NZ724911B2 - Compositions and methods for identifying ehrlichia species - Google Patents

Abstract

The invention provides methods, kits, compositions, and devices useful for detection of antibodies that bind to Ehrlichia antigens and/or for differentiation of certain Ehrlichia species from others. In particular, the invention provides methods and kits useful for identifying species of Ehrlichia using populations of isolated peptides. In one embodiment, the method of identifying species of Ehrlichia as either E. ewingii, E. canis or E. chaffeensis involves three different antigenic peptide populations, named ECHEW1, EE13 and EE12EW1 in a multi-peptide assay. The method involves contacting a sample from the subject with the different peptide populations. Depending on which antibody-peptide complexes form, the specific species of Ehrlichia is identified. For example, formation of first and second sets of complexes with the ECHEW1 and EE13 peptides indicates an E. ewingii infection, whereas formation of just the first ECHEW1 peptide but not the second EE13 peptide population indicates an E. canis and/or E. chaffeensis infection. sing populations of isolated peptides. In one embodiment, the method of identifying species of Ehrlichia as either E. ewingii, E. canis or E. chaffeensis involves three different antigenic peptide populations, named ECHEW1, EE13 and EE12EW1 in a multi-peptide assay. The method involves contacting a sample from the subject with the different peptide populations. Depending on which antibody-peptide complexes form, the specific species of Ehrlichia is identified. For example, formation of first and second sets of complexes with the ECHEW1 and EE13 peptides indicates an E. ewingii infection, whereas formation of just the first ECHEW1 peptide but not the second EE13 peptide population indicates an E. canis and/or E. chaffeensis infection.

Description

COMPOSITIONS AND METHODS FOR IDENTIFYING EHRLICHIA S CROSS-REFERENCE TO RELATED ATIONS This application claims the benefit of US. Provisional Application No. 61/975,581, filed April 4, 2014, which is hereby orated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY The contents of the text file submitted electronically herewith are orated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: ABAX_043_0lWO_SeqList_ST25.txt, date recorded April 1, 2015, file size 86 kilobytes).

FIELD OF THE INVENTION The present invention relates generally to compositions and methods for detecting bacterial infection and identifying bacteria species. In particular, the invention relates to peptide compositions, methods, and kits for detecting dies against bacterial antigens (e. g. antigens from Ehrlichz'a spp.).

BACKGROUND OF THE INVENTION Ehrlichz'a bacteria are obligate intracellular ens that infect ating lymphocytes in ian hosts. The most natural mode ofEhrlichia transmission is via a variety of tick vectors. Ehrlichz'a canis (E. canis) and Ehrlichz'a chafleensz’s (E. chafleensz’s) are members of the same sub-genus group ofEhrlichia that infect canines and humans and cause canine monocytic ehrlichiosis (CME) and human monocytic ehrlichiosis (HME), tively. Another species of Ehrlichia known as Ehrlichz'a ewz’ngz’z’ (E. ewz’ngz’z) has tropism for granulocytes and causes granulocytic ehrlichiosis. The canine disease is characterized by fever, epilepsy, incoordination, lethargy, bleeding episodes, lymphadenopathy, weight loss, and pancytopenia. In humans the disease is terized by fever, headache, myalgia, and leukopenia.

WO 53949 Indirect immunofluorescence assays (IPA) and enzyme-linked immunosorbent assays (ELISA) have typically been used in the diagnosis of these diseases. These assays measure or otherwise detect the binding of anti-Ehrlichz'a antibodies from a subj ect's blood, plasma, or serum to infected cells, cell lysates, or partially purified whole Ehrlichz’a proteins.

However, currently known assays for detecting anti-Ehrlichz'a dies or nts thereof are severely limited in usefulness e of sensitivity and specificity issues directly related to the impure nature of the Ehrlichz'a antigen(s) used in these tests.

The diseases caused by bacteria belonging to different Ehrlichz’a species manifest differently and e te management routine (Thomas, R.J., et al.; Expert Rev Anti Infect Ther. 2009 August; 7(6): 709—722). It is, therefore, important to identify the hz’a species that causes a particular infection. The tly known immunoassays use mixtures of many whole Ehrlichz'a antigens or antigens that are not species specific. PCR methods, which may be useful to identify Ehrlichz'a species, are useable only if the tick is red and/or the tissue from host is tested soon after infection. Furthermore, ation of bacteria from the infection site, another method which may be useful to identify Ehrlichz'a species, is not only technically complex but also requires freshly infected tissue. In addition, a cultivation method for the species E. ewz’ngz’z’ has not yet been developed.

Accordingly, there remains a need in the art for additional assays for detecting Ehrlichz’a antigens and serodiagnosis of monocytic ehrlichiosis and granulocytic ehrlichiosis.

In particular, there remains a need for an assay for identifying hz'a species, especially an assay that can be used in a variety of stances and for various samples, including samples that do not require isolation from freshly infected s. The present invention provides methods, compositions, and kits to facilitate the diagnosis, the species identification, and the treatment of the various types of Ehrlichia infections.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery that particular mixtures, or populations, ofEhrlichia peptides or their variants have preferential binding affinity for antibodies elicited by antigens from particular Ehrlichz'a species. The inventors have found that a particular combination of these peptide mixtures or tions can be used to identify the Ehrlichz’a species inducing the antibody response. ingly, the present invention provides a method for identifying the s ichia infecting a subject.

In certain embodiments, the method for identifying the species ofEhrlichia infecting a subject comprises: contacting a sample from the subject with a first population of isolated peptides; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; contacting said sample with a second population of isolated peptides; and detecting formation of a second set of complexes comprising an antibody and one or more es in the second population, wherein formation of both the first and second sets of complexes indicates that the subject is infected with E. ewz‘ngz'z', and wherein formation of the first but not the second set of xes indicates that the subject is infected with E. canis and/or E. chafieensz’s. In some embodiments, the first population of isolated peptides comprises at least three different peptides, each comprising a sequence of S-X2-K-E-X5-K-Q- Xg-T-Xlo-Xl1-X12-X13-G-L-K-Q-Xlg-W-X20-G-X22-X23-X24-X25-X26-G-G-G-G-G-N-F-S-AK-E-E-ng-A-E-T-R-X44-T-F-G-L-X49-K-Q-Y-D-G-A-X56-I-X5g-E-N-Q-V-Q-N-K-F-T-I-S- N-C (SEQ ID NO: 1) or a fragment thereof, wherein X2 is an amino acid selected from the group ting ofA and V, X5 is an amino acid selected from the group ting of E and D, X8 is an amino acid selected from the group ting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid ed from the group consisting of G and A, X12 is an amino acid selected from the group ting of L and V, X13 is an amino acid selected from the group consisting ofY and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23 is an amino acid selected from the group ting of A, S, and T, X24 is an amino acid selected from the group consisting ofA and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X44 is any amino acid, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid. In d embodiments, the second population of isolated peptides ses at least three different peptides, each comprising a sequence of F-S-A-K- E-E-X7-A-E-T-R-X12-T-F-G-L-X17-K-Q-Y-D-G-A-X24-I-X26-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 2) or a fragment thereof, wherein X7 is any amino acid, X12 is any amino acid, X17 is any amino acid, X24 is any amino acid, and X26 is any amino acid In certain other embodiments, the method comprises: contacting a sample from the subject with a first population of isolated peptides as described herein; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; ting said sample with a third population of isolated es; and ing formation of a third set of complexes comprising an antibody and one or more es in the third population, wherein formation of both the first and third sets of antibody-peptide complexes indicates that the subject is infected with E. canis and/or E. chafieensis, and wherein formation of the first but not the third set of antibody-peptide complexes indicates that the subject is infected with E. ewz’ngz’z’. In n embodiments, the third population of isolated peptides ses at least three different peptides, each comprising a sequence of S-X2-K-E-X5-K-Q-Xg-T-X10-X11-X12-X13-G-L-K-Q-Xlg-W-X20-G- X22-X23-X24-X25-X26-G-G-G-G-G-N-F-S-A-K-E-E-X39-A-X41-T-R-X44-T-F-G-X4g-X49-K-QY-D-G-A-X56-I-X5g-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 3) or a fragment thereof, wherein X2 is an amino acid selected from the group consisting ofA and V, X5 is an amino acid selected from the group consisting of E and D, X8 is an amino acid selected from the group ting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid selected from the group consisting of L and V, X13 is an amino acid selected from the group consisting ofY and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23 is an amino acid ed from the group consisting of A, S, and T, X24 is an amino acid selected from the group consisting ofA and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X41 is an amino acid selected from the group consisting of D and N, X44 is any amino acid, X48 is an amino acid selected from the group consisting ofV and A, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid.

In certain embodiments, the method comprises: contacting a sample from the subject with a first population of isolated peptides as described herein; detecting formation of a first set of xes comprising an antibody and one or more peptides in the first population; contacting said sample with a second population of isolated peptides as described herein; detecting formation of a second set of complexes comprising an antibody and one or more peptides in the second population; contacting said sample with a third population of isolated peptides as described herein; and detecting ion of a third set of complexes comprising an antibody and one or more peptides in the third population, wherein formation of both the first and second sets of complexes but not the third set indicates that the subject is infected with E. ewz’ngz'z', and wherein formation of both the first and third sets of complexes but not the second set indicates that the subject is infected with E. canis and/or E. chafi’eensis.

In some embodiments of the methods, one or more peptides in the first tion of peptides ses a fragment of SEQ ID NO: 1. The fragment of SEQ ID NO: 1 may comprise at least 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 1. In n embodiments, the fragment of SEQ ID NO: 1 comprises amino acids 33 to 71 of SEQ ID NO: 1. In particular embodiments, each e in the first population comprises a sequence of SEQ ID NO: 1.

In certain other embodiments of the methods, one or more peptides in the second population of peptides comprises a fragment of SEQ ID NO: 2. The nt of SEQ ID NO: 2 may comprise at least 15, 20, 25, 30, or 35 uous amino acids from SEQ ID NO: 2. In some embodiments, each peptide in the second population comprises a sequence of SEQ ID NO: 2.

In other embodiments of the methods, one or more peptides in the third population of peptides comprises a fragment of SEQ ID NO: 3. The fragment of SEQ ID NO: 3 may comprise at least 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 3. In certain embodiments, the fragment of SEQ ID NO: 3 comprises amino acids 33 to 71 of SEQ ID NO: 3. In particular embodiments, each peptide in the third population comprises a sequence of SEQ ID NO: 3.

In some embodiments of the s, the sample is fiarther analyzed with at least one assay to determine whether the infecting species is E. canis or E. chafleensz’s.

In certain embodiments, at least one of the ing steps in any of methods described herein may comprise: (i) ming an ELISA assay; (ii) running a lateral flow assay; (iii) performing an ination assay; (iV) performing a Western blot, slot blot, or dot blot assay; (V) performing a wavelength shift assay; (Vi) running the sample through an analytical or centrifugal rotor; or (Vii) running a microarray assay. In some embodiments, one or more of the detecting steps comprises spinning the sample in an analytical or centrifugal rotor. In other embodiments, one or more of the detecting steps comprises analyzing the sample with an electrochemical sensor, an optical sensor, chemiluminescence sensor or an opto-electronic sensor. In particular embodiments, one or more of the detecting steps comprises performing an ELISA assay or a lateral flow assay.

Certain embodiments of the method further comprise reporting ion results. The reporting can be done electronically, in g, or verbally. It can be done Via a machine such as a computer.

In another aspect, the invention provides kits for detecting antibodies that bind to hia antigens and/or identifying the species of Ehrlichia infecting a subject. In certain embodiments, the kit comprises one, two, or three different populations of peptides of the invention as bed herein. In certain embodiments, the kits further comprise an instruction for using the peptide populations to identify the species of Ehrlichia in a ical sample. In some embodiments, the kit further comprises one or more labeling reagents.

In certain embodiments of the methods or the kits of inventions, the peptides in the populations of isolated peptides are attached to or immobilized on a solid support.

Additional aspects and embodiments of the invention will be apparent from the detailed description that follows.

BRIEF PTION OF THE DRAWINGS Figure l is a diagram of an ment of a method for identifying Ehrlichia species. The abbreViation "EAL" represents ELISA score for an ELISA assay using peptide tion EEl3 (SEQ ID NO: 2), while "CAL" represents ELISA score for an ELISA assay using e population EE12EW1 (SEQ ID NO: 3). In this embodiment, a whole blood sample is tested in an ELISA assay, ELISA ECHEWl, using peptide population ECHEWl (SEQ ID NO: 1), which comprises a first population of peptides as bed herein. If the result of ELISA ECHEWl is positive, the sample then undergoes another ELISA assay, ELISA EEl3, using peptide tion EEl3, which comprises a second population of peptides as described herein, and oes yet another ELISA assay, ELISA EE12EW, using peptide population , which comprises a third population of peptides as described herein. A positive result of ELISA EEl3 combined with negative result of ELISA , or a higher EAL than CAL, indicates that the sample is infected with E. ewingii. A positive result of ELISA EEl2EW combined with negative result of ELISA EEl3, or a higher CAL than EAL, indicates that the sample is infected with E. canis and/or E. chafieensis. If the sample is identified to be infected with E. canis and/or E. chafieensz’s, the sample then undergoes another assay, in this example an IFA assay for E. canis or E. chafleensz’s, rently or non-concurrently, to determine whether the sample is infected with E. canis or E. chafleensz’s.

