WO2000017381A1 - Pcr methods and materials - Google Patents

Abstract

In broad terms, the present invention includes materials and methods useful to distinguish between and among species of a genus. The present methods utilize the differences in PCR amplicon sizes to specifically identify a given species.

Description

PCR Methods and Materials

This application claims priority to U.S. Provisional Patent Application Serial Number 60/101,023, filed September 18, 1998

BACKGROUND OF THE INVENTION The present invention is concerned with speciation of organisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish between or among species have not always met the expectations of consumers because they are either too costly, cumbersome or unavailable.

Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among species. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. One assay uses several sets of species-specific primers instead of digestion with restriction enzymes, with identification of the PCR products made by size. Minnick and Barbian, 31 J Microb Meth 51 (1997).

One genus of microorganisms, Bartonella, causes a variety of species-dependent disease states in humans, and is therefore important to speciate prior to treatment. Humans infected with bacteria from the genus Bartonella display a variety of pathogies, and appropriate treatment has been surmised as dependant on the species causing the pathology. For instance, the species B. henselae (relatively common in flea-infested areas) presents as cat scratch disease or atypical cat scratch disease, and health care professionals continue to debate the appropriate antibiotic treatment. Bass et al, 16 Pediatr. Infect. Dis. J, 163 (1997). B. clarridgeiae, another causative agent of cat scratch disease, can be treated with antibiotics, but it is not clear which are the most appropriate, ibid.

B. bacilliformis is the causative agent for Carrion's disease (Oroya fever), and is typically treated with chloramphenicol, penicillins or tetracyclines. ibid. Another species, B. elizabethae has been associated with cardiac valve abnormalities, and is so rare that appropriate antibiotics have yet to be determined, ibid. B. quintana causes trench fever (rare except for unsanitary living conditions or in the immunocompromised), and has been successfully treated with penicillins, tetracyc lines and cephalosporins. Kordick et al, 35(1) J. Clin. Microb. 1813 (1997). B. vinsonii sub vinsonii and B. vinsonii sub berkhoffii have only been found in dogs and voles.

Available Bartonella PCR diagnostics require several steps, and are therefore inconvenient for laboratory analysis of samples. For instance, PCR assays on the basis of differences in citrate synthase sequences have been performed using a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes, followed by gel electrophoresisis to distinguish among species. Joblet et al, 33(7) J. Clin. Microb. 1879 (1995); Norman et al, 33(7) J. Clin. Microb. 1797 (1995). PCR assays on the basis of differences in 16S rRNA sequences have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129 FEMS Microbiol. Letters 261 (1995).

Likewise, primers have been used to amplify the region between the 16S and 23 S genes (called "the intergenic region") of Bartonella. In those assays, restriction enzymes were also used to cut and distinguish the PCR products. Matar et al. , 31 (7) J. Clin. Microb. 1730 (1993) and Roux and Raoult, 33(6) J. Clin. Microb. 1573 (1995). In Roux, a difference in size of PCR products (prior to digestion by restriction enzymes) was noted (page 1576); however, the differences are so small as to be indistinguishable on a gel. Moreover, no suggestion is made in Roux to use the pre-digestion PCR product size differences for the purpose of differentiation. In Matar, page 1732 that all three species had "an approximately 1 ,600-bp fragment" and bacilliformis had a 1 ,000 bp fragment prior to digestion.

In a different approach, Minnick and Barbian, 31 J Microb Meth 51 (1997) designed one set of primers from the 16S/23S intergenic region of Bartonella, and amplified fragments from 5. bacilliformis, B. elizabethae, B. henselae andB. vinsonii. The fragments were of different, but indistinguishable sizes (Figure 2), and the researchers therefore conducted a second, species-specific amplification using different sets of primers for each species represented (Figure 3). Minnick, at 55 (1997).

Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.

SUMMARY OF THE INVENTION

The present invention requires only a single step to generate amplicons which identify a specific species.

It is therefore an object to provide a simplified assay for distinguishing between or among species of a genus.

It is a specific object to provide a simplified assay for distinguishing between or among Bartonella species. It is yet another object to provide materials (nucleic acids, vectors, cells, etc.) related to the methods disclosed, including primers and the full sequence of the 16S/23S region of two species of Bartonella.

In all of the above embodiments, it is an object to provide methods to diagnose disease using the materials and methods provided. It is also an object to provide methods for identifying primers useful to conduct

PCR assays which capitalize on the species-specific size differences in an intergenic region of a prokaryote.

It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the species-specific size differences in the 16S/23S intergenic region of Bartonella.

Finally, it is an object of the invention to provide a kit for convenient use of the materials and methods herein provided.

Definitions: For the purposes of the present invention, the following terms shall have the following meanings:

"Amplicon(s)" shall mean a nucleic acid(s) produced through use of primers in PCR. "Genus-specific primer(s)" shall mean primers capable of amplifying an amplicon from at least a portion of the 16S/23S intergenic region of at least two species of the same genus, and no other genera, and wherein the size of the amplicon is unique to the species.

"Bartonella genus-specific primer(s)" shall mean primers capable of amplifying an amplicon from at least a portion of the 16S/23S intergenic region of at least two Bartonella species, and no other genera, and wherein the size of the amplicon is unique to the species.

When the term "Genus-specific primer(s)" or "Bartonella genus-specific primer(s)" is used to describe primers used in PCR assays, it is assumed that said primers are also being in amounts sufficient to amplify at least one ascertainable fragment.

A "set" of primers means at least one forward and at least one reverse primer, that, when used in a PCR assay in appropriate amounts, is capable of amplifying an amplicon.

Brief Description of the Figures Figure 1. Nucleotide sequence alignment of a portion of the 16S-23S rRNA intergenic region of B. bacilliformis (GenBank accession #L26364), B. elizabethae (#L35103), B. henselae (#L35101), B. quintana (#L35100), and _5. vinsonii (baker strain) (#L35102). Corresponding GenBank nucleotide numbers are indicated at the beginning and end of the sequences. Arrows designate PCR primer positions. Figure 2. PCR-based identification of Bartonella species. An ethidium bromide- stained agarose gel (3%) demonstrating amplified products from DNA template derived from Bartonella species. Bartonella bacilliformis, B. elizabethae, B. henselae, and B. quintana yielded expected products of 186 bp, 216 bp, 147 bp, and 132 bp, respectively. Template DNA from B. vinsonii (subspecies berkhoffii) yielded a PCR product of approximately 235 bp. PCR amplification of the B. clarridgeiae template DNA yielded no product. First and last lanes contain 20 base pair ladder.

Figure 3. Phylogenetic comparison of 16S-23S rRNA intergenic region sequences for Bartonella species. Calculated matching percentages are indicated at each branch point of the dendrogram. The lengths of horizontal and vertical lines are not significant. Figure 4. Nucleotide sequence alignment of a portion of the 16S-23S rRNA intergenic region of B. bacilliformis (GenBank accession #L26364), B. clarridgeiae (GenBank accession #AF167989), B. elizabethae (#L35103), B. henselae (#L35101), B. quintana (#L35100), B. vinsonii (baker strain) (#L35102), and B. vinsonii (subspecies berkhoffii) (#AF 167988). Corresponding GenBank nucleotide numbers are indicated at the beginning and end of the sequences. Arrows designate PCR primer positions. Non- conserved B. clarridgeiae nucleotide located at the 3 ' end of initial PCR primer used (see Figure 1) denoted by an asterisk (*).

Figure 5. PCR-based identification of Bartonella species. An ethidium bromide- stained agarose gel (3%) demonstrating amplified products from DNA template derived from Bartonella species. Bartonella bacilliformis, B. clarridgeiae, B. elizabethae, B. henselae, B. quintana, and B. vinsonii (subspecies berkhoffii) yielded expected products of 211, 154 bp, 241 bp, 172 bp, 157 bp, and 260 bp, respectively. First and last lanes contain 20 base pair ladder. Figure 6. PCR-based identification of Bartonella species from animals known to be infected with either B. clarridgeiae (Sample A), B. henselae (Sample B), or B. vinsonii (subspecies berkhoffii) (Sample C). DNA was extracted from 200 1 of blood, eluted in a final volume of 200 1, then 5 1 of template DNA was used in each PCR amplification. After amplification, PCR products were electrophoresed on a 3% agarose gel and stained with ethidium bromide. Amplified control template DNA derived from isolated B. clarridgeiae, B. henselae, and B. vinsonii (subspecies berkhoffii) yielded expected products of 154 bp, 172 bp, and 260 bp, respectively. First and last lanes contain 20 base pair ladder. DETAILED DESCRIPTION OF THE INVENTION

In broad terms, the present invention includes materials and methods useful to distinguish between and among species of a genus. The methods simplify and are therefore more cost-effective than previous methods. In addition, because the present methods are simpler than previous methods, the risk of operator error, contamination, or any other technical problem is reduced, making the present invention inherently more reliable than previous methods.

