Effect of tick salivary gland extract on the cytokine production by mouse epidermal cells

Previous studies have demonstrated that both tick saliva and Borrelia burgdorferi sensu lato antigens modulate the cytokine response of the host. In this paper, the effect of salivary gland extract (SGE) from Ixodes ricinus (L., 1758) ticks on cytokine production by primary cultures of mouse epidermal cells stimulated with Borrelia afzelii Canica, Nato, du Merle, Mazie, Baranton et Postic, 1993 spirochetes was analysed. Epidermal cells were derived from C3H/HeN mice, susceptible to Lyme disease, and BALB/c mice, which are resistant. In cultures from C3H/HeN mice, SGE down regulated production of tumour necrosis factor alpha (TNF-alpha) and up regulated Th2 cytokine, interleukin 4 (IL-4). Cultures from BALB/c mice produced higher basal levels of monitored cytokines, but their production was affected by SGE a different way. While Th2 cytokines IL-6 and IL-10 were down regulated, the effect on TNF-alpha and IL-4 was ambiguous. These results indicate that the effect of tick saliva on the epidermal cells of Lyme disease-susceptible C3H/HeN mice mirrors its effect on other cells of the immune system.     

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.     

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.     

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.     

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.     

Prevalence of borreliae in Ixodes ricinus ticks from southern Moravia, Czechoslovakia

A total of 378 adult Ixodes ricinus ticks were collected by flagging vegetation in four localities of two districts (Breclav, Znojmo) in south Moravia and examined microscopically. Borreliae were identified in Giemsa-stained midgut smears from 32 (i.e. 8.5%) ticks (9.4% females, 7.2% males); the infection rate varied between 0.0 and 11.4% in the four localities examined. Among female ticks, significantly more were found to be infected in autumn (19.7%) than in spring (5.8%). Dark-field (DF) and Giemsa-stained smears (GS) examinations were compared for their sensitivity in detecting borreliae in 128 field-collected ticks; GS method showed a little higher sensitivity (11.7% ticks were positive) than DF procedure (9.4% ticks positive). Two strains of Borrelia burgdorferi were isolated from a total of 150 adult I. ricinus ticks cultured in BSK medium.     

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.     

Studies on the transovarial transmission of Borrelia burgdorferi sensu lato in the taiga tick Ixodes persulcatus

The possibility of vertical transmission of Borrelia burgdorferi sensu lato in Ixodes persulcatus Schulze, 1930 ticks was studied in the progeny of 20 females collected from the vegetation in an active focus of ixodid tick-borne borrelioses (ITBB) located in the Perm oblast, Russia, where Borrelia garinii and B. afzelii are circulating. The presence of Borrelia DNA was detected by the PCR method after feeding and egg laying in 16 engorged females (80.0%), as well as in 36.5 +/- 7.2% samples containing 20 eggs each and in 21.4 +/- 4.2% samples containing 10 eggs each. The respective rates of individual egg infection were 0.4-8.0% and 0.5-23.0%. PCR analysis of 370 eggs (one egg per sample) and 781 unfed larvae hatched from the same egg masses (1, 10, 20, 40, and 50 larvae per sample) failed to reveal the presence of Borrelia DNA. Negative results were also obtained in experiments on inoculating the BSK II medium with the egg and larval materials. Microscopic analysis of 1,683 smear preparations of eggs and 1,416 preparations of unfed daughter larvae revealed spirochete-like cells in 7 (0.4 +/- 0.3%) and 13 (0.9 +/- 0.5%) preparations, respectively; typical Borrelia cells were found in seven preparations of larvae (0.5 +/- 0.4%). Only 1 out of 16 infected females transmitted Borrelia vertically, through the eggs to the larval progeny. The infection rate in this progeny was about 7%, and the prevalence of Borrelia in individual larvae was 0.4-0.8 cells per 100 microscopic fields. These data do not allow the conclusion that transovarial transmission of B. burgdorferi sensu lato in the I. persulcatus tick is an established fact. However, they show that, even if such transmission is possible, its probability is very low.     

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.     

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.     

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.     

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.     