Figure 2 is a graphical representation of anti-Ehrlichz'a antibody scores of plasma samples drawn at various times from dogs infected with the ted hz'a species. Dogs were experimentally infected with various species of Ehrlichia, and plasma samples were drawn on various days post infection as indicated in the graphs. ELISA assays were med on the samples using each of the three populations of peptides, ECHEWl, EE12EW, and EEl3. The top left and bottom left panels show the results from samples separately taken from two dogs infected with E. canis. The top right panel shows the results from samples taken from a dog infected with E. chafi’eensis. Antibody scores were calculated using methods described .

DETAILED DESCRIPTION As used herein, the following terms shall have the following meanings: The term "antigen," as used herein, refers to a molecule capable of being recognized by an antibody. An antigen can be, for e, a peptide or a modified form thereof. An antigen can comprise one or more epitopes.

The term "epitope," as used herein, is a n of an antigen that is specifically recognized by an antibody. An epitope, for example, can comprise or consist of a portion of a peptide (e.g., a peptide of the ion). An epitope can be a linear epitope, sequential epitope, or a conformational epitope. In certain embodiments, epitopes may comprise non-contiguous regions.

The term "OMP-l protein" refers to any of the Outer Membrane Protein 1 paralogs ichia, ing, but not limited to, E. canis P-30, E. canis P30-l, E. chafleensz’s P28, E. chafleensz’s OMP-lC, E. chafleensz's OMP-lD, E. nsz’s , and E. chafleensz’s OMP-lF.

The term "MSP4 protein" refers to any member of the Surface Antigen MSP4 family of Ehrlichia, including, but not limited to, E. canis MSP4, P30-5, and P28-l. OMP and MSP are allelic variants.

The terms ic acid," "oligonucleotide" and "polynucleotide" are used hangeably herein and encompass DNA, RNA, cDNA, whether single stranded or double stranded, as well as chemical modifications thereof.

Single letter amino acid abbreviations used herein have their rd meaning in the art, and all peptide sequences bed herein are written according to convention, with the N—terminal end to the left and the C-terminal end to the right.

The term "score" as used herein refers to a relative value, level, strength, or degree of an assay result. It can be artificially created by a person of skill in the art or by using an algorithm, sometimes using samples with known analytes, e. g., antigens or antibodies, optionally using samples with known concentrations or titers of the known analytes. It can be a number assigned manually by a person of skill in the art or generated with a formula or thm. It can also be a symbol, e.g., "-", "+", or "++". A score can be generated from calculation with a formula or algorithm, or can be assigned by visual inspection, measurement, or estimation of the assay result. When using samples with known concentrations or titers of known analytes, such samples can be assayed in diluted and undiluted conditions, and a range of scores or a standard curve of scores can be generated, which can be used to assign or estimate the scores of unknown samples assayed for the same analytes, preferably with the same .

Additional terms shall be defined, as required, in the detailed description that follows.

WO 53949 2015/024208 The present invention is based, in part, on the discovery that particular mixtures, or populations, ofEhrlichia peptides or their variants have preferential binding affinity for antibodies elicited by ns from particular Ehrlichz'a species. The inventors have found that a particular combination of these peptide es or populations can be used to identify the Ehrlichz'a s inducing the antibody response. Accordingly, the present invention provides a method for identifying the species ofEhrlichia infecting a subject, if present.

In certain embodiments, the method for identifying the species ofEhrlichia infecting a subject, if t, comprises: ting a sample from the subject with a first population of isolated peptides as described herein; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; contacting said sample with a second population of isolated es as bed herein; and detecting formation of a second set of complexes comprising an antibody and one or more peptides in the second population, wherein formation of both the first and second sets of complexes indicates that the subject is infected with E. ewz‘ngz'z', and wherein formation of the first but not the second set of complexes indicates that the subject is infected with E. canis and/or E. chafleensz's.

In other embodiments, the method for identifying the species ofEhrlichia infecting a subject, if present, comprises: contacting a sample from the subject with a first tion of isolated peptides as described herein; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; contacting said sample with a third population of isolated peptides as described herein; and detecting formation of a third set of complexes comprising an antibody and one or more peptides in the third tion, wherein formation of both the first and third sets of antibody-peptide complexes indicates that the t is infected with E. canis and/or E. chafieensis, and wherein formation of the first but not the third set of antibody-peptide complexes tes that the subject is infected with E. ewz’ngz’z’.

In yet other embodiments, the method for fying the species ofEhrlichia infecting a subject, if present, ses: contacting a sample from the t with a first population of isolated peptides as described ; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; contacting said sample with a second population of isolated peptides as described herein; detecting formation of a second set of complexes comprising an antibody and one or more peptides in the second population; contacting said sample with a third population of isolated peptides as described ; and detecting formation of a third set of complexes comprising an antibody and one or more peptides in the third tion, wherein formation of both the first and second sets of xes but not the third set indicates that the subject is infected with E. ewz’ngz'z', and wherein formation of both the first and third sets of complexes but not the second set indicates that the subject is infected with E. canis and/or E. chafi’eensis.

In particular embodiments of the methods of the invention, the first population of isolated peptides is e of specifically binding to antibodies against antigens from multiple species of Ehrlichia, including E. canis, E. chafleensz’s, and E. ewz’ngz’z’. In certain embodiments, the first population of isolated peptides comprises at least three different peptides, each comprising a sequence S-X2-K-E-X5-K-Q-Xg-T-X10-X11-X12-X13-G-L-K-Q- X1g-W-XZO-G-Xzz-ng-X24-X25-X26-G-G-G-G-G-N-F-S-A-K-E-E-X39-A-E-T-R-X44-T-F-G- L-X49-K-Q-Y-D-G-A-X56-I-X58-E-N-Q-V-Q-N-K-F-T-I-S-N—C (SEQ ID NO: 1) or a fragment thereof, wherein X2 is an amino acid selected from the group consisting ofA and V, X5 is an amino acid selected from the group ting of E and D, X8 is an amino acid selected from the group consisting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid ed from the group consisting of L and V, X13 is an amino acid selected from the group ting ofY and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23 is an amino acid selected from the group consisting of A, S, and T, X24 is an amino acid ed from the group consisting ofA and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X44 is any amino acid, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid.

In certain embodiments, X39 in SEQ ID NO: 1 is K. In some embodiments, X44 in SEQ ID NO: I is K or R, and/or X49 in SEQ ID NO: I is E or D. In certain embodiments, X56 in SEQ ID NO: I is K or Q, and/or X58 in SEQ ID NO: I is E or T.

In certain other embodiments, the fragment of SEQ ID NO: 1 comprises at least 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 1. In certain embodiments, the fragment of SEQ ID NO: 1 comprises amino acids 33 to 71 of SEQ ID NO: 1. In particular embodiments, each e in the first population ses a sequence of SEQ ID NO: 1.

In some embodiments, the first population of isolated peptides comprises at least one ce, or a fragment thereof, selected from the group consisting of: S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K—Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S- A-K—E-E-K—A-E-T-R-K—T-F-G-L-E-K—Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K—F-T-I-S-N-C (SEQ ID NO: 4); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K—Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-SA-K —E-E-K—A-E-T-R-R—T-F-G-L-E-K—Q-Y-D-G-A-K—I-E-E-N-Q-V-Q-N-K—F-T-I-S-N-C (SEQ ID NO: 5); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K—Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-S- A-K—E-E-K—A-E-T-R-R-T-F-G-L-D-K-Q-Y-D-G-A-K—I-E-E-N-Q-V-Q-N-K—F-T-I-S-N-C (SEQ ID NO: 6); E-E-K—Q-T-T-T-G-L-Y-G-L-K—Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-SA-K —E-E-K—A-E-T-R—R-T-F-G-L-E-K—Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K—F-T-I-S-N-C (SEQ ID NO: 7); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N —K-F-T-I-S-N-C (SEQ ID NO: 8); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R—K-T-F-G-L-D-K-Q-Y-D-G-A-Q-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 9); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 10); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N —K-F-T-I-S-N-C (SEQ ID NO: 11); S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N -N-K-F-T-I-S-N-C (SEQ ID NO: 12); S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 13); S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 14); S-V-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 15); S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 16); S-A-K-E-E-K-Q-P-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 17); S-A-K-E-E-K-Q-P-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 18); E-E-K-Q-P-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 19); E-E-K-Q-T-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 20); S-A-K-E-E-K-Q-T-T-T-A-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 21); S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N —K-F-T-I-S-N-C (SEQ ID NO: 22); S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 23); S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-N—W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 24); S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-N-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 25); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-N—W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 26); S-A-K-E-E-K—Q-T-T-T-G-L-Y-G-L-K-Q-N—W-N—G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 27); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N—G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 28); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 29); WO 53949 S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-S-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 30); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-T-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 31); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 32); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N -I-S-N-C (SEQ ID NO: 33); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-I-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 34); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-I-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 35); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 36); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 37); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-N—G-G-G-G-G-N-F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 38); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-K-G-G-G-G-G-N-F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 39); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N—G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 40); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 41); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N—G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R—R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 42); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R—R-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 43); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-R —A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 44); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R-Q-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 45); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R -G-L-Q-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 46); E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-N-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 47); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R —K-T-F-G-L-E-K-Q-Y-D-G-A-R-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 48); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-E-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 49); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-E-T-R-K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-D-E-N-Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 50); and S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-E-T-R —K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 51).

In some embodiments, the first population of isolated peptides comprises at least two or three different sequences, or fragments f, selected from the group consisting of SEQ ID NOs: 4-5 1.

In particular embodiments of the methods, the second population of isolated es is capable of specifically or entially binding to antibodies against antigens from E. z’z’. In some embodiments, the second population of isolated peptides does not bind or minimally binds to antibodies against antigens from E. canis or E. chafleensz’s. In certain embodiments, the second population of isolated peptides comprises at least three different peptides, each comprising a sequence of F-S-A-K-E-E-X7-A-E-T-R-X12-T-F-G-L- X17-K-Q-Y-D-G-A-X24-I-X26-E-N-Q-V-Q-N-K-F-T-I-S-N—C (SEQ ID NO: 2) or a fragment thereof, wherein X7 is any amino acid, X12 is any amino acid, X17 is any amino acid, X24 is any amino acid, and X26 is any amino acid.

In certain embodiments of the second population of isolated peptides, X7 in SEQ ID NO: 2 is K. In some ments, X12 in SEQ ID NO: 2 is K or R, and/or X17 in SEQ ID NO: 2 is E or D. In certain embodiments, X24 in SEQ ID NO: 2 is K or Q, and/or X26 in SEQ ID NO: 2 is E or T.

In certain other ments, the fragment of SEQ ID NO: 2 comprises at least 15, 20, 25, 30, or 35 contiguous amino acids from SEQ ID NO: 2. In some embodiments, each peptide in the second tion comprises a sequence of SEQ ID NO: 2.

In particular embodiments, the second population of isolated peptides comprises at least one sequence, or a nt thereof, selected from the group consisting of: F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F- T-I-S-N—C (SEQ ID NO: 52); F-S-A-K-E-E-K-A-E-T-R—R—T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F- T-I-S-N—C (SEQ ID NO: 53); F-S-A-K-E-E-K-A-E-T-R—R—T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N—K-F- T-I-S-N—C (SEQ ID NO: 54); F-S-A-K-E-E-K-A-E-T-R—R—T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N—K-F- T-I-S-N—C (SEQ ID NO: 55); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 56); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-Q-I-T-E-N-Q-V-Q-N-K-F- N-C (SEQ ID NO: 57); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-T-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 58); K-E-E-R—A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 59); F-S-A-K-E-E-K-A-E-T-R—Q-T-F-G-L-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 60); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-Q-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 61); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-N-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 62); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-R—I-E-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 63); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-E-I-E-E-N-Q-V-Q-N-K-F-T- I-S-N-C (SEQ ID NO: 64); F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-D-E-N—Q-V-Q-N-K-F- T-I-S-N—C (SEQ ID NO: 65); and F-S-A-K-E-E-K-A-E-T-R—K-T-F-G-L-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N-K-F- T-I-S-N-C (SEQ ID NO: 66).

In some embodiments, the second population of isolated peptides comprises at least two or three ent sequences, or fragments thereof, selected from the group consisting of SEQ ID NOs: 52-66.

In yet other embodiments of the method, the third population of isolated peptides is capable of specifically or preferentially binding to antibodies against ns from E. canis and E. nsz’s. In some ments, the third population of isolated peptides does not bind or minimally binds to antibodies against antigens from E. ewz’ngz’z’. In some embodiments, the third population of isolated peptides comprises at least three different peptides, each comprising a sequence of S-X2-K-E-X5-K-Q-Xg-T-X10-X11-X12-X13-G-L-K-Q- X1g-W-XZO-G-Xzz-ng-X24-X25-X26-G-G-G-G-G-N-F-S-A-K-E-E-X39-A-X41-T-R—X44-T-F-G- X48-X49-K-Q-Y-D-G-A-X56-I-X5g-E-N-Q-V-Q-N-K—F-T-I-S-N-C (SEQ ID NO: 3) or a nt thereof, wherein X2 is an amino acid selected from the group consisting ofA and V, X5 is an amino acid selected from the group consisting of E and D, X8 is an amino acid selected from the group consisting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid selected from the group consisting of L and V, X13 is an amino acid selected from the group consisting ofY and F, X18 is an amino acid ed from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23 is an amino acid selected from the group consisting of A, S, and T, X24 is an amino acid ed from the group consisting ofA and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X41 is an amino acid ed from the group consisting of D and N, X44 is any amino acid, X48 is an amino acid selected from the group consisting ofV and A, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid.

