Pergamon 0022-1910(94)00107-3

J. Insect Physiol. Vol. 41, No. 4, 369-375, pp. 1995 Copyright 0 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0022-1910195 $9.50 + 0.00

A Secreted Calreticulin Protein in Ixodid Tick (Amblyomma americanum) Saliva DEBORAH C. JAWORSKI,*t FRANK A. SIMMEN,$ WILLIAM LAMOREAUXg LEWIS B. COONS,g MARK T. MULLER,l GLEN R. NEEDHAM*

Received 21 Jury 1994; revised 19 August 1994

A complementary DNA clone from salivary glands of feeding female Amblyomma americanum ticks has been characterized as encoding caheticulin. Calreticulin, a major endoplasmic reticulum (ER) calcium-binding protein, appears to be secreted in Amblyomma and Dermacentor saliva. Evidence is accummulating that calreticulin performs roles unrelated to calcium storage. Unlike most known calreticulins, tick-secreted calreticulin lacks the ER retention signal, KDEL. This is the first molecular cloning of a specific tick salivary gland protein. The finding of a secreted calreticulin in tick saliva suggests a role for calreticulin in blood feeding through host immunosuppression or antihemostasis.

Calreticulin Tick Saliva Antihemostasis Immunosuppression

INTRODUCTION

Tick salivary secretions produced during a lengthy feeding period contain a variety of components that modulate events at the attachment site including the transmission of pathogens (Kaufman, 1989; Jones et al., 1989; Ribeiro, 1989a,b; Ribeiro, 1987). While some components of salivary secretions have been identified, none have been well characterized (Gordon and Allen, 1991; Ribeiro et al., 1990; Ribeiro et al., 1988; Ribeiro et al., 1985; Willadsen and Riding, 1979). The specific impact of any one component in the feeding lesion has been difficult to determine due to the small amounts of saliva available from the tick. Our purpose was to identify and clone specific salivary gland proteins that could be produced in a bacterial expression system. To do this, we performed immuno-screening of a salivary gland cDNA expression library prepared from fed lone star ticks. A polyspecific antiserum to 3-day feeding salivary gland extracts (A. americanum females) was used (Jaworski et al., 1990). We previously determined that this

*Acarology Laboratory, Department of Entomology, The Ohio State University, 484 W. 12th Avenue, Columbus, OH 43210, U.S.A.

TPresent address: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore, 655 W. Baltimore Street, Baltimore, MD 2/201, U.S.A.

$Department of Dairy and Poultry Sciences and Animal and Cell Biology Program, University of Florida, P.O. Box 110920, Gainesville, FL 32611, U.S.A.

§Center for Electron Microscopy, Department of Biology, University of Memphis, Memphis, TN 38152, U.S.A.

7Department of Molecular Genetics, The Ohio State University, 484 W. 12th Avenue, Columbus, OH 43210, U.S.A.

antiserum recognizes gland polypeptides that are feeding-specific. Here, we have cloned a specific salivary gland protein that is homologous to the calcium-binding protein, calreticulin. This protein appears to be secreted during the feeding process and is detected in dopamine/ theophylline-stimulated saliva.

MATERIALS AND METHODS

Ticks

Colony specimens of A. americanurn (lone star ticks) were reared as described by Patrick and Hair (1975). Immatures were fed on chickens and adults on rabbits or sheep. While off the host, ticks were confined in cartons within a Plexiglas chamber and in a relative humidity of 93 + 3% generated by a saturated KNO, solution. The temperature was maintained at 26 f 2°C with a 1ight:dark cycle of 14:lO. Female A. americanum ticks used for preparing cDNA were fed on the back of a Dorset ewe (1.5 years old) in the presence of males. Females were removed on the fourth day of infestation for dissection. Staggered feedings of ticks on the sheep over a 3-week period were necessary to obtain a sufficient quantity of glands for isolation of mRNA. In total, 550 females were dissected to yield 1100 glands.

