Novel ectoparasite saliva proteins and apparatus to collect such proteins

Abstract

The present invention is directed to a novel product and method for isolating ectoparasite saliva proteins, and a novel product and method for detecting and/or treating allergic dermatitis in an animal. The present invention includes a saliva protein collection apparatus capable of collecting ectoparasite saliva proteins substantially free of contaminating material. The present invention also relates to ectoparasite saliva proteins, nucleic acid molecules having sequences that encode such proteins, and antibodies raised against such proteins. The present invention also includes methods to obtain such proteins and to use such proteins to identify animals susceptible to or having allergic dermatitis. The present invention also includes therapeutic compositions comprising such proteins and their use to treat animals susceptible to or having allergic dermatitis.

What is claimed is:  
     
         1 . A formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts.  
     
     
         2 . A formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein represented as a protein peak in FIG. 2.  
     
     
         3 . A formulation comprising at least one isolated ectoparasite saliva product substantially free of contaminating material, said formulation being produced by a process comprising: 
 (a) collecting ectoparasite saliva products on a collection means within a saliva collection apparatus containing ectoparasites, said apparatus comprising: 
 (i) a housing operatively connected to a chamber, said chamber having an ambient temperature warmer than said housing thereby forming a temperature differential between said housing and said chamber, said housing being capable of retaining ectoparasites; and  
 (ii) an interface between said housing and said chamber, said interface comprising ((a)) a means capable of collecting at least a portion of saliva products deposited by ectoparasites retained in said apparatus and ((b)) a barrier means capable of substantially preventing contaminating material from contacting said collection means, wherein said temperature differential attracts ectoparasites retained in said housing to attempt to feed through said barrier means and collection means and, thereby, deposit saliva products on said collection means; and  
   (b) extracting said saliva products from said collection means to obtain said formulation.    
     
     
         4 . A formulation comprising an ectoparasite saliva product, wherein said formulation, when submitted to Tris glycine SDS-PAGE, comprises a fractionation profile as depicted in a figure selected from the group consisting of FIG. 1B, lane 13 and FIG. 1B, lane 14.  
     
     
         5 . A therapeutic composition for treating allergic dermatitis comprising a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts.  
     
     
         6 . An assay kit for testing if an animal is susceptible to or has allergic dermatitis, said kit comprising: 
 (a) a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts; and    (b) a means for determining if said animal is susceptible to or has allergic dermatitis, wherein said means comprises use of said formulation to identify animals susceptible to or having allergic dermatitis.    
     
     
         7 . An apparatus to collect ectoparasite saliva products for use in a formulation that are substantially free of contaminating material, said apparatus comprising: 
 (a) a housing operatively connected to a chamber, said chamber having an ambient temperature warmer than said housing thereby forming a temperature differential between said housing and said chamber, said housing being capable of retaining ectoparasites; and    (b) an interface between said housing and said chamber, said interface comprising (i) a means capable of collecting at least a portion of saliva products deposited by ectoparasites retained in said apparatus and (ii) a barrier means capable of substantially preventing contaminating material from contacting said collection means, wherein said temperature differential attracts ectoparasites retained in said housing to attempt to feed through said barrier means and collection means and, thereby, deposit saliva products on said collection means.    
     
     
         8 . A method to produce a formulation comprising ectoparasite saliva products, wherein said formulation is substantially free of contaminating material, said method comprising: 
 (a) collecting ectoparasite saliva products on a collection means within a saliva collection apparatus containing ectoparasites, said apparatus comprising: 
 (i) a housing operatively connected to a chamber, said chamber having an ambient temperature warmer than said housing thereby forming a temperature differential between said housing and said chamber, said housing being capable of retaining ectoparasites; and  
 (ii) an interface between said housing and said chamber, said interface comprising ((a)) a means capable of collecting at least a portion of saliva products deposited by ectoparasites retained in said apparatus and ((b)) a barrier means capable of substantially preventing contaminating material from contacting said collection means, wherein said temperature differential attracts ectoparasites retained in said housing to attempt to feed through said barrier means and collection means and, thereby, deposit saliva products on said collection means; and  
   (b) extracting said products from said collection means to form said formulation.    
     
     
         9 . A method to identify an animal susceptible to or having allergic dermatitis, said method comprising: 
 (a) administering to a site on said animal a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts, and administering to a different site on said animal a control solution selected from the group consisting of positive control solutions and negative control solutions; and    (b) comparing a reaction resulting from administration of said formulation with a reaction resulting from administration of said control solution, wherein said animal is determined to be susceptible to or to have allergic dermatitis if said reaction to said formulation is at least as large as said reaction to said positive control solution, and wherein said animal is determined not to be susceptible to or not to have allergic dermatitis if said reaction to said formulation is about the same size as said reaction to said negative control solution.    
     