In certain embodiments of the third population of isolated es, X39 in SEQ ID NO: 3 is K. In certain embodiments, X44 in SEQ ID NO: 3 is K or R, and/or X49 in SEQ ID NO: 3 is E or D. In certain embodiments, X56 in SEQ ID NO: 3 is K or Q, and/or X58 in SEQ ID NO: 3 is E or T.

In certain other ments, the fragment of SEQ ID NO: 3 comprises at least 20, 25, 30, 35, or 40 contiguous amino acids from SEQ ID NO: 3. In certain embodiments, the fragment of SEQ ID NO: 3 comprises amino acids 33 to 71 of SEQ ID NO: 3. In particular embodiments, each peptide in the third population comprises a ce of SEQ ID NO: 3.

In particular embodiments, the third population of isolated peptides comprises at least one sequence, or a fragment thereof, selected from the group consisting of: S-A-K—E-E-K—Q-T-T-T-G-L-Y-G-L-K—Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N-F-SA-K —E-E-K—A-D-T-R-K-T-F-G-V-E-K—Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 67); 2015/024208 S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-R-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 68); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-R-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 69); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-R-T-F-G-V-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 70); E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 71); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-Q-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 72); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-Q-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 73); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 74); S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 75); S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-T-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 76); S-V-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 77); S-V-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 78); S-A-K-E-D-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N -N—K-F-T-I-S-N-C (SEQ ID NO: 79); S-A-K-E-E-K-Q-P-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 80); S-A-K-E-E-K-Q-P-T-V-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 81); S-A-K-E-E-K-Q-P-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 82); S-A-K-E-E-K-Q-T-T-V-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 83); S-A-K-E-E-K-Q-T-T-V-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 84); S-A-K-E-E-K-Q-T-T-T-A-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 85); S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 86); S-A-K-E-E-K-Q-T-T-T-G-V-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 87); S-A-K-E-E-K-Q-T-T-T-G-V-Y-G-L-K-Q-N—W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 88); S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 89); S-A-K-E-E-K-Q-T-T-T-G-L-F-G-L-K-Q-N—W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N -N—K-F-T-I-S-N-C (SEQ ID NO: 90); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-N—W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 91); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N—G-S-A-A-T-S-G-G-G-G-G-N—F-S- E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 92); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N—G-S-S-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 93); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-N—G-S-T-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 94); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 95); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-S-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 96); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-V-T-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 97); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-S-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 98); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-T-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 99); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-N—G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 100); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-K-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 101); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-I-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 102); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 103); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-N—G-G-G-G-G-N-F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 104); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-P-K-G-G-G-G-G-N-F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 105); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-N—G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N -N-K-F-T-I-S-N-C (SEQ ID NO: 106); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-K-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 107); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-N —T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 108); E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-N—T-R-K-T-F-G-A-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 109); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-N—T-R-K-T-F-G-V-D-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 110); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-A-E-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 111); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-A-D-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N —K-F-T-I-S-N—C (SEQ ID NO: 112); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-R-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 113); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-Q-T-F-G-V-E-K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 114); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-Q-K-Q-Y-D-G-A-K-I-E-E-N —Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 115); E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-N —K-Q-Y-D-G-A-K-I-E-E-N-Q-V-Q-N—K-F-T-I-S-N—C (SEQ ID NO: 116); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-R-I-E-E-N —Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 117); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-E-I-E-E-N—Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 118); S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-SA-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-D-E-N-Q-V-Q-N —K-F-T-I-S-N—C (SEQ ID NO: 119); and S-A-K-E-E-K-Q-T-T-T-G-L-Y-G-L-K-Q-D-W-D-G-S-A-A-T-S-G-G-G-G-G-N—F-S- A-K-E-E-K-A-D-T-R-K-T-F-G-V-E-K-Q-Y-D-G-A-K-I-S-E-N-Q-V-Q-N—K-F-T-I-S-N-C (SEQ ID NO: 120).

In some ments, the third population of isolated es comprises at least two or three different sequences, or fragments thereof, selected from the group ting of SEQ ID NOs: 67-120.

In certain embodiments, the populations of isolated peptides used in the method comprise a fragment of a peptide sequence described herein. For example, in certain ments, the populations of isolated peptides comprise a fragment of a sequence selected from the group consisting of SEQ ID NOs: 1-120. The nt can be, e.g., at least , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids in length. The fragment can be contiguous or can include one or more deletions (e.g., a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues). In some embodiments, the fragments comprise amino acids 1 to 26 of a sequence selected from the group consisting of SEQ ID NOs: 1-120. In other embodiments, the fragments comprise amino acids 33 to 71 of a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4-51, and 67-120. In certain embodiments, the fragments comprise an epitope of a e sequence selected from the group consisting of SEQ ID NOs: 1-120.

In some embodiments, one or more of the peptides in the first and/or third tion of peptides used in the method are no longer than 71, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length. In particular embodiments, at least three peptides in the first and/or third population ofpeptides are no longer than 71, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length. In certain embodiments, each peptide in the first and/or third population of peptides is no longer than 71, 75, 80, 85, 90,95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length.

In some other embodiments, one or more of the es in the second population of peptides used in the method are no longer than 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length. In ular embodiments, at least three peptides in the second population ofpeptides are no longer than 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in . In certain embodiments, each peptide in the second population of peptides is no longer than 39, 40,45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length.

In particular embodiments, each peptide in the first and third population of peptides is no longer than 71, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length, and each peptide in the second population ofpeptides is no longer than 39, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, or 2000 amino acids in length.

In yet other embodiments, the populations of isolated peptides can comprise the peptides disclosed in US Application No. 14/052,296 and/or US Patent Application Publication No. 201 1/0124125A1, the contents of which are hereby incorporated by nce in their entirety.

In some embodiments, the peptides in the populations of isolated peptides used in the method can comprise a sequence that is at least about 80, 85, 90, 95, 98, or 99% identical to a ce selected from SEQ ID NOs: 1-120. Percent sequence ty has an art recognized meaning and there are a number of methods to measure identity between two ptide or polynucleotide sequences. See, e. g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, ; 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 Heinj e, ce Analysis In Molecular Biology, Academic Press, (1987); and Gribskov & Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, . Methods for aligning polynucleotides or polypeptides are codified 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 ), and Bestfit program (Wisconsin Sequence Analysis e, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) which uses the local homology algorithm of Smith and Waterman (Adv. App. Math., 489 ). For example, the computer program ALIGN which s the FASTA algorithm can be used, with an affine gap search with a gap open penalty of —12 and a gap extension penalty of —2.

When using any of the sequence ent programs to determine whether a particular sequence is, for instance, about 95% identical to a reference sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference ptide and that gaps in identity of up to 5% of the total number of amino acids in the reference polypeptide are allowed.

Variants of the peptide sequences can be readily selected by one of skill in the art, based in part on known properties of the sequence. For example, a variant peptide can include amino acid substitutions (e.g., conservative tutions with naturally occurring amino acids, non-naturally occurring amino acids, or amino acid analogs) and/or deletions (e.g., small, single amino acid deletions, or deletions encompassing 2, 3, 4, 5, 10, 15, 20, or more contiguous amino . Thus, in certain embodiments, a variant of a native peptide sequence is one that differs from a naturally-occurring sequence by (i) one or more (e.g., 2, 3, 4, 5, 6, or more) conservative amino acid substitutions, (ii) deletion of 1 or more (e.g., 2, 3, 4, , 6, or more) amino acids, or (iii) a combination thereof. Deleted amino acids can be contiguous or non-contiguous. Conservative amino acid substitutions are those that take place within a family of amino acids that are related in their side chains and chemical properties. These e, e.g., (l) acidic amino acids: aspartate, glutamate; (2) basic amino acids: lysine, arginine, histidine; (3) nonpolar amino acids: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) uncharged polar amino acids: glycine, gine, glutamine, cysteine, serine, threonine, tyrosine; (5) aliphatic amino acids: glycine, alanine, , e, isoleucine, serine, threonine, with serine and ine optionally grouped separately as aliphatic-hydroxyl; (6) aromatic amino acids: phenylalanine, tyrosine, tryptophan; (7) amide amino acids: asparagine, glutamine; and (9) sulfur-containing amino acids: cysteine and methionine. See, e.g., Biochemistry, 2nd ed., Ed. by L. , W H Freeman and Co.: 1981. Methods for confirming that variant peptides are suitable are conventional and routine.

Variants of the peptide sequences encompass variations on previously defined peptide sequences. For example, a previously described peptide sequence comprising a known epitope may be lengthened or shortened, at one or both ends (e.g., by about 1-3 amino acids), and/or one, two, three, four or more amino acids may be substituted by conservative amino acids, etc. Furthermore, if a region of a protein has been identified as containing an epitope of interest, an investigator can " the region of interest (e.g., by about 5 amino acids in either ion) from the endpoints of the original rough region to optimize the activity.

In some embodiments, the peptides in the tions of isolated peptides used in the method can further comprise an additional N—terminal peptide sequence, an additional C-terminal e sequence, or a combination thereof.

In certain ments, the additional N—terminal peptide sequence can comprise l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids and can be either a native or non- WO 53949 native sequence. In other embodiments, the additional inal peptide ce can comprise l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids and can be either a native or non- native sequence.

The additional N—terminal or C-terminal peptide sequence can be a native sequence. As used herein, a "native" sequence is a peptide sequence from a naturally- occurring Ehrlichz'a OMP-l sequence, or a variant thereof. In certain embodiments, the peptide sequence is a fragment of a naturally-occurring Ehrlichz'a OMP-l sequence. The peptide sequence can be, e.g., from a conserved or non-conserved region of OMP-l. The peptide sequence can comprise, e.g., an epitope, such as an dominant epitope or any other epitope recognizable by a host (e.g., human, dog, etc.) immune system. OMP-l proteins and peptides thereof have been described, e.g., in US. Patent Nos. 6,544,517, 6,893,640, 6,923,963, 7,063,846, and 770, US. Patent Applications 265333 and 2009/0075368, and European Patent No. 1026949, the contents of each of which are incorporated herein by reference in their entirety.

In certain embodiments, the onal inal or inal peptide sequence is a non-native sequence. As used herein, a "non-native" sequence is any protein sequence, Whether from an Ehrlichz'a protein or otherwise, other than a native OMP-l peptide SGQUGHCG.

In certain embodiments, the additional N—terminal or C-terminal peptide sequence can comprise or consist of another peptide having a sequence, or a fragment thereof, selected from SEQ ID NOs: l-l20.

In some embodiments, the additional N—terminal or C-terminal peptide sequence can be linked to the peptides in the populations of isolated peptides through one or more linking amino acids (e.g. glycine, serine, or cysteine residues).

The isolated peptides in the populations may be isolated by chemical synthesis and/or purification. In some embodiments, the peptides are produced biologically (i.e., by cellular machinery, such as a ribosome) and then isolated. As used herein, an "isolated" e is a peptide that has been ed either synthetically or ically and then purified, at least partially, from the als and/or cellular machinery used to produce the peptide. In certain embodiments, an isolated peptide of the invention is substantially purified.

The term "substantially purified," as used herein, refers to a molecule, such as a peptide, that is substantially free of cellular material (proteins, , carbohydrates, nucleic acids, etc.), culture medium, chemical precursors, chemicals used in synthesis of the e, or combinations thereof. A peptide that is ntially purified has less than about 40%, 30%, %, 20%, 15%, 10%, 5%, 2%, l% or less of the cellular material, culture medium, other polypeptides, chemical precursors, and/or chemicals used in synthesis of the peptide.

Accordingly, a substantially pure molecule, such as a peptide, can be at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, by dry weight, the molecule ofinterest. An isolated peptide or population of peptides can be in water, a buffer, or in a dry form awaiting reconstitution, e. g., as part of a kit.

In n embodiments, one or more peptides in the populations are conjugated to a ligand. For e, in certain embodiments, the peptides are biotinylated.

In other embodiments, the peptides are conjugated to streptavidin, avidin, or neutravidin. In other embodiments, the peptides are conjugated to a carrier protein (e.g, serum albumin, keyhole limpet hemocyanin (KLH), or an immunoglobulin Fc domain). In still other embodiments, the peptides are conjugated to a dendrimer and/or are part of a multiple antigenic peptide system . The peptides may also be conjugated to colloidal gold, quantum dots or other nanoparticles and/or to latex particles. In still another embodiment, the peptides may be conjugated to s, fluorescent or chemi-luminescent markers.

