OUTER MEMBRANE PROTEIN OF EHRLICHIA CANIS AND EHRLICHIA CHAFFEENSIS

Abstract: The present invention relates to diagnostic tools for veterinary and human use which are used for serodiagnosing ehrlichiosis in mammals, particularly in members of the Canidae family and in humans. The present invention also provides polynucleotides which encode the outer membrane proteins of E. chafeensis. The polynucleotides encode an OMP-1 family of proteins of E. chafeensis and P30 family of proteins of E. canis. The present invention also provides the following isolated proteins of E. chafeensis OMP-1, OMP-1A, OMP-1B, OMP-1C, OMP-1D, OMP-1E, OMP-1F, OMP-1R, OMP-1S, OMP-1T, OMP-1U, OMP-1V, OMP-1W, OMP-1X, and OMP-1Z, referred to hereinafter collectively as the "OMP family". The present invention also provides the following isolated proteins of E. canis P30, P30-a, P30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, and P30-10, referred to hereinafter as the P30 family. The present invention also relates to an assay for diagnosing ehrlichiosis in humans using a recombinant outer membrane protein of E. chafeensis, particularly OMP-1. The present invention also relates to an assay for diagnosing ehrlichiosis in humans and members of the family Canidae using a recombinant outer membrane protein of E. canis, particularly P30.

Description: OUTER MEMBRANE PROTEIN OF EHRLICHIA CANIS AND EHRLICHIA CHAFFEENIS This work was supported by grant ROl AI40934 from National Institutes of Health. The government has certain rights in this invention. BACKGROUND OF THE INVENTION The ehrlichiae are obligate intracellular bacteria that infect circulating leucocytes. Ehrlichia chafeensis infects the monocytes and macrophages in humans and causes human monocytic ehrlichiosis. The clinical manifestations of ehrlichiosis in humans are nonspecific and similar to Rocky Mountain spotted fever. The clinical manifestations include fever, chills, headache mylagia or vomiting and weight loss. Most patients have a history of tick exposure. Ehrlichia canis infects and causes ehrlichiosis in animals belonging to the family Canidae. Canine ehrlichiosis consists of an acute and a chronic phase. The acute phase is characterized by fever, serous nasal and ocular discharges, anorexia, depression, and loss of weight. The chronic phase is characterized by severe pancytopenia, epistaxis, hematuria, blood in feces in addition to more severe clinical signs of the acute disease. If treated early during the course of the disease, dogs respond well to doxycycline. However, chronically infected dogs do not respond well to the antibiotic. Therefore, early diagnosis is very important for treating canine ehrlichiosis. The primary diagnostic test for diagnosing canine ehrlichiosis and human ehrlichiosis is the indirect fluorescent antibody (IFA) test. This test uses the etiologic agent Ehrlichia canis to diagnose canine ehrlichiosis. The IFA test uses Ehrlichia chafeensis as antigen for diagnosing human ehrlichiosis. The IFA test has, however, serious limitations. The IFA test is subject to false positives because the antigens are made of whole infected cells which comprise many nonspecific proteins which will cross-react with sera from some patients. The IFA test is also subject to false negatives because IFA antigens are unstable and may become inactivated during storage. In addition the IFA test requires a special equipment to perform the test. For example, the IFA test requires a tissue culture system for growing the bacterium that are used to prepare the antigen slides, a fluorescent microscope, and trained persons to evaluate the serum reactivity to the bacterial antigen on the slide. Tools which permit simpler, more rapid, and objective serodiagnosis of canine ehrlichiosis or human ehrlichiosis are desirable. SUMMARY OF THE INVENTION The present invention relates to improved diagnostic tools for veterinary and human use which are used for serodiagnosing ehrlichiosis in mammals, particularly in members of the Canidae family and in humans. The present invention also provides polynucleotides or nucleic acids which encode the outer membrane proteins of E. chafeensis. The OMP-1 polynucleotide encodes an OMP-1 protein of E. chafeensis having a molecular weight of about 27.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG.3B, SEQ ID NO: . The OMP-1B polynucleotide encodes an OMP-1B protein of E. iafeensis having a molecular weight of about 28.2 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 4B, SEQ ID NO: . The OMP-IC polynucleotide encodes an OMP-IC protein of E. chafeensis having a molecular weight of about 27.6 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 5B, SEQ ID NO: . The OMP-ID polynucleotide encodes an OMP-ID protein of E. chafeensis having a molecular weight of about 28.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 6B, SEQ ID NO: . The OMP-IE polynucleotide encodes an OMP-IE protein of E. chafeensis having a molecular weight of about 27.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 7B, SEQ ID NO: . The OMP-IF polynucleotide encodes an OMP-IF protein of E. chafeensis having a molecular weight of about 27.9 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 8B, SEQ ID NO: . The OMP-1A polynucleotide encodes an OMP-1A protein of E. chafeensis having a molecular weight of about 29.6 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 9B, SEQ ID NO: . The OMP-IR polynucleotide encodes an OMP-IR protein of E. chafeensis having a molecular weight of at least 23 kDa and comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 10B, SEQ ID NO: . The OMP-I S polynucleotide encodes an OMP-IS protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 1 I B, SEQ ID NO: . The OMP-IT polynucleotide encodes an OMP-IT protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 12B, SEQ ID NO: . The OMP-IU polynucleotide encodes an OMP-IU protein of E. chafeensis having a molecular weight of about 30.6 kDa and an amino acid sequence which is at least 85% homologous to amino acid sequence shown in FIG. 13B, SEQ ID NO: . The OMP-IV polynucleotide encodes an OMP-IV protein of E. chafeensis having a molecular weight of about 28.0 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 14B, SEQ ID NO: . The OMP-IW polynucleotide encodes an OMP-IW protein of E. chafeensis having a molecular weight of about 28.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 15B, SEQ ID NO: _. The OMP-IX polynucleotide encodes an OMP-IS protein of E. chafeensis having a molecular weight of about 27.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 16B, SEQ ID NO: . The OMP-1Y polynucleotide encodes an OMP-1Y protein of E. chafeensis having a molecular weight about 28.