The present invention includes methods to detect at least one prokaryotic species in a test sample, comprising the following steps: a.) conducting polymerase chain reaction using starting materials comprising a test sample and at least one set of genus- specific primers; and b.) detecting the prokaryotic species in the event that a species- specific sized amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s) is within the scope of the present invention.

The present invention also includes methods to detect Bartonella species in a test sample, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus-specific primers; and b.) detecting Bartonella species in the test sample in the event a Bartonella-sized amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s), (e.g. size-differentiating chromatography) is within the scope of the present invention.

For instance, the above method can be used to identify both the specific presence, or the specific absence of a certain species of Bartonella. As an example, the present method could be used to test a sample using a primer set (for instance, one forward sequence, one reverse sequence, in amounts necessary to conduct PCR) designed to amplify, both B. bacilliformis and B. quintana, although the size of the amplicons would differ. In that instance, it is possible that the primers would amplify a fragment unique for B. quintana, and not B. bacilliformis. The result would indicate the presence of 5. quintana as well as the absence of B. bacilliformis. In another example, the present method could be used to test a sample using a primer set designed to amplify uniquely-sized amplicons from each and every known Bartonella species. Amplicons resulting from use of the genus-specific primer set would identify, by their size or absence, the species of Bartonella present and/or absent in the sample. For instance, if 5. elizabethae and B. henselae-sizεd amplicons were present and B. quintana, B. bacilliformis and B. clarridgeiae-sϊzed amplicons were absent, then the result would indicate the presence of B. elizabethae and B. henselae and the absence of B. quintana, B. bacilliformis and B. clarridgeiae. In fact, methods as described, wherein the primers are capable of amplifying uniquely-sized amplicons for every Bartonella species is a preferred embodiment of the present invention. However, methods wherein the primers are capable of amplifying uniquely-sized amplicons from

B. henselae and B. clarridgeiae (the species which have been associated with cat scratch disease) are also preferred.

Moreover, the present invention is not limited to the use of only one set of genus-specific or Bartonella genus-specific primers. The methods herein also include those wherein a second set of primers is used, for example, for nested PCR. However, methods wherein PCR is conducted using one set of genus-specific or Bartonella genus- specific primers is preferred.

Methods which utilize primers designed using conserved sequences in or flanking the Bartonella 16S/23S intergenic region are within the scope of the present invention. The regions which span the consensus sequences in the Bartonella 16S/23S intergenic sequences from nucleotides 1-100, 130 -150 and 300-350 (nucleotide numbers for B. henselae, Genbank Accession Number L35101) are particularly useful for designing forward primers for the present methods. A preferred region for designing forward primers for the present invention is the region spanning bases 351 through 402. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences.

The regions which span the consensus sequences in the Bartonella 16S/23S intergenic sequences from nucleotides 430-530, 860-940 and 1000-1035 (nucleotide numbers for B. henselae, Genbank Accession Number L35101) are particularly useful for designing reverse primers for the present methods. A preferred region for designing reverse primers for the present invention is the region spanning bases 552 through 652.

Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences.

Methods as above wherein the Bartonella genus-specific forward primer is selected from the group consisting of: SEQ ID NO 5; SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 1 1 ; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22 are preferred.

More preferred are SEQ ID NO 5; SEQ ID NO 11 ; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22 . Methods as described in the previous paragraph wherein the Bartonella genus-specific reverse primer is selected from the group consisting of: SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 19; and SEQ ID NO 21 are preferred. SEQ ID NO 6; SEQ ID NO 10; SEQ ID NO 13;

SEQ ID NO 19; and SEQ ID NO 21 are more preferred. The most preferred forward primer for use in a diagnostic assay is SEQ ID NO 20, and the most preferred reverse primer for use in a diagnostic assay is SEQ ID NO 19.

Also provided in the present invention are methods to detect Bartonella-caxised disease in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus- specific primers; and b.) detecting Bartonella-caused disease in the test sample in the event a Bαrtonellα-sized amplicon is present. A method as in this paragraph, wherein the Bαrtonellα-caused disease is bacilliary angiomatosis or cat scratch disease is preferred.

Specifically the present invention also provides methods to detect cat-scratch disease in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus- specific primers capable of amplifying B. henselae and/or B. clarridgeiae nucleic acid; and b.) detecting cat-scratch disease in the test sample in the event a B. henselae- and/or B. clarridgeiae-sized amplicon is present.

Despite the focus of the preceding paragraphs on the ability of the present invention to distinguish products from Bartonella species, the present invention is not limited thereto. The general concept of using intergenic sequences (or other variable regions) to conduct PCR so as to generate PCR products of distinguishable and distinguishing size is within the scope of the present invention. For example, the intergenic sequences, of certain Mycobacterium species are known to be variable, and primers common to the intergenic sequences of these organisms (and which would result in size-distinguishing products) would eliminate the extra step of having to conduct restriction enzyme digests on the PCR products. Moreover, even for those organisms for which an acceptable assay exists, the present invention is useful in that it easy and convenient to conduct.

Intergenic sequences of organisms are generally available through journal publications, or through Genbank or NIH blast database. The most used database can be found at http://www.ncbi.nlm.nih.gov/. A search for intergenic sequences would typically include searching on either a known sequence or the name of the organisms to be distinguished.

The primers for the above assay can be designed using the 16S/23S intergenic sequence from B. henselae (Genbank accession number L35101); B. bacilliformis (Genbank accession number L26364); B. quintana (Genbank accession number L35100); B. vinsonii sub vinsonii (Genbank accession number L35102); B. elizabethae

(Genbank accession number L35103) and the sequence information herein provided. Moreover, it is known in the art that primers are preferrably G-C rich, ideally more than 50% of the bases being G or C. The length of the primer is usually chosen to minimize the chances of amplifying non-target nucleic acid, as well as minimize self- hybridization. Primers are typically 17 to 30 bases in length, although there are no absolute rules with regard to length or G-C content. For the purposes of the present invention, other parameters may take precedent over the length or constitution of the primers. Certain computer programs (such as MacVector) are helpful in primer design and PCR condition optimization. The assays described herein comprise both a PCR step and an amplicon size- determination step. PCR can be conducted according to techniques known to those of skill in the art, including for example, thermocycle PCR and isothermal PCR. A number of printed publications describe these procedures. For instance Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989); Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates, Inc., 1993); and Walker et al, 89 Proc Natl Acad Sci USA 392 (1992) describe typical parameters. Moreover, journal articles by investigators studying the organisms of interest will typically contain details about PCR amplification of the organisms' nucleic acid.

For example, thermocycle PCR can be conducted as follows: a sample is taken for amplification. Then, a thermocycler is used (at alternatingly high and low temperatures) to promote a.) dissociation of double stranded nucleic acid; and b.) hybridization of the primers to any sample nucleic acid and c.) subsequent synthesis of complementary nucleic acid. When the primers are bound to a nucleic acid in the test sample, the polymerase synthesizes a nucleic acid complementary to the sample nucleic acid, and when the primers are not bound, no synthesis takes place. A suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition. A bodily fluid refers to any fluid that can be collected (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milk and feces.

A second step in the described methods of the present invention is a size- determination of the PCR products generated. Size determination can be carried out according to any acceptable method, with gel electrophoresis being preferred. Methods for determining size of PCR products are described in Sambrook, supra and Ausubel, supra. Use of a control (identity known) sample or a sizing ladder is particularly helpful as well.

The present invention also includes kits useful for distinguishing between or among species of the same genus, comprising at least one set of genus-specific primers, said primers being capable of amplifying uniquely-sized fragments from at least a portion of an intergenic region of at least two species of said genus. The present kits preferably further comprise a gel material, such as, but not limited to, agarose or acrylamide.

Specifically, the present invention includes kits useful for distinguishing between or among Bartonella species, comprising at least one set of Bartonella genus- specific primers. The present kits preferably further comprise a gel material, such as, but not limited to, agarose or acrylamide.

Nucleic acid compounds are also provided by the present invention. Specifically, compositions of matter comprising forward and reverse Bartonella genus-specific primers as described herein are included in the present invention. Particular forward Bartonella genus-specific primers selected from the group consisting of: SEQ ID NO 5;

SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 11 ; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22 are included, with SEQ ID NO 5; SEQ ID NO 11; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22 being preferred. More preferred is SEQ ID NO 20. Particular reverse Bartonella genus-specific primers selected from the group consisting of: SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 13;

SEQ ID NO 15; SEQ ID NO 19; and SEQ ID NO 21 are also included, with SEQ ID NO 6; SEQ ID NO 10; SEQ ID NO 13; SEQ ID NO 19; and SEQ ID NO 21 being preferred. More preferred is SEQ ID NO 19.

The sequences described in the sequence listing can be shortened from the 5' end, provided that the resulting sequence does not result in loss of specificity when the shortened sequence is used as a primer. Those shortened primers are also useful as a part of a genus-specific primer set. For example, those primers wherein the 5' terminus is shortened by 1-10 bases are also within the scope of the present invention. Primers wherein the 5' terminus is shortened by 1-8 bases are preferred. Primers which are 14 bases in length and include at least one differentiating codon are most preferred. Any of these sequences can be used as primers in the methods described.

