Methods for detecting Ehrlichia canis and Ehrlichia chaffeensis in vertebrate and invertebrate hosts

BACKGROUND OF THE INVENTION

The Ehrlichiae are obligate intracellular bacteria found in circulating leukocytes of infected animals.

Ehrlichia canis

(

E. canis

) infects monocytes and causes ehrlichiosis in animals belonging to the family Canidae.

E. canis

is transmitted by the brown dog tick,

Rhipicephalus sanguineus.

Canine monocytic ehrlichiosis (CME) 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.

Human monocytic ehrlichiosis (HME) is a tick-borne, emerging infectious disease that is caused by the rickettsial pathogen,

Ehrlichia chaffeensis

(

E. chaffeensis

). The course of HME begins with an asymptomatic pre-patent period, followed by an acute phase where the vertebrate host suffers pyrexia, anorexia, weight loss, cytopenia and even death. Non-human hosts that survive the acute phase typically undergo partial recovery and suffer mild chronic infections, during which they could be persistent carriers that are capable of infecting tick vectors. Both dogs and white tailed deer are susceptible to infection with

E. chaffeensis,

and both of these hosts are suspected reservoirs of this pathogen.

E. chaffeensis

appears to be transmitted by

Amblyomma. americanum,

and appears to be endemic to the southern U.S. where this tick is indigenous. Symptoms of human monocytic ehrlichiosis (HME) are similar to those of canine monocytic ehrlichiosis (CME) that is also known as tropical canine pancytopenia. (Hildebrandt, P. K., D. L. Huxsoll, et al. (1973 (1973). Pathology of canine ehrlichiosis (tropical canine pancytopenia).

Am J Vet Res

34(10): 1309-20.; Kuehn, N. F. and S. D. Gaunt (1985). Clinical and hematologic findings in canine ehrlichiosis.

J Am Vet Med Assoc

186(4): 355-8.; Eng, T. R., J. R. Harkess, et al. (1990). Epidemiologic, clinical, and laboratory findings of human ehrlichiosis in the United States, 1988.

Jama

264(17): 2251-8.; McDade, J. E. (1990). Ehrlichiosis—a disease of animals and humans.

J Infect Dis

161(4): 609-17.) The etiologic agents of CME and HME,

E. canis

and

E. chaffeensis,

respectively, have been placed in the same genogroup based on 16S rRNA sequences and antigenic cross-reactivity Anderson, B. E., J. E. Dawson, et al. (1991).

Ehrlichia chaffeensis,

a new species associated with human ehrlichiosis.

J Clin Microbiol

29(12): 2838-42.

The primary test for diagnosing CME or HME is the indirect fluorescent antibody (IFA) test. This test uses the etiologic agents

Ehrlichia canis

or

E. chaffeensis,

respectively, to diagnose infection. The IFA test, however, has serious limitations. The IFA test is subject to false positives because the antigens are whole infected cells which comprise many nonspecific proteins that can 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.

Serodiagnosis is another method which has been developed to diagnose canine or human ehrlichiosis. The method involves testing the blood of the animal for antibodies immunoreactive with outer membrane proteins of these pathogens. Serodiagnosis cannot be used until the infected subject has produced such antibodies. Accordingly, serodianosis cannot be used early during the course of infection. Moreover, serodiagnosis cannot be used for detecting an ongoing infection.

Accordingly, it is desirable to have additional methods and tools which can be used for diagnosing canine and human ehrlichiosis, particularly methods and tools which can be used to detect an ongoing infection. Methods and tools which can be used to detect

E. canis

and

E. chaffeensis

in the invertebrate vectors which transmit these pathogens to their respective vertebrate hosts are also desirable.

SUMMARY OF THE INVENTION

The present invention provides tools and methods for detecting the presence of

E. canis

and

E. chaffeensis

in a sample obtained from an animal, particularly from a member of the Canidae family. The method for detecting

E. canis

comprises providing a p30 primer set comprising a first primer having a sequence which is complementary to a sequence on the

E. canis

p30 gene sense strand and a second primer which is complementary to a sequence which is complementary to a sequence on the

E. canis

p30 gene of antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p30 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of the region of the p30 gene which is located between the nucleotide sequences to which the first p30 primer and the second p30 primer bind is indicative of the presence of

E. canis

in the sample.

The present invention also relates to the p30 primer sets. Each p30 primer set comprises a first p30 primer and the second p30 primer, both of which are from 15 to 35 nucleotides in length. The first p30 primer, i.e., the forward primer, comprises a sequence which is complementary to a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTTTTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTTACTGTTCCAA AC. SEQ ID NO.1. The second p30 primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2. Such primers are useful for detecting the presence of

E. canis

in members of the Canidae family. Such primers are also useful for detecting the presence of

E.canis

DNA in samples obtained from ticks or other invertebrate carriers which feed on the vertebrate hosts.

The method for detecting

E. chaffeensis

comprises providing a p28 primer set comprising a first primer comprising a sequence which is complementary to a sequence on the

E. chaffeensis

p28 gene sense strand and a second primer which is complementary to a sequence on the

E. chaffeensis

p28 gene antisense strand, amplifying DNA in the sample using a polymerase chain reaction and the p28 primer set, and determining the length or sequence of the PCR products, wherein the presence of a PCR product having a sequence or length which corresponds to the sequence or length of that portion of the p28 gene which is located between the sequence to which the first p28 primer and second p28 primer bind is indicative of the presence of

E. chaffeensis

DNA in the sample.

The present invention also relates to the primers in the p28 primer set. The first p28 and second p28 primers are from 15 to 35 nucleotides in length. The first p28 primer, i.e. the forward primer, comprises a sequence which is substantially identical to the complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within the following sequence :A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO.3.

The second primer, i.e. the reverse primer, comprises a sequence which is complementary to the inverse complement of a consecutive sequence, preferably of at least 14 nucleotides in length, within one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO.4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO.5. Such primers are useful for detecting the presence of

E. chaffeensis

in samples obtained from vertebrate animals such as humans or dogs, or from the invertebrate vectors which transmit this pathogen from one vertebrate animal to another.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

shows the nucleotide sequence of the p30 gene (SEQ ID NO. 47) of the Jake strain of

E. canis.

(Genbank Accession No. AF082744.)

FIG. 2

is a consensus open reading frame (ORF) sequence for the

E. chaffeensis

p28 gene (SEQ ID NO. 48). The consensus sequence was derived from the p28 genes of the Sapulpa, Jacksonville and Arkansas isolates.

FIG. 3

shows the results of a PCR assay of peripheral blood from dogs during acute and chronic experimental CME. Three dogs were tested with the nested p30-based PCR described in the text. Buffy coat DNA preparations were assayed from two dogs prior to (lanes 1 and 3) and 21 d after (Lanes 3 and 4) inoculation with

E. canis

Ebony isolate-infected blood from an experimentally infected dog. The third dog (lane 5) was from the experimentally infected donor blood. The molecular size standard (M) is a 100 bp ladder (Life Technologies).

FIG.

4

. Confirmation of the

E. canis

nested target amplicon sequence. Multiple sequence alignment of the amplicon (nucleotides 409-583 of SEQ ID NO. 47) derived from nested p30-based PCR of DNA from

E. canis

(Ebony isolate), the

E. canis

p30 target DNA sequence (Jake Isolate; nucleotides 384-593 of SEQ ID NO. 47) and primer set 1 (ECA30-384S, SEQ ID NO. 6 and ECA30-583A, reverse complement of SEQ ID NO. 7). The DNA sequence for ECA30-583A is the reverse complement of that oligonucleotide. The sequences were aligned and compared with the Pileup (GCG, version 10) and Boxshade programs. White letters surrounded by a black box represent bases that are identical in each sequence of the alignment.

FIG.

