Putative salivary allergens of the cat flea, Ctenocephalides felis felis.

The cat flea, Ctenocephalides felis felis, is the major initiator of flea bite hypersensitivity in dogs. Previous analyses of whole extracts of the flea and flea salivary secretions have failed to identify the allergens responsible. We dissected >2000 salivary glands from adult female fleas, extracted them into buffered saline containing protease inhibitors and fractionated the extract using gel permeation HPLC. Dogs were classified as hypersensitive to fleas (flea-feeding positive, FF+) or insensitive (flea-feeding negative, FF-) using a provocative test with live fleas. The allergenicity of the components of the salivary gland extract was tested by intradermal injection of samples of the column eluates. Dogs were also injected intradermally with a sample of whole salivary gland extract, and with histamine as a positive control. Negative control injections consisted of eluate from the column collected prior to fractions containing any protein. The skin of FF- dogs either did not respond or had a minimal response (a bleb approximately 2 mm larger than the injection blebs at the negative control injection sites) to all fractions and to the whole extract; histamine control injections produced positive responses (defined as wheals 5 mm greater than the blebs at the negative control injection sites) in all dogs. The skin of three of the nine FF+ dogs reacted positively to injection of a fraction containing protein/s with apparent MW 40k. Five other FF+ dogs reacted positively to the fractions containing proteins with apparent MW 12-8k. A single dog responded with very large, red wheals to injection of both the approximately MW 40k and MW12-8k fractions. These findings suggest that proteins with apparent MW 40k and MW 12k-8k are important in flea bite hypersensitivity. This work also supports a previous finding that mice which had been exposed to flea bites had antibodies to proteins with approximately MW 40k that were detected in salivary secretions of the flea.

Arthropod vaccines.

Antigens located in the midgut of the tick are hidden from the host's immune system. Egg production of ticks can be reduced when ticks are fed on animals vaccinated with midgut antigens of the tick, and a subunit vaccine formulated with the recombinant antigen Bm86 is now available that can reduce the number of ticks infesting cattle grazing on pasture. Midgut antigens used in vaccines against insects that transmit pathogenic organisms to humans have not been as effective in reducing insect fecundity and an alternative approach may be necessary. Transmission-blocking vaccines directed at interfering with the vector-pathogen interaction could result in loss of vector competence and block the spread of disease-causing organisms.

Biodegradable implants for the delivery of veterinary vaccines: design, manufacture and antibody responses in sheep.

Biodegradable implants made from cholesterol and lecithin (C:L) were used to deliver a recombinant antigen (recombinant Dichelobacter nodosus pili) and adjuvant (Quil A) to sheep. Implants (5.5- x 1.8-mm) were placed subcutaneously and compared to a conventional vaccination regime (2 injections, 4 weeks apart) for antibody responses and tissue compatibility. Release profiles of antigen and adjuvant were also studied in vitro and in vivo. The presence of Quil A in vaccine implants had a marked effect on the rate at which antigen was released with 29 and 44% being released in the first 24 h from implants containing pili alone and pili with Quil A, respectively. Sheep produced significant levels of antibody when immunized with implants, however the response was short-lived and of significantly lower intensity than the response stimulated by two injections of antigen with Quil A (P < 0.01). A second implant system was developed where implants coated with C:L to delay antigen release, were used in combination with uncoated implants to deliver a priming dose and boosting dose of antigen. Antibody titres stimulated by the 4 double implant system were equivalent to those stimulated by a conventional regime of two injections (four weeks apart) for the first six weeks of the experiment. After this time, antibody levels in the groups which received implants dropped significantly. In vitro studies revealed that some of the implant coatings had caused a delay in the release of antigen (the rate of release peaked at 72 h), however this was not long enough to provide a significant boosting effect. In all cases, implants were well tolerated by sheep and caused less local reaction than injected vaccines.

Mapping the T cell epitopes of the Babesia bovis antigen 12D3: implications for vaccine design.