In certain embodiments, peptides in the populations of isolated peptides are modified. The peptides of the ion may be modified by a variety of techniques, such as by denaturation with heat and/or a detergent (e.g. , SDS). Alternatively, peptides of the invention may be modified by association with one or more further moieties. The association can be covalent or non-covalent, and can be, for example, via a terminal amino acid linker, such as lysine or cysteine, a chemical coupling agent, or a peptide bond. The additional moiety can be, for example, a , a ligand or, a fusion partner, a detectable label, an , or a substrate that lizes the peptide.

In addition, the peptides in the populations of isolated peptides may be modified to e any of a variety ofknown chemical groups or molecules. Such modifications e, but are not limited to, glycosylation, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment to polyethylene glycol (e.g., PEGylation), covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, de bond formation, demethylation, formation of covalent cross-links, formation of cystine, ion of pyroglutamate, ation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, ubiquitination, ations with fatty acids, transfer-RNA mediated addition of amino acids to proteins such as arginylation, etc. Analogues of an amino acid (including unnatural amino acids) and peptides with tuted linkages are also included.

Modifications as set forth above are well-known to those of skill in the art and have been described in great detail in the scientific literature. l particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in many basic texts, such as Proteins-Structure and Molecular Properties, 2nd ed., T. e. Creighton, W.H.

Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C.

Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth. Enzymol. 182:626-646 and Rattan et al. (1992) Ann. NY. Acad. Sci. 663:48-62.

In certain embodiments, one or more or all peptides in a population of peptides is attached to or immobilized on a substrate, such as a solid or semi-solid support. The attachment can be covalent or non-covalent, and can be facilitated by a moiety associated with the peptide that enables covalent or valent binding, such as a moiety that has a high affinity to a component attached to the carrier, support or e. For example, the peptide can be associated with a ligand, such as biotin, and the component associated with the surface can be a corresponding ligand receptor, such as avidin. In some embodiments, the peptide can be associated with a fusion partner, e.g., bovine serum albumin (BSA), which facilitates the attachment of the peptide to a ate. In other ments, the es of the invention are attached to or immobilized on a ate via a metallic nanolayer such as a localized surface plasmon nce spectroscopy (LSPR) surface. In one embodiment, the metallic nanolayer is comprised of cadmium, zinc, mercury, or a noble metal, such as gold, silver, copper, and um. The peptide or population of peptides can be attached to or immobilized on the substrate either prior to or after the on of a sample containing antibody during an immunoassay.

In certain embodiments, the substrate is a bead, such as a colloidal particle (e.g., a colloidal rticle made from gold, silver, platinum, copper, cadmium, metal composites, other soft metals, core-shell ure particles, or hollow gold nanospheres) or other type of le (e.g., a magnetic bead or a particle or nanoparticle comprising silica, latex, polystyrene, rbonate, rylate, or PVDF). Such particles can comprise a label (e.g, a colorimetric, chemiluminescent, or fluorescent label) and can be useful for visualizing the location of the peptides during immunoassays. In certain embodiments, a al cysteine of a peptide of the ion is used to bind the e directly to the nanoparticles made from gold, , platinum, copper, cadmium, metal composites, or other soft metals, or metallic nanoshells (e.g., gold hollow spheres, gold-coated silica ells, and -coated gold shells).

In certain embodiments, the substrate is a dot blot or a flow path in a lateral flow immunoassay device. For example, the peptides can be attached or immobilized on a porous membrane, such as a PVDF membrane (e.g, an ImmobilonTM membrane), a nitrocellulose membrane, polyethylene membrane, nylon membrane, or a similar type of membrane.

In n embodiments, the substrate is a flow path in an analytical or centrifugal rotor. In other embodiments, the substrate is a tube or a well, such as a well in a plate (e.g., a microtiter plate) suitable for use in an ELISA assay. Such substrates can comprise glass, cellulose-based materials, thermoplastic polymers, such as polyethylene, polypropylene, or polyester, sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers), or cast membrane fllm composed of nitrocellulose, nylon, polysulfone, or the like. A substrate can be sintered, fine particles of polyethylene, commonly known as porous polyethylene, for example, 0.2-15 micron porous polyethylene from x Corporation (Albuquerque, NM). All of these substrate materials can be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like.

In one embodiment, the methods of invention involve detecting the presence of naturally occurring antibodies against one or more Ehrlichz'a antigens (e.g., the antigen of a enic hz'a, such as E. chafieensz’s, E. mum's, E. ewz’ngz’z’, or E. canis) which are produced by the infected subj ect's immune system in its biological fluids or tissues, and which are e of binding specifically to one of more peptides in a population of peptides and, optionally, one or more suitable additional antigenic polypeptides or peptides.

For example, in one aspect, the present invention provides a method of detecting in a sample from a subject the presence of dies against antigens from E. chafleensis, E. mum's, E. ewingii, and/or E. canis comprising ting the sample with a population of peptides comprising at least three different peptides, wherein each peptide comprises a sequence of SEQ ID NO: 1; and detecting formation of complexes comprising an antibody and one or more peptides in the population, wherein formation of the complexes tes the presence of antibodies against antigens from E. chafieensis, E. mum's, E. ewz’ngz'z', and/or E. canis. In some ments, the population of peptides comprises at least two or three different sequences selected from the group consisting of SEQ ID NOs: 4-51.

In other embodiments, the present invention provides a method of detecting in a sample from a subject the presence of dies against antigens from E. ewz’ngz’z’ comprising contacting the sample with a population of peptides comprising at least three different peptides, wherein each peptide comprises a sequence of SEQ ID NO: 2; and detecting formation of xes comprising an antibody and one or more peptides in the population, wherein formation of the complexes indicates the presence of antibodies against antigens from E. ewz’ngz'z'. In some embodiments, the population of peptides comprises at least two or three different ces selected from the group ting of SEQ ID NOs: 52- In certain embodiments, the present invention provides a method of detecting in a sample from a subject the ce of dies against antigens from E. chafieensis and/or E. canis comprising contacting the sample with a population of peptides sing at least three different peptides, wherein each peptide ses a sequence of SEQ ID NO: 3; and detecting formation of complexes comprising an antibody and one or more peptides in the population, n formation of the complexes indicates the presence of antibodies t antigens from E. chafieensis and/or E. canis. In some embodiments, the population of WO 53949 peptides comprises at least two or three different sequences selected from the group consisting of SEQ ID NOs: .

There are a number of different assays that may be used to detect formation of antibody-peptide complexes comprising one or more peptides in the methods of the invention. For example, the detecting step can comprise performing an ELISA assay, ming an immunofiuorescence assay, performing a lateral flow immunoassay, performing an agglutination assay, performing a wavelength shift assay, performing a Western blot, slot blot, or dot blot, analyzing the sample in an analytical or centrifugal rotor, or analyzing the sample with an electrochemical, l, or opto-electronic sensor. These different assays are described herein and/or are well-known to those skilled in the art.

Suitable immunoassay methods typically e: receiving or ing (e.g., from a patient) a sample of body fluid or tissue likely to contain antibodies; contacting (e.g., incubating or reacting) a sample to be assayed with a population of peptides, under conditions effective for the formation of a specific peptide-antibody complex (e.g., for c binding of the peptide to the antibody); and assaying the contacted (reacted) sample for the presence of an antibody-peptide reaction (6.g. the amount of an antibody-peptide , determining complex). The presence of an elevated amount of the antibody-peptide complex tes that the subject was exposed to and infected with an infectious Ehrlichz’a s. A peptide, including a modified form thereof, which "binds specifically" to (e.g., "is specific for" or binds rentially" to) an antibody against an Ehrlichz'a antigen interacts with the antibody, or forms or undergoes a physical association with it, in an amount and for a sufficient time to allow detection of the antibody. By fically" or "preferentially," it is meant that the peptide has a higher affinity (e.g., a higher degree of selectivity) for such an antibody than for other antibodies in a sample. For example, the peptide can have an affinity for the antibody of at least about l.5-fold, 2-fold, ld, 3-fold, or higher than for other dies in the sample. Such y or degree of specificity can be determined by a variety of routine procedures, including, e.g., competitive binding studies. In an ELISA assay, a positive response is defined as a value 2 or 3 standard ions greater than the mean value of a group of healthy controls. In some embodiments, a second tier assay is required to provide an unequivocal serodiagnosis of monocytic and/or granulocytic ehrlichiosis.

Phrases such as "sample containing an antibody" or "detecting an antibody in a sample" are not meant to exclude samples or determinations (e.g., detection attempts) where no antibody is contained or detected. In a general sense, this invention involves assays to determine whether an dy produced in response to infection with an infectious Ehrlichz'a is present in a sample, irrespective of whether or not it is detected.

Conditions for reacting peptides and antibodies so that they react specifically are well-known to those of skill in the art. See, e.g., Current Protocols in Immunology (Coligan et al., editors, John Wiley & Sons, Inc).

In some ments, the methods comprise receiving or obtaining a sample ofbody fluid or tissue likely to n antibodies from a subject. The antibodies can be, e.g., of IgG, IgE, IgD, IgM, or IgA type. Generally, IgM and/or IgA antibodies are ed, e.g., for detection at early stages of infection. IgG antibodies can be detected when some of the additional peptides discussed above are used in the method (e.g., peptides for the ion of flagellum proteins). The sample is preferably easy to obtain and may be whole blood, plasma, or serum derived from a venous blood sample or even from a finger prick. Tissue from other body parts or other bodily fluids, such as cerebro-spinal fluid (CSF), saliva, gastric secretions, mucus, urine, etc., are known to contain antibodies and may be used as a source of the sample. The sample may also be a tissue extract or a cell lysate.

Once a population of peptides and sample antibody are permitted to react in a suitable medium, an assay is performed to determine the presence or absence of an antibody- peptide reaction. Among the many types of le assays, which will be t to a skilled worker, are immunoprecipitation and agglutination assays performed with or without ement.

The protocols for immunoassays using antigens for detection of specific dies are well known in art. For example, a conventional sandwich assay can be used, or a tional competitive assay format can be used. For a discussion of some suitable types of assays, see Current Protocols in Immunology (supra). In certain embodiments, a peptide of the invention is immobilized on a solid or semi-solid e or carrier by means of covalent or valent binding, either prior to or after the on of the sample containing antibody.

Devices for performing specific binding assays, especially immunoassays, are known and can be readily adapted for use in the present methods. Solid phase assays, in l, are easier to perform than heterogeneous assay methods which require a separation step, such as precipitation, centrifugation, filtration, chromatography, or magnetism, because separation of reagents is faster and simpler. Solid-phase assay devices include iter plates, flow-through assay devices (e.g., lateral flow immunoassay devices), dipsticks, and immunocapillary or immunochromatographic immunoassay devices.

In some embodiments of the invention, the solid or semi-solid surface or carrier ed to the populations of peptides is the floor or wall in a microtiter well, a filter surface or membrane (e.g., a nitrocellulose membrane or a PVDF inylidene fluoride) membrane, such as an ImmobilonTM membrane), a hollow fiber, a beaded chromatographic medium (e.g., an agarose or polyacrylamide gel), a magnetic bead, a fibrous cellulose matrix, an HPLC matrix, an FPLC matrix, a substance having molecules of such a size that the molecules with the peptide bound thereto, when dissolved or sed in a liquid phase, can be retained by means of a filter, a substance capable of forming micelles or participating in the formation of micelles allowing a liquid phase to be changed or ged without entraining the micelles, a soluble polymer, or any other suitable carrier, support or surface.

In some embodiments of the invention, a population of peptides is ed with a suitable label which enables detection. Conventional labels may be used which are capable, alone or in t with other compositions or compounds, of providing a detectable signal. Suitable labels include, but are not limited to, enzymes (e.g., HRP, beta- galactosidase, alkaline phosphatase, etc.), fluorescent labels, radioactive labels, colored latex particles, and metal-conjugated labels (e.g., metallic nanolayers, metallic nanoparticle- or metallic ell-conjugated labels). Suitable ic nanoparticle or metallic nanoshell labels e, but are not limited to, gold les, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells. Metallic yers suitable for detectable layers include yers sed of cadmium, zinc, mercury, and noble metals, such as gold, silver, copper, and platinum. le ion methods include, e.g., detection of an agent which is tagged, directly or indirectly, with a colorimetric assay (6.g. , for detection of HRP or beta- galactosidase activity), Visual inspection using light microscopy, immunofluorescence microscopy, including confocal microscopy, or by flow cytometry (FACS), autoradiography (e.g., for detection of a radioactively labeled agent), electron microscopy, immunostaining, lular fractionation, or the like. In one embodiment, a radioactive element (e.g., a radioactive amino acid) is incorporated directly into a peptide chain; in another embodiment, a fluorescent label is associated with a peptide via biotin/avidin interaction, association with a fiuorescein conjugated antibody, or the like. In one embodiment, a detectable specific binding partner for the antibody is added to the mixture. For example, the binding partner can be a detectable secondary antibody or other binding agent (e.g., protein A, n G, protein L, chimeric proteins A/G, A/G/L, A/L, G/L or ations thereof) which binds to the first dy. This secondary antibody or other binding agent can be labeled, e.g., with a radioactive, enzymatic, fluorescent, luminescent, chemi-luminescent, metallic rticle or ic nanoshell (e.g. colloidal gold), or other detectable label, such as an avidin/biotin, avidin/streptavidin or avidin/polystreptavidin system. In another embodiment, the binding r is a peptide of the invention, which can be conjugated directly or indirectly (e.g. via biotin/avidin or biotin/streptavidin interaction) to an enzyme, such as horseradish peroxidase or alkaline phosphatase or other signaling moiety. In such embodiments, the detectable signal is produced by adding a substrate of the enzyme that produces a detectable signal, such as a chromogenic, fiuorogenic, or chemiluminescent substrate.