Also, with regard to the nucleic acid compounds herein provided, are isolated nucleic acid compounds comprising a 16S/23S intergenic sequence of Bartonella clarridgeiae, or a fragment thereof. Preferred nucleic acid compounds comprise SEQ ID NO 1, or a fragment thereof, and the antisense compound thereof, which is SEQ ID

NO 2, or a fragment thereof. Moreover, vectors and cells comprising SEQ ID NO 1 and SEQ ID NO 2 or fragments thereof are also provided.

Isolated nucleic acid compounds comprising a 16S/23S intergenic sequence of Bartonella vinsonii, subspecies berkhoffii, or fragments thereof are also specifically provided. Preferred nucleic acid compounds comprise SEQ ID NO 3, or a fragment thereof, and the antisense compound thereof, which is SEQ ID NO 4, or a fragment thereof. Moreover, vectors and cells comprising SEQ ID NO 3 and SEQ ID NO 4 or fragments thereof are also provided.

A "fragment" as used herein, is a nucleic acid molecule which is subset of the referent compound, and which is at least 12 bases in length. Preferably the fragments herein are at least about 20, 30, 40 or 50 or 100 bases in length.

The vectors of the present invention can be any vector, including those derived from prokaryotes, eukaryotes or viruses. The vectors can be synthetic or hybrids of any of the materials described. The regulatory regions can be any promoter, terminator, enhancer or other regulatory signal or sequence. Cells of the present invention can be any cells, including bacterial, fungal and/or eukaryotic cells.

Included within the scope of the present invention, with particular regard to the nucleic acids above, are allelic variants, degenerate sequences and homologues. By "nucleic acid(s)" is meant any allelic variant, hybrids, and fragments thereof. "Allelic variant" is meant to refer to a sequence that occurs at essentially the same locus (or loci) as ther referent sequence (eg. SEQ ID NOs 1 through 4), but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants are well known to those skilled in the art and would be expected to be found within intergenic sequences. The present invention also includes variants due to laboratory manipulation, such as, but not limited to, variants produced during polymerase chain reaction amplification or site directed mutagenesis.

A nucleic acid sequence homologous to a nucleic acid herein is characterized by the ability to hybridize to the exemplified nucleic acid compounds (or allelic variants or degenerates thereof) under stringent conditions. Stringent hybridization conditions are described in Sambrook, et al, Molecular Cloning: A Laboratory Manual, at 9.47-9.51 (Cold Spring Harbor Laboratory Press, 1989).

In another embodiment of the present invention, there are provided methods to detect Bartonella clarridgeiae in a test sample, comprising: conducting polymerase chain reaction using starting materials which comprise genus- or species-specific primers constructed from SEQ ID NO 1 and/or SEQ ID NO 2, under conditions which allow production of an amplicon in the event that Bartonella clarridgeiae is present in the test sample; and detecting Bartonella clarridgeiae in the event that an amplicon is present.

Additionally, there are provided methods to detect Bartonella vinsonii, subspecies berkhoffii in a test sample, comprising: conducting polymerase chain reaction using starting materials which comprise genus- or species-specific primers constructed from SEQ ID NO 3 and/or SEQ ED NO 4, under conditions which allow production of an amplicon in the event that Bartonella vinsonii, subspecies berkhoffii is present in the test sample; and detecting Bartonella vinsonii, subspecies berkhoffii in the event that an amplicon is present.

Lastly, the present invention includes methods to design a set of primers capable of amplifying a uniquely-sized fragment from at least two species of a genus, comprising: identifying genomic fragments from at least two species of the same genus, said genomic fragments having differences in absolute size, and said genomic fragments being defined at least on the periphery by alignable conserved sequences; and identifying a forward and reverse sequence on the periphery which are common to each of the species for which primers are desired.

Examples Example 1 Comparison of 16S-23S rRNA intergenic sequences of Bartonella species.

The 16S-23S rRNA intergenic sequences for B. bacilliformis (Genbank accession #L26364, 25), B. elizabethae (#L35103, 30), B. henselae (#L35101, 30), B. quintana (#L35100, 30), and B. vinsonii (baker strain) (#L35102, 30) were aligned using the DNA analysis computer program, DNAsis (Hitachi Software Engineering America Ltd., South San Francisco, CA). Figure 1 illustrates alignment of approximately 200 nucleotides in the 5' region of the 16S-23S intergenic sequences. In this region, a non- conserved area is bordered by two areas of high homology. Individual Bartonella species differ in the non-conserved region primarily due to sequence insertions and/or deletions. The extent of variation suggested that PCR primers designed to amplify across the non-conserved region would generate amplified products of different sizes for each species of Bartonella. A PCR assay was designed to amplify the region shown in

Figure 1. Template DNA was obtained from B. bacilliformis, B. clarridgeiae, B. elizabethae, B. henselae, B. quintana, and B. vinsonii (subspecies berkhoffii). B. koehlerae was not available for analysis at the time this work was performed. Further analysis of 5. vinsonii (baker strain) was not included because this Bartonella species has not been associated with disease in either humans or domestic animals. Template

DNA was amplified using 5'-(C/T)CTTCGTTTCTCTTTCTTCA-3' (B. henselae nts 302-321, SEQ ID NO 14) and 5'-GGATAAACCGGAAAACCTTC-3' (B. henselae nts 448-429, SEQ ID NO 7)) as forward and reverse primers, respectively. The 16S-23S rRNA intergenic sequences predict that these primers should amplify products of 186 bp (B. bacilliformis), 216 bp (B. elizabethae), 147 bp (B. henselae), and 132 bp (B. quintana). A predicted product size could not be determined for B. clarridgeiae or B. vinsonii (subspecies berkhoffii) because sequence of the 16S-23S rRNA intergenic region for these Bartonella species has not been reported.

Example 2 Materials used in PCR Bacterial strains. B. bacilliformis (ATCC #35685), B. clarridgeiae (ATCC

#51734 and #700095), B. elizabethae (ATCC #49927), B. quintana (ATCC #51694), B. vinsonii (subspecies berkhoffii) (ATCC #51572) were obtained from the American Type Culture Collection (Rockville, MD). B. henselae isolates; Houston-1 (ATCC #49882), Oklahoma (ATCC # 49793), Marseilles, MO-2, SA-1, CA-4, Tiger-2, and Lassiter were kindly provided by Russell Regnery, Viral and Rickettsial Diseases Branch, Centers for

Disease Control and Prevention, Atlanta, GA.

Clinical samples. Blood was obtained using aseptic procedures from the jugular vein of cats or dogs and placed in EDTA anti-coagulant tubes. Molecular characterization of B. henselae, B. clarridgeiae, and 5. vinsonii (subspecies berkhoffii) isolates from these naturally-infected cats and dogs has been previously reported.

Example 3 DNA extraction and PCR amplification of the 16S- 23S rRNA intergenic region. DNA for PCR amplification was prepared from pure cultures of each bacterial strain using the QIAamp DNA Mini Kit (QIAGEN Inc., Valencia, CA) and from blood using the QIAamp DNA Blood Mini Kit. PCR amplifications were performed in 50 1 containing 10 mM Tris, pH 8.3, 50 mM KC1, 3.5 mM MgCl₂, 200 M each dATP, dCTP, and dGTP, 400 μM dUTP, 1 M each primer, and 2.5 units Amplitaq Gold DNA polymerase (PE Applied Biosystems, Foster City, CA). Amplification buffer was optimized with dUTP for use with Uracil glycosylase to prevent PCR amplification product carryover. Optimum primer annealing temperatures were determined in a RoboCycler® Gradient Temperature Cycler (Stratagene, La Jolla, CA). Amplifications were performed in a GeneAmp PCR System 9700 thermal cycler (PE Applied Biosystems) using a time-release PCR protocol (13) as follows; 10 minute incubation at

20C followed by 2 minutes denaturation at 95 °C then 45 cycles of 1 minute denaturation at 95 °C, 1 minute annealing at 60°C, and 30 second extension at 72C. PCR amplification products were identified by ethidium bromide fluorescence after electrophoresis in 3% agarose gels.

Example 4 Identifying Organisms Present

After amplification according to Example 3, PCR products were electrophoresed on a 3% agarose gel, stained with ethidium bromide and photographed. As shown in Figure 2, the expected product size was amplified from B. bacilliformis, B. elizabethae, B. henselae, and B. quintana template DNA. The template DNA from B. vinsonii (subspecies berkhoffii) yielded a PCR product of approximately 235 bp rather than the

172 bp product predicted from the B. vinsonii (baker strain) sequence. PCR amplification of the B. clarridgeiae template DNA yielded no product using these primers.

