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WO1992003156A1 - Vaccins antipuces derives de membranes - Google Patents

Vaccins antipuces derives de membranes Download PDF

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Publication number
WO1992003156A1
WO1992003156A1 PCT/US1991/005852 US9105852W WO9203156A1 WO 1992003156 A1 WO1992003156 A1 WO 1992003156A1 US 9105852 W US9105852 W US 9105852W WO 9203156 A1 WO9203156 A1 WO 9203156A1
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WO
WIPO (PCT)
Prior art keywords
flea
membrane
fleas
supernatant
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1991/005852
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English (en)
Inventor
Joan P. Opdebeeck
Robyn E. Boreham
Ann E. Arfsten
Andrew W. Heath
Miles K. Yamanake
Lynnor B. Stevenson
Beverly Dale
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Queensland UQ
Paravax Inc
Original Assignee
University of Queensland UQ
Paravax Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Queensland UQ, Paravax Inc filed Critical University of Queensland UQ
Publication of WO1992003156A1 publication Critical patent/WO1992003156A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/4359—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from fleas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to prevention of flea infestation in mammals and birds. More particularly, the invention concerns vaccines derived from membranous elements of fleas which are used to immunize mammalian and avian subjects against flea infestation.
  • Fleas are insects which behave as ectoparasites for birds and mammals. They are a serious nuisance both in the raising of animals which are sources of food and fiber and in the nurture of pet cats and dogs. The problem in the latter situation is particularly serious because the infestation also becomes a source of annoyance for the pet owner who may find his or her home generally contaminated with fleas which feed on the pets, and these parasites can induce an allergic reaction in both the pets and humans.
  • the prevalence of flea aller- gic dermatitis (FAD) constitutes the foremost veterinary der atological problem in the U.S. (Kwochka, K. . , Vet Clin North Am (1987) 12:1235-1262).
  • flea i.e., insect
  • helminths which are worms
  • ticks which are arachnids
  • control of all forms of parasitism has generally involved internal or external applications of chemicals.
  • Commonly encountered approaches to control the flea problem are generally focused on use of insecti ⁇ cides in formulations such as sprays, shampoos, dusts, dips, or foams, or in pet collars. None are notably successful.
  • IGRs insect growth regulators
  • PCT application W087/05513 (US 4,814,170) assigned to Apht&n Corporation describes antiendo- or antiectoparasite vaccines in general, which are derived from endocrine products, such as juvenile hormones.
  • PCT application WO86/02839 suggests vaccines against helminths which contain suspensions, homogenates or extracts of nonparasitic nematode species. Presuma ⁇ bly, these nonparasitic species are closely enough related to the parasitic forms to engender appropriate antibodies.
  • a similar approach with respect to protozoa is disclosed in PCT application WO83/03199.
  • British application 1580539A published in 1980 suggests an antiparasitic vaccine derived from secretions of the parasite. None of these approaches are specifically directed to protection against flea infestation.
  • the invention provides a vaccine useful to immunize mammalian and avian subjects in such a manner as to lower infestation by the insect ectoparasites which are responsible for the problems associated with extensive flea infestation, and to lower the population of fleas in the surrounding environment.
  • the vaccine employs antigens which are associated with membranes of the major parasitic fleas of dogs and cats, e.g.. Ctenoceoha1ides felis. and provides nonallergic protection against this nuisance.
  • the invention is directed to a vaccine for protection of a subject against infestation by fleas, which vaccine comprises an amount of flea membrane or flea membrane extract, or an effec ⁇ tive antigenic component of said membrane or extract, which is effective to confer resistance to the infesta ⁇ tion, either by killing the insects or by incapacitating them in some way, or both.
  • the successful application of the vaccine also lowers the flea population in the animal's surroundings.
  • the invention is also directed to antibodies specifically immunoreactive with these anti ⁇ gens.
  • the invention is directed to a method to protect a subject against infestation by fleas and to reduce the flea population in its environ ⁇ ment which comprises administering the vaccine of the invention.
