Parasite Immunology 1991, 13,605-6 16

Vaccination with ‘concealed’ antigens: myth or reality? P.WILLADSEN & R.V.McKENNA CSIRO Division of Tropical Animal Production, Private Bag 3, Indooroopilly, Queensland, Australia 4068 Accepted for publication, 9 May 1991 Summary Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be ‘concealed‘ from the host’s immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms.

Keywords: Boophihis microplus, tick, antigen, ‘concealed’ antigen, glycoprotein. Western blotting, ELISA

Introduction

It is now well established that cattle can be vaccinated against the tick Boophilus microplus using material from tick gut (Johnston et al. 1986, Kemp et al. 1986, Willadsen & Kemp 1988, Opdebeeck et al. 1988). Ingestion of the blood meal by ticks feeding on vaccinated cattle leads, of necessity, to the uptake of antibodies and other components of the hosts’ immune system, resulting in damage to the gut. As a result, the number of ticks engorging, their average weight and their ability to lay eggs may all be adversely affected. Although Boophilus microplus is the best established example of this approach to immunological control, it is by no means unique. Similar effects appear to be obtained, for example, with other ticks (Wikel 1988), lice (Ben-Yakir & Mumcuoglu 1988), flies (Schlein & Lewis 1976) and mosquitoes. Thus there appears to be encouraging evidence Correspondence: P. Willadsen.

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that this approach to immunological control of blood-feeding ectoparasites may be applicable in a variety of systems. We have also considered it valuable to distinguish this approach to immunological control from the more traditional one of mimicking naturally-acquired immunity to the ectoparasites. Typically, for ticks, such immunity is attributed to immunological reaction with salivary gland antigens or components of tick attachment cement, that is, with those components of the tick which the host experiences during normal infestation (Willadsen 1980). For vaccination against internal organs such as the gut, we have suggested the term vaccination against ‘concealed antigens’ (Willadsen & Kemp 1988). The validity of the idea of ‘concealed antigens’ has recently been called into question (Brown 1988, Opdebeeck & Daly 1990). Experimentally, the reason for this is that cattle, whether vaccinated with adult gut material or simply infested with ticks, possess antibodies that react with a variety of tick tissues and subcellular fractions, including membrane material from adult tick salivary glands, adult guts and larvae. The assertion is therefore that ‘tick gut membranes are therefore not so-called “concealed” antigens’ (Opdebeeck & Daly 1990). We would regard this as an oversimplified interpretation of the antigenic nature of tick proteins and of the relationship between the production of antibody and immunity to the parasite. It is more meaningful to ask a series of three related questions. Firstly, do cattle on exposure to Boophilus microplus produce antibody to proteins that one would not intuitively expect to be involved in a normal host-parasite interaction? Secondly, are the antibodies monospecific for these proteins or could the immunoreactivity observed be simply the result of a fortuitous immunological cross-reactivity? Thirdly, do the antibodies produced in this way contribute to immunity? We have now examined these questions with respect to a heterogeneous mixture of intrinsic -membrane glycoproteins as well as to a defined tick gut antigen, the Bm86 antigen. This antigen is available not only in native form but also in the form of effective recombinant antigens (Willadsen er al., 1991, in press). Materials and methods P R E P A R A T I O N OF A N T I G E N S

Native Bm86 was obtained from preparations of membrane glycoproteins. These were isolated from detergent extracts of semi-engorged adult female Boophilus microplus as described previously (Willadsen et al. 1988, Willadsen et al. 1989). To isolate Em86 from this material, the glycoproteins were passed down columns of affinity purified antibody to a recombinant form of Bm86 (Courtesy of D. Smith, Biotech Australia). The antigen was essentially pure after elution from the affinity columns, but was rechromatographed on a Mono Q ion exchange column to ensure homogeneity. Purity was checked by SDS gel electrophoresis and silver staining (Willadsen et al. 1988). A recombinant form of Bm86, rec-Bm86, was produced in E. coliand contained 640 of the 650 amino acids of the mature, native tick protein. It was a gift from Biotech Australia as was another recombinant form of the same antigen, produced in a eukaryotic expression system. The glycoprotein mixtures referred to as intrinsic membrane glycoproteins (IMG)

