Aetiology of skeletal muscle ''cramps'' during exercise: a novel hypothesis

Secretions of the tick salivary glands are essential to the successful completion of the prolonged feeding of these ectoparasites as well as the conduit by which most tick-borne pathogens are transmitted to the host. In ixodid ticks the salivary glands are the organs of osmoregulation, and excess water from the bloodmeal is returned via saliva into the host. Host blood must continue to flow into the feeding lesion as well as remain fluid in the tick mouthparts and gut. The host's haemostatic mechanisms are thwarted by various anti-platelet aggregatory, anticoagulatory and anti-vasoconstrictory factors in tick saliva. Saliva components suppress the immune and inflammatory response of the host permitting the ticks to remain on the host for an extended period of time and, adventitiously, enhancing the transmission and establishment of tick-borne pathogens. Over the years much work has been done on the numerous enzyme and pharmacological activities found in the tick saliva. The present article reviews the most recent work on salivary gland secretions with special emphasis on how they favour pathogen transmission.     

Studies on the development of Theileria annulata Dschunskowsky and Luhs, 1904 in the tick--Hyalomma anatolicum anatolicum Koch, 1844

The endogenous development of Theileria annulata in the vector tick--Hyalomma anatolicum anatolicum has been described. Parasites within and outside the erythrocytes were seen in the gut contents of larvae and nymphs immediately after their removal from the infected cattle but not after 24 hours. Two bodies resembling the intra-erythrocytic stage of the parasite, lying close to each other were seen near the gut epithelium of a larva on the 4th day of its dropping off the infected host. Neither multiplication nor union of the parasite resulting in zygote formation in the gut of the tick, was noticed. Next stage of the parasite was seen in the salivary glands of nymphs and adults. The pre-infective stage characterized by indistinct chromatin dots (nuclei) surrounded with cytoplasm was seen in alveolar cells of the salivary glands of nymphs and adult ticks from the first to the third day of their feeding of the host. This stage progressed to the infective stage consisting of distinct chromatin particles (the infective particles) surrounded with cytoplasm in the salivary glands of nymphs and adult ticks after the second and first day, respectively of feeding on the host. The infective particles were also seen within ducts of the salivary glands.     

A novel neuropeptide-endocrine interaction controlling ecdysteroid production in ixodid ticks

Ixodid (hard) ticks are blood-feeding arthropods that require a blood meal to complete each stage of development. However, the hormonal events coordinating aspects of feeding and development are only poorly understood. We have delineated a new neuropeptide-endocrine interaction in the adult tick, Amblyomma hebraeum, that stimulates the synthesis of the moulting hormones, the ecdysteroids. In adult female ticks, ecdysteroid synthesis could be demonstrated in integumental tissue incubated in vitro with a synganglial (central nervous system) extract, but not in its absence. Stimulation by the synganglial extract is both time- and dose-dependent, but is completely abolished by trypsin treatment, suggesting that the activity is due to a peptide/protein. Integumental tissue ecdysteroidogenesis is also stimulated by elevation of the cAMP concentration using forskolin and 3-isobutyl-l-methyl-xanthine, or by 8-bromo-cAMP. This suggests the involvement of at least a cAMP second messenger system in the neuropeptide-ecdysteroidogenesis axis, without precluding a role for other second messengers as well. Despite involving a quite different steroidogenic tissue, the foregoing system has some parallels with the known prothoracicotropic hormone (neuropeptide)-prothoracic gland endocrine axis of insects.     

Importance of localized skin infection in tick-borne encephalitis virus transmission

Arboviruses are transmitted to vertebrates by the "bite" of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels > or = 2.0 log10 ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission.     

