BRPI0920387B1 - COMPOSITIONS AND METHODS FOR DETECTING SPECIFIC ANTIBODIES FOR ANAPLASMA PHAGOCYTOPHILUM (APH) AND ANAPLASMA PLATYS (APL) - Google Patents

Abstract

COMPOSITIONS AND METHODS FOR DETECTING SPECIFIC ANTIBODIES FOR ANAPLASMA PHAGOCYTOPHILUM (APH) AND ANAPLASMA PLATYS (APL). The present invention relates to methods and compositions for detecting and treating anaplasmaphagocytophilum and Anaplasma platys infection.

Description

PRIORITY

[001] This patent application claims the benefit of U.S. Application No. 61/103,743 filed October 8, 2008, which is incorporated by reference into this application in its entirety.

BACKGROUND OF THE INVENTION

[002] Anaplasmosis occurs in mammals, including, for example, humans, horses, sheep, dogs, cats, deer and ruminants and is caused by infection of granulocytic cells with the tick-borne agent Anaplasma phagocytophilum ("Aph") (formerly known as Ehrlichia equi). Common clinical symptoms include fever, lethargy, lameness, thrombocytopenia, swollen lymph nodes and anorexia, all of which are non-specific for anaplasmosis. Therefore, specific testing is important for correct diagnosis.

[003] Anaplasma platys ("Apl") (formerly known as Ehrlichia platys) is a very closely related species. Apl can cause infectious canine cyclic thrombocytopenia (ICCT). Infected dogs are usually asymptomatic in the U.S., but can become clinically ill in other parts of the world. Current serologic tests for Anaplasma cannot differentiate the two species because of significant cross-reactivity. Methods for detecting Aph and Apl and methods for differentiating between the two infections are needed in the art.

[004] The onset of clinical symptoms occurring during the acute phase of anaplasmosis may precede the advent of measurable levels of antibodies against some Anaplasma antigens. Thus, there is a need for a rapid, sensitive and reliable immunological test for Aph infection, Apl infection, or both, for example, in mammals exhibiting the clinical symptoms of acute anaplasmosis.

SUMMARY OF THE INVENTION

[005] One embodiment of the invention provides a composition comprising one or more purified polypeptides consisting of an amino acid sequence set forth as SEQ ID NO:8 or SEQ ID NO:10; or consisting of: amino acids 1 to 45 of SEQ ID NO:10; amino acids 41 to 89 of SEQ ID NO:10; amino acids 85 to 130 of SEQ ID NO:10; amino acids 126 to 160 of SEQ ID NO:10; amino acids 129 to 146 of SEQ ID NO:10; amino acids 144 to 160 of SEQ ID NO:10; or amino acids 136 to 155 of SEQ ID NO:10; or consisting of at least 17 contiguous amino acids of an amino acid sequence set forth as SEQ ID NOs:1 to 21; or consisting of a polypeptide having at least about 94% identity to SEQ ID NOs:1 to 21. The one or more purified polypeptides may be linked to an indicator reagent, a signal sequence, a stop transfer sequence, an amino acid spacer, a transmembrane domain, a protein purification linker, a heterologous polypeptide, a moiety that enhances an immune response, a moiety that facilitates purification, a moiety that facilitates polypeptide stability, one or more additional polypeptides comprising SEQ ID NOs:1 to 21, or a combination thereof. Another embodiment of the invention provides an isolated polynucleotide encoding one or more of the purified polypeptides.

[006] Yet another embodiment of the invention provides a method of detecting antibodies that specifically bind to an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both in a test sample. The method comprises contacting the composition comprising one or more purified polypeptides with the test sample under conditions that allow polypeptide/antibody complexes to form. Polypeptide/antibody complexes are detected. Detection of the polypeptide/antibody complexes is an indication that antibodies specific for an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both are present in the test sample. The composition comprising one of the more purified polypeptides may consist of at least 17 contiguous amino acids of an amino acid sequence set forth as SEQ ID NOs:8 or 19. Detection of the polypeptide/antibody complexes is an indication that antibodies specific for Anaplasma platys are present in the test sample. The composition comprising one of the most purified polypeptides may consist of at least 17 contiguous amino acids of an amino acid sequence set forth as SEQ ID NO:10; amino acids 41 to 89 of SEQ ID NO:10; or amino acids 85 to 130 of SEQ ID NO:10; or may consist of a polypeptide having at least approximately 94% identity to SEQ ID NOs:10, amino acids 41 to 89 of SEQ ID NO:10, or amino acids 85 to 130 of SEQ ID NO:10. Detection of the polypeptide/antibody complexes is an indication that antibodies specific for an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both are present in the test sample. The composition comprising one of the most purified polypeptides may consist of at least 17 contiguous amino acids from amino acids 1 to 45 of SEQ ID NO:10; amino acids 126 to 160 of SEQ ID NO:10; amino acids 129 to 146 of SEQ ID NO:10; amino acids 144 to 160 of SEQ ID NO:10; amino acids 136 to 155 of SEQ ID NO:10; or may consist of a polypeptide having at least about 94% identity to amino acids 1 to 45 of SEQ ID NO:10; amino acids 126 to 160 of SEQ ID NO:10; amino acids 129 to 146 of SEQ ID NO:10; amino acids 144 to 160 of SEQ ID NO:10; amino acids 136 to 155 of SEQ ID NO:10. Detection of polypeptide/antibody complexes is an indication that antibodies specific for an Anaplasma phagocytophilum polypeptide are present in the test specimen. The complexes may be contacted with an indicator reagent prior to detection. The amount of antibodies in the test sample can be determined. One or more purified polypeptides can be bound to a substrate.

[007] Yet another embodiment of the invention provides an antibody that specifically binds to a polypeptide consisting of SEQ ID NOs:1 to 21. The antibody can be a monoclonal antibody, polyclonal antibody, a Fab fragment, a Fab' fragment, Fab'-SH fragment, F(ab')2 fragment, Fv fragment, or a single chain antibody.

[008] Yet another embodiment of the invention provides a method of detecting an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both in a sample. The method comprises contacting one or more antibodies that specifically bind to one or more purified polypeptides of the invention with the sample under conditions that allow polypeptide/antibody complexes to form. The polypeptide/antibody complexes are detected. Detection of the polypeptide/antibody complexes is an indication that an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both are present in the sample. The one or more purified polypeptides may consist of at least 17 contiguous amino acids from amino acids 1 to 45 of SEQ ID NO:10, amino acids 126 to 160 of SEQ ID NO:10; amino acids 129 to 146 of SEQ ID NO:10; amino acids 144 to 160 of SEQ ID NO:10; or amino acids 136 to 155 of SEQ ID NO:10. Detection of polypeptide/antibody complexes is an indication that Anaplasma phagocytophilum polypeptides are present in the test specimen. The one or more purified polypeptides may consist of at least 17 contiguous amino acids of an amino acid sequence set forth as SEQ ID NOs:8 or 19. Detection of polypeptide/antibody complexes is an indication that Anaplasma platys polypeptides are present in the test specimen. The one or more purified polypeptides may consist of at least 17 contiguous amino acids of an amino acid sequence set forth as SEQ ID NOs:10; amino acids 41 to 89 of SEQ ID NO:10; or amino acids 85 to 130 of SEQ ID NO:10. Detection of polypeptide/antibody complexes is an indication that an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide is present in the test specimen. The one or more antibodies may be monoclonal antibodies, polyclonal antibodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, or single-chain antibodies.

[009] In another embodiment of the invention, a composition comprising purified polypeptides of the invention further comprises a pharmaceutically acceptable or veterinarily acceptable carrier and/or an adjuvant.

[0010] Yet another embodiment of the invention provides a method of treating or ameliorating Anaplasma platys infection, Anaplasma phagocytophilum infection, or both, to a mammalian subject comprising administering to the mammalian subject a therapeutically effective amount of a composition comprising one or more of the purified polypeptides of the invention.

[0011] Yet another embodiment of the invention provides a method of inducing an immune response in a mammal comprising administering an immunologically effective amount of a composition comprising one or more of the purified polypeptides of the invention.

[0012] The invention therefore provides methods and compositions for detecting and treating Apl and/or Aph infection.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 shows the results of complete assays with polypeptides comprising SEQ ID NOs: 12 to 18 and 10.

[0014] Figure 2 shows the results of complete assays with polypeptides comprising SEQ ID NOs:15, 19, and 10.

[0015] Figure 3 shows the results of complete assays with polypeptides comprising SEQ ID NOs:15, 19, and 10.

[0016] Figure 4 shows the results of complete time course assays with polypeptides comprising SEQ ID NOs:15, 19, and 10.

[0017] Figure 5 shows the results of complete time course assays with polypeptides comprising SEQ ID NOs:15 and 10.

[0018] Figure 6 shows the results of complete assays with polypeptides comprising SEQ ID NOs:12 to 15.

[0019] Figure 7A-B shows the results of complete assays with polypeptides comprising SEQ ID NO:20.

DETAILED DESCRIPTION OF THE INVENTION POLYPEPTIDES OF THE INVENTION

[0020] As used in this application, the singular forms "a", "the", "a" and "one" include the plural referents unless the context clearly dictates otherwise.

[0021] A polypeptide is a polymer of two or more amino acids covalently linked by amide bonds. A polypeptide may be post-translationally modified. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used in the synthesis of the polypeptide, or combinations thereof. A polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used in the synthesis of the polypeptide, etc., has less than approximately 30%, 20%, 10%, 5%, 1% or more of other polypeptides, culture medium, chemical precursors, and/or other chemicals used in the synthesis. Therefore, a purified polypeptide is approximately 70%, 80%, 90%, 95%, 99% or more pure. A purified polypeptide does not include cell extracts or mixtures of unpurified or semi-purified polypeptides that are less than 70% pure.

[0022] The term "polypeptides" may refer to one or more than one type of polypeptide (set of polypeptides). "Polypeptides" may also refer to mixtures of two or more different types of polypeptides (a polypeptide mixture). The terms "polypeptides" or "polypeptide" may also each mean "one or more polypeptides".