-

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were prepared from adult tick membrane material (Willadsen 1988). The membrane material was extracted in Triton X-I14 essentially as described by Pryde & Phillips (1986) and the detergent rich fraction isolated after phase separation. The glycoproteins used in ELlSA assays were obtained from this mixture by sequential chromatography on both lentil lectin and wheat germ lectin affinity columns as described previously (Willadsen et d.1989). Subfractions from glycoprotein isolates were produced by chromatography on Mono Q columns in 0.05 M Tris chloride pH 7.50.1 '%,(w/v) N-tetradecyl-N,N-dimethyl3-ammonio- I -propane sulphonate (Boehringer, Mannheim) using gradients in sodium chloride. Low molecular weight, tick-derived carbohycirate was prepared from a mixture of glycoproteins isolated by chromatography on either wheat germ lectin or lentil lectin. The mixture, at 0.38 mg/ml. was incubated at pH 7.3 in 5 mM DTT with 0.5 mg/ml trypsin and 0.6 mg/ml chymotrypsin for 22 h, then with 1 mg/ml pronase for44 h. It was finally heated in a boiling water bath for 10 min. No residual glycoprotein was detectable on a silver stained SDS gel after this treatment, but as a further precaution, the digest was passed through a 10 kD cutoff filter (Centricon 10, Amicon, Danvers, MA) to remove any high molecular weight material, including residual proteolytic enzymes. ANTISERA

Sera obtained from cattle before and after tick infestation as well as sera from cattle vaccinated with native or recombinant Bm86 were from 12 to 18 month old female Hereford (Bos tourus) animals raised in a tick free area of New South Wales. Field antisera from cattle with extended tick exposure were again from Hereford animals, aged 18-24 months, raised in a tick-infested area of central Queensland. Bovine antisera to glycoprotein fractions and Bm86 were produced as described previously (Willadsen eta/. 1989). Rabbit antisera to both native and recombinant Bm86 were raised with an initial injection in Freund's complete adjuvant, followed by booster injections in Freund's incomplete adjuvant. ELlSA AND WESTERN BLOTTING PROTOCOLS

Antibody concentrations were estimated using a direct, non-competitive ELISA. Antigens were diluted in carbonate/bicarbonate buffer pH 9.6 to a final concentration of I pg/ml (recombinant antigens) or 0.2 pg/ml (glycoprotein antigens). Aliquots of 200 p1 were added to each well of a microtitre plate. Following overnight incubation at 4 C. plates were washed with PBS containing0.05'!4 (w/v) Tween 20. The same buffer was used for subsequent coupling and washing steps. Primary antibody was diluted in PBS-Tween 20 and allowed to react for 1 h at 37' C. For the assays with bovine sera, this was followed by incubation with a 1 :500 dilution of 0.5 mg/ml peroxidase labelled goat anti-bovine IgG ( H + L ) (Kirkegaard & Perry, Gaithersburg, MD). For assays of rabbit antisera, reaction with the primary antiserum was followed by a 45 min reaction with a I : 1200 dilution of biotinylated donkey anti-rabbit IgG (Amersham International, UK), then 45 min with 1 : 1200 peroxidase-coupled streptavidin (Amersham International, U K). Peroxidase activity was measured with I mg/ml 5-aminosalicylic acid and 1.7 mM hydrogen peroxide in 20 mM phosphate buffer pH 6.7. Absorbance values were measured using a Titertek Multiscan Plus Mk I1 ELISA reader interfaced with an IBM compatible

