JMD ASIP MEMBERSHIP
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sirigireddy, K. R.
Right arrow Articles by Ganta, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sirigireddy, K. R.
Right arrow Articles by Ganta, R. R.
JMD 2005, Vol. 7, No. 2
Copyright © 2005 American Society for Investigative Pathology & Association for Molecular Pathology

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Kamesh R. Sirigireddy and Roman R. Ganta

From the Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen’s genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several rickettsial agents of the family Anaplasmataceae cause severe, tick-borne pathogen infections in a wide range of vertebrate host species.1 They include emerging infections in humans with Ehrlichia chaffeensis, Ehrlichia ewingii, and Anaplasma phagocytophilum and canine infections with E. canis, E. ewingii, E. chaffeensis, A. platys, and A. phagocytophilum.1, 2, 3, 4, 5, 6, 7, 8, 9 Human E. canis infection is also reported in an isolated case from Venezuela, South America.10 Several Ehrlichia and/or Anaplasma species also infect many domestic and wild animals, including dogs, cattle, and horses.1 Co-infections with two or more rickettsiales and other tick-transmitted pathogens are common in vertebrate and tick hosts.6, 9, 11, 12, 13, 14, 15, 16, 17 Persistent subclinical infections of Ehrlichia and Anaplasma species are well documented in the hosts recovered from a clinical disease.18, 19, 20, 21

Because rickettsiales are able to infect a broad range of hosts, and multiple pathogens can co-exist in both vertebrate and invertebrate hosts, the availability of a rapid, highly sensitive, and specific test that can diagnose one or more pathogens, including co-infections, in a test sample will be valuable for timely diagnosis and treatment. Such a test will be useful for monitoring and controlling the spread of infections from ticks. Moreover, a multiplex molecular test will be valuable in studies to assess the impact of co-infections on the disease outcome. Similarly, it will be useful in studies to evaluate vaccines and therapeutics.

In this study, we described the development of a rapid, two-step, species-specific multiplex molecular test to detect one or more infections with three Ehrlichia and two Anaplasma species. We also reported a novel cloning strategy to generate the positive controls needed to establish the test. The molecular test was used to detect natural infections, including co-infections in dogs with E. chaffeensis, E. canis, E. ewingii, A. platys, and A. phagocytophilum. The dog was chosen as the model system to evaluate the test utility because it is known to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RNA Isolation
E. chaffeensis Arkansas isolate and E. canis Oklahoma isolate were grown in a canine macrophage cell line, DH82, as described previously.22, 23 A 0.5-ml sample of a culture pellet was centrifuged at 13,000 x g for 15 minutes at 4°C and used to isolate total RNA using the Tri-reagent method (Sigma Chemical Co., St. Louis, MO) as per the manufacturer’s instructions. The RNA recovered was dissolved in 100 µl of nuclease-free water and stored at –70°C in the presence of 40 U of RNase inhibitor, RNasin (Ambion Inc., Austin, TX). Total RNA from blood samples was also isolated according to the Tri-reagent method by using 0.25 ml of blood collected in ethylenediamine tetraacetic acid (EDTA) and the final pellet was resuspended in 100 µl of nuclease-free water. A capture method was also used to isolate Ehrlichia/Anaplasma species 16S ribosomal RNA (rRNA) (described below).

DNA Isolation
Five ml of 80 to 100% Ehrlichia-infected DH82 culture was used to isolate genomic DNA by the sodium dodecyl sulfate, proteinase K, phenol, chloroform, isoamyl alcohol method.24 A. phagocytophilum genomic DNA, isolated from in vitro cultures, was provided by Dr. J. Stephen Dumler, The Johns Hopkins Medical Institutions, Baltimore, MD.