[0023] An embodiment of the invention provides one or more of the following polypeptides: p44-3 MEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAK 46 (SEQ ID NO:3)Aph p44-4 YLLAKELAYDVVTGQTDNLAAALAKTSGKDIVQFA 35 (SEQ ID NO:4)Aph p44-4-l AKELAYDVVTGQTDNLAA 18 (SEQ ID NO:5)Aph p44-4-2 LAAALAKTSGKDIVQFA 17 (SEQ ID NO:6)Aph p44-4-3 YLLAKELAYDVVTGQTDKLTAALAKTSGKDFVQFA 35 (SEQ ID NO:9)Aph rp44GSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGETKAVYPYLKDGKSV KLESHKFDWNTPDPRIGFKDNMLVAMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSK EDEADTVYLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFANAVKISSPTIDGKVCS GDHAAIVSTKGKDYKADPKESGNNGHETSQCSGLSSS 213 (SEQ ID NO: 10)

[0024] In one embodiment of the invention, the X at position 143 of SEQ ID NO:10 is K or N; the X at position 145 is T or A; and the X at position 156 is F or I.

[0025] One embodiment of the invention provides the following polypeptides: 1. Amino acids 1 to 45 of SEQ ID NO:10 (Aph p44-1) (which have the same reactivities as SEQ ID NOs:1 and 12). 2. Amino acids 41 to 89 of SEQ ID NO:10 (Aph p44-2) (which have the same reactivities as SEQ ID NOs:2 and 13). 3. Amino acids 85 to 130 of SEQ ID NO:10 (Aph p44-3) (which have the same reactivities as SEQ ID NOs:3 and 14). 4. Amino acids 126 to 160 of SEQ ID NO:10 (Aph p44-4) (which have the same reactivities as SEQ ID NOs:4, 9, 11, 15, and 20). 5. Amino acids 129 to 146 of SEQ ID NO:10 (Aph p44-4-1)(which have the same reactivities as SEQ ID NOs:5 and 16).6. Amino acids 144 to 160 of SEQ ID NO:10 (Aph p44-4-2)(which have the same reactivities as SEQ ID NOs:6 and 17).7. Amino acids 136 to 155 of SEQ ID NO:10 (Aph p44-3)(which have the same reactivities as SEQ ID NOs:7 and 18).

[0026] Furthermore, SEQ ID NOs:8 and 9 have the same reactivities, i.e., the polypeptides specifically bind to antibodies specific for Anaplasma antigens in substantially the same manner.

[0027] In one embodiment of the invention, the polypeptides have an N-terminal C-residue. For example:Aph p44-l CGSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGE (SEQ ID NO: 12)Aph p44-2 CESNGETKAVYPYLKDGKSVKLESHKFDWNTPDPRIGFKDNMLVA MEGSV (SEQ ID NO: 13)Aph p44-3 CMEGSVGYGIGGARVELEIGYERFKTKGIRDSGSKEDEADTVYLLAK (SEQ ID NO: 14)Aph p44-4 CLAAALAKTSGKDIVQFA(SEQ ID NO: 17)Aph p44-4-3 CVVTGQTDNLAAALAKTSGKD(SEQ ID NO: 18)Apl p44-4 CAKKLPHTLVSDQSDKFLEELKNTKAAEIVKFA(SEQ ID NO: 19)p44-4-V CYLLAKELAYDVVTGQTDKLTAALAKTSGKDFVQFA(SEQ ID NO:20)Aph rp44CGSDVRAHDDVSALETGGAGYFYVGLDYSPAFSKIRDFSIRESNGETKAVYPYLKDGKS VKLESHKFDWNTPDPRIGFKDNMLVAMEGSVGYGIGGARVELEIGYERFKTKGIRDSGS KEDEAADTVYLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFANAVKISSPTIDGKVC SGDHAAIVSTKGKDYKADPKESGNNGHETSQCSGLSSS (SEQ ID NO:21).

[0028] In one embodiment of the invention, the X at position 144 of SEQ ID NO:21 is K or N; the X at position 146 is T or A; and the X at position 157 is F or I.

[0029] SEQ ID NOs:4 to 7 and 9 can be aligned as follows:Aph p4 4~4 YLLAKELAYDWTGQTDNLAAALAKTSGKDIVQFA (SEQ ID NO: 4)Aph p44-4-l AKELAYDWTGQTDNLAA (SEQ ID NO: 5)Aph p44-4-2 LAAALAKTSGKDIVQFA (SEQ ID NO:6)Aph p44~4~3 WTGQTDNLAAALAKTSGKD (SEQ ID NO:7)Aph p44-4-V YLLAKELAYDWTGQTDKLTAALAKISGKDFVQFA (SEQ ID NO: 9)

[0030] A consensus sequence of SEQ ID NOs:4 through 7 and 9 is shown in SEQ ID NO:11:SEQ ID NO:11 YLLAKELAYDVVTGQTDXLXAALAKTSGKDXVQFA

[0031] In one embodiment of the invention, the X at position 18 of SEQ ID NO:11 is N or K; the X at position 20 is T or A; and the X at position 31 is F or I. One embodiment of the invention comprises amino acids 4 to 21 of SEQ ID NO:11; amino acids 19 to 34 of SEQ ID NO:11; or amino acids 11 to 30 of SEQ ID NO:11.

[0032] One embodiment provides a purified polypeptide consisting of less than approximately 46, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 contiguous amino acids (or any range between approximately 46 and approximately 6 amino acids) of SEQ ID NOs:1-9 and 11-21. One embodiment provides a purified polypeptide consisting of greater than approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, or 46 contiguous amino acids (or any range between approximately 46 and approximately 6 amino acids) of SEQ ID NOs:1 through 9 and 11 through 12. Naturally occurring Aph or Apl amino acids are any polypeptide naturally produced by an Aph or Apl organism. A purified polypeptide may comprise less than a certain number of contiguous naturally occurring amino acids from Anaplasma (e.g., approximately less than 200, 175, 150, 125, 100, 75, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids (or any range between approximately 200 and approximately 6 amino acids)) of SEQ ID NOs:1 through 21. That is, the purified polypeptide is smaller than the full-length polypeptide. The fact that these polypeptides are smaller than a full-length Anaplasma polypeptide is important because smaller polypeptides may have greater specificity and/or sensitivity than full-length polypeptides in Aph and/or Apl assays. Additionally, these smaller polypeptides may be less expensive to produce, and may be obtained in higher purity, than the full-length polypeptide.

[0033] Another embodiment provides a purified polypeptide consisting of less than approximately 200, 175, 150, 125, 100, 75, 50, 25, 20, 15, 10, 6 or less contiguous naturally occurring amino acids from Anaplasma (or any range between approximately 200 and approximately 6 amino acids) of SEQ ID NO:10. Another embodiment provides a purified polypeptide consisting of greater than approximately 6, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200 or more contiguous naturally occurring amino acids from Anaplasma (or any range between approximately 6 and approximately 200 amino acids) of SEQ ID NO:10.

[0034] Variant polypeptides that are at least approximately 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the polypeptide sequences set forth in SEQ ID NOs:1 to 21 are also polypeptides of the invention. For example, a variant polypeptide of SEQ ID NO:1 can be at least approximately 98% (approximately 1 amino acid modification), 96% (approximately 2 amino acid modifications), 93% (approximately 3 amino acid modifications), 91% (approximately 4 amino acid modifications), 89% (approximately 5 amino acid modifications), 87% (approximately 6 amino acid modifications), 84% (approximately 7 amino acid modifications), 82% (approximately 8 amino acid modifications), or 80% (approximately 9 amino acid modifications) identical to SEQ ID NO:1. A variant polypeptide of SEQ ID NO:2 can be at least approximately 98% (approximately 1 amino acid modification), 96% (approximately 2 amino acid modifications), 94% (approximately 3 amino acid modifications), 92% (approximately 4 amino acid modifications), 90% (approximately 5 amino acid modifications), 88% (approximately 6 amino acid modifications), approximately 86% (approximately 7 amino acid modifications), 84% (approximately 8 amino acid modifications), 82% (approximately 9 amino acid modifications), or 80% (approximately 10 amino acid modifications) identical to SEQ ID NO:2. A variant polypeptide of SEQ ID NO:3 can be at least approximately 98% (approximately 1 amino acid modification), 96% (approximately 2 amino acid modifications), 94% (approximately 3 amino acid modifications), 91% (approximately 4 amino acid modifications), 89% (approximately 5 amino acid modifications), 87% (approximately 6 amino acid modifications), 85% (approximately 7 amino acid modifications), 83% (approximately 8 amino acid modifications), or 80% (approximately 9 amino acid modifications) identical to SEQ ID NO:3. A variant polypeptide of SEQ ID NOs:4 and 9 can be at least approximately 97% (approximately 1 amino acid modification), 94% (approximately 2 amino acid modifications), 91% (approximately 3 amino acid modifications), 89% (approximately 4 amino acid modifications), 86% (approximately 5 amino acid modifications), 83% (approximately 6 amino acid modifications), or 80% (approximately 7 amino acid modifications) identical to SEQ ID NOs:4 and 9. A variant polypeptide of SEQ ID NO:5 can be at least approximately 94% (approximately 1 amino acid modification), 89% (approximately 2 amino acid modifications), or 83% (approximately 3 amino acid modifications) identical to SEQ ID NO:5. A variant polypeptide of SEQ ID NO:6 can be at least approximately 94% (approximately 1 amino acid modification), 88% (approximately 2 amino acid modifications), or 82% (approximately 3 amino acid modifications) identical to SEQ ID NO:6. A variant polypeptide of SEQ ID NO:7 can be at least approximately 95% (approximately 1 amino acid modification), 90% (approximately 2 amino acid modifications), 85% (approximately 3 amino acid modifications), or 80% (approximately 4 amino acid modifications) identical to SEQ ID NO:7. A variant polypeptide of SEQ ID NO:8 can be at least approximately 97% (approximately 1 amino acid modification), 94% (approximately 2 amino acid modifications), 91% (approximately 3 amino acid modifications), 88% (approximately 4 amino acid modifications), 84% (approximately 5 amino acid modifications), or 81% (approximately 6 amino acid modifications) identical to SEQ ID NO:8.

[0035] Variant polypeptides have one or more conservative amino acid variations or other minor modifications and retain biological activity, i.e., they are biologically functional equivalents. A biologically active equivalent has substantially equivalent function when compared to the corresponding wild-type polypeptide. In one embodiment of the invention, a polypeptide has approximately 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or fewer conservative amino acid substitutions.

[0036] Percent sequence identity has a recognized meaning in the art and there are several methods for measuring the identity between two polypeptide or polynucleotide sequences. See, for example, Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin & Griffin, Eds., Computer Analysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991). Methods for aligning polynucleotides or polypeptides are encoded in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wisconsin 53711) that uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 2:482–489 (1981)). For example, the ALIGN computer program that employs the FASTA algorithm can be used, with an affine gap search with an open gap penalty of -12 and a gap extension penalty of -2.