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personal computer. Data were processed using the Kinetic Linked Immunosorbent Assay (KELA) essentially as described previously (Barlough et al. 1983) to yield a kinetic estimate of the peroxidase activity. ELISA reactions were analysed using two parameters (Malvano et al. 1982). For the screening of antisera, the KELA rate at a single antiserum dilution was used. For a more accurate reflection of relative antibody concentration, antisera were serially diluted and the reciprocal of the serum dilution giving a KELA rate of 0.05 Abslmin obtained by regression analysis. Western blots were obtained from antigen preparations separated by SDS pofyacrylamide gel electrophoresis on 4% to 1 8Y0 gradient gels and transferred to nitrocellulose in an LKB Multiphor I1 Novablot semi-dry system according to the manufacturer’s instructions, using a transfer buffer containing 0.025 M ethanolamine, 0.04 M glycine and 20% (v/v) methanol. After blocking in 2% gelatin in Tris buffered saline, the blots were developed using either bovine or rabbit sera essentially as described for the ELISAs. Peroxidase label was detected with a solution of4 mg luminol, 10 mg iodophenol and 50 p1 of 30% hydrogen peroxide in 100 ml Tris buffered saline using Kodak X-Omat film.

Results A N T I B O D Y TO I N T R I N S I C MEMBRANE GLYCOPROTEINS ( I M G )

To investigate the production of antibodies to IMG following tick infestation, cattle were chosen for which sera were available both before tick infestation and following infestation with approximately 20 000 Boophilus microplus over six weeks. Seventeen pairs of such sera were tested at a dilution of 1 in 100 in an ELISA assay, using IMG as the antigen. In all sera, there was an increase in ELISA reaction following tick exposure. The mean KELA rate pre-tick exposure was 0.073+0.023, while after tick exposure it was 0.26 0.084. The difference was highly significant (t = 7.28; P < 0.001). ANTIBODY PRODUCED TO

BM86

The same 17 pairs of antisera as above, collected before and after tick exposure, were reacted in an ELISA at a 1 in 100 dilution with rec-Bm86 tick gut antigen. This antigen was produced in Escherichia coli and contained 98% of the amino acid sequence of the antigen Bm86. The activity of both native Bm86 and rec-Bm86 as protective antigens has been reported previously (Willadsen et al. 1989, Willadsen et al. in press). Here, the KELA rates before tick exposure had a mean value of 0.043f0.018, while after tick exposure the rates were 0.054 +0.022. Although this difference in means is not significant ( t = 1.533, P 0.1). Thus extended exposure to ticks did not increase the amount of antibody reacting with the recombinant antigen. Nevertheless, the increase in reaction with the rec-Bm86 antigen after first tick exposure appeared to be real, if small. To estimate the significance of the apparent increase in ELISA reactions, 12 pairs of sera were selected for further study. Thkse included the nine showing the greatest increase in ELISA reaction with recombinant antigen after tick exposure. The same group included the sera showing the strongest

Figure 1. Western blots against native Bm86, using bovine sera before (B) and after (A) exposure to 20 000 ticks. Sera (V) were from cattle vaccinated with 0.007 mg of native Bm86 as positive controls.