5

. shows: Sensitivity of the p30-based nested PCR assay. The 16S rDNA (panel A) and p30 (panel B) PCR assays were compared to determine relative sensitivities. Buffy coat DNA was isolated from an

E. canis

carrier dog and subjected to a dilution series for simultaneous amplification with the two assays. Lanes 1-5, respectively, are assays of the template dilutions of 1×10

0

, 10

−1

, 10

−2

, 10

−3

and 10

−4

for both panels.

FIG.

6

. shows: Specificity of the p30-based nested PCR assay. Lane 1 is the template-free control, lanes 2-3 represent amplification of 50 ng of

E. canis

Ebony and Oklahoma isolates, respectively, and lanes 4-6 represent assay of 50 ng of DNA isolated from

E. chaffeensis,

HGE agent and

E. muris,

respectively. The 100 bp ladder (M) serves as the molecular size standard.

FIG.

7

. shows: Preparation of blood samples for the p30-based nested PCR assay. Blood was collected with heparin from an

E. canis

-infected carrier dog, divided into 0.5 ml aliquots and subjected to different preparation procedures performed in duplicate as described in the text. Samples assayed with the nested p30-based PCR included buffy coats treated with DNAzol (Lanes 1 and 2), buffy coats treated with DNAzol BD (Lanes 3 and 4), whole blood treated with DNAzol BD (Lanes 5 and 6), buffy coats subjected to proteinase K digestion followed by phenol extraction and ethanol precipitation (Lanes 7 and 8) and DNA extracted from PBMC with Qiagen spin columns (Lanes 9 and 10). A 100 bp DNA ladder (M) was used as a molecular size standard.

FIG.

8

. Analysis of oligonucleotide primer sets derived from the

E. canis

p30 gene sequence. In the primer site plot (A), the horizontal lines represent the portion of the p30 open reading frame analyzed for optimal primer pairs. Each horizontal line represents the p30 ORF; vertical bars above the line represent the location of forward primers and vertical bars below each line represent reverse primers. The truncated multiple sequence (B) demonstrates similarity between the sequences of the forward primers and the

E. chaffeensis

p28 sequence clusters I (nucleotides 93-272 of SEQ ID NO. 48), II (nucleotides 93-272 of SEQ ID NO. 48) and III to the

E. canis

p30 sequence (nucleotides 241-417 of SEQ ID NO. 47). Letters surrounded by a black box represent bases identical to the

E. canis

sequence. Gray letters surrounded by a black box represent bases identical to

E. canis

and all three

E. chaffeensis

gene clusters. The same analysis was performed for reverse primers. The percent identical bases between the different primers and the

E. chaffeensis

cluster sequences were determined and are displayed in Table 1. Other sequences and their SEQ ID NOs. are as follows: F1

—

384-408, SEQ ID NO. 6; F2

—

387-408, SEQ ID NO. 8; F3

—

390-412, SEQ ID NO. 10; F4

—

351-371, SEQ ID NO. 11; F5

—

350-371, SEQ ID NO. 13; F6

—

356-377, SEQ ID NO. 15; F7

—

356-377, SEQ. ID NO. 15; F8

—

388-412, SEQ ID NO. 16; F9

—

282-306, SEQ ID NO. 18; F10

—

356-377, SEQ ID NO. 15; F11

—

355-377, SEQ ID NO. 21; F12

—

353-377, SEQ ID NO. 23; F13

—

278-301, SEQ ID NO. 24.

FIG.

9

. Analysis of oligonucleotide primer sets derived from the

E. chaffeensis

p28 consensus sequence. The primer site plot (A) represents the location of forward and reverse primers within p28; vertical bars above the line represent the location of forward primers and vertical bars below each line represent reverse primers. The truncated multiple sequence alignments demonstrate identity between the sequences of the forward (B) and reverse (C) primers. Gray letters surrounded by a black box represent bases identical to

E. canis

and all three

E. chaffeensis

gene clusters. The percent identical bases between the different primers and the

E. canis

sequences were determined and are displayed in Table 5. In (B), sequences and their SEQ ID NOs. are as follows:

E. chaffeensis

I, II, and III, nucleotides 57 to 101 of SEQ ID NO. 48; echF58-78, nucleotides 58-78 of SEQ ID NO. 48; echF58-79, nucleotides 58-79 of SEQ ID NO. 48; echF59-79, nucleotides 59-79 of SEQ ID NO. 48; echF59-79, nucleotides 59-79 of SEQ ID NO. 48; echF59-80, nucleotides 59-80 of SEQ ID NO. 48; echF60-80 of SEQ ID NO. 48; echF61-82, nucleotides 61-82 of SEQ ID NO. 48; echF67-85, nucleotides 67-85 of SEQ ID NO. 48; echF78-99, nucleotides 78-99 of SEQ ID NO. 48;

E. canis,

nucleotides 202-249 of SEQ ID NO. 47. In (C), sequences and their SEQ ID NOs. are as follows:

E. chaffeensis

I, II, and III, reverse complement of nucleotides 291-352 of SEQ ID NO. 48; echR313-293, reverse complement of nucleotides 293-313 of SEQ ID NO. 48; ecbR314-293, reverse complement of nucleotides 293-314 of SEQ ID NO. 48; echR314-294, reverse complement of nucleotides 294-314 of SEQ ID NO. 48; echR315-294, reverse complement of nucleotides 294-315 of SEQ ID NO. 48; echR320-302, reverse complement of nucleotides 302-320 of SEQ ID NO. 48; ecbR321-302, reverse complement of nucleotides 302-321 of SEQ ID NO. 48; echR322-302, reverse complement of nucleotides 302-322 of SEQ ID NO. 48; echR339-319, reverse complement of nucleotides 319-339 of SEQ ID NO. 48; echR341-320, reverse complement of nucleotides 320-341 of SEQ ID NO. 48; echR352-331, reverse complement of nucleotides 331-352 of SEQ ID NO. 48;

E. canis,

reverse complement of nucleotides 436-497 of SEQ ID NO. 47.

FIG.

10

. shows: Detection of

E. chaffeensis

(Arkansas isolate) with the p28-based PCR assay. Panel A demonstrates amplicons from duplicate non-optimized PCR in the presence of 50 ng of DNA from

E. chaffeensis

-infected DH82 cells and various primer sets that are specific to the Arkansas isolate (11

A

, 13

A

and 15

A

) or universal to all three p28 clusters (3

U

and 1

U

), which are described in Table X. Panel B consists of male ticks of four species known to parasitize dogs that were intrastadially exposed to

E. chaffeensis

by allowing them to feed on on a rickettsemic dog followed by holding for 10 days in a humidity chamber. Both 1° and 2° optimized PCR assays are shown for

A. americanum.

PCR-positive ticks are indicated by a white “+” at the top of each lane. Note that

E. chaffeensis

infection was detected in 20-40% of each tick species tested. A 100 bp ladder served as the molecular size standard (M).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect,the present invention provides methods and tools for detecting the presence of

Ehrlichia canis

in samples obtained from a vertebrate or invertebrate animal. The method comprises amplifying the DNA contained within the sample using a primer set comprising primers which comprise sequences that are complementary to select regions of the p30 gene of

E. canis

and a polymerase chain reaction (PCR) to provide a pool of PCR products, and then assaying the pool for the presence or absence of a PCR product whose length or sequence indicates that PCR product corresponds to the region of the p30 gene that is flanked by the nucleotide sequences which are complementary to the first and second members of the p30 primer set. The tools are the members of the p30 primer sets. The

E. canis

p 30 gene belongs to the omp-1 multiple gene family and has the sequence which is set forth in Genbank as Accession No. AF082744.1 (Jake isolate), Accession No. AF082750.1 (Florida isolate), Accession No. AF082749.1 (Fuzzy isolate), Accession No. AF082748.1m (DJ isolate), Accession No. AF082747.1 (Arkansas isolate), Accession No. AF082746.1 (Oklahoma isolate) and Accession No. AF082745.1 (Louisiana isolate of

E. canis.

) The

E. canis

p30 gene encodes the immunodominants ehrlichial outer membrane protein P30.