  • the invention is directed to methods to prepare the effective vaccines.
  • Figure 1 shows a diagram of flea anatomy indi ⁇ cating the internal organs of interest.
  • Figure 2 is a halftone copy of a photograph showing the results of an SDS gel run on a total flea membrane and total flea supernatant antigens prepared as described in Example 1A.
  • Figure 3 is a halftone copy of a photograph showing the results of an SDS gel run on a total flea membrane and total flea supernatant antigens prepared as described in Example IB.
  • Figure 4 is a halftone copy of a photograph showing the results of an SDS gel run on a gut flea membrane antigen preparation.
  • the invention vaccines are preparations which are or contain antigens associated with internal flea membranes.
  • the membranes may be obtained as total membrane preparations, or the insect may be dissected and an individual membrane type ⁇ used as a source for the antigen used in the vaccine.
  • the vaccines are formulated in conventional ways, optionally using adjuvants either in or along with the formulation.
  • Low molecular weight antigens are also conjugated to carriers or to themselves if necessary to enhance immunogenicity.
  • fed fleas By “fed” fleas is meant that the fleas from which the preparations are derived have been allowed to consume a blood meal for a 24-48 hour period either on animals or by artificial feeding prior to membrane preparation. In the case of "unfed” fleas, newly emerged fleas that have never taken a blood meal are used in the membrane preparations.
  • Total flea membrane (TFM) preparations are obtained by a number of alternative procedures.
  • frozen (about -70°C) unfed or fed male and/or fe ale fleas are disrupted by homogenization in buffer, e.g., 0.15M PBS, pH 7.2, 1 mM EDTA.
  • a suitable proportion of fleas to buffer is about 2500 frozen fleas in 10 ml buffer.
  • Homogenization can be conducted by shaking in the presence of 1/8" stainless steel beads at 4°C for 4 minutes, or by grinding the fleas to a powder with a mortar and pestle in the presence of liquid nitrogen prior to the addition of buffer. It may be helpful to sonicate the preparation, for example using a soniprep 150 (MSE) at an amplitude of 22 for a total of 3 1/2 minutes on ice (30 second bursts cooling on ice between bursts) .
  • MSE soniprep 150
  • Debris is then removed from the homogenate, usually by filtration through coarse filters or by spinning at low speed, for example 600 x g for 10 minutes at 4°C.
  • the pellet may be rehomogenized and resonicated to obtain additional membrane extract.
  • the total supernatant is then freed of organ- elles and cells by centrifugation at moderate speed, for example at 15,000 x g for 20 minutes (at 4°C) to remove these nonmembranous portions, such as cells, mitochondria, lysosomes and microbodies.
  • the supernatant from this spin (which contains the membrane fraction) is then centrifuged at very high speed, -100,000 x g for 2 hours at 4°C.
  • the pellet from this harvest contains the membrane fragments including cell membranes and ribosomal and endoplasmic reticulum membrane fragments.
  • the membrane pellet is then homogenized in a suitable buffer and assayed for protein content if desired.
  • the midgut is removed by dissection under a binocular microscope, and immediately placed in a tube of the buffer on ice.
  • the tubes are eventually stored at -70°C. It appears that -10,000 midguts are required to obtain a convenient preparation, although of course by modifying the procedure to a micro scale, smaller numbers could be used.
  • the harvested midguts are thawed, pooled and homogenized in a glass/teflon homogenizer.
  • the procedure described above for total membrane preparations may be applied, or alternatively the homogenate may be transferred to siliconized 1.5 ml icrofuge tubes and spun in an Eppendorf centrifuge at a setting 3 for 10 minutes at 4°C.
  • the supernatants are transferred to other sili ⁇ conized tubes and spun at setting 13 for 15 minutes at 4°C.
  • the supernatants are transferred to a Beckman 8.5 ml conical heat-seal tube overlaid with mineral oil and spun in an SW28 rotor at 100,000 x g for 2 hrs.
  • the pellet of this spin is the membrane fraction and is assayed for protein content if desired, and the individual antigenic elements isolated if desired.
