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 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.     

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.     

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.     

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.     

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.     

Studies on ticks of veterinary importance in Nigeria. VIII. Differences observed in the biology of ticks which fed on different domestic animal hosts

Ticks of the species Amblyomma variegatum (Fabr.), Boophilus decoloratus (Koch), Boophilus geigyi Aeschl. et Morel, and Hyalomma rufipes Koch were detached from cattle, sheep and horses and the influence of these various hosts on the biology of ticks was investigated. No A. variegatum was found in horses. The parameters studied were preoviposition and oviposition periods, ovipositional capacity, eclosion period, hatching patterns, egg sizes and temperature effect. Although the preoviposition and eclosion periods were similar in each tick species irrespective of the host from which the adults were detached, the oviposition period was longest among ticks detached from cattle and least among those detached from horses. The ticks engorged on cattle also laid the highest number of eggs and those which engorged on horses laid the least number. The hatching pattern of the eggs in any tick species was not influenced by the host from which the female was detached. The lengths of eggs of the ticks whose adults were detached from horses were generally smaller than those detached from cattle and sheep. Temperature affected the adult females detached from cattle, sheep and horses equally and this was also true of the larvae they produced. The practical field applications of these results are discussed.     

Studies on ticks of veterinary importance in Nigeria XVII. Resistance of rabbits to infestation with Amblyomma variegatum and Boophilus decoloratus (Acari: Ixodidae)

Rabbits exposed to larvae, nymphs and adults of Amblyomma variegatum acquired resistance to the infestation with adults of this tick. The degree of acquired resistance was lowest in the rabbits exposed once previously to larvae and highest in those exposed twice previously to adults. Rabbits also acquired resistance after primary infestation with Boophilus decoloratus; their resistance became very high during the thrice-repeated feeding. New terms are introduced for the quantification of the degree of resistance of animals to tick infestation. Rabbits were protected against infestation with B. decoloratus by intravenous injection of serum from the rabbits on which the ticks had fed three times. The relevance of the results to integrated tick control strategies is discussed.     

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.     

Studies on ticks of veterinary importance in Nigeria: intrinsic factors influencing oviposition and egg-hatch of Amblyomma variegatum under natural conditions

During the peak of rains of 1983, 1984 and 1985 in the forest zone of Nigeria, female Amblyomma variegatum engorged to various weights were subjected to different experiments for the purpose of observing the intrinsic factors which influence oviposition and egg-hatch under natural conditions. Two types of oviposition patterns were observed. New terms were introduced to quantify the relationships between the daily weight loss and daily number of eggs oviposited by a tick. These terms are "actual mass", "convertible blood mass", "oviposition efficiency" and "mass conversion rate". A major advantage of adopting the "oviposition efficiency" and "mass conversion rate" is that they relate directly to the measure of the metabolic activity utilized by the tick for the process of oviposition. The entity "convertable blood mass" also enables recognition of individual capability in the utilization of blood mass for oviposition. Investigation on the relationship of weight of eggs with sequence of oviposition showed that eggs were heavier when few eggs were laid than when numerous eggs were oviposited. The heaviest eggs were laid towards the end of oviposition. Two types of relationship were found to exit between the sequence of oviposition and the eclosion period of eggs. Adult A. variegatum engorged to more than 2.49 x were affected by immersion in water for longer than 7 days. Such ticks died without ovipositing and the water in which they were submerged became dark-red. Eggs immersed in water for periods ranging from 1-7 days hatched in about the same number of days as control eggs and their viability in terms of percent-hatch was not affected.     

Tick repellents: Past, present, and future

Ticks are important vectors of human and animal diseases. One important protective measure against ticks is the use of personal arthropod repellents. Deet and the synthetic pyrethroid permethrin currently serve as the primary personal protective measures against ticks. Concern over the safety of deet and its low repellency against some tick species has led to a search for new user-approved, efficacious tick repellents. In this article, we review the history and efficacy of tick repellents, discovery of new repellents, and areas in need of attention such as assay methodology, repellent formulation, and the lack of information about the physiology of repellency.     