A "detection system" for detecting bound peptide, as used herein, may comprise a detectable binding partner, such as an antibody specific for the peptide. In one embodiment, the binding partner is labeled directly. In another embodiment, the g r is attached to a signal ting reagent, such as an enzyme that, in the presence of a suitable substrate, can produce a able signal. A surface for immobilizing the peptide may optionally any the detection system.

In some embodiments of the ion, the detection procedure comprises visibly ting the antibody-peptide complex for a color change, or inspecting the antibody-peptide x for a physical-chemical change. Physical-chemical changes may occur with oxidation reactions or other chemical reactions. They may be detected by eye, using a spectrophotometer, or the like.

A very useful assay format is a lateral flow immunoassay format. Antibodies to human or animal (e.g., dog, mouse, deer, etc.) immunoglobulins, or staph A, G, or L proteins, can be labeled with a signal generator or reporter (e.g. , colloidal gold) that is dried and placed on a glass fiber pad (sample application pad or conjugate pad). The diagnostic peptide is lized on membrane, such as nitrocellulose or a PVDF (polyvinylidene fluoride) membrane (e.g., an ImmobilonTM membrane). When a solution of sample (blood, serum, etc.) is d to the sample application pad (or flows through the conjugate pad), it dissolves the labeled reporter, which then binds to all antibodies in the sample. The resulting complexes are then transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by ary action. If antibodies against the diagnostic peptide are present, they bind to the diagnostic peptide striped on the membrane, thereby generating a signal (6.g. , a band that can be seen or visualized). An additional antibody specific to the labeled antibody or a second labeled antibody can be used to produce a control signal.

An alternative format for the lateral flow immunoassay comprises the populations of ed peptides being conjugated to a ligand (e.g., biotin) and complexed with labeled ligand receptor (e.g., streptavidin-colloidal gold). The labeled peptide complexes can be placed on the sample application pad or conjugate pad. Anti-human IgG/IgM or anti-animal (e.g., dog, mouse, deer) IgG/IgM antibodies or other peptides of the invention are immobilized on a ne, such as ellulose of PVDF, at a test site (e.g., a test line). When a sample is added to the sample ation pad, antibodies in the sample react with the labeled peptide complexes such that dies that bind to peptides of the invention become indirectly labeled. The antibodies in the sample are then transported into the next membrane (PVDF or nitrocellulose containing the diagnostic peptide) by capillary action and bind to the immobilized anti-human M or anti-animal IgG/IgM antibodies (or protein A, protein G, protein A/G fusion proteins, protein L, or combinations thereof) or immobilized peptides of the invention. If any of the sample antibodies are bound to the labeled peptides of the invention, the label associated with the peptides can be seen or ized at the test site. In r embodiment of this type of lateral flow device (in which the peptides of the invention are used both as the immobilized capture agent at a test site and as a soluble d complex to react with antibodies in a sample), to amplify the detection signal, protein A, protein G, and/or protein A/G fusion proteins conjugated to a detectable label (e.g., metallic nanoparticle or nanoshell, HRP, ALP, fluorophore, colored latex particle) may be applied to the test site where they will bind to the Fc region of any dies to Ehrlichz'a antigens captured by the lized peptides of the invention. Suitable controls for this assay can include, e.g., a chicken IgY-colloidal gold conjugate d at the sample application pad or conjugate pad, and an anti-chicken IgY antibody immobilized at a control site located al to the test site. Other suitable controls can include chicken anti-Protein A, mouse IgG or any other ns capable of binding to Protein A/G/L. In at least some of the lateral flow immunoassays performed in the methods of invention and described herein, chicken anti-Protein A was used as the control line.

Another assay for the screening of blood products or other physiological or biological fluids is an enzyme linked immunosorbent assay, z'.e., an ELISA. Typically in an ELISA, isolated peptides or mixtures or populations of peptides are adsorbed directly, or ing conjugation to a carrier n, to the surface of a microtiter well directly or through a e matrix (e.g., an antibody). Residual, non-specific protein-binding sites on the surface are then blocked with an appropriate agent, such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (a buffered solution of nonfat dry milk which also contains a preservative, salts, and an antifoaming agent). The well is then incubated with a biological sample suspected of containing specific anti-Ehrlichz'a (e.g., anti- E. chafleensz’s, anti-E. z’z’, or anti-E. canis) antibody. Such biological sample can be a serum, , or other type of sample. The sample can be applied neat, or more often it can be diluted, usually in a buffered solution which contains a small amount (0. l-l0.0% by weight) of protein, such as BSA, NGS, or BLOTTO. After incubating for a sufficient length of time to allow specific binding to occur, the well is washed to remove d protein and then incubated with an optimal tration of an appropriate anti-immunoglobulin antibody (e.g., for human subjects, an anti-human immunoglobulin (0tHng) from another animal, such as dog, mouse, cow, etc.) or another peptide of the invention that is conjugated to an enzyme or other label by standard procedures and is dissolved in blocking buffer. The label can be chosen from a variety of enzymes, including adish peroxidase (HRP), beta-galactosidase, alkaline phosphatase (ALP), glucose oxidase, etc. In certain embodiments, Protein A or Protein G-HRP is used in the methods of invention. Sufficient time is allowed for specific binding to occur again, then the well is washed again to remove d conjugate, and a suitable ate for the enzyme is added. Color is allowed to develop and the optical density of the ts of the well is determined visually or instrumentally red at an appropriate wave length). The cutoff OD value may be defined as the mean OD+3 standard ions (SDs) of at least 50 serum samples collected from duals from an area where ehrlichiosis is not endemic, or by other such conventional def1nitions. In the case of a very specific assay, OD+2 SD can be used as a cutoff value.

In another embodiment, the methods comprise an agglutination assay. For example, in certain embodiments, metallic nanoparticles or metallic nanoshells (e.g., colloidal gold, etc.) or latex beads are conjugated to the populations of isolated peptides.

Subsequently, the ical fluid is incubated with the bead/peptide conjugate, y g a on mixture. The reaction mixture is then ed to ine the presence of the antibodies. In certain embodiments, the agglutination assays comprise the use of a second population of particles, such as metallic nanoparticles or metallic nanoshells (e.g., dal gold, etc.) or latex beads, conjugated to (1) antibodies specific to the peptides of itions of the invention, in the case of a competition assay, or (2) antibodies e of detecting sample dies (e.g., anti-human IgG or IgM antibodies, anti-dog IgG or IgM antibodies, anti-cat IgG or IgM antibodies, etc.), in the case of a sandwich assay. Suitable agglutination methods can comprise centrifiJgation as a means of assessing the extent of agglutination.

In still other embodiments, the populations of isolated peptides are electro- or dot-blotted onto nitrocellulose paper. Subsequently, a sample, such as a biological fluid (e.g., serum or plasma) is incubated with the d antigen, and antibody in the biological fluid is allowed to bind to the antigen(s). The bound dy can then be detected, e.g., by standard immunoenzymatic methods or by visualization using metallic nanoparticles or nanoshells coupled to secondary antibodies or other antibody binding agents, such as protein A, n G, protein A/G fusion proteins, protein L, or combinations thereof.

In still other embodiments, peptide or compositions of the invention are electro- or dot-blotted onto nitrocellulose paper. Subsequently, a sample, such as a biological fluid (e.g., serum or plasma) is incubated with the blotted n, and antibody in the biological fluid is allowed to bind to the antigen(s). The bound antibody can then be ed, e.g., by standard immunoenzymatic methods or by visualization using metallic nanoparticles or nanoshells coupled to secondary antibodies or other antibody binding agents, such as protein A, protein G, n A/G fusion proteins, protein L, or combinations thereof.

In still other ments, a protein microarray (or protein chip) is used in the methods. For example, in certain embodiments, the microarray or chip comprises a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, which are conjugated to an array of capture proteins comprising a population of peptides as described above. Samples, optionally labeled with a fluorescent dye, are added to the array. Specif1c binding n the antibodies in the samples, if present, and the immobilized protein emits a fluorescent signal that is read by a laser scanner. Unlabeled antibodies bound to the peptides of invention may also be subsequently labeled with quantum dot-labeled Protein A, A/G, etc.

Microarrays of isolated peptides may also be used in a microchip chip format in a centrifugal analyzer. Protein microarrays are high-throughput, rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents.

It should be understood by one of skill in the art that any number of conventional protein assay formats, particularly immunoassay formats, may be designed to utilize the tions of isolated peptides for any of the methods described herein. This invention is thus not limited by the selection of the particular assay , and is believed to encompass all suitable assay formats that are known to those of skill in the art.

Using any of the suitable assay s described herein or otherwise known to those of skill in the art, formation of complexes sing an antibody and one or more peptides in the populations of isolated peptides can be ed. By a "set" of complexes, it refers to complexes formed between one population of isolated peptides and any antibodies in a sample. When a detection result is described as formation of one but not another set of complexes, it includes a range of results that can be obtained with two different populations of isolated peptides. By "formation of the first but not the second set of complexes", e.g., it can include a clearly positive result obtained with the first tion of isolated peptides and a clearly negative result with the second population of isolated peptides. It can also include a very high score of the result obtained with the first tion of isolated peptides and a very low score of the result ed with the second population of ed peptides. It can further include any relatively higher score of the result obtained with the first population than the second tion of isolated peptides.

For any of the assay formats described herein, a score can be assigned to the assay result of each sample. Such score refers to a relative value, level, strength, or degree of an assay result. It can be artificially created by a person of skill in the art or by using an algorithm, mes using s with known analytes, e. g., antigens or dies, optionally using samples with known trations or titers of the known analytes (which can be called "standards" or "calibrators"). A score can be a number manually assigned by a person of skill in the art or generated with a formula or computer algorithm, e. g., from zero for a negative control to any positive number for a positive control (e.g., l, 2, 3, 4, 5, 10, 20, , 40, 50, 60, 80, 100, 120, 150, 200, 300, 400, 500, 1000, etc.). It can also be represented by symbols, e.g., "-" for a negative control, and "+", "++", "+++", etc., for positive controls.

A score can be determined by calculation with a a or by automatic processing with a computer algorithm, or can be determined by visual inspection, measurement, or estimation of the assay result. When using samples with known concentrations or titers of known analytes (the standards or calibrators), such standards/calibrators can be assayed in diluted and undiluted ions, and a range of scores or a standard curve of scores can be generated, which can be used to determine the scores of unknown samples assayed for the same analytes, preferably with the same assays and in the same assay runs.

In certain embodiments, the method uses a combination of immunochemical assays and three populations of peptides to identify whether a sample is infected with one, two or all three of the ing hz'a species: E. canis, E. chafleensz’s, and E. ewz’ngz'z'.

In some embodiments of the method, a standard sample that has a known titer of antibodies t a certain species (e.g., E. ewz’ngz’z), or a certain combination of species (e.g., E. canis and E. chafleensz’s, or E. canis, E. chafleensz’s, and E. ewz’ngz’z) is . In certain ments, the standard sample is diluted to a series of standards/calibrators.

Calibrators may be prepared using purified antibodies. Calibrators may also be prepared by selecting, pooling and/or diluting antisera/antibody samples with various levels of dy titer. A person of skill in the art should know how to te suitable calibrators. Scores and a standard curve can be generated for the series of standards/calibrator. In some embodiments, a cutoff is generated for a sample to be classified as positive for comprising antibodies against certain species of Ehrlichia (e. g., a cutoff for antibodies against E. ewz’ngz'z', a cutoff for dies against E. canis and E. chafieensz’s, and another cutoff for antibodies against E. canis, E. chafieensz’s, and E. ewz’ngz’z’). A sample can be classified as a low, medium, or high sample. A low sample is y just above the limit of detection, and a very high sample shows most se in a given population. Calibrators for ELISA are often prepared to represent the ranges from low to very high samples.

In certain embodiments, an unknown sample is tested with the same assays as the standards. In some embodiments, a score of the unknown sample is generated against the scores or the standard curve of each standard, e.g., a score of the unknown sample can be ted against a standard that has a known titer of antibodies against E. ewingii; and another score of the unknown sample can be generated against a rd that has a known titer of antibodies against both E. canis and E. chafleensz’s.

Scores can be compared among samples assayed for a same analyte or for different analytes.

When comparing scores of samples assayed for a same analyte, the scores can be determined from and compared t the same range of scores or the same standard curve of scores generated from the standards/calibrators, if all the samples are assayed in the same experiment under the same conditions as the standards/calibrators. The scores can also be determined from ent ranges of scores or different standard curves of scores from the standards/calibrators, if the samples are assayed in different experiments along with the same standards/calibrators. Then relative scores of the assayed samples in relation to the same standards/calibrators can be determined and compared to each other.