To detect and differentiate medically relevant Bartonella species, new primers complementary to 16S-23S rRNA intergenic region sequences shared by all of the

Bartonella species were selected for PCR amplification (Figure 4). Amplification of template DNA using 5'-(C/T)CTTCGTTTCTCTTTCTTCA-3' (B. henselae nts 302- 321, SEQ ID NO 14) and 5'-AACCAACTGAGCTACAAGCC-3' (B. henselae nts 473- 454, SEQ ID NO 19) as forward and reverse primers, respectively, resulted in amplified products corresponding to the predicted size, namely, 211 bp (B. bacilliformis), 154 bp

(B. clarridgeiae), 241 bp (B. elizabethae), 172 bp (B. henselae), 157 bp (B. quintana), and 260 bp (B. vinsonii subspecies berkhoffii) (Figure 5). Amplification of template DNA derived from the CA-4, MO-2, SA-1 , Houston, Lassiter, Marseilles, Oklahoma, and Tiger-2 isolates of 5. henselae yielded the same size amplification product, demonstrating conservation of this target region amongst different isolates of B. henselae (data not shown). Amplification of template DNA derived from Clostridium perfringens, Enterobacter cloacae, Escherichia coli, Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia equi, Ehrlichia ewingii, Ehrlichia risticii, Fusobacterium necrophorum, Klebsiella pneumoniae, Salmonella choleraesuis, and Staphylococcus intermedius did not result in product amplification (data not shown).

Example 5 Sequencing of the 16S-23S rRNA intergenic region for B. clarridgeiae and B. vinsonii (subspecies berkhoffii).

PCR amplification of the entire 16S-23S rRNA intergenic region was accomplished using primers described by Matar et al, 31(7) J. Clin. Microb. 1730

(1993) and Roux and Raoult, 33(6) J. Clin. Microb. 1573 (1995). PCR products amplified from the 16S-23S rRNA intergenic regions of 5. clarridgeiae and 5. vinsonii (subspecies berkhoffii) were sequenced using an ABI PRISM™ Model 377 with XL upgrade DNA Sequencer (PE Applied Biosystems) after product labeling using the PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied

Biosystems) following the manufacturer's protocol. Sequence alignments and phylogenetic comparisons were done with the DNA analysis computer program, DNAsis (Hitachi Software Engineering America Ltd., South San Francisco, CA).

To investigate the failure to amplify product from B. clarridgeiae and the discrepancy between the amplification product from B. vinsonii (subspecies berkhoffii) and the size predicted from the B. vinsonii (baker strain) sequence, the 16S-23S rRNA intergenic regions from B. clarridgeiae and B. vinsonii (subspecies berkhoffii) were sequenced (Genbank #'s AF167989 and AF167988, respectively). The 16S-23S rRNA intergenic sequence of 5. clarridgeiae and B. vinsonii (subspecies berkhoffii) were compared with the reported sequences of B. bacilliformis, B. elizabethae, B. henselae, B. quintana, and B. vinsonii (baker strain) (Figure 3). As expected, B. vinsonii (subspecies berkhoffii) is most closely related to B. vinsonii (baker strain). Alignment of the B. vinsonii (subspecies berkhoffii) 16S-23S rRNA intergenic region revealed 63 bp inserted in the target region relative to the B. vinsonii (baker strain) sequence (Figure 4). Based on 16S-23S rRNA intergenic region sequences, B. clarridgeiae is most closely related to B. bacilliformis (Figure 3). Analysis of the B. clarridgeiae 16S-23S rRNA intergenic region sequence revealed that the 3' nucleotide of the reverse PCR primer sequence is not conserved with other Bartonella species, thus explaining the inability to amplify a

PCR product (Figure 4). The 16S-23S rRNA intergenic sequences for B. clarridgeiae and B. vinsonii (subspecies berkhoffii), have been submitted to GenBank under accession no. AF167989 and AF167988, respectively

Example 6. PCR detection in clinical samples.

To evaluate the utility of this assay for detection of Bartonella species in clinical samples, DNA was prepared from blood of animals known to be infected with either B. henselae, B. clarridgeiae, ov B. vinsonii (subspecies berkhoffii). Briefly, DNA was extracted from 200 1 of blood using the QIAamp® Blood Kit (QIAGEN Inc., Santa Clarita, CA) and eluted in a final volume of 200 1 per the manufacturer's protocol.

Samples (5 1 of template DNA) were amplified using the primers described above. After amplification, PCR products were electrophoresed on a 3% agarose gel, stained with ethidium bromide and photographed. As illustrated in Figure 6, the single-step PCR assay is capable of detecting and differentiating infections with B. henselae, B. clarridgeiae, and B. vinsonii (subspecies berkhoffii) in clinical samples derived from naturally-infected animals.

Example 7. Sensitivity of PCR versus blood culture for detection of B. henselae.

To determine the sensitivity of the single-step PCR assay relative to blood culture we purified template DNA from 200 1 blood containing 10 to 100 CFU/ml of 5. henselae derived from experimentally-infected cats. DNA was eluted in 200 1 buffer and 5 1 was used as template in the single-step PCR assay as described above. As shown in Table 1, the single-step PCR assay detected B. henselae in 100% of blood samples with 50-100 CFU/ml, 85% of blood samples with 30 CFU/ml, 75% of blood samples with 20 CFU/ml, and 75% of blood samples with 10 CFU/ml.

Table 1. Sensitivity of single-step PCR assay

CFU's Single-step PCR assay

50-100 12/12 (100%) 30 6/7 (85%)

20 3/4 (75%)

10 3/4 (75%)

Haemobartonella PCR Methods and Materials

This application claims priority to U.S. Provisional Patent Application Serial

Number 60/100,987, filed September 18, 1998.

BACKGROUND OF THE INVENTION The present invention is concerned with speciation of organisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish between or among species have not always met the expectations of consumers because they are either too costly, cumbersome or unavailable.

Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among species. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. One assay uses several sets of species-specific primers instead of digestion with restriction enzymes, with identification of the PCR products made by amplicon size. Minnick and Barbian, 31 J Microb Meth 51 (1997).

Haemobartonella felis, which causes infectious feline anemia, has two known subspecies: the California subspecies and the Ohio/Florida (herein called "Ohio") subspecies. Other organisms also cause anemia (eg. Bartonella and Ehrlichia), but treatment of the anemia is ideally directed to the causative organism. PCR technology has been used to detect Haemobartonella felis, although distinguishing between anemia- causing subspecies has not been accomplished.

In Rikihisa et al, 35 (4) J. Clin. Microb. 823 (1997), there is disclosed the use of portions of conserved 16S sequences as primers in order to sequence the 16S genes of H. felis California and H. felis Ohio, and evolutionarily compare them to each other as well as to other organisms. It also discloses a method for distinguishing H. felis strains from one another, which method requires restriction enzyme cleavage and gel electrophoresis. In Messick et al, 36 (2) J. Clin. Microb. 462 (1998), there are disclosed primers useful to identify (selectively) H. felis Ohio. It does not identify an assay for distinguishing H. felis Ohio and H. felis California or other organisms from H. felis Ohio.

In another organism, Bartonella, PCR assays have been discussed which use differences in citrate synthase sequences. These assays use a first step of conducting PCR and a second step of digesting the PCR products with restriction enzymes to distinguish among species. Joblet et al, 33(7) J. Clin. Microb. 1879 (1995); Norman et al, 33(7) J. Clin. Microb. 1797 (1995). PCR assays on the basis of differences in 16S rRNA sequences in Bartonella have also been conducted, using restriction enzymes to distinguish among species. Birtles, 129 FEMS Microbiol Letters 261 (1995).

Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.

SUMMARY OF THE INVENTION

The present invention requires only a single step to generate amplicons which identify a specific species.

It is therefore an object to provide a simplified assay for distinguishing between or among Haemobartonella species. It is yet another object to provide materials related to the methods disclosed, including primer sets.

In all of the above embodiments, it is an object to provide methods to diagnose disease using the materials and methods provided.

It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the species-specific size differences in the 16S region of

Haemobartonella.

Finally, it is an object of the invention to provide a kit for convenient use of the materials and methods herein provided. Defmitions: For the purposes of the present invention, the following terms shall have the following definitions:

"Amplicon(s)" shall mean a nucleic acid produced through use of primers in PCR.

"Genus-specific primer(s)" shall mean primers being capable of amplifying an amplicon from at least a portion of the 16S region of at least two Haemobartonella species, and no other genera, and wherein the size of the amplicon is unique to the species.

When the term "Genus-specific primer(s)" is used to describe primers used in PCR assays, it is assumed that said primers are also being in amounts sufficient to amplify at least one ascertainable fragment.

A "set" of primers means at least one forward and at least one reverse primer, that when used in a PCR assay in appropriate amounts and in the presence of amplifiable nucleic acid, is capable of amplifying nucleic acid.

"Species" means any species or subspecies, or other subset of species or subspecies.

DETAILED DESCRIPTION OF THE INVENTION

In broad terms, the present invention includes materials and methods useful to distinguish between and among species of a genus. The methods simplify and are therefore more cost-effective than previous methods. In addition, because the present methods are simpler than previous methods, the risk of operator error, contamination, or any other technical problem is reduced, making the present invention inherently more reliable than previous methods.

The present invention also includes methods to detect Haemobartonella species in a test sample, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Haemobartonella species in the test sample in the event a Haemobartonella-sized amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s) (e.g. size-differentiating chromatography) is within the scope of the present invention.