  • analogous preparations may be made from other internal organs, including ovary, testis, nerve cord and Malpighian tubules. Dissection according to the diagrammatic representation of internal flea anatomy in Figure 1 permits use of these various sources of internal organ membranes.
  • midgut preparations include the proventriculus (1) , the true midgut (2), the pylorus (6), and the hindgut (4). Also noted in Figure 1 are the locations of the Malpighian tubules (3) , the ovaries (5) , and the nerve cord (8) .
  • the preparation of suitable membrane-containing fractions involves homogenization of the organ desired or of the whole insect, removal of large components, and finally, removal of membranes from the soluble components by high speed centrifugation. Once harvested, the membrane fraction is useful as a source of purified antigen components which can be purified using immunoaffinity columns wherein the affinity ligand comprises antibodies raised by administration of the membranes, or other conventional separation techniques. Antigen-antibody complexes formed during such affinity-based purifications can also be used for immunization and protection.
  • Suitable carriers include, for example, keyhole limpet hemocyanin (KLH) , animal or human serum albumin, and diphtheria or tetanus toxoid. Conjugation is by conven ⁇ tional means, depending on the nature of the antigen.
  • the antigen can often be coupled directly to carrier, but more frequently a linker, such as those commercially available from Pierce Chemical Company, Rockford, IL, is used.
  • the linkages employed are those conventionally used, such as amide bonds, thioethers, and disulfides.
  • membrane antigens may be polymerized or crosslinked, for example, with gluteraldehyde, enhancing immunogenicity by increasing perceived molecular weight or presenting novel immunogenic epitopes.
  • mRNA prepared from the whole organism or the various tissues is available for reverse transcription into cDNA encoding said membrane antigens.
  • Clones expressing this cDNA can be detected using the above antibodies; the cDNA can be ligated into expression vectors such as the lambda-gt system and the raised antibodies used to detect the production of antigen from these vectors. The induction of antibodies is described hereinbelow.
  • the antigenic composition is administered in an immune protocol of multiple dosages at an appropriate protein concentration of 10-1000 ⁇ g per dose, preferably 100-250 ⁇ g, for total flea membrane (TFM) and at an appropriate dose, usually about 10-100 ⁇ g per dose for individual organ membranes such as midgut flea membrane (GFM) preparations.
  • adjuvants such as Quil A (saponin) , RIBI adjuvant, complete or incomplete Freund's adjuvant (CFA or IFA) , aluminum phosphate/alum, or one or more of the ISCOMs, or muramyl dipeptide (MDP) , depending on the subject.
  • Quil A for administration of the vaccine to sheep, Quil A at l mg/ml in a total volume of 2.5 ml per injection is preferred.
  • saponin at concentrations to 400 ⁇ g/ml or RIBI at suitable concentrations are used in a total volume of 0.5-1 ml per injection.
  • Standard formulations for vaccines in general are usable and such formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton PA, latest edition.
  • Administration is generally systemic by injection, usually intramuscular or subcutaneous injec ⁇ tion. Intravenous injection is, for the most part, impractical for veterinary use. Suitable subjects are those animals who are targets of flea parasitism.
  • mice may also be immunized specifically for antibody production.
  • the precise nature of the immunization regimen is readily determined using routine optimization procedures, and the general parameters for administration to a specific subject are well known to those of ordinary skill in the art.
  • the dosage levels and the nature of the formulation varies with the nature of the subject and the severity of the infestation, as well as the precise vaccine active ingredient employed.
  • the results of vaccination as described above are severalfold.
  • the vaccinated animals are protected against flea infestation in that the number of fleas found feeding on the animals directly subsequent to vaccination, even after challenge by artificially introducing a flea population, is greatly reduced. Also, feeding fleas that survive may have markedly reduced fecundity. Thus, the animals per se are protected from the parasitism of the insects.