[0037] Using any of the sequence alignment programs to determine whether a particular sequence is, for example, approximately 95% identical to a reference sequence, the parameters are set such that the percent identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.

[0038] Variant polypeptides can generally be identified by modifying one of the polypeptide sequences of the invention, and evaluating the properties of the modified polypeptide to determine whether it is a biological equivalent. A variant is a biological equivalent if it reacts substantially the same as a polypeptide of the invention in an assay, such as an immunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), enzyme-linked immunosorbent assay, or a western blot assay, for example, and has 90 to 110% of the activity of the original polypeptide. In one embodiment, the assay is a competition assay in which the biologically equivalent polypeptide is capable of reducing the binding of the polypeptide of the invention to a corresponding reactive antigen or antibody by up to 80, 95, 99, or 100%. An antibody that specifically binds to a corresponding wild-type polypeptide also specifically binds to the variant polypeptide.

[0039] A conservative substitution is one in which an amino acid is replaced by another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative modifications: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

[0040] A polypeptide of the invention may further comprise a signal (or leader) sequence that co-translationally or post-translationally directs transfer of the protein. The polypeptide may also comprise a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an Fc region of immunoglobulin or bovine serum albumin.

[0041] A polypeptide may be covalently or non-covalently linked to an amino acid sequence with which the polypeptide is not normally associated in nature, i.e., a heterologous amino acid sequence. A heterologous amino acid sequence may be from a non-Anaplasma phagocytophilum, non-Anaplasma platys organism, a synthetic sequence, or an Anaplasma phagocytophilum sequence or Anaplasma platys sequence not normally located at the carboxy or amino terminus of a polypeptide of the invention. Additionally, a polypeptide may be covalently or non-covalently linked to compounds or molecules other than amino acids, such as indicator reagents. A polypeptide may be covalently or non-covalently linked to an amino acid spacer, an amino acid linker, a signal sequence, a stop-transfer sequence, a transmembrane domain, a protein purification linker, or a combination thereof. A polypeptide may also be linked to a moiety (i.e., a functional group which may be a polypeptide or other compound) that enhances an immune response (e.g., cytokines such as IL-2), a moiety that facilitates purification (e.g., affinity tags such as a six-histidine tag, trpE, glutathione, maltose-binding protein), or a moiety that facilitates polypeptide stability (e.g., polyethylene glycol; amino-terminal protecting groups such as acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl, or t-butyloxycarbonyl; carboxyl-terminal protecting groups such as amide, methylamide, and ethylamide). In one embodiment of the invention, a protein purification linker may be one or more C amino acid residues, for example, at the amino terminus or carboxyl terminus of a polypeptide of the invention. An amino acid spacer is a sequence of amino acids that is not associated with a polypeptide of the invention in nature. An amino acid spacer can comprise approximately 1, 5, 10, 20, 100, or 1,000 amino acids.

[0042] If desired, a polypeptide of the invention may be part of a fusion protein, which may also contain other amino acid sequences, such as amino acid linkers, amino acid spacers, signal sequences, TMR transfer stop sequences, transmembrane domains, as well as linkers useful in protein purification, such as glutathione-S-transferase, histidine tag, and Staphylococcus protein A or combinations thereof. Other amino acid sequences may be present at the C- or N-terminus of a polypeptide of the invention to form a fusion protein. More than one polypeptide of the invention may be present in a fusion protein. Fragments of polypeptides of the invention may be present in a fusion protein of the invention. A fusion protein of the invention may comprise one or more polypeptides of the invention, fragments thereof, or combinations thereof.

[0043] The polypeptides of the invention may be in a multimeric form. That is, a polypeptide may comprise one or more copies of a polypeptide of the invention or a combination thereof. A multimeric polypeptide may be multiple antigen peptide (MAP). See e.g., Tam, J. Immunol. Methods, 196:17-32 (1996).

[0044] Polypeptides of the invention may comprise an antigenic determinant that is recognized by an antibody specific for Anaplasma phagocytophilum or Anaplasma platys or for both Anaplasma phagocytophilum and Anaplasma platys. The polypeptide may comprise one or more epitopes (i.e., antigenic determinants). An epitope may be a linear epitope, a sequential epitope, or a conformational epitope. Epitopes within a polypeptide of the invention may be identified by a variety of methods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186 (1988). For example, a polypeptide of the invention may be isolated and screened. A series of short peptides, which together span an entire polypeptide sequence, may be prepared by proteolytic cleavage. Starting with, for example, 30-mer polypeptide fragments, each fragment can be tested for the presence of recognized epitopes in an ELISA. For example, in an ELISA assay, an Anaplasma phagocytophilum polypeptide, such as a 30-mer polypeptide fragment, is bound to a solid support, such as the wells of a multiwell plastic plate. A population of antibodies is labeled, added to the solid support, and allowed to bind to the unlabeled antigen under conditions where nonspecific absorption is blocked, and any unbound antibody and other proteins are washed away. Antibody binding is detected, for example, by a reaction that converts a colorless substrate to a colored reaction product. Progressively smaller and overlapping fragments can then be tested from an identified 30-mer to map the epitope of interest.

[0045] A polypeptide of the invention may be produced recombinantly. A polynucleotide encoding a polypeptide of the invention may be introduced into a recombinant expression vector, which may be expressed in a suitable host cell expression system using techniques well known in the art. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system may be used. Optionally, a polynucleotide encoding a polypeptide may be translated in a cell-free translation system. A polypeptide may also be chemically synthesized or obtained from Anaplasma cells.

[0046] An immunogenic polypeptide of the invention may comprise an amino acid sequence set forth in SEQ ID NOs:1 to 21 or fragments thereof. An immunogenic polypeptide can elicit antibodies or other immune responses (e.g., T cell responses of the immune system) that recognize epitopes of a polypeptide having SEQ ID NOs: 1 to 21. An immunogenic polypeptide of the invention can also be a fragment of a polypeptide having an amino acid sequence in SEQ ID NOs: 1 to 21. An immunogenic polypeptide fragment of the invention can be approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more amino acids in length. An immunogenic polypeptide fragment of the invention may be approximately 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or fewer amino acids in length.

ANAPLASMA POLYNUCLEOTIDES

[0047] Polynucleotides of the invention contain less than an entire microbial genome and may be single or double stranded nucleic acids. A polynucleotide may be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA, or combinations thereof. Polynucleotides may be purified free of other components, such as proteins, lipids, and other polynucleotides. For example, the polynucleotide may be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. A nucleic acid molecule existing among hundreds and millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a restriction digest of genomic DNA should not be considered a purified polynucleotide. Polynucleotides of the invention encode the polypeptides of the invention described above. In one embodiment of the invention, the polynucleotides encode a polypeptide set forth in SEQ ID NOs:1 to 21 or fragments thereof.

[0048] Polynucleotides of the invention may consist of less than approximately 639, 450, 300, 225, 147, 138, 135, 105, 96, 60, 54, 51, 45, 30, 20, 10 or fewer contiguous polynucleotides, whether naturally occurring (i.e., Aph or Apl polynucleotides) or non-naturally occurring. The polynucleotides of the invention may consist of more than approximately 10, 20, 30, 45, 51, 54, 60, 96, 105, 135, 138, 147, 225, 300, 450, 639 or more contiguous polynucleotides, either naturally occurring (i.e., Aph or Apl polynucleotides) or non-naturally occurring. Purified polynucleotides may comprise additional heterologous nucleotides (i.e., nucleotides that are not from Aph or Apl) and even additional Aph or Apl nucleotides as long as they do not naturally occur contiguously with the polynucleotides. The polynucleotides of the invention may comprise other nucleotide sequences, such as coding for linker sequences, signal sequences, TMR transfer stop sequences, transmembrane domains, or linkers useful in protein purification, such as glutathione-S-transferase, histidine tag, and Staphylococcus protein A.

[0049] The polynucleotides of the invention may be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not immediately contiguous with one or both of the 5' and 3' flanking genomic sequences with which it is naturally associated. An isolated polynucleotide may be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid sequences found naturally immediately flanking the recombinant DNA molecule in a naturally occurring genome are removed or absent. Isolated polynucleotides also include nucleic acid molecules that do not occur naturally.

[0050] Polynucleotides of the invention may also comprise fragments encoding immunogenic polypeptides. Polynucleotides of the invention may encode full-length polypeptides, polypeptide fragments, and fusion or variant polypeptides.

[0051] Degenerate nucleotide sequences encoding polypeptides of the invention, as well as homologous nucleotide sequences that are at least approximately 80, or approximately 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to polynucleotide sequences of the invention and complements thereof are also polynucleotides of the invention. Percent sequence identity can be calculated as described in the "Polypeptides" section. Degenerate nucleotide sequences are polynucleotides that encode a polypeptide of the invention or fragments thereof, but differ in nucleic acid sequence from the wild-type polynucleotide sequence, due to the degeneracy of the genetic code. Complementary DNA (cDNA) molecules, species homologues and variants of polynucleotides of the invention that encode biologically functional polypeptides are also polynucleotides of the invention.

[0052] Polynucleotides of the invention can be obtained from nucleic acid sequences present in, for example, a biological sample, such as blood, serum, saliva or tissue from an infected individual. Polynucleotides can also be synthesized in the laboratory, for example, using an automated synthesizer. An amplification method, such as PCR, can be used to amplify polynucleotides from genomic DNA or cDNA encoding the polypeptides.

[0053] Polynucleotides of the invention may comprise coding sequences of naturally occurring polypeptides or may encode altered sequences that do not occur in nature. If desired, polynucleotides may be cloned into an expression vector comprising expression control elements, including, for example, origins of replication, promoters, enhancers, or other regulatory elements that direct expression of the polynucleotides of the invention in host cells. An expression vector may be, for example, a plasmid, such as pBR322, pUC, or ColE1, or an adenovirus vector, such as adenovirus Type 2 vector or adenovirus Type 5 vector. Optionally, other vectors may be used, including but not limited to Sindbis virus, simian virus 40, alphavirus vectors, poxvirus vectors, and cytomegalovirus vectors, and retroviral vectors such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus. Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, virus particles, virus-like particles, cosmids (plasmids into which the COS sites of phage lambda have been inserted), and replicons (genetic elements that are capable of replication under their own control in a cell) may also be used.

[0054] Methods for preparing polynucleotides operatively linked to an expression control sequence and expressing them in a host cell are well known in the art. See, e.g., U.S. Patent No. 4,366,246. A polynucleotide of the invention is operatively linked when it is positioned adjacent or proximate to one or more expression control elements, which direct transcription and/or translation of the polynucleotide.

