Developmental changes in morphology and toxin content of the salivary gland of the australian paralysis tick Ixodes holocyclus

Binnington K.C. and Stone B.F. 1981. Developmental changes in morphology and toxin content of the salivary gland of the Australian paralysis tick Ixodes holocyclus.International Journal for Parasitology11: 343–351. Histological study of the salivary gland of female I. holocyclus has shown that 2 of the 4 cell types present are richest in granules in the unfed stage but have discharged these granules after an attachment period of 24 h. The presence of a toxin in homogenates of salivary glands from unfed females and its absence after 24 h of attachment may be associated with the loss of granules from these 2 cell types shortly after attachment. Another cell type shows a gradual increase in granule content throughout feeding and a fourth, a peak in granule content after an attachment period of 120 h. The latter cell type may produce the paralysing toxin since ticks do not paralyse the host until they have fed for about this time and homogenised glands are most toxic at 120–144 h.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Vector competence of the tick Ixodes ricinus for transmission of Bartonella birtlesii

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Transcriptome and toxin family analysis of the paralysis tick, Ixodes holocyclus

The Australian paralysis tick (Ixodes holocyclus) secretes neuropathic toxins into saliva that induce host paralysis. Salivary glands and viscera were dissected from fully engorged female I. holocyclus ticks collected from dogs and cats with paralysis symptoms. cDNA from both tissue samples were sequenced using Illumina HiSeq 100?bp pair end read technologies. Unique and non-redundant holocyclotoxin sequences were designated as HT2–HT19, as none were identical to the previously described HT1. Specific binding to rat synaptosomes was determined for synthetic HTs, and their neurotoxic capacity was determined by neonatal mouse assay. They induced a powerful paralysis in neonatal mice, particularly HT4 which produced rapid and strong respiratory distress in all animals tested. This is the first known genomic database developed for the Australian paralysis tick. The database contributed to the identification and subsequent characterization of the holocyclotoxin family that will inform the development of novel anti-paralysis control methods.     

Identification of Four Novel Rhipicephalus annulatus Upregulated Salivary Gland Proteins as Candidate Vaccines

The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1–4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.     

Role of an aquaporin in the sheep tick Ixodes ricinus: Assessment as a potential control target

Ticks undergo tremendous osmoregulatory stress as they take on up to 100 times their body weight in blood, returning about 75% of the ingested water and ions via their saliva into the host. We postulated that water channels, or aquaporins, involved in this mass water transport might be good targets for acaricide development. An aquaporin (IrAQP1) identified in the sheep tick, Ixodes ricinus, was present only in tissues involved in mass water flux, namely the gut, rectal sac and especially abundant in the salivary glands. IrAQP1 was localised by in situ hybridisation in specific cell and acini types, possibly Type III acini, but absent from the type I acini that are responsible for rehydration of ticks in the non-feeding phase. Gene knockdown of IrAQP1 in isolated salivary glands completely inhibited dopamine-stimulated secretion. Further, IrAQP1 knockdown adult females had 50% reduced body weight gains over the first 5 days feeding on an artificial feeding apparatus and 21% at the point of engorgement on hosts. Haemolymph osmolarity was increased in the IrAQP1-knockdown ticks. Importantly, the blood volume ingested per body weight was reduced by 30%. Overall, it would appear that water passage from the gut to the saliva was disrupted and tick guts were simply too “full” to ingest more blood. However, double-stranded RNA interference of IrAQP1 did not affect mortality of the ticks which successfully fed to detachment at day 9. Overall, our data indicate that IrAQP1 plays a pivotal role in blood meal water handling through the gut and salivary gland, and although its disruption by double-stranded RNA interference dramatically affects feeding performance, ticks remained feeding on the host with subsequent potential pathogen transmission and, therefore, IrAQP1 is not a suitable candidate target for tick control.     

Theileria parva: expression of a sporozoite surface coat antigen

A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A--colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process.     

Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americanum-derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies

Guinea pigs immunized by subcutaneous injection of an emulsion of incomplete Freund's adjuvant (IFA) containing tick salivary gland extract antigens (SGA) from partially fed female ticks expressed a significant level of tick rejection when challenged 17 days later. This level of tick rejection was similar to animals actively sensitized by tick feeding and challenged at the same time. SGA emulsified with complete Freund's adjuvant (CFA) or administered with saline was ineffective. However, ticks that fed on animals immunized with SGA+IFA or SGA+CFA expressed significant reductions in engorgement weight. SGA was active when prepared with or without protease inhibitors. The minimum effective immunizing dose of SGA was between 100 and 280 micrograms per animal. Extracts made from salivary gland-derived cement material (CA) from partially fed female ticks administered at 50 micrograms in IFA induced levels of tick rejection comparable to animals immunized with 280 micrograms of SGA+IFA. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) of 35S- and 125I-radiolabeled SGA and CA extracts immunoprecipitated by guinea pig anti-tick serum that transferred immune resistance demonstrated a unique protein of 20,000 m.w. Serum from animals immunized with SGA+IFA (successful immunization) recognized this same protein, whereas serum from animals immunized with SGA+CFA (unsuccessful immunization) did not. The results of this study suggest that a 20,000 m.w. protein derived from the tick salivary gland may be responsible for the induction and perhaps elicitation of host immune resistance responses to Amblyomma americanum ticks.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

Amblyomma americanum (L): Tick macrophage migration inhibitory factor peptide immunization lengthens lone star tick feeding intervals in vivo

Immunizations of New Zealand White rabbits with specific macrophage migration inhibitory factor (MIF) tick peptide (PEP) produced circulating anti-tick PEP antibodies in the hosts. Antibody titers of greater than 1:5000 to tick MIF peptide were observed for crude sera from PEP-immunized rabbits. PEP- and BSA-vaccinated rabbits were infested with Amblyomma americanum adults. Feeding intervals, female weights, egg masses and percent egg hatch were measured for ticks feeding on control and immunized hosts. Feeding intervals were significantly lengthened to 13.3 days for PEP-vaccinated hosts compared to BSA-vaccinated controls at 12.4 days, while female engorgement weights and egg masses were unchanged. By immunizing hosts using specific tick PEP, we were able to alter the length of time the ticks fed on their hosts.     

Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.     

Brain factor induced formation of inositol phosphates in tick salivary glands

A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.     

Microarray analysis of gene expression changes in feeding female and male lone star ticks,Amblyomma americanum (L)

A collection of EST clones from female tick Amblyomma americanum salivary glands was hybridized to RNA from different feeding stages of female tick salivary glands and from unfed or feeding adult male ticks. In the female ticks, the expression patterns changed dramatically upon starting feeding, then changed again towards the end of feeding. On beginning feeding, genes possibly involved in survival on the host increased in expression as did many housekeeping genes. As feeding progressed, some of the survival genes were downregulated, while others were upregulated. When the tick went into the rapid feeding phase, many of the survival genes were downregulated, while a number of transport‐associated genes and genes possibly involved in organ degeneration increased. In the males, the presence of females during feeding made a small difference, but feeding made a larger difference. Males showed clear differences from females in expression, as well. Protein synthesis genes were expressed more in all male groups than in the partially fed females, while the putative secreted genes involved in avoiding host defenses were expressed less. © 2009 Wiley Periodicals, Inc.     

Tick-host interaction: a synthesis of current concepts

Ixodid ticks are recognized world-wide as major vectors of arboviruses, rickettsiae, spirochaetes and parasitic protozoa of man and domestic animals. Some ticks also inject a debilitating, sometimes fatal, paralytic toxin in their saliva. All these factors are transmitted via the salivary glands and mouthparts of the feeding tick. Tick feeding is a prolonged and complex process. Major developmental events occurring within the tick during feeding, as well as extensive tick-host interactions, all influence the likelihood of pathogen transmission. In this review, Reuben Kaufman discusses the sequence and complexity of these interactions.