     
         10 . A method to identify an animal susceptible to or having allergic dermatitis by measuring the presence of antibodies indicative of allergic dermatitis in said animal, said method comprising: 
 (a) contacting a formulation with a body fluid from said animal under conditions sufficient for formation of an immunocomplex between said formulation and said antibodies, if present, in said body fluid, said formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts; and    (b) determining the amount of immunocomplex formed, wherein formation of said immunocomplex indicates that said animal is susceptible to or has allergic dermatitis.    
     
     
         11 . A method to desensitize a host animal to allergic dermatitis, comprising administering to said animal a therapeutic composition comprising a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts.  
     
     
         12 . A method for prescribing treatment for allergic dermatitis, comprising: 
 (a) identifying an animal that is susceptible to or has allergic dermatitis by an in vivo or in vitro assay comprising a formulation comprising at least one isolated ectoparasite saliva protein, wherein said ectoparasite saliva protein comprises at least a portion of an amino acid sequence, wherein said portion is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule that encodes a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts; and    (b) prescribing a treatment comprising administering said formulation to said animal.    
     
     
         13 . The invention of  claim 1 ,  2 ,  5 ,  6 ,  9 ,  10 ,  11  or  12 , wherein said a flea saliva protein is selected from the group consisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3.  
     
     
         14 . The invention of  claim 1 ,  2 ,  5 ,  6 ,  9 ,  10 ,  11  or  12 , wherein said flea saliva protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:56.  
     
     
         15 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  61 ,  62 ,  63  or  65 , wherein said ectoparasite is selected from the group consisting of fleas, flies, mosquitoes, ticks, mites, lice, spiders, ants and true bugs.  
     
     
         16 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  61 ,  62 ,  63  or  65 , wherein said ectoparasite comprises a flea.  
     
     
         17 . The invention of  claim 16 , wherein said flea is of a species selected from the group consisting of  Ctenocephalides canis  and  Ctenocephalides felis.    
     
     
         18 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  9 ,  10 ,  11 ,  12 ,  61 ,  62 ,  63  or  65 , wherein said ectoparasite saliva protein or product is selected from the group consisting of ectoparasite saliva proteins having molecular weights ranging from about 6 kilodaltons to about 65 kilodaltons as determined by Tris-glycine SDS-PAGE.  
     
     
         19 . The invention of  claim 1 ,  3 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises a flea saliva extract selected from the group consisting of FS-1 flea saliva extract, FS-2 flea saliva extract, FS-3 flea saliva extract and mixtures thereof.  
     
     
         20 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  61 ,  62 ,  63 ,  64  or  65 , wherein said formulation or a formulation comprising an expression product of said nucleic acid molecule, comprises at least a portion of at least one flea saliva protein selected from the group consisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3.  
     
     
         21 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises an ectoparasite saliva protein selected from the group consisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2, fspN3, and mixtures thereof.  
     
     
         22 . The invention of  claim 1 ,  2 ,  4 ,  5 ,  6 ,  9 ,  10 ,  11  or  12 , wherein said formulation is produced by a process comprising: 
 (a) collecting ectoparasite saliva products on a collection means within a saliva collection apparatus containing ectoparasites, said apparatus comprising: 
 (i) a housing operatively connected to a chamber, said chamber having an ambient temperature warmer than said housing thereby forming a temperature differential between said housing and said chamber, said housing being capable of retaining ectoparasites; and  
 (ii) an interface between said housing and said chamber, said interface comprising ((a)) a means capable of collecting at least a portion of saliva products deposited by ectoparasites retained in said apparatus and ((b)) a barrier means capable of substantially preventing contaminating material from contacting said collection means, wherein said temperature differential attracts ectoparasites retained in said housing to attempt to feed through said barrier means and collection means and, thereby, deposit saliva products on said collection means; and  
 
 (b) extracting said products from said collection means to form said formulation.  
 
     
     
         23 . The invention of  claim 22 , wherein the ambient temperature in said chamber ranges from about 20° C. to about 45° C. and the ambient temperature in said housing ranges from about 5° C. to about 35° C.  
     
     
         24 . The invention of  claim 22 , wherein the relative humidity in said chamber ranges from about 50% relative humidity to about 100% relative humidity and the relative humidity in said housing ranges from about 40% relative humidity to about 60% relative humidity.  
     