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reaction with the glycoprotein antigens. To this group were added three sera showing a weak reaction with glycoprotein antigen in ELISAs. These sera were then titrated in two-fold serial dilutions against three antigens: the IMG antigen, rec-Bm86 and an irrelevant, non-tick recombinant produced in E. coli. Significanceof titres was measured using a paired t- statistic for sera before and after tick exposure. Titres against rec-Bm86 increased significantly after tick exposure ( t = 5.59, df= I 1, P < 0.001) though again, the biological significance of this is questionable. Although 9 of the 12 sera had been chosen to show the largest increase in titre to this antigen, the mean increase in titre for the group was still only 3.8-fold. More importantly, this small increase was also non-specific. The effect was shown also by ‘antibody’ to the non-tick recombinant. The titres of the pre-tick sera to both recombinants were not significantly different (t=0.53, P > 0-5) and neither were they different after tick exposure ( t = 0.82, P > 0.1). In fact, the titres against the irrelevant recombinant antigen increased slightly more than did those against the tick recombinant (4.2-fold cf. 3.8-fold). Further, it appeared that the effect was not specific to E. coli recombinant proteins. Similar behaviour was seen in ELISA assays on wells coated with horse serum at a concentration of 200 p g per well. At a serum dilution of 1 in 25 or 1 in 100, there were increases in KELA rate in the antisera after tick exposure, though the reactions were too low to estimate an antibody titre accurately. In summary, there is no evidence from the ELISA assays that cattle produce detectable antibody to the rec-Bm86 antigen after tick infestation. By comparison, the reactions with glycoprotein were significant. For the group of 12 sera, the geometric mean titres increased from 90 prior to tick exposure to 3540 after exposure. Sera from 6 cattle which developed high titre antibody to membrane glycoprotein after tick exposure were used in immunoblots against native Bm86. The results are shown in Figure I , with sera from two animals vaccinated with the antigen for comparison. Four of the six cattle exposed to ticks in a natural infestation produced detectable antibody to the native Bm86, though none of the six produced detectable antibody to rec-Bm86. ANTIBODY PRODUCED TO CARBOHYDRATE EPITOPES

To test the possibility that much of the antibody to glycoproteins produced by tickexposed cattle was due to carbohydrate rather than polypeptide epitopes, several sera giving the highest titres against IMG were re-assayed against IMG in the presence and absence of low molecular weight tick carbohydrate. This was produced, as described in Materials and methods, by exhaustive proteolytic digestion of tick membrane glycoproteins, followed by ultrafiltration. It was added at the same time as primary antibody to ELISA assays, at a concentration equivalent to 50 pg/ml of the original glycoprotein. As is shown in Figure 2, this produced a significant reduction in ELISA titre. To show that the effect was not due to a non-specific interaction or the presence of residual proteinases, parallel assays were carried out using rec-Bm86 as antigen and sera from cattle vaccinated with rec-Bm86. There was no effect of carbohydrate in these assays (Figure 2). Evidence that a number of tick glycoproteins share carbohydrate epitopes is shown in Figure 3. Native Bm86, IMG and five other mixtures of membrane glycoproteins were reacted in immunoblots with rabbit antisera to: rec-Bm86 (A and B), eukaryotic expressed Bm86 ( C )and native Bm86 (D and E). The antisera to the recombinants reacted specifically with native Bm86 in these samples, while the antiserum to affinity purified

Vaccination with ‘concealed’antigens IMG Animal 101

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Figure 2. Effect of tick carbohydrate (CHO) on the KELA rates against IMG using sera from three tick exposed cattle (54,94 and 101) -0-, Pre-tick KELA rate; -0-, Pre-tick KELA rate with CHO; -=-, Post-tick KELA rate; -0-, Post-tick KELA rate with CHO. Effect of CHO on sera from two cattle (436, 437) vaccinated with rec-Bm86. -0-, 436 KELA rate; 436 -0-, 436 437 KELA rate; -0-, 437 KELA rate with CHO. KELA rate with CHO; -B-,

native Bm86 showed extensive cross-reactivity to other glycoproteins. The effect was not due to differences in antibody titre to the protein portion of the Bm86 antigen. Titres against rec-Bm86 of the two antisera to non-glycosylated recombinants, eukaryotic recombinant and native protein were 290000, 86 000, 98 000 and 69000 respectively. Similar results were obtained with other rabbit antisera and bovine antisera to native and recombinant Bm86. PROTECTIVE EFFECT OF ANTI-CARBOHYDRATE EPITOPES