In another aspect, the present invention provides methods and tools for detecting the presence of

Ehrlichia chaffeensis

in samples obtained from vertebrate or invertebrate animals. The method comprises amplifying the DNA contained within the sample using a p28 primer set whose members comprise a sequence which is complementary to select regions of the p28 gene of

E. chaffeensis

and a polymerase chain reaction (PCR) to provide a pool of PCR products, and then assaying the pool for the presence of a PCR product whose length or sequence indicates that PCR product corresponds to the region of the p28 gene that comprises and is flanked by the nucleotide sequences to which the members of the p28 primer set bind. The tools are the members of the p28 primer sets. The

E. chaffeensis

p28 gene belongs to the omp-1 multiple gene family and has the sequence which is set forth in Genbank as (Accession No. AF077735.1 (St. Vincent isolate), Accession No. AF077734.1 (Sapulpa isolate), Accession No. AF077733.1 (Jax isolate), Accession No. AF077732.1 (91HE17 isolate) and Accession No. AF068234 (Arkansas isolate). The

E. chaffeensis

p28 gene encodes the immunodominants ehrlichial outer membrane protein P28.

Primer

The primers in the p30 primer set are based upon select sequences in the p30 gene of

E. canis.

The p30 gene encodes the major outer membrane protein of

E. canis.

The sequences of the first and second primers in the p30 primer set are distinct from sequences found in the closely related p28 gene in

E. chaffeensis.

The first primer in the p30 primer set is an oligonucleotide of from 15 to 35 nucleotides in length, preferably from 18 to 30 nucleotides in length. The second p30 primer in the

E. canis

primer set is an oligonucleotide of from 15 to 35 nucleotides, preferably from 18 to 30 nucleotides in length. The first p30 primer, i.e., the forward primer, comprises a sequence which is substantially identical to the complement of consecutive sequence located between nucleotides 278 and 412 of the sense strand of the open reading frame sequence of the p30 gene of

E. canis.

The sequence of the sense strand in this region is as follows: CCA AGTGTCTCAC ATTTTGGTAG CTTCTCAGCT AAAGAAGAAA GCAAATCAAC TGTTGGAGTT TTTGGATTAA AACATGATTG GGATGGAAGT CCAATACTTA AGAATAAACA CGCTGACTTT ACTGTTCCAA AC. SEQ ID NO.1 As used herein the term “substantially identical” means that the sequence is at least 90% identical, preferably at least 95% identical, more preferably 100% identical to a particular reference sequence, e.g., to the complement of a consecutive sequence contained within SEQ ID NO. 1. The second p30 primer, i.e., the reverse primer, comprises a sequence which is substantially identical to and the inverse of a consecutive sequence located between nucleotides 465 through 597 of the sense strand of the p30 gene of

E. canis.

The sequence of the second p30 primer is substantially identical to the complement of the inverse complement of a consecutive sequence contained within the following sequence: GTTACT CAATGGGTGG CCCAAGAATA GAATTCGAAA TATCTTATGA AGCATTCGAC GTAAAAAGTC CTAATATCAA TTATCAAAAT GACGCGCACA GGTACTGCGC TCTATCTCAT CACACATCGG CAGCCAT, SEQ ID NO.2.

In specific embodiments, the first and second primers in the p30 primer set comprise the sequences shown in Table 1 below. The first and second primers may also comprise sequences which are shorter by one or two oligonucleotides than the sequences shown in Table 1 below. The first and second primers of the

E. canis

primer set may also comprise a sequence which is longer than the sequences shown in Table 1 below. Such sequences have one or two additional nucleotides attached to the 5′ end of the above-listed sequences. The additional nucleotides are selected from the group consisting of adenylic acid, guanylic acid, and combinations thereof

The sequence of the thirteen

E. canis

p30 primer sets shown in Table 1 below are based upon a comparison of the open-reading frame sequences of the seven

E. canis

and five

E. chaffeensis

isolates. It is expected that such primer sets will specifically amplify the target sequence of multiple

E. canis

isolates, but not the

E. chaffeensis

isolates. It is expected that the primers shown in Table 1 below are both species-universal and species-specific for

E. canis.

In accordance with the present invention, it has been determined that amplification of DNA isolated from

E. chaffeensis, E. muris,

and the HGE agent with these primer sets failed to produce a PCR product that is diagnostic of

E. canis.

TABLE 1

Oligonucleotide sequences of

E. canis

p30 primer sets.

Set

Forward

Reverse

1

ECA30-384S (SEQ ID NO.6)

ECA30-583A (SEQ ID NO.7)

(ATAAACACGCTGACTTTACTGTTCC)

(GTGATGAGATAGAGCGCAGTACC)

2

ECA30-387S (SEQ ID NO.8)

ECA30-597A (SEQ ID NO.9)

(AACACGCTGACTTTACTGTTCC)

(ATGGCTGCCGATGTGTGATG)

3

ECA30-390S (SEQ ID NO.1O)

ECA30-597A (SEQ ID NO.9)

(ACGCTGACTTTACTGTTCCAAAC)

(ATGGCTGCCGATGTGTGATG)

4

ECA30-351S (SEQ ID NO.11)

ECA30-591A (SEQ ID NO.12)

(AACATGATTGGGATGGAAGTC)

(GCCGATGTGTGATGAGATAG)

5

ECA30-350S (SEQ ID NO.13)

ECA30-59lbA (SEQ ID NO.14)

(AAACATGATTGGGATGGAAGTC)

(GCCGATGTGTGATGAGATAGAG)

6

ECA30-356S (SEQ ID NO.15)

ECA30-591A (SEQ ID NO.12)

(GATTGGGATGGAAGTCCAATAC)

7

ECA30-356S (SEQ ID NO.15)

ECA30-59lbA (SEQ ID NO.14)

8

ECA30-388S (SEQ ID NO.16)

ECA30-597bA (SEQ ID NO.17)

(ACACGCTGACTTTACTGTTCCAAAC)

(ATGGCTGCCGATGTGTGATGAG)

9

ECA30-282S (SEQ ID NO.18)

ECA30-486A (SEQ ID NO.19)

(GTGTCTCACATTTTGGTAGCTTCTC)

(CTTGGGCCACCCATTGAGTAAC)

10

ECA30-356S (SEQ ID NO.15)

ECA30-589A (SEQ ID NO.20)

(CGATGTGTGATGAGATAGAGC)

11

ECA30-355S (SEQ ID NO.21)

ECA30-589bA (SEQ ID NO.22)

(TGATTGGGATGGAAGTCCAATAC)

(CGATGTGTGATGAGATAGAGCG)

12

ECA30-353S (SEQ ID NO.23)

ECA30-597A (SEQ ID NO.9)

(CATGATTGGGATGGAAGTCCAATAC)

(ATGGCTGCCGATGTGTGATG)

13

ECA30-278S (SEQ ID NO.24)

ECA30-484A (SEQ ID NO.25)

(CCAAGTGTCTCACATTTTGGTAGC)

(TGGGCCACCCATTGAGTAAC)

The predicted annealing scores and specificity values, as compared to the p28 of

E. chaffeensis,

of these primer sets are shown in Table 2 below.

TABLE 2

Ranks of

E. canis

p30 primer sets with predicted annealing and specificity values.

