Tick Extracts Research Articles

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus ( Boophilus) annulatus salivary gland

Immunoscreening of a cDNA expression library of the Rhipicephalus ( Boophilus) annulatus tick with purified rabbit anti- R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661 bp sequence. The sequence includes an open reading frame of 543 bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51 kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100 °C, whereas extracts from cells not expressing the protein were heat-sensitive at 60 °C.

First Molecular Evidence of Potentially ZoonoticBabesia microtiandBabesiasp. EU1 inIxodes ricinusTicks in Belgium

We report the first molecular evidence of the presence of Babesia sp. EU1 and Babesia microti in Ixodes ricinus ticks in Belgium. A 1-year national survey collected 1005 ticks from cats and dogs. A polymerase chain reaction technique amplifying a part of the 18S rRNA gene detected Babesia spp. in 11 out of 841 selected and validated tick extracts. Subsequent sequencing identified Ba. microti (n=3) and Babesia sp. EU1 (n=6). This study has demonstrated a low infection rate (1.31% with 95% CI: 0.65-2.33) of Babesia spp. carriage in I. ricinus ticks in Belgium but, for the first time, reports two potentially zoonotic species belonging to this genus. Coinfection with Ba. microti and Borrelia burgdorferi sensu stricto also was demonstrated. In addition, this study clearly demonstrates that inhibitors of polymerase chain reaction amplification are present in engorged ticks.

Detection of Vector-Borne Agents in Lone Star Ticks, <I>Amblyomma americanum</I> (Acari: Ixodidae), From Mississippi

In this study, we evaluated Amblyomma americanum (lone star tick) in Mississippi for the presence of Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis; Ehrlichia ewingii, causative agent of human and canine granulocytic ehrlichiosis; Borrelia lonestari, putative agent of southern tick-associated rash illness; Francisella tularensis, the agent of tularemia; and Rickettsia spp., particularly R. amblyommii, a suspected pathogen. We collected adult A. americanum from four regions of Mississippi: Northeast, Northwest, Southeast, and East. Of the ticks collected, 192 were dissected and DNA was extracted for nested polymerase chain reaction (PCR) assays to detect the above bacteria. In all, 3% of tick extracts had evidence of Borrelia sp., 4% for E. chaffeensis, 6% for E. ewingii, and 44% for a Rickettsia species. As determined by sequencing, most Rickettsia spp. were R. amblyommii. In addition, extracts from 42 pools (total of 950) of larval A. americanum collected in Southwest Mississippi were tested for the presence of E. chaffeensis and Rickettsia species. Of these extracts from pools, nine of 37 (24%) were PCR positive for a Rickettsia sp., most often, R. amblyommii; none had evidence of E. chaffeensis, supporting the ability of lone star ticks to transovarially transmit R. amblyommii, but not E. chaffeensis. This study demonstrates E. chaffeensis, E. ewingii, "B. lonestari", and R. amblyommii in A. americanum by PCR for the first time in Mississippi. Understanding the prevalence and epidemiology of these agents in Mississippi should increase awareness of tick-borne disease in the medical community.

Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface

Conidial germination and the formation of appressoria are important events in the interactions between entomopathogenic fungi and their arthropod hosts. In this study, we demonstrate the effects of lipids extracted from tick epicuticle and the surface of a mammalian host (calf) on conidial germination and the development of appressoria in two subspecies of Metarhizium anisopliae, M. anisopliae var. anisopliae ( M. an. an.-7) and M. anisopliae var. acridum ( M. an. ac.-5), which have different levels of virulence toward ticks. Pentane extracts of epicuticles of ticks susceptible and resistant to fungal infection always stimulated the germination of M. an. an.-7 conidia and the development of their appressoria; whereas the effects of dichloromethane (DCM) extracts of tick epicuticle varied depending on the tick. The DCM extracts from most of the tick species and developmental stages stimulated conidial germination and/or the formation of appressoria in M. an. an.-7. However, a DCM extract of lipids from the most resistant tick, engorged Hyalomma excavatum female, inhibited the germination of M. an. an.-7 conidia. Conidia of the non-virulent M. an. ac.-5 did not germinate on agarose amended with any of the examined tick extracts. However, when the tick extracts were placed on bactoagar, conidial germination increased 7- to 8-fold. Extracts from the skin, hair and ear secretions of a calf stimulated conidial germination and the formation of appressoria in M. an. an.-7, but not M. an. ac.-5. This study demonstrates that lipids from tick epicuticles and mammalian skin selectively affect the germination of conidia of entomopathogenic fungi. The effects of these lipids may explain the variability in tick control these fungi provide for different hosts.