Salivary gland preparation

During dissections, ticks were perfused with cold 0.1 M Tris-HCl buffer with 20 mM EGTA to minimize Ca2+-stimulated proteolysis (Mane et al., 1986), and

369

370 DEBORAH C.JAWORSKl efd.

a

1 / 1 ccc ACG AGC gly thr ser 61 / 21 CM CAT GCA glu asp ala 121 / 41 ACC CTG GTG thr leu val 181 / 61 GTG AAG CTC val lys leu 241 / 81 ATC ATG TTC ile met ph. 301 / 101 TAC AAG CCC tw lys gly 361 / 121 CAC CTG TAC his leu tyr 421 / 141 GTC CCC GAG val ala glu 481 / 161 GAC CCC GAG asp pro glu 541 / 181 GAC MC MC asp lys lys 601 / 201 CCC GAG GAC pro glu asp 661 / 221 GAG TAC MC glu tyr lys 721 / 241 GTG CAC CCC val his pro 781 / 261 GAG CTG TGC glu leu cys 841 / 281 CTG CTC ATC leu leu ile 901 / 301 CTC AAG GAC leu lys asp 961 / 321 AAG AAG GAG lys lys glu 1021 / 341 MC GAG MC lys glu lys 1081 / 361 GM CTG TGA glu leu OPA 1141 / 381 ttg ttt att