Identity Scores to

E. chaffeensis

a

Total

Primer

Cluster I

Cluster II

Cluster III

Annealing

Set

Forward

Reverse

Forward

Reverse

Forward

Reverse

Mean

b

Score

c

Rank

d

1

0.64

0.74

0.48

0.87

0.56

0.78

0.68

52

2

2

0.59

0.45

0.55

0.65

0.55

0.60

0.56

57

1

3

0.74

0.45

0.61

0.65

0.70

0.60

0.62

58

3

4

0.81

0.55

0.86

0.75

0.71

0.70

0.73

63

5

5

0.82

0.55

0.82

0.77

0.73

0.73

0.74

64

6

6

0.91

0.55

0.86

0.75

0.77

0.70

0.76

64

7

7

0.91

0.55

0.86

0.77

0.77

0.73

0.76

65

9

8

0.68

0.45

0.60

0.68

0.68

0.64

0.62

66

4

9

0.64

0.86

0.64

0.95

0.72

0.91

0.79

67

12

10

0.91

0.62

0.86

0.81

0.77

0.71

0.78

67

10

11

0.87

0.64

0.83

0.82

0.74

0.73

0.77

68

11

12

0.88

0.45

0.84

0.65

0.80

0.70

0.72

68

8

13

0.67

0.85

0.67

0.95

0.71

0.90

0.79

68

13

a

Identity Score = No. oligonucleotide bases identical to aligned template ÷ total No. oligonucleotide bases.

b

Mean identity score for both primers of each set to all three

E. chaffeensis

clusters.

c

Determined by GCG (version 10) Prime program. Sum of annealing scores for primer secondary structure, non-specific primer binding to the template sequence and self-complementarity of individual primers and complementarity between primer pairs; the lowest scores indicate the primer sets with least complementarity to sequences other than the target binding site.

d

Rank determined by product of annealing score and mean identity score; lower values have higher rank.

The primers in the

E. chaffeensis

p28 primer set are based upon select sequences in the p28 gene of

E. chaffeensis.

The p28 gene encodes a major outer membrane protein of

E. chaffeensis.

The sequences of the first and second primers in the p28 primer set are distinct from sequences found in the closely related p30 gene in

E. canis.

The first primer in the p28 primer set is an oligonucleotide of from 15 to 35 nucleotides in length., preferably from 18 to 30 nucleotides in length. The second primer in the

E. chaffeensis

p 28 primer set is an oligonucleotide of from 15 to 35 nucleotides, preferably from 18 to 30 nucleotides in length. The first p28 primer, i.e., the forward primer, comprises a sequence which is substantially identical to the complement of consecutive sequence located between nucleotides 15 and 101 of the sense strand of the open reading frame sequence of the p28 gene of

E. chaffeensis.

The sequence of the sense strand in this region is as follows: A GTTTTCATAA CAAGTGCATT GATATCACTA ATATCTTCTC TACCTGGAGT ATCATTTTCC GACCCAACAG GTAGTGGTAT TAACGG, SEQ ID NO. 3.

The second p28 primer, i.e., the reverse primer, comprises a sequence which is substantially identical to and the inverse of a consecutive sequence located between nucleotides 341 through 365 or nucleotides 641 through 674 of the sense strand of the p28 gene of

E. chaffeensis.

The sequence of the second p28 primer is substantially identical to the complement of the inverse complement of a consecutive sequence contained within one of the following two sequences: CAT TTCTAGGTTT TGCAGGAGCT ATTGGCTACT CAATGGATGG TCCAAGAATA GAGCTTGAAG TATCTTATGA, SEQ ID NO. 4, or C AAGGAAAGTT AGGTTTAAGC TACTCTATAA GCCCAGA, SEQ ID NO. 5.

In specific embodiments, the first and second primers in the

E. chaffeensis

p28 primer set comprise the sequences shown in Table 3 below. The first and second primers may also comprise sequences which are shorter by one or two oligonucleotides than the sequences shown in Table 3 below. The first and second primers of the

E. chaffeensis

p28 primer set may also comprise a sequence which is longer than the sequences shown in Table 3 below. Such sequences have one or two additional nucleotides attached to the 5′ end of the above-listed sequences. The additional nucleotides are selected from the group consisting of adenylic acid, guanylic acid, and combinations thereof

TABLE 3

Product: 1

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

84 AGGTAGTGGTATTAACGG

101 (SEQ ID NO. 26)

reverse primer (20-mer):

360 AGATACTTCAAGCTCTATTC

341 (SEQ ID NO. 27)

forward

reverse

primer % GC:

44.4

35.0

primer Tm (degrees Celsius):

44.4

43.6

PRODUCT

product length:

277

product % GC:

31.0

product Tm:

70.2 degrees Celsius

difference in primer Tm:

0.8 degrees Celsius

annealing score:

57

optimal annealing temperature:

47.3 degrees Celsius

Product: 2

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

84 AGGTAGTGGTATTAACGG

101 (SEQ ID NO. 26)

reverse primer (20-mer):

365 TCATAAGATACTTCAAGCTC

346 (SEQ ID NO. 28)

forward

reverse

primer % GC:

44.4

35.0

primer Tm (degrees Celsius):

44.4

44.0

PRODUCT

product length:

282

product % GC:

30.9

product Tm:

70.2 degrees Celsius

difference in primer Tm:

0.5 degrees Celsius

annealing score:

57

optimal annealing temperature:

47.4 degrees Celsius

Product: 3

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (19-mer):

50 CTTCTCTACCTGGAGTATC

68 (SEQ ID NO. 29)

reverse primer (21-mer):

669 GCTTATAGAGTAGCTTAAACC

649 (SEQ ID NO. 30)

forward

reverse

primer % GC:

47.4

38.1

primer Tm (degrees Celsius):

44.8

45.5

PRODUCT

product length:

620

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

0.8 degrees Celsius

annealing score:

61

optimal annealing temperature:

48.3 degrees Celsius

Product: 4

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

83 CAGGTAGTGGTATTAACG

100 (SEQ ID NO. 31)

reverse primer (19-mer):

364 CATAAGATACTTCAAGCTC

346 (SEQ ID NO. 32)

forward

reverse

primer % GC:

44.4

36.8

primer Tm (degrees Celsius):

43.6

42.2

PRODUCT

product length:

282

product % GC:

31.2

product Tm:

70.3 degrees Celsius

difference in primer Tm:

1.4 degrees Celsius

annealing score:

63

optimal annealing temperature:

47.0 degrees Celsius

Product: 5

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

83 CAGGTAGTGGTATTAACG

100 (SEQ ID NO. 31)

reverse primer (19-mer):

359 GATACTTCAAGCTCTATTC

341 (SEQ ID NO. 33)

forward

reverse

primer % GC:

44.4

36.8

primer Tm (degrees Celsius):

43.6

41.9

PRODUCT

product length:

277

product % GC:

31.4

product Tm:

70.3 degrees Celsius

difference in primer Tm:

1.7 degrees Celsius

annealing score:

63

optimal annealing temperature:

46.9 degrees Celsius

Product: 6

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (19-mer):

50 CTTCTCTACCTGGAGTATC

68 (SEQ ID NO. 29)

reverse primer (20-mer):

669 GCTTATAGAGTAGCTTAAAC

650 (SEQ ID NO. 34)

forward

reverse

primer % GC:

47.4

35.0

primer Tm (degrees Celsius):

44.8

42.8

PRODUCT

product length:

620

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

2.0 degrees Celsius

annealing score:

63

optimal annealing temperature:

47.7 degrees Celsius

Product: 7

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (19-mer):

55 CTACCTGGAGTATCATTTTC

74 (SEQ ID NO. 35)

reverse primer (21-mer):

670 GGCTTATAGAGTAGCTTAAAC

650 (SEQ ID NO. 36)

forward

reverse

primer % GC:

40.0

38.1

primer Tm (degrees Celsius):

44.7

45.5

PRODUCT

product length:

616

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

0.9 degrees Celsius

annealing score:

64

optimal annealing temperature:

48.3 degrees Celsius

Product: 8

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

45 AATATCTTCTCTACCTGG

62 (SEQ ID NO. 37)

reverse primer (19-mer):

359 GATACTTCAAGCTCTATTC

341 (SEQ ID NO. 33)

forward

reverse

primer % GC:

38.9

36.8

primer Tm (degrees Celsius):

40.8

41.9

PRODUCT

product length:

315

product % GC:

32.1

product Tm:

70.9 degrees Celsius

difference in primer Tm:

1.1 degrees Celsius

annealing score:

65

optimal annealing temperature:

47.0 degrees Celsius

Product: 9

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

15 AGTTTTCATAACAAGTGC

32 (SEQ ID NO. 38)

reverse primer (19-mer):

