A cement protein of the tick Rhipicephalus appendiculatus,located in the secretory e cell granules of the type III salivary gland acini,induces strong antibody responses in cattle

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.     

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.     

Tick antigens recognized by serum from a guinea pig resistant to infestation with the tick Rhipicephalus appendiculatus

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the cattle disease East Coast Fever, was induced in a guinea pig by repeated tick infestation. This resistance is expressed as the ability of the host to interfere with tick feeding. Resistance to ixodid tick feeding is an acquired response mediated by host antibody. We report the use of antibodies from a resistant host animal, in immunoblotting, to characterize the tick antigens recognized. The major tick antigens identified had molecular weights of 120,000, 94,000, 88,000, 77,000, 58,000, 46,000, 35,000, 31,000, 28,000, 25,000, 20,000 and 16,000. Most of these antigens were found in tick salivary glands. The presence and concentration of many tick salivary antigens appeared to vary with relation to the tick feeding cycle. Many of the antigens present in salivary glands were also detected in tick cement. Tick gut extract, although a poorer source of antigens, contained more of the 31,000 dalton antigen than salivary glands. Larval and nymphal tick extract lacked many of the antigens present in adult ticks. The data suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens.     

Genes transcribed in the salivary glands of female Rhipicephalus appendiculatus ticks infected with Theileria parva

We describe the generation of an auto-annotated index of genes that are expressed in the salivary glands of four-day fed female adult Rhipicephalus appendiculatus ticks. A total of 9162 EST sequences were derived from an uninfected tick cDNA library and 9844 ESTs were from a cDNA library from ticks infected with Theileria parva, which develop in type III salivary gland acini. There were no major differences between abundantly expressed ESTs from the two cDNA libraries, although there was evidence for an up-regulation in the expression of some glycine-rich proteins in infected salivary glands. Gene ontology terms were also assigned to sequences in the index and those with potential enzyme function were linked to the Kyoto encyclopedia of genes and genomes database, allowing reconstruction of metabolic pathways. Several genes code for previously characterized tick proteins such as receptors for myokinin or ecdysteroid and an immunosuppressive protein. cDNAs coding for homologs of heme-lipoproteins which are major components of tick hemolymph were identified by searching the database with published N-terminal peptide sequence data derived from biochemically purified Boophilus microplus proteins. The EST data will be a useful resource for construction of microarrays to probe vector biology, vector-host and vector-pathogen interactions and to underpin gene identification via proteomics approaches.     

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Immune suppression induced by the brown ear tick Rhipicephalus appendiculatus Neumann, 1901

The brown ear tick Rhipicephalus appendiculatus suppressed in vivo and in vitro immune responses in the rabbit. Humoral responses were suppressed during infestation; however, suppression was transient and was unrelated to previous exposure of hosts to similar tick infestations. Immune suppression was caused by putative lymphocytotoxic factor(s) in tick salivary secretions as evidenced by in vitro lymphocytotoxicity assays. In an identical study of Rhipicephalus zambeziensis, a closely related rhipicephalid, the immune response was not suppressed, indicating that immune suppression is not common to the genus Rhipicephalus.     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Skin test to detect resistance of cattle to Rhipicephalus appendiculatus ticks

The evaluation of a skin test to detect acquired resistance to Rhipicephalus appendiculatus Neumann ticks is described. An extract of salivary glands of partially fed female R. appendiculatus was prepared by dissection, sonication and filtration and used for intra-dermal injection. Tests were carried out on twelve calves of known resistance to R. appendiculatus and on twelve naive calves to establish threshold values for positive reactions. Four rabbits naive to ticks were skin tested repeatedly to assess immunogenicity of the test. Reactions to the test at 1 h and at 24 h after injection were significantly correlated with resistance. The correlation was higher with the reactions at 24 h. Rabbits were immunized by the test but the reactions never exceeded the positive threshold. Further development by field testing is recommended.     

Survival of unfed Rhipicephalus appendiculatus and Rhipicephalus zambeziensis adults

Two ixodid tick species, Rhipicephalus appendiculatus (Neumann) and Rhipicephalus zambeziensis (Walker et al.) (Acari: Ixodidae), both originating from the southern province of Zambia, were used to study the survival time of adults at a range of different humidities and temperatures. In general, the experiment clearly demonstrates the different survival times of the two species in relation to the climatic conditions tested: survival of R. zambeziensis was better under more extreme conditions of temperature and humidity. These findings offer an explanation for the different distribution ranges of the two tick species. Rhipicephalus appendiculatus is more confined to cooler and wetter conditions, whereas R. zambeziensis is adapted to hotter and drier areas.     