For instance, the above method can be used to identify both the specific presence, or the specific absence of a certain species of Haemobartonella. As an example, the present method could be used to test a sample using a primer set (one forward sequence, one reverse sequence, in amounts necessary to conduct PCR) designed to amplify, both H felis Ohio and H felis California, although the size of the amplicons would differ. In that instance, it is possible that the primers would amplify an amplicon unique for H. felis Ohio, and not H. felis California. The result would indicate the presence of H. felis Ohio as well as the absence of H. felis California. In fact, methods as described, wherein the primers are capable of amplifying uniquely-sized amplicons from H. felis Ohio and H. felis California is a preferred embodiment of the present invention. However, methods wherein the primers are capable of amplifying uniquely-sized amplicons for every Haemobartonella species are also preferred.

Moreover, the present invention is not limited to the use of only one set of genus-specific primers. The methods herein also include those wherein a second set of primers is used, for example, for nested PCR. However, methods wherein PCR is conducted using one set of genus-specific primers is preferred.

Methods which utilize primers designed using conserved sequences in or flanking the Haemobartonella 16S region are within the scope of the present invention. A preferred region for designing forward primers for the present invention is the region spanning nucleotides 175-425. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences. A preferred region for designing reverse primers for the present invention is the region spanning nucleotides 455-700. Not all bases are identical in these regions, but those in the art are aware of primer design strategy in light of non-identical sequences. Methods as above wherein the genus-specific forward primer comprises a sequence selected from the group consisting of SEQ ID NO 23 and SEQ ID NO 25 are most preferred. Methods as described in the previous paragraph wherein the Haemobartonella genus-specific reverse primer comprises a sequence selected from the group consisting of SEQ ID NO 24 and SEQ ID NO 26 are most preferred. Also provided in the present invention are methods to detect Haemobartonella- caused disease in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus- specific primers; and b.) detecting Haemobartonella-caused disease in the test sample in the event a Haemobartonella-sϊzed amplicon is present. A method as in this paragraph, wherein the Haemobartonella-caused disease is anemia is preferred.

Specifically the present invention also provides methods to detect anemia in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise at least one set of genus-specific primers capable of amplifying H. felis Ohio and H felis California nucleic acid, and a test sample; and b.) detecting feline infectious anemia in the test sample in the event a H. felis Ohio or a H. felis California- sized amplicon is present.

The genus-specific primers for the above assay can be designed using the H. felis Ohio (Genbank Accession Number 95297) and H. felis California (Genbank Accession Number 88564). The assays described herein comprise both a PCR step and an amplicon size- determination step. PCR can be conducted according to techniques known to one of skill in the art, including, for example, thermocycle PCR and isothermal PCR. A number of printed publications describe these procedures. For instance Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989), Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates,

Inc., 1993) and Walker et al, 89 Proc Natl Acad Sci USA 392 (1992) describe typical parameters. Moreover, journal articles by investigators studying the organisms of interest will typically contain details about PCR amplification of the organisms' nucleic acid. For example, thermocycle PCR is conducted as follows: a sample is taken for amplification. Then, a thermocycler is used (at alteraatingly high and low temperatures) to promote a cycle between a.) dissociation of double stranded nucleic acid; and b.) hybridization of the primers to any sample nucleic acid; and c.) subsequent synthesis of complementary nucleic acid. When the primers are bound to a nucleic acid in the test sample, the polymerase synthesizes a nucleic acid complementary to the sample nucleic acid, and when the primers are not bound, no synthesis takes place. A suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition. A bodily fluid refers to any fluid that can be collected (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milk and feces.

The second step in the described methods of the present invention is a size- determination of the PCR products generated. Size determination can be carried out according to any acceptable method, with gel electrophoresis being preferred. Methods for determining size of PCR products are described in Sambrook, supra and Ausubel, supra. Use of a control (identity known) sample or a sizing ladder is particularly helpful as well.

The primers of the present invention can be designed by aligning 16S regions from at least two Haemobartonella species and identifying primers which would amplify an amplicon having differences in absolute size as well as capable of priming polymerase chain reaction. Moreover, it is known in the art that primers are preferrably G-C rich, ideally more than 50% of the bases G or C. The length of the primer is usually chosen to minimize the chances of amplifying non-target nucleic acid, as well as minimize self-hybridization. Primers are typically 17 to 30 bases in length, although there are no absolute rules with regard to length or G-C content. For the purposes of the present invention, other parameters may take precedent over the length or constitution of the primers. Certain computer programs (such as Mac Vector) are helpful in primer design and PCR condition optimization.

The present invention includes kits useful for distinguishing between or among Haemobartonella species, comprising at least one set of genus-specific primers. The present kits preferably further comprise a gel material, such as, but not limited to, agarose or acrylamide.

Nucleic acid compounds are also provided by the present invention. Specifically, compositions of matter comprising a set of genus-specific primers as described herein are included in the present invention. A particular forward Haemobartonella genus-specific primers comprising a sequence selected from the group consisting of SEQ ID NO 23 and SEQ ID NO 25 are preferred. Particular reverse Haemobartonella genus-specific primers comprising a sequence selected from the group consisting of SEQ ID NO 24 and SEQ ID NO 26 are also preferred.

The sequences described in the sequence listing can be shortened from the 5' end, provided that the resulting sequence does not result in loss of specificity when the shortened sequence is used as a primer. Those shortened primers are also useful as a part of a genus-specific primer set. For example, those primers wherein the 5' terminus of SEQ ID NO 23 or SEQ ID NO 24 is shortened by 1-10 bases are also within the scope of the present invention. Primers wherein the 5' terminus of SEQ ID NO 23 or SEQ ID NO 24 is shortened by 1-8 bases are preferred. SEQ ID NO 25 and SEQ ID NO 26 are most preferred. Any of these sequences can be used as primers in the methods described.

Examples Example 1 Identification of Suitable Primers

The 16S gene sequences for H. felis Ohio, H felis California, B. henselae, B. clarridgeae, M. felis, H. muris, E. coli, S. enteritis, K. pneumoniae and M. muris were aligned to identify regions of homology. The goals of primer selection were to identify primers which would 1) be specific for H. felis (i.e. would not amplify product from other cat pathogens), 2) amplify product from both known isolates of H. felis (subspecies

Ohio and California), and 3) be able to differentiate between the two known isolates of H. felis.

The region in the 16S gene identified as a candidate amplicon contained an approximately 25 base pair deletion in the H. felis, subspecies H. felis Ohio, sequence as compared to the subspecies California sequence. This deletion is the basis for differentiating between H. felis subspecies Ohio and California. A forward primer, 5'-

(ACGAAAGTCTGATGGAGCAATA-3' (nucleotide numbers 363-384 H. felis, subspecies Ohio, Genbank #95297) (SEQ ID NO 23), and a reverse primer, 5'- ACGCCCAATAAATCCG(A/G)ATAAT-3' (nucleotide numbers 532-51 1 H. felis, subspecies Ohio, Genbank #95297) (SEQ ID NO 24), were selected to specifically amplify H. felis DNA. The specificity is derived from the 3 ' end of each primer which is complementary to the 16S gene sequence for H. felis (both subspecies) and incompatible for annealing to the "other pathogen" 16S gene sequences. These primers were designed to amplify 170 and 193 bp products from H. felis subspecies Ohio and California, respectively. This difference in PCR product size allowed easy differentiation of the H. felis subspecies by agar gel electrophoresis.

Example 2 Conducting PCR

DNA was extracted from 200 μl of blood collected from H. e/.s-infected cats, or from relevant microorganisms obtained from the American Type Culture Collection (Rockville, MD), using the Qiagen QiaAmp Blood Kit (Qiagen, Valencia, CA). PCR reactions were run in 50 μl volumes in the Perkin Elmer GeneAmp 9700 Thermocycler (PE Applied Biosystems (PEAB), Foster City, CA). The PCR reaction (50 μl) contained 10 mM Tris-ΗCl, pΗ 8.3, 50 mM KC1, 3.5 mM MgCl₂, 400 μM dUTP, 200 μM dATP, 200 μM dCTP, and 200 μM dGTP, 2.5 units Taq polymerase (PE Amplitaq Gold, PEAB), 1 unit of Uracil DNA Glycosylase (PEAB), 1.0 μM "forward" (SEQ ID

NO 23) and "reverse" (SEQ ID NO 24) primers and 5 μl of Rediload (a commercial loading buffer, Research Genetics, Ηuntsville AL) and 5μl of template DNA. PCR reactions were optimized using dUTP for prevention of PCR product carryover contamination. Prior to amplification, samples were digested with Uracil DNA Glycosylate for 10 minutes at 20°C, followed by 2 minutes at 95 °C. The thermocycling profile was repeated for 45 cycles as follows: denaturation for 1 minute at 95 °C, annealing for 1 minute at 60°C, and extension for 30 seconds + 1 second/cycle at 72°C. Example 3 Identifying Organisms Present

PCR products were separated based on size by electrophoresis through 2.5% agarose containing 0.65 μg/ml ethidium bromide. Base-pair markers were used for size reference. Results were documented using the Bio-Rad Insta-Doc Gel Documentation System. The primers amplified 170 and 193 bp products from H. felis Ohio and H. felis

California, respectively. This difference in PCR product size allowed easy differentiation of the H. felis subspecies by agar gel electrophoresis.