Capture Primer, Polymerase Chain Reaction (PCR) Primer, and TaqMan Probe Design for the Multiplex Reverse Transcriptase (RT)-PCR Assay
The 16S rRNA gene sequences for several Ehrlichia/Anaplasma species, available in the GenBank nucleotide sequence database, were downloaded and aligned by using the University of Wisconsin’s Genetic Computer Group programs Pileup and Pretty.25 Genera-specific regions were used to design a capture primer to facilitate capturing of 16S rRNA of all Ehrlichia and Anaplasma species from a sample. PCR primers for the real-time RT-PCR assay development were also designed from the genera-specific region (Figure 1Go and Table 1Go ). Species-specific regions were used from the alignment to design TaqMan probes for use in the real-time, species-specific pathogen detection of E. canis, E. chaffeensis, E. ewingii, A. phagocytophilum, and A. platys. Fluorescent reporter dyes and quencher molecules on the TaqMan probes were carefully selected to facilitate the multiplex assay (Table 1)Go . The primers and TaqMan probes were custom synthesized from Nucleic Acid Facility, University of Pennsylvania, Philadelphia, PA, or from Integrated DNA Technology Inc., Coralville, IA.



View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Sequence alignment of the 16S rRNA genes and the design of RT-PCR primers, capture primer, and TaqMan probes. The 16S rRNA gene sequences available in the GenBank database for Ehrlichia and Anaplasma species were analyzed using the Genetic Computer Group programs, PILEUP and PRETTY.25 GenBank numbers for the sequences are M73222, E. chaffeensis; M73221, E. canis; M73227, E. ewingii; X61659, E. ruminantium; M82801, A. platys; M73220, A. phagocytophilum; and M60313, A. marginale. The complete sequence of E. chaffeensis from nucleotides 1 to 151 and 426 to 482 are presented. The sequences for the other species are shown only when they differ from the E. chaffeensis sequence. A dot indicates identity with the E. chaffeensis sequence. Dashes refer to the gaps introduced by the PILEUP program to obtain the best alignment. The PCR primers and the capture primer were identified with their names. Lines with arrowheads refer to the orientation of the primers. The genera- and species-specific TaqMan probes are identified with shaded text.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Primers and Probes Used in the Multiplex Molecular Test Development

 
Establishment of Positive Control Plasmids for Ehrlichia and Anaplasma Species
Genomic DNA of E. chaffeensis, E. canis, and A. phagocytophilum were used as the templates to amplify a 0.48-kb 16S rRNA gene segment by using Ehrlichia/Anaplasma common TaqMan forward primer and Ehrlichia/Anaplasma common reverse primer. The PCRs were performed with 200 ng of genomic DNA using the Ampli TaqPCR reagent kit (Applied Biosystems, Foster City, CA). The thermal cycles include the initial denaturation for 3 minutes at 94°C, 45 cycles of 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 60 seconds, followed by one cycle of 72°C for 3 minutes. The PCR products were resolved on a 1% agarose gel in 1x Tris-acetate EDTA buffer (40 mmol/L Tris-acetate, 1 mmol/L EDTA, pH 8.0) containing 0.1 µg/ml of ethidium bromide, and were visualized under UV light.24 The amplicons were blunt-ended by using T4 DNA polymerase and were ligated into the EcoRV site of the plasmid, pBlue Script SK+. Subsequently, the ligation mix was transformed into E. coli, XL1 Blue (Stratagene, La Jolla, CA), the transformants containing the recombinant plasmids were selected and plasmid DNA was isolated and used for sequencing with a Thermo-sequencing reaction kit (USB Corp., Cleveland, OH).24

Because our attempts to obtain genomic DNA for A. platys and E. ewingii were unsuccessful, we could not use the approach just described for developing positive controls for these two organisms. A novel method for generating positive control plasmids without the need of genomic DNA for these two species was designed (Figure 2)Go . The regions of the 16S rRNA gene segment selected for preparing the positive control plasmid shares extensive homology between E. ewingii and E. chaffeensis, except for one variable region located in the middle (Figure 1)Go . Similarly, A. phagocytophilum and A. platys differ mostly at the central variable region (Figure 1)Go . This information was used to design long forward primers having central variable regions specific for each species, plus Ehrlichia/Anaplasma conserved 5' and 3' overhangs. E. ewingii-specific primer was 96 bases, whereas the A. platys-specific primer was 88 bases in length (Table 1)Go . These primers were used in combination with the Ehrlichia/Anaplasma common reverse primer in the PCRs, with E. chaffeensis or A. phagocytophilum-positive control plasmids as the templates (Figure 2)Go . Because the long primers of E. ewingii and A. platys anneal to the E. chaffeensis and A. phagocytophilum templates only at the 5' and 3' ends, the amplicons are expected to contain E. ewingii- and A. platys-specific sequences, respectively. The PCR products were cloned into the plasmid, Blue Script, and the insert sequences were verified by performing DNA sequence analysis as previously described.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Schematic representation of the molecular strategy used to generate E. ewingii-positive control. Alignment shown in Figure 1Go reveals that E. ewingii 16S rRNA sequence differs from E. chaffeensis mostly at the central variable region where the TaqMan probes were designed. The schematic representation of the sequences in this figure shows variable sequences as different shaped lines. To generate an E. ewingii 16S rRNA gene segment, a long forward primer was designed. It contains the Ehrlichia species common sequence at the 5' and 3' ends and E. ewingii-specific sequence in the middle. This primer and a genera-specific reverse primer, together with the E. chaffeensis 16S rRNA gene segment as the template were used in the PCR to generate amplicons containing E. ewingii sequence. The amplicons were cloned into a plasmid. A similar strategy was also used to clone A. platys-positive control plasmid using the A. phagocytophilum DNA segment as the template.