The Babesia bovis antigen 12D3 was analysed to identify potential T-cell epitopes. Two predictive algorithms identified 13 possible sites but there was minimal agreement between the different predictive methods. Experimental determination of the T-cell epitopes recognized by nine cattle was achieved using a panel of overlapping peptides which identified seven different epitopes, five of which were clustered together around residues 210-320 of the molecule. No T cell epitopes were located within the tightly disulphide bonded core of 12D3. Using a series of truncated peptides, the location of two of the epitopes was mapped to residues 35-43 and 266-275. The sequences of these two epitopes was compared with a database of previously described binding motifs for MHC II alleles and each epitope was found to contain three sequence motifs recognized by HLA-DR alleles. The BoLA-DRB3 alleles occurring in these cattle were determined by a sequence specific oligonucleotide hybridization assay. Within those cattle whose T cells proliferated in response to 12D3, there was a consistent pattern of epitope recognition and presence of particular DRB3 alleles. The implications for effective vaccine design are discussed.

Salivary antigens of the cat flea, Ctenocephalides felis felis.

The cat flea, Ctenocephalides felis felis, is the major cause of flea bite hypersensitivity (FBH) in dogs and cats, yet little progress has been reported on identifying the antigens responsible. We obtained flea salivary antigens by washing secretions from containers probed by the mouthparts of fleas, and by extracting whole flea salivary glands. Mice were exposed to feeding fleas to generate antibodies to salivary antigens injected in vivo. The sera were tested for antibodies against the salivary antigens described and against a whole flea extract; in indirect ELISA, antibodies to salivary secretions were detected in 60% of the sera from mice exposed to feeding fleas. These sera identified four protein bands at apparent MW 56, 54, 42 and 40 K which corresponded to prominent protein bands in whole salivary gland extracts identified by protein staining after SDS-PAGE. Fixed sections of whole fleas exposed to the antisera showed that only structures within the salivary glands were identified. The salivary secretions and gland extracts are now being used to study immune responses of dogs suffering from FBH.

Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus.

OBJECTIVE: To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter Nodosus (strain A). PROCEDURE: Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded. RESULTS: The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects. CONCLUSION: Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus.

Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B. microplus, and which were subsequently protected (84% and 94% respectively) against challenge with B. microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens form the midgut of B. microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QU13. Larval membrane antigen extracted from N-strain B. microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B. microplus. It was concluded that protective antigens recognized by QU13 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.

The effects of continuous and intermittent delivery of antigens of Boophilus microplus on the development of murine antibodies.

Protective antigens solubilised from the membranes of the midgut (LI-GM) of the adult cattle tick Boophilus microplus were delivered to mice either continuously for up to 4 weeks from osmotic pumps implanted subcutaneously or intermittently in a pulsatile fashion by injections or by a combination of both modes of delivery. The effects of delivery profile on antigen specific antibody levels and avidity were compared. LI-GM delivered either by three injections (weeks 0, 2 and 4), or continuously from osmotic pumps (over 4 weeks) induced similar levels of antibodies in mice. The mode of delivery of LI-GM when in the presence of the adjuvant Quil A did not generally affect either the level or the avidity of the antibody response; indeed a single injection of LI-GM in the presence of Quil A stimulated an immune response similar to that induced by several combinations of pulsatile and of continuous delivery where mice were exposed to antigen and adjuvant for up to 4 weeks. LI-GM incubated at 37 degrees C in vitro and in vivo for periods from 4 h to 14 days was partly degraded into low molecular weight (less than 29 kDa) components. The immunogenicity of LI-GM incubated in vitro for 4 h was significantly decreased, although its antigenicity was not affected after incubation for up to 14 days. In conclusion, delivery of LI-GM continuously from osmotic pumps demonstrated that single-step immunisation of mice with tick antigens was feasible. However, it was also demonstrated that the continuous delivery of antigen was only advantageous (i.e. potential for a decrease in the number of times an animal must be handled) compared with delivery by injections when no adjuvant was used. Further work is necessary to establish the effect antigen degradation has in limiting the immune response resulting from continuous delivery of antigen from osmotic pumps.