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 17B, SEQ ID NO: . The OMP-IZ polynucleotide encodes an OMP-IZ protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 18B, SEQ ID NO: . The outer membrane proteins from E. chaffeensis, particularly a recombinant form of OMP-1, are immunogenic and, thus are useful for preparing antibodies. Such antibodies are useful for immunolabeling isolates of E. chafeensis and for detecting the presence of E. chafeensis in body fluids, tissues, and particularly in monocytes and macrophages. The isolated outer membrane proteins, particularly OMP-1, are also useful for detecting antibodies to E. chafeensis in the blood of patients with clinical signs of ehrlichiosis. The isolated outer membrane protein, particularly OMP-1, are also useful immunogens for raising antibodies that are capable of reducing the level of infection in an immunized mammal that has been infected with E. chafeensis. The isolated membrane proteins are also useful in a vaccine for protecting against infection with E. chafeensis. The present invention also relates to isolated polynucleotides which encode 30 kDa outer membrane proteins from Ehrlichia canis. The proteins are designated P30 and P30a. The proteins, particularly P30, are immunogenic and are, thus, useful for preparing antibodies that are useful for immunolabeling isolates of E. canis. The P30 protein is also useful for diagnosing canine ehrlichiosis in mammals, particularly in members of the family Canidae, most particularly in dogs and for diagnosing infections with E. chafeensis in humans. The P30 protein is also a useful immunogen for raising antibodies that reduce the level of infection in an immunized mammal that has been infected with E. canis. The P30 protein is also useful in a vaccine for protecting animals against infection with E. canis. The present invention also provides the following isolated proteins of E. chafeensis OMP-1 (also known as p28), OMP-1 A, OMP-1B, OMP-IC, OMP-ID, OMP-IE, OMP-IF, OMP-IR, OMP-IS, OMP-IT, OMP -1U, OMP-IV, OMP-I W, OMP-IX, and OMP-IZ, referred to hereinafter collectively as the "OMP family". The present invention also provides the following isolated proteins of E. canis P30, P30-a, P30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, and P30-10, referred to hereinafter as the P30 family. The present invention also relates to an assay for diagnosing ehrlichiosis in humans using a recombinant outer membrane protein of E. chafeensis, particularly OMP-1. The present invention also relates to an assay for diagnosing ehrlichiosis in humans and members of the family Canidae using a recombinant outer membrane protein of E. canis, particularly P30. Brief Description of the Figures FIG. 1. shows the DNA sequence of and the amino acid sequence encoded by the E. chafeensis (p28) gene cloned in pCRII/?26\ The N-terminal amino acid sequence of native omp-1 protein (P28) determined chemically is underlined. Five amino acid residues at the N terminus of P28 which were not included in the p28 gene, are indicated by boldface. Arrows indicate annealing positions of the primer pair designed for PCR FIG. 2. shows the restriction map of 6.3-kb genomic DNA including the omp-1 gene copies in E. chafeensis. The four DNA fragments were cloned from the genomic DNA (pPS2.6, pPS3.6, pEC2.6, and pEC3.6). A recombinant plasmid pPS2.6 has an overlapping sequence with that of pEC3.6. The closed boxes at the bottom show PCR-amplified fragments from the genomic DNA for confirmation of the overlapping area. Open boxes at the top indicate open reading frames (ORF) of omp-1 gene copies with direction by arrows. Open boxes at the bottom show DNA fragments subcioned for DNA sequencing. FIG. 3B shows one embodiment of the OMP-1 protein; FIG. 3A shows one embodiment of the OMP-1 polynucleotide. FIG. 4B shows one embodiment of the OMP-1B protein, FIG. 4A shows one embodiment of the OMP-1B polynucleotide FIG. 5 A shows one embodiment of the OMP-IC polynucleotide; FIG 5B shows one embodiment of the OMP-IC protein. FIG. 6B shows one embodiment of the OMP-ID protein; FIG. 6A shows one embodiment of the OMP-ID polynucleotide. FIG. 7 A shows one embodiment of the OMP-IE protein; FIG 7B shows one embodiment of the OMP-IE polynucleotide. FIG. 8 A shows one embodiment of the OMP-IF protein; FIG 8 B shows one embodiment of the OMP-IF polynucleotide. FIG. 9B shows one embodiment of the OMP-1 A protein, FIG 9 A shows one embodiment of the OMP-1 A polynucleotide. FIG. 10 B shows one embodiment of a portion of the OMP-IR protein, FIG 10A shows one embodiment of an OMP-IR polynucleotide encoding such polypeptide. FIG. 11 B shows one embodiment of a portion of the OMP-IS protein, FIG 1 1A shows one embodiment of the OMP-I S polynucleotide encoding such polypeptide. FIG. 12 B shows one embodiment of a portion of the OMP-IT protein, FIG 12A shows one embodiment of the OMP-IT polynucleotide encoding such polypeptide. FIG. 13 B shows one embodiment of the OMP-IU protein, FIG 13 A shows one embodiment of the OMP- IU polynucleotide. FIG. 14 B shows one embodiment of the OMP-IV protein, FIG 14A shows one embodiment of the OMP- IV polynucleotide. FIG. 15 B shows one embodiment of the OMP-IW protein, FIG 15A shows one embodiment of the OMP- IW polynucleotide. FIG. 16 B shows one embodiment of the OMP-IX protein, FIG 16A shows one embodiment of the OMP- IW polynucleotide. FIG. 17 B shows one embodiment of the OMP-1Y protein, FIG 17A shows one embodiment of the OMP- 1Y polynucleotide. FIG. 18 B shows one embodiment of the OMP-IZ protein, FIG 18A shows one embodiment of the OMP- IZ polynucleotide. FIG. 19 B shows one embodiment of the P30 protein, FIG 19A shows one embodiment of the P30 polynucleotide. FIG. 20 B shows one embodiment of the P30a protein, FIG 20A shows one embodiment of the p30A polynucleotide. FIG. 21 B shows one embodiment of the P30-1 protein, FIG 21A shows one embodiment of the p30-l polynucleotide. FIG. 22 B shows one embodiment of the P30-2 protein, FIG 22 A shows one embodiment of the p30-2 polynucleotide. FIG. 23 B shows one embodiment of the P30-3 protein, FIG 23 A shows one embodiment of the p30-3 polynucleotide. FIG. 24 B shows one embodiment of the P30-4 protein, FIG 22 A shows one embodiment of the p30-4 polynucleotide. FIG. 25 B shows one embodiment of the P30-5 protein, FIG 22 A shows one embodiment of the p30-5 polynucleotide. FIG. 26 B shows one embodiment of the P30-6 protein, FIG 26 A shows one embodiment of the p30-6 polynucleotide. FIG. 27 B shows one embodiment of the P30-7 protein, FIG 27 A shows one embodiment of the p30-7 polynucleotide. FIG. 28 B shows one embodiment of the P30-8 protein, FIG 28 A shows one embodiment of the p30-8 polynucleotide. FIG. 29 B shows one embodiment of a portion of the P30-9 protein, FIG 29 A shows one embodiment of the p30-9 polynucleotide encoding such polypeptide. FIG. 30 B shows one embodiment of a portion of the P30-10 protein, FIG 30 A shows one embodiment of the p30-10 polynucleotide encoding such polypeptide. FIG. 31 depicts the amino acid sequences alignment of seven E. chafeensis OMP-ls and Cowdria ruminantium MAP-1. Aligned positions of identical amino acids with OMP-IF are shown with dots. The sequence of C. ruminantium MAP-1 is from the report of Van Vliet et al (1994) Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium. Infect. Immun. 62:1451-1456. Gaps indicated by dashes were introduced for optimal alignment of all proteins. Bars indicates semivariable region (SV) and three hypervariable regions (HV1, HV2, and HV3). DETAILED DESCRIPTION OF THE INVENTION Isolated Polynucleotides Encoding OMP-1.OMP-1 A. OMP- I B, OMP-IC. OMP-ID. OMP-IF and the OMP from E. Canis In one aspect, the present invention, provides isolated polynucleotides that encode the outer membrane proteins, OMP-1 (or p28), OMP-1B, OMP-IC, OMP- ID, OMP- IE, OMP-IF, OMP-1A, OMP-IR, OMP-I S, OMP- IT, OMP -1U, OMP-IV, OMP-IW, OMP-IX, OMP-1Y and OMP-IZ from E. chafeensis and the outer membrane proteins P30, P30-a, P-30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, and P30-10 from E. Canis or an immunogenic fragment thereof. The polynucleotide is single stranded or double stranded. The polynucleotide may be a DNA or RNA molecule, preferably a DNA molecule, and comprises a sequence which codes for the respective outer membrane protein. Preferably, the polynucleotide encodes at least the mature form of outer membrane protein. The polynucleotide optionally further comprises a leader sequence and encode an outer membrane preprotein that is processed in the cell to form the mature protein. The polynucleotide of the present invention may also be fused in frame to a marker sequence which allows for purification of the corresponding outer membrane protein. The OMP-1 polynucleotide encodes an OMP-1 protein of E. chafeensis having a molecular weight of about 27.