31 / 11 GAC GCT GAG AAG asp ala glu lys

91 / 31 TCC AAG TTC GAG ser lys phe glu

151 / 51 CAC GAG CAG MC his glu gln asn

211 / 71 CAG ACG CAG ATG gln thr gln met

271 / 91 ccc GGC ACC AAG pro gly thr lys

331 / 111 MC GAG ATC CCC lys glu ile l rg

391 / 131 GAC AAC ACG TAC asp asn thr tyr

451 / 151 GAC TGG TCC TTC asp trp ser phe

511 / 171 TGG GAC GAC CCC trp asp asp arg

571 / 191 CCC GAG TAC ATC pro glu tyr ile

631 / 211 CCC GAG TGG GAG gly glu trp glu

691 / 231 CAG ATC GAC MC gln ile asp l sn

751 / 251 TAC ACG CCC GAC tyr thr ala asp

811 / 271 TGG CAG GTG MC trp gln val lys

871 / 291 GCG CCC GTG CAC ala arg val his

931 / 311 GAG AAG CAG GAG glu lys gln glu

991 / 331 GAC GAG TTC GAA asp glu phe glu

1051 / 351 CCC CCC CCC GAG ala ala pro glu

1111 / 371 ggg act ctt ttt

1171 / 391 ttc tea tgt ttt

CCC GGCMG TTC TAC GGT ala @Y lYS phe tyr gly

AGC ser

CTG CAG ACC TCC leu gln thr ser

CCC TTC TAC GGC ATC TCC

-3 phe tyr !3lY lie ser

CTG CM; TTC ACC GTC MC val gln phe thr val lys

ccc pro

TTC TCC MCGAGGGCMG

phe ser asn glu gly lys

ATT GAC FCC GGCGGCGGCTAC ile asp cys gly gly gly tyr

GAC TGC asp cys

CTG GAC leu asp

TTC CAC CCC GAG TCC CCC TAC MG

his CllY glu ser pro tyr lys set

ccc CCC GAC ATC TGC CCC pro pro asp ile CYS glY

MG GTC CAC GTC ATT TTC MC lYS val his val ile phe asn

MC MC CAC CTC ATC MC

lYS l sn his leu ile l sn TGC MC GAC GAC GTG TTC ACC CYS lys asp asp val phe thr

ACG CTG ATC GTG MG ccc thr leu ile val lys pro

MC GGCCAC CTC GAG AGC lYS glY glu leu glu ser

GTG GTC MG val val lys

ATC GAC MC GAG

ile asp asn glu

CTG CCC CCC MC AX ATC AAG leu pro pro lys lys ile lys

GCC MGMG ccc GAG GAC ala lys lys pro glu asp

ccc GAG GAC TGG GAC MC pro glu asp trp asp lys

CCC MC ATC GAC GAC CCC GAG

ala lys ile asp asp pro glu

CCC GAC CCC GAC CCC ACC AAG

pro asp pro asp ala thr lys

GAC GAC GAC ATG GAC a8p asp asp met l 8p

ccc CCC CAG ATC AAC AAC CCC pro pro gln ile l 8n l sn pro

CCC

pro CCC TAC ala tyr

MC GGC CCC TGG lys gly ala trp

trp

GGT GAG TGG AM CCC MC

9lY glu trp lYS pro lys

GAG ATC GAC MC CCC GAG

glu ile asp l sn pro glu

ACC ATC GGC TTC GAC CTG thr ile gly Me asp leu

ccc pro

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MC CTG lyr leu

CCC ACC gly thr

TAC CAC TTC CCC tyr his phe pro

ATC TTC GAC MC ile phe asp asn

ACC GAC GAC GAG GAG TAC thr asp asp PlU glu tyr

GAG GAG ACG TGG CCC CCC glu glu thr trp ala ala

GAG

glu GAG MA glu lys

GAC CCC asp ala

MC

lYS

MC

lYS

ATG MG met lys

GAC GAG arp glu

GAG

glu GM GAG

glu glu GAC CCC MC AGC

arp ala ly8 ser

GAC GM GAG AAG GAA GAG GAC aw glu glu lys glu glu asp

GAC

asp

GM GAC GAG GAG ACC ACC

PlU asp glu PlU thr thr GAC GAC GAC CAC AX CAC GAG

asp asp asp his lys his glu

Q9sI tgg cca ccg ctg gtc gta eag gca tct ctc att ttt

tct ctc tea tee gw P+t ctc tee ccc cca ccc cca ccc

1201 / 401 1231 / 411 ccc att gtg gtq tag caa aga gaa atg gtt gat tta aaa aaa l aa aaa aaa

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372 DEBORAH C. JAWORSKI et al.

100 PM phenyl methane-sulfonyl fluoride (PMSF) and cl-Zmacro globulin (20 pl/ 100 ~1 tick buffer; carrier fixed; binds 13 nM protease/ml; Boehringer-Mannheim). Glands were stored at -20°C until enough glands were available for mRNA isolation.

RNA pur$kation

RNA was purified from 1100 tick salivary glands according to Pharmacia’s mRNA purification kit (Piscataway, NJ). To avoid losing any mRNA from the gland sample, only one passage over oligo (dT)-cellulose was done. Optical density (O.D.) of the sample was performed at A,,, to determine the amount of mRNA present in the sample.

Salivary gland cDNA expression library

A salivary gland cDNA expression library was prepared from the mRNA described above. This library was made with Uni-ZAP XR (Stratagene), an engineered bacteriophage vector (lambda gt 11 derivative). Uni-ZAP XR vector was double digested with EcoRI and Xho I, and salivary gland DNA inserts larger than 500 nucleotides were ligated into this site. With this vector the inserts were ligated in the 5’ to 3’ direction eliminating the possibility of inserts in the opposite direction (3’ to 5’). Therefore, the expression products had a greater probability of being present (one per three clones, rather than one per six). Uni-ZAP XR contains a pBluescript plasmid that can be easily excised from the bacteriophage. Additionally, pBluescript is ampicillin-resistant for colony selection and contains the Lac promoter for fusion protein production.

Immunological screening

Phage were plated out in E. coli XR Blue cells at 103-10’ pfu per 150 mm agar plate and lytic infection was induced at 42°C according to standard methods (Stratagene). Expression of /I-galactosidase_cDNA fusion proteins was then induced by isopropyl fi-D-thiogalactopyranoside (IPTG) and bacterial protein was transferred to nitrocellulose filters by the overlay technique of Young and Davis (1983). Antigen-positive clones were identified by reactions on duplicate nitrocellulose filters with anti- serum followed by iodinated Protein A (ICN). Antiserum was preabsorbed with lysates of XR Blue E. coli (Stratagene) to reduce binding to the E. coliportion of the fusion protein. Antiserum to partially feeding glands was used to screen the library. Positive clones were detected by autoradiography at - 70°C overnight. Clones reacting on duplicate lifts were picked and rescreened at a lower density. Finally, these clones were purified a third time by the method described above. Phage stocks for positive clones were made (plaque core with 500 ~1 SM media and 20 ~1 chloroform) and were stored at 4°C.

Plasmid DNA preparation

Plasmid stocks were made from these phage particles by in vivo excision using helper phage (Stratagene). Plasmid

stocks were stored in glycerol at - 20°C according to the procedure of Maniatis et al. (1982). Minipreparations were done using the alkaline-lysis method (Maniatis et al., 1982). On the basis of fusion protein analysis described below and minipreparations of plasmid DNA from positive clones, plasmid 161A (p161A) was chosen for the experiments to follow and a rudimentary map of this clone was made.