New Approaches to Detection and Identification of <I>Rickettsia africae</I> and <I>Ehrlichia ruminantium</I> in <I>Amblyomma variegatum</I> (Acari: Ixodidae) Ticks From the Caribbean

Imported from Africa in the 1700s and despite frequent modern eradication efforts, Amblyomma variegatum (F.) spread through the Caribbean by cattle transport, small ruminants, and migrating birds. A. variegatum is a vector for Rickettsia africae, the causative agent of African tick bite fever, and Ehrlichia ruminantium, the causative agent of heartwater. We examined 95 A. variegatum and six Rhipicephalus (Boophilus) microplus (Canestrini) collected from cattle at an abattoir in Antigua. Engorged tick extracts adsorbed on Nobotu filter paper strips and new nested polymerase chain reaction (PCR) assays for E. ruminantium and Dermatophilus congolensis were used to evaluate these ticks for the presence of these pathogenic bacteria. Amblyomma ticks (62.4%) contained R. africae DNA by PCR/restriction fragment length polymorphism analysis and DNA sequencing of the OmpA and 17-kDa antigen genes. Twenty Amblyomma and two Rh. microplus contained E. ruminantium DNA. No E. chaffeensis, Anaplasma phagocytophilum, Coxiella burnetii, or D. congolensis DNA was detected in these ticks. The continued presence of Am. variegatum in the Caribbean poses a significant risk of infection in cattle with E. ruminantium and in humans by R. africae. Eradication efforts are essential to prevent the further spread of Am. variegatum.

Comparative IgG recognition of adult tick extracts by sera of experimentally infested bovines

Comparative IgG recognition of adult tick extracts by sera of experimentally infested bovines

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus ( Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.

Identification of Proteins, Enzymes and Potential Vaccine Candidates from the Aust Paralysis Tick, Ixodes Holocyclus Using 2D-PAGE and Equalizer Technology

Ticks are blood-feeding arthropods that are vectors for many diseases of humans and animals caused by viral, rickettsial, bacterial, fungal, protozoal and nematode pathogens. Extensive studies of the antihaemostatic molecules (anticoagulants, vasodilators, platelet aggregation inhibitors) in the saliva of blood-feeding arthropods (Rhodnius prolixus, bed bugs, sandflies, mosquito) have been reported (Champagne,2005) but very little is known about the proteins involved in the attachment and feeding processes of ticks and this is particularly so for Ixodes holocyclus. To date, no complete genome has been published for any tick species and EST libraries are available for only four species (Boophilus microplus, Amblyomma variegatum, Ripecephalus appendiculatus and Ixodes scapularis). Thus a targeted proteomic approach to the investigation of I.holocyclus was initiated seeking to identify the enzymes secreted into the feeding chamber and enzymes involved into the digestion of haemoglobin. Enzymes were detected in whole, unengorged female tick extracts and whole, engorged female tick extracts whose proteins had been equalized using Proteominer beads, a combinatorial hexapeptide ligand library bound to chromatographic beads that aims to embrace all proteins present in a sample (Boschetti et al, 2007). The proteins in the extracts were then separated by 2D-PAGE and visualised by the fluorescent peptide substrate assay method developed by Zhao and Russell (2003). The method uses commercially available, enzyme specific, peptide substrates containing 4-methyl-coumaryl -7-amide that

Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus

A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936 bp cloned fragment was sequenced and showed an open reading frame of 516 bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8 kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.

Molecular characterization of a myosin alkali light chain-like protein, a “concealed” antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti- H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti- H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti- H. qinghaiensis saliva immune could not. This proved Hq02 protein was a “concealed” antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.

Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis

Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HlLAP). Endogenous HlLAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HlLAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HlLAP was induced by a blood-feeding process. A functional recombinant HlLAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the K m value 0.19 ± 0.011 mM and V max value 157.2 ± 3.17 nmol/min/mg protein) and a methionyl substrate (with the K m value 0.12 ± 0.0052 mM and V max value 171.9 ± 2.31 nmol/min/mg protein). Enzyme activity was found to be optimum at pH 8 and 35 °C. The recombinant HlLAP enzyme activity was strongly dependent on metal divalent cations, Mn 2+, and was inhibited by bestatin. These results indicate that HlLAP play an important role for host’s blood digestion process.

A single-run, real-time PCR for detection and identification ofBorrelia burgdorferi sensu latospecies, based on thehbbgene sequence

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.

Comparison of Preservation Methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for Reliable DNA Amplification by PCR

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 degrees C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.

Cutaneous hypersensitivity induced in rabbits by extracts of the tick Amblyomma cajennense (Acari: Ixodidae).

The cutaneous hypersensitivity test was used to correlate host resistance to ticks and type of reactions elicited by Aomblyomma cajennense (Fabricius, 1787) tick extract in rabbits. Rabbits were divided into 3 groups of 2 animals each: naive, pre-infested and control. Cutaneous hypersensitivity was induced by intradermal inoculation of 25 microg extract in 0.03 ml of phosphate buffered saline (PBS) in rabbit ears. Control rabbits were inoculated with PBS only. The ear thickness was measured with a Mitutoyo device before and 10 min, 1, 2, 4, 18, 24, 48, 72 and 96 h post-inoculation (PI). Pre-infested rabbits showed an immediate type reaction within the 1st 10 min PI (60% increase in ear thickness) and a delayed reaction (18 h) (85% increase), whereas the naive rabbits showed only the immediate reaction within the 1st 4 h (60% increase). PBS induced only mild reactions. These results point out the crucial role of the cellular immune response of rabbits in the expression of resistance to A. cajennense.