Although the efficacy of recombinant antigens in inducing protection against the tick (Willadsen er al., 199I , in press) shows that carbohydrate is not essential for the protective effect of native Bm86, such results leave open the question of whether the carbohydrate could have some protective function. During the identification of protective Boophilus microplus antigens, many tick glycoproteins have been tested as potential antigens but have shown no protective effect at all (Willadsen et al. 1988). Antisera from some cattle vaccinated with these non-protective mixtures of glycoprotein antigens reacted strongly with native Bm86. Table 1 shows a comparison of the ELISA rates for sera from three groups of cattle against two antigens. The antigens were the native and the recombinant Bm86. The cattle groups were (a) non-vaccinated control cattle (b) cattle vaccinated with heterogeneous mixtures of glycoproteins that failed to protect against tick infestation but whose sera reacted strongly with native Bm86 and (c) cattle vaccinated with purified native Bm86. As the results show, cattle that did not produce antibody to the polypeptide portion of the Bm86 molecule, as measured by the reaction with rec-Bm86, were not protected despite the high antibody titre to the native Bm86 molecule.

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Figure 3. Western blots (A-E) against native Bm86 (1) and various mixtures of tick membrane glycoproteins (2-7). The amounts of protein per lane were 5.5, 440, 86, 210, 28 and 116 ng respectively. Rabbit antisera were raised against rec-Bm86 (A and B), recombinant Bm86 from a eukaryotic expression system (C) and native Bm86 (E). D shows a pre-vaccination control serum from rabbit E. The figure also shows a silver stain of the same samples at four-fold higher amounts. The right hand lane contains the Pharmacia molecular weight standards.

Discussion At first sight, some of the data presented above support the contention that antigens located in the gut of Boophilus microplus are exposed t o the host during a normal tick

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Table 1. Kinetic ELISA of protected and non-protected, vaccinated cattle with two antigens ~~

Number of cattle rec-Bm86 native Bm86

~

~~

~~

Control

Non-protected

Protected

12 0~046+0-011 0.025 k0.0 15

13 0.049t0.018 0.203 0.049

7 0.206_+0.104 0.191 0.126

The results are the means and standard deviations of kinetic ELISA rates. Assays were with bovine sera diluted 1 in 1000. Control cattle had been vaccinated only with adjuvant. Non-protected cattle were vaccinated with mixtures of glycoproteins but failed to show any difference from control cattle on first infestation with ticks. Protected cattle were vaccinated with native Bm86.

infestation and that they stimulate an immunological response. The ELISA assays establish that cattle exposed to ticks develop antibodies that react with intrinsic membrane glycoproteins. This is consistent with the findings of Opdebeeck & Daly (1990) and it is, superficially, a surprising finding. One would not expect such membrane proteins to be secreted into the host. The question whether there is immunological contact between the host and proteins of the tick gut during a natural infestation is better explored by using a defined antigen rather than a complex and heterogeneous mixture. For this purpose, we have used the Bm86 antigen, a tick gut antigen of known protective activity which is, moreover, available both as a native tick protein and as recombinants (Willadsen & Kemp 1988, Willadsen et al. 1989, Rand et al. 1989, Willadsen et al., 1991, in press). ELISA assays show that there is no evidence at all that cattle exposed to ticks under natural conditions for either a relatively short or extended time develop detectable antibody to the recombinant, non-glycosylated antigen, rec-Bm86. The very small increase in ELISA reaction shown by sera after tick infestation is at least 1000-fold less than the antibody titres produced by vaccination with microgram amounts of the native antigen. Moreover, this increase in reaction is non-specific, since it also occurs to an unrelated, non-tick recombinant protein. Probably it relates, at least in part, to the increase in total IgG levels reported for other tick species after tick infestation (Rechav 1987). In apparent contradiction to these observations, some sera from cattle naturally exposed to ticks do recognize native Bm86 of Western blots. The conclusion must be that the native Bm86 carries epitopes that are not present on the recombinant protein. In theory, these unique epitopes could be either polypeptide or carbohydrate. Several facts make the first of these possibilities very unlikely. There is a high level of cross-reactivity between native Bm86 and rec-Bm86. Vaccination with the native antigen results in hightitre antibodies against rec-Bm86, while vaccination with rec-Bm86 induces anti body that reacts strongly with native Bm86. In fact rec-Bm86 can be as effective in producing immunity as the native Bm86 (Willadsen et al., 1991, in press). How then can we explain