Fusion protein analysis

Fusion protein extracts of each positive clone were prepared. Briefly, cells with plasmid were grown overnight at 37°C then IPTG was added to induce protein expression. Bluescript plasmid without an insert was used as a control. Cells were lysed to release protein, incubated on ice for 10 min and Triton X-100 was added to a final concentration of 0.1%. This solution was incubated on ice for 10 min, centrifuged for 1 h at 20 K and 1 ml of supernatant was added to 2 x Laemmli sample buffer. Samples were boiled for 2 min and loaded on 7.5% SDS-polyacrylamide gels, Gels were immunoblotted and probed with anti-p-galac- tosidase monoclonal antibody (from Promega) and anti-feeding gland antiserum (antiserum from original immunoscreening).

Fusion protein was cut from nitrocellulose transfer of an entire gel and used to immunize a New Zealand White (NZW) rabbit. The nitrocellulose strip containing fusion protein was minced with complete Freund’s adjuvant and injected with an 18-gauge needle subcutaneously. Nitrocellulose minced with incomplete Freund’s was used to boost the animal with the fusion protein 2 weeks later. Blood was drawn from the rabbit, allowed to clot to separate serum, centrifuged to remove any remaining red blood cells (5 min at 1000 rpm), and frozen in aliquots at -20°C for subsequent immunoblotting. Immunoblots of tick salivary glands and midgut tissues taken at unfed (0), 1, 2, 3, and 6 days using the anti-fusion protein antibody (anti-161A) were performed as previously described (Jaworski et al., 1990).

DNA sequencing

Dideoxysequencing of plasmid 161A was done using standard methods (Sanger et al., 1977) provided with the Sequenase Version 2.0 kit (United States Biochemical, Cleveland, OH). Sequence data were analyzed using the National Institutes of Health Genbank Database accessed through the National Center for Biotechnology Information (NCBI) and the BLAST program (Altschul et al., 1990). The sequence for p161A has been deposited at NCBI, accession number UO7708.

Protein ident@cation

We hypothesized that the tick salivary gland fusion protein was likely to be a tick calreticulin based on preliminary sequence data. To test this, we obtained antibody to rabbit calreticulin from Dr Marek Michalak, Faculty of Medicine, University of Alberta, Canada.

CALRETICULIN IN TICK SALIVA 373

Immunoblots were performed with this antibody using the avidin-biotin system of labeling (Vectastain ABC, Vector Laboratories, Ingold, CA).

Art$cially induced saliva

Saliva was collected from ticks weighing between 64 and 75 mg by four 10 ~1 injections of 1 mM dopamine, 1 mM theophylline and 3% DMSO in tick saline/MOPS (pH 7.0) at 15-min intervals (Needham and Sauer, 1979). Saliva was immediately processed in SDS sample buffer for gel electrophoresis.

RESULTS

Clone 161A was identified using the antiserum described above, and subsequently, subcloned and sequenced [Fig.l(a)]. Amino acid sequence is shown in Fig. 1 (a) and is compared to other calreticulins in Fig. 1 (b).

We used immunoblots to confirm that 161A is calreticulin,andtodeterminewhetheritisasecretedprotein in ticks. Authentic antibody to purified rabbit calreticulin reacts with a 58 kDa protein in tick salivary glands and a 64 kDa polypeptide fusion protein from clone 161A

[Fig.2(a)]. Calreticulin was identified in the saliva and migrated to a similar position as purified calreticulin in side-by-side gel lanes [Fig.2(b)]. Calreticulin was present in the saliva of both A. americanurn and D. variabilis

[Fig.2(c)]. To characterize further the calreticulin in the salivary

glands during tick feeding, we first prepared an antibody to tick calreticulin. The fusion protein was immobilized on nitrocellulose membrane, excised, and directly implanted into a rabbit [Fig.3(a)]. The resulting polyclonal antibody detects a single band in our fusion protein extract [Fig.3(b), lane 61, and in purified rabbit skeletal muscle calreticulin (not shown). Calreticulin was evident in salivary glands during feeding [Fig.3(b), lanes 2-51, but was in low abundance in non-feeding females [Fig.3(b), lane 11. When ticks were fed on rabbits immunized with tick calreticulin-fusion protein, the feeding sites were necrotic and hemorrhagic with visible cavities in the epidermis (not shown).