  • the immunized animals are useful as sources of antibodies immunoreactive with the specific antigens in the membrane extracts. Antisera are recovered from these animals and used directly, for example, as affinity purification ligands or as detection reagents to analyze for the presence or absence .
  • the immortalized cell lines can be screened using conventional immunoassays employing, as test antigen, the appropriate membrane extract or fraction or purified component thereof.
  • TFM Vaccine A Total flea membrane antigen (TFM) was prepared as described generally hereinabove. Briefly, about 5,000 frozen unfed C_ felis fleas were suspended in 20 ml of 0.15 M PBS, pH 7.2, containing 1 mM EDTA, and homogenized by shaking in the presence of 1/8-inch stainless steel beads at 4°C for 4 min. The beads were removed, and the mixture was then sonicated using a Soniprep 150 (MSE) at an amplitude of 22 for 3-1/2 min on ice using 30-sec bursts and cooling back to 6°C between bursts. The suspension was centrifuged at low speed (600 x g) at 4°C and the pellet was again treated as above to obtain additional extract. This extract was also recovered by centrifugation at low speed.
  • MSE Soniprep 150
  • the combined supernatants were then centrifuged at moderate speed (about 15,000 x g) for 20 min at 4°C and the pellet was discarded.
  • the supernatant was centrifuged at about 100,000 x g for 2 hr at 4°C and the pellet was recovered.
  • the supernatant from this centrifugation step is designated TFS for total flea supernatant.
  • the pellet is designated total flea membrane (TFM) .
  • TFM pellet was then homogenized in an additional 5 ml of 0.15 M PBS, pH 7.2, 1 mM EDTA.
  • the final concentration of protein in the buffer was
  • TFS and TFM preparations were subjected to gel electrophoresis on polyacrylamide gels using SDS detergent followed by Coomassie blue staining for protein visualization. The results of this gel are shown in Figure 2. Lane 1 shows total adult flea extract after the low speed spin. Lane 2 shows the TFM preparation. Lane 3 shows the TFS preparation.
  • TFS total flea supernatant
  • TFM total flea supernatant
  • PBS 0.15M, pH 7.2 with 1 mM EDTA total flea membrane
  • TFM total flea membrane
  • This procedure yielded 35 ml of TFS containing 1-2 mg/ml protein, and 1 ml of TFM containing -2 mg/ml of protein.
  • Portions of TFS and TFM were subjected to electrophoresis on polyacrylamide gels using SDS detergent followed by Coomassie blue staining for protein visualization (Figure 3).
  • Lane 1 shows total adult flea extract after the low speed spin.
  • Lanes 2 and 3 are TFS samples from two different preparations. Lanes
  • TFM-KLH keyhole limpet hemocyanin
  • Vaccines were formulated with adjuvants recommended by manufacturer or known in the art.
  • Example 2 Protection of Sheep with TFM Vaccines Two groups of 4 sheep each were vaccinated with either 250 ⁇ g TFM, as prepared in Example 1A, in 2.5 ml vaccine containing l mg/ml Quil A, or with a control vaccine of 2.5 ml volume containing 1 mg/ml Quil A alone. The sheep were bled throughout the immunization regimen and antibody titer against flea membrane antigen was determined in a standard solid state ELISA, using the TFM preparation as antigen. After 6 immunizations the sheep were challenged as follows.
  • the back of the animal was shaved and a cup containing 30 fleas was affixed to the shaved area with glue.
  • the fleas were allowed to feed for seven days after which time the contents of the cups were analyzed for the number of living and dead fleas. (In general, only 50% of fleas on sheep feed.)
  • Example 3 Efficacy of TFM Vaccine in Cats
  • Conditioned adult cats of mixed breed and sex were subjected to a "prechallenge" with 100 unrestrained fleas in order to determine the number of fleas each individual cat would carry through a seven-day challenge period prior to any treatment such as immunization.
  • Two groups of four cats were vaccinated with TFM, prepared as in Example IB, using 200 ⁇ g protein per 0.5 ml dose and either saponin or RIBI adjuvants for the primary injection, followed by 100 ⁇ g of protein for all subsequent immunizations.