When comparing scores of samples assayed for different analytes, e.g., antibodies against different species of hia, each species or combination of species (such as E. canis and E. chafleensz’s) has its own calibrators. The limit of detection for each set of ators and assay is determined by generating a cutoff normally assigned by adding 2—3 standard deviations to the mean of the samples known to be negative for the antibodies being detected. The calibrators for the population of isolated peptides ing all of the Ehrlichz'a species contain a e of dies to different Ehrlichz'a species in appropriate ratios, for example, at least 5% each of anti-cams, anti-chafieensz’s, and anti-ewz’ngz'z', so that there is enough of each species and the assay does not miss any of the assayed species. The calibrators for the population of peptides detecting both E. canis and E. chafleensz’s, contain a mixture of anti-canis and anti-chafleensz’s samples in appropriate ratios, for example, at least % of each species. The calibrators for the population of peptides ing only E. ewz’ngz’z’ contain only anti-ewz’ngz’z’ samples. The calibrators for the population of peptides detecting E. canis/E. chafieensis and for the population of peptides ing E. ewz’ngz’z’ are assigned the same range of scores which are limited to the linear portion of the respective standard .

In some embodiments, scores are generated from detecting the formation of xes comprising antibodies and es in the populations of peptides as described herein. In certain embodiments, a score is generated from ing the formation of complexes comprising antibodies in a sample, if present, and peptides in a first, second, or 2015/024208 third population of peptides as described herein, resulting in a first score, a second score, or a third score, respectively.

In some embodiments, the second score is compared to the third score, manually or by using a computer. In particular embodiments, a sample is identified or classified to be infected with E. ewz’ngz’z’ if the second score is higher than the third score. In other embodiments, a sample is identified or classified to be infected with E. canis and/or E. chafieensis if the third score is higher than the second score.

In yet other embodiments, the method fiarther ses a step to determine whether the infecting species is E. canis or E. chafieensz’s. For example in one such ment an assay is med to detect dies against E. canis but not E. chafleensz’s, to generate a score for E. canis. Another assay can be performed to detect antibodies against E. chafieensis but E. canis, to generate a score for E. chafleensz’s. The assay results, optionally the scores, are compared to each other to determine whether the infecting species is E. canis or E. chafleensz’s. In some embodiments, if the score for E. canis is higher than the score for E. chafi’eensz’s, the sample is classified as infected with E. canis but not E. chafieensz’s. In other embodiments, if the score for E. chafi’eensz’s is higher than the score for E. canis, the sample is classified as infected with E. nsis but not E. canis. In some embodiments, if the two scores are identical, the sample is classified as infected with both E. chafieensis and E. canis or as undetermined.

In certain embodiments, the sample used in the methods is from a wild animal (e.g., a deer or rodent, such as a mouse, chipmunk, el, etc.). In other embodiments, the sample is from a lab animal (e.g., a mouse, rat, guinea pig, rabbit, monkey, primate, etc.). In other embodiments, the sample is from a domesticated or feral animal (e.g., a dog, a cat, a horse). In still other embodiments, the sample is from a human. In other ments, the sample is from a canine or feline subject. In some embodiments, the sample is a bodily fluid.

In particular embodiments, the sample is a blood, serum, plasma, al spinal fluid, mucus, urine, or saliva sample. In certain embodiments, the sample is a whole blood sample.

In other embodiments, the sample is a tissue (e.g., a tissue homogenate), tissue extract, or a cell lysate.

Much of the preceding discussion is directed to the detection of antibodies against pathogenic hz'a. However, it is to be understood that the discussion also applies to the detection of primed T-cells, either in vitro or in viva.

It is expected that a ediated immune se (e.g., a T-helper response) is generated, since IgG is produced. It is therefore expected that it will be possible to determine the immunological reactivity between primed T-cells and a population of peptides as described herein. In vitro this can be done by ting T-cells isolated from the subject with the population of peptides and measuring the immunoreactivity, e.g., by measuring subsequent T-cell proliferation or by measuring release of cytokines from the T-cells, such as IFN—y. These methods are well-known in the art.

When a method of the invention is carried out in viva, any of a variety of conventional assays can be used. For example, one can m an assay in the form of a skin test, e. g., by intradermally injecting, in the subject, a population of peptides as described herein. A positive skin reaction at the on of injection indicates that the subject has been exposed to and infected with the Ehrlichz'a species that the population of peptides is specific to. The species of Ehrlichia ing the subject can be identified using the method of invention with the populations of peptides as described herein. This or other in viva tests rely on the detection of a T-cell response in the subject. n embodiments of the method further comprise reporting detection results. The reporting can be done onically, in g, or verbally. It can be done via a machine such as a computer.

In yet another aspect, the invention es kits. In some embodiments, the kits comprise at least one population of isolated peptides as described herein. In particular embodiments, a kit comprises at least two or three different populations of peptides. In some embodiments, a kit comprises a first, second, and/or third populations of peptides as described herein. In certain embodiments, the kits fithher comprise an instruction.

In some embodiments, the kit is a kit for detecting antibodies that bind to hz'a antigens and/or identifying the species of Ehrlichia ing a subject, if present.

In certain embodiments, the kit comprises: a first population of isolated es as bed herein; a second population of isolated peptides as described herein; a third population of isolated peptides as described ; and an ction for using the first, second, and third populations of peptides to identify the species ofEhrlz'chz'a in a biological sample, if present.

In particular embodiments of the kits, the first population of ed peptides is capable of cally binding to antibodies against antigens from multiple species of Ehrlichia including E. canis, E. chafleensz’s, and E. ewz’ngz’z’. In other ments, the first population of ed peptides ses at least three different peptides, each comprising a sequence of SEQ ID NO: 1 or a fragment thereof as described herein. Specific examples of the peptide sequences with SEQ ID NO: 1 that can be used in the kits have been described above, e.g., those with specific amino acids at ons that can have various amino acids.

Some specific examples are SEQ ID NOs: 4-51. Fragments of SEQ ID NO: 1 that can be used in the kits have also been described above.

In other particular embodiments of the kits, the second population of isolated peptides is capable of specifically or preferentially binding to antibodies against antigens from E. ewz’ngz’z’, but not to or not preferentially to antibodies against antigens from E. canis or E. chafleensz’s. In other embodiments, the second population of isolated peptides comprises at least three different peptides, each comprising a sequence of SEQ ID NO: 2 or a fragment thereof as described . Specific examples of the peptide sequences with SEQ ID NO: 2 that can be used in the kits have been described above, e.g., those with specific amino acids at locations that can have various amino acids. Some specific examples are SEQ ID NOs: 52-66. nts of SEQ ID NO: 2 that can be used in the kits have also been described above.

In yet other embodiments of the kits, the third tion of isolated peptides is capable of specifically or preferentially binding to antibodies against antigens from E. canis and E. chafleensz’s, but not to or not preferentially to antibodies against antigens from E. ewz’ngz’z’. In other embodiments, the third population of isolated peptides comprises at least two or three different es, each comprising a sequence of SEQ ID NO: 3 or a fragment thereof as described herein. c examples of the peptide sequences with SEQ ID NO: 3 that can be used in the kits have been described above, e. g., those with c amino acids at locations that can have various amino acids. Some specific examples are SEQ ID NOs: 67- 120. Fragments of SEQ ID NO: 3 that can be used in the kits have also been described above.

In certain embodiments of the kits, the e tions are attached to or immobilized on a solid support. In some embodiments, the peptide populations are attached to or immobilized on a solid support through a metallic nanolayer (e.g., cadmium, zinc, mercury, gold, silver, copper, or platinum nanolayer). In certain embodiments, the solid support is a bead (e.g., a colloidal particle or a metallic nanoparticle or nanoshell), a flow path in a lateral flow immunoassay device, a flow path in an analytical or centrifugal rotor, a tube or a well (e.g., in a plate), or a sensor (e.g., an electrochemical, optical, or opto- electronic ).

Reagents for particular types of assays can also be provided in kits of the invention. Thus, the kits can include a population of beads (e. g., suitable for an agglutination assay or a lateral flow assay), or a plate (e.g., a plate suitable for an ELISA assay). In other embodiments, the kits se a device, such as a lateral flow immunoassay device, an analytical or centrifugal rotor, a Western blot, a dot blot, a slot blot, or an electrochemical, optical, or opto-electronic sensor. The tion of beads, the plate, and the devices are useful for performing an immunoassay. For example, they can be useful for detecting formation of an antibody-peptide complex sing an antibody from a sample and a peptide of the invention. In certain embodiments, a peptide, a mixture of different peptides (1.6. population of peptides) of the invention, or a e composition of the invention is attached to or immobilized on the beads, the plate, or the device.

In on, the kits can include various diluents and buffers, labeled conjugates or other agents for the detection of specifically bound antigens or antibodies (e.g. labeling reagents), and other -generating reagents, such as enzyme substrates, cofactors and chromogens. In some embodiments, the kit comprises an anti-human, anti-canine, or anti-feline IgG/IgM antibody conjugated to a able label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, hore, m dot, colored latex particle, or enzyme) as a labeling reagent. In other embodiments, the kit comprises protein A, protein G, protein A/G fiJsion proteins, protein L, or combinations thereof conjugated to a detectable label (e.g., a metallic nanoparticle, metallic nanoshell, metallic nanolayer, fluorophore, colored latex le, or enzyme) as a labeling reagent. An exemplary protein A/G fiJsion protein combines four Fc-binding domains from protein A with two from protein G. See, WO 53949 e. g., Sikkema, , Amer. h. Lab, 7:42, 1989 and on et al., J. Biol. Chem. 263, 4323-4327, 1988, both which are hereby incorporated by reference in their ties.

Other components of a kit can easily be determined by one of skill in the art.

Such components may include coating reagents, polyclonal or monoclonal capture antibodies specific for a tion of peptides as described , purified or semi-purified extracts of these antigens as standards, monoclonal antibody detector antibodies, an anti-mouse, anti- dog, anti-cat, anti-chicken, or anti-human dy conjugated to a detectable label, tor charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, a sample preparatory cup, etc. In one embodiment, a kit comprises buffers or other reagents appropriate for constituting a reaction medium allowing the formation of a peptide-antibody complex.

In certain embodiments, the kits comprise an instruction indicating how to use the first, second, and/or third populations of isolated peptides as described herein to detect an antibody to an Ehrlichz'a antigen and/or to fy the species of Ehrlichia infecting a subject, if present. In certain embodiments, the kits comprise an instruction indicating how to use a population of beads, a plate, or a device (e.g., comprising a peptide or a population of peptides of the invention) to detect an antibody to one or more hz'a ns and/or to identify the species of Ehrlichia. In particular embodiments, the instruction comprises directions to identify the species of Ehrlichia ing a subject, if present, according to the methods described herein. In certain embodiments, the instruction comprises directions to contact a biological sample with the first, second, and third populations of peptides separately. In particular embodiments, the instruction comprises directions to contact a biological sample with the first, second, and third populations of peptides sequentially.

Such kits provide a convenient, efficient way for a clinical laboratory to diagnose infection by a pathogenic Ehrlichz'a and/or identifying the species of Ehrlichia infecting a subject.

In another aspect, the invention provides compositions useful for identifying the species of Ehrlichia ing a t, if present. In some embodiments, the composition comprises at least one population of isolated peptides as described . In certain embodiments, the invention provides a combination of itions comprising the first, second, and third populations of peptides, respectively.

In another aspect, the invention provides devices useful for identifying the s of Ehrlichia infecting a subject, if present. In some ments, the device comprises at least one population of isolated peptides as defined above. In certain embodiments, the device ses the first, second, and third populations of es.

In certain embodiments, the devices are useful for performing an immunoassay. For e, in certain embodiments, the device is a lateral flow immunoassay device. In some embodiments, the device is a slide comprised of a plurality of beads to which a e or population of peptides is attached. In other embodiments, the deVice is an analytical or centrifugal rotor. In other embodiments, the deVice is a dot blot, slot blot, or Western blot. In other embodiments, the device is a tube or a well, e.g., in a plate suitable for an ELISA assay. In still other embodiments, the deVice is an electrochemical sensor, an optical sensor, an opto-electronic sensor, an X-ray film, chemi-luminescence imager or a photon detection equipment.

The methods, kits, compositions, and deVices of the invention offer a number of advantages. For example, they allow for simple, inexpensive, rapid, sensitive and accurate ion of antibodies against Ehrlichz'a and identification of the species of Ehrlichia infecting a subject, if present. They also avoid serologic cross-reactivity with other conditions with similar symptoms. This allows for an accurate sis of the bacteria and s, thereby facilitates timely and appropriate treatment that may be needed for the particular species of Ehrlichia.

The following examples illustrate various aspects of the invention. The examples should, of , be understood to be merely illustrative of only certain embodiments of the invention and not to constitute limitations upon the scope of the invention.

EXAMPLES Example 1 - Experimental Infection of Dogs with Ehrlichia and ion of Species- specific Anti-Ehrlichia Antibodies with ELISA This example shows that antibodies specific to particular Ehrlichz'a species were generated and found reactive to the tions of peptides as described herein. 2015/024208 A number of dogs were experimentally infected with E. canis, E. chafieensis or E. ewz’ngz’z’ (four dogs for each Ehrlichz'a species) for the purpose of studying the course of pathological changes and antibody production. The animals were infected using cultures of E. canis and E. chafleensz’s, respectively, and blood stabilates of E. ewz’ngz’z’ (E. ewz’ngz’z’ has not been sfully cultured, and thus no slides for ting IFA are currently ble for this species.) Plasma samples were drawn from the ed dogs at various time points to generated the "dog plasma samples".