364 CATAAGATACTTCAAGCTC

346 (SEQ ID NO. 32)

forward

reverse

primer % GC:

33.3

36.8

primer Tm (degrees Celsius):

42.5

42.2

PRODUCT

product length:

350

product % GC:

31.4

product Tm:

70.9 degrees Celsius

difference in primer Tm:

0.3 degrees Celsius

annealing score:

66

optimal annealing temperature:

47.4 degrees Celsius

Product: 10

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

15 AGTTTTCATAACAAGTGC

32 (SEQ ID NO. 38)

reverse primer (19-mer):

359 GATACTTCAAGCTCTATTC

341 (SEQ ID NO. 33)

forward

reverse

primer % GC:

33.3

36.8

primer Tm (degrees Celsius):

42.5

41.9

PRODUCT

product length:

345

product % GC:

31.6

product Tm:

70.9 degrees Celsius

difference in primer Tm:

0.6 degrees Celsius

annealing score:

66

optimal annealing temperature:

47.3 degrees Celsius

Product: 11

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (25-mer):

50 CTTCTCTACCTGGAGTATCATTTTC

74 (SEQ ID NO. 39)

reverse primer (26-mer):

662 GAGTAGCTTAAACCTAACTTTCCTTG

637 (SEQ ID NO. 40)

forward

reverse

primer % GC:

40.0

38.5

primer Tm (degrees Celsius):

50.9

52.1

PRODUCT

product length:

613

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

1.2 degrees Celsius

annealing score:

67

optimal annealing temperature:

50.1 degrees Celsius

Product: 12

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (19-mer):

50 CTTCTCTACCTGGAGTATC

68 (SEQ ID NO. 29)

reverse primer (22-mer):

662 GAGTAGCTTAAACCTAACTTTC

641 (SEQ ID NO. 41)

forward

reverse

primer % GC:

47.4

36.4

primer Tm (degrees Celsius):

44.8

46.6

PRODUCT

product length:

613

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

1.8 degrees Celsius

annealing score:

67

optimal annealing temperature:

48.3 degrees Celsius

Product: 13

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

45 AATATCTTCTCTACCTGG

62 (SEQ ID NC. 37)

reverse primer (18-mer):

365 TCATAAGATACTTCAAGC

348 (SEQ ID NO. 42)

forward

reverse

primer % GC:

38.9

33.3

primer Tm (degrees Celsius):

40.8

40.2

PRODUCT

product length:

321

product % GC:

31.8

product Tm:

70.8 degrees Celsius

difference in primer Tm:

0.6 degrees Celsius

annealing score:

68

optimal annealing temperature:

46.7 degrees Celsius

Product: 14

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

45 AATATCTTCTCTACCTGG

62 (SEQ ID NO. 37)

reverse primer (19-mer):

364 CATAAGATACTTCAAGCTC

346 (SEQ ID NO. 32)

forward

reverse

primer % GC:

38.9

36.8

primer Tm (degrees Celsius):

40.8

42.2

PRODUCT

product length:

320

product % GC:

31.9

product Tm:

70.9 degrees Celsius

difference in primer Tm:

1.4 degrees Celsius

annealing score:

69

optimal annealing temperature:

47.0 degrees Celsius

Product: 15

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (24-mer):

51 TTCTCTACCTGGAGTATCATTTTC

74(SEQ ID NO. 50)

reverse primer (26-mer):

670 GGCTTATAGAGTAGCTTAAACCTAAC

645 (SEQ ID NO. 51)

forward

reverse

primer % GC:

37.5

38.5

primer Tm (degrees Celsius):

49.8

51.3

PRODUCT

product length:

620

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

1.5 degrees Celsius

annealing score:

69

optimal annealing temperature:

49.8 degrees Celsius

Product: 16

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (22-mer):

53 CTCTACCTGGAGTATCATTTTC

74 (SEQ ID NO. 43)

reverse primer (22-mer):

670 GGCTTATAGAGTAGCTTAAACC

649 (SEQ ID NO. 49)

forward

reverse

primer % GC:

40.9

40.9

primer Tm (degrees Celsius):

47.7

48.1

PRODUCT

product length:

618

product % GC:

30.1

product Tm:

71.2 degrees Celsius

difference in primer Tm:

0.4 degrees Celsius

annealing score:

71

optimal annealing temperature:

49.2 degrees Celsius

Product: 17

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (22-mer):

53 CTCTACCTGGAGTATCATTTTC

74 (SEQ ID NO. 43)

reverse primer (22-mer):

662 GAGTAGCTTAAACCTAACTTTC

641 (SEQ ID NO. 41)

forward

reverse

primer % GC:

40.9

36.4

primer Tm (degrees Celsius):

47.7

46.6

PRODUCT

product length:

610

product % GC:

30.0

product Tm:

71.1 degrees Celsius

difference in primer Tm:

1.1 degrees Celsius

annealing score:

71

optimal annealing temperature:

48.8 degrees Celsius

Product: 18

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

57 ACCTGGAGTATCATTTTC

74 (SEQ ID NO. 44)

reverse primer (18-mer):

674 TCTGGGCTTATAGAGTAG

657 (SEQ ID NO. 45)

forward

reverse

primer % GC:

38.9

44.4

primer Tm (degrees Celsius):

42.6

43.0

PRODUCT

product length:

618

product % GC:

30.1

product Tm:

71.2 degrees Celsius

difference in primer Tm:

0.4 degrees Celsius

annealing score:

73

optimal annealing temperature:

47.7 degrees Celsius

Product: 19

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (19-mer):

50 CTTCTCTACCTGGAGTATC

68 (SEQ ID NO. 29)

reverse primer (18-mer):

673 CTGGGCTTATAGAGTAGC

656 (SEQ ID NO. 46)

forward

reverse

primer % GC:

47.4

50.0

primer Tm (degrees Celsius):

44.8

45.1

PRODUCT

product length:

624

product % GC:

30.3

product Tm:

71.2 degrees Celsius

difference in primer Tm:

0.4 degrees Celsius

annealing score:

73

optimal annealing temperature:

48.4 degrees Celsius

Product: 20

[DNA] = 50.000 nM [salt] = 50.000 mM

PRIMERS

5′

3′

forward primer (18-mer):

55 CTACCTGGAGTATCATTTTC

74 (SEQ ID NO. 35)

reverse primer (19-mer):

673 CTGGGCTTATAGAGTAGC

656 (SEQ ID NO. 46)

forward

reverse

primer % GC:

40.0

50.0

primer Tm (degrees Celsius):

44.7

45.1

PRODUCT

product length:

619

product % GC:

30.2

product Tm:

71.2 degrees Celsius

difference in primer Tm:

0.5 degrees Celsius

annealing score:

75

optimal annealing temperature:

48.3 degrees Celsius

Sample

For the vertebrate hosts the sample is a tissue sample or bodily fluid, such as for example, buffy coats and peripheral blood mononuclear cells. For the invertebrate vectors which may transmit the pathogen from one vertebrate host to another, the sample can be dissected from ticks (e.g., midgut, salivary glands and hemolymph), ticks can be cut into pieces, and ticks can be frozen and smashed in preparation for PCR assay. Further preparation of tick tissues may involve just heating the sample, digesting the samples with proteinase or isolating pure DNA from the tick tissues.

Methods

Optionally, DNA is extracted from the sample using standard methods. For example, DNA may be extracted from fluids using commercially available PCR filters such as, for example, the Isocode filters which are available from Schleicher and Schuell, New Hampshire Methods of extracting DNA from tissue samples, are also described in Maniatis, T., J. Sambrook, and E. F. Fritsch. 1989. Molecular Cloning: a Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., which is specifically incorporated herein by reference. An optimal method for vertebrates involves collection of whole blood in the presence of an anticoagulant such as heparin, collecting the buffy coat fraction from the whole blood, removal of host hemoglobin by hypotonic lysis of erythrocytes remaining with the buffy coat followed by several washings of the buffy coat prior to preparation of the template for PCR assay. For detecting the pathogens in the invertebrate vectors, DNA is extracted from tick halves, salivary glands and midgut by digestion with proteinase K in the presence of nonionic detergents such as Tween-20 and NP-40 followed by heat inactivation of the proteinase and an optional procedure to extract proteins with phenol and chloroform followed by DNA precipitation by salt and ethanol.