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 3B SEQ ID NO: ; Figure 3B shows one embodiment of the OMP-1 protein, Figure 3A shows one embodiment of the OMP-1 polynucleotide. The OMP- IB polynucleotide encodes an OMP- IB protein of E. chafeensis having a molecular weight of about 28.2 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 4B SEQ ID NO: ; Figure 4B shows one embodiment of the OMP- IB protein, Figure 4 A shows one embodiment of the OMP- IB polynucleotide. The OMP-IC polynucleotide encodes an OMP-IC protein of E. chafeensis having a molecular weight of about 27.6 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 5B SEQ ID NO: ; Figure 5B shows one embodiment of the OMP-IC protein, Figure 5 A shows one embodiment of the OMP-IC polynucleotide. The OMP-ID polynucleotide encodes an OMP-ID protein of E. chafeensis having a molecular weight of about 28.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 6B SEQ ID NO: ; Figure 6B shows one embodiment of the OMP-ID protein, Figure 6 A shows one embodiment of the OMP-ID polynucleotide. The OMP-IE polynucleotide encodes an OMP-IE protein of E. chafeensis having a molecular weight of about 27.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 7B SEQ ID NO: __; Figure 7B shows one embodiment of the OMP-IE protein, Figure 7A shows one embodiment of the OMP-IE polynucleotide. The OMP-IF polynucleotide encodes an OMP-IF protein of E. chafeensis having a molecular weight of about 27.9 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 8B SEQ ID NO: ; Figure 8B shows one embodiment of the OMP-IF protein, Figure 8 A shows one embodiment of the OMP-IF polynucleotide. The OMP-1 A polynucleotide encodes an OMP-1A protein of E. chafeensis having a molecular weight of about 29.6 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 9B SEQ ID NO: ; Figure 9B shows one embodiment of the OMP-1A protein, Figure 9A shows one embodiment of the OMP-1 A polynucleotide. The OMP-IR polynucleotide encodes an OMP-IR protein of E. chafeensis having a molecular weight of at least 23 kDa and comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 10B SEQ ID NO: ; Figure 10B shows one embodiment of a portion of the OMP-IR protein, Figure 10A shows one embodiment of the OMP-IR polynucleotide encoding such polynucleotide. The OMP-IS polynucleotide encodes an OMP-IS protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 1 IB SEQ ID NO: ; Figure 1 IB shows one embodiment of a portion of the OMP-IS protein, Figure 11A shows one embodiment of the OMP-IS polynucleotide encoding such polypeptide. The OMP-IT polynucleotide encodes an OMP-IT protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG.12B SEQ ID NO: ; Figure 12B shows one embodiment of a portion of the OMP-IT protein, Figure 12B shows one embodiment of a polynucleotide encoding such polypeptide. The OMP-IU polynucleotide encodes an OMP-IU protein of E. chafeensis having a molecular weight of about 30.6 kDa and an amino acid sequence which is at least 85% homologous to amino acid sequence shown in FIG. 13B SEQ ID NO: ; Figure 13B shows one embodiment of the OMP-IU protein, Figure 13A shows one embodiment of the OMP-IU polynucleotide. The OMP-IV polynucleotide encodes an OMP-IV protein of E. chafeensis having a molecular weight of about 28.0 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 14B SEQ ID NO: ; Figure 14B shows one embodiment of the OMP-IV protein, Figure 14A shows one embodiment of the OMP-IV polynucleotide. The OMP-IW polynucleotide encodes an OMP-IW protein of E. chafeensis having a molecular weight of about 28.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 15B SEQ ID NO: ; Figure 15B shows one embodiment of the OMP-IW protein, Figure 15A shows one embodiment of the OMP-IW polynucleotide. The OMP-IX polynucleotide encodes an OMP-IS protein of E. chafeensis having a molecular weight of about 27.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 16B SEQ ID NO: ; Figure 16B shows one embodiment of the OMP-IX protein, Figure 16A shows one embodiment of the OMP-IX polynucleotide. The OMP-1 Y polynucleotide encodes an OMP-1 Y protein of E. chafeensis having a molecular weight about 28.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 17B SEQ ID NO: ; Figure 17B shows one embodiment of the OMP-1Y protein, Figure 17A shows one embodiment of the OMP- 1 Y polynucleotide. The OMP-IZ polynucleotide encodes an OMP-IZ protein of E. chafeensis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 18B SEQ ID NO: ; Figure 18B shows one embodiment of a portion of the OMP-IZ protein, Figure 18A shows one embodiment of an OMP-IZ polynucleotide encoding such polypeptide. The p30 polynucleotide encodes a P30 protein of E. canis having a molecular weight of about 28.8 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 19B SEQ ID NO: ; Figure 19B shows one embodiment of the P30 protein, Figure 19A shows one embodiment of the p30 polynucleotide. The p30A polynucleotide encodes a P30a protein of E. canis having a molecular weight of about 29.1 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 20B SEQ ID NO: ; Figure 20B shows one embodiment of the P30a protein, Figure 20A shows one embodiment of the p30A polynucleotide. The p30-l polynucleotide encodes a P30-1 protein of E. canis having a molecular weight of about 27.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 21B SEQ ID NO: ; Figure 2 IB shows one embodiment of the P30-1 protein, Figure 21 A shows one embodiment of the p30-l polynucleotide. The p30-2 polynucleotide encodes a P30-2 protein of E. canis having a molecular weight of about 28.0 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 22B SEQ ID NO: ; Figure 22B shows one embodiment of the P30-2 protein, Figure 22A shows one embodiment of the p30-2 polynucleotide. The p30-3 polynucleotide encodes a P30-3 protein of E. canis having a molecular weight of about 28.7 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 23B SEQ ID NO: ; Figure 23B shows one embodiment of the P30-3 protein, Figure 23A shows one embodiment of the p30-3 polynucleotide. The p30-4 polynucleotide encodes a P30-4 protein of E. canis having a molecular weight of about 28.0 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 24B SEQ ID NO: ; Figure 24B shows one embodiment of the P30-4 protein, Figure 24A shows one embodiment of the p30-4 polynucleotide. The p30-5 polynucleotide encodes a P30-5 protein of E. canis having a molecular weight of about 29.4 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 25B SEQ ID NO: ; Figure 25B shows one embodiment of the P30-5a protein, Figure 25A shows one embodiment of the p30-5a polynucleotide. The p30-6 polynucleotide encodes a P30-6 protein of E. canis having a molecular weight of about 29.5 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 26B SEQ ID NO: ; Figure 26B shows one embodiment of the P30-6 protein, Figure 26A shows one embodiment of the p30-6 polynucleotide. The p30-7 polynucleotide encodes a P30-7 protein of E. canis having a molecular weight of about 29.9 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 29B SEQ ID NO: ; Figure 29B shows one embodiment of the P30-7 protein, Figure 29A shows one embodiment of the p30-7 polynucleotide. The p30-8 polynucleotide encodes a P30-8 protein of E. canis having a molecular weight of about 30.3 kDa and an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 28B SEQ ID NO: ; Figure 28B shows one embodiment of the P30-8 protein, Figure 28A shows one embodiment of the p30-8 polynucleotide. The p30-9 polynucleotide encodes a P30-9 protein of E. canis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 29B SEQ ID NO: ; Figure 29B shows one embodiment of a portion of the P30-9 protein, Figure 29A shows one embodiment of the p30-9 polynucleotide encoding such polypeptide. The p30-10 polynucleotide encodes a P30-10 protein of E. canis comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence shown in FIG. 30B SEQ ID NO: ; Figure 30B shows one embodiment of a portion of the P30-10 protein, Figure 30A shows one embodiment of the p30-10 polynucleotide encoding such polypeptide. The polynucleotides encoding an E. chafeensis outer membrane protein or an E. canis outer membrane protein have a sequence that is at least 85%, preferably at least 90%, more preferably at least 95% homologous to or similar to the amino acid sequences shown in Figures 3B through 30B, and thus embrace polynucleotides encoding outer membrane proteins from different strains of E. chafeensis and E. canis. The polynucleotides encode an outer membrane protein whose conserved regions collectively are at least 90%, preferably at 95%, more preferably at least 97% homologous to the conserved regions of the amino acid sequences of the present invention. The outer membrane proteins of E. chafeensis and E. canis have six conserved regions, which are separated by one semivariable region and three hypervariable regions. The conserved regions of the outer membrane proteins OMP- 1, OMP-1 A, OMP- IB, OMP1-C, OMP-ID, OMP1-F are depicted in Fig. 31. Preferably, the amino acid sequence of the outer membrane proteins of E. chafeensis and E. canis are at least 30% divergent from the amino acid sequence of MAP-1. Such sequences include allelic, strain variants and other amino acid sequence variants (e.g., including "muteins" or "mutant proteins"), whether naturally-occurring or biosynthetically produced. As used herein, "amino acid sequence homology" is understood to mean amino acid sequence similarity, and homologous sequences share identical or similar amino acids, where similar amino acids are conserved amino acids as defined by ϋayoff et al, Atlas of Protein Sequence and Structure; vol. 5, Supp. 3, pp. 345-362 (M. O. Dayoff, ed., Natϊ BioMed. Research Fdn., Washington D.C. 1978.) Thus, a candidate sequence sharing 85% amino acid sequence homology with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 85% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence, or constitute a conserved amino acid change thereto. "Amino acid sequence identity" is understood to require identical amino acids between two aligned sequences. Thus, a candidate sequence sharing 85% amino acid identity with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 85% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence. As used herein, all homologies and identities are calculated using the amino acid sequences shown in the cited Figure or SEQ ID NO as the reference sequence. Thus, to determine whether an amino acid sequence is 85% homologous to OMP-1, one uses the amino acid sequence shown in Fig. , SEQ ID NO: as a reference. Also as used herein, sequences are aligned for homology and identity calculations using the method of the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, MD) which employs the method of Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410. Identities are calculated by the Align program (DNAstar, Inc.) In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are ignored when making the homology/identity calculation. In another aspect, the present invention provides a nucleotide sequence encoding a polypeptide which comprises a fragment of the OMP1 protein, hereinafter referred to as "rP28" . The rP28 polypeptide weighs approximately 31 kDa and comprises all but of the first 5 amino acids of mature OMP-1 protein. The rP28 polypeptide comprises the amino acid sequence extending from amino acid 6 through amino acid 251 of the amino acid sequence shown in Fig.l, SEQ ID NO. The present invention also embraces polypeptides where one or more of the amino acids in the sequence extending from amino acid 1 or 6 through amino acid 251 Fig. 1 are replaced by conservative amino acid residues. The present invention also relates to derivatives of rP28 that have an amino acid sequence identity of at least 85%, more preferably at least 90%, and most preferably of at least 95% with the amino acid sequence extending from amino acid 1 or 6 through amino acid 251 of the protein and which derivative binds to antibodies in sera from humans infected with E. chafeensis. The polynucleotides are useful for producing the outer membrane proteins of E. chafeensis and E. canis. For example, an RNA molecule encoding the outer membrane protein OMP-1 is used in a cell-free translation systems to prepare OMP-1. Alternatively, a DNA molecule encoding the outer membrane protein is introduced into an expression vector and used to transform cells. Suitable expression vectors include for example chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40, bacterial plasmids, phage DNAs; yeast plasmids, vectors derived from combinations of plasmids and phage DNAs, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. The DNA sequence is introduced into the expression vector by conventional procedures. Accordingly, the present invention also relates to recombinant constructs comprising one or more of the polynucleotide sequences. Suitable constructs include, for example, vectors, such as a plasmid, phagemid, or viral vector, into which a sequence that encodes the outer membrane protein has been inserted. In the expression vector, the DNA sequence which encodes the outer membrane protein is operatively linked to an expression control sequence, i.e., a promoter, which directs mRNA synthesis. Representative examples of such promoters, include the LTR or SV40 promoter, the E xoli lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or in viruses. The promoter may also be the natural promoter of the outer membrane protein coding sequence. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Preferably, the recombinant expression vectors also include an origin of replication and a selectable marker, such as for example, the ampicillin resistance gene of E. coli to permit selection of transformed cells, i.e. cells that are expressing the heterologous DNA sequences. The polynucleotide sequence encoding the outer membrane protein is incorporated into the vector in frame with translation initiation and termination sequences. Optionally, the sequence encodes a fusion outer membrane protein which includes an N-terminal or C-terminal peptide or tag that stabilizes or simplifies purification of the expressed recombinant product. Representative examples of such tags include sequences which encode a series of histidine residues, the Herpes simplex glycoprotein D, or glutathione S-transferase. Polynucleotides which encode portions of the outer membrane proteins of E. chafeensis and E. canus are useful as probes for isolating and identifying E. chafeensis genes and E. canis genes, particularly full-length genes from new strains or isolates of E. chafeensis and E. canis. The Outer Membrane Proteins of E. chafeensis and E. Canis In addition to the outer membrane proteins OMP-1, OMP- IB, OMP-IC, OMP-ID, OMP-1 E, and OMP- IF, OMP-IR, OMP-I S, OMP-IT, OMP-IU, OMP-IV, OMP-IW, OMP-IX, OMP-1Y, and OMP-IZ from E. chafeensis and the proteins P30, P30A, P30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, and P30-10 from E. Canis, the present inventions embraces non-naturally occurring allelelic forms or derivatives of the outer membrane proteins, where one or more of the amino acids have been replaced by conservative amino acid residues, typically by using direct synthesis or recombinant techniques. Preparing the Outer Membrane Proteins The outer membrane proteins of the present invention are synthetically produced by conventional peptide synthesizers. The outer membrane proteins are also produced using cell-free translation systems and RNA molecules derived from DNA constructs that encode the outer membrane protein. Alternatively, the outer membrane protein is made by transfecting host cells with expression vectors that comprise a DNA sequence which encodes the outer membrane protein and then inducing expression of the outer membrane protein in the host cells. The outer membrane protein is expressed in suitable host cells, preferably bacteria, under the control of suitable promoters. Host cells are transformed with the expression vectors of this invention and cultured in conventional nutrient media. Such media optionally contains additional compounds, such as for example compounds that induce promoters, such as for example isopropyl-β-D-thiogalactoside which induces the Lac promoter, or compounds, such as for example, ampicillin, which allows for selection of transformants. Following transformation of the suitable host strain and growth of the host strain to an appropriate cell density, the cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification of the outer membrane protein. Such purification usually involves salting-out of the protein fraction, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, and high performance liquid chromatography (HPLC). Preparation of Antibodies The isolated outer membrane proteins, particularly the recombinant forms of the outer membrane proteins, are used as immunogens to produce antibodies immunospecific for the corresponding protein. The term "immunospecific" means the antibodies have substantially greater affinity for the protein used as an immunogen than for other proteins. Such antibodies are generated using conventional techniques by administering the respective outer membrane protein or a portion thereof, i.e., the recombinant polypeptide, to an animal, preferably a nonhuman. collecting blood from the immunized animals and isolating the serum and/or the IgG fraction from the blood. Monoclonal antibodies are prepared by injecting animals with the immunogens, extracting antibody- producing B cells from the animal, fusing the B cells with a myeloma cells to produce hybridomas, obtaining the monoclonal antibodies from the hybridomas. Antibodies to the outer membrane proteins of E. chafeensis and E. canis are useful research tools for identifying cells, particularly monocytes, infected with E.chafeensis or E. canis and for purifying the corresponding outer membrane protein of E.chafeensis or E. Canis from partially purified preparations by affinity chromatography. Such antibodies are also useful for identifying bacterial colonies, particularly colonies of genetically-engineered bacteria, that are expressing the major outer membrane protein . Diagnostic Method The present invention also provides a method for detecting antibodies to the E. chafeensis or E.canis in a sample of a bodily fluid from a patient. The method comprises providing an isolated outer membrane protein of E. chafeensis or E. canis, particularly a recombinant form of the isolated protein, contacting the outer membrane protein or polypeptide with a sample taken from the patient; and assaying for the formation of a complex between the outer membrane protein or polypeptide and antibodies in the sample. For ease of detection, it is preferred that the isolated protein or polypeptide be attached to a substrate such as a column, plastic dish, matrix, or membrane, preferably nitrocellulose. The sample may be a tissue or a biological fluid, including urine, whole blood, or exudate, preferably serum. The sample may be untreated, subjected to precipitation, fractionation, separation, or purification before combining with the isolated protein or peptide. Interactions between antibodies in the sample and the isolated protein or peptide are detected by radiometric, colorimetric, or fluorometric means, size-separation, or precipitation. Preferably, detection of the antibody-outer membrane protein complex is by addition of a secondary antibody that is coupled to a detectable tag, such as for example, an enzyme, fluorophore, or chromophare. Formation of the complex is indicative of the presence of anti-E chafeensis or anti-E canis antibodies, either IgM or IgG, in the patient. Thus, the method is used to determine whether a patient is infected with E. chafeensis or E. canis. Preferably, the method employs an enzyme-linked immunosorbent assay (ELISA) or a Western immunoblot procedure . Such methods are relatively simple to perform and do not require special equipment as long as membrane strips are coated with a high quality antigen. Accordingly, it is more advantageous to use a recombinant form of the outer membrane protein of E. chafeensis or E. canis since such proteins, typically, are more pure and consistent in quality than a purified form of such protein. Immunogenic Composition The present invention also relates to immunogenic compositions comprising one or more of the isolated outer membrane proteins of E. chafeensis and a pharmaceutically acceptable adjuvant and to immunogenic compositions comprising an isolated P30 protein of E. canis and a pharmaceutically acceptable adjuvant, which, preferably, enhances the immunogenic activity of the outer membrane protein in the host animal. Preparation of a Polynucleotide which Encodes OMP-KP28) A. Isolation of the Outer Membrane Proteins E. chafeensis Arkansas strain and E. canis Oklahoma strain were cultivated in the DH82 dog macrophage cell line and purified by Percoll density gradient centrifugation. Purified ehrlichiae (100 μg) were suspended with 10 mM sodium phosphate buffer, pH 7.4, containing 0.1% Sodium ΛMauroyl sarcosine (Sarkosyl) [Sigma, St. Louis, MO], 50 μg/ml each Dnase I (Sigma) and Rnase A (Sigma), and 2.5 mM MgCl 2 . After incubation at 37° for 30 min, the sample was separated by centrifugation at 10,000 x g for 1 h into the soluble supernatant and the insoluble precipitate. The insoluble pellet was resuspended 2 to 3 times with 0.1% Sarkosyl and centrifuged. The final pellet was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and by electron microscopy. Transmission electron microscopy revealed that the purified ehrlichial fraction consists of a mixture of