Attenuation of Schistosoma mansoni cercarial infectivity to albino mice by methanol extract of some plant species

This study elucidates the activity of certain plants’ methanol extract: Anagallis arvensis, Solanum nigrum (green fruits), Chenopodium ambrosioides, Calendula officinalis and Sesbania sesban, on the infectivity of S. mansoni cercariae to albino mice. Then, some parasitological parameters, e.g. the worm load/mouse, number of ova/g tissue in liver and intestine and the developmental stages of ova in the small intestinal wall (Oogram) of infected mice were determined. In addition, certain biochemical parameters of serum from infected mice (total protein, albumin, the activities of AlT, AsT, AcP and AkP enzymes) were, also, recorded.The results showed that exposure of S. mansoni cercariae for 30 min to the tested plants’ methanol extract before mice infection has a higher suppressive effect on their infectivity to albino mice then those exposed to this extract during mice infection. The number of worms recovered/infected mouse and the number of ova/g tissue from liver and intestine of mice groups infected with cercariae exposed to the tested plants’ methanol extract either pre- or during mice infection were less than those of infected control groups (e.g. the reduction rates of worm load/mouse and number of ova/g tissue in the intestine were 46.1% and 76.8%, respectively, for mice infected with cercariae exposed to 5 ppm of A. arvensis during mice infection).The results, also, indicated that exposing S. mansoni cercariae to methanol extract of the experimental plants either pre- or during mice infection reduced the activities of the enzymes AlT, AsT, AcP and AkP that were elevated in mice infected with untreated cercariae, meanwhile, the concentrations of total protein and albumin were increased in the serum of mice infected with these treated cercariae in comparison with those of mice group infected with untreated cercariae.     

Experimental infection of immunocompetent and immunodeficient mice with Encephalitozoon cuniculi

An experimental infection with the microsporidian Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 was studied using a model of immunocompetent BALB/c mice and immunodeficient SCID mice. The course of infection after intraperitoneal inoculation of E. cuniculi spores was evaluated using the presence of spores in peritoneal macrophages as a criterion. First significant decrease in the proportion of infected cells was recorded on day 9 post infection (p.i.) in BALB/c mice. From day 14 p.i. no spores were observed in macrophages from BALB/c mice, while the number of infected macrophages from SCID mice increased until the death of the mice. The natural killer (NK) cell activity of mouse splenocytes was compared with the production of interferon gamma (IFN-gamma) by these cells. While in BALB/c mice NK activity peaked on days 9 and 14 p.i., in SCID mice the marked increase of NK activity was recorded close before death of mice, on day 21 p.i. in correlation with the production of IFN-gamma. Production of specific antibodies was demonstrated from day 9 p.i. in sera from BALB/c mice. It is concluded that intraperitoneal infection of SCID mice with spores of E. cuniculi results in the marked increase in the number of peritoneal exudate cells and in the percentage of infected cells close before death of mice. Neither high activity of NK cells nor increased production of IFN-gamma are sufficient for the recovery of SCID mice from an E. cuniculi infection.     

Putative effector genes detected in Fusarium oxysporum from natural ecosystems of Australia

The Fusarium oxysporum species complex (FOSC) causes disease in plants and animals, but is also widely dispersed in natural ecosystems without evidence of disease. The present study screened a population representing natural ecosystems across the Australian continent for the putative effector genes pisatin demethylase 1 (PDA1), pectate lyase (pelD), secreted gene expression (SGE1) and secreted in xylem (SIX). The genes pelD and SGE1 were prevalent in the natural isolates, PDA1 was present at an intermediate level, whereas SIX genes were detected at low levels. Phylogenies of these putative effector genes were compared to the EF‐1α species phylogeny to determine the likely modes of gene transmission: vertical gene transfer (VGT) and horizontal gene transfer (HGT). There was evidence of both modes of gene transmission within the F. oxysporum isolates. PDA1, pelD and SGE1 were likely to be only vertically inherited, whereas the SIX genes had evidence for both VGT and HGT. The phylogenetic relationships of SIX genes in isolates from natural ecosystems and formae speciales from agro‐ecosystems were also established. These findings have important implications for the evolution of effectors in the FOSC.     

