriptus Pallas). We reviewed the literature for earlier records of ticks from Guinea-Bissau. The regional distributions of the 21 tick species hitherto recorded in Guinea-Bissau are listed.

Comparison of plant products and pyrethroid-treated bed nets for protection against mosquitoes (Diptera: Culicidae) in Guinea Bissau, West Africa.

In Guinea Bissau, West Africa, the shrub Hyptis suaveolens Poit. (Lamiaceae) and smoke of the bark of the tree Daniellia oliveri Rolfe (Caesalpiniaceae) traditionally are used to reduce numbers of mosquitoes indoors at night. We recorded the numbers of mosquitoes in the bedrooms of 30 households in a rural village, Mandinka-Rá in central Guinea Bissau. Each household was provided with bed nets and allocated randomly to 1 of 6 treatments as follows: (1) lambda-cyhalothrin-treated bed nets (10 mg/m2), (2) permethrin-treated bed nets (500 mg/m2), (3) burning of H. suaveolens, (4) burning of the bark of D. oliveri, (5) fresh H. suaveolens, and (6) control group using untreated bed nets and no plant products. In treatment groups 1-4, the mean number of mosquitoes was significantly less than the mean of the control. These results indicated that the use of burning H. suaveolens or D. oliveri indoors at night repelled endophagic mosquitoes and may contribute significantly to reducing the prevalence of diseases caused by pathogens transmitted by house-frequenting, nocturnally blood-feeding arthropods.

Rickettsia helvetica in Ixodes ricinus ticks in Sweden.

In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group Rickettsia isolated from Ixodes ricinus ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of Rickettsia helvetica. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme AluI. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of R. helvetica from the known positions where the AluI enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for R. helvetica. Investigation of 125 living I. ricinus ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.

Plant products used as mosquito repellents in Guinea Bissau, West Africa.

By standardized interviews of people in 23 rural villages, in the Oio region of Guinea Bissau, we collected data on which plant species and plant derived products or methods people use to reduce mosquito biting activity. The following plants were used to reduce numbers of mosquitoes indoors at night: fresh or smouldering Hyptis suaveolens Poit. (Lamiaceae), smoke of the bark of Daniellia oliveri Rolfe (Caesalpiniaceae), smoke of the infructescence of Elaeis guineensis Jacq. (Arecaceae), smoke of the seed capsules of Parkia biglobosa (Jacq.) Benth. (Mimosaceae), smoke of the leaves of Azadirachta indica A.Juss. (Meliaceae) and Eucalyptus sp. (Myrtaceae), fresh Ocimum canum Sims (Lamiaceae), and fresh Senna occidentalis (L.) Link (Caesalpiniaceae). In two field experiments we estimated the 'repellent activity' of certain of these plants and compared their efficacies with those of two commercially available mosquito repellents, i.e. 'positive' controls. In the first experiment we tested: smouldering H. suaveolens (85.4% repellency); fresh H. suaveolens (73.2%); burning of the bark of D. oliveri (74.7%); and smoke of the leaves of Eucalyptus (72.2%). In the second experiment we tested: smouldering H. suaveolens (83.6% repellency); fresh H. suaveolens (66.5%); burning of the bark of D. oliveri (77.9%); smoke of the leaves of A. indica (76.0%); smoke of the infructescence of E. guineensis (69.0%); fresh O. canum (63.6%); and fresh S. occidentalis; (29.4%). All the products tested, except S. occidentalis were significantly more effective than the negative control.

Increasing geographical distribution and density of Ixodes ricinus (Acari: Ixodidae) in central and northern Sweden.

