Detection of tick-borne pathogens in ticks from migratory birds in the Baltic region of Russia.

We report the finding of tick-borne encephalitis (TBE)-virus in indigenous Ixodes ricinus (L.), 'Candidatus Neoehrlichia mikurensis' in exotic Ixodes frontalis (Panzer) and Rickettsia aeshlimannii in exotic Hyalomma marginatum Koch subadult ticks detached from 18.5% (107/577) infested migratory birds in the Baltic region of Russia. This is the first record of human pathogenic 'Candidatus N. mikurensis' in I. frontalis ticks. Moreover, seven other pathogens were identified in I. ricinus ticks. Spotted Fever Group rickettsiae were the predominant pathogen group and were detected only in nymphs. Future investigations are warranted to further characterize the role of birds in the epizootiology of tick-borne pathogens in this region.

Evidence of ehrlichiosis agents found in ticks (Acari: Ixodidae) collected from migratory birds.

Two Ehrlichia pathogens were found in immature Ixodes ricinus (L.) ticks collected from migratory passerine birds in the Curonian Spit area of the Baltic Region of Russia (Kaliningrad enclave). During the spring and fall of 2000, 1,606 passerine birds (eight species) were collected; 6.8% of them (110/1,606) were infested by ticks, and 51.8% (57/110) of tick clusters contained various human pathogenic microorganisms. Human monocytic ehrlichiosis (HME) and human granulocytic ehrlichiosis (HGE) agents were found in 14% (8/57) of cases. Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto were found in 92.9% (53/57) of the ticks. In five out of eight cases, infection of both Ehrlichia and Borrelia were obtained. In one case, a single nymph contained HME, B. afzelii, and B. garinii. Borrelia burgdorferi s.s. and B. afzelii were found together in one pool of four nymphs and one larva. All agents were identified using polymerase chain reaction and species-specific primers. In 8.8% of the ticks collected from birds in the fall and 22% in the spring, pathogens were isolated from attached co-feeding nymphs and larvae. These data demonstrate that Ehrlichia exchange could occur between co-feeding ticks on animals without systemic infection.

Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia.

The presence and distribution of Ehrlichia spp. and Borrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinus and 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carried Ehrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and an Ehrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded a Borrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.

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.

Abiotic parameters and diel and seasonal activity of Borrelia-infected and uninfected Ixodes persulcatus (Acarina: Ixodidae).

During the 1997 season of Ixodes persulcatus Schulze tick activity, 579 specimens were collected by flagging in the vicinity of St. Petersburg (Morskaja, Lisy Nos). Collections were made from April to July, and at each collection time the following measurements were recorded: light intensity (in lux); temperature of soil 1-1.5 cm below leaf litter, at the soil surface, and in the air 1.5 m above the soil surface; and relative humidity. The gradient between soil at a depth of 1-1.5 cm and soil surface temperatures appeared to be the main physical parameter that initiated tick migration out of the leaf litter. At soil temperature intervals of 5-10 degrees C and 21-26 degrees C, ticks were found only when the soil temperature 1-1.5 cm below the soil surface was colder than at its surface. When temperature ranged from 11 to 20 degrees C, 24% of the ticks were collected when the surface temperature was colder than that below the soil surface. Sixty percent of the ticks, especially nymphs, were collected when the soil surface and subsoil temperature difference ranged from 0 to 2.0 degrees C. The gradient between soil temperature and relative humidity appeared to increase the importance of the gradient between the soil and surface temperature at which time the maximum number of ticks were caught. Borrelia burgdorferi sensu lato infected ticks were collected only after 1100 hours. Prior to and just after 1100 h the mean difference between soil temperatures was 4.7 degrees C, whereas the mean difference between soil and surface temperatures was 6.4 degrees C at this time. The most important factors that influenced the appearance of Borrelia-infected ticks were the temperature gradients that existed between the surface and soil temperatures and the soil and air relative humidity. Greater numbers of infected nymphs were collected when temperature intervals ranged from 10 to 14 degrees C and lower numbers were collected between 15 and 20 degrees C. No Borrelia-infected nymphs were captured when the temperature interval ranged from 21 to 26 degrees C. Infected adults appeared to be more tolerant of higher temperatures than Borrelia-infected nymphs. A significant relationship was found between the activity of Borrelia-infected I. persulcatus and the temperature of the air, soil below the surface, at the soil surface, and relative humidity.

Sexual transmission of Borrelia garinii by male Ixodes persulcatus ticks (Acari, Ixodidae).

We investigated the transmission of Borrelia garinii and Borrelia afzelii between male and female Ixodes persulcatus ticks. For this purpose the infection rate of partners from tick couples was determined by polymerase chain reaction and reverse line blot. In couples, where the male tick was infected with B. garinii, four out of nine female partners carried B. garinii. In eight couples, male ticks had a dual infection of B. afzelii and B. garinii and three female partners were infected by Borrelia spirochetes. Two female ticks carried B. garinii, and one female tick had a dual infection. No evidence for transmission of B. afzelii from male to female ticks was found among seven couples. In 45 couples where the female tick was infected, not one male tick carried spirochetes. The difference in the B. garinii infection rate between male and female ticks among these couples is highly significant. Our data suggest that transmission of B. garinii from male ticks to female ticks does occur. Sexual transmission of this pathogen may play an important role in the maintenance of B. garinii in I. persulcatus.

Tick-borne borrelioses pathogen identification in Ixodes ticks (Acarina, Ixodidae) collected in St. Petersburg and Kaliningrad Baltic regions of Russia.