Effect of fast protein liquid chromatography fractionated salivary gland extracts from different ixodid tick species on interleukin-8 binding to its cell receptors

Interleukin-8 plays a critical role in inflammatory processes. Hence generation of molecules with anti-IL-8 activity is likely to be important for successful feeding and for survival of the ticks. Anti-IL-8 activity was studied in saliva of three ixodid tick species--Dermacentor reticulatus (Fabricius, 1794), Rhipicephalus appendiculatus Neumann, 1901, and Amblyomma variegatum (Fabricius, 1794). The greatest activity was shown in saliva prepared from D. reticulatus. The activity was attributed to tick salivary gland molecules that bind to IL-8, preventing binding of the chemokine to its specific receptor, rather than to occupation of the IL-8 cell receptor by the tick molecules. The distribution of anti-IL-8 activity in fast protein liquid chromatography (FPLC) fractions of salivary gland extracts (SGE) derived from adult female D. reticulatus, R. appendiculatus and A. variegatum was compared directly by both ELISA and receptor-binding inhibition assays. The correspondence in results with fractions of SGE from ELISA is consistent with detection of tick molecules that inhibit IL-8 binding to its receptor. As IL-8 is an important chemoattractant and activator of neutrophils, the presence of an anti-IL-8 activity in tick saliva indicates that neutrophils play an important role in the host response to parasitism by ticks.     

Occurrence of the tick Ixodes ricinus (L.) under the conditions of anthropopressure

The authors report on their findings of the tick Ixodes ricinus under urban conditions of Prague. The concrete data were taken as a basis for a general scheme of possible occurrence of this species in various town parts, determined by the state of vegetation and presence of large mammals. Discussed are also conditions for I. ricinus maintenance in another type of anthropogenous landscape, the spoil banks resulting from brown coal exploitation.     

Cytokine response to infection with the microsporidian, Encephalitozoon cuniculi.

The production of three cytokines, interferon gamma (IFN-gamma), interleukin 10 (IL-10) and interleukin 12 (IL-12), was measured after intraperitoneal infection of immunocompetent Balb/c mice and immunodeficient SCID mice with the microsporidian, Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923. High levels of IFN-gamma were detected in ex vivo cultures of peritoneal exudate cells (PEC) of Balb/c mice, a lower, but earlier IFN-gamma response was observed in PEC from SCID mice. The early IL-10 response was detected in ex vivo cultures of splenocytes from Balb/c but not from SCID mice, explaining a delay in the IFN-gamma response in Balb/c mice. IL-12 was detected in PEC cultures from SCID mice, indicating an alternative pathway of IFN-gamma production by NK cells stimulated by IL-12 derived from macrophages.     

Influence of thymic preparations on the result of experimental infection with Taenia crassiceps (Zeder, 1800) in ICR mice

Thymalin (Thymarin) and T-activin--thymic preparations of polypeptidic character--were used for influencing the parasitic infection in a model system mouse--Taenia crassiceps. A single subcutaneous application of 100 micrograms of Thymalin per mouse at the day of infection resulted in a decrease (by 54.9%) in the number of cysticerci in peritoneal cavity of experimental mice compared with the controls. Administration of Thymalin with T. crassiceps larval homogenate at various intervals before and after infection resulted in a statistically significant increase of the level of specific antibodies in the serum of infected mice, this increase, however, did not correlate with the corresponding protective effect. Immunosuppressant azathioprine, injected subcutaneously from 7th to 3rd day preceding infection at a dose of 100 micrograms resulted in a significant increase in the number of T. crassiceps larvae in the peritoneal cavity of experimental mice compared with the controls (by 48.7%). T-activin, injected subcutaneously to mice, immunosuppressed by azathioprine, led to a restoration of resistance of mice to T. crassiceps infection. Subcutaneous application of T-activin alone had a significant protective effect (decrease in cysticerci number by 53.7% in comparison with the controls). Correlation of the level of specific antibodies in the serum of infected mice, value of spleen index and number of T. crassiceps cysticerci in peritoneal cavity of mice was not detected.