 
In Vitro Transcription
In vitro transcripts from the inserts of all five plasmids were prepared for use in the molecular test development. The recombinant plasmids were digested with BamHI restriction enzyme (a site for BamHI is located at the 3' end of the insert in the multiple-cloning sites region of the plasmid). BamHI was chosen because it does not have a recognition sequence within the inserts, thus allowing the plasmids to linearize downstream to inserts to facilitate the synthesis of the transcripts by using T7 polymerase. BamHI-digested plasmid DNAs (~3 µg) were used to generate recombinant transcripts by using the T7 MEGAscript high-yield transcription kit as outlined in the kit protocol (Ambion Inc.). The recombinant transcripts were purified free of plasmid DNA by treating with DNase I and using the RNA purification kit, MEGAclear (Ambion Inc.). The quality, quantity, and length of the transcripts were determined in a Bio-analyzer (Agilent Technologies, Palo Alto, CA).

Magnetic Capture of Ehrlichia/Anaplasma 16S rRNA
A magnetic capture technique was developed to isolate Ehrlichia/Anaplasma 16S rRNA from in vitro cultures or blood by following a strategy similar to the one reported for Chlamydia trachomatis,26 but with several modifications (Figure 3)Go . An Ehrlichia and Anaplasma genera-specific capture primer was designed from the complementary sequence of 16S rRNA that is conserved in all known Ehrlichia/Anaplasma species. A 12-nucleotide-long dC tail and biotin molecule were added to the capture primer at the 5' end to facilitate the capturing of rRNA (Table 1)Go . The capture primer, in combination with streptavidin-coated magnetic beads and a magnetic separation rack (New England Biolabs Inc., Beverly, MA), was used to isolate the Ehrlichia/Anaplasma species 16S rRNA. Magnetic beads coated with 170 pmol of streptavidin were washed twice with a wash buffer (0.5 mol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA) and were separated with a magnetic rack. The washed beads were incubated at room temperature for 5 minutes with 25 µl of 0.8 µmol/L capture primer.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Schematic representation of the 16S rRNA capture method. A sample containing Ehrlichia/Anaplasma species organisms is lysed in the GTC buffer and added to the conjugate of the avidin-coated magnetic particles coupled with the biotin-linked capture primer. A magnetic stand is used to capture and purify 16S rRNA from the lysed sample.

 
Affinity between biotin on the capture primer and streptavidin on the magnetic beads allowed the beads and probe to form a complex. The complex was washed twice with the wash buffer to remove excess unbound capture primer. Twenty-five µl in vitro culture of E. canis or E. chaffeensis (5 to 10 million of 80 to 90% infected DH82 cells/ml) and 25 µl of uninfected canine blood were mixed, lysed by adding an equal volume of GTC buffer (0.4 mol/L Tris-HCl, pH 7.85 containing 5 mol/L guanidinium thiocyanate, 0.5 mol/L sarkosyl, and 20 mmol/L EDTA), incubated at 65°C for 5 minutes, transferred to a tube containing the magnetic beads-capture primer complex, and stored at room temperature for 10 minutes. This step allows the formation of Ehrlichia/Anaplasma 16S rRNA and capture primer hybrids (Figure 3)Go . The mixture was then washed twice with the wash buffer and once with a low-salt wash buffer (0.15 mol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, and 1 mmol/L EDTA). Ehrlichia 16S rRNA was eluted by adding 50 µl of preheated (70°C) TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) and was stored at –80°C in the presence of RNasin until further use.