Example 4 Prevalance of H. felis Polymerase chain reaction. Primers that detect a segment of the 16S rRNA gene common to both sequenced strains (California and Ohio) of Η. felis were utilized in either a nested PCR or timed-release PCR.

Study group. To solicit participation in this study, veterinarians in different regions of the country were contacted by phone and letter. Blood samples were collected from client-owned cats, placed into EDTA, and transported to the laboratory by overnight courier frozen on dry ice or on a cold pack for PCR testing. Samples from cats with suspected haemobartonellosis (suspect cats) were submitted based on presence of fever, anemia, or cytologic evidence of infection. Samples from cats without clinical suspicion of haemobartonellosis (control cats) were submitted as a cohort to a case with suspected haemobartonellosis or were selected from samples for which a complete blood cell count was performed at the laboratory for other reasons.

Complete blood cell count and cytologic assessment. Complete blood cell count (CBC) data was available for some cats. For samples from cats in Colorado, the CBC was performed at the laboratory and thin blood smears were examined for the presence of H. felis by one of the authors (WR). CBC information from other cats in the study was solicited from the referring veterinarian. Samples for which an entire CBC was present were assumed to have had cytologic examination for hemoparasites. For some samples, thin blood smears were submitted for cytologic examination of red blood cells, but not complete blood cell count. Due to variation in laboratories and reporting methods, only the packed cell volume (PCV) or hematocrit (ΗCT) and cytologic presence or absence of H. felis were assessed in this study. Samples with a PCV or ΗCT

> 25 were considered normal; samples with PCV or ΗCT < 25 were considered anemic. Statistical evaluation. Based on PCR results, cats were defined as Ηflg infected, Ηfsm infected, Ηflg and Ηfsm infected, or H. felis naive (both variants). Frequency distributions for each PCR based category was calculated for cats with and without anemia as well as for cats with suspected H. felis infection and control cats. Results were compared by chi square analysis with statistical significance defined as P < 0.05.

Results. A total of 220 blood samples were assessed in the study; 82 cats were suspected to have haemobartonellosis and 138 cats were included as controls. Of the samples, the majority came from cats that resided in Colorado. Based on PCR results, 10 cats (4.5%) were infected with Ηflg, 28 cats (12.7%) were infected with Ηfsm, and 5 cats (2.3%) were infected with Ηflg and Ηfsm for a overall H felis prevalence of 19.5%. Overall, cats with suspected haemobartonellosis (28.0%) were more likely (P = 0.0142) than control cats (14.5%) to be H felis infected (Table 1). Significantly greater numbers of cats suspected to have haemobartonellosis were Ηflg infected (P < 0.0005) or Ηflg and Ηfsm infected (P

= 0.0456) than control cats (Table 1). There was no difference in the prevalence of Ηfsm infection between the suspect and control groups. CBC were available for 156 cats; 28 cats were anemic and 128 cats were normal. Of the CBC, the majority were performed at the reference laboratory. Based on overall PCR results, a similar number (P = 0.1339) of anemic cats (28.6%) and healthy cats (16.4%) were infected with Η. felis (Table 2). However, significantly more anemic cats were Hflg infected (P = 0.0057) or Hflg and Hfsm infected (P = 0.0264) than normal cats (Table 2).

Comparisons of PCR results and red blood cell cytologic examination results are listed in Table 3. Only cats with suspected haemobartonellosis were positive on cytologic examination. Each of these 7 cats was concurrently PCR positive.

Conversely, there were 26 samples that were PCR positive but negative on cytologic examination. Table 1. Prevalence of Haemobartonella felis infections in cats

Suspect (n = 82) Control (n = 138)

PCR result # positive (%) # positive (%) Chi square P value

OH + 10(12.2) 0(0) 17.631 <0.0001*

CA + 9(11.0) 19(13.8) 0.361 0.5479

OH/CA + 4 (4.9) 1 (0.7) 3.995 0.0456*

Total 23 (28.0) 20(14.5) 6.011 0.0142*

*StatisticalIy significant

Table 2. Distribution Haemobartonella. felis PCR results in cats with and without anemia

Anemia (n = 28) Normal (n = 128)

PCR result # positive (%) # positive (%) Chi square P value

OH + 4(14.3) 3 (2.3) 7.645 0.0057*

CA + 2(7.1) 17(13.3) 0.809 0.3683

OH/CA + 2(7.1) 1 (0.8) 4.93 0.0264*

Any OH + 6(21.4) 4(3.1) 12.83 0.0003*

Any CA + 4(14.3) 18(14.1) 0.001 0.9755

Total 8 (28.6) 21 (16.4) 2.247 0.1339

"Statistically significant Table 3. Comparison of PCR and cytologic examination of blood smears for diagnosis of haemobartonellosis.

Suspect (n = 53) Control (n = 112) Total (n= 165)

Result # positive (%) # positive (%) # positive . %)

Smear +, OH + 5 (9.4) 0(0) 5 (3.0)

Smear +, CA + 1 (1.9) 0(0) 1 (0.6)

Smear +, OH/CA + 1(1.9) 0(0) 1 (0.6)

Smear +, Any PCR + 7(13.2) 0(0) 7 (4.2)

Smear -, OH + 4(7.5) 0(0) 4 (2.4)

Smear -, CA + 3 (5.7) 16(14.3) 19(11.5)

Smear -, OH/CA + 2 (3.8) 1 (0.9) 3(1.8)

Smear -, Any PCR + 9(17.0) 17(15.2) 26(15.8)

Smear -, All PCR - 37 (69.8) 95 (84.8) 132(80.0)

Although the present invention has been fully described herein, it is to be noted that various changes and modifications are apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention.

Ehrlichia PCR Methods and Materials

BACKGROUND OF THE INVENTION

The present invention is concerned with distinguishing among genera of pathogenic canine microorganisms, for the purpose of improving differential diagnosis of disease. The assays currently available to distinguish among canine genera have not always met the expectations of consumers because they are either too costly or unavailable.

Polymerase chain reaction (PCR) and serological assays are currently used to distinguish among genera of canine pathogens. Serological tests present problems with cross-reactivity and available PCR tests are complicated. Typically, PCR-based assays require three steps: 1) conducting PCR using a primer set which distinguishes among members of different genera, but not among members of the same genus; 2) digesting the PCR products with restriction enzymes and 3) distinguishing among species on the basis of restriction digest patterns. The present invention reduces the steps to a total of two: 1) conducting PCR using at least one primer set which amplifies nucleic acids from all members of the Ehrlichia genus and which does not amplify nucleic acids from other common canine pathogens; and 2) identifying if an amplicon is present.

Ehrlichia species cause nonspecific febrile illnesses, usually associated with observable leukopenia. The organism is known to be tick-borne, and delayed treatment

(usually antibiotics) can result in death. Several species of Ehrlichia are known: E. sticii; E. sennetsu; E. canis; E. chafeensis; E. ewingii; E. platys; a yet-unnamed species which causes human granulocyte Ehrlichia (HGE); E. equi and E. phagocytophilia. A review of modes of transmission, pathology, serological diagnosis, and PCR diagnosis using species-specific primers from the 16S sequence is: Dawson et al, 156 Arch Intern Med 137 (1996).

In Warner and Dawson, Genus and Species-Level Identification of Ehrlichia Species by PCR and Sequencing, Protocol 2 from ASM Press, Washington D.C. (1996), the typical protocol for identifying Ehrlichia is described. It requires an initial "all bacteria" amplification, with a second set of species-specific primers for each of the

Ehrlichia species. The protocol requires two rounds of PCR.

In Dawson et al., Polymerase Chain Reaction Evidence of Ehrlichia chafeensis, an etiologic agent of human ehrlichiosis, in dogs from southeast Virginia, 57(8) Amer. J. Vet. Res. I ll 5 (1996), the above two-PCR method was used to determine geographical disctribution of Ehrlichia species.

In Dawson et al, 57(1) Am J Trop MedHyg 109 (1997), there is disclosed the use of repetitive element PCR (rep-PCR) to distinguish amongst Ehrlichia species. Rep-PCR uses relatively non-specific outward-facing primers to generate PCR products which reflect differences in distances between adjacent DNA repeats. Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.

SUMMARY OF THE INVENTION It is therefore an object to provide a simplified assay for all Ehrlichia species from other common canine pathogens.

It is yet another object to provide materials related to the methods disclosed, including primer sets.

In all of the above embodiments, it is an object to provide methods to diagnose disease using the materials and methods provided.

It is also an object to provide methods for identifying primers useful to conduct PCR assays which capitalize on the genus-specific size differences in the 16S region of Ehrlichia and other common canine pathogens.

Finally, it is an object of the invention to provide a kit for convenient use of the materials and methods herein provided.