Variation in body size in the tick complex Rhipicephalus appendiculatus/Rhipicephalus zambeziensis.

We examined the relationship between body size and the phenology of the tick complex Rhipicephalus appendiculatus/Rhipicephalus zambeziensis. These ticks transmit Theileria parva in cattle. In Africa, the body size of R. appendiculatus increases with latitude while the body size of the morphologically similar Rhipicephalus zambeziensis is constant at two different latitudes. A larger body size is necessary once survival becomes a constraint. The most plausible explanation for the smaller R. appendiculatus in equatorial Africa is the cost to produce a larger egg. The consequences of these findings for the introduction of R. appendiculatus in new environments are discussed. New field observations from southern Zambia indicate that R. appendiculatus body size does not vary seasonally as compared to eastern Zambia. This is an additional indication of the presence of a single diapausing population of larger ticks.     

Fipronil-induced cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

The tick Rhipicephalus sanguineus is currently considered an urban plague. For this reason many studies are intended to find methods to control these ectoparasites. Thus, the present study analyzed the ultrastructural modifications of the salivary glands cells of semi-engorged females of R. sanguineus resulting from their exposition to Fipronil (active ingredient of Frontline®). The studied individuals were divided into four groups. Group 1 was exposed to distilled water (control) and groups 2, 3 and 4 were exposed to 1, 5 and 10 ppm of Fipronil, respectively. The salivary gland of ticks subjected to the acaricide showed accelerated process of cell death by atypical apoptosis, as well as augmented cell damages as the concentration of the chemical compound was increased. The acaricide toxicity at cellular level was demonstrated by remarkable changes of elements of the cytoskeleton and spherocrystals (extremely hard inorganic structures). However, tick defense mechanisms, such as the observed autofagic vacuoles proved the cells attempt to preserve their integrity and minimize the devastating action of this chemical compound on the salivary glands.     

Rhipicephalus appendiculatus (Acari: Ixodidae): dynamics of Thogoto virus infection in female ticks during feeding on guinea pigs

Engorged nymphs (Rhipicephalus appendiculatus) were inoculated parenterally with Thogoto (THO) virus (approximately 1 microl per nymph; 10(6)-10(7) PFU/ml). The adult females which resulted were used as the source of infected ticks for this study. Hemolymph, salivary glands, synganglion, gut, ovary, and Malpighian tubules were collected on each day of the blood meal and titrated for THO virus by plaque assay. The percent of tissues infected with virus was 16% or less on the day of attachment. Percent infection rose for all tissues throughout 6-7 days of feeding, reaching 40-100% infection during the rapid phase of engorgement. For the first 4 days of feeding, virus titer in the synganglion was higher than in salivary glands (means of 6.4-34.7 PFU/synganglion and 1.6-8.8 PFU/salivary gland pair). From days 5-7, virus titer was generally higher in the salivary gland than the synganglion (means of 422, 408, and 817 PFU/gland pair and means of 62, 811, and 9 PFU/synganglion). However, because a salivary gland pair is much heavier than a synganglion, the virus concentration in the synganglion was much higher than in the salivary gland during the slow phase of feeding. During the rapid phase of feeding, the difference in virus titer between the synganglion and salivary gland reduced. This difference between the early and late stages of feeding may explain why a previous study [J. Gen. Virol. 70 (1989) 1093], using immunofluorescence and immuno-gold labelling, failed to detect virus in the salivary gland early in feeding. These data provide evidence to explain that R. appendiculatus can transmit THO virus within 24h of attachment, an important epidemiological finding.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

Immunomodulatory arsenal of nymphal ticks

Ticks have developed their own immunomodulatory mechanisms to inhibit the host inflammatory response. One of them involves the ability to subvert the cytokine network at the site of tick feeding by secreting cytokine binding molecules. Most studies have focused on the immunomodulatory prowess of adult female ticks. Here we describe anti-cytokine activity in salivary gland extracts (SGEs) prepared from 2-day-fed nymphs of Dermacentor reticulatus Fabricius, Ixodes ricinus L., Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius. Anti-CXCL8 activity was detected in nymphs of all species. Relatively high activity against CCL2, CCL3 and CCL11 was observed in SGEs of R. appendiculatus and A. variegatum nymphs, whereas SGEs of I. ricinus nymphs showed comparatively high anti-interleukin-2 (-IL-2) and anti-IL-4 activities. These data show that nymphs, which epidemiologically are usually more important than adults as disease vectors, possess a range of anti-cytokine activities that may facilitate pathogen transmission.     