The geographical distribution and density of Ixodes ricinus (L.) in the 2 northern regions, Svealand and Norrland, in Sweden were studied by using a questionnaire in Swedish magazines for house owners and dog owners, and in provincial newspapers. Analysis of the approximately 1,200 answers revealed that ticks are present in all parts of Svealand (except northern Värmland and northern and western Dalarna), the southeastern part of Norrland (i.e., Gästrikland and Hälsingland), and along the Baltic Sea coast of central and northern Norrland. The proportion of answers reporting ticks and the estimated tick density (i.e., the number of ticks infesting dogs and cats) decreased from south to north. The answers to the questionnaire and data from field sampling of ticks suggest that tick density decreased distinctly along a narrow boundary zone, coinciding with the biological limit of Norrland (Limes Norrlandicus), crossing Sweden through southern Värmland, southeastern Dalarna, and central Gästrikland. The results of the questionnaire suggest that I. ricinus ticks are more widespread today than in the early 1980s, especially in Värmland, western and central Dalarna, Hälsingland, and the coastal areas of Medelpad, Angermanland, and Västerbotten, and that the proportion of the human population at risk for tick-borne pathogens in Svealand and Norrland is increasing.

The palpal ratio method compared with PCR to distinguish between Anopheles gambiae s.s. and A. melas from Guinea Bissau, West Africa.

We compared the palpal ratio method with the polymerase chain reaction (PCR) to distinguish between Anopheles gambiae s.s. and A. melas. At the end of the rainy season of 1995, female mosquitoes were collected indoors in the Antula area of Bissau, Guinea Bissau. A subsample of 354 mosquitoes were identified first with PCR and then with the palpal ratio method (study A). Subsequently, another 195 mosquitoes were identified first with the palpal ratio method and then with PCR (study B). The highest percentage (100%, n = 16) of correctly identified A. melas was obtained at the palpal ratio cut-off point of 0.83. However, at this point 4.0% (14/347) and 11.3% (21/186) of the A. gambiae were erroneously identified as A. melas in study A and B, respectively. This suggests that the palpal ratio method is not sufficiently reliable to distinguish between A. gambiae and A. melas from the Bissau area.

Lyme borreliosis awareness.

A Lyme borreliosis information leaflet has been produced to promote awareness amongst the general public. It was designed to provide a framework for similar material throughout Europe and complements a questionnaire produced to measure awareness of Lyme borreliosis. This questionnaire can be used to determine the impact of educational campaigns using material such as the leaflet. Feasibility studies showed that the questionnaire successfully highlighted predictable differences between sample groups and also that the leaflet performed well in increasing knowledge in low-awareness groups.

Ixodes ricinus strains in Europe.

An analysis of cuticular hydrocarbons (CH) of unfed adult Ixodes ricinus ticks collected throughout Europe showed that there are 10 distinct I. ricinus groups. Studies on the seasonal and annual consistency of CH composition and possible effects of host and environmental factors suggested that CHs may be used as a genuine genetic marker for I. ricinus. Preliminary studies compared the vector competence of ticks from three of the most separated I. ricinus groups and the results suggested that there may be significant differences in tick susceptibility to Borrelia afzelii.

European reservoir hosts of Borrelia burgdorferi sensu lato.

Existing knowledge on reservoir hosts of Borrelia burgdorferi sensu lato was collated and reviewed and several species, particularly birds, were identified as reservoir competent. At the present time, 9 small mammals, 7 medium-sized mammals and 16 bird species, including passerines, sea birds and pheasants, appear to be capable of transmitting spirochaetes to ticks and thus of participating in the natural circulation of B. burgdorferi s.l. in Europe. The house mouse, Mus musculus is strongly suspected of reservoir competence and many other small rodent species, particularly in eastern Europe and Russia, have been implicated. Ungulates are not thought to play a major role as reservoir hosts, though co-feeding transmission may permit some tick infection. The criteria for establishment of reservoir status are outlined and a method for identification of host blood meals of previous instars of unfed ticks, developed in a participant laboratory, is briefly described.

Lyme borreliosis habitat assessment.

Tick ecologists throughout Europe provided descriptions of Lyme borreliosis habitats according to a standardised format and data for 105 habitats in 16 countries were received. The data showed that high risk situations, as defined by the presence of large numbers of B. burgdorferi sensu lato-infected nymphal I. ricinus, occur in heterogeneous deciduous woodland, usually with a recreational function and with a diverse fauna, usually including deer. Large numbers of ticks occurred in some other habitats, but infection prevalence was usually low. The situation for adult I. ricinus was similar but less clearly defined. Tick infection rates were found to be lower in western Europe than in the east, and the infection rate in I. persulcatus, the most easterly vector species, was markedly higher than in I. ricinus. In the vast majority of habitats the infection rate in adult I. ricinus was greater than in nymphs. Larvae were rarely found to be infected.