     
         25 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation is produced by a process comprising: 
 (a) collecting ectoparasite saliva products on a collection means within a saliva collection apparatus containing ectoparasites, said apparatus comprising: 
 (i) a housing operatively connected to a chamber, said chamber having an ambient temperature warmer than said housing thereby forming a temperature differential between said housing and said chamber, said housing being capable of retaining ectoparasites; and  
 (ii) an interface between said housing and said chamber, said interface comprising ((a)) a means capable of collecting at least a portion of saliva products deposited by ectoparasites retained in said apparatus and ((b)) a barrier means capable of substantially preventing contaminating material from contacting said collection means, wherein said temperature differential attracts ectoparasites retained in said housing to attempt to feed through said barrier means and collection means and, thereby, deposit saliva products on said collection means;  
 
 (b) extracting said products from said collection means with a solution to form an ectoparasite extract;  
 (c) fractionating said ectoparasite extract to obtain separated peak fractions; and  
 (d) recovering at least one of said peak fractions substantially free of the remaining fractions to obtain said formulation.  
 
     
     
         26 . The invention of  claim 25 , wherein separated peak fractions obtained in step (c) comprise a fractionation profile as depicted in FIG. 2.  
     
     
         27 . The invention of  claim 1 ,  2 ,  5 ,  6 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises an ectoparasite saliva protein produced by a process comprising culturing a recombinant cell transformed with at least one nucleic acid molecule encoding at least one of said ectoparasite saliva proteins to produce said formulation.  
     
     
         28 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  61 ,  62 ,  63 ,  64  or  65 , wherein said formulation or a formulation comprising an expression product of said nucleic acid molecule, when administered to a host animal, is capable of substantially desensitizing said animal to allergic dermatitis.  
     
     
         29 . The invention of  claim 28 , wherein said allergic dermatitis is selected from the group consisting of flea allergy dermatitis, mosquito allergy dermatitis and Culicoides allergy dermatitis.  
     
     
         30 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  61 ,  62 ,  63 ,  64  or  65 , wherein said formulation or a formulation comprising an expression product of said nucleic acid molecule is used to identify animals susceptible to or having allergic dermatitis.  
     
     
         31 . The invention of  claim 30 , wherein said allergic dermatitis is selected from the group consisting of flea allergy dermatitis, mosquito allergy dermatitis and Culicoides allergy dermatitis.  
     
     
         32 . An isolated antibody capable of selectively binding to an ectoparasite saliva protein or product as set forth in  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 .  
     
     
         33 . The invention of  claim 1 ,  2 ,  4 ,  5 ,  6 ,  9 ,  10 ,  11  or  12 , wherein said formulation is substantially free of contaminating material.  
     
     
         34 . The invention of  claim 33 , wherein said contaminating material comprises material selected from the group consisting of blood proteins, fecal material, larval culture medium, and mixtures thereof.  
     
     
         35 . The invention of  claim 2  or  62 , wherein said peak is selected from the group consisting of peak A, peak B, peak C, peak D, peak E, peak F, peak G, peak H, peak I, peak J, peak K, peak L, peak M and peak N.  
     
     
         36 . The invention of  claim 1 ,  2 ,  3 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  61 ,  62 ,  63 ,  64  or  65 , wherein said formulation or a formulation comprising an expression product of said nucleic acid molecule, when submitted to Tris glycine SDS-PAGE, comprises a fractionation profile as depicted in a figure selected from the group consisting of FIG. 1B, lane 13 and FIG. 1B, lane 14.  
     
     
         37 . The invention of  claim 1 ,  2 ,  3 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises less than about 50 percent of contaminating material.  
     
     
         38 . The invention of  claim 1 ,  2 ,  3 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises less than about 10 percent of contaminating material.  
     
     
         39 . The invention of  claim 5 ,  6 ,  9 ,  10 ,  11 ,  12  or  63 , wherein said composition further comprises at least one component selected from the group consisting of an excipient, an adjuvant and a carrier.  
     
     
         40 . The invention of  claim 5 ,  11  or  63 , wherein said composition comprises a controlled release composition.  
     
     
         41 . The invention of  claim 6 , wherein said means of determining is selected from the group consisting of in vivo tests and in vitro tests.  
     
     
         42 . The invention of  claim 41 , wherein said in vivo test comprises a skin test comprising: 
 (a) administering to a site on said animal said formulation and administering to a different site on said animal a control solution selected from the group consisting of positive control solutions and negative control solutions; and    (b) comparing a reaction resulting from administration of said formulation with a reaction resulting from administration of said control solution, wherein said animal is determined to be susceptible to or to have allergic dermatitis if said reaction to said formulation is at least as large as said reaction to said positive control solution, and wherein said animal is determined not to be susceptible to or not to have allergic dermatitis if said reaction to said formulation is about the same size as said reaction to said negative control solution.    
     
     
         43 . The invention of  claim 6 ,  9 ,  10  or  12 , wherein said kit or said method detects hypersensitivity selected from the group consisting of immediate hypersensitivity and delayed hypersensitivity.  
     