Real-Time Quantitative PCR and RT-PCR
TaqMan-based real-time amplification27, 28 was performed by using the Smart Cycler system (Cepheid, Sunnyvale, CA). Because the Smart Cycler system has the capability to detect the fluorescent emission from only four unique probes, the test procedure to detect the five pathogens was split into two parts. Part 1 of the test included the detection format for E. chaffeensis, E. canis, and E. ewingii. Part 2 of the test was designed to detect A. platys and A. phagocytophilum. The mixture for PCR assay is 25 µl in volume, containing 10 pmol each of the TaqMan forward and reverse primers, 10 nmol of dNTPs, 125 nmol MgCl2, 4 U of platinum TaqDNA polymerase (Invitrogen Technologies, Carlsbad, CA) and varying concentrations of TaqMan probes for each pathogen. They are 7.5 pmol for E. chaffeensis; 3.75 pmol for E. canis; 8.75 pmol for E. ewingii; 6.0 pmol for A. platys; and 3.75 pmol for A. phagocytophilum. The concentrations of the TaqMan probes were chosen after the standardization experiments to yield optimal results. The temperature cycles used for the assay are: initial heating for 3 minutes at 95°C, followed by 45 cycles of 95°C for 15 seconds, 50°C for 30 seconds, and 60°C for 60 seconds. The PCR product formation was monitored in real-time by measuring the emitted fluorescence in the extension phase of the PCR cycles with the Smart Cycler system. The machine qualifies a reaction positive for the presence of a template when it detects 10 fluorescent units for each fluorescent emission channel. The PCR cycle at which this occurs is regarded as the Ct value and it is template concentration-dependent. Similarly, the real time RT-PCR was performed in a 25-µl reaction, but containing 1 µl of SS-III and Taq mix (SuperScript-III, one-step RT-PCR system with platinum TaqDNA polymerase; Invitrogen Technologies). Thermal cycles for RT-PCR included an additional initial step at 48°C for 30 minutes to generate the cDNA. The optimal assay conditions for species specificity and multipathogen detection were established by using the plasmid DNA of cloned 16S rRNA gene segments. Ten-fold serial dilutions of the positive control plasmids or in vitro synthesized transcripts (ranging from 1 billion to 1 molecule) were made from the known quantities of the plasmid DNA or RNA. The samples were used in real-time PCR and RT-PCR analysis to determine the Ct values to establish detection limits of the multiplex molecular test.

Collection of Blood from Dogs Clinically Suspected of Canine Ehrlichiosis
Clinicians from several regions within the Unites States were contacted by phone, fax, and/or mail to collect blood samples from clinically suspected canine ehrlichiosis cases. The criterion of a clinical ehrlichiosis in a dog was at the discretion of the clinician examining a case. The case reports were also received and archived at the K-State diagnostic laboratory. A total of 95 samples were collected in EDTA tubes during 2003 from Arkansas, Arizona, Connecticut, Florida, Georgia, Kansas, Kentucky, Missouri, New Mexico, New York, and US Virgin Islands. Typically, the samples were received in ice packs by overnight shipment. Within 3 days of receipt, plasma was separated and stored at –80°C for indirect fluorescent antibody (IFA) analysis. RNA from 0.25 ml of the plasma-free blood was isolated and resuspended in 100 µl of nuclease-free water by following the Tri-reagent RNA isolation method (described above). RNA also was isolated from 50 µl each of clinically suspected canine blood samples by the magnetic capture. The final RNA was eluted in 50 µl of TE buffer. RNA recovered from an equivalent of 6.5 µl of whole blood was used for evaluating samples for the presence of Ehrlichia/Anaplasma species. The assays included reaction-positive and -negative controls. Similarly, the RNA purification method included a cross contamination control that followed through all extraction procedures.

E. canis IFA
The plasma samples were assessed for antibodies against E. canis by the IFA test29 with a commercial kit per the protocol using fluorescein isothiocyanate-conjugated secondary antibody (VMRD Inc., Pullman, WA). E. canis IFA titers were determined by using 1:128 or higher (up to 1:4096) diluted plasma. A limited number of samples were also tested for the presence of E. canis antibodies by SNAP 3Dx test (IDEXX Laboratories, Westbrooke, ME).