Transmission of mixed malaria species and strains by mosquitoes, as detected by PCR, in a study area in Guinea-Bissau.

Parasites present in blood samples of asymptomatic carriers and in the midgut of mosquitoes collected within a few days from the same households, have been analysed by PCR. A high prevalence (32%) of infected mosquitoes was observed and, in half of these, two parasite species were found simultaneously. The distribution of parasite species in the mosquito correlated with that found in the infected persons. Genotype patterns of Plasmodium falciparum populations were however found to be different in the two sets of samples. These results and the potential of PCR are discussed with reference to investigations of the dynamics of malaria transmission.

Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.

Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in south-central Sweden.

Infestation by Ixodes ricinus ticks on rodents, hares and cervids was examined at Bogesund, 10 km north of Stockholm, in south-central Sweden during 1991-1994 and on varying hares (Lepus timidus) at Stora Karlsö and Gotska Sandön in the Baltic Sea during 1992-1993. At Bogesund, there were great differences between two consecutive years in the number of I. ricinus larvae infesting bank voles (Clethrionomys glareolus). The seasonal pattern of infestation by I. ricinus larvae and nymphs on bank voles was unimodal in 1991, with peaks in June-July and bimodal in 1992, with peaks in June and August. Male bank voles, compared to females and older voles, compared to young voles, harboured greater numbers of I. ricinus ticks. Apodemus mice, compared to bank voles, harboured greater numbers of I. ricinus ticks. Ixodes ricinus larvae engorged on Apodemus mice were heavier than larvae engorged on bank voles and resulted in larger nymphs. However, there was no difference in the proportions of viable nymphs resulting from larvae engorged on mice or voles. The ranges in the numbers of I. ricinus ticks infesting individual hosts were 1-451 for rodents, 16-2374 for hares and 428-2072 for roe deer (Capreolus capreolus). These ranges of tick numbers are estimated to represent potential blood losses from individual hosts of approximately 0.2-65% for rodents, 0.2-13% for hares and 0.3-9.0% for roe deer. Within the populations of all host species examined, the distributions of all stages of I. ricinus were clumped, with most host individuals harbouring few ticks and only a few individuals harbouring many ticks. The data suggest that, even though a small proportion of tick hosts may be severely affected, the direct effects of feeding by I. ricinus are unlikely to play an important role on mammal population dynamics.

Relationship between Ixodes ricinus density and prevalence of infection with Borrelia-like spirochetes and density of infected ticks.

During 1994-1995 we examined the density of questing Ixodes ricinus (L.) nymphs and their prevalence of infection with Borrelia-like spirochetes at 43 localities in south-central Sweden with median nymphal densities ranging from 0.1 to > 50/100 m2. The general pattern was that the infection prevalence in nymphs increased with nymphal density at < 10 nymphs per 100 m2, peaked (20-30%) at 10-20 nymphs per 100 m2, and decreased at higher nymphal densities. A low infection prevalence (7-10%) in nymphs at 4 of the 5 localities with the highest nymphal densities (> 35 nymphs per 100 m2) at the small island of Bedarön was most likely related to the presence of large numbers of fallow deer, Dama dama (L.), which are reservoir-incompetent for Borrelia burgdorferi s.l. Johnson, Schmid, Hyde, Steigerwalt & Brenner. However, because the increase in nymphal density at Bedarön was greater than the decrease in infection prevalence, 5 of the 6 highest densities of infected nymphs occurred in the 5 localities at Bedarön. We suggest that reservoir-incompetent cervids, being more important hosts for adult 1. ricinus than for the larvae, indirectly increase the density of infected nymphal ticks by feeding large numbers of adult ticks, thereby increasing the number of larvae feeding on reservoir-competent shrews, rodents, and hares.