The effect of various adjuvants on the humoral immune response of sheep and cattle to soluble and membrane midgut antigens of Boophilus microplus.

Sheep and cattle were immunized with soluble and membrane midgut antigens of the cattle tick, Boophilus microplus in association with various adjuvants, and antibody levels to soluble and membrane midgut extracts were measured by enzyme-linked immunosorbent assay (ELISA). The adjuvants used were Quil A, Freund's incomplete adjuvant (FIA) and aluminium hydroxide (A1(OH)3). Significant antibody levels to midgut membrane antigen (GM) were not detected in the sera of sheep immunized with GM plus A1(OH)3. However, membrane and soluble midgut antigens combined with Quil A generated significantly higher levels of antibodies in sheep than these antigens emulsified in FIA (P < 0.05). In cattle, although soluble midgut antigen (GS) plus Quil A induced significantly higher levels of antibodies compared with GS in FIA (P < 0.05), neither preparation provided significant protective immunity against challenge with B. microplus (46% and 22%, respectively). A soluble form of membrane midgut antigen extracted by low ionic strength buffer (LI-GM) when combined with Quil A provided 83% and 87% protection against challenge with B. microplus in two separate experiments. Quil A was clearly superior to FIA and A1(OH)3 as an adjuvant for these tick antigens.

Anti-idiotypic antibodies as surrogate antigens for vaccinating against the cattle tick, Boophilus microplus.

QU13, a monoclonal antibody (mAb) raised against midgut (GM) antigens from Boophilus microplus and shown to recognise antigens which protect cattle from tick challenge was used to immunise cattle and rabbits to produce anti-idiotypic antibodies (AIA). Polyclonal antisera against mAb QU13 were produced in rabbits and cattle. AIA were purified from these antisera by affinity chromatography procedures. These purified AIA were found to block mAb QU13 binding to GM in enzyme-linked immunosorbent assay (ELISA). AIA purified from bovine antiserum elicited an immune response in cattle to antigens extracted by detergent from the midgut of B. microplus (TXGM) after the fourth vaccination (P = 0.06) compared with the bovine immunoglobulin (Ig) control. The mean antibody level in the group of experimental cattle vaccinated with AIA purified from rabbit antisera was significantly higher (P < 0.03) than that of bovine Ig control cattle after the fourth vaccination and an anamnestic response (P < 0.11) occurred in the rabbit AIA vaccinated group of cattle when a single booster dose of 300 micrograms of TXGM was given after the first tick challenge. The positive control group of cattle vaccinated with TXGM were significantly protected (P < 0.05) against tick infestation after the booster dose of 300 micrograms of TXGM. The AIA vaccinated groups of cattle were not protected against challenge with 20,000 larval ticks either before or after the booster injection of 300 micrograms of TXGM.

Antigens identified by monoclonal antibodies in tissue sections of Boophilus microplus.

A panel of monoclonal antibodies (mAbs) raised against midgut antigens of Boophilus microplus were used to probe various stages and organs of the tick. One of the monoclonal antibodies in this panel (QU13) has previously been shown to recognize protective antigens. Of the 18 mAbs tested, all except two (QU5 and QU12) reacted with sections of adult midgut and Malpighian tubules using an avidin-biotin alkaline phosphatase method for immunostaining. MAbs QU1, QU2, QU3, QU4, QU12, QU13, and QU18 reacted specifically with the lumenal surfaces of type III acini of the salivary gland. These seven mAbs also stained the midgut in larval sections indicating that the antigens recognized were not stage specific. However, none of the seven mAbs tested recognised antigens in either the adult ovary or the developing egg. Antigens which were immunogold labelled by mAbs QU1, QU4, QU11, QU13, and QU15 in electron microscopy were located either on or near the surface of the microvilli of digestive cells from the midgut of the adult tick. We conclude that common antigens are present on the lumenal surfaces of the adult midgut, type III acini of the salivary gland, and the Malpighian tubules and that these antigens are also located in the larval gut.