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the lack of antibody to rec-Bm86 but the existence of antibody to native Bm86 in tickexposed cattle? Could it be due to a unique polypeptide epitope in the native protein? This is an unlikely explanation, since cattle would have had to respond to this unique epitope on tick infestation but not to any of the cross-reactive epitopes. More probably, tick-exposed cattle produce antibody to carbohydrate epitopes. That this occurs is shown by the fact that the reaction of sera from such animals with glycoprotein is partially inhibited by low molecular weight tick carbohydrate. It is not surprising that the antibody binding is not totally inhibited in view of the low concentration and low molecular weight of the blocking agent. The probable reason for the reaction of bovine sera from tick infested cattle with native Bm86 is shown by the fact that, while rabbit antisera raised to rec-Bm86 or a eukaryotic recombinant of the same protein are monospecific when tested, against mixtures of tick glycoproteins, rabbit antisera to the native Bm86 react widely with other glycoproteins. The obvious explanation for this is that tick glycoproteins share crossreactive carbohydrate epitopes. It is easy to conceive that cattle would produce antibody to carbohydrate epitopes after tick infestation since the attachment cement, for example, is partially carbohydrate (Stone, Binnington & Court 1977). In view of such crossreactivity however, the existence of antibody in tick exposed cattle which binds to native Bm86 is not an indication that the animal has been exposed to this antigen. The only reliable evidence, the experiments with rec-Bm86, suggest strongly that such exposure does not occur. This does not mean that natural exposure of a host to gut antigens cannot occur, but it does demonstrate that the minimum evidence for such exposure would be the production of monospecific antibody by the host. In more general terms, the results demonstrate that neither positive ELISA titres nor positive immunoblots are conclusive evidence that a host has been exposed to a particular parasite antigen, at least if it is a glycoprotein. Though apparently trivial, this is a fact seemingly too often forgotten. The important practical question is whether such anti-carbohydrate antibodies are protective in the Boophilus microplus-bovine host system. The fact that recombinant antigen produced in E. coli is protective a t least shows that the carbohydrate determinants in Bm86 are not essential for protection (Willadsen et ai. in press). In addition, it has been shown that some cattle vaccinated with heterogeneous mixtures of tick glycoproteins may produce high titre antibodies to the native Bm86 but not to the recombinant version of the same antigen. This can best be explained by production of antibody to the cross-reactive carbohydrate determinants already shown to be present in this antigen. The fact that such cattle are not protected against tick infestation is evidence that antibody to carbohydrate determinants of this antigen is not protective. Finally, is immunity to 'concealed antigens' a reality or not? As yet, there is no evidence against the validity of the concept. The strongest evidence for two independent mechanisms of immunity in the case of Boophilus micropius is the fact that ticks from vaccinated cattle show types of damage that are not seen in ticks maturing on cattle with naturally acquired, immunologically mediated resistance (Kemp et al. 1986). Supporting evidence is supplied by the fact that the immunological mechanisms, as far as they are currently understood, appear to be different (Willadsen, 1987).There is thus considerable evidence that the principle offers a useful approach to the induction of a novel form of immunity against ectoparasitic arthropods.

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Acknowledgements T h e a u t h o r s a r e indebted t o Biotech Australia f o r t h e gift o f recombinant antigens. T h i s w o r k was supported in part under the Generic Technology c o m p o n e n t of t h e Industry Research and Development Act, 1986.