Genetic variation in the body weight of ticks of the Rhipicephalus appendiculatus/zambeziensis complex

Sixteen strains of ticks of the Rhipicephalus appendiculatus/zambeziensis complex were created by cross-breeding for one to three generations. The body-weights of adult male ticks of these consanguineous lines were compared. A significant genetic variation was distinguished between the groups. Three classes whose mean weights varied around, respectively, 2, 3 and 4 mg/adult male tick, were found. This variation in weight may be explained by hybridization between R. appendiculatus Neumann and R. zambeziensis Walker, Norval & Corwin. Hybrid ticks were undersized whereas R. appendiculatus and R. zambeziensis groups had significantly higher weights.     

Factors influencing the distribution of questing ticks and the prevalence estimation of T. parva infection in brown ear ticks in the Tanga region, Tanzania.

Questing ticks from various districts and agro-ecological zones (AEZ) in the Tanga Region of Tanzania were studied for a two-year period between September 1999 and July 2001. Collections of both nymphal and adult ticks occurred at 29 sites using a blanket or white cloth dragging technique. The species recorded in the order of decreasing abundance were Rhipicephalus appendiculatus, Boophilus spp., and Ambylomma variegatum. Rhipicephalus appendiculatus field infestation levels varied across administrative districts and seasons, with Amani and the cool-to-dry season having lower tick counts ( = -2.09, SE = 0.71, P < 0.001 and = -1.54, SE = 0.56, P = 0.006 for Amani and cool to dry season, respectively). Based on the polymerase chain reaction technique, Theileria parva infection prevalence in adult R. appendiculatus was estimated to be 2.6%. Implications of these findings in light of the spatial and temporal distribution of ticks are discussed for the accurate diagnosis of multiple tick-borne diseases, the infected vector tick, the endemic status of T. parva in the region, and the implemention of control strategies.     

An immunocytochemical marker for the complex granules of tick salivary glands which traces e-granule shedding to interstitial labyrinthine spaces

An antigenic protein (12C), previously isolated from salivary glands of Rhipicephalus appendiculatus, as a possible factor in host resistance to repeated tick infestation, is labeled by protein A-gold immunocytochemistry in adult feeding ticks. It is a component of the gland's complex a-, d- and e-granules and also appears within the chitinous walls of intercalated ducts. In females, between days 4 and 7 of feeding, labeled e-granules appear also within the labyrinthine spaces of acinus type III, apparently released from e-cells as the abluminal interstitial cells initiate formation of a basolateral labyrinth. Granules thus shed are fragmented by interstitial cell processes, some fragments being phagocytized, others disintegrating to single point label scattered throughout the labyrinth. The latter possibly may pass into the acinar lumen. By the eighth day the label is gone from the labyrinth.     

Engorgement of Rhipicephalus haemaphysaloides ticks blocked by silencing a protein inhibitor of apoptosis

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.

     

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.     

Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses

ABSTRACT. Experimental studies were undertaken to ascertain the vector potential of Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius for Thogoto (THO) and Dhori (DHO) viruses, candidate members of the Orthomyxoviridae. In the first set of experiments, ticks were infected orally by feeding on viraemic hamsters. THO virus replicated in R.appendiculatus and A.variegatum, persisted trans-stadially in both tick species, and was transmitted to susceptible hosts during feeding. In contrast, both R. appendiculatus and A. variegatum were refractory to per os infection by DHO virus. In the second set of experiments, engorged R. appendiculatus and A. variegatum nymphs were parenterally inoculated with DHO virus. The virus persisted transstadially in both tick species and was transmitted by bite to susceptible hosts. These results indicate that the midgut acts as a barrier to per os infection of R.appendiculatus and A. variegatum by DHO virus. However, when this barrier is bypassed, i.e. by parenteral inoculation, both R.appendiculatus and A.variegatum can serve as efficient vectors of DHO virus.