Natural infection of the tick Dermacentor reticulatus (Fabr.) with the microsporidian Nosema slovaca Weiser et Rehácek in Slovakia

A total of 6,199 adult ticks (Haemaphysalis intermis, Dermacentor reticulatus, D. marginatus and Ixodes ricinus) collected in three localities in Slovakia were examined for microsporidian infection. The spores of Nosema slovaca were found in six D. reticulatus (0.097%). The microsporidian experimentally inoculated into the hemocele of half-engorged females of D. reticulatus caused acute infection and death of the host. The infection can be transmitted also to other tick species from the same region. The yield of spores from one tick ranges from 3 X 10(5) to 52 X 10(7) depending on the infection dose and tick species. Peroral infection of some butterfly larvae with the microsporidian pores was unsuccessful. The importance of Nosema infection in ticks in nature is discussed.     

Francisella tularensis from ixodid ticks in Czechoslovakia

A total of 26,478 ixodid ticks (935 pools) were examined by intracerebral inoculation of suckling mice. Six species of ticks were tested: Ixodes ricinus (23,470 individuals), I. trianguliceps (12), Haemaphysalis punctata (831), H. concinna (39), Dermacentor reticulatus (69) and D. marginatus (2,057). The ticks were collected largely by flagging vegetation, a substantial minority (4%) from animals. Three strains of Francisella tularensis were isolated, one each from I. ricinus (males, district Breclav, southern Moravia), D. reticulatus (males, district Breclav) and D. marginatus (engorged females collected from sheep in Roznava district, eastern Slovakia). D. marginatus and D. reticulatus represent new vector species for Czechoslovakia.     

Detection of Anaplasma dna in Ixodes ricinus ticks: pitfalls

A total of 150 nymphal Ixodes ricinus (L., 1758) (Acari: Ixodidae) from the Czech Republic were examined for Anaplasma phagocytophilum (Foggie, 1949) Dumler et al., 2001 by PCR using EHR521/747 primers: 22 of 50 pools were positive (minimum prevalence, 14.7%). However, sequencing of the PCR products did not show complete homology with A. phagocytophilum (91%) while the closest relationship (95%) was found to "Candidatus Ehrlichia walkefii". The results indicate a need for care in interpretation of Anaplasma PCR results and for PCR optimization for detecting A. phagocytophilum in ticks.     

Peculiarities of behaviour of taiga (Ixodes persulcatus) and sheep (Ixodes ricinus) ticks (Acarina: Ixodidae) determined by different methods

A comparison of the behavioural peculiarities of Ixodes persulcatus Schulze, 1930 (north-western population, Russia) and Ixodes ricinus (L., 1758) from western Russia and Denmark was determined by using two methods. Method 1 involved a sojourn of ticks on vertical plastic sticks and showed that the questing behaviour of I. ricinus nymphs was dependent on temperature and relative humidity (RH). A significantly greater number of nymphs quested at 22 degrees C and 100% RH than at 18 degrees C. When the humidity was reduced to 30% all of the nymphs departed. In the second method, the activity of ticks on an inclined "ticksdrome" was estimated. The activity of I. ricinus adults from the Danish population was 1.2 times greater than that of ticks from Russia. Females of the species studied and specimens from all study areas were more active than all other stages of development. The locomotor activity of both adult and immature I. ricinus that were infected with Borrelia burgdorferi sensu lato was suppressed when compared with uninfected specimens. The locomotor activity of I. persulcatus females infected by borreliae with exoskeleton anomalies was 1.3 times greater (P<0.05) than that of infected ticks without anomalies. Our data showed that infected females with exoskeleton anomalies could crawl faster on a human and reach uncovered parts of the body that are vulnerable for attachment and feeding. A study of locomotor activity and questing behaviour may be useful for comparing the risk for different tick species and populations to transmit tick-borne pathogens.     