electron dense and light forms of E. chafeensis with slight disintegration of inner membrane . Ehrlichiae were not surrounded with the host inclusion membrane. Various sizes of membrane vesicles (< 1 μm) without significant ribosomes or nuclear materials were observed in the Sarkosyl-insoluble fraction from the organism. Succinic dehydrogenase (inner membrane marker enzyme of gram negative bacteria) activities were at less than the detection limit (1 n moles / min / mg of protein) in the Sarkosyl-insoluble fraction compared to approximately 10 n moles / min / mg of protein in the Percoll-purified organisms, suggesting that the insoluble fraction primarily consisted of the outer membrane of E. chafeensis. Analysis of the Sarkosyl-soluble, and insoluble fraction of E. chafeensis by SDS-PAGE suggested that proteins of 30-kDa range in the insoluble fraction represent the major outer membrane proteins of this organism. Analysis of the Sarkosyl-soluble, and insoluble fraction of E. canis by SDS-PAGE suggested that proteins of 30- kDa range in the insoluble fraction represent the major outer membrane proteins of this organism also. E. canis was antigenically cross reactive with E. chafeensis. These findings indicate that the 30-kDa range proteins represent the major outer membrane proteins of these two Ehrlichia spp. To improve resolution of the outer membrane proteins, proteins in the Sarkosyl-insoluble pellet prepared from 400 μg of purified E. chafeensis were separated by a reversed-discontinuous (Rd) SDS-PAGE (2.5-cm-long 17% gel on top of 11-cm-long 12% gel). At least five proteins of 30-kDa range in E. chafeensis (P23, P25, P27, P28, and P29) were resolved from the Sarkosyl-insoluble proteins. B. Cloning and sequencing of the p28 gene The portion of the membrane containing bound proteins was excised and analyzed with an Applied Biosystems protein sequencer (Model 470). The N-terminal amino acid sequence of P28 was determined as D P A G S G I N G N F Y S G K Y M P, SEQ IN NO . Based on 6th to 12th amino acids of this sequence, a forward primer, FECH1, having the sequence: 5'- CGGGATCCGAATTCGGfA/T/G/C)ATfA/T/C)AA(T/C1GGfA/T/G/C1AAfT/C1TTfT/OTA-3'. SEQ ID NO was designed. Amino acids at the 1 to 5 positions of the N terminus of P28 were not included in this primer design. For insertion into an expression vector, a 14-bp sequence (underlined) was added at the 5' end of primer to create an EcoBΛ and a BamWl site. The reverse primer, RECH2, which includes a Noll site at the 5' end for ligation into an expression vector had the sequence : 5'-AGCGGCCGCTTA(A/G)AA(T/C)A(C/G) (A/G)AA (C/T)CT T(C/G)C TCC-3\ SEQ ID NO . Genomic DNA of E. chafeensis was isolated from purified organisms. PCR amplification with FECH1 and RECH2 primers was performed using a Perkin-Elmer Cetus DNA Thermal Cycler (model 480). A 0.8-kb amplified product was cloned in the pCRII vector of a TA closing kit, as described by the manufacturer (Invitrogen Co., San Diego, CA). The clone obtained was designated pCRII/?2o\ Both strands of the inserted DNA were sequenced by a dideoxy-termination method with an Applied Biosystems 373A DNA sequencer. The 0.8-kb DNA fragment, cloned in pCRIIp2ό\ had an open reading frame (ORF) of 756 bp encoding a 251 -amino acid recombinant protein (including both PCR primer regions) with a molecular mass of 27,685 Da. The nucleotide sequence of the open reading frame, SEQ ID NO: , and the amino acid sequence of the polypeptide of the OMP-1 protein, SEQ ID NO , are shown in Figs and , respectively. A DNA fragment comprising the p30 gene was prepared in a similar manner, i.e., by PCR amplification of genomic DNA of E. canis with the FECH1 and RECH2 primers. Preparation of Polynucleotides which encode OMP 1A. OMP1B. OMPl-C, OMP-ID. OMP-IF. and OMP1-E A. Southern blot analysis. Genomic DNA extracted from the purified E. chafeensis (200 ng each) was digested with restriction endonucleases, electrophoresed, and transferred to Hybond-N + nylon membrane (Amersham, Arlington Heights, IL), by a standard method. The 0.8-kb p28 gene fragment from the clone pCRllp28 was labeled with [α- 32 P]dATP by the random primer method using a kit (Boehringer Mannheim, Indianapolis, IN) and the labeled fragment was used as a DNA probe. Hybridization was performed at 60°C in rapid hybridization buffer (Amersham) for 20 h. The nylon sheet was washed in 0.1 x SSC (1 x SSC containing 0.15M sodium chloride and 0.015M sodium citrate) with 1% SDS at 55°C and the hybridized probes were exposed to Hyperfilm (Amersham) at -80°C. Genomic Southern blot analysis with several restriction enzymes resulted in one or more DNA fragment(s) of E. chafeensis which hybridized to 32 P-labeled p28 gene probe. The restriction enzymes used did not cut within the p28 gene portion of the pCRl!p28 insert. Xba I, Bgl II, and Kpn I produced two bands, Spe I generated three bands, and EcoR V and Pst I produced multiple bands with different densities. EcoR I generated a broad band of 2.5 to 4kb. These p28 homologous genes are designated as omp-1 (outer membrane protein-1) family. B. Cloning and sequencing of genomic copies of E. chafeensis p28 gene. The EcoR I and Pst 1 fragments of DNA, detected by genomic Southern blot analysis as described above, were inserted into pBluescript II KS (+) vectors, and the recombinant plasmids were introduced into E. coli DH5α. Using the colony hybridization method with the 32 P-labeled p28 gene probe, four positive clones were isolated from the transformant. The positive clones were designated pEC2.6, pEC3.6, pPS2.6, and pPS3.6. These contained the ehrlichial DNA fragments of 2.6-kb (EcoR I), 3.6 kb (EcoR I), 2.6 kb (Pst I), and 3.6 kb (Pst I), respectively. The inserts of the clones pEC3.6 and pPS2.6 overlapped as shown in Fig. . The overlapping area was further confirmed by PCR of E. chafeensis genomic DNA with two pairs of primer sets interposing the junctions of the four clones. The 1.1- to 1.6-kb DNA fragments of i /ndIII-///ndIII, //wdlll-iϊcσRI, or Xhol-EcoRl in the pEC2.6 and pEC3.6 were subcloned for sequencing. DNA sequencing was performed with suitable synthetic primers by dideoxy-termination method as described above. Four DNA fragments from 2.6 to 3.6 kb were cloned from the iscoRI-digested and the Λ/I-digested genomic DNA of E. chafeensis by colony hybridization with radiolabeled p28 gene probe. The inserted DNA of the two recombinant clones, pEC3.6 and PPS2.6, were overlapped as shown in Fig. 7. Sequencing revealed one 5'- truncated ORF of 243 bp (designated omp-lA) and five complete ORF of 836-861 bp (designated omp-l to omp- 7F), which are tandemly-arrayed and are homologous to the p28 gene (but are not identical), in the ehrlichial genomic DNA of 6,292 bp. The intergenic spaces were 581 bp between omp-IA. and omp-lB and 260-308 bp among others. Putative promoter regions and ribosome-binding sites were identified in the noncoding regions. Sequence analysis and GenBank accession number. Nucleotide sequences were analyzed with the DNASIS program (Hitachi Software Engineering Co., Ltd., Yokohama, Japan). A homology search was carried out with databases of the GenBank, Swiss Plot, PDB and PIR by using the software basic local alignment search tool in the BLAST network service (the National Center for Biotechnology Information, Bethesda, MD). Phylogenetic analysis was performed by using the PHYLIP software package (version 3.5). An evolutional distance matrix, generated by using the Kimura