Although all of the infected animals showed the presence of bacterial DNA by PCR, in the time period allowed for the study, only one of the E. chafieensz’s-infected dogs and two of the E. infected dogs showed the presence of anti-bacterial antibodies as determined by reactivity with SNAP 4DX PlusTM (manufactured by IDEXX Laboratories, Inc., which detects antibodies against E. canis, E. ewz’ngz’z’ and E. chafi’eensz’s). All the dog plasma samples from the infection study found positive on SNAP 4DX PlusTM were also positive in the ELISA assays performed according to the method described below, using the first population of peptides as described below (ECHEWl).

Three different populations of peptides were synthesized using standard synthesis procedures. Each e in the first population of peptides (ECHEWl) contained a sequence of SEQ ID NO: 1, which comprises a chimeric e encompassing two ent sequences that bind antibodies elicited to the following Ehrlichz'a antigens: msp4, p30 or p30- 1 from canis/chafi’eensz’s and 28kD from ewz’ngz'z'. The ECHEWl population of peptides specifically binds to antibodies elicited by multiple Ehrlichz'a spp. (E. canis, E. chafleensz’s, and E. ewz’ngz’z’). Each peptide in the second population of peptides (EEl3) contained a sequence of SEQ ID NO: 2. The EEl3 population of peptides specifically binds to antibodies ed primarily by E. ewz’ngz’z’ with some low cross-reactivity to E. canis and E. chafleensz’s.

Each peptide in the third population of peptides (EElZEWl) contained a sequence of SEQ ID NO: 3. The l population of peptides ically binds to antibodies elicited primarily by E. canis and E. chafieensis with some low cross-reactivity to E. ewz’ngz'z'.

ELISA Method 1. Coating Antigen 1.1. The d number of wells in l plates (Thermo Scientific NuncTM MaXiSorp" Microplates) were coated with 1-20 ug/mL of AbaXis Ehrlichz'a antigen population ECHEWl, EElZEWl, or EEl3, each conjugated to BSA and diluted in 0.1 M sodium carbonate/bicarbonate buffer (pH . Coating was performed by adding 0.1mL of the antigen to each well and incubating the plate on a micro plate shaker at 25 0-300 rpm at room temperature for approximately one hour. 1.2. The coating solution was removed, followed by dabbing the plates on paper towels to eliminate any hanging droplets. 0.3 mL of deionized water was added to each well, and the plates were shaken at 25 0-300 rpm for 5 minutes. The liquid was d as above. 1.3. The wash step as in 1.2 was repeated twice. 2. Blocking of the Plate 2.1. The coated plate wells were blocked by treating with the ng solution ting of 30g of t milk in 100 mL of deionized water. Each well was filled with 0.3 ml of blocking solution and the plates were placed on a shaker at 250-300 rpm for approximately one hour. 2.2. Blocking solution was removed and the plate was dabbed on a paper towel to remove hanging droplets. 3. Sample/Calibrator Incubation 3.1 Anti-Ehrlichz'a antibody calibrators were ted from canine plasma by making a pool of high titer plasma samples against known species. Species was determined by SNAP 4DX PlusTM and SNAP 3DxTM (manufactured by IDEXX, which detects antibodies against E. canis and E. chafleensz’s, but not E. ewz’ngz'z') differential testings and IFA. The pool was then assigned an arbitrary score and diluted to various levels in a negative canine plasma diluent. The score scaled linearly with the dilution: for example, if a sample with score 40 was diluted 2 fold the resulting score would be 20.

A set of five Ehrlichz'a calibrators was run on each plate for the hz'a ELISA.

One set was comprised of plasma samples that were positive to E. canis, E. chafieensis and E. ewz’ngz'z', and was used with an ECHEWl-coated plate. One set was comprised of anti-E. canis/E. nsz’s-positive samples, using samples that show close titers in the IFA for canis and nsis, respectively, and was used with an ECHEWl-coated plate and a EE12EW1-coated plate. Another set was comprised of anti-E. ewz’ngz’i-positive s and was used with a EE13-coated plate. Each dog plasma sample or calibrator was diluted 25 0-fold in the blocking solution. Aliquots of 0.1 mL of each of the diluted calibrators and the dog plasma samples were added to the wells and plates were placed on the shaker at 250-300 rpm for one hour. Both the calibrators and dog plasma samples were run in duplicate and the results reported are the average of the two readings. 3.2. The sample solution was removed and the plate was washed in the washing buffer containing 50mM Trizma base (Sigma-Aldrich T1503) and 0.05% CHAPS detergent (pH 8.0) (Sigma-Aldrich C3023). The washing step was carried out by adding 0.3mL of the washing buffer and g the plate at 250-300 rpm for 5 minutes. The washing solution was removed by inverting the plate and then dabbing on a paper towel to eliminate any hanging droplets. 3.3 The above washing step was repeated twice. 4. ate Incubation 4.1. Protein A-HRP conjugate (Bio-Rad 170-6522) was diluted 8000-fold in the blocking solution (described in 2.1 above) and 0.1 mL of the diluted ate was added to each well. The plates were then incubated with shaking at 25 0-300 rpm at room temperature for approximately one hour. 4.2. The conjugate was removed and the plates were dabbed on a paper towel to remove hanging droplets. The plates were washed thrice as described above in 3.2 and 3.3.

Finally, the plates were washed with 0.3 mL of distilled water per well. 4.3. The bound conjugate was d by adding 0.1 mL of the substrate TMB solution (Millipore E8022). The substrate was allowed to react for 10 min at room temperature before OD 650 nm readings were taken on a plate reader ramax 340 PC.).

Plasma samples from the infected dogs were drawn at several time points and assayed with the Ehrlichz'a ELISA method as described above using ECHEWl, EE13, and 1, tively. The results are shown in Figure 2. These results show the reactivity of ECHEWl and EE12EW1 with antibodies produced in se to E. canis and E. chafleensis. The antibodies produced in response to E. canis did not react with E. ewingii—specific peptide population EE13. A very slight cross-reactivity of the 42 day- post-infection sample from the E. chafleensis-infected dogs with EE13 was noted. 2015/024208 Example 2 - Detection of Presence of and Species-Specific Antibodies from Additional Known Anti-Ehrlichia-Positive or Ne ative Sam les Usin the Po ulations of Pe tides This example shows the detection of the presence of hrlichz'a antibodies and, if t, the species-specific antibodies from onal samples that were identified by reference methods to be hrlichz'a-positive or negative, using the ECHEWl, EEl3, and EElZEWl populations of peptides in ELISA. It shows that the ELISA results agree with reference method results.

Each peptide in the three populations, ECHEWl, EEl3, and EElZEWl, was linked separately to the carrier protein bovine serum albumin (BSA) using thio-ether chemistry. The resulting BSA-peptide conjugates were used as capture entities in 96-well ELISA plates to create three separate ELISA assays (one tion of peptides per plate).

The plates were then blocked to prevent undesirable non-specific binding.

A total of 224 anti-Ehrlichz'a-positive samples (dog plasma samples positive to E. Cam's, E. chafieensis, or E. z’z’ as determined by IPA and SNAP 4DX PlusTM/SNAP 3DXTM) and 264 anti-Ehrlichz'a-negative samples (244 dog plasma samples and 20 dog whole blood samples negative to E. Cam's, E. chafieensis, and E. ewz’ngz’z’ as determined by the same reference methods) were incubated with the immobilized peptide populations in each of the three ELISA plates. After one hour incubation, the unreacted materials were removed by washing the micro wells. The specifically captured dog IgG or IgM were detected by reaction with HRP-labeled Protein A. HRP was assayed using a cial TMB substrate.

The optical density of each well was read at 650 nm with a plate . A summary of the results separated by "Sample Status", from IPA and SNAP tests, is shown in Table 1 below.

Table l - ELISA Results of Known Ehrlichia-Positive or Negative Samples ELISA Resultz Sample 1 ECHEWl - ECHEW1 - ECHEWl - Positive with Positive with Negative EElZEWl>EEl3 EEl3>EE12EWl ———-_- --—"_ Posmve, es 23 1 24 mdetermmate 1The Sample Status was determined from the results of IFA and SNAP tests. 2An ELISA Result for ECHEWl was classified as "positive" if it had a score 23 or "negative" if it had a score <3.

Of the 224 anti-Ehrlichz'a-positive s (determined by IFA and SNAP tests), 209 were identified positive by our ELISA assay using the peptide population ECHEWl. Thus, the percent sensitivity of the ELISA ECHEWl was 93.3%. Of the 264 anti-Ehrlichz'a-negative samples, 259 were identified ve by our ELISA assay. Thus, the percent specificity of the ELISA ECHEWl was 98.1%. Furthermore, of the 108 samples that were classified as anti-E. cams-specific or anti-E. chafieensz’s-specific by IFA and SNAP tests, 99 ("ECHEWl-Positive with EE12EW1>EE13") were correctly identified by our ELISA detection process. Of the 92 samples that were classified as anti-E. z’i—specific by IFA and SNAP tests, 80 ("ECHEWl-Positive with EE13>EE12EW1") were identified by our ELISA detection process. Therefore, our ELISA methods are in good agreement with the reference methods.

In addition, of the 25 anti-Ehrlichz'a-positive samples whose species information could not be determined by IFA or SNAP assays, our ELISA identified them to be either E. Cam's/E. chafleensz’s specific (if the EE12EW1 score was greater than the EE13 score) or E. ewz’ngz’z’ specific (if the EE13 score was greater than the EE12EW1 , with fairly high confidence.

In some embodiments, lateral flow immunoassays can be used in place of the ELISA assays in methods described above. ore, other assay formats ing the populations of peptides as described herein can be used in the methods of the invention to identify Ehrlichz'a species.

Example 3 - tion of Standard Curves and Identification of Three Unknown Samples This example demonstrates in detail how standard curves for the three populations of isolated peptides, ECHEWl, EE13, and 1, could be generated, as well as how three unknown samples were classified according to the methods of the invention.

An ELISA assay was performed according to the method described in Example 1. In ular, a set of five Ehrlichz'a ators generated from known canine plasma samples as described in Example 1 was run on each plate for the Ehrlichz'a ELISA.

One set was comprised of E. E. chafieensis positive samples and was used with an ECHEWl-coated plate and an EE12EW1-coated plate. Another set was comprised of E. ewz’ngz’z’ positive samples and was used with an EE13-coated plate.

Each of three unknown canine plasma samples was diluted 250, 500 and 1000- fold in the blocking solution. Aliquots of 0.1 mL of each of the diluted calibrators and the unknown samples were then added to the wells and plates were placed on the shaker at 250- 300 rpm for one hour.

Both the calibrators and unknown samples were run in duplicate and the results reported are the average of the two readings.

Data Analysis A rd curve for each population of es was prepared by using the respective ELISA calibrators with scores (ECHEWl Score for all species, EElZEWl Score for canis and/or chafleensz’s, and EE13 Score for ewz’ngz'z') on the x-axis and optical density (OD) on the y axis. The Ehrlichz'a scores of the unknown samples were interpolated from this standard curve. The ECHEWl Score, EElZEWl Score, or EE13 Score for an unknown sample was determined by using the OD from a dilution that falls within the calibration CUTVG .

Results The ELISA results (OD 650 nm readings) of the calibrators are shown in Table 2: Table 2 - rd Curves (Assigned Scores and OD gs of Calibrators) OD 650 nm OD 650 nm OD 650 nm Score ECHEWl EE12EW1 EE13 _——-!_ Standard curves were calculated as follows: ECHEWl: OD = 0.0088 (ECHEWl Score)+ 0.0027 EElZEWl: OD = 0.0055 (EElZEWl Score)+ 0.005 EE13: OD = 0.0069 (EE13 Score)+ 0.0029 The ELISA results of the unknown samples are shown in Table 3: Table 3 - ELISA OD Readings of Unknown Samples Sample Name OD 650 nm OD 650 nm OD 650 nm ECHEWl EE12EW1 EE13 0.0003 0.012 0.002 Scores of the unknown samples were calculated by the following formula: (SCORE): (OD - B)/A Where B is the intercept of the standard curve and A is the slope.

For each score the OD and the constants used come from the peptide population in question.

The scores calculated for each unknown sample are as s: 1.) Unknown 1 (ECHEWl Score)= 0.0027)/0.0088 = 47 (EElZEWl = (0.03-0.005)/0.0055 = 5 (EE13 Score): (0 .34-0.0029)/0.0069 = 49 The ECHEWl Score was used to determine if the sample is ve or negative for infection with any species from E. canis, E. nsis, and E. ewz’ngz’z’. Then the EE13 Score was compared to the EElZEWl Score to determine the species of the infection. In this case, for n 1, ECHEWl Score is positive, and EE13 Score >> EE12EWl Score, so the sample is positive for E. ewz’ngz'z'. 2.) Unknown 2 (ECHEWl Score): (0.48-0.0027)/0.0088 =54 (EE12EW1 Score)= 0.005)/0.0055 =55 (EE13 Score): (0.01-0.0029)/0.0069 = l EE12EWl Score >> EE13 Score, so the sample is positive for E. canis/E. chafi’eensz’s. 3.) Unknown 3 l Score): (0.003-0.0027)/0.0088 = 0 W1 Score): —0.005)/0.0055 = 1 (EE13 Score) = (0.002-0.0029)/0.0069 = 0 All three scores are very low so the sample is negative for all three Ehrlichz'a species.