[0055] Polynucleotides of the invention can be used, for example, as probes or primers, e.g., PCR primers, to detect the presence of Anaplasma polynucleotides in a test sample, such as a biological sample. Probes are molecules capable of interacting with a nucleic acid target, typically in a sequence-specific manner, e.g., through hybridization. Primers are a subset of probes that can withstand enzymatic manipulation and that can hybridize to a target nucleic acid such that enzymatic manipulation occurs. A primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with enzymatic manipulation.

[0056] A probe or primer may be approximately 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more contiguous nucleotides that encode polypeptides of the invention.

[0057] Hybridization of nucleic acids is well understood in the art and discussed in this application. Typically a probe can be produced from any combination of nucleotides or nucleotide derivatives or analogs available in the art. The ability of such probes and primers to specifically hybridize to polynucleotide sequences of Anaplasma phagocytophilum or Anaplasma platys will allow them to be used in detecting the presence of complementary sequences in a given test sample. Polynucleotide probes and primers of the invention can hybridize to complementary sequences in a test sample, such as a biological sample, including saliva, sputum, blood, plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate or tissue. Polynucleotides from the sample, for example, can be subjected to gel electrophoresis or other size separation techniques or can be immobilized without size separation. Polynucleotide probes and primers may be labeled. Suitable labels, and methods for labeling probes and primers, are known in the art, and include, for example, nick-translationally or kinase-incorporated radioactive labels, biotin labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, metal chelator labels, and enzymatic labels. The sample polynucleotides are contacted with the probes or primers under hybridization conditions of suitable stringency.

[0058] Depending on the application, varying hybridization conditions may be used to achieve varying degrees of probe or primer selectivity toward the target sequence. For applications requiring high selectivity, relatively stringent conditions may be used, such as low salt conditions and/or high temperatures, such as a salt concentration of approximately 0.02 M to approximately 0.15 M salt at temperatures ranging from approximately 50°C to approximately 70°C. For applications requiring less selectivity, less stringent hybridization conditions may be used. For example, salt conditions of approximately 0.14 M to approximately 0.9 M salt, at temperatures ranging from approximately 20°C to approximately 55°C. The presence of a hybridized complex comprising the probe or primer and a polynucleotide complementary to the test sample indicates the presence of an Apl or Apl polynucleotide sequence in the sample.

ANTIBODIES

[0059] Antibodies of the invention are antibody molecules that specifically bind to an Anaplasma phagocytophilum polypeptide or Anaplasma platys polypeptide or variant polypeptide of the invention or fragment thereof. An antibody of the invention can be specific for an Aph polypeptide or Apl polypeptide or a variant polypeptide or a combination thereof, for example, an antibody specific for one or more of SEQ ID NOs:1 to 21. In another embodiment of the invention, an antibody is specific for an Anaplasma phagocytophilum polypeptide (for example, an antibody specific for SEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20). In another embodiment of the invention, an antibody is specific for an Anaplasma platys polypeptide (for example, an antibody specific for SEQ ID NOs:8 or 19). In another embodiment of the invention, an antibody is specific for both an Anaplasma phagocytophilum polypeptide and an Anaplasma platys polypeptide (e.g., an antibody specific for SEQ ID NOs:2, 3, 10, 13, 14, or 21). One of skill in the art can readily determine whether an antibody is specific for an Anaplasma phagocytophilum polypeptide or Anaplasma platys polypeptide using assays described in this application. An antibody of the invention can be a polyclonal antibody, a monoclonal antibody, a single chain antibody (scFv), or antigen-binding fragment of an antibody. Antigen-binding fragments of antibodies are a portion of an intact antibody comprising the antigen-binding site or variable region of an intact antibody, wherein the portion is free of the heavy chain constant domains of the Fc region of the intact antibody. Examples of antibody antigen-binding fragments include Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments.

[0060] An antibody of the invention can be any class of antibody, including, for example, IgG, IgM, IgA, IgD, and IgE. An antibody or fragment thereof binds to an epitope of a polypeptide of the invention. An antibody can be produced in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. Means for preparing and characterizing antibodies are well known in the art. See, for example, Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev. Immunol. 12:125-68 (1992). For example, polyclonal antibodies can be produced by administering a polypeptide of the invention to an animal, such as a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma, for example, by precipitation with ammonium sulfate, followed by chromatography, such as affinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art.

[0061] "Specifically binds" or "specific for" means that a first antigen, e.g., a polypeptide from Anaplasma phagocytophilum or Anaplasma platys, recognizes and binds to an antibody of the invention with greater affinity than to other, nonspecific molecules. A nonspecific molecule is an antigen that does not share any common epitope with the first antigen. In a preferred embodiment of the invention, a nonspecific molecule is not derived from Anaplasma sp. An Anaplasma sp. is any species of the genus Anaplasma. For example, an antibody raised against a first antigen (e.g., a polypeptide) to which it binds more efficiently than to a nonspecific antigen can be described as specifically binding to the first antigen. In one embodiment, an antibody or antigen-binding fragment thereof specifically binds to a polypeptide of SEQ ID NOs:1 to 21 or fragments thereof when it binds with a binding affinity Ka of 107 L/mol or greater. Specific binding can be tested using, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or western blot assay methodology well known in the art.

[0062] Antibodies of the invention include antibodies and antigen-binding fragments thereof that (a) compete with a reference antibody for binding to SEQ ID NOs:1 to 21 or antigen-binding fragments thereof; (b) bind to the same epitope of SEQ ID NOs:1 to 21 or antigen-binding fragments thereof as a reference antibody; (c) bind to SEQ ID NOs:1 to 21 or antigen-binding fragments thereof with substantially the same Kd as a reference antibody; and/or (d) bind to SEQ ID NOs:1 to 21 or fragments thereof at substantially the same rate as a reference antibody, wherein the reference antibody is an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of SEQ ID NOs:1 to 21 or antigen-binding fragments thereof with a binding affinity Kd of 107 L/mol or more.

[0063] Additionally, monoclonal antibodies directed against epitopes present in a polypeptide of the invention can also be readily produced. For example, normal B cells from a mammal, such as a mouse, that has been immunized with a polypeptide of the invention can be fused, for example, with HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas that produce Anaplasma-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semisolid agar or by limiting dilution. Clones that produce Anaplasma-specific antibodies are isolated by another round of screening. Monoclonal antibodies can be screened for specificity using standard techniques, for example, by binding a polypeptide of the invention to a microtiter plate and measuring monoclonal antibody binding by an ELISA assay. Techniques for producing and processing monoclonal antibodies are known in the art. See, for example, Kohler & Milstein, Nature, 256:495 (1975). Particular isotypes of a monoclonal antibody may be prepared directly, by initial fusion selection, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of a different isotype using a parent selection technique to isolate class switch variants. See Steplewski et al., P.N.A.S. U.S.A. 82:8653 1985; Spria et al., J. Immunolog. Meth. 74:307, 1984. Monoclonal antibodies of the invention may also be recombinant monoclonal antibodies. See, e.g., U.S. Patent No. 4,474,893; U.S. Patent No. 4,816,567. Antibodies of the invention may also be chemically constructed. See, e.g., U.S. Patent No. 4,676,980.

[0064] Antibodies of the invention can be chimeric (see, e.g., U.S. Patent No. 5,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986); Reichmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992)), caninized, canine, or human antibodies. Human antibodies can be made, e.g., by direct immortalization, phage display, transgenic mice, or Trimera methodology, see, e.g., Reisener et al., Trends Biotechnol. 16:242-246 (1998).

[0065] Antibodies that specifically bind to Anaplasma phagocytophilum antigens to the exclusion of Anaplasma platys antigens (e.g., SEQ ID NOs: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20), are particularly useful for detecting the presence of Anaplasma phagocytophilum antigens in a specimen, such as a serum, blood, plasma, urine, fecal, tissue, cellular, or saliva specimen from an animal infected with Anaplasma phagocytophilum.

[0066] Antibodies that specifically bind to Anaplasma platys antigens to the exclusion of Anaplasma phagocytophilum antigens (e.g., SEQ ID NOs:8 or 19), are particularly useful for detecting the presence of Anaplasma platys antigens in a specimen, such as a serum, blood, plasma, urine, fecal, tissue, or saliva sample from an animal infected with Anaplasma platys.

[0067] Antibodies that specifically bind to Anaplasma platys and Anaplasma phagocytophilum antigens (e.g., SEQ ID NOs:2, 3, 10, 13, 14, or 21), are particularly useful for detecting the presence of Anaplasma platys and Anaplasma phagocytophilum antigens in a specimen, such as a serum, blood, plasma, urine, fecal, tissue, or saliva sample from an Anaplasma platys-infected or Anaplasma phagocytophilum-infected animal.

[0068] An immunoassay for an antigen for Anaplasma may utilize one antibody or multiple antibodies. An immunoassay for an antigen for Anaplasma may utilize, for example, a monoclonal antibody specific for an Anaplasma epitope, a combination of monoclonal antibodies specific for epitopes of an Anaplasma polypeptide, monoclonal antibodies specific for epitopes of different Anaplasma polypeptides, polyclonal antibodies specific for the same Anaplasma antigen, polyclonal antibodies specific for different Anaplasma antigens, or combinations of monoclonal and polyclonal antibodies. Immunoassay protocols may be based, for example, on competition, direct reaction, or using sandwich assays, for example, labeled antibody. Antibodies of the invention may be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzymatic, colloidal metal, radioisotope, and bioluminescent labels. Antibodies of the invention may specifically bind to Aph antigens only, Apl antigens only, or Apl antigens and Apl antigens.

[0069] Antibodies of the invention or fragments thereof may be bound to a support and used to detect the presence of Apl and/or Aph antigens. Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magletite.

[0070] Antibodies of the invention can further be used to isolate Apl and/or Aph organisms or antigens by immunoaffinity columns. The antibodies can be affixed to a solid support by, for example, adsorption or by covalent binding so that the antibodies retain their immunoselective activity. Optionally, spacer groups can be included so that the antigen-binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind Anaplasma organisms or Anaplasma antigens from a sample, such as a biological sample including saliva, serum, sputum, blood, urine, stool, cerebrospinal fluid, amniotic fluid, wound exudate or tissue. The bound Anaplasma organisms or Anaplasma antigens are recovered from the column matrix, for example, by a change in pH.