The DNA is then amplified with the one of the primer sets at PCR conditions equivalent to or comparable to denaturation for 0.5 to 1.0 minute at 94° C. in a solution, followed by annealing at about 50-72° C. for 0.5 to 1.0 minutes. As recognized in the art, suitable stringency conditions can be attained by varying a number of factors such as the length and nature, i.e., DNA or RNA, of the primer; the length and nature of the target sequence; the concentration of the salts and other components of the PCR solution; the temperature and time of each step, e.g., denaturation, annealing, and elongation. All of these factors may be varied to generate conditions of stringency which are equivalent to the conditions listed above. Changes in stringency are accomplished primarily through the manipulation of annealing temperature and time.

Thereafter, the size of the PCR products is determined. Preferably the size of the PCR products is determined using gel electrophoresis and a plurality of DNA standards of varying sizes. The PCR products and DNA standards that are present on the gel are visualized using standard techniques, such as ethidium bromide staining. Optionally, the PCR products may be separated, preferably by gel electrophoresis and the sequence of the PCR products determined using standard techniques.

To further enhance the sensitivity and specificity of the method for detecting

E. canis,

a nested PCR assay which employs two different

E. canis

primer sets is used. In such assay, the sample is first amplified with a primer set comprising two external primers, i. e. ECA30-351S and ECA30-591A, ECA30-350S and ECA30-59lbA, ECA30-356S and ECA30-591A, ECA30-356S and ECA30-591Ba, ECA30-356S and ECA30-589A, ECA30-355S and ECA30-589bA, and ECA30-353S and ECA30-597A. Thereafter, the products of the first PCR are amplified with a second primer set that comprises internal primers, eg. ECA30-384S and ECA30. A highly preferred primer set for this second amplification comprises a first primer comprising the sequence 5′-ATAAACACGCTGACTTTACTGTTCC-3′ (SEQ ID NO. 6) and a second primer of comprising the sequence 5′-GTGATGAGATAGAGCGCAGTACC-3′ (SEQ ID NO. 7). Following the second amplification, the size or sequence of the PCR products are determined to detect a PCR product which indicates that a DNA molecule having a sequence which corresponds to the region of the p30 gene that is located between the consecutive sequences to which the internal primers bind is present in the sample.

To further enhance the sensitivity and specificity of the method for detecting

E. chaffeensis,

a nested PCR assay which employs two different

E. chaffeensis

primer sets is used. In such assay, the sample is first amplified with a primer set that comprises external primers, i.e.

E. chaffeensis

primer set 3. A highly preferred primer set for this first amplification comprises a first primer comprising the sequence CTTCTCTACCTGGAGTATC (SEQ ID NO. 29) and a second primer comprising the sequence GCTTATAGAGTAGCTTAAACC (SEQ ID NO. 30). Thereafter, the products of the first PCR are amplified with a second primer set that comprises two internal primers, e.g.

E. chaffeensis

primer set 1. A highly preferred primer set for this second amplification comprises a first primer comprising the sequence AGGTAGTGGTATTAACGG (SEQ ID NO. 26) and a second primer which comprises the sequence AGATACTTCAAGCTCTATTC (SEQ ID NO. 27). Following the second amplification, the size or sequence of the PCR products are determined to detect a PCR product which indicates that a DNA molecule having a sequence which corresponds to 277 bp of the p28 gene is present in the sample.

EXAMPLES

The following examples are for purposes of illustration only and are not intended to limit the scope of the invention as defined in the claims which are appended hereto. The references cited in this document are specifically incorporated herein by reference.

Example 1

A. Obtaining DNA from Blood Samples of Infected Animals

Two colony-reared Beagle dogs that were seronegative for exposure to

E. canis

by IFA were inoculated with heparinized blood from a donor dog infected with

E. canis

(Ebony isolate). Heparinized blood was collected from each animal and split into 0.5 ml aliquots and subjected to the following treatments: (1) DNAzol extraction of buffy coat, (2) DNAzol-BD (Molecular Research Center) extraction of whole blood, (3) DNAzol-BD extraction of buffy coat, (4) spin-column purification of peripheral blood mononuclear cell DNA with a QIAamp Kit (Qiagen) and (5) phenol/chloroform extraction followed by ethanol precipitation of buffy coat samples. All sample preparation methods were done in duplicate and in two trials. Buffy coats were isolated from the heparinized whole blood after centrifugation at 1000×g for 20 min, except for those to be purified with spin-columns, which were isolated after isopycnic centrifugation as described in Iqbal, Z., and Y. Rikihisa. 1994. Application of the polymerase chain reaction for the detection of

Ehrlichia canis

in tissues of dogs. Vet. Microbiol. 42:281-7. All DNA isolations with commercial kits were performed in strict accordance to the manufacturer's instructions. DNAzol and DNAzol-BD isolations were performed in the presence of polyacryl carrier (Molecular Research Center) as recommended by the manufacturer.

The remaining method was a modification of a protocol that was previously described in Stich, R. Wet al. 1991. Preliminary development of a polymerase chain reaction assay for

Anaplasma marginale

in ticks. Biotechnol. Techniq. 5:269-274.; and Stich, R. W., et al 1993. Detection of

Anaplasma marginale

(Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of

Dermacentor andersoni

(Acari: Ixodidae) with the polymerase chain reaction J. Med. Entomol. 30:789-94. Briefly, the buffy coats were removed from whole blood and transferred to a 1.5 ml microfuge tube. Erythrocytes remaining in the buffy coat were lysed with two volumes of TE (10 mM Tris HCI, pH 8.0, 1 mM EDTA), and the hemoglobin was removed by washing pelleted cells (13,000×g for 1 min) three times in TE. The final cell suspension was in 400 μl of RPMI 1640 containing 100 μg/ml proteinase K and 0.45% (v/v) NP-40 and 0.45% (v/v) Tween-20, and the protein was digested for 1-2 hr at 55° C. Digests were extracted one time each with equal volumes of buffer-saturated phenol (pH>7.5) (Life Technologies), phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1). DNA was precipitated by adding 1/10 volume of 3 M sodium acetate and 2.5 volumes of absolute ethanol (−20° C.) and stored overnight at −20° C. The DNA samples were centrifuged at 13,000×g for 20 min at 4° C., allowed to air dry in a sterile, bleach-treated cabinet and resuspended in 25 μl of HPLC grade H

2

O. The samples containing

E. canis

DNA were diluted empirically throughout the optimization process.

B. Nested PCR

The primers used in the first PCR reaction is set 12 shown in Table 1 above. The primers used in the second PCT reaction is set 1 shown in Table 1 above. PCR was performed with a Perkin-Elmer 2400 thermal cycler. Master mixes, made with the PE Biosystems Reagents (Foster City, Calif.), were divided into 50 or 25 μl final reaction volumes containing PCR Gold buffer, 0.8 mM dNTP mix and specified amounts of MgCl

2

, primers, Amplitaq-Gold DNA Polymerase and 10% (v/v) template. The reaction profile, except for stated exceptions, consisted of 95° C. for 10 min followed by 35 cycles of 94° C. for 1.0 min, 60° C. for 0.5 min, and 72° C. for 0.5 min followed final extension at 72° C. for 7.0 min.

The following PCR parameters were progressively optimized: (1) annealing temperature (37°, 50°, 55°, 60°, 65° and 70° C. with 1.5 mM MgCl

2

, 0.5 μM primers and 0.025 U/μl Amplitaq-Gold), (2) MgCl

2

concentration (1.0-4.0 mM MgCl

2

in 0.5 mM increments), (3) primer concentration (0.1-1.0 μM in 0.1 μM increments), (4) Amplitaq-Gold concentration (0.01-0.1 U/μl in 0.01 U/μl increments) and (5) cycle number (25-65 cycles in 5 cycle increments).