Babesia microti (Piroplasmida: Babesiidae) in nymphal Ixodes ricinus (Acari: Ixodidae) in the Czech Republic

A total of 350 nymphs of the common tick Ixodes ricinus (Linnaeus, 1758) were collected in an endemic focus of Lyme borreliosis (South Moravia, Czech Republic) and examined for the presence of the protozoan Babesia microti (Fran?a, 1909) by polymerase chain reaction (PCR), using primers specific for the B. microti gene encoding small subunit rRNA. The assay revealed five positive pools (out of 70 pools examined); the corresponding prevalence rate was about 1.5%. Sequence analysis of the PCR products confirmed their 100% homology with that of B. microti. The study represents the first evidence of B. microti in ixodid ticks in the Czech Republic.     

The prevalence of Borrelia burgdorferi sensu lato in Ixodes persulcatus and I. ricinus ticks in the zone of their sympatry

A total of 7210 unfed adult Ixodes persulcatus Schulze, 1930 and I. ricinus (L., 1758) ticks were collected from the vegetation by flagging in 35 study sites located in the zone of their sympatry (mainly in Leningrad region, Russia). Borrelia infection in ticks was estimated by the dark-field microscopic analysis of gut contents in standard vital preparations at a magnification of x600. No correlation was revealed between the series of parameters characterising the abundance of each tick species (tau = -0.13) and between the series of these parameters and the prevalence of Borrelia in each vector. It is concluded that in the broad zone of I. persulcatus and I. ricinus sympatry, the presence and proportion of one vector in the ecosystem does not have any significant effect on the extensity of infection and on the epizootic and epidemic significance of the other vector. Each tick species has its independent (of the other species) and relatively original functional role in the focal ecosystem.     

Probability models of the rate of infection with tick-borne encephalitis virus in Ixodes persulcatus ticks.

A total of 3,254 adults of Ixodes persulcatus tick were collected in a taiga forest habitat situated in the Amgun river basin (Khabarovsk region, the Far East, USSR) and examined individually for the presence and amount of tick-borne encephalitis virus. The over-all proportion of infected ticks was 6.6% and it varied between 3.4% and 9.4% in the years 1982 to 1985. The amount of virus per tick was approximated by the gamma distribution determining a probability that the number of plaque-forming units (PFU) per tick is not greater than a selected value. The frequency distribution of infected ticks followed a model of the negative binomial distribution, enabling the estimation of probability of the occurrence of a given number of infected ticks in the area. However, the parameters of both probability models (i.e., the PFU content per tick, and the frequency of infected ticks) varied for particular years.     

Bartonella infections in fleas (Siphonaptera: Pulicidae) and lack of bartonellae in ticks (Acari: Ixodidae) from Hungary

Fleas (95 Pulex irritans, 50 Ctenocephalides felis, 45 Ctenocephalides canis) and ixodid ticks (223 ixodes ricinus, 231 Dermacentor reticulatus, 204 Haemaphysalis concinna) were collected in Hungary and tested, in assays based on PCR, for Bartonella infection. Low percentages of P. irritans (4.2%) and C. felis (4.0%) were found to be infected. The groEL sequences of the four isolates from P. irritans were different from all the homologous sequences for bartonellae previously stored in GenBank but closest to those of Bartonella sp. SE-Bart-B (sharing 96% identities). The groEL sequences of the two isolates from C. felis were identical with those of the causative agents of cat scratch disease, Bartonella henselae and Bartonella clarridgeiae, respectively. The pap31 sequences of B. henselae amplified from Hungarian fleas were identical with that of Marseille strain. No Bartonella-specific amplification products were detected in C. canis, I. ricinus, D. reticulatus and H. concinna pools.     