Cypermethrin inhibits egg development in the ixodid tick, Amblyomma hebraeum

The pyrethroid insecticide, cypermethrin (CyM), stimulates vitellogenesis in Ornithodoros moubata (an argasid tick) by stimulating the release of the normal vitellogenesis-inducing factor (a neuropeptide) and subsequent release of the vitellogenic hormone [19]. Here we examine the effects of CyM on egg development in the ixodid tick, Amblyomma hebraeum. Ovary weight, oocyte size, and vitellin content of the ovary were measured after CyM treatment; in partially fed ticks, none of these parameters were affected significantly. However, CyM treatment caused an inhibition of ovary development, as well as reduction of both hemolymph 20-hydroxyecdysone (20E; the vitellogenic hormone in this species) and vitellogenin (Vg)-concentrations in engorged ticks. In addition, the degree of salivary gland degeneration (which is triggered by 20E) was slightly reduced in CyM-treated engorged ticks. These results indicate that CyM acts differently in Amblyomma compared to Ornithodoros. Instead of stimulating vitellogenesis, CyM inhibits egg development perhaps in part as a result of inhibiting release of 20E.     

An in vitro feeding assay to test acaricides for control of hard ticks

Animal husbandry could not be practised over large areas of the planet without acaricides. The prevention of tick bite and the transmission of diseases requires the use of pesticides, but this contributes to the development of tick resistance against acaricides. This drives the quest for new molecules that target physiological processes crucial to tick survival. In vivo trials involve multiple repetitions because of inherent variations between host animals, requiring large amounts of test products and ticks. An in vitro alternative should permit the testing of the ability of a product to restrict attachment and feeding by ticks at precise doses. In this paper an in vitro feeding system is described where the European tick Ixodes ricinus L. feeds on blood through a cellulose rayon-reinforced silicone membrane. The membrane Shore hardness is modified to imitate the elastic retraction forces of skin that ensure the closing of tick penetration sites on the membrane to prevent bleeding. Tick attachment (75-100%) is achieved by adding chemical and mechanical stimuli to the membrane. Survival curves for different doses of fipronil and ivermectin tested with the method showed highly reproducible acaricide effects within 5-7 days. Significant effects are recorded down to ppb levels in blood. Standardised tests can be made with blood from the same donor animal or culture medium under the membrane.     

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.     

Rickettsiae of the spotted fever group in Hungary

Rickettsiae were detected by haemocyte test in 7.2% of Dermacentor marginatus and 4.7% of D. reticulatus ticks collected in Hungary. Six strains of rickettsiae were isolated from positive specimens. These rickettsiae according to CFR are closely related, probably identical with R. slovaca and they represent the first rickettsiae of the Spotted Fever group found in that country.     

Effects of permethrin and amitraz on gas exchange and water loss in unfed adult females of Amblyomma americanum (Acari: Ixodidae)