Two isolated Baltic seashore populations of Ixodes ticks were studied as vectors of different Borrelia genospecies in Russia by using darkfield microscopy and modified polymerase chain reaction (PCR). In the Kalinigrad region (Kurish Spit, forests near the settlements of Lesnoye and Rybachy), 788 Ixodes ricinus (L.) adults and nymphs were collected by flagging and studied by darkfield microscopy during 1995-1996. There were 88 darkfield microscopy positive specimens (11.2%) of which 69 were also analyzed by PCR. Borrelia afzelii and B. garinii were found individually and together in ticks. In this region, on the Kurish Spit, 7 patients with tick borrelioses were observed: 2 in the Russian part of Spit and 5 in the Lithuanian part. A significant difference was found between Borrelia prevalence during the spring and fall peaks of tick abundance. Specimens that were darkfield microscopy positive prevailed in the fall (25.15%) in comparison with the spring peak (7.3%). The number of specimens with identified genospecies prevailed in the spring: 22 out of 35 versus 4 out of 31 in the fall. Among 29 PCR positive I. ricinus, 21 contained B. afzelii, 3 had B. garinii, and 2 had dual infection. In 1995, only B. afzelii infected specimens were observed. In the vicinity of St. Petersburg (the seashore of the northern Gulf of Finland, in forests near Lisy Nos, Morskaja) during 1992-1996, 31 patients with a tick-borne borrelioses were registered. We collected 487 Ixodes persulcatus Schulze by flagging and studied them by darkfield microscopy in 1995-1996 of which 144 ticks (29.6%) were darkfield microscopy positive. Sixty darkfield-positive specimens were analyzed by PCR, and in 88.3% of cases genospecies were identified. B. afzelii and B. garinii were identified individually and together in ticks. In 1995, I. persulcatus with dual infection prevailed with 11 out of 21 (52.4% positive), whereas in 1996, most I. persulcatus ticks contained B. garinii (81.2%). Dual infection was observed in 4 of 32 (12.5%) ticks. Dual infections in I. persulcatus females increased within the seasonal peak of tick activity as was observed in 1995 and in 1996. Many patients not only had erythema migrans, but also exhibited early neurological symptoms that coincided with the number of tick vectors that had dual infections in June, indicating that these patients were bitten by female ticks that had dual infections. A significant difference existed between levels of infection in I. ricinus and I. persulcatus, with all 3 types of Borrelia infection observed 2 times more often in I. persulcatus than in I. ricinus and dual infection occurred in I. persulcatus 3.7 times more often. It appeared that I. persulcatus is a much more dangerous vector of tick-borne borrelioses than I. ricinus.

Preliminary studies on virus and spirochete accumulation in the cement plug of ixodid ticks.

We provide evidence that tick-borne encephalitis virus and Borrelia burgdorferi s.l. are accumulated in the cement plug in the host skin within the first few hours after tick attachment. Extirpation of the tick without the cement plug, even very soon after the attachment, did not prevent the transmission by Ixodes ricinus, Ixodes persulcatus or Dermacentor reticulatus to mice. This was within 1 hour in the case of the TBE virus and after 20-22 h of attachment, in the case of Borrelia and I. persulcatus. The epidemiological significance of these findings is discussed.

Exchange of Borrelia burgdorferi between Ixodes persulcatus (Ixodidae:Acarina) sexual partners.

Borrelia burgdorferi sensu lato infection rate in Ixodes persulcatus Schulze maintained at different relative humidity gradients in male and females pairs, separated by sex, and in ticks of both sexes having either normal or abnormal exoskeleton were compared. Ticks were collected in the St. Petersburg Region of Russia during 1992 and 1994. We observed that the infection rate among the ticks maintained as sexual pairs was 1.75-2.00 times higher than that among ticks maintained singles, indicating a borreliae interchange between sexual partners. This pathogen interchange was thought to result from a venereal or omovampiric (cannibalistic) mode of borreliae transmission. Borrelia burgdorferi s.l. was determined to be present in 22.9% (112 infected specimens of 489 total), whereas infection occurred in 17.4% of single females and 16.5% of single males. The data indicate the importance of isolating ticks sexually during quantitative disease investigations with borreliae as well as tick-borne encephalitis virus and other tick-borne pathogens.

Venereal and cannibalistic ways of Borrelia burgdorferi sensu lato exchange between males and females of Ixodes persulcatus (Ixodidae, Acarina).

Borrelia burgdorferi sensu lato (s. l.) infection rate in Ixodes persulcatus Shulze maintained in different relative humidity gradient in male and female pairs and separately by sex were compared. Ticks collected in the St. Petersburg Region of Russia during 1992-1994 temperate and 1995 hot seasons. We observed that the infection rate among specimens maintained as sexual pairs were 1.2-2.5 times higher than among ticks maintained separately. Rate of borrelia infection in tick couples was a bit higher among specimens collected within a hot spring-summer period than among ticks collected during the seasons with temperate temperatures. This pathogen interchange was thought to result from a venereal or cannibalistic (omovampiric) mode of borreliae transmission. Traces of male bites (scars) on the female bodies were checked more often during a hotter seasons. borrelia burgdorferi s.l. was determined to be present in 22.9-29.3% whereas infection occurred in 12-20% of single specimens. Our data indicate the importance of isolating ticks sexually during disease investigations with borreliae as well as tick-borne encephalitis and probably other tick-borne pathogens.