C. Analysis of PCR Products

PCR product (20 μl) was added to 5 μl of loading buffer (40% (w/v) sucrose, 89 mM Tris, 89 mM boric acid, 2 mM EDTA) and electrophoresed on a 1.5% (w/v) agarose gel with 0.5 μg/ml ethidium bromide in 1×TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 60-80 V for 1-1.5 hr. A 100 bp ladder (Life Technologies, Rockville, Md.) was used as the molecular weight standard. DNA bands were visualized with ultraviolet light and documented using an Alpha Innotech 2000 gel imaging system.

RESULTS

Amplification of the 200 bp Target Sequence by Nested PCR of

E. canis

-infected Peripheral Blood from Dogs with Acute Experimental CME

A 200 bp band was amplified from DNA prepared from blood samples collected from three dogs during acute or chronic experimental CME (FIG.

1

). This band was not observed from blood collected prior to infection of the two dogs assayed during acute CME. The DNA sequence of this amplicon was found to be identical to the 200 bp p30 target sequence and the reverse complement of ECAR1583A (FIG.

2

).

Optimum Conditions for Isolating DNA from Blood Samples

Several approaches to blood preparation for PCR assay were compared with duplicate samples in two trials. Venous blood was collected with heparin from an experimentally infected

E. canis

carrier, equal aliquots that were subjected to different protocols for the preparation of template for PCR. Inhibition of PCR by hemoglobin is well known, thus removing hemoglobin from the blood sample is a major concern of any procedure used to assay these samples with PCR. Commercially available chemical and spin column kits for DNA isolation did provide some positive results with buffy coat and whole blood samples, but these treatments were inconsistent, failing to result in amplification of DNA in both duplicates of both trials. However, buffy coat isolation followed by lysing residual erythrocytes, removal of hemoglobin, protein digestion, protein extraction and DNA precipitation was the only procedure that resulted in amplification of the 200 bp target in both duplicates of both trials (FIG.

5

). Moreover, this method has been used to detect

E. canis

in peripheral blood of a carrier dog for over one year (data not shown).

Optimization of the p30-based PCR Assay for

E. canis

Initial optimization experiments with primer set 2 resulted in the amplification of multiple bands, thus this primer set is less preferred. The remaining assays employed primer sets 1 and 12. For the primary reaction with set 12, optimum PCR conditions were determined to be 1.5 mM MgCl

2

, 0.2 μM of each primer, 0.04 units/μl of Amplitaq-Gold and, after enzyme activation at 95° C. for 10 min, 55 cycles with 94, 65 and 72° C. denaturation, annealing and extension temperatures for 30 sec each. For the secondary or nested PCR with internal primer set 1, optimum reaction conditions were determined to be 2.5 mM MgCl

2

, 0.5 μM of each primer, 0.03 units/μl of Amplitaq-Gold, 10% (vol/vol) of the appropriate primary reaction with primer set 12 and 40 cycles with 94, 60 and 72° C. denaturation, annealing and extension temperatures for 30 sec each.

To confirm that the 200 bp amplicon associated with

E. canis

infection originated from the target DNA sequence, several p30-based nested PCR assays of buffy coat DNA from an

E. canis

-infected dog were pooled and the amplicons isolated with the QIAquik PCR Purification Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. The purified amplicon was submitted to The Ohio State University Neurobiotechnology DNA Sequencing Facility for cycle sequencing in the presence of the primer, ECAF1384S.

The optimum number of cycles for both reactions, particularly the primary reaction, were somewhat greater than expected. One explanation for this may be the enzyme used, Amplitaq-Gold, which requires activation at 95° C. for 10 min prior to the amplification cycles. It is possible that a portion of this enzyme remains inactive until the amplification cycles are underway; thus additional active enzyme becomes available in the course of the reaction, increasing the number of cycles required for optimal PCR.

Specificity and Sensitivity

The sensitivity of this p30-based assay was compared to that of the previously reported 16S rDNA-based assay as described in Wen, B., Y. Rikihisa, J. M. Mott, R. Greene, H. Y. Kim, N. Zhi, G. C. Couto, A. Unver, and R. Bartsch. 1997. Comparison of nested PCR with immunofluorescent-antibody assay for detection of

Ehrlichia canis

infection in dogs treated with doxycycline. J. Clin. Microbiol. 35:1852-5.The buffy coat fraction was removed from 5 ml of heparinized blood from an experimentally infected dog during acute CME, and subjected to DNA isolation with DNAzol (Molecular Research Center) according to the manufacturer's instructions. A tenfold dilution series of the buffy coat DNA was then tested with both the p30- and the 16S rDNA-based PCR assays.

Specificity of the p30-based assay was tested by attempts to amplify 50 ng samples of DNA isolated from

E. muris, E. chaffeensis

and HGE agent in experimentally infected host cells. DNA.

E. chaffeensis

and

E. muris

are the two known species most closely related to

E. canis

(based on 16S rDNA sequence homology) Faint bands at sizes other than that of the target sequence were occasionally observed, indicating that similar omp-1 sequences may be amplified under these conditions, but the robust 200 bp band was only observed with

E. canis

template.

Example 2

E. chaffeensis

p28 ORF sequences were compared to identify prospective oligonucleotide primer sequences for a PCR assay. Design of the primers for this PCR assay was similar to that described for

E. canis,

except that identities of these primers to the three clusters of p28 sequences among the

E chaffeensis

isolates were also considered by dividing the product of the annealing and

E. canis

identity scores by the mean of the three

E.chaffeensis

cluster identity scores. Primers were designed from the

E. chaffeensis

Arkansas isolate p28 sequence. These primer sequences were aligned with the homologous portions of the

E. canis

p30 geme and the three p28 clusters and the identity of these sequences determined Total annealing scores, identity to the different p28 clusters and identity to p30 were then used to rank these primer sets. A second approach to primer design involved the design of primers to the consensus sequence of all three

E. chaffeensis

p28 clusters through less stringent annealing score parameters. These primers, which are shown in Table 3 above are expected to be universal for all five

E. chaffeensis

isolates that composed these three clusters.

Primers for

E. chaffeensis

were designed with the Wisconsin Package (GCG) Version 10 software suite (Madison, Wis.). This was accomplished with a multiple sequence alignment of the target template from isolates representing all three clusters of

E. chaffeensis

of and the

E. canis

p30 sequence. Initially optimal primer sequences could not be obtained from the consensus of all three p28 sequences, thus primers were designed from the

E. chaffeensis

Arkansas isolate p28 sequence since this is the isolate to be used in the proposed investigation. Twenty-five sets of primers were then identified. These primer sequences were aligned with the homologous portions of p30 and the three p28 clusters and the identity of these sequences determined. Total annealing scores, identity to the different p28 clusters and identity to p30 were then used to rank these primer sets. Three primer sets were chosen for development of a p28-based assay by virtue of their overall rank and relative positions flanking or internal to other primer sets for nested PCR and found to amplify

E. chaffeensis

DNA from infected canine cells. Two of the

E. chaffeensis

Arkansas isolate-specific primer sets were optimized and used as a nested PCR assay to detect the pathogen in intrastadially infected male ticks of four species known to parasitize dogs.

TABLE 4

Ranks of

E. chaffeensis

p28 primer sets with predicted annealing and specificity values.

a

Aneal-

Identity Scores to

E. chaffeensis

Identity Scores to

Primers

ing

Cluster I

Cluster II

Cluster III

E. canis

Set

Fwd.

Rev.

Score

Fwd.

Rev.

Fwd.

Rev.

Fwd.

Rev.

Mean

Fwd.

Rev.