[0071] Antibodies of the invention can also be used in immunolocalization studies to analyze the presence and distribution of a polypeptide of the invention during various cellular events or physiological conditions. Antibodies can also be used to identify molecules involved in passive immunization and to identify molecules involved in the biosynthesis of non-protein antigens. Identification of such molecules can be useful in vaccine development. Antibodies of the invention, including, for example, monoclonal antibodies and single chain antibodies, can be used to monitor the course of improvement of a disease caused by Apl and/or Aph. By measuring the increase or decrease of antibodies specific for Apl, and/or Aph in a test sample from an animal, it can be determined whether a particular therapeutic regimen aimed at improving the disorder is effective. Antibodies can be detected and/or quantified using, for example, direct binding assays, such as RIA, ELISA, or western blot assays.

DETECTION METHODS

[0072] The methods of the invention can be used to detect antibodies or specific binding fragments thereof specific for Anaplasma antigens, Apl antigens, Aph antigens, Apl polynucleotides, Aph polynucleotides or a combination thereof in a test sample, such as a biological sample, an environmental sample or a laboratory sample. A test sample can potentially comprise Apl polynucleotides, Aph polynucleotides, Apl polypeptides, Anaplasma sp. polypeptides, Aph polypeptides, antibodies specific for Anaplasma sp., antibodies specific for Apl, and/or antibodies specific for Aph, unrelated polynucleotides, polypeptides, antibodies or antigens, combinations of the above or none of the above. A biological sample can include, for example, sera, blood, cells, plasma, saliva, urine, feces or tissue from a mammal, such as a horse, cat, dog or human. The test sample may be untreated, precipitated, fractionated, separated, diluted, concentrated or purified.

[0073] In one embodiment, methods of the invention comprise contacting one or more polypeptides of the invention with a test sample under conditions that allow polypeptide/antibody complexes, i.e., immune complexes, to be formed. That is, the polypeptides of the invention specifically bind to antibodies specific for Apl and/or Aph antigens located in the sample. In one embodiment of the invention, one or more polypeptides of the invention (e.g., SEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, 20 or fragments thereof) specifically bind to antibodies that are specific for Aph antigens and do not specifically bind to Apl antigens. In another embodiment of the invention, one or more polypeptides of the invention (e.g., SEQ ID NOs:2, 3, 10, 13, 14, 21 or fragments thereof) specifically bind to antibodies that are specific for both Aph and Apl antigens. In one embodiment of the invention, one or more polypeptides of the invention (e.g., SEQ ID NOs:8, 19 or fragments thereof) specifically bind to antibodies that are specific for Apl antigens and do not specifically bind to Aph antigens. One of skill in the art is familiar with assays and conditions that are used to detect antibody/polypeptide complex binding. The formation of a complex between polypeptides and anti-Apl and/or anti-Aph antibodies in the sample is detected. Formation of antibody/polypeptide complexes is an indication that Anaplasma phagocytophilum polypeptides and/or Anaplasma platys polypeptides are present in the sample. Lack of detection of polypeptide/antibody complexes is an indication that Anaplasma phagocytophilum polypeptides and/or Anaplasma platys polypeptides are not present in the sample.

[0074] Antibodies of the invention can be used in a method of diagnosing Apl and/or Aph infection by obtaining a test sample, for example, from a human or animal suspected of having an Apl and/or Aph infection. The test sample is contacted with antibodies of the invention under conditions that allow the formation of antibody-antigen complexes (i.e., immune complexes). One skilled in the art is aware of conditions that allow and are suitable for the formation of antigen/antibody complexes. The amount of antibody-antigen complexes can be determined by methodology known in the art. A level that is higher than that formed in a control sample indicates an Apl and/or Aph infection. A control sample is a sample that does not comprise Aph and/or Apl polypeptides or antibodies specific for Aph and/or Apl. In one embodiment of the invention, the control does not contain Anaplasma sp. polypeptides or antibodies specific for Anaplasma sp. In one embodiment of the invention, an antibody is specific only for Aph antigens. In another embodiment of the invention, an antibody is specific for both Aph and Apl antigens. In another embodiment of the invention, an antibody is specific only for Apl antigens. Alternatively, a polypeptide of the invention may be contacted with a test sample. Antibodies specific for Apl, and/or Aph in a positive test sample will form antibody-antigen complexes under suitable conditions. The amount of antibody-antigen complexes may be determined by methods known in the art.

[0075] In one embodiment of the invention, infection by Anaplasmaphagocytophilum and/or Anaplasma platys can be detected in a subject. A biological sample is obtained from the subject. One or more purified polypeptides comprising SEQ ID NOs:1 to 21 or other polypeptides of the invention are contacted with the biological sample under conditions that allow polypeptide/antibody complexes to form. The polypeptide/antibody complexes are detected. Detection of the polypeptide/antibody complexes is an indication that the mammal has an infection by Anaplasma platys and/or Anaplasma phagocytophilum. Lack of detection of the polypeptide/antibody complexes is an indication that the mammal does not have an infection by Anaplasma phagocytophilum or an infection by Anaplasma platys.

[0076] Since SEQ ID NOs:2, 3, 10, 13, 14, and 21 are specific for both anti-Apl and anti-Aph antibodies, the detected infection may be Aph infection, Apl infection, or both Apl and Aph infection. Since SEQ ID NOs:1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, and 20 are specific for anti-Aph antibodies, the detected infection is Aph infection. Since SEQ ID NOs:8 and 19 are specific for anti-Apl antibodies, the detected infection is Apl infection. The lack of detection of polypeptide/antibody complexes is an indication that the individual does not have an Anaplasma phagocytophilum or Anaplasma platys infection.

[0077] In one embodiment of the invention, Apl and/or Aph infection can be detected in a subject up to 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or more after the subject acquires Apl and/or Apl infection. In one embodiment of the invention, Apl and/or Aph infection can be detected in a subject up to 21 days, 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days or less after the subject acquires Apl and/or Apl infection.

[0078] In one embodiment of the invention, the polypeptide/antibody complex is detected when an indicator reagent, such as an enzyme conjugate, that is linked to the antibody, catalyzes a detectable reaction. Optionally, an indicator reagent comprising a signal generating compound may be applied to the polypeptide/antibody complex under conditions that allow the formation of a polypeptide/antibody/indicator complex. The polypeptide/antibody/indicator complex is detected. Optionally, the polypeptide or antibody may be labeled with an indicator reagent prior to the formation of a polypeptide/antibody complex. The method may optionally comprise a positive or negative control.

[0079] In one embodiment of the invention, one or more antibodies of the invention are bound to a solid phase or substrate. A test sample potentially comprising a protein comprising a polypeptide of the invention is added to the substrate. One or more antibodies that specifically bind to polypeptides of the invention are added. The antibodies may be the same antibodies used in the solid phase or may be from a different source or species and may be bound to an indicator reagent, such as an enzyme conjugate. Washing steps may be performed prior to each addition. An enzyme substrate or chromophore is added and the color is allowed to develop. The color reaction is stopped and the color may be quantified using, for example, a spectrophotometer.

[0080] In another embodiment of the invention, one or more antibodies of the invention are bound to a solid phase or substrate. A test sample potentially comprising a protein comprising a polypeptide of the invention is added to the substrate. Second antispecies antibodies that specifically bind to polypeptides of the invention are added. These second antibodies are of a different species than the solid phase antibodies. Third antispecies antibodies that are added that specifically bind to the second antibodies and that do not specifically bind to the solid phase antibodies are added. The third antibodies may comprise an indicator reagent, such as an enzyme conjugate. Washing steps may be performed prior to each addition. The enzyme substrate or chromophore is added and the color is allowed to develop. The color reaction is stopped and the color can be quantified using, for example, a spectrophotometer.

[0081] Assays of the invention include, but are not limited to, those based on competition, direct reaction or sandwich type assays, including, but not limited to, enzyme-linked immunosorbent assay (ELISA), western blot, IFA, radioimmunoassay (RIA), hemagglutination (AI), fluorescence polarization immunoassay (FPIA), and microtiter plate assays (any assay performed in one or more wells of a microtiter plate). An assay of the invention comprises a reversible flow chromatographic binding assay, e.g., a SNAP® assay. See U.S. Patent No. 5,726,010.

[0082] Assays may use solid phases or substrates or may be performed by immunoprecipitation or any other method that does not use solid phases. Where a solid phase or substrate is used, one or more polypeptides of the invention are directly or indirectly bound to a support or solid substrate, such as a microtiter well, magnetic bead, non-magnetic bead, column, matrix, membrane, fibrous mat composed of synthetic or natural fibers (e.g., glass- or cellulose-based materials or thermoplastic polymers such as polyethylene, polypropylene or polyester), sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature). In one embodiment of the invention, a substrate is sintered, fine particles of polyethylene, commonly known as porous polyethylene, e.g., 10 to 15 micron porous polyethylene from Chromex Corporation (Albuquerque, NM). All of these substrate materials may be used in suitable forms, such as films, sheets or plates, or may be coated or bonded or laminated to suitable inert carriers, such as paper, glass, plastic films or fabrics. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent and the like interactions.

[0083] In one type of assay format, one or more polypeptides may be coated onto a solid phase or substrate. A test sample suspected of containing an anti-Apl, and/or anti-Aph antibody or antigen-binding fragment thereof is incubated with an indicator reagent comprising a signal generating compound conjugated to an antibody or antigen-binding antibody fragment specific for Apl and/or Aph for a period and under conditions sufficient to form antigen/antibody complexes of antibodies of the test sample to the solid phase polypeptides or of the indicator reagent compound conjugated to an antibody specific for Apl and/or Aph to the solid phase polypeptides. The reduction in binding of the indicator reagent conjugated to an anti-Apl and/or anti-Aph antibody to the solid phase can be measured quantitatively. A measurable reduction in signal compared to the signal generated from the confirmed Apl-negative and/or confirmed Aph-negative test sample indicates the presence of anti-Apl and/or anti-Aph antibodies in the test sample. This type of assay can quantify the amount of anti-Apl and/or anti-Aph antibodies in a test sample.

[0084] In another type of assay format, one or more polypeptides of the invention are coated onto a support or substrate. A polypeptide of the invention is conjugated to an indicator reagent and added to a test sample. This mixture is applied to the support or substrate. If antibodies specific for Apl and/or Aph are present in the test sample, they will bind to one or more polypeptides conjugated to an indicator reagent and to one or more polypeptides immobilized on the support. The polypeptide/antibody/indicator complex can then be detected. This type of assay can quantify the amount of anti-Apl and/or anti-Aph antibodies in a test sample.