DISCUSSION

Clone 16 1 A is over 69% homologous to rabbit skeletal muscle calreticulin (Fliegel et al., 1989). These two

1234

a-CRT a-CRT a-CRT FIGURE 2. Analysis of calreticulin in tick glands and saliva using Western blotting. Samples were prepared as indicated below,

analyzed by SDSpolyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (Schleicher & Schuell Inc.) by electroblotting. The blot was probed with antibody to rabbit skeletal muscle calreticulin (anti-CRT, from M. Michalak) and

reactions were visualized using an avidin-biotin system and peroxidase labeling (Vectastain Inc.). The arrow and number indicate

the location and molecular mass in kilodaltons of the tick fusion protein (161A). Positions of corresponding molecular markers

are indicated in the figure in kilodaltons (kDa). Approximately 10 pg of protein was loaded per lane. (a)Calreticulin identified

from tick fusion protein and tick salivary glands. Lane 1, salivary glands dissected from 4-day fed female ticks. Lane 2, fusion

proteins (after IPTG induction) prepared from lysogens of pBluescript containing no insert. Lane 3, fusion proteins (after IPTG induction) prepared from lysogens of pBluescript containing tick cDNA 161A. The tick fusion protein is a chimeric protein

containing a small portion of the /I-galactosidase protein in addition to the tick protein. This accounts for the observed difference

in molecular weights for calreticulin in salivary glands and fusion protein. (b)Calreticulin identified in tick saliva. Lane 1, glutathione-S-transferase-calreticulin fusion protein (from M. Michalak). Lane 2, purified calreticulin (from M. Michalak)

prepared without 2-mercaptoethanol. Lane 3, tick saliva from 6-day fed A. americanurn females. Proteins from tick saliva were

prepared from the collection of dopamine (1 mM) plus theophylline (1 mM)-stimulated saliva of female lone star ticks that had fed for 6 days. Lane 4, purified calreticulin prepared with 2-mercaptoethanol. Lane 5, extracts of salivary glands from 6-day fed

females. (c)Calreticulin identified in tick saliva of two ixodid tick species, the lone star tick (Amb&vwnu americunum) and the

American dog tick (Dermacentor nariubilis). Saliva proteins were prepared from the collection of dopamine (I mM) plus

theophylline (1 mM)-stimulated saliva from female ticks that had fed on rabbits for 4 days. Gland extracts were prepared from females that had fed for 4 days. Lane 1, purified calreticulin migrating to 45 kDa. Please note, calreticulin migrates to different

positions in SDS-PAGE gels depending on the amount ofcalcium it binds. In our hands, purified calreticulin degraded to smaller subunits after boiling. Lane 2, saliva from D. variabilis. Lane 3, saliva from A. americanurn. Lane 4, salivary gland extracts from

D. variu6ilis females.

314 DEBORAH C. JAWORSKI et al.

a b 1 2 3 4

92.5

69

45

30

,64

69 4 .64

30 a-p -Gall a -FedSEG a-161A

FIGURE 3. Analysis of fusion protein 161A. (a)Characterization of fusion protein from cDNA clone 161A by Western blotting.

Fusion proteins (after IPTG induction) were prepared from lysogens of pBluescript containing tick cDNA 161A (lanes 1 and 3)

and pBluescript containing no insert (lanes 2 and 4). Samples were loaded onto SDS-polyacrylamide gel. The polypeptides were

transferred to a nitrocellulose membrane (Schleicher & Schuell Inc.) by electroblotting. The blot was divided in half and lanes

1 and 2 were probed with a monoclonal antibody directed against /3-galactosidase (anti-b-GAL, Boehringer-Mannheim Corp.),

while lanes 3 and 4 were probed with a rabbit polyclonal antisera prepared against salivary glands from feeding ticks

(anti-FedSEG), followed by 9 Protein A. Extra bands visible in lanes 3 and 4 are the result of non-specific antibody binding

to E. coli proteins released from lysogens. (b)Appearance of calreticulin in salivary glands of female ticks shown by Western

blotting. Salivary glands of female ticks and were dissected at intervals of non-feeding through 6 days of feeding, prepared for

electrophoresis, and transferred to a nitrocellulose membrane (Schleicher & Schuell Inc.) by electroblotting. Protein 16lA was

detected using a rabbit polyclonal antibody described in text. Lane 1, unfed salivary glands. Lane 2, salivary glands at 24 h. Lane