Data Analysis
Paired t-test analysis was performed using the Statview statistical software package (SAS Institute Inc., Cary, NC). A P value <0.01 is considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The 16S rRNA as the Multiplex Molecular Test Target
The 16S rRNA was selected as the target for the Ehrlichia and Anaplasma species multiplex pathogen diagnosis because it is expected to be present in multiple copies in a cell. Genera- and species-specific sequences were identified from the16S rRNA gene segments of Ehrlichia and Anaplasma species to prepare primers and probes used for the molecular test development (Figure 1)Go . To aid in establishing the test, recombinant plasmids containing the 16S rRNA gene segments were prepared using conventional cloning strategies for E. chaffeensis, E. canis, and A. phagocytophilum. A novel molecular strategy was used to generate positive control plasmids for E. ewingii and A. platys pathogens (Figure 2)Go .

Multiplex, Species-Specific Pathogen Detection
A TaqMan-based, quantitative, real-time RT-PCR method is used in developing the multiplex Ehrlichia/Anaplasma molecular test. The species-specific probes for the five pathogens detected templates only from their respective species and did not cross-react with templates of the other four species. Serial dilution of the 16S rRNA recombinant transcripts made from the positive control plasmids aided in determining the analytical sensitivity and linearity (Figure 4)Go . The minimum number of transcripts detected by the test was 100 molecules for all five pathogens. The test was also linear with differing concentrations of transcripts up to 1 billion molecules (Figure 4)Go . To examine whether nonequivalent molar ratios can be similarly detected a fixed concentration of one of the three species of Ehrlichia or Anaplasma recombinant transcripts and differing concentrations of the other transcripts were tested for the detection throughout a range of 1000 to 100,000 molecules. The assay identified the transcripts when the difference in the concentration among the templates is up to 100-fold. Beyond this, only the template having the highest concentration was tested positive. The sensitivity of detection by real-time RT-PCR assay for the RNA recovered by the magnetic capture method was also compared with a commercially available Tri-reagent RNA isolation method. RNA was isolated from either cultured E. chaffeensis or E. canis organisms, or from plasma-free or whole blood from a healthy dog spiked with cultured organisms for this analysis. Data for E. chaffeensis RNA recovery by these methods are reported in Table 2Go . The capturing method using the cultures spiked in whole blood resulted in significantly less recovery of RNA than from the Tri-reagent method. The RNA isolated from in vitro cultures or cultures spiked in plasma-free blood had no significant differences in the recovery efficiency (Table 2)Go .



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. Multiplex molecular test’s detection sensitivity and linearity with RNA concentration. Serial 10-fold dilutions of the RNA transcripts, made from the positive control plasmids, were analyzed at equivalent molar concentrations of the three Ehrlichia species (A) and two Anaplasma species (B). The fluorescent emission from serial dilution templates for one species each (E. canis and A. platys) is shown in the insets. The average Ct values from three independent experiments for each template concentration of the three Ehrlichia species and two Anaplasma species were plotted against the log number of RNA molecules in A and B, respectively.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Comparison of RNA Recovery Efficiency of Magnetic Capture and Tri-Reagent Methods

 
Ratio of 16S rRNA to Its Gene Copies
It is uncertain how many 16S rRNA molecules are present in Ehrlichia and Anaplasma species. Because the assay consistently detected 100 16S rRNA transcripts, the minimum detection sensitivity can be one organism provided that there are at least 100 molecules of 16S rRNA present per Ehrlichia/Anaplasma organism. Genomic DNA and total RNA from equal volumes of E. chaffeensis cultures were isolated and were used to determine the ratio between 16S rRNA and its gene targets in the genome by real-time PCR and RT-PCR analysis. The analysis was performed four times, using different batches of in vitro cultures. The analysis revealed a 100- to 180-fold difference in the rRNA to rDNA targets.