[0085] In another type of assay format, one or more polypeptides of the invention are coated onto a support or substrate. The test sample is applied to the support or substrate and incubated. Unbound components of the sample are washed off the solid support with a wash solution. If Apl-specific and/or Aph-specific antibodies are present in the test sample, they will bind to the polypeptide coated onto the solid phase. This polypeptide/antibody complex can be detected using a second species-specific antibody that is conjugated to an indicator reagent. The polypeptide/antibody/antispecies indicator antibody complex can then be detected. This type of assay can quantify the amount of anti-Apl and/or anti-Aph antibodies in a test sample.

[0086] The formation of a polypeptide/antibody complex or a polypeptide/antibody/indicator complex can be detected, for example, by radiometric, colorimetric, fluorometric, size separation, or precipitation methods. Optionally, detection of a polypeptide/antibody complex is by the addition of a secondary antibody that is coupled to an indicator reagent comprising a signal generating compound. Indicator reagents comprising signal generating compounds (labels) associated with a polypeptide/antibody complex can be detected using the methods described above and include chromogenic agents, catalysts such as enzyme-conjugated fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridines, phenanthridiniums, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. Selection of a particular label is not critical, but it will be capable of producing a signal by itself or in conjunction with one or more additional substances.

[0087] The formation of the complex is indicative of the presence of anti-Apl and/or anti-Aph antibodies in a test sample. Therefore, the methods of the invention can be used to diagnose Apl and/or Apl infection in a patient.

[0088] The methods of the invention can also indicate the amount or quantity of anti-Apl and/or anti-Aph antibodies in a test sample. With many indicator reagents, such as enzyme conjugates, the amount of antibody present is proportional to the signal generated. Depending on the type of test sample, it can be diluted with a suitable buffered reagent, concentrated, or contacted with a solid phase without any manipulation. For example, it is typically preferred to test serum or plasma samples that have been previously diluted, or concentrated specimens, such as urine, in order to determine the presence and/or amount present of antibody.

[0089] The invention further comprises assay kits (e.g., articles of manufacture) for detecting anti-Apl and/or anti-Aph antibodies or antibody fragments, Apl polypeptides, and/or Aph polypeptides in a sample. A kit comprises one or more polypeptides of the invention and means for determining binding of the polypeptide to anti-Apl antibodies and/or anti-Aph antibodies or antigen-binding antibody fragments in the sample. A kit or article of manufacture may also comprise one or more antigen-binding antibodies or antibody fragments of the invention and means for determining binding of the antibodies or antigen-binding antibody fragments to Apl polypeptides, and/or Aph polypeptides in the sample. A kit may comprise a device containing one or more polypeptides or antibodies of the invention and instructions for use of the one or more polypeptides or antibodies, e.g., for identifying Apl and/or Apl infection in a mammal. The kit may also comprise packaging material comprising a label indicating that one or more polypeptides or antibodies of the kit may be used for the identification of Apl and/or Apl infection. Other components, such as buffers, controls and the like, known to those of ordinary skill in the art, may be included in such test kits. The polypeptides, antibodies, assays and kits of the invention are useful, for example, in the diagnosis of individual cases of Apl and/or Apl infection in a patient, as well as epidemiological studies of Apl and/or Aph rashes.

[0090] Polypeptides and assays of the invention may be combined with other polypeptides or assays to detect the presence of Anaplasma along with other organisms. For example, polypeptides and assays of the invention may be combined with reagents that detect heartworm and/or Borrelia burgdorferi and/or Ehrlichia canis.

[0091] Polynucleotides of the invention can be used to detect the presence of Apl and/or Aph polynucleotides in a sample. The polynucleotides can be used to detect Apl and/or Aph polynucleotides in a sample by a simple hybridization reaction and can also be used in, for example, polymerase chain reactions (PCR), such as a real-time PCR reaction. Methods and compositions of the invention can also be used to differentially detect the presence of Aph from other Anaplasma sp., such as Apl.

[0092] PCR assays are well described in the art, including, for example, U.S. Patent Nos. 4,683,195; U.S. Patent No. 4,683,202; U.S. Patent No. 4,965,188. Generally, polynucleotide primers are annealed to denatured strands of a target nucleic acid. Primer extension products are formed by the polymerization of deoxynucleoside triphosphates by a polymerase. PCR then involves repetitive cycles of template nucleic acid denaturation, primer annealing, and extension of the annealed primers by the action of a thermostable polymerase. The process results in the exponential amplification of target Anaplasma sp. nucleic acids in the test sample, which allows for the detection of target polynucleotides existing at very low concentrations in a sample.

[0093] Real-time PCR assays are based on the detection of a signal, e.g., a fluorescent reporter signal. This signal increases in direct proportion to the amount of PCR product in a reaction. Real-time PCR is any amplification technique that allows monitoring the progress of a continuous amplification reaction. See, Quantitation of DNA/RNA Using Real-Time PCR Detection, Perkin Elmer Applied Biosystems (1999); PCR Protocols (Academic Press New York, 1989). By recording the amount of fluorescence emission in each cycle, it is possible to monitor the PCR reaction during the exponential phase where the first significant increase in the amount of PCR product is in correlation with the initial amount of target template. The higher the initial copy number of the target nucleic acid, the faster a significant increase in fluorescence is observed.

[0094] One embodiment of the invention provides a method for detecting and/or quantifying Apl and/or Aph polynucleotides in a test sample. Sense primers and antisense primers can be added to a test sample under conditions suitable for a polymerase chain reaction. The primers hybridize to Apl and/or Aph polynucleotides such that an amplification product is formed if Apl and/or Aph polynucleotides are present in the test sample. Amplification products are detected and the presence and/or amount of Apl and/or Aph polynucleotides is determined. Amplification products can be detected with a polynucleotide probe that hybridizes, under conditions suitable for a polymerase chain reaction, to an Apl and/or Aph polynucleotide sequence. The amplification product can be quantified by measuring a detection signal from the probe and comparing said detection signal to a second probe detection signal from a quantification standard. The quantification standard can be extracted in parallel with the test sample.

[0095] One embodiment of the invention provides a method for differentially detecting Anaplasma phagocytophilum polypeptides from Anaplasma platys in a sample. The method comprises: (a) contacting one or more antibodies that specifically bind to a polypeptide consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20 with a sample under conditions that allow polypeptide/antibody complexes to form and detecting the polypeptide/antibody complexes; and (b) contacting one or more antibodies that specifically bind to a polypeptide consisting of SEQ ID NO: 8 or 19 with the sample under conditions that allow polypeptide/antibody complexes to form and detecting the polypeptide/antibody complexes.

[0096] If polypeptide/antibody complexes are detected in step (a) and step (b) then the sample contains Anaplasma phagocytophilum polypeptides and contains Anaplasma platys polypeptides. If polypeptide/antibody complexes are detected in step (a) and are not detected in step (b) then the sample contains Anaplasma phagocytophilum polypeptides and does not contain Anaplasma platys polypeptides. If polypeptide/antibody complexes are not detected in step (a) and are detected in step (b) then the sample contains Anaplasma platys polypeptides and does not contain Anaplasma phagocytophilum polypeptides. If polypeptide complexes are not detected in step (a) and are not detected in step (b) then the sample does not contain Anaplasma platys polypeptides and does not contain Anaplasma phagocytophilum polypeptides.

[0097] Another embodiment of the invention provides a method of detecting antibodies that specifically bind to an Anaplasma platys polypeptide, an Anaplasma phagocytophilum polypeptide, or both an Anaplasma platys polypeptide and an Anaplasma phagocytophilum polypeptide. The method comprises: (a) contacting one or more purified polypeptides comprising SEQ ID NOs: 1, 4, 5, 6, 7, 9, 11, 12, 15, 16, 17, 18, or 20 with a test sample under conditions that allow polypeptide/antibody complexes to form and detecting the polypeptide/antibody complexes; and(b) contacting one or more purified polypeptides comprising SEQ ID NO:8 or 19, wherein the purified polypeptide is contacted with a test sample, under conditions that allow polypeptide/antibody complexes to form and detection of the polypeptide/antibody complexes.

[0098] If polypeptide/antibody complexes are detected in step (a) and step (b) then the sample contains antibodies that specifically bind to Anaplasma phagocytophilum polypeptides and Anaplasma platys polypeptides (i.e., antibodies that are capable of specifically binding to both Anaplasma platys and Anaplasma phagocytophilum polypeptides). If polypeptide/antibody complexes are detected in step (a) and are not detected in step (b) then the sample contains antibodies that specifically bind Anaplasma phagocytophilum polypeptides and does not contain antibodies that specifically bind Anaplasma platys polypeptides. If polypeptide/antibody complexes are not detected in step (a) and are detected in step (b) then the sample contains antibodies that specifically bind Anaplasma platys polypeptides and does not contain antibodies that specifically bind Anaplasma phagocytophilum polypeptides. If polypeptide complexes are not detected in step (a) and are not detected in step (b) then the sample does not contain antibodies specific for Anaplasma platys polypeptides and does not contain antibodies specific for Anaplasma phagocytophilum polypeptides.

Methods of Treating, Improving or Preventing a Disease Caused by Aph and/or Apl

[0099] Polypeptides, polynucleotides and antibodies of the invention can be used to treat, ameliorate or prevent a disease caused by Apl and/or Aph. For example, an antibody, such as a monoclonal antibody of the invention or antigen-binding fragments thereof, can be administered to an animal, such as a human or dog. In one embodiment of the invention, an antibody or antigen-binding fragment thereof is administered to an animal in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. A pharmaceutical composition comprises a therapeutically effective amount of an antibody or antigen-binding fragments thereof. A therapeutically effective amount is an amount effective in alleviating the symptoms of an Apl and/or Apl infection or in reducing the amount of Apl and/or Aph organisms in an individual.

[00100] Polypeptides or polynucleotides of the invention may be present in an immunogenic composition and used to elicit an immune response in a host. An immunogenic composition or immunogen is capable of inducing an immune response in an animal. The immunogenic polynucleotide or polypeptide composition of the invention is particularly useful in sensitizing an immune system of an animal such that, as a result, an immune response is produced that ameliorates or prevents the effect of infection by Apl and/or Aph. Elicitation of an immune response in the animal model may be useful for determining, for example, optimal doses or routes of administration. Elicitation of an immune response may also be used to treat, prevent or ameliorate a disease or infection caused by Apl and/or Aph. An immune response includes humoral immune responses or cell-mediated immune responses or combinations thereof. An immune response may also comprise the promotion of a generalized host response, for example, by promoting the production of defensins.

[00101] One embodiment of the invention provides an immunogen comprising a polypeptide of the invention and one or more additional regions or portions covalently joined to the polypeptide at the carboxyl terminus or amino terminus. Each region or portion, for example, may enhance the immune response, facilitate purification of the immunogen, or facilitate polypeptide stability.