3, glands at 2 days. Lane 4, salivary glands at 3 days. Lane 5, salivary glands dissected at day 6. Lane 6, tick fusion protein prepared

as indicated above. The tick fusion protein is a chimeric protein containing a small portion of the j-galactosidase protein in

addition to the tick protein. This accounts for the observed difference in molecular weights for calreticulin in salivary glands and fusion protein. The arrow and number indicate location and molecular mass (kDa) of the tick fusion protein (161A). Positions

of corresponding molecular weight markers are indicated on the figure in kilodaltons. Approximately 10 pg of protein was loaded

per lane.

sequences are more than 80% homologous in the amino-terminal domain; and 41% homologous in the carboxy-terminal region. Molecular mass for tick calreticulin was 58 kDa and within the range of reported values for calreticulins, 55-63 kDa, as measured by migration in SDS-polyacrylamide gels (Michalak et al., 1992). Calreticulin was identified in the saliva of both Amblyomma and Dermacentor females. The finding of a secreted calreticulin is novel, and its presence is not limited to one species or genus of tick.

Calreticulin was most evident in the salivary glands of ticks fed 3 and 6 days and it is recovered from pharmacologically-induced saliva. Rabbits immunized with tick calreticulin-fusion protein exhibited necrotic feeding lesions where ticks were allowed to feed. This local host response was very different from that of unimmunized hosts where overt bite-site reaction is usually not observed. It appears that antibody to the tick calreticulin-fusion protein binds calreticulin thereby initiating a localized immune reaction to tick infestation. The immune reaction may disrupt the feeding cycle. Collectively, these data indicate that calreticulin is synthesized and secreted into the host during the tick feeding interval.

A major difference between 161A and other calreticulins is a lack of the ER retention signal,

KDEL-161A ends with HEEL. Other parasite calreti- culins contain a variant of KDEL (HDEL, Schistosoma mansoni, Khalife et al., 1993) or no sequence (Onchocerca volvulus, Unnasch et al., 1988). The Onchocerca volvulus calreticulin (RAL-1) has been localized to the surface of the infective larvae, which was not expected for an ER-resident protein (R. Tuan and T.R. Unnasch, personal communication). Two putative calcium-binding regions have been identified in rabbit skeletal muscle calreticulin (Michalak et al., 1992). The first is located in the P-domain and is retained in tick calreticulin. The second region, found in the C-terminal domain, is divergent for tick calreticulin; however, acidic residues predominate and calcium-binding could be retained. On the basis of our screening with immune host sera and the missing KDEL sequence, we suggest that 16 1 A represents a calreticulin that is routed through a secretory pathway rather than being retained in the ER.

Calreticulin appears to be a multifunctional protein (Michalak et al., 1992). Recently, its role in binding nuclear receptor sites has been shown (Burns et al., 1994; Dedhar et al., 1994). A non-secreted form of calreticulin should exist in tick cells. However, our data point clearly to secreted calreticulin, which suggests a novel role for this protein. What is the role for a secreted calreticulin during tick feeding? Arthropod blood feeding has been linked to

CALRECTICULININ IN TICK SALIVA 375

ADP-degrading enzymes like apyrase (Ribeiro et al., 1985) which require calcium as a cofactor. Calcium in the lesion could activate such enzymes impairing platelet function and augmenting bleeding (Mustard and Packman, 1977). Calreticulin has been shown to have antithrombotic activity; and it is involved in the nitric oxide pathway (Benedict et al., 1993). Calreticulin in saliva could serve to increase nitric oxide in the feeding lesion leading to increased vascular permeability. Calreticulin may also be unrecognized by the host as a foreign antigen. Shared antigens exist between endopara- sites and hosts (Braun et al., 1991; Clegg et al., 1971). However, the possibility of a shared antigen between a tick and its vertebrate host has not been investigated. This would be a major adaptation favoring parasite success. More detailed in vivo and in vitro experiments should clarify the role of calreticulin in tick feeding.

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Acknowledgements~This project was supported by a grant from The

Ohio State University Office of Research to GRN. Goat anti-rabbit

calreticulin antibody and purified rabbit skeletal muscle calreticulin and GST-fusion protein were the generous gifts of Dr Marek Michalak,

Cardiovascular Disease Research Group, Faculty of Medicine,

University of Alberta, Edmonton, Alberta, Canada T6G 252. Lone star

saliva was provided by Dr John R. Sauer, Department of Entomology,

Oklahoma State University, Stillwater, OK 74078, U.S.A.