  • Four adjuvant control cats all received saponin alone.
  • the animals were again challenged with 100 unrestrained fleas after the fourth immunization.
  • cat pans and cats were visually checked for fleas daily.
  • living fleas were combed off and enumerated.
  • individual cat ova-cultures were set up from the contents of each pan.
  • the suspension was sonicated 7 times for 20 sec with 2 min rests for cooling.
  • the sonicate was spun at 15,000 x g for 20 min at 4°C to obtain a second supernatant (S2) .
  • S2 second supernatant
  • the pellets from the 15,000 x g spins were reextracted for additional protein. Otherwise they were discarded.
  • the two supernatants SI and S2 above were combined and centrifuged at 100,000 x g for 2 hr.
  • the resulting supernatant is designated gut flea supernatant (GFS) and has a volume of 96 ml at 82 ⁇ g protein/ml, i.e. 7.9 mg total protein.
  • the pellet from the 100,000 x g spin is designated gut flea membrane (GFM) and is resuspended by glass homogenizer in 2 ml buffer. From 11,000 midguts, this pellet (diluted to 6 ml) gave 928 ⁇ g protein of which, after withdrawal of aliquots for assay, 836 ⁇ g remained.
  • GFM gut flea membrane
  • GFM was prepared with a single centrifugation step. Two hundred tubes of 100 dissected unfed midguts each were thawed on ice and centrifuged in an Eppendorf microfuge for 5 minutes at maximum speed at 4°C. Approximately 100 ⁇ l of supernatant was removed from each tube, pooled, and designated "low speed supernatant". Each pellet was resuspended in 50 ⁇ l of flea antigen buffer (1 x PBS, 1 mM EDTA) . In groups of 1000 organs, the suspended pellets were transferred to a 1 ml conical glass/glass homogenizer and homogenized on ice to a smooth suspension (-17 strokes) .
  • each prep was sonicated in an ice water bath for 2 minutes in 15 second bursts at 40% power using a microprobe.
  • the sonicated material was centrifuged in 2 aliquots (10,000 organs each) in a Beckman type 65 rotor for 2 hours at -100,000 x g (4°C) .
  • the "high speed" supernate from the 100,000 x g spin was added to the low speed supernatant for a total volume of 35 ml. This was designated gut flea supernatant (GFS) .
  • Total protein in GFS from 20,000 organs was 2.88 mg.
  • the two membrane pellets were resuspended in 200 ⁇ l flea antigen buffer each and frozen. Pellets were thawed, resuspended in a total volume of 2 ml, sonicated at 40% power for 2 minutes in 15 second bursts in ice water bath, and diluted to a final volume of 5 ml. This preparation was designated GFM. The total GFM protein yield from 20,000 organs was 2.5 mg.
  • Figure 4 shows a silver-stained polyacrylamide gel of GFS (lane 1) and GFM (lane 2) .
  • the first immmunization (week 0) contained 10 ⁇ g GFM as described in example 4A with 500 ⁇ g Quil A adjuvant in 2 mis total volume.
  • the second immunization (week 4) contained 10 ⁇ g GFM with 1 mg Quil A adjuvant in 2 ml total volume.
  • the third immunization (week 8) contained 25 ⁇ g GFM with RIBI adjuvant (prepared per manufacturer) in 1 ml total volume.
  • Four subsequent boosts contained varying amounts of GFM with RIBI adjuvant in 1 ml volumes.
  • the cats were challenged with flea infestation as described in Example 3. The results were evaluated in terms of the percentage of fleas recovered after vaccination in comparison with the percentage of fleas recovered prior to vaccination in vaccinates and the control groups. The percentage of fleas combed off after vaccination were, on the average, 32% lower (p ⁇ 0.1) than the percentage recovered before, whereas in the control groups the fleas combed off at a similar time were only 7% lower than the previous percentage. In comparing the flea counts of vaccinated versus unvaccinated cats at comparable "postvaccination" times, the vaccinates harbored, on the average, 33% fewer fleas.