Evaluation of Blood Samples from Dogs with Suspected Ehrlichiosis
To examine the utility of the test for routine pathogen diagnosis, 95 blood samples were collected from dogs clinically suspected for ehrlichiosis in 2003 and were analyzed by the multiplex test. Twenty-three samples tested positive by the molecular test for at least one of the five pathogens (Table 3)Go . Of the 23 samples, 9 samples were positive for E. canis (39.16%), 6 were positive for E. ewingii (26.1%), 6 were positive for A. platys (26.1%), 2 were positive for E. chaffeensis (8.7%), and 1 was positive for A. phagocytophilum (4.4%). The RNA molecules detected in test-positives ranged from 1.60 x 103 to 3.05 x 107per ml of blood. One sample tested positive for both E. canis and A. platys. E. canis real-time RT-PCR-positives were detected from samples obtained from Florida, Kansas, Oklahoma, New Mexico, and Arizona. E. ewingii-positives were identified from Kansas, New Mexico, Arkansas, Missouri, and Florida samples. E. chaffeensis-positives were found in samples obtained from Kansas and Missouri. A. platys-positives were detected in samples from New Mexico and Missouri whereas A. phagocytophilum was identified only in a sample from New York.


View this table:
[in this window]
[in a new window]
  		Table 3. Canine Blood Samples Analyzed by the Multiplex Molecular Test and for E. canis Antibody Titers

 
Because the molecular test detected two or more templates only if they are present in the range of up to 100-fold concentration difference (described above), we randomly re-evaluated 12 of the 23 positive test samples for other species by performing simplex real-time RT-PCRs. We also evaluated these samples by using an Ehrlichia/Anaplasma common TaqMan probe to further confirm the molecular test-positives. All single-infection test-positives also tested positive with the Ehrlichia/Anaplasma common TaqMan probe, but were found to be negative for the remaining four species by the simplex RT-PCR analysis.

Plasma from 84 blood samples was analyzed for E. canis antibody titers. The samples that tested positive are included in Table 3Go . Thirty-eight samples tested positive for E. canis antibody, with titers ranging from 1:128 to 1:4096. All nine samples positive for E. canis by molecular testing also contained antibody titers. Three E. ewingii molecular test-positives were positive for the E. canis antibody titer. Only one A. platys real-time RT-PCR-positive tested positive for E. canis IFA. One E. chaffeensis real-time RT-PCR-positive also had detectable E. canis antibodies. Antibody data for two A. platys and one each of the E. ewingii and E. chaffeensis molecular test-positives were not available. A sample positive for both A. platys and E. canis also had the antibody titer for E. canis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we describe a multiplex molecular test having the ability to detect single or co-infections with up to five tick-transmitted rickettsiales; E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys. The molecular test procedure includes a simplified and rapid magnetic-capture technique to purify 16S rRNA; its enrichment by genera-specific RT-PCR assay and species-specific pathogen detection by real-time monitoring with species-specific TaqMan probes. The whole procedure takes ~1.5 hours of hands-on time plus 2 hours of thermocycler analysis time.

The magnetic-capture method for rapid purification of pathogens’ rRNA described in this study has the potential for automation for high-throughput sample processing. The multiplex molecular test development included the preparation of in vitro transcripts for 16S rRNA after gene segments for all five pathogens were cloned. The transcripts were used in defining the minimum number of RNA molecules needed to identify a test-positive. This information, together with the determination of the ratio between the rRNA and rDNA allowed us to estimate the detection limit of the test. The test has the ability to detect as few as one infected host cell in a test sample. Our data are in agreement with a previous report suggesting that an RT-PCR method using 16S rRNA as the target is 100 times more sensitive than a PCR method.30

Many Ehrlichia and Anaplasma species cause infection in widely different vertebrate hosts.1 Co-infections with two or more pathogens are also reported in vertebrates and ticks.6, 11, 12, 13, 14, 15, 17, 31 The ability of this test to detect all known Ehrlichia and Anaplasma species causing infections in a host, including co-infections, will be valuable for infection monitoring in ticks and several vertebrate hosts, including humans. However, our analysis suggested that the multiplex assay detected only templates having the highest concentration when nonequivalent molar ratios of two or more pathogen templates are present at a ratio greater than 100-fold. This can be a potential limitation for detecting multipathogen-infected sample when two or more pathogens are differing in their bacteremia by greater than 100-fold. This will not have any bearing in initiating a treatment regime as all Ehrlichia/Anaplasma pathogen infections respond to the same antibiotic treatment. However, as we described in the current study for clinical samples, this limitation may also be overcome by repeating the sample analysis on positives after omitting the probes that yield positives.