Changes in blood sugar and ammonium levels after ligation of the afferent hepatic vessels

Certain aspects of the development of Ehrlichia canis, causative agent of canine ehrlichiosis (tropical canine pancytopenia) in Rhipicephalus sanguineus ticks were studied. It was found that partial feeding of nymphs infected as larvae with E canis was a desirable, if not necessary, preliminary treatment for successful infection of dogs with ground-up ticks. It remains unclear whether feeding increased the number or altered the virulence of ehrlichiae within tick tissues. Ehrlichia canis organisms were detected by immunofluorescent microscopy in the midgut and hemocytes and by electron microscopy in the midgut and salivary glands of partially engorged adult ticks which had been infected as larvae and nymphs. Organisms were not observed in the ovary. Intracytoplasmic inclusions contained 1 to 80 elementary bodies, each provided with 2 distinct membranes. Infection of the midgut and salivary gland was confirmed by injecting homogenates of these tissues into susceptible dogs. Staining of gut smears of partially engorged adult ticks by fluorescein-conjugated anti-E canis antibody was found to be a reliable indicator of the infection.     

Ethanol administration delays recovery from acute pancreatitis induced by exocrine hyperstimulation

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.     

Cardiovascular dysfunction related to threat, avoidance, and vigilant work: application of event-related potential and critique

Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide. The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal. The inclusion is composed of one or more types of delta-endotoxins (Cry and Cyt proteins). Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa. The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia. The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms. The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.     

Effects of the feeding process of Ixodes scapularis (Acari: Ixodidae) on embryonic development of its parasitoid, Ixodiphagus hookeri (Hymenoptera: Encyrtidae)

The chalcid wasp Ixodiphagus hookeri (Howard) is a parasitoid of several ixodid ticks including the blacklegged tick, Ixodes scapularis Say. We evaluated effects of the feeding process of nymphal I. scapularis on the embryonic development of I. hookeri. Potentially wasp-parasitized nymphal I. scapularis were collected on Prudence Island, RI. Subsamples of the questing nymph cohort were allowed to feed on laboratory white mice. Both the body length and the scutal length of ticks were measured individually for questing nymphs and for feeding nymphs that were removed from hosts at time intervals of 12, 24, 36, 48, and 72 h after attachment. The diameters of wasp eggs they contained were also measured for each designated time interval. There was a positive relationship between the mean scutal index (ratio between body length and scutal length) of ticks and the mean diameter of wasp eggs during 72 h of tick feeding (P < 0.05). Moreover, it appeared that within 24 h of tick attachment, the scutal index of ticks remained unchanged. However, after that period, the scutal index increased significantly (P < 0.05). Diameters of wasp eggs increased continuously during tick feeding and at 72 h after attachment, enclosed eggs and completely formed larvae were found in several ticks. We conclude that factors related to the feeding process of nymphal I. scapularis are necessary to initiate the embryonic development of wasps.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31-35 kDa and 18-25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Vaccines against blood-sucking arthropods

This paper provides selected personal insights on the development of vaccines against blood-sucking arthropods, with particular emphasis on vaccines against ticks. The emergence of novel or concealed antigens of haematophagous ectoparasites as candidate vaccine antigens is reviewed and the effect of feeding by the parasite on the expression of protective antigens is considered. The distribution of protective antigens through life cycle stages, the stage of the life cycle targeted by protective responses, and the nature of these responses, are commented on briefly. Concealed antigens of the gut, including the peritrophic membrane, and other internal organs, are evaluated for the role they play in induction of immunity artificially. Some of the work carried out to purify and characterise protective antigens of tick guts is described. A commentary is developed on vaccines that combine both "concealed" and "exposed" antigens. Some of the problems associated with the infestation and challenge of vaccinated hosts in the field are identified and the delivery of parasite antigens as vaccines that are both protective and "user-friendly" is emphasised as a major problem to be solved.     