Mean

Rank

1

b

67-85

341-320

55

1.0

1.0

0.90

0.95

1.0

1.0

0.98

0.68

0.91

0.80

8

2

58-78

321-302

58

1.0

0.95

0.95

0.95

1.0

1.0

0.98

0.57

0.90

0.74

7

3

58-78

314-294

58

1.0

0.90

0.95

0.90

1.0

1.0

0.96

0.57

0.86

0.72

5

4

61-82

339-319

58

1.0

0.90

0.91

0.95

1.0

1.0

0.96

0.64

0.86

0.75

10

5

61-82

315-294

59

1.0

0.91

0.91

0.91

1.0

1.0

0.96

0.64

0.86

0.75

15

6

61-82

320-301

59

1.0

1.0

0.91

0.95

1.0

1.0

0.98

0.64

0.90

0.77

16

7

60-80

314-294

60

1.0

0.90

0.95

0.90

1.0

1.0

0.96

0.62

0.86

0.74

13

8

59-79

314-294

60

1.0

0.90

0.95

0.90

1.0

1.0

0.96

0.62

0.86

0.74

14

9

58-79

322-302

60

1.0

0.95

0.95

0.95

1.0

1.0

0.98

0.59

0.90

0.75

11

10

58-78

320-301

60

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.57

0.90

0.74

9

*11

b

58-78

341-320

61

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.57

0.91

0.74

12

12

59-79

320-301

62

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.62

0.90

0.76

19

13

b′

78-99

314-294

62

1.0

0.90

0.95

0.90

1.0

1.0

0.96

0.41

0.86

0.64

2

14

59-80

320-301

62

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.64

0.90

0.77

20

15

58-78

352-331

62

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.57

0.77

0.67

3

16

58-78

339-319

64

1.0

0.90

0.95

0.95

1.0

1.0

0.97

0.57

0.86

0.72

18

17

78-99

341-320

64

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.41

0.91

0.66

4

18

58-78

313-293

65

1.0

0.95

0.95

0.90

1.0

1.0

0.97

0.57

0.95

0.76

25

19

60-80

341-320

64

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.62

0.91

0.77

21

20

78-99

352-331

65

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.41

0.77

0.59

1

21

61-82

314-293

65

1.0

0.91

0.91

0.91

1.0

1.0

0.96

0.64

0.86

0.75

24

22

59-79

341-320

65

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.62

0.91

0.77

23

23

59-80

352-331

66

1.0

1.0

0.95

0.95

1.0

1.0

0.98

0.64

0.77

0.71

17

24

b′

78-99

314-293

66

1.0

0.91

0.95

0.91

1.0

1.0

0.96

0.41

0.86

0.64

6

25

60-80

339-319

66

1.0

0.90

0.95

0.95

1.0

1.0

0.97

0.62

0.86

0.74

22

a

All values determined as described for Table 1, except for Rank, which was determined by the product of annealing score and mean

E. canis

identity score, divided by mean

E. chaffeensis

annealing score; lower values have higher rank.

b,b′

Nested primer sets.

*Primer sets chosen for optimization.

Primers derived from both approaches were chosen for use in a p28-based assay by virtue of their overall rank and relative positions flanking or internal to other primer sets for nested PCR. These candidate primer were synthesized and they were all tested with 50 ng of DNA from

E. chaffeensis

-infected DH82 cells and Life Technologies reagents at concentrations of 1×PCR buffer, 1.5 mM MgCl

2

, 0.5 μM of each primer and 0.025 U/μl Platinum Taq DNA Polymerase at an initial incubation period at 94° C. for 2 min followed by 25 cycles of 94° C. for 30 sec, 50° C. for 30 sec and 72° C. for 1 min 7 min final extension step at 72° C. Each primer set tested amplified a clean, robust amplicon of the expected size under unoptimized conditions. Primer sets F58/R341 and F78/R314 were chosen first for optimization due to the more stringent parameters used for their selection.

Optimum parameters for primer sets F58/R341 and F78/R314 were then used to assay canine and tick host samples. Dog number 146 was inoculated iv. with DH82 cells that were infected in vitro with

E. chaffeensis

(Arkansas isolate). Once the dog was PCR-positive by the 16S rDNA-based PCR assay and seroconverted, it was infested with 150 adult males of

A. americanum,

and 10 each of

A. maculatum, D. variabilis

and

R. sanguineus.

These ticks were allowed to simultaneously acquisition feed seven days prior to their removal and incubation in a humidity chamber for another 10 days. The held ticks were then placed at 37° C. at 100% rh for 80 hr before they were aseptically bisected and digested with proteinase K in the presence of nonionic detergents. These ticks (2.5 μl) were then assayed with

E. chaffeensis

p28-specific nested primer sets 11

A

(F58/R341)and 13

A

(F78/R314).

Samples from each tick species fed tested PCR-positive at a frequency of 20-40% with the nested assay. These results are the first to our knowledge to demonstrate detection of

E. chaffeensis

in experimentally infected ticks, only the second time to detect

E. chaffeensis

in individual ticks and the first detection of this pathogen in

A. maculatum

or

R. sanguineus.

Further sequence analysis with less stringent criteria did reveal 20 primer sequences complementary to the p28 consensus sequence of all three clusters. Two sets of the consensus primers (See * in Table 5 below) were also tested and found to amplify robust amplicons from

E. chaffeensis

DNA.

TABLE 5

Ranks of universal

E. chaffeensis

p28 primer

sets with predicted values.

Aneal

Identity Scores to

Primers

-ing

E. canis

Set

Fwd.

Rev.

Score

Fwd.

Rev.

Mean

Rank

*1

84-101

360-341

57

0.5

0.8

0.65

1

2

84-101

365-346

57

0.5

0.8

0.65

1

*3

50-68

669-649

61

0.58

0.67

0.62

3

4

83-100

364-346

63

0.44

0.79

0.62

5

5

83-100

359-341

63

0.44

0.79

0.62

5

6

50-68

669-650

63

0.58

0.65

0.61

4

7

55-74

670-650

64

0.65

0.67

0.66

7

8

45-62

359-341

65

0.61

0.79

0.70

10

9

15-32

364-346

66

0.78

0.79

0.78

19

10

15-32

359-341

66

0.78

0.79

0.78

19

11

50-74

662-637

67

0.64

0.73

0.69

11

12

50-68

662-641

67

0.58

0.68

0.63

8

13

45-62

365-348

68

0.61

0.78

0.69

13

14

45-62

364-346

69

0.61

0.79

0.70

15

15

51-74

670-645

69

0.67

0.58

0.62

9

16

53-74

670-649

71

0.69

0.64

0.66

12

17

53-74

662-641

71

0.67

0.72

0.69

17

18

57-74

674-657

73

0.67

0.72

0.69

14

19

50-68

673-656

73

0.58

0.72

0.65

14

20

55-74

673-656

75

0.65

0.72

0.69

18

Number	Date	Country
WO9913720	Mar 1999	WO
WO0032745	Jun 2000	WO

Methods for detecting Ehrlichia canis and Ehrlichia chaffeensis in vertebrate and invertebrate hosts

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Foreign Referenced Citations (2)

Non-Patent Literature Citations (4)

Entry
McBride et al. Molecular Cloning of the Gene for a Conserved Major Immunoractive 28-Kilodalton Protein of Ehrlichia canis: a Potential Serodiagnostic Antigen Clinical and Diagnostic Laboratory Immunology May 1999 p. 392-399.*
Ohashi et al. Cloning and Characterization of Muligenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis Journal of Clinical Microbiology Sep. 1998 p. 26.*
“Detection of Ehrlichia canis in Rhipicephalus sanguineus with a p30-based PCR Assay” by Grover, et al. Seventy-ninth Conference of Research Workers in Animal Diseases, Chicago, Illinois, Nov. 7-9, 1999.
“A Polymerase Chain Reaction Assay for Ehlrichia Canis” by Stich, et al., 3rd Annual Conference, Tick an Tick-Borne Pathogens: Into the 21st Centure, High Tatra Mountains, Slovakla, Aug. 30—Sep. 3, 1999.