References BARLOUGH, J.E., JACOBSON, R.H., DOWNING, D.R., MARCELLA, K.L., LYNCH,T.J. & SCOTT,F.W. ( 1983) Evaluation of a computer-assisted, kinetics based enzyme-linked immunosorbent assay for the detection of coronavirus antibodies in cats. Journal of Clinical Microbiology 17, 202 BEN-YAKIR, D. & MUMCUOGLU, Y.K. (1988) Host resistance to the human body louse (Pediculus humanus) induced by immunization with louse extracts. In Proceedings of the XVIIIth International Congress of Entomology, Vancouver, p. 282 (Abstr.) BROWN,S.J. (1988) Evidence for regurgitation by Amblyornma americanum. Veterinary Parasitology 28,335 JOHNSTON L.A.Y., KEMPD.H. & PEARSON, R.D. (1986) Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: effects of induced immunity on tick populations. International Journa1,for Parasitology 16,27 KEMPD.H., AGBEDER.I.S.. JOHNSTONL.A.Y. & GOUGHJ.M. (1986) Immunization of cattle against Boophilus microplus using extracts derived from adult female ticks: feeding and survival of the parasite on vaccinated cattle. International Journal for Parasitology 16, 115 MALVANO, R., BONIOLO, A., DOVIS,M. & ZANNINO, M. (1982) ELISA for antibody measurement: aspects related to data expression. Journal of Immunological Methods 48, 51 OPDEREECK, J.P., WONG,J.Y.M., JACKSON, L.A. & DOBSON,C. (1988) Hereford cattle immunized and protected against Boophilus microplus with soluble and membrane-associated antigens from the midgut of ticks. Parasite Immunology 10,405 OPDEBEECK, J.P. & DALY,K.E. ( 1 990) Immune responses of infested and vaccinated Hereford cattle to antigens of the cattle tick, Boophilus microplus. Veterinary Immunology and Immunopathology 25,99 PRYDE,J.G. & PHILLIPS, J.H. (1986) Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114.Biochemical Journal 233, 525 A., SPRINGK., TELLAM R., WILLADSEN P. & COBONG.S. RANDK.N., MOORET., SRISKANTHA (1989) Cloning and expression of a protective antigen from the cattle tick Boophilus microplus. Proceedings of the National Academy qf Sciences U.S.A. 86, 9657 RECHAV, Y. (1987) Resistance of Brahman and Hereford cattle to African ticks with reference to serum gamma globulin levels and blood composition. E.uperimental and Applied Acarology 3, 219 SCHLEIN Y. & LEWISC.T. (1976) Lesions in hematophagous flies after feeding on rabbits immunized with fly tissues Physiological Entomology 1, 55 STONE,B.F., BINNINGTON, K.C. & COURT,R.D. (1977) The attachment cement of the cattle tick Boophilus microplus: formation and composition. World Association for the Advancement of Veterinary Parasitology. Abstract TI WIKEL,S.K. (1988) Immunological control of hematophagous arthropod vectors: utilization of novel antigens. Veterinary Parasitology 29, 235. WILLADSEN, P. (1980) Immunity to ticks. Advances in Parasitology 18,293. WILLADSEN, P. ( 1 987) Immunological approaches to the control of ticks. International Journcil for Parasitology 17, 67 1.

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WILLADSEN P. & KEMP D.H. (1988) Vaccination with ‘concealed’ antigens for tick control. Parasitology Today 4, 196 WILLADSEN P., MCKENNAR.V. & RIDINGG.A. (1988) Isolation from the cattle tick, Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host. International Journalfor Parasitology 18, 183. WILLADSEN P., RIDINGG.A., MCKENNAR.V.et al. (1989). Immunological control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus. The Journal of Immunology 143, 1346. WILLADSEN, P., KEMP,D.H. & COBON,G. (1991) Towards the development of a commercial vaccine against Boophilus microplus. In Modern Acarology, eds F. Dusbabek & V. Buvka, SPB Academic Publishing, The Hague and Academia, Prague

Vaccination with 'concealed' antigens: myth or reality?

Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well chara...
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