Comparison of the protein profiles of salivary gland extracts derived from three species of unfed and partially fed ixodid ticks analysed by SDS-PAGE

Salivary gland extracts (SGE) from unfed and 5 days fed adult female Ixodes ricinus (Linnaeus, 1758); Haemaphysalis inermis (Birula, 1895) and Dermacentor reticulatus (Fabricius, 1794) ticks were prepared. The protein content after feeding increased by 10.6, 8.7 and 6.8 times, respectively. Extracts were equilibrated to the same protein content and submitted to SDS-polyacrylamide gel electrophoresis followed by computer analysis of the scanned gels. Relative differences in protein profiles of extracts obtained from unfed and partially fed ticks were found in all species and some of them were similar in all three species used in the study. Results demonstrate that the increase of the protein content in salivary glands during the feeding does not occur proportionally. Some proteins are synthesised preferentially (67.1 kDa, 13.5 kDa) but other bands (in range of 15-16 kDa) present in the SGE derived from unfed ticks are less discernible in that of fed ticks.     

Modulation of human lymphocyte proliferation by salivary gland extracts of ixodid ticks (Acari: Ixodidae): effect of feeding stage and sex

Ixodid ticks remain attached to their hosts for several days to weeks. During this extended feeding process new proteins involved in the modulation of host immune responses are expressed in tick salivary glands. In our study a stimulatory or inhibitory effect of salivary gland extracts (SGE) of unfed and partially fed female Ixodes ricinus (Linnaeus, 1758), female and male Amblyomma variegatum (Fabricius, 1794) and Rhipicephalus appendiculatus Neumann, 1901 ticks on human lymphocyte proliferation induced by Concanavalin A (ConA) and phytohaemagglutinin (PHA), respectively, was investigated. SGE of all female ticks examined suppressed proliferation of ConA-induced lymphocytes; highly significant suppression was observed in the presence of unfed I. ricinus and 9-day fed A. variegatum SGE. SGE of partially fed A. variegatum and I. ricinus females suppressed PHA responses of lymphocytes. Lymphocytes showed reduced PHA and ConA responses in the presence of SGE of unfed and 2-day fed R. appendiculatus females, while SGE of 6-day fed females enhanced PHA responses, but reduced their ConA responses; generally SGE of 2-day fed females displayed the strongest inhibition. Amblyomma variegatum male SGE slightly enhanced PHA, but significantly reduced ConA responses of lymphocytes and their inhibitory effect increased during feeding. SGE of unfed and 2-day fed R. appendiculatus males enhanced PHA and ConA responses and those of 6-day fed males suppressed lymphocyte proliferation. The results suggest that (i) species- and sex-specific differences exist in the effects of tick salivary gland antigens on human lymphocyte proliferation and (ii) effect of SGE on human lymphocyte responses to mitogens varies depending on the tick feeding status.     

Comparison of virulence of Coxiella burnetii isolates from bovine milk and from ticks.

Laboratory animals (mice and guinea pigs) were infected with the isolates of Coxiella burnetii (Derrick, 1939) obtained from bovine milk (M18 and M35) and the ticks Ixodes ricinus (Linnaeus, 1758) and Dermacentor marginatus (Sulzer, 1776) (Kl3 and Kl6, respectively), and with the reference strain Nine Mile. Neither mortality nor lethality occurred with the mice. Antibody response in mice infected with isolates from milk was lower (1:16-512) than that from ticks (1:32-4096). Onset of seropositivity also occurred later - on the 10th day post-infection (p.i.) for M18 and M35 in comparison with the 7th day for Kl3 and Kl6. In guinea pigs, infection manifested by fever. The fever was less evident in guinea pigs infected with isolates from milk (39.5-40.1 degrees C) than in guinea pigs infected with isolates from ticks (39.5-40.6 degrees C). Partially engorged females of Dermacentor reticulatus (Fabricius, 1794) were inoculated with isolates M18 and Kl3. No differences in the multiplication of C. burnetii in haemocytes between these two isolates were ascertained.     