Ixodid ticks infesting rodents and sheep in diverse biotopes of southern India

A total of 127 rodents were trapped in southern India. Examination of these rodents revealed the presence of 2 species of ticks, Haemaphysalis spinigera and Rhipicephalus ramachandrai. The former species is the principal vector of Kyasanur Forest Disease (KFD) in India, and the latter's role, if any, is unknown. Sheep grazing in 1 of the study area were infested with another ixodid tick. Haemaphysalis intermedia, which is a vector of Bhanja virus in India. The presence of H. spinigera on domestic rats is important from the standpoint of KFD enzootiology. This tick shows a narrow habitat preference but a wide host range. In peridomestic situations, the field rodent Bandicota bengalensis did not harbor any tick species. Contiguity of feral and domestic biotopes in some areas contributed to the transfer of R. ramachandrai from its preferred wild rodent host, Tatera indica, to domestic rats Rattus sp.     

Influence of caecectomy and dietary protein concentration on apparent excreta amino acid digestibility in adult cockerels

Infestation with ixodid tick stimulates the immune regulatory and effector pathways of the hosts involving antigen presenting cells, T-lymphocytes, B-lymphocytes, basophils, mast cells, eosinophils and a variety of bioactive molecules like cytokines, antibodies and complement. Tick-mediated immunosuppression has been investigated using cells derived from infested animals and by exposing cells from uninfected animals to tick salivary gland molecules. Tick-induced suppression of host immune defences is characterized by reduced ability of lymphocytes from infested animals to proliferate in vitro in the presence of concanavalin A (Con A), diminished primary antibody responses to T-cell dependent antigen, and decreased elaboration of macrophage (IL-1 and TNF-alpha) and Th1-lymphocyte cytokines (IFN-gamma), whereas Th2 cytokines production (IL-4, IL-5 and IL-10) is enhanced. It is known that IL-10 inhibits Th1 cell development and also reduces the in vitro T-lymphocyte proliferative response to Con A stimulation. Proteins which inhibited T-lymphocyte in vitro responsiveness to Con A were also isolated from tick salivary glands.     

Amblyomma variegatum (Acari: Ixodidae): mechanism and control of arbovirus secretion in tick saliva

Saliva is considered to be the conduit by which pathogens are transmitted from blood-sucking arthropod vectors to their vertebrate hosts, but supporting evidence for this is fragmentary. To determine if Thogoto (THO) virus, a tick-borne member of the influenza virus family, is transmitted via tick saliva, and whether virus replication is a prerequisite for such transmission, two experimental conditions were compared: (1) "biological transmission" and (2) "mechanical transmission." In (1), THO virus was allowed to infect and replicate in a natural vector, Amblyomma variegatum: virus was detected in saliva collected from 3/22 (14%) ticks. In (2), virus was inoculated directly into the hemocoel with the drug used to induce salivation and saliva was collected immediately to preclude the possibility of virus replication: virus was detected in saliva collected from 31/170 (18%) ticks. The results demonstrate that THO virus is secreted in tick saliva and that virus can pass from the hemolymph to the salivary glands independently of viral replication within the tick. The comparatively low numbers of ticks that yielded virus-positive saliva samples together with the results from assays of serial saliva samples suggested that virus secretion may not be a continuous process during salivation. Ticks in which THO virus had established an infection showed an impaired secretory response compared with uninfected ticks and ticks used for mechanical transmission.     

Preliminary characterization and pathogenicity studies of a virus isolated from ticks (Ornithodoros coriaceus) and from tick-exposed cattle

Several viral isolates from ticks (Ornithodoros coriaceus) and from the blood of cattle which aborted after exposure to these ticks were found to be identical by reciprocal cross serum-neutralization tests. Characterization studies indicate that the virus is a member of the Togaviridae family, although specific identification is still incomplete. Whether its natural host is the tick or bovine animals is also unknown. Pregnant cows inoculated with the agent by all conventional parenteral routes, including intrafetal, delivered healthy calves at term. It was concluded, therefore, that it was not a bovine pathogen and that the abortions which occurred after tick-exposure were due to a 2nd agent in O coriaceus ticks which also harbor the virus. While several ciruses believed to be tick-borne have been isolated from cattle in various parts of the world, it is believed that the present report describes the first isolation in the Western Hemisphere for a viral agent from Argasid ticks which has been demonstrated to replicate in cattle.     