formula in the PROTDIST, was used for construction of a phylogenetic tree by using the unweighted pair-group method analysis (UPGMA) (Felsenstein, J. 1989. PHYLIP-phylogeny inference package (version 3.3). Cladistics 5:164-166). The data were also examined using parsimony analysis (PROTPARS in PHYLIP). A bootstrap analysis was carried out to investigate the stability of randomly generated trees by using SEQBOOT and CONSENSE in the same package. The nucleotide sequence of the p28 gene and its gene copies has been assigned GenBank accession numbers U72291 and AF021338, respectively. Proteins of the E. chafeensis omp-1 Family. Five complete omp-1 gene copies (omp-lB to omp-lΕ) encode 279 to 287-amino acid proteins with molecular masses of 30,320 - 31,508 Da. Omp-1 A encodes an 82-amino acid partial protein (9,243 Da) which lacks the N-terminal region. The 25-amino acid sequence at the N-terminus of OMP- 1 B to OMP- 1 F (encoded in omp-lB to omp-lΕ) is predicted to be a signal peptide because three carboxyl-terminal amino acids of the signal peptides (Ser-X-Ala in OMP- IB, Leu-X-Ser for OMP-C, and Ser-X-Ser for OMP-ID and OMP-IF) are included in the preferred amino acid sequence of signal peptidase at the processing sites proposed by Oliver .. The calculated molecular masses of the mature OMP- IB to OMP-IF from the predicted amino acid sequences are 28,181 Da for OMP-1B, 27,581 Da for OMP-IC, 28,747 Da for OMP-ID, 27,776 Da for OMP-IE, and 27,933 Da for OMP-IF. The estimated isoelectric points are 4.76-5.76 in the mature OMP-1B to OMP-IF. An amino acid sequence in omp- 1Ε gene (the 80th to 94th amino acids) was identical to the N-terminal amino acid sequences of E. chafeensis native P23 protein as determined chemically, which indicates that P23 is derived from the omp-1 F gene. Amino acid sequences identical to the N-terminal sequences of P25, P27, and P29 were not found in those from omp-1 gene copies cloned in this study. Alignment of predicted amino acid sequences of the E. chafeensis OMP-1 family and Cowdria ruminantium, revealed substitutions or deletions of one or several contiguous amino acid residues throughout the molecules. The significant differences in sequences among the aligned proteins are seen in the regions indicated SV (semivariable region) and HV (hypervariable region) 1 to 3 in Fig 31. Computer analysis for hydropathy revealed that protein molecules predicted from all omp-1 gene copies contain alternative hydrophilic and hydrophobic motifs which are characteristic of transmembrane proteins. The HV1 and HV2 were found to locate in the hydrophilic regions. The amino acid sequences of 5 mature proteins without signal peptides (OMP-IC to OMP-IF and a P28) were similar to one another (71-83%) but the sequence of OMP-1B was dissimilar to those of the 5 proteins (45- 48%). The amino acid sequences of the 5 proteins showed an intermediate degree of similarity with that of C. ruminantium MAP-1 (59-63%), but the similarity between that of the OMP- IB and the C. ruminantium MAP-1 was low (45%). These relations are shown in a phylogenetic tree which was obtained based on the amino acid sequence alignment by UPGMA method in the PHYLIP software package (Fig. 10). Three proteins (P28, OMP-ID, and OMP-IF) and two proteins (OMP-IC and OMP-IE) formed two separate clusters. The OMP- IB was located distantly from these two clusters. The C. ruminantium MAP-1 was positioned between the OMP- IB and other members in the OMP-1 family. Preparation of a Recombinant form of OMP-1 and P30 The 0.8-kb p28 gene was excised from the clone pCRllp28 by EcoRl-Notl double-digestion, ligated into EcoRl-Notl sites of a pET 29a expression vector, and amplified in Escherichia coli BL21 (DE3)pLysS (Novagen, Inc., Madison, WI). The clone (designated pET29p28) produced a fusion protein with a 35-amino acid sequence carried from the vector at the N terminus. The amino acid sequence of the OMP-1 portion of the fusion protein is depicted in Fig. [ . An expression vector comprising the p30 gene was used to prepare the recombinant form of P30. The following examples are for purposes of illustration only and are not intended to limit the scope of the claims which are appended hereto. Preparation of anti rP28 (anti-OMPP antibody The (r) P28 antigen was prepared by excising the gel band corresponding to the rP28 in SDS-PAGE, mincing the band in phosphate-buffered saline (PBS), p 7.4, and mixing with an equal volume of Freund's incomplete adjuvant (Sigma). The rP28 mixture (1 mg of protein each time) was subcutaneously injected into a rabbit every 2 weeks four times. A serum sample was collected from the rabbit to provide the anti-rP28 antibody The anti-rP28 antibody was examined by western immunoblots analysis. The results indicated that the rabbit anti-rP28 antibody recognized not only rP28 (31 kDa) and P28, but also P29 and P25 of E. chafeensis and P30 of E. canis . These results indicate that P28 shares antigenic epitopes with P25 and P29 in E. chafeensis and P30 of E. canis. Example 1. Assaying for the presence of anti-OMP-1 antibody in a Patient Convalescent-phase serum from a patient with clinical signs of human ehrlichiosis was used. Western blot analyses using the rP28 protein as antigen was performed with 1 :1,000 dilutions of this serum. Alkaline phosphatase-conjugated affinity-purified anti-human, anti-rabbit or anti-mouse immunoglobulin G (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) were used at a 1: 1,000 or 1:2,000 dilution as secondary antibodies. Results indicated that serum from a patient with clinical signs of human ehrlichiosis reacted strongly to rP28 (31 kDa). Example 2 Assaying for the presence of anti-OMP- 1 antibody in a Patient Convalescent-phase serum from a patient with clinical signs of human ehrlichiosis was reacted with the rP30 protein of E.canis as described in Example 1. The serum reacted strongly to rP30. These results indicate the rP30 is useful for diagnosing an infection with E. chafeensis in human patients. Example 3. Identifying E. chafeensis-infected cells using anti-rP 28 antibody E. chafeensis-m ' ϊected DH82 cells were sonicated and centrifuged at 400 x g for 10 min. The supernatant was then centrifuged at 10,000 x g for 10 min to obtain ehrlichia-enriched pellet. The pellet was resuspended and incubated with rabbit anti-rP28 antibody or normal rabbit serum (1:100 dilution) at 37°C for lh in PBS containing 1% bovine serum albumin (BSA-PBS). After washing, the ehrlichiae was incubated with gold-conjugated protein G (20 nm), Sigma) at 1:30 dilution for 1 h at room temperature in BSA-PBS. After washing again, the specimen was fixed with 1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% trinitrophenol in 0.1 M cacodylate buffer (pH 7.4) for 24h and postfixed in 1% osmium- 1.5% potassium ferricyanide for 1 h (34). The section was then embedded in PolyBed 812 (Polysciences, Warraington, Pa). The specimen was ultrathin sectioned at 60 nm, stained with uranyl acetate and lead citrate, and observed with a Philips 300 transmission electron microscope at 60 kV. Transmission immunoelectron microscopy with colloidal gold-conjugated protein G and rabbit anti-rP28 antibody revealed gold particles bound to E. chafeensis surface. The distribution of the particles was random, close to the surface, and appeared as if almost embedded in the membrane, suggesting that the antigenic epitope protrudes very little from the lipid bilayer. Nonetheless, the