Effects of two acaricides, permethrin and amitraz, with different modes of action, on gas exchange and water balance of the lone star tick, Amblyomma americanum, were examined using a flow-through carbon dioxide (CO₂) and water vapor analyzer. Untreated adult female ticks exhibited a distinct discontinuous gas exchange pattern (DGEP) with no measurable water loss. Similarly, ticks with acetone (a solvent) showed little change in gas exchange and water loss. Topical treatment of ticks with a lethal dose of permethrin caused immediate major water loss and long-lasting high frequency DGEP, with increased amplitude of CO₂ release. A small water loss spike was observed for each CO₂ release. Treatment of ticks with amitraz abolished the DGEP and elicited a long-lasting continuous gas exchange pattern, indicating significantly increased tick metabolism. No detectable water loss was observed during amitraz-elicited continuous gas exchange. Treatment of ticks with a mixture of sub-lethal doses of permethrin and amitraz caused a similar initial major water loss, as in permethrin-treated ticks, followed by a second period of water loss activity at 10–14 h after treatment. Mixture of the two acaricides caused immediate onset of major chaotic CO₂ release activities with a significantly elevated baseline CO₂ emission level. The standard metabolism rate (SMR, VCO₂) of acetone-treated ticks (0.452 μl h⁻¹) was similar to that of the untreated ticks (0.461 μl h⁻¹). Compared to untreated control ticks, permethrin-, amitraz-, and the acaricide mixture-treated ticks exhibited significantly increased SMR (1.054, 1.392 and 1.520 μl h⁻¹ respectively (P < 0.05). Results obtained from this study clearly demonstrated the detrimental effects of permethrin and amitraz on CO₂ gas exchange and water balance in A. americanum ticks. The data also revealed subtle differences between permethrin and amitraz in their effects on tick metabolism, specifically gas exchange and water loss. In addition, the study may provide insight into the modes of action of these two acaricides in tick physiology. The results may also help understand the mechanism of synergism between permethrin and amitraz that was reported previously in other ticks and insects.     

Chemical control of cattle ticks—resistance problems

Chemical control, by dipping or spraying infested cattle with ixodicidal compounds, is the only practicable method of dealing with the cattle tick problem in the short term. Widespread exposure to ixodicidal compounds, often at subeffective concentrations, has resulted in the selection of resistant strains from tick populations. A situation has now been reached where cattle ticks in widely separated parts of the world have shown a capacity to develop resistance to most of the currently available chemical classes of ixodicide. There is a very real probability that these resistant strains will eventually replace susceptible tick populations, particularly in those areas where the cattle tick problem is already most acute. Research and development of new chemical groups of compounds with high ixodicidal activity and low mammalian toxicity is a matter of some urgency. In this review, the biological background of the tick problem is outlined. A brief history of the development of tick resistance to arsenic, organochlorine, organophosphorus and carbamate compounds is given. Reference is made to the underlying biochemical mechanisms involved. Some of the human factors affecting the development of tick resistance are examined and future prospects are assessed.     

Effects of the avermectin, MK-243, on ovary development and salivary gland degeneration in the ixodid tick, Amblyomma hebraeum

Injection of the avermectin analogue, MK-243, into engorged female Amblyomma hebraeum Koch resulted in reduced ovary weight, oocyte length, and ovary vitellin content. There was no significant reduction in hemolymph vitellogenin concentration in MK-243 treated ticks. Although MK-243 was previously shown to markedly reduce hemolymph 20E-concentration, injection of 20E, the vitellogenic hormone in this tick, did not reverse the effects of MK-243 on ovary development. These data suggest that MK-243 may exert its inhibition of egg development more at the level of vitellogenin uptake than vitellogenin synthesis. MK-243 also reversed salivary gland degeneration slightly, probably via its inhibitory effect on 20E-synthesis.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

A standardised in vivo and in vitro test method for evaluating tick repellents

The threat of transmission of Lyme borelliosis and tick-borne encephalitis by ixodid ticks has resulted in an increasing number of tick repellents coming onto the market. To allow proper evaluation of the efficacy of different types of compounds and their formulations, there is a need for standardised methods for testing ticks repellents. Ticks show a marked negative geotactic response following contact with a potential host, i.e., they climb up in order to locate attachment and feeding sites, whereas exposing ticks to repellents induces positive geotaxis, i.e., ticks walk downwards or drop off the treated host or substrate. We describe here complementary tests that employ these geotactic responses to evaluate repellents: one in vitro on a warm glass plate and the other on the lower human leg (shin). The compounds tested were DEET, EBAAP, icaridin, capric acid, lauric acid, geraniol, citriodiol, citronella essential oil and lavender essential oil, all non-proprietary ingredients of widely distributed tick repellent formulations.