[00102] The generation of an antibody titer by an animal against Apl and/or Aph can be important in protection from infection and clearance of infection. Detection and/or quantification of antibody titers after delivery of a polypeptide or polynucleotide can be used to identify epitopes that are particularly effective in eliciting antibody titers. Epitopes responsible for a strong antibody response to Apl and/or Aph can be identified by eliciting antibodies directed against Apl and/or Aph polypeptides of different lengths. Antibodies elicited by a particular polypeptide epitope can then be tested using, for example, an ELISA assay to determine which polypeptides contain epitopes that are most effective in generating a strong response. Polypeptides or fusion proteins containing these epitopes or polynucleotides encoding the epitopes can then be constructed and used to elicit a strong antibody response.

[00103] A polypeptide, polynucleotide or antibody of the invention can be administered to a mammal, such as a mouse, rabbit, guinea pig, monkey, baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, or to animals, such as chickens or ducks, to elicit antibodies in vivo. Injection of a polynucleotide has the practical advantages of simplicity of construction and modification. Furthermore, injection of a polynucleotide results in the synthesis of a polypeptide in the host. In this way, the polypeptide is presented to the host's immune system with post-translational modifications, native structure and conformation. A polynucleotide can be delivered to an individual as "naked DNA".

[00104] Administration of a polynucleotide, polypeptide, or antibody may be by any means known in the art, including intramuscular, intravenous, intrapulmonary, intramuscular, intradermal, intraperitoneal, or subcutaneous injection, by aerosol, intranasal, infusion pump, suppository, mucosal, topical, and oral, including injection using a biological ballistic weapon ("gene weapon"). A polynucleotide, polypeptide, or antibody may be accompanied by a protein carrier for oral administration. A combination of administration methods may also be used to elicit an immune response. Antibodies may be administered in a daily dose of approximately 0.5 mg to approximately 200 mg. In one embodiment of the invention, antibodies are administered in a daily dose of approximately 20 to approximately 100 mg.

[00105] Pharmaceutically acceptable carriers and diluents and veterinarily acceptable carriers and diluents for therapeutic use are well known in the art and are described in, for example, Remington’s Pharmaceutical Sciences, Mack Publishing Co (A.R. Gennaro ed. (1985)). The carrier should not induce the production of antibodies harmful to the host. Such carriers include, but are not limited to, large, slowly metabolized macromolecules such as proteins, polysaccharides such as SEPHAROSE® functionalized latex, agarose, cellulose, cellulose beads and the like, polylactic acids, polyglycolic acids, polymeric amino acids such as polyglutamic acid, polylysine and the like, amino acid copolymers, peptoids, lipitoids and inactive, avirulent virus particles or bacterial cells. Liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules and bioadhesives can also be used as a vehicle for a composition of the invention.

[00106] Pharmaceutically acceptable salts may also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates or benzoates. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid and other proteins well known to those skilled in the art. Compositions of the invention may also contain liquids or excipients such as water, saline, phosphate buffered saline, Ringer's solution, Hank's solution, glucose, glycerol, dextrose, malodextrin, ethanol or the like, alone or in combination, as well as substances such as wetting agents, emulsifying agents, tonicity adjusting agents, detergent or pH buffering agents. Additional active agents such as bacteriocidal agents may also be used.

[00107] If desired, costimulatory molecules, which enhance immunogen presentation to lymphocytes, such as B7-1 or B7-2, or cytokines, such as MIP1α, GM-CSF, IL-2 and IL-12, may be included in a composition of the invention. Optionally, adjuvants may also be included in a composition. Adjuvants are substances that may be used to not specifically enhance a particular immune response. Generally, an adjuvant and a polypeptide of the invention are mixed prior to presentation to the immune system, or presented separately, but are presented at the same site in the animal. Adjuvants may include, for example, oily adjuvants (e.g., Freund's complete and incomplete adjuvants), mineral salts (e.g., AlK(SO4)2; AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, and Ca3(PO4)2), polynucleotides (i.e., Poly IC and Poly AU acids), and certain natural substances (e.g., wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella. Adjuvants that may be used include, but are not limited to, MF59-0, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to as nor- MDP), n-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall scaffold (MPL+TDM+CWS) in 2% squalene/TWEEN® 80 (polysorbate) emulsion.

[00108] The compositions of the invention may be formulated into ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafer-type discs, injectable formulations, mouthwashes, dentifrices and the like. The percentage of one or more polypeptides, polynucleotides or antibodies of the invention in such compositions and preparations may range from 0.1% to 60% of the weight of the unit.

[00109] Administration of polypeptides, polynucleotides, or antibodies can elicit an immune response in the animal that lasts for at least 1 week, 1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immune response can be maintained in an animal by providing one or more injections that stimulate the polypeptide, polynucleotide, or antibodies at 1 month, 3 months, 6 months, 1 year, or longer after primary injection. If desired, costimulatory molecules or adjuvants can also be provided before, after, or in conjunction with the compositions.

[00110] A composition of the invention comprising a polypeptide, polynucleotide, antibody or a combination thereof is administered in a manner compatible with the particular composition used and in an amount that is effective to elicit an immune response as detected, for example, by an ELISA. A polynucleotide can be injected intramuscularly into a mammal, such as a baboon, chimpanzee, dog or human, at a dose of 1 ng/kg, 10 ng/kg, 100 ng/kg, 1000 ng/kg, 0.001 mg/kg, 0.1 mg/kg or 0.5 mg/kg. A polypeptide or antibody can be injected intramuscularly into a mammal at a dose of 0.01, 0.05, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

[00111] Polypeptides, polynucleotides or antibodies, or a combination thereof can be administered to an animal that is not infected with Apl and/or Aph or can be administered to an animal infected with Apl and/or Aph. An immunologically effective amount or therapeutically effective amount means that administration of that amount to a subject, in a single dose or as part of a series, is effective for treating, ameliorating, or preventing Apl and/or Aph infection. Particular dosages of polynucleotide, polypeptide, or antibody in a composition will depend on many factors including, but not limited to, the species, age, gender, concurrent medication, general condition of the mammal to which the composition is administered, and the mode of administration of the composition. An effective amount of the composition of the invention can be readily determined using routine experimentation alone.

[00112] All patents, patent applications and other scientific or technical writings referred to anywhere in this application are incorporated by reference into this application in their entirety. The invention illustratively described in this application may properly be practiced in the absence of any element or elements, limitation or limitations not specifically described in this application. Thus, for example, in each example in this application any one of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced by any two other terms, retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and it is not intended that in the use of such terms and expressions any equivalent of the features shown and described or portions thereof be excluded, but it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts disclosed in this application may appeal to those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

[00113] Furthermore, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also hereby described in terms of any individual member or subgroup of members of the Markush group or other group.

EXAMPLES Example 1 Detection of anti-Aph and anti-Apl antibodies with polypeptides derived from the Aph p44 protein

[00114] The polypeptides shown in SEQ ID NOs:12 through 18 and 10 were coated at 0.5 μg/mL onto 4 Immulon® microtiter plates in pH 9.6 carbonate buffer overnight. For all examples, the polypeptide shown in SEQ ID NO:10 has an N at position 143, an A at position 145, and an I at position 156.

[00115] Plates were washed twice with PetChek® wash buffer. Plates were blocked with 2% TWEEN® 20 (polysorbate)/2.5% sucrose in 0.1M Tris pH 7.6 for 2h and then dried with desiccant overnight. A 1:200 dilution of test samples in the conjugate diluent was added to the plates and incubated for 40 minutes. Plates were washed 6 times with PetChek® wash buffer. HRP-conjugated rabbit anti-dog IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA; Cat. No. 304-035-003), diluted 1:2000 in the conjugate diluent, was added to the plates and incubated for 40 minutes. Plates were washed 6 times with PetChek® wash buffer. 60 μl of 3,3’,5,5’-tetramethylbenzidine (“TMB”) was added to the plates and incubated for 10 minutes. 50 μl of Stop solution was added and A650 was determined. Test samples were as follows:

[00116] APL_13DPI: dog experimentally infected with Apl 13 days post-infection

[00117] APL_78DPI: dog experimentally infected with Apl 78 days post-infection

[00118] APH: pooling of samples from 7 Aph-infected Minnesota dogs

[00119] neg: sample pooling of 7 random healthy dogs

[00120] The results are shown in Table 1 and Figure 1.TABLE 1. TABLE 1. -CONTINUED-

[00121] All 8 peptides tested showed positive reactivity to the pool of sera from 7 Aph-infected Minnesota dogs. rp44, p44-2, and p44-3 showed cross-reactivity to sera from an experimentally Apl-infected dog at 2 different time points of infection. Reactivities of p44-1, p44-4, p44-4-1, p44-4-2, and p44-4-3 with sera from the experimentally Apl-infected dog were close to reference levels. Therefore, the p44-1, p44-4, p44-4-1, p44-4-2, and p44-4-3 polypeptides are not cross-reactive with sera from Apl-infected dogs.

Example 2 Species-specific detection of Aph or Apl in field samples from endemic areas

[00122] Polypeptides (Aph p44-4 and Apl p44-4 at 0.5 μg/mL; Aph rp44 at 0.25 μg/mL) were coated onto 4 Immulon® plates in carbonate buffer pH 9.6 overnight. Plates were washed 2x with PetChek® wash buffer and then blocked with 2% TWEEN® 20 (polysorbate)/2.5% Sucrose in 0.1M Tris pH 7.6 for 2 hours. Plates were dried with desiccant overnight. A 25 μL test sample was mixed with 50 μL of peptide:HRP conjugate (0.5 μg/mL of p44-4-Aph (SEQ ID NO:15):HRP conjugate, 1 μg/mL of p44-4-Apl (SEQ ID NO:19):HRP conjugate, and 3 μg/mL of Aph rp44 conjugate) and incubated in the microtiter plate (incubation time was 1 hour in the experiment shown in Figure 2, and 1 hour and 45 minutes in the experiment shown in Figure 3. The plates were washed 6 times with PetChek® wash buffer. 60 μL of TMB was added to the plates and incubated for 10 minutes. 50 μL of Stop solution was added, and A650 was determined. The cutoff value was determined based on reactivities for 10 negative samples; cutoff = mean + 2x SD (standard deviations).