antigenic epitope was surface-exposed, and thus, could be recognized by rabbit anti-rP28 antibody. No gold particles were observed on host cytoplasmic membrane or E. chafeensis incubated with normal rabbit serum. Example 4. Immunization of mice and E. chafeensis challenge. The rP28 band in SDS- PAGE was excised, minced, and mixed with an equal volume of Freund's incomplete or complete adjuvant. Nine BALB/c male mice (6 weeks old) were divided into two groups. Five mice were intraperitoneally immunized a total of four times at 10-day intervals; twice with a mixture of the minced gel with the vP28 (30 to 40 μg of protein per mouse each time) and incomplete adjuvant, and twice with a mixture of the recombinant protein (the same amount as before) and complete adjuvant. Four mice were intraperitoneally injected with a mixture of the minced gel without protein and the respective adjuvants. For ehrlichia-challenge, approximately 1 x 10 7 DH82 cells heavily-infected with E. chafeensis were disrupted by sonication in serum-free DMEM (GIBCO-BRL) and centrifuged at 200 x g for 5 min. The supernatant was diluted to a final volume of 5 ml, and 0.3 ml was inoculated intraperitoneally into each mouse 10 days after the last immunization. Before challenge, all 5-immunized mice had a titer of 1: 160 against E. chafeensis antigen by IFA and all 4-nonimmunized mice were negative. At day 5 post-challenge, approximately 1 ml of blood was collected in an EDTA tube from each mouse and protection was assessed by PCR detection of E. chafeensis 16S rDNA in the buffy coat of the collected blood. E. chafeensis could not be reisolated in cell culture at day 10 postinfection . Day 5 post challenge is the optimum time at which establishment of ehrlichial infection can be examined by PCR without the influence of residual DNA from the ehrlichiae used as the challenge before the spontaneous clearance of organisms take place. The E. chafeensis-speciϊic DNA fragment was observed in all nonimmunized mice but not in any immunized mice, indicating that immunization of rP28 apparently protects mice from ehrlichial infection and indicating that the P28 is a potential protective antigen Example 5 Assaying for the presence of anti-P30 antibody in Dogs The rP30 protein was used as an antigen in a Western immunoblot analysis and dot blot analysis to detect the presence of antibody to E. canis in serum from E-canis infected dogs. The results of the Western immunoblot analysis indicated that reactivity of the sera with rP30 was stronger than the reactivity that was observed when purified E.canis was used as antigen. The results of the dot blot assay indicated that rP30 is a useful and sensitive tool for serodiagnosis of canine ehrlichiosis.

Claims: CLAIMS What is claimed is: 1. An isolated polynucleotide encoding an outer membrane protein of E. chafeensis or an immunogenic fragment thereof, wherein the outer membrane protein is selected from the group consisting of OMP-1, OMP- IB, OMP-IC, OMP-ID, OMP-IE, OMP-IF, OMP-IR, OMP-IS, OMP-IT, OMP-IU OMP-IV, OMP-IW OMP-IX OMP-1 Y, and OMP-IZ. 2. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP-1 protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 3B, SEQ. ID NO . 3. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP- IB protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 4B, SEQ. ID NO . 4. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP-IC protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig.5B, SEQ. ID NO . 5. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP-ID protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 6B , SEQ. ID NO . 6. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP-IE protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 7B, SEQ. ID NO . 7. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an OMP-IF protein comprising a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 8B, SEQ. ID NO . 8. The isolated polynucleotide of claim 1 wherein the polynucleotide encodes an immunogenic fragment of the OMP-1 protein, said fragment comprising a sequence which is at least 85% homologous to the amino acid sequence extending from amino acid 6 through amino acid 251 as shown in Fig. 1, SEQ. ID NO . 9. An isolated polynucleotide encoding an outer membrane protein of E. canis or an immunogenic fragment thereof, wherein the outer membrane protein is selected from the group consisting of P30, P30-A, P30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, P30-10. 10. The isolated polynucleotide of claim 9 wherein said P30 protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig, 19 SEQ ID NO. 11. An isolated outer membrane protein of E. chafeensis or an immunogenic fragment thereof, wherein said protein is selected from the group consisting of OMP-1, OMP- IB, OMP-IC, OMP-ID, OMP-IE, OMP-IF, OMP- IR, OMP-IS, OMP-IT, OMP-IU OMP-IV, OMP-IW OMP-IX OMP-1Y, and OMP-IZ. 12. The isolated OMP-1 protein of claim 1 1, wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 4B, SEQ. ID NO . 13. The isolated OMP- IB protein of claim 11 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 5B, SEQ. ID NO . 14. The isolated OMP-IC protein of claim 11 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 6B, SEQ. ID NO . 15. The isolated OMP-ID protein of claim 11 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 7B, SEQ. ID NO . 16. The isolated OMP-IE protein of claim 11 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 8B, SEQ. ID NO . 17. The isolated OMP-IF protein of claim 11 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig. 9B, SEQ. ID NO . 18. The isolated immunogenic fragment of the OMP-1 protein of claim 11, said fragment comprising a sequence which is at least 85% homologous to the amino acid sequence extending from amino acid 6 through amino acid 251 as shown in Fig. 1, SEQ. ID NO . 19. An isolated outer membrane protein of E. canis or an immunogenic fragment thereof, wherein the outer membrane protein is selected from the group consisting of P30, P30-A, P30-1, P30-2, P30-3, P30-4, P30-5, P30-6, P30-7, P30-8, P30-9, P30-10. 20. The isolated P-30 protein of claim 19 wherein said protein comprises a sequence which is at least 85% homologous to the amino acid sequence shown in Fig 19, SEQ ID NO. . 21. A method for diagnosing an infection with E. chafeensis in a patient comprising the steps of: (a) providing a serum sample from the patient; (b) providing an outer membrane protein selected from the group consisting of a protein of claim 11, a protein of claim 19, and mixtures thereof; (c) contacting the serum sample with the outer membrane protein; and (d) assaying for the formation of a complex between antibodies in the serum sample and the outer membrane protein, wherein formation of said complex is indicative of infection with E. chafeensis. 22. A method for diagnosing an infection with E. canis in a Canidae patient comprising the steps of: (a) providing a serum sample from the patient ; (b) providing an outer membrane protein of claim 19; (c) contacting the serum sample with the outer membrane protein; and (d) assaying for the formation of a complex between antibodies in the serum sample and the outer membrane protein, wherein formation of said complex is indicative of infection with E. canis.

Inventors: OHASHI NORIS, RIKIHISA YASUKO, OHASHI, NORIS, RIKIHISA, YASUKO
Applicants: UNIV OHIO, THE OHIO STATE UNIVERSITY
Application year: 1998
Main classification: A01N43
IPC codes: A61K39, A61P31, C07K14