Replication of tick-borne encephalitis (TBE) virus in Ixodes ricinus ticks

The influence of external factors on virus carriage of Ixodes ricinus ticks in laboratory and in nature was studied. In laboratory experiment, only one nymph was positive for the presence of virus on 120th day after metamorphosis. The virus titer was 10(2) mouse i.c. LD50/0.03 ml. Transmission experiments were negative. The nymphs were positive on 75th, 111th and 159th day after metamorphosis, always chilling in the field experiment. The titres of virus varied from the lowest detectable amount value to 10(3.6) mouse i.c. LD50/0.03 ml. The transmission of virus was positive in two cases.     

Two ways of experimental infection of Ixodes ricinus ticks (Acari: Ixodidae) with spirochetes of Borrelia burgdorferi sensu lato complex

A previously reported procedure for the introduction of Borrelia spirochetes into tick larvae by immersion in a suspension of spirochetes was tested on Ixodes ricinus (L.) ticks and three of the most medically important European Borrelia genomic species, B. burgdorferi sensu stricto, B. garinii and B. afzelii. The procedure was compared with "classical" infection of nymphs by feeding on infected mice. Both methods yielded comparable results (infection rate 44-65%) with the exception of B. afzelii, which produced better results using the immersion method (44%) compared with feeding on infected mice (16%). Nymphs infected by the immersion method at the larval stage were able to transmit the infection to na?ve mice as shown by serology and PCR detection of spirochetal DNA in organs. The immersion method is faster than feeding on infected mice and provides more reproducible conditions for infection. It can be exploited for studies on both pathogen transmission and Borrelia-vector interactions.     

Effect of salivary gland extract from Ixodes ricinus ticks on the proliferation of Borrelia burgdorferi sensu stricto in vivo

Saliva-activated transmission (SAT) of Borrelia burgdorferi sensu stricto was demonstrated using real-time PCR and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. C3H/HeN mice were injected intradermally with 1.5 x 10(3) spirochetes mixed with 40 microg of SGE per mouse. The control group was inoculated with the same dose of spirochetes without SGE. The accelerating effect of SGE on spirochete proliferation was demonstrated on day 1 post infection, when a 4.2-fold increase in spirochetes was found in the skin and a 10-fold increase in the blood, compared with control mice. The data represent the first direct evidence of a SAT effect of I. ricinus SGE on infection with the Lyme disease agent B. burgdorferi.     

Experimental infection of ticks Ixodes ricinus with tick-borne encephalitis virus under different microclimatic conditions

The influence of environmental temperature and relative air humidity on the development of ticks Ixodes ricinus and their infection rate with tick-borne encephalitis virus was studied. It was found that the temperature influenced significantly the development of ticks, but it did not influence their infection rate. To the contrary, the relative air humidity influenced the infection rate. It is assumed that the intrinsic factors play a primary role in the infection of ticks, while extrinsic factors have a secondary role; they are probably important at border values only.     

Tick salivary gland extract-activated transmission of Borrelia afzelii spirochaetes

Saliva-activated transmission of Borrelia afzelii Canica, Nato, du Merle, Mazie, Baranton et Postic, 1993 was demonstrated using salivary gland extract (SGE) from Ixodes ricinus (L., 1758) ticks and C3H mice. Injection of Borrelia spirochaetes together with SGE increased the level of bacteraemia and accelerated the appearance of bacteria in the urinary bladder, compared with the injection of spirochaetes alone. More I. ricinus nymphs became infected when feeding on mice inoculated with B. afzelii plus SGE. Analysis of cytokines produced by cells of draining lymph nodes from SGE-treated mice showed a suppression of proinflammatory cytokines IFN-gamma, IL-6 and GM-CSF following a transient upregulation in comparison with the control mice infected without SGE.     

Occurrence of ixodid ticks--the main vectors of tick-borne encephalitis virus in urbanized territory

The influence of urbanization on ticks I. ricinus and I. persulcatus--the main vectors of tick-borne encephalitis virus is discussed on the basis of concrete examples. It has been revealed that under favourable conditions the populations of these tick species can exist for a long time not only in towns and new housing estates, but in the old residential districts as well. The necessity of studying the ecology of urban populations of ixodid ticks is pointed out.     