Tularemia transmitted by arthropod bites

The authors present four patients affected with tularaemia in Koprivnica-Krizevci region. The disease occurred in the period from April to September 1994, in 3 males and 1 female patient, aged 9-49 years. In all affected persons the infections were transmitted after bites of arthropods, ticks or other insects. The disease was clinically manifested as ulceroglandular form, without complications and relapses. The diagnosis was established by anamnestic and epidemiological data, clinical picture, the course of the disease, laboratory findings, tularin test and specific agglutination test (two samples). All patients were successfully treated with doxycyclin.     

Ultrastructural nucleolar alterations induced by an ametantrone--poly r(A-U) complex

Several studies have described changes in the expression of proteins, especially of OspA and OspC, of B. burgdorferi sensu stricto during tick feeding. In this study, the expression of OspA and OspC of B. afzelii in unfed and feeding I. ricinus nymphs and in the subsequent adults was followed by means of the immunofluorescence test. Spirochaetes expressing OspA and OspC were observed in 70% and 80%, respectively of the unfed nymphs. In feeding and in fully engorged ticks, spirochaetes expressed OspC, while OspA disappeared 24 hours after the beginning of the blood meal. Spirochaetes expressing OspC in salivary glands were observed in one engorged tick. After molting, in unfed adults spirochaetes again expressed OspA and OspC but did so less frequently (6% and 13%, respectively). The mouse strain (AKR/N or BALB/C) on which ticks had their infectious blood meal influenced OspC expression in the following tick stage. In the skin of AKR/N mice, at the tick feeding site, B. afzelii expressed OspC only, as was shown by immunostaining.     

Mx1-based resistance to thogoto virus in A2G mice is bypassed in tick-mediated virus delivery

The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus, Mx1(+) A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.     

Determining the duration of Ixodes scapularis (Acari: Ixodidae) attachment to tick-bite victims

The duration of tick attachment is one factor associated with risk for human infection caused by several tick-borne pathogens. We measured tick engorgement indices at known time intervals after tick attachment and used these indices to determine the length of time that ticks were attached to tick-bite victims in selected Rhode Island and Pennsylvania communities where the agents of Lyme disease and human babesiosis occur. The total body length and width as well as the length and width of the scutum were measured on nymphal and adult female Ixodes scapularis Say removed from laboratory animals at 0, 12, 24, 36, 48, 60, and 72 h after their attachment. Three engorgement indices were calculated at each time interval. In addition, engorgement indices measurements were recorded for 504 ticks submitted to a commercial laboratory for pathogen detection testing between 1990 and 1992. No detectable change was observed in the average engorgement indices for either nymphal or adult ticks between 0 and 24 h of attachment using any of the engorgement indices. After 24 h of tick attachment, all engorgement indices continuously increased: average indices for nymphs attached 36, 48, and 60 h were significantly different from those attached < or = 24 h and from each other. Similarly, average engorgement indices for adult ticks attached < or = 36 h were significantly different from those attached for 48 h or more. More than 60% of tick-bite victims removed adult ticks by 36 h of attachment, but only 10% found and removed the smaller nymphal ticks within the first 24 h of tick feeding. The duration of tick attachment may serve as a useful predictor of risk for acquiring various infections, such as Lyme disease and babesiosis, transmitted by I. scapularis. Regression equations developed herein correlate tick engorgement indices with duration of feeding. A table containing specific engorgement index prediction intervals calculated for both nymphs and adults will allow the practitioner or clinical laboratory to use easily measured tick engorgement indices to predict transmission risk by determining the duration of feeding by individual ticks.     

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340-348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd-1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [32P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A > Mur A > Mak A > 20E > ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG 10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions-5, + 2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.     

The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects

The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects. Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2. The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A. While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3a binding is restricted to gut cells of susceptible insects. Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells. These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality. Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.