Immunization of Rabbits with Antigens from the Tick Rhipicephalus Appendiculatus (Neumann, 1901)
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The behaviour of larvae and nymphs of B. appendiculatus infested on naive rabbits displayed a sequence of events when released on the ears. Complete attachment was attained after 18-24 hours of tick application. Engorged ticks were observed as early as the end of 48 hours and complete drop off of the replete ticks took place after 60 hours of tick application Rabbits acquired resistance to larvae and nymphs of the ixodid tick B. appendiculatus after four successive infestations. Resistance was characterized by delayed attachment, time to drop off the host and reduced engorgement weights. The resistant rabbit rejected 80-90% of the tick instars applied compared to the controls which allowed more than 95% of the applied ticks to complete engorgement successfully. After the second and subsequent infestations, the number of successfully attaching and engorging ticks decreased with each infestation. Detachment/attachment process by the ticks to new sites on the periphery of the earlier skin bite was observed. Some ticks appeared engorged with host tissue fluids rather than erythrocytes and others were observed dead while still attached on ,the host or engulfed in serous exudate. Initial erythematous reactions followed by the development of diffuse papules with substantial oedema were also recorded. Some of the skin reactions consisted of plaques which persisted for almost one month before subsiding. Antigens isolated from the salivary glands extract by fractionation with Sephadex G75 yielded two major peaks. Only the first peak was shown to stimulate in vivo delayed skin reactivity when administered intradermally to tick resistant rabbits. Maximum skin reactivity occurred after 48 hours of test and completely subsided by 108 hours. The histopathology studies revealed infiltration of large numbers of polymorphonuclear and mononuclear leukocytes. Rabbits which were immunized with unfed whole tick tissue homogenates derived from eggs, larvae, nymphs and adults of B. appendiculatus in Freund's Complete Adjuvant (FCA) showed high antibody titres. The antibody titres were detected by passive haemagglutination test and reached peak by day 28 post inoculation. The patterns of the antibody response were typically the same and no group of animals ,showed a decrease in the antibody titre during the 42 weeks period of study. The antibody responses were maintained for overly 3 years. Immunodiffusion tests showed a minimum of 3 to 4 precipitin lines,with the antiserum from rabbits immunized with egg extracts appearing more potent. When larval ticks were fed on all the immunized rabbits at the peak of high antibody response, there were no immediate adverse effects observed on the ticks. Blood haematological parameters of the animals immunized with the unfed tick instars was also studied and compared with that from successively infested rabbits with larvae and nymphs. There were no significant changes in the blood components in the animals immunized with unfed whole tick extracts but there were greater changes in the blood picture in the rabbits successively infested with larval and nymphal ticks. When immune sera to the unfed whole tick homogenates was inoculated to fully engorged nymphs there were no adverse effects observed on them. The nymphs moulted to adults which also performed well when fed on naive rabbits. Rabbits immunized with antigen extract from fully fed adult ticks showed dramatic adverse effects on adult ticks feeding on them. There was initially delayed attachment process, reduced engorgement weights, prolonged time to drop off the host, reduced egg production, egg weights and reduced egg viability. Mortalities were also observed in some of the hatched larvae. These results suggest the presence of the target antigen(s) in antigen extract from fed ticks and absence of these antigen(s) in the unfed whole tick homogenates. Adult ticks of R. appendiculatus were also fed on rabbits immunized with precipitin lines derived from immunodiffusion in agarose with lyophilized fed tick homogenate or purified soluble antigen extracts and antiserum from rabbits immunized with fed tick homogenates. Adverse effects on reproductive potential of ticks feeding on the immunized rabbits were observed. The ticks displayed attachment, prolonged engorgement and extended periods to drop off the host. There was a reduction in egg hatchability greater than 80% in each of the experimental animals as compared to the controls which showed a reduction of less than 7%. Some of the larvae which hatched from the two groups of animals immunized against the different types of immunogens were observed dead within one -month of hatching but this varied from one tick batch to another. Both the lyophilized fed tick extract and purified soluble antigen extracts, precipitin line derived immunogens, showed more or less the same trend of results but rabbits immunized with purified soluble antigen extract precipitin line showed slightly higher values of the observed adverse effects. Antibodies from resistant animals by successive adult tick infestations and by immunization with fed tick antigen extract were used to isolate, enumerate and characterize the tick antigens recognized as an approach to identificationof the target antigen(s) in feeding ticks. Ouchterlony immunodiffusion tests showed 5 precipitin lines. In crossed immuno electrophoresis only one antigen was detected using sera from animals made resistance by multiple infestations. Sera from extract immunized animals detected this antigen and nine others. Al though the tick antigen detected by both sets of sera in crossed immuno electrophoresis was radiolabelled with 35S aminoacids it was not detected by Staphylococcus aureus mediated immune precipitation with sera from hosts made resistant by multiple infestations. Antibodies from rabbits immunized with fully fed tick extracts detected by immune precipitation nine proteins in the aureus system. The molecular weights of these antigens as assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were: 180,000; 140,000; 130,000; 98,000; 94,000; 92,000; 88,000; 85,000 and 82,000 daltons. The rate of synthesis of these antigens appeared to vary with relation to the feeding cycle. The proteins increased with each day of feeding reaching maximum by day 6 to 8 and then decreased gradually throughout the post engorgement to preoviposition period. Quantitation and characterization of antigenic constituents of salivary gland, reproductive system, gut and the haemolymph derived from day five fed female ticks of R. appendiculatus were also carried out. This was achieved almost completely by solubilizing the tissues in 0.1% Sodium dodecyl sulphate and 0.1% Nonidet P-40 respectively and the proteins separated simultaneously into their components in terms of electrophoretic mobility and molecular weight. There were 50-55 protein bands demonstrated in the salivary glands 40 in the reproductive system, 44 in the gut and about 32 protein bands that could be demonstrated by SDSpolyacrylamide gel electrophoresis. The protein bands ranged from 11,000 to 250,000 daltons. There were common protein bands shared by all the four tissue organs of about the same molecular weight but not necessarily the 'same molecular size. The immunological relationship of the possible target antigen(s) being produced by the four tissues to each other was shown by immunoprecipitation. Immune serum to• fed tick antigen extract demonstrated a minimum of 9-11 specifically immune precipitated proteins with molecular weights ranging from 82,000 to 180,000 daltons from each of the four tissues. These were possibly some of the specific antigen(s) being contributed by the tissue organs and possibly the ones that were involved in the protective immune response(s) against ticks, feeding on rabbits immunized with fed tick tissue homogenates. A simple procedure for the isolation and characterization Of. the possible target tick antigen (s) from whole tick homogenates using Staphylococcus aureus protein A immunoadsorbent chromatography is also described. The elution pattern on the Protein A-SeP0arose Cl-4B showed the presence of one peak as eluted by a 0.1M glycine pH 3.0. Analysis of the eluted antibody-antigen complexes on the SDS-PAGE continuous buffer system showed comparable results with those protein bands recognized, by the same immune serum on immunoprecipitation, employing S. aureus alone as an immunoadsorbent. The protein bands resolved in this system appeared sharper with considerable reduction in background of the non specifically recognized antigens.