Establishment ofHarmonia axyridis on Citrus and Some Data on Its Phenology in Greece

In September 1993, a colony ofHarmonia axyridis Pallas (Coleoptera:Coccinellidae) was imported from France into Greece. In 1994, insectary-reared adults were released in 11 citrus orchards in four citrus-growing areas of Greece. Between May 19 and June 8, 1994,H. axyridis was recovered from a total of seven localities in three of these areas. This species was established on orange, mandarin and sour orange trees heavily infested withToxoptera aurantii, Aphis spiraecola andA. gossypii; its absence from the remaining four localities may have been the result of low prey densities. Twenty-three days after the initial releases,H. axyridis larvae comprised 57.9% and 83.3%, respectively, of the aphidophagous coccinellid larval populations in two localities (on Chios Island). In samples taken at Leonidion 43 days after the introduction release, both adult and larval populations ofH. axyridis represented approximately one-third of aphidophagous coccinellid adults and larvae found, whereas the indigenousAdalia bipunctata comprised about one-half of the population. In cages placed outside the Athens laboratory,H. axyridis completed four overlapping generations annually; average longevities of 56.2, 66.8, 78.9 and 102.2 days, respectively, were recorded for the successive generations. Adults of the 3rd and 4th generations overwintered, giving rise to the following year’s 1st generation. Oviposition began in April and emergence of 1st generation adults occurred in mid-May. The egg-laying activity of the females throughout the warm period of the year indicates thatH. axyridis does not diapause in summer. From December until March, small aggregations (2-4 individuals) were observed within the cages at protected sites.     

Lyme borreliosis: insights into tick-/host-borrelia relations

Lyme borreliosis (LB) is a serious infectious disease of humans and some domestic animals in temperate regions of the Northern Hemisphere. It is caused by certain spirochetes in the Borrelia burgdorferi sensu lato (s.l.) species complex. The complex consists of 11 species (genospecies). Borrelia burgdorferi sensu stricto (s.s.), Borrelia garinii and Borrelia afzelii are the major agents of human disease. Borrelia burgdorferi s.l. species are transmitted mainly by ticks belonging to the Ixodes ricinus species complex plus a few additional species not currently assigned to the complex. B. burgdorferi infections may produce an acute or chronic disease with a wide array of clinical symptoms such as erythema migrans (EM), carditis, arthritis, neuroborreliosis, and acrodermatitis chronica atrophicans (ACA). Differences in LB spirochetes 'genospecies' and strains/isolates determine the occurrence and severity of this multi-system disease. Accurate and reliable identification of the LB spirochetes in ticks as well as knowledge of their prevalence are essential for prevention against the disease and development of an effective vaccine. An overview of the knowledge of molecular factors with emphasis on potential protein-carbohydrate interactions in the tick-borrelia system is the main focus of this review.     

A predictive model for early-warning of Septoria leaf blotch on winter wheat

Disease–weather relationships influencing Septoria leaf blotch (SLB) preceding growth stage (GS) 31 were identified using data from 12 sites in the UK covering 8 years. Based on these relationships, an early-warning predictive model for SLB on winter wheat was formulated to predict the occurrence of a damaging epidemic (defined as disease severity of 5% or > 5% on the top three leaf layers). The final model was based on accumulated rain > 3 mm in the 80-day period preceding GS 31 (roughly from early-February to the end of April) and accumulated minimum temperature with a 0°C base in the 50-day period starting from 120 days preceding GS 31 (approximately January and February). The model was validated on an independent data set on which the prediction accuracy was influenced by cultivar resistance. Over all observations, the model had a true positive proportion of 0.61, a true negative proportion of 0.73, a sensitivity of 0.83, and a specificity of 0.18. True negative proportion increased to 0.85 for resistant cultivars and decreased to 0.50 for susceptible cultivars. Potential fungicide savings are most likely to be made with resistant cultivars, but such benefits would need to be identified with an in-depth evaluation.