Definitions: For the purposes of the present invention, the following terms shall have the following definitions: "Amplicon(s)" shall mean a nucleic acid produced through use of primers in PCR.

"Genus-specific primer(s)" shall mean primers being capable of amplifying amplicons from at least a portion of the 16S region of all Ehrlichia species, and not capable of amplifying amplicons from Clostridium perfringens,

Enterobacter cloacae, Fusobacterium necrophorum, Klebsiella pneumoniae, Salmonella chloleraesuis, Staphylococcus intermedius, Escherichia coli, Streptococcus sp, Hemobartonella felis (California (CA) and Ohio (OH)) or Bartonella henselae. When the term is used to describe primers used in PCR assays, it is assumed that said primers are also being in amounts sufficient to amplify at least one ascertainable fragment. A "set" of primers means at least one forward and at least one reverse primer that when used in a PCR assay in appropriate amounts and in the presence of amplifiable nucleic acid, is capable of amplifying nucleic acid.

"Species" means any species or subspecies, or other subset of species or subspecies.

DETAILED DESCRIPTION OF THE INVENTION In broad terms, the present invention includes materials and methods useful to distinguish among canine pathogenic genera. The methods simplify and are therefore more cost-effective than previous methods. In addition, because the present methods are simpler than previous methods, the risk of operator error, contamination, or any other technical problem is reduced, making the present invention inherently more reliable than previous methods.

The present invention includes methods to detect Ehrlichia species in a test sample, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Ehrlichia species in the test sample in the event an amplicon is present. A method as described, wherein step b.) comprises gel electrophoresis is preferred, although any method for detecting amplicon(s) is within the scope of the present invention.

For instance, the above method can be used to identify the presence or absence of Ehrlichia. Moreover, the present invention is not limited to the use of only one set of genus-specific primers. The methods herein also include those wherein a second set of primers is used, for nested PCR. The second set of primers can either be capable of amplifying uniquely-sized amplicons, or be simple species-specific primers. If desired, a second, species-specific primer set may also be utilized so as to confirm the existence or absence of a suspected species or subspecies.

Methods as above wherein the genus-specific primers are selected from the group of sets consisting of: SEQ ID NO 27 and SEQ ID NO 28; and SEQ ID NO 27 and SEQ ID NO 29 are most preferred.

Also provided in the present invention are methods to detect Ehrlichia-c&used disease in a mammal, comprising: a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and b.) detecting Ehrlichia-caused disease in the test sample in the event an amplicon is present. A method as in this paragraph, wherein the Ehrlichiα-caused disease is anemia is preferred.

The genus-specific primers for the above assay can be designed using the E. risticii (Genbank Accession Number M21290); E. sennetsu (Genbank Accession Number M73225); E. cαnis (Genbank Accession Number M73221); E. chαfeensis

(Genbank Accession Number M73222); E. ewingii (Genbank Accession Number M73227); E. plαtys (Genbank Accession Number M82801); E. equi (Genbank Accession Number M73223) (possible causative agent of a yet-unnamed species which causes human granulocyte Ehrlichia (HGE) from human (Genbank Accession Number U02521) and E. Phagocytophilia (Genbank Accession Number M73224).

The assays described herein comprise both a PCR step and an amplicon determination step. PCR can be conducted according to one of skill of the art. A number of printed publications describe these procedures. For instance Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al, Current Protocols in Molecular Biology (Greene Publishing Associates, Inc., 1993) describe typical parameters. Moreover, journal articles by investigators studying the organisms of interest will typically contain details about PCR amplification of the organisms' nucleic acid.

For example, thermocycle PCR is conducted as follows: a sample is taken for amplification. Then, a thermocycler is used (at altematingly high and low temperatures) to promote a cycle between a.) dissociation of double stranded nucleic acid; and b.) hybridization of the primers to any sample nucleic acid; and c.) subsequent synthesis of complementary nucleic acid. When the primers are bound to a nucleic acid in the test sample, the polymerase synthesizes a nucleic acid complementary to the sample nucleic acid, and when the primers are not bound, no synthesis takes place. A suitable biological sample includes, but is not limited to, a bodily fluid composition or a cellular composition. A bodily fluid refers to any fluid that can be collected (i.e., obtained) from an animal, examples of which include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, cerebrospinal fluid (CSF), saliva, lymph, nasal secretions, milk and feces. The primers of the present invention can be designed by aligning 16S regions of canine pathogens and identifying primers which are capable of both amplifying only Ehrlichia amplicon(s) as well as capable of priming polymerase chain reaction. Moreover, it is known in the art that primers are preferrably G-C rich, ideally more than 50% of the bases G or C. The length of the primer is usually chosen to minimize the chances of amplifying non-target nucleic acid, as well as minimize self-hybridization.

Primers are typically 17 to 30 bases in length, although there are no absolute rules with regard to length or G-C content. For the purposes of the present invention, other parameters may take precedent over the length or constitution of the primers. Certain computer programs (such as MacVector) are helpful in primer design and PCR condition optimization.

The present invention includes kits useful for distinguishing Ehrlichia from other canine pathogens, comprising at least one set of genus-specific primers. The present kits preferably further comprise a gel material, such as, but not limited to, agarose or acrylamide. Nucleic acid compounds are also provided by the present invention,

Specifically, compositions of matter comprising a set of genus-specific primers as described herein are included in the present invention. Particular sets of genus-specific primers selected from the sets consisting of: SEQ ID NO 27 and SEQ ID NO 28; and SEQ ID NO 27 and SEQ ID NO 29 are also included. Primers sets can be synthesized commercially by Gibco (on the internet at www.lifetech.com/). The sequences described in the sequence listing can be shortened from the 5' end, provided that the resulting sequence does not result in loss of specificity when the shortened sequence is used as a primer. Those shortened primers are also useful as a part of a genus-specific primer set. For example, those primers wherein the 5' terminus of SEQ ID NO 27, SEQ ID NO 28 or SEQ ID NO 29 is shortened by 1-10 bases are also within the scope of the present invention. Primers wherein the 5' terminus of SEQ ID NO 27 SEQ ID NO 28 or SEQ ID NO 29 is shortened by 1-8 bases are preferred. SEQ ID NO 30 and SEQ ID NO 31 are most preferred. Any of these sequences can be used as primers in the methods described.

Examples Example 1 Identification of Suitable Primers

Primers were designed based on consensus sequence in the 16S gene of the Ehrlichia genus. Primers were designed in such a way that they would amplify a fragment only from Ehrlichia spp. and not from other bacteria (Fig. 1). One forward primer 16S

Ehrlichia genus TRLS-F [5' CTG GGG ACT ACG GTC GCA AGA CT 3'] (SEQ ID NO 27) and two reverse primers 16S Ehrlichia genus TRLS-R#1 [5' CGA CAA CCA TGC AGC ACC TGT G 3'] (SEQ ID NO 28) and TRLS-R#2 (SEQ ID NO 29) were designed. Initial studies showed that TRLS-F and TRLS-R#2 [5' TTG ACG GTC ACT ATT TGA CCT C 3 '] were highly specific for Ehrlichia genus. All further experiments were carried out with TRLS-F and TRLS-R#2.

Example 2 Obtaining a Test Sample

DNA prepared from 5 Ehrlichia sp. were used for initial standardization of the assay. Canine clinical samples were obtained from Animal Diagnostic Laboratory (Phoenix, Arizona) and Dr. Michael Lappin (Colorado State University, Fort Collins, Colorado). DNA was extracted from bacteria using the Qiagen QiaAmp tissue kit. DNA from blood samples were extracted using the Qiagen QiaAmp blood kit. DNA was eluted 2 times in 100 μl of elution buffer (QIAamp Blood Kit, Qiagen, Inc.) to boost sensitivity of detection.

Example 3 Conducting PCR

PCR annealing temperature was determined by a temperature-gradient experiment (from 55°C to 65°C) conducted for all the 5 Ehrlichia species. The optimum temperature for annealing was found to be 60°C.

Thermocycle profile (Time-released

Pre-PCR (1 cycle) 20°C 20 min

95°C 10 min

PCR (45 cycles) Denaturation 95°C 1 min

Annealing 60°C 1 min

Extension 72°C 30 sees, with additional second per cycle

Post-PCR (1 cycle) 72°C 10 min

Soak 4°C

The PCR assay was standardized using 5 Ehrlichia sp. as templates (E. canis, E. ewingii, E. risticii, E. chaffeensis, E. equi). A 299-300 bp PCR product was amplified from each species. PCR products were sequenced and identified to be the respective species. The 300-bp amplified product from all 5 species were aligned to identify nucleotide differences in that region. There are unique nucleotide differences that will enable us to distinguish species if required.

For specificity evaluation, DNA from the following samples were tested using primers TRLS-F AND TRLS-R#2 in the PCR assay described above: Clostridium perfringens; Enterobacter cloacae; Fusobacterium necrophorum; Klebsiellapneumoniae; Salmonella choleraesuis; Staphylococcus intermedius; Escherichia coli; Streptococcus sp.;

Hemobartonella felis-CA; Hemobartonella felis-OH; Bartonella henselae.