Cutoff The cutoff for the ELISA test method was calculated on the basis of analysis of 294 samples, 128 negatives and 166 ves. These samples were classified by use of SNAP 4Dx Plus and E. Cam's and E. Chafieensz’s IFA titers. The samples used in this study were any for which both methods agreed, i.e., both SNAP and IFA were Positive or both were ve. In this case ver IFA titer was higher was the value used. Each of these 294 samples was tested using ELISA assays according to the procedure described in Example 1, and an antibody level score was assigned to each assay result for each sample. Positive and negative status for a sample run through this ELISA assay were determined on the basis of ECHEWl Score alone so all the calculations here were concerning the ECHEWl score for these samples.

The cutoff was set at three standard deviations above the negative mean. For this sample set that is: Mean of Negative samples 0.37 Standard Deviation tive samples 0.82 Mean + 3x{StDev} 2.84 In this Example all scores were rounded to the nearest integer so any sample with an ECHEWl Score >=3 were considered a positive. At an ELISA score of 3, one would expect 99.2% Specificity and 95.8% Sensitivity.

To the extent that any definitions in documents incorporated by nce are inconsistent with the definitions provided herein, the definitions provided herein are controlling. Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various changes and modifications, as would be obvious to one skilled in the art, can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the ing claims.

The disclosures, including the claims, figures and/or drawings, of each and every patent, patent application, and ation cited herein are hereby incorporated herein by reference in their entireties.

Claims (24)

What is claimed:

1. A method for identifying the species of Ehrlichia infecting a subject, if present, the method comprising: contacting a sample from the subject with a first population of ed peptides comprising at least three different peptides, each comprising a sequence of S-X2-K-E-X5-KQ-X8-T-X10-X11-X12-X13-G-L-K-Q-X18-W-X20-G-X22-X23-X24-X25-X26-G-G-G-G-G-N-F-SA-K-E-E-X39-A-E-T-R-X44-T-F-G-L-X49-K-Q-Y-D-G-A-X56-I-X58-E-N-Q-V-Q-N-K-F-T-IS-N-C (SEQ ID NO: 1) or a fragment f, n the fragment comprises amino acids 33-71 of SEQ ID NO: 1 and further comprises a deletion of no more than 5 amino acids of SEQ ID NO: 1, wherein the fragment is capable of specifically g to one or more antibodies against antigens from hia ewingii (E. ewingii) and one or more antibodies against antigens from Ehrlichia canis (E. canis) and/or Ehrlichia chaffeensis (E. chaffeensis); wherein X2 is an amino acid selected from the group consisting of A and V, X5 is an amino acid ed from the group consisting of E and D, X8 is an amino acid selected from the group consisting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid selected from the group consisting of L and V, X13 is an amino acid selected from the group consisting of Y and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23.is an amino acid selected from the group consisting of A, S, and T, X24 is an amino acid ed from the group consisting of A and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X44 is any amino acid, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid; detecting formation of a first set of complexes comprising an antibody and one or more peptides in the first population; contacting said sample with a second population of isolated peptides comprising at least three different peptides, each comprising a ce of K-E-E-X7-A-E-T-R-X12- T-F-G-L –X17-K-Q-Y-D-G-A-X24-I-X26-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 2), or a fragment thereof, wherein the fragment ses a deletion of no more than 5 amino acids of SEQ ID NO: 2, wherein the fragment is capable of specifically binding to one or more antibodies against antigens from E. ewingii, wherein X7 is any amino acid, X12 is any amino acid, X17 is any amino acid, X24 is any amino acid, and X26 is any amino acid; and detecting formation of a second set of complexes comprising an antibody and one or more peptides in the second population, wherein formation of both the first and second sets of xes indicates that the subject is infected with Ehrlichia i (E. ewingii), and wherein formation of the first but not the second set of xes indicates that the subject is infected with Ehrlichia canis (E. canis) and/or Ehrlichia chaffeensis (E. chaffeensis).

2. A method for identifying the s of hia infecting a subject, if present, the method comprising: ting a sample from the subject with the first population of isolated es as defined in claim 1; detecting formation of a first set of xes comprising an antibody and one or more peptides in the first population; contacting said sample with a third population of isolated peptides comprising at least three different peptides, each comprising a sequence of S-X2-K-E-X5-K-Q-X8-T-X10-X11-X12- X13-G-L-K-Q-X18-W-X20-G-X22-X23-X24-X25-X26-G-G-G-G-G-N-F-S-A-K-E-E-X39-A-X41- 4-T-F-G-X48-X49-K-Q-Y-D-G-A-X56-I-X58-E-N-Q-V-Q-N-K-F-T-I-S-N-C (SEQ ID NO: 3) or a fragment thereof, wherein the fragment comprises amino acids 33-71 of SEQ ID NO: 3 and further comprises a deletion of no more than 5 amino acids of SEQ ID NO: 3, wherein the fragment is capable of specifically binding to one or more antibodies against antigens from E. canis and/or E. chaffeensis and is not capable of binding to one or more antibodies against antigens from E. ewingii, wherein X2 is an amino acid selected from the group consisting of A and V, X5 is an amino acid ed from the group consisting of E and D, X8 is an amino acid selected from the group consisting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid selected from the group consisting of L and V, X13 is an amino acid selected from the group consisting of Y and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid ed from the group consisting of S and V, X23 is an amino acid selected from the group consisting of A, S, and T, X24 is an amino acid selected from the group consisting of A and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X41 is an amino acid selected from the group consisting of D and N, X44 is any amino acid, X48 is an amino acid selected from the group consisting of V and A, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid; and detecting ion of a third set of complexes comprising an antibody and one or more peptides in the third population, wherein formation of both the first and third sets of antibody-peptide complexes indicates that the subject is infected with E. canis and/or E. chaffeensis, and wherein formation of the first but not the third set of antibody-peptide complexes indicates that the subject is infected with E. i.

3. The method of claim 1, wherein: X39 in SEQ ID NO: 1 is K; X44 in SEQ ID NO: 1 is K or R; X49 in SEQ ID NO: 1 is E or D; X56 in SEQ ID NO: 1 is K or Q; X58 in SEQ ID NO: 1 is E or T; X7 in SEQ ID NO: 2 is K; X12 in SEQ ID NO: 2 is K or R; X17 in SEQ ID NO: 2 is E or D; X24 in SEQ ID NO: 2 is K or Q; and/or X26 in SEQ ID NO: 2 is E or T.

4. The method of claim 1, wherein: each peptide in the first tion comprises a sequence of SEQ ID NO: 1.

5. The method of claim 1, wherein at least one of said detecting steps comprises: (i) performing an ELISA assay; (ii) running a lateral flow assay; (iii) performing an agglutination assay; (iv) performing a n blot, slot blot, or dot blot assay; (v) performing a wavelength shift assay; (vi) g the sample through an analytical or centrifugal rotor; or (vii) running a microarray assay.

6. The method of claim 1, wherein said sample is from a human, canine, or feline subject.

7. The method of claim 1, wherein said sample is a blood, serum, plasma, cerebral spinal fluid, tissue extract, urine, or saliva sample.

8. The method of claim 1, further comprising reporting detection results.

9. The method of claim 2, n: X39 in SEQ ID NO: 1 is K; X44 in SEQ ID NO: 1 is K or R; X49 in SEQ ID NO: 1 is E or D; X56 in SEQ ID NO: 1 is K or Q; X58 in SEQ ID NO: 1 is E or T; X39 in SEQ ID NO: 3 is K; X44 in SEQ ID NO: 3 is K or R; X49 in SEQ ID NO: 3 is E or D; X56 in SEQ ID NO: 3 is K or Q; and/or X58 in SEQ ID NO: 3 is E or T.

10. The method of claim 2, wherein: each peptide in the first population comprises a sequence of SEQ ID NO: 1; and/or each peptide in the third tion comprises a sequence of SEQ ID NO: 3.

11. The method of claim 2, wherein at least one of said detecting steps comprises: (i) performing an ELISA assay; (ii) running a l flow assay; (iii) performing an agglutination assay; (iv) performing a Western blot, slot blot, or dot blot assay; (v) performing a wavelength shift assay; (vi) running the sample through an analytical or centrifugal rotor; or (vii) running a rray assay.

12. The method of claim 2, wherein said sample is from a human, canine, or feline subject.

13. The method of claim 2, wherein said sample is a blood, serum, plasma, al spinal fluid, tissue extract, urine, or saliva sample.

14. The method of claim 2, further comprising reporting detection results.

15. The method of claim 2, wherein the sample is further analyzed with at least one assay to determine whether the infecting species is E. canis or E. chaffeensis.

16. The method of claim 15, wherein said at least one assay is an indirect immunofluorescence assay (IFA), a dot blot assay, a l flow assay, ELISA, or a n Blot.

17. The method of claim 1 further comprising: contacting said sample with a third population of isolated peptides comprising at least three different peptides, each comprising a ce of S-X2-K-E-X5-K-Q-X8-T-X10-X11-X12- X13-G-L-K-Q-X18-W-X20-G-X22-X23-X24-X25-X26-G-G-G-G-G-N-F-S-A-K-E-E-X39-A-X41- T-R-X44-T-F-G-X48-X49-K-Q-Y-D-G-A-X56-I-X58-E-N-Q-V-Q-N-K-F-T-I-S-N-C C (SEQ ID NO: 3) or a fragment thereof, wherein the fragment comprises amino acids 33-71 of SEQ ID NO: 3 and r comprises a deletion of no more than 5 amino acids of SEQ ID NO: 3, n the fragment is e of specifically binding to one or more antibodies against ns from E. canis and/or E. chaffeensis and is not capable of binding to one or more antibodies against ns from E. ewingii, wherein X2 is an amino acid selected from the group consisting of A and V, X5 is an amino acid selected from the group consisting of E and D, X8 is an amino acid selected from the group consisting of T and P, X10 is an amino acid selected from the group consisting of T and V, X11 is an amino acid selected from the group consisting of G and A, X12 is an amino acid selected from the group consisting of L and V, X13 is an amino acid selected from the group consisting of Y and F, X18 is an amino acid selected from the group consisting of D and N, X20 is an amino acid selected from the group consisting of D and N, X22 is an amino acid selected from the group consisting of S and V, X23 is an amino acid selected from the group consisting of A, S, and T, X24 is an amino acid selected from the group consisting of A and I, X25 is an amino acid selected from the group consisting of T and P, X26 is an amino acid selected from the group consisting of S, N, and K, X39 is any amino acid, X41 is an amino acid selected from the group consisting of D and N, X44 is any amino acid, X48 is an amino acid selected from the group consisting of V and A, X49 is any amino acid, X56 is any amino acid, and X58 is any amino acid; and detecting formation of a third set of xes sing an antibody and one or more peptides in the third population, wherein formation of both the first and second sets of xes but not the third set indicates that the subject is infected with E. ewingii, and wherein formation of both the first and third sets of complexes but not the second set indicates that the subject is infected with E. canis and/or E. chaffeensis.

18. The method of claim 17, wherein: X39 in SEQ ID NO: 1 is K; X44 in SEQ ID NO: 1 is K or R; X49 in SEQ ID NO: 1 is E or D; X56 in SEQ ID NO: 1 is K or Q; X58 in SEQ ID NO: 1 is E or T; X7 in SEQ ID NO: 2 is K; X12 in SEQ ID NO: 2 is K or R; X17 in SEQ ID NO: 2 is E or D; X24 in SEQ ID NO: 2 is K or Q; X26 in SEQ ID NO: 2 is E or T, X39 in SEQ ID NO: 3 is K; X44 in SEQ ID NO: 3 is K or R; X49 in SEQ ID NO: 3 is E or D; X56 in SEQ ID NO: 3 is K or Q; and/or X58 in SEQ ID NO: 3 is E or T.

19. The method of claim 17, wherein: each peptide in the first population comprises a sequence of SEQ ID NO: 1; and/or each peptide in the third population comprises a sequence of SEQ ID NO: 3.

20. The method of claim 17, wherein said sample is from a human, canine, or feline subject.

21. The method of claim 17, wherein said sample is a blood, serum, plasma, cerebral spinal fluid, tissue extract, urine, or saliva sample.

22. The method of claim 17, wherein at least one of said detecting steps comprises: (i) performing an ELISA assay; (ii) running a lateral flow assay; (iii) ming an agglutination assay; (iv) performing a Western blot, slot blot, or dot blot assay; (v) performing a wavelength shift assay; (vi) g the sample through an analytical or centrifugal rotor; or (vii) running a microarray assay.

23. The method of claim 17, further comprising ing detection results.

24. The method of claim 17, wherein the sample is further analyzed with at least one assay to determine whether the infecting species is E. canis or E. chaffeensis.