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz- l-Arg-pNA at the rate of 30.2 μmol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.

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.

Antigens from Rhipicephalus sanguineus ticks elicit potent cell-mediated immune responses in resistant but not in susceptible animals

In the present study we compared the immunological reactions between Rhipicephalus sanguineus tick-infested susceptible (dogs and mice) and tick-resistant hosts (guinea pigs), elucidating some of the components of efficient protective responses against ticks. We found that T-cells from guinea pigs infested with adult ticks proliferate vigorously in the presence of concanavalin A (ConA), whereas ConA-induced cell proliferation of tick-infested mice and dogs was significantly decreased at 43.1 and 94.0%, respectively, compared to non-infested controls. Moreover, cells from mice and dogs submitted to one or three successive infestations did not exhibit a T-cell proliferative response to tick antigens, whilst cells from thrice tick-infested guinea pigs, when cultured with either a tick extract or tick saliva, displayed a significant increase in cell proliferation. Also, we evaluated the response of tick-infested mice to a cutaneous hypersensitivity test induced by a tick extract. Tick-infested mice developed a significant immediate reaction, whereby a 29.9% increase in the footpad thickness was observed. No delayed-type hypersensitivity (DTH) reaction was detected. Finally, the differential cell count at the tick attachment site in repeatedly infested mice exhibited a 6.6- and 4.1-fold increase in the percentage of eosinophils and neutrophils, respectively, compared to non-infested animals, while a decrease of 77.0–40.9 in the percentage of mononuclear cells was observed. The results of the cutaneous hypersensitivity test and the cellular counts at the tick feeding site for mice support the view that tick-infested mice develop an immune response to R. sanguineus ticks very similar to dogs, the natural host of this species of tick, but very different from guinea pigs (resistant host), which develop a DTH reaction in addition to a basophil and mononuclear cell infiltration at the tick-attachment site. In conclusion, saliva introduced during tick infestations reduces the ability of a susceptible animal host to respond to tick antigens that could stimulate a protective immune response. As a consequence, the animals present a lack of DTH response and disturbed cellular migration to tick feeding site, which can represent a deficient response against ticks.

Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis

A cDNA expression library prepared from mRNA of Haemaphysalis longicornis ( H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30 kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128–148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue.

Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis.

Antioxidant enzymes in eukaryotes play an important role in protection against the oxygen radicals generated during aerobic metabolism. Here we report the cloning and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin from the hard tick Haemaphysalis longicornis (HlPrx). HlPrx is 939 bp long and contains a 101 bp non-translated sequence at the 5' end and a polyadenylation singnal followed by a poly(A) tail at the 3' end. HlPrx encodes a full-length protein with a predicted molecular mass of 26 kDa that possesses one cysteine residue at amino acid 49 that is conserved among Prx proteins of various species. GenBanktrade mark analysis showed that the deduced amino acid sequence had significant similarity to mammalian and plant Prxs at the amino acid level. A DNA-nicking assay revealed that Escherichia coli-expressed recombinant HlPrx (rHlPrx) inhibited oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse antirHlPrx serum showed reaction with a major constituent protein spot in extracts of adult ticks. In addition, immunoblot analysis showed that rHlPrx was immunoreacted with serum from rabbits repeatedly infested with H. longicornis. Localization analysis using mouse antirHlPrx serum revealed that native HlPrx was highly expressed in the salivary gland of the tick. Moreover, Northern blot analysis showed that the level of HlPrx transcripts was increased during blood sucking. The present results indicate that HlPrx may be an important detoxifying enzyme during the normal life span as well as during blood sucking in ticks.

Characterization of an 84 kDa protein inducing an immediate hypersensitivity reaction in rabbits sensitized to Haemaphysalis longicornis ticks

An immunogenic 84 kDa protein was isolated and purified from whole tick extracts of Haemaphysalis longicornis larvae by a combination of ion exchange, reverse phase and hydrophobic interaction chromatographies. The protein, when injected intradermally into rabbits exposed to repeated tick feeding, induces an immediate cutaneous hypersensitivity reaction. It has been purified to homogeneity as shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. Amino acid sequences for two peptides derived from proteolytic cleavage of p84 were scanned against known proteins on the SWISS-PROT database. A 7 residue motif, ISGWGNT present in one of the two peptides appeared conserved in both vertebrate and invertebrate trypsin-like serine proteinases, while another 7 amino acid motif, HVPAGQI present in the second peptide showed homology to an Escherichia coli ATP-binding protein. We have discussed our findings in relation to isolation and characterization of target antigens for tick vaccine candidates.