     
         44 . The invention of  claim 9  or  42 , wherein said reaction is selected from the group consisting of a wheal, induration, erythema, and combinations thereof.  
     
     
         45 . The invention of  claim 41 , wherein said in vitro test comprises a method for measuring the presence of antibodies indicative of allergic dermatitis in said animal, said method comprising: 
 (a) contacting said formulation with a body fluid from said animal under conditions sufficient for formation of an immunocomplex between said formulation and said antibodies, if present, in said body fluid; and    (b) determining the amount of immunocomplex formed, wherein formation of said immunocomplex indicates that said animal is susceptible to or has allergic dermatitis.    
     
     
         46 . The invention of  claim 6  or  10 , wherein said formulation is immobilized on a substrate.  
     
     
         47 . The invention of  claim 10  or  45 , wherein said antibodies comprise immunoglobulin IgE antibodies.  
     
     
         48 . The invention of  claim 10  or  45 , wherein said kit detects immediate hypersensitivity in said animal.  
     
     
         49 . The apparatus of  claim 3 ,  7  or  8 , wherein said collection means comprises a membrane made of material capable of binding said products in such a manner that said products can be eluted from said membrane.  
     
     
         50 . The apparatus of  claim 3 ,  7  or  8 , wherein said membrane comprises a material selected from the group consisting of polyvinyl difluoride, cellulose esters, nitrocellulose, nylon, polysulfone, and polytetrafluoroethylene.  
     
     
         51 . The apparatus of  claim 3 ,  7  or  8 , wherein said membrane comprises a Durapore™ membrane.  
     
     
         52 . The apparatus of  claim 3 ,  7  or  8 , wherein said membrane comprises a DE-81 chromatography paper membrane.  
     
     
         53 . The apparatus of  claim 3 ,  7  or  8 , wherein said barrier means comprises a material selected from the group consisting of plastic, teflon, cloth, paper, paraffin and wax.  
     
     
         54 . The apparatus of  claim 3 ,  7  or  8 , wherein said barrier means comprises Parafilm™.  
     
     
         55 . The apparatus of  claim 3 ,  7  or  8 , wherein said apparatus further comprises a blotting means capable of maintaining a humidity suitable for survival of said ectoparasite.  
     
     
         56 . An isolated nucleic acid molecule that encodes the ectoparasite saliva protein of  claim 1 ,  2  or  3 .  
     
     
         57 . An isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene encoding a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts.  
     
     
         58 . A method for producing at least one ectoparasite saliva protein, comprising: 
 (a) culturing a cell transformed with at least one nucleic acid that hybridizes under stringent hybridization conditions with a gene encoding a flea saliva protein present in a flea saliva extract selected from the group consisting of FS-1, FS-2 and FS-3 flea saliva extracts to produce said protein; and    (b) recovering said ectoparasite saliva protein.    
     
     
         59 . The invention of  claim 56 ,  57  or  58 , wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule encoding a flea saliva protein selected from the group consisting of fspA, fspB, fspC1, fspC2, fspD1, fspD2, fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3.  
     
     
         60 . The invention of  claim 56 ,  57  or  58 , wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:50, SEQ ID NO:52 and SEQ ID NO:55.  
     
     
         61 . A recombinant molecule comprising at least one isolated nucleic acid molecule as set forth in  claim 56 ,  57  or  58 , operatively linked to at least one transcription control sequence.  
     
     
         62 . A recombinant cell comprising a cell having at least one isolated nucleic acid molecule as set forth in  claim 56 ,  57  or  58 , said cell being capable of expressing said nucleic acid molecule.  
     
     
         63 . A therapeutic composition comprising at least one isolated nucleic acid molecule as set forth in  claim 56 ,  57  or  58 .  
     
     
         64 . The invention of  claim 4 , wherein said formulation comprises a flea saliva extract selected from the group consisting of FS-1 flea saliva extract, FS-2 flea saliva extract and mixtures thereof.  
     
     
         65 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises at least a portion of at least one flea saliva protein selected from the group consisting of fspE, fspF, fspG1, fspG2, fspG3, fspH, fspI, fspJ1, fspJ2, fspK, fspL1, fspL2, fspM1, fspM2, fspN1, fspN2 and fspN3.  
     
     
         66 . The invention of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11  or  12 , wherein said formulation comprises at least a portion of at least one flea saliva protein selected from the group consisting of fspG1, fspG2, fspG3, fspH, fspM1, fspM2, fspN1, fspN2 and fspN3.  
     
     
         67 . The invention of  claim 10  or  45 , wherein said body fluid is pretreated to remove non-IgE antibodies from said fluid.