  • Example 4B 0.33 ml of the 5 ml total GFM preparation described in Example 4B with 1.7 ml of flea antigen buffer (1 x PBS with EDTA) was combined in one vial (x3) of RIBI and vortexed for three min. One ml (approximately 80 ⁇ g protein) was administered intramuscularly to each of 6 cats.
  • ml approximately 80 ⁇ g protein
  • 2 ml of the total 35 ml combined super tant preparation of Example 4B was added to one vial (x3) of RIBI and vortexed for 3 min.
  • One ml (approximately 80 ⁇ g protein) was administered intramuscularly to each of 6 cats.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Insects & Arthropods (AREA)
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  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Vaccin servant à protéger les oiseaux et les mammifères contre l'infestation par des puces et comprenant des membranes ou des extraits de membranes de puces, ou des constituants de ceux-ci. Ce vaccin a aussi pour effet de réduire les populations de puces dans l'environnement du sujet. Les anticorps formés par ces vaccins sont également utiles pour l'assainissement et la diagnose. On donne une préférence particulière aux membranes provenant d'intestins de puce et aux constituants desdites membranes.
PCT/US1991/005852 1990-08-22 1991-08-15 Vaccins antipuces derives de membranes Ceased WO1992003156A1 (fr)

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US57125790A 1990-08-22 1990-08-22
US571,257 1990-08-22

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WO1992003156A1 true WO1992003156A1 (fr) 1992-03-05

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011271A1 (fr) * 1994-10-07 1996-04-18 Heska Corporation Nouvelles proteines de la salive ectoparasites et appareil de collecte de ces proteines
WO1996014089A1 (fr) * 1994-11-03 1996-05-17 Zoogen, Inc. Procedes et compositions pour diagnostiquer des allergies aux puces
US7629446B2 (en) 1994-10-07 2009-12-08 Heska Corporation Nucleic acid molecules encoding a novel ectoparasite saliva protein
EP3415010A1 (fr) 2017-06-13 2018-12-19 Agrosavfe Nv Polypeptides anti-insectes et procédés

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993018788A1 (fr) * 1992-03-24 1993-09-30 The University Of Queensland Antigene de diagnostic et de desensibilisation concernant la dermatite allergique due aux mouches chez l'animal

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
IMMUNOLOGY, Volume 67, issued 1989, OPDEBEECK et al., "Hereford cattle protected against Boophilus microplus with antigens purified by immunoaffinity, chromatography from larval and adult ticks", pages 388-393. *
PARASITE IMMUNOLOGY, Volume 10, issued 1988, OPDEBEECK et al., "Hereford cattle immunized and protected against Boophilus microplus with soluble and membrane-associated antigens from the midgut of ticks", pages 405-410. *
PHYSIOLOGICAL ENTOMOLOGY, Volume 1, issued 1976, SCHLEIN et al., "Lesions in hematophagous flies after feeding on rabbits immunized with fly tissues", pages 55-59. *
THE JOURNAL OF IMMUNOLOGY, Vol. 143, No. 4, issued 15 August 1989, WILLADSEN et al., "Immunologic Control of a parasitic arthropod", pages 1346-1351. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011271A1 (fr) * 1994-10-07 1996-04-18 Heska Corporation Nouvelles proteines de la salive ectoparasites et appareil de collecte de ces proteines
JP2007181463A (ja) * 1994-10-07 2007-07-19 Heska Corp 新規な外部寄生生物唾液タンパク質及びそのようなタンパク質を収集する装置
US7629446B2 (en) 1994-10-07 2009-12-08 Heska Corporation Nucleic acid molecules encoding a novel ectoparasite saliva protein
WO1996014089A1 (fr) * 1994-11-03 1996-05-17 Zoogen, Inc. Procedes et compositions pour diagnostiquer des allergies aux puces
EP3415010A1 (fr) 2017-06-13 2018-12-19 Agrosavfe Nv Polypeptides anti-insectes et procédés

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