Immunohistochemical localization of excretory/secretory antigens in adult Ancylostoma caninum using monoclonal antibodies and infected human sera.

Human eosinophilic enteritis (EE) may result from hypersensitivity to the excretory/secretory (ES) antigens of adult Ancylostoma caninum. The origin of several antigens were identified by probing adult A. caninum with mouse monoclonal antibodies (MoAbs), sera from mice vaccinated with ES antigens and sera from human EE patients. Six MoAbs (AC/ES 1-6) were produced against ES antigens, two being IgG3 and four IgM. Western blots demonstrated four different antigen specificities: MoAb AC/ES 1 bound strongly to an ES product at about 30 kDa; AC/ES 2 recognized a broad band ranging from 50-200 kDa; AC/ES 3, AC/ES 5 and AC/ES 6 reacted at about 68 kDa, and AC/ES 4 at about 97 kDa. Sections of formalin-fixed, paraffin embedded adult A. caninum were then incubated with these MoAbs and immunostained by the peroxidase-anti-peroxidase (PAP) technique. The target epitope of MoAb AC/ES 1 was found mainly in the oesophageal, amphidial and excretory glands; AC/ES 2 reacted weakly with many structures in the sections; AC/ES 3, AC/ES 5 and AC/ES 6 were specific for excretory glands only, and AC/ES 4 bound to amphidial glands. Sera from immunized mice reacted with all three (especially the excretory) glands and the cuticle. In an indirect assay, worm sections probes with three human EE patient sera demonstrated maximal staining in the amphidial glands. Our findings confirm that ES products of A. caninum include immunogenic glandular secretions which may be involved in the pathogenesis of human EE.

Vaccines against blood-sucking arthropods.

This paper provides selected personal insights on the development of vaccines against blood-sucking arthropods, with particular emphasis on vaccines against ticks. The emergence of novel or concealed antigens of haematophagous ectoparasites as candidate vaccine antigens is reviewed and the effect of feeding by the parasite on the expression of protective antigens is considered. The distribution of protective antigens through life cycle stages, the stage of the life cycle targeted by protective responses, and the nature of these responses, are commented on briefly. Concealed antigens of the gut, including the peritrophic membrane, and other internal organs, are evaluated for the role they play in induction of immunity artificially. Some of the work carried out to purify and characterise protective antigens of tick guts is described. A commentary is developed on vaccines that combine both "concealed" and "exposed" antigens. Some of the problems associated with the infestation and challenge of vaccinated hosts in the field are identified and the delivery of parasite antigens as vaccines that are both protective and "user-friendly" is emphasised as a major problem to be solved.

Flea allergy dermatitis in dogs diagnosed by intradermal skin tests.

Thirty-three dogs were categorised according to their clinical signs of flea allergy dermatitis and reactivity to feeding fleas (Ctenocephalides felis felis). A soluble extract of whole fleas (FS), fractions of this extract separated by chromatography, and a commercially available flea antigen extract were used in intradermal skin tests (IDST) to establish the presence or absence of type I and type IV hypersensitivity. The reactions were measured and the results were analysed using three grading systems commonly reported in the literature. The results of the IDST for the groups of dogs varied according to the grading system used. FS, the most effective of the antigen preparations, identified 94 per cent of dogs which reacted to feeding fleas when a result was considered positive if the mean diameter of the wheal at the antigen injection site exceeded the diameter of the wheal at the negative control site by five mm at 15 and/or 30 minutes after injection.

Quil A and ISCOMs as adjuvants for midgut membrane antigens of Boophilus microplus.