[00123] Figure 2 demonstrates results using A. platys positive samples from dogs living in A. platys endemic areas (HP: Arizona, P: Bahamas). rp44 (SEQ ID NO:10) gave positive results from 4 of 5 "HP" samples, and positive results from 7 of 7 "P" samples. Aph p44-4 (SEQ ID NO:15) gave positive results from 0 of 5 "HP" samples, and from 0 of 7 "P" samples. Apl p44-4 (SEQ ID NO:19) gave positive results from 5 of 5 "HP" samples, and 7 of 7 "P" samples. Therefore, Aph p44-4 does not cross-react with sera from Apl infected dogs.

[00124] Figure 3 demonstrates results using positive samples from A. phagocytophilum dogs living in an Aph-endemic area (Minnesota "ME"). rp44 (SEQ ID NO:10) gave strong positive results from 21 of 22 ME samples. Aph p44-4 (SEQ ID NO:15) gave strong positive results from 20 of 22 ME samples. Apl p44-4 (SEQ ID NO:19) gave negative results from 18 of 22 samples and very weak positive results from 4 of 22 ME samples. Therefore, Apl p44-4 can be considered not to cross-react with sera from Aph-infected dogs.

Example 3 Time Course Response in an Experimental Apl Infection Model

[00125] Polypeptides (Aph p44-4 and Apl p44-4 at 0.5 μg/mL; Aph rp44 at 0.25 μg/mL) were coated onto 4 Immulon® microtiter plates in carbonate buffer pH 9.6 overnight. Plates were washed 3 times with PetChek® wash buffer. Plates were blocked with 2% TWEEN® 20 (polysorbate)/2.5% sucrose in 0.1M Tris pH 7.6 for 2h and allowed to dry overnight. A 25 μL test sample was mixed with 50 μL of peptide:HRP conjugate (0.5 μg/mL Aph p44-4 conjugate, 1 μg/mL Apl p44-4 conjugate, and 3 μg/mL Aph rp44 conjugate) and incubated in the microtiter plate for 1 hour. The plates were washed 6 times with PetChek® wash buffer. 60 μL of TMB was added and allowed to incubate for 10 minutes. 50 μL of Stop solution was added and the A650 was determined. The results are shown in Figure 4.

[00126] The results demonstrate that SEQ ID NO:19 (Apl p44-4) can be used to detect Apl infections. SEQ ID NO:19 can detect Apl infection at approximately 10 days post-infection. Aph p44-4 (SEQ ID NO:15) shows no cross-reactivity with sera from Apl infected dogs. Aph rp44 (SEQ ID NO:10) cross-reacts with sera from Apl infected dogs at approximately 14 days and can be used to detect Apl and Aph infection.

Example 4 Time Course Response in an Experimental Aph Infection Model

[00127] Aph p44-4 (SEQ ID NO:15) and Aph rp44 (SEQ ID NO:10) were tested for their reactivity with sera from dogs experimentally infected with Aph at a series of time points. 11 random field samples from healthy dogs were also tested. The polypeptides were coated onto 4 Immulon® microtiter plates (Aph p44-4 at 0.5 μg/mL; Aph rp44 at 0.25 μg/mL) in carbonate buffer pH 9.6 overnight. The plates were washed four times with PetChek® wash buffer. The plates were blocked with 2% TWEEN® 20 (polysorbate)/2.5% Sucrose in 0.1 M Tris pH 7.6 for 2 hours and then dried with desiccant overnight. A 25 μL test sample was mixed with 50 μL of peptide:HRP conjugate (0.5 μg/mL of p44-4-Aph (SEQ ID NO:15):HRP conjugate and 3 μg/mL of Aph rp44 conjugate) and added to the plates. The plates were incubated for 1 hour. The plates were washed 6 times with PetChek® wash buffer. TMB was added to the plates and incubated for 10 minutes. 50 μL of Stop solution was added and A650 was determined. The results are shown in Figure 5. Aph p44-4 (SEQ ID NO:15) was able to detect Aph infection at approximately 10 to 14 days post-infection. Aph rp44 (SEQ ID NO:10) was able to detect Aph infection at approximately 10 days post-infection.

Example 5 Detection of acute Aph infection

[00128] Polypeptides were coated at 0.5 μg/mL or 1.0 μg/mL onto 4 Immulon® plates in carbonate buffer pH 9.6 overnight. Plates were washed 4 times with PetChek® wash buffer and then blocked with 2% TWEEN® 20 (polysorbate)/2.5% sucrose in 0.1M Tris pH 7.6 for 2 hours. Plates were dried with desiccant overnight. 100 μL of a test serum sample at 1:200 dilution in sample diluent was incubated for 45 minutes. Plates were washed 6 times with PetChek® wash buffer. 100 μL of HRP-conjugated rabbit anti-dog IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA; Cat. No. 304-035-003) at 1:2000 dilution in sample diluent was added and incubated for 45 minutes. Plates were washed 6 times with PetChek® wash buffer. 60 μL of TMB was added to the plates and incubated for 10 minutes. 50 μL of Stop solution was added and A650 was determined. Test samples were as follows:

[00129] Positive ("+") represents a pool of seven sera from dogs infected in the last stage by Aph

[00130] Negative ("-") represents a pool of 7 random healthy dog serum samples

[00131] VML8, VML14, VML21 and VML156 represent serum samples from four dogs that were IFA positive for Aph and had acute clinical signs of anaplasmosis. The results are shown in Figure 6. Aph p44-4 reacted with all 4 sera. Aph p44-1 and Aph p44-2 did not react with all 4 sera, while Aph p44-3 reacted weakly with all 4 sera. Therefore, Aph p44-4 and Aph p44-3 can be used to detect Aph in individuals with acute clinical signs of anaplasmosis.

Example 6 Performance of p44-4-v Peptide

[00132] Aph p44-4-v (SEQ ID NO:20) was tested with the samples listed in Table 2.TABLE 2.

[00133] Polypeptides were coated at 0.5 μg/mL onto 4 Immulon® plates in carbonate buffer pH 9.6 overnight. Plates were washed twice with PetChek® wash buffer and then blocked with 2% TWEEN® 20 (polysorbate)/2.5% sucrose in 0.1 M Tris pH 7.6 for 2 hours. Plates were dried with desiccant overnight. Test sample at a 1:200 dilution of the conjugate diluent was added to the plates and incubated for 40 minutes. Plates were washed 6 times with PetChek® wash buffer. HRP-conjugated rabbit anti-dog IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA; Cat. No. 304-035-003) at a 1:2000 dilution of the conjugate diluent was added to the plates and incubated for 40 minutes. The plates were washed 6 times with PetChek® wash buffer. 60 μL of TMB was added to the plates and incubated for 10 minutes. 50 μL of Stop solution was added and the A650 was determined. The results are shown in Figure 7 and represent the mean of duplicate experiments. p44-4-v gave positive results for the following samples: VML21; ILS73, APH, and +ve; and negative results for the following samples: APL and -ve. Therefore, Aph p44-4-v can detect Aph infection in acute cases, and at least as early as 14 days post-infection. Aph p44-4-v is not cross-reactive with sera from Apl-infected dogs.

Claims (14)

1. Composition, characterized by the fact that it comprises one or more purified polypeptide(s) consisting of an amino acid sequence presented as SEQ ID NO: 1, 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: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, in which the one or more purified polypeptide(s) is (are) linked to an indicator reagent, a heterologous signal sequence, a heterologous transfer stop sequence, a heterologous protein purification linker, a heterologous polypeptide, a heterologous portion that enhances an immune response, a heterologous portion that facilitates purification, a heterologous portion that facilitates polypeptide stability, or a combination thereof. 2. A method for detecting antibodies that specifically bind to an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both in a test sample, comprising: (a) contacting the composition comprising one or more purified polypeptide(s) consisting of an amino acid sequence set forth as SEQ ID NO: 1, 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: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 with the test sample under conditions that allow polypeptide/antibody complexes to be formed; (b) detecting the polypeptide/antibody complexes; wherein detection of the polypeptide/antibody complexes is an indication that antibodies specific for a polypeptide of Anaplasma phagocytophilum or an Anaplasma platys polypeptide or both are present in the test specimen. 3. The method of claim 2, wherein the one or more purified polypeptide(s) is/are linked to an indicator reagent, a signal sequence, a transfer stop sequence, an amino acid spacer, a protein purification linker, a heterologous polypeptide, a moiety that enhances an immune response, a moiety that facilitates purification, a moiety that facilitates polypeptide stability, one or more additional polypeptides comprising SEQ ID NOs:1, 4, 5, 6, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20, or a combination thereof. 4. Method according to claim 2, characterized by the fact that the composition comprising one or more purified polypeptide(s) consists of an amino acid sequence presented as SEQ ID NOs:8 or 19; and in which the detection of the polypeptide/antibody complexes is an indication that antibodies specific for Anaplasma platys are present in the test sample. 5. The method of claim 2, wherein the composition comprising one or more purified polypeptide(s) consists of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, and wherein detection of the polypeptide/antibody complexes is an indication that antibodies specific for an Anaplasma phagocytophilum polypeptide are present in the test sample. 6. Method according to claim 2, characterized by the fact that it further comprises contacting the complexes of step (a) with an indicator reagent prior to carrying out step (b). 7. Method according to claim 2, characterized in that the amount of antibodies in the test sample is determined. 8. Method according to claim 2, characterized in that the one or more purified polypeptide(s) is/are bound to a substrate. 9. A method of detecting an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide or both in a sample, comprising: (a) contacting one or more antibodies that specifically bind to one or more purified polypeptides consisting of an amino acid sequence set forth as SEQ ID NO: 1, 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: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 with the sample under conditions that allow polypeptide/antibody complexes to form; (b) detecting the polypeptide/antibody complexes; wherein detection of the polypeptide/antibody complexes is an indication that an Anaplasma phagocytophilum polypeptide or an Anaplasma platys polypeptide is present. an Anaplasma platys polypeptide or both are present in the sample. 10. The method of claim 10, wherein the purified polypeptides consist of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and wherein detection of the polypeptide/antibody complexes is an indication that Anaplasma phagocytophilum polypeptides are present in the test sample. 11. The method of claim 9, wherein the purified polypeptides consist of SEQ ID NOs:8 or 19; and wherein detection of the polypeptide/antibody complexes is an indication that Anaplasma platys polypeptides are present in the test sample. 12. Method according to claim 9, characterized in that the one or more antibodies are monoclonal antibodies, polyclonal antibodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments or single chain antibodies. 13. Composition according to claim 1, characterized in that it further comprises a pharmaceutically acceptable or veterinarily acceptable vehicle. 14. Composition according to claim 13, characterized in that it further comprises an adjuvant.