None of these samples tested positive by the Ehrlichia PCR assay. Example 4 Identifying Organisms Present

Clinical samples from Arizona and Panama were tested by the Ehrlichia PCR Assay. 18 of 42 Arizona samples tested positive while only 2 of 19 Panama samples tested positive. Some of the Arizona samples that had high IFA titer tested negative in the PCR assay. However, preliminary statistical analysis of the results indicated a good correlation between PCR positivity and IFA titer (Fig. 2) All negative samples were tested by the traditional nested PCR assay and were found to be negative.

DNA templates from two positive clinical samples were diluted from 10^"' to 10^"6 before using in the PCR assay. PCR products could be detected from templates diluted up to 10^"3 times. PCR products from 3 Arizona and 2 Panama samples were sequenced and were identified to be E. canis.

Although the present invention has been fully described herein, it is to be noted that various changes and modifications are apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention.

WHAT IS CLAIMED IS (Part One):

1. A method to detect Bartonella species in a test sample, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus-specific primers; and

b.) detecting Bartonella species in the test sample in the event a Bartonella-sized amplicon is present.

2. A method of claim 1, wherein step b.) comprises gel electrophoresis.

3. A method of claim 1 , wherein the primers are capable of amplifying uniquely-sized fragments for every Bartonella species.

4. A method of claim 1, wherein the forward primer is designed from the region spanning from about base 300 through about base 350, and the reverse primer is designed from the region spanning from about base 430 through about base 530 of a Bartonella henselae 16S/23S intergenic region.

5. A method of claim 1, wherein the forward primers are selected from the group consisting of: SEQ ID NO 5; SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22.

6. A method of claim 1, wherein the reverse primers are selected from the group consisting of: SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 19; and SEQ ID NO 21.

7. A method of claim 2, wherein the primers are capable of amplifying uniquely-sized amplicons from B. henselae and B. clarridgeiae. 8. A method to detect Bartonella-caused disease in a mammal, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus-specific primers; and

b.) detecting Bartonella-caused disease in the test sample in the event a Bαrtonellα-sized amplicon is present.

9. A method of claim 8, wherein the Bαrtonellα-caused disease is bacilliary angiomatosis.

10. A method of claim 8, wherein the Bαrtonellα-caused disease is cat scratch disease.

11. A method to detect cat-scratch disease in a mammal, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of Bartonella genus-specific primers; and

b.) detecting cat-scratch disease in the test sample in the event an amplicon is present, which amplicon is the size appropriate for a species chosen from the group consisting of: B. henselae; and B. clarridgeiae.

12. A kit useful for distinguishing between or among Bartonella species, comprising at least one set of Bartonella genus-specific primers.

13. A kit of claim 12, which further comprises gel material. 14. A composition of matter comprising a set of Bartonella genus-specific primers.

15. A set of Bartonella genus-specific primers of claim 14, wherein the forward primers are selected from the group consisting of: SEQ ID NO 5; SEQ ID NO 8; SEQ ID NO 9; SEQ ID NO 11; SEQ ID NO 14; SEQ ID NO 20; and SEQ ID NO 22.

16. A set of Bartonella genus-specific primers of claim 14, wherein the reverse primers are selected from the group consisting of: SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 10; SEQ ID NO 12; SEQ ID NO 13; SEQ ID NO 15; SEQ ID NO 19; and SEQ ID NO 21.

17. An isolated nucleic acid compound comprising a 16S/23S intergenic sequence of Bartonella clarridgeiae, or a fragment thereof.

18. A nucleic acid compound of claim 17, wherein the nucleic acid compound is selected from the group consisting of: SEQ ID NO 1 ; and SEQ ID NO

2.

19. A vector comprising the nucleic acid compound of claim 17.

20. A cell comprising the nucleic acid compound of claim 17.

21. An isolated nucleic acid compound comprising a 16S/23S intergenic sequence of Bartonella vinsonii, subspecies berkhoffii.

22. A nucleic acid compound of claim 21 , wherein the nucleic acid compound is selected from the group consisting of: SEQ ID NO 3; and SEQ ID NO

4. 23. A vector comprising the nucleic acid compound of claim 21.

24. A cell comprising the nucleic acid compound of claim 21.

25. A method to detect Bartonella clarridgeiae in a test sample, comprising:

conducting polymerase chain reaction using starting materials which comprise species-specific primers designed from a compound of claim 17, under conditions which allow production of an amplicon in the event that Bartonella clarridgeiae is present in the test sample; and

detecting Bartonella clarridgeiae in the event that an amplicon is present.

26. A method to detect Bartonella vinsonii, subspecies berkhoffii in a test sample, comprising:

conducting polymerase chain reaction using starting materials which comprise species-specific primers designed from a compound of claim 21, under conditions which allow production of an amplicon in the event that Bartonella vinsonii, subspecies berkhoffii is present in the test sample; and

detecting Bartonella vinsonii, subspecies berkhoffii in the event that an amplicon is present. 27. A method to design a set of primers capable of amplifying a uniquely- sized fragment from at least two species of a genus, comprising:

identifying genomic fragments from at least two species of the same genus, said genomic fragments having differences in absolute size, and said genomic fragments being defined at least on the periphery by alignable conserved sequences; and

identifying a forward and reverse sequence on the periphery which are common to each of the species for which primers are desired.

28. A method to detect at least one prokaryotic species in a test sample, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise at least one set of genus-specific primers; and

b.) detecting at least one of said species on the basis of amplicon size.

29. A method of claim 28, wherein step b.) comprises gel electrophoresis.

Claims

WHAT IS CLAIMED IS (Part Two):

1. A method to detect Haemobartonella species in a test sample, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and

b.) detecting Haemobartonella species in the test sample in the event a Haemobartonella-sized amplicon is present.

2. A method of claim 1, wherein step b.) comprises gel electrophoresis.

3. A method of claim 1 , wherein the primers are capable of amplifying uniquely-sized fragments for every Haemobartonella species.

4. A method of claim 1, wherein the forward primer comprises a sequence selected from the group consisting of SEQ ID NO 23 and SEQ ID NO 25.

5. A method of claim 4, wherein the reverse primers comprises a sequence selected from the group consisting of SEQ ID NO 24 and SEQ ID NO 26.

6. A method of claim 1, wherein the primers are capable of amplifying uniquely-sized amplicons from Haemobartonella felis Ohio and Haemobartonella felis California.

7. A method to detect Haemobartonella-caxised disease in a mammal, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and

b.) detecting Hαemobαrtonellα-caused disease in the test sample in the event a Hαemobαrtonellα-sized amplicon is present.

8. A method of claim 7, wherein the Hαemobαrtonellα-caused disease is anemia.

9. A method to detect anemia in a mammal, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and

b.) detecting anemia in the test sample in the event an amplicon is present, which amplicon is the size appropriate for a species chosen from the group consisting of: Haemobartonella felis Ohio; and Haemobartonella felis California.

10. A kit useful for distinguishing between or among Haemobartonella species, comprising at least one set of genus-specific primers.

11. A kit of claim 10, which further comprises gel material.

12. A composition of matter comprising a set of genus-specific primers.

13. A set of genus-specific primers of claim 12, wherein the forward primer comprises a sequence selected from the group consisting of SEQ ID NO 23 and SEQ ID NO 25.

14. A set of genus-specific primers of claim 12, wherein the reverse primer comprises a sequence selected from the group consisting of SEQ ID NO 24 and SEQ ID NO 26.

WHAT IS CLAIMED IS (Part Three):

1. A method to detect Ehrlichia species in a test sample, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and

b.) detecting Ehrlichia species in the test sample in the event an amplicon is present.

2. A method of claim 1, wherein step b.) comprises gel electrophoresis.

3. A method of claim 1, wherein the starting materials comprise two sets of genus-specific primers.

4. A method of claim 1, wherein the primers are selected from the group of pairs consisting of: SEQ ID NO 27 and SEQ ID NO 28; and SEQ ID NO 27 and SEQ ID NO 29.

5. A method of claim 1, wherein the primers are capable of amplifying amplicons from E. canis, E. ewingii, E. chaffeenisis, E. risticii, and E. equi.

6. A method to detect Ehrlichia-caused disease in a mammal, comprising:

a.) conducting polymerase chain reaction using starting materials which comprise a test sample and at least one set of genus-specific primers; and

b.) detecting Ehrlichiα-causcd disease in the test sample in the event an amplicon is present.

7. A method of claim 6, wherein the Ehrlichiα-caused disease is a nonspecific febrile illness.

8. A kit useful for distinguishing Ehrlichia from other canine pathogens, comprising at least one set of genus-specific primers.

9. A kit of claim 8, which further comprises gel material.

10. A composition of matter comprising a set of genus-specific primers.

11. A set of genus-specific primers of claim 10, which primers are selected from the sets consisting of: SΕQ ID NO 27 and SΕQ ID NO 28; and SΕQ ID NO 27 and SΕQ ID NO 29.