Quil A used with Boophilus microplus gut membrane antigen (GM) had a significant effect on antibody levels induced in sheep (P < 0.05) since GM alone did not induce a significant level of antibodies. Injection of a vaccine containing GM and Quil A, either subcutaneously or intramuscularly, induced similar levels of antibodies in sheep. However, Quil A injected subcutaneously induced acute inflammatory reaction. The amount of Quil A for use with GM was determined to be 1000 micrograms/ml. Immunostimulating complexes (ISCOMs) incorporating detergent-solubilized membrane midgut antigens (TX-GM) failed to induce an immune response in cattle without the addition of Quil A. The addition of Quil A to the ISCOMs containing TX-GM did not stimulate antibody responses greater than those stimulated by TX-GM plus Quil A, and protection in vaccinated cattle was 86% and 74%, respectively.

IgG and IgE antibodies against antigens of the cat flea, Ctenocephalides felis felis, in sera of allergic and non-allergic dogs.

Antigens from a soluble extract of cat fleas (FS) were separated using SDS-PAGE, and transferred to nitrocellulose paper. Sera from dogs shown by use of a provocative flea feeding test to be either allergic (23 dogs) or non-allergic (20 dogs) to flea bites, were used in Western blots to identify flea antigens that react with canine IgG or IgE. The sera were also tested in ELISA against FS to quantify levels of IgG and IgE antibodies. Antibodies present in the sera of both flea allergic and non-allergic dogs identified multiple antigens. There was a great diversity of responses within each group, and there was no pattern of reactivity that distinguished dogs with flea allergy from dogs not allergic to fleas. IgG and IgE antibodies were not significantly different between the two groups of dogs. These results demonstrated that there is little association between particular antibody responses and allergic reactivity of dogs to fleas.

Tick infestations on cattle vaccinated with extracts from the eggs and the gut of Boophilus microplus.

Extracts prepared from the membranes of eggs (EM) and guts (GM) of Boophilus microplus were used to immunise cattle which were then infested twice with 20,000 larval ticks 1 week apart. EM antigens did not protect cattle against challenge with ticks, despite high levels of anti-egg antibodies in the sera of the vaccinated cattle, detected by an indirect enzyme-linked immunosorbent assay (ELISA). Cattle vaccinated with GM, however, had high levels of antibodies against GM and were protected significantly against challenge with B. microplus. Anti-EM and anti-GM antibodies in the sera of cattle cross-reacted significantly with GM and EM respectively on ELISA and recognised both specific and common antigens in extracts of the eggs and guts of B. microplus on Western blots. Exposure of cattle to field infestation with ticks during vaccination with gut antigens did not adversely affect the levels of antibodies generated.

Immunopotentiation and delivery systems for antigens for single-step immunization: recent trends and progress.

The use of adjuvants for immunopotentiation has been investigated since the 1920s and a number of comprehensive reviews and monographs have been published on this subject. A recent trend in immunopotentiation has been the use of delivery systems which allow for sustained or controlled release of antigens and which induce prolonged immunity following a single dose. This concept has been termed either single-step or single-shot immunization. The delivery system has been modulated to potentiate the immune response either by delivering the antigen (and perhaps an adjuvant or adjuvants) either over a prolonged period of time or in a predetermined sequence or by incorporating substances with immunoadjuvant properties (e.g., lecithin and certain biodegradable polymers) as carriers within the delivery system. This Review focuses on the progress made in the design of delivery systems for immunopotentiation. Particular emphasis is given to delivery systems designed to achieve single-step immunization.

An attempt to protect cats against infestation with Ctenocephalides felis felis using gut membrane antigens as a vaccine.

Cats (n = 5) were vaccinated with membrane antigens extracted from the gut of unfed fleas (Ctenocephalides felis felis) together with Quil A and RIBI as adjuvants. Five unvaccinated cats were retained as controls. All the cats were infested on 6 separate occasions with fleas (46-250 per challenge). Protection was assessed from the number of fleas retrieved and the fecundity of the female fleas, measured as the number of developed oocytes contained in the reproductive tract. Cats injected with gut membrane antigens had significantly elevated levels of anti-flea antibodies in their sera, but they were neither protected significantly against infestation with fleas nor was the apparent fecundity of fleas which had fed on vaccinated cats decreased. The possible reason why gut membrane antigens failed to protect cats against fleas are discussed.