Many PCR- and RT-PCR-based methods to detect infections with Ehrlichia and Anaplasma species are described.30, 32, 33, 34, 35, 36, 37, 38, 39 A PCR followed by the reverse line blot hybridization assay to detect species-specific amplified products is described recently for the simultaneous detection of Ehrlichia and Anaplasma species in ruminants and dogs.40, 41 In this assay, a 16S rRNA gene segment was amplified with a genera-specific primer set, and the products are hybridized to a membrane coated with species-specific oligonucleotide probes. The PCR assays described in the literature require additional protocols after a PCR or RT-PCR step. TaqMan-based real-time PCR methods that eliminate the need for analysis after amplification are also described for E. chaffeensis and A. phagocytophilum infection detection.42, 43 All of the reported methods, including the real-time PCR assays, are designed to detect only one pathogen at a time. The current article is the first report that describes a simplified multipathogen rRNA recovery and detection protocol for Ehrlichia and Anaplasma species infections.

The multiplex molecular test was used to evaluate infections in 95 canine blood samples suspected of ehrlichiosis. The test identified 23 positives, which included 22 for single-pathogen infections of the five rickettsials and one positive sample for co-infection with E. canis and A. platys. These results support previous reports that dogs can acquire infections with E. canis, E. chaffeensis, E. ewingii, A. platys, and A. phagocytophilum.6, 7, 8 The rRNA detected in the test-positives ranged from 1.60 x 103 to 3.05 x 107 molecules per ml of blood. Because the pathogens have a minimum of 100 rRNA molecules per each genomic DNA target, the test-positives ranged from 1.60 x 101 to 3.05 x 105 organisms per ml of infected blood. Infection rates in canine samples seem similar for E. canis, E. ewingii, and A. platys. E. chaffeensis and A. phagocytophilum were detected in fewer samples.

Antibody-based diagnostic procedures, such as the IFA or enzyme-linked immunosorbent assay, are commonly used for the diagnosis of rickettsial infections.44, 45, 46, 47 For example, IFA for E. canis is the most commonly used technique to monitor canine ehrlichiosis infections.29, 47 Sensitivity of the E. canis-specific IFA to the multiplex molecular test was compared by evaluating the clinical samples. All E. canis molecular test-positives were detected by the IFA test. Some molecular test-negative samples also had high antibody titers. These may represent samples from dogs that had an E. canis infection in the past, but cleared the pathogen, or may represent carrier animals. The majority of the E. ewingii, A. platys, and A. phagocytophilum molecular test-positives were negative by E. canis IFA. This is not surprising because they are not expected to have high antibody titers against E. canis. E. canis antibody-positives were also detected in five animals that tested positive by the molecular test for E. ewingii, E. chaffeensis, or A. platys. These may represent samples from dogs having prior exposure to E. canis with persistent antibody titers. Although it is possible, E. canis antibody titers in this group may not have resulted from the cross-reactions of antibodies against E. ewingii, E. chaffeensis, or A. platys, because some molecular test-positives with these organisms were also negative for E. canis antibody titers (Table 3)Go . Comparison of the IFA and molecular test results demonstrate that the E. canis-specific IFA fails to detect infections with four other Ehrlichia/Anaplasma species. These data demonstrate that the multiplex molecular test has added advantage over simplex test in detecting infections with closely related organisms that do not share antibody cross-reactivity.

The molecular test can be adapted to serve as a valuable diagnostic tool for monitoring human infection because E. chaffeensis, E. ewingii, and A. phagocytophilum also infect people.1, 2, 3, 4, 5, 9 Infections with a foreign animal disease agent, E. ruminantium, is of increasing concern to the ruminant population on the mainland United States, because of its presence in the Caribbean and its likely introduction through exotic animals and ticks.48, 49 Likewise, bovine anaplasmosis, caused by A. marginale, is an endemic disease in the United States and other parts of the world.21 By incorporating additional species-specific probes, the molecular test described here can be adapted for infection monitoring with these closely related pathogens.

In conclusion, we established a multiplex, molecular test useful to rapidly diagnose single or co-infections with up to five tick-borne rickettsial pathogens. The test serves as a new tool to monitor tick-borne infections in dogs and can be adapted for screening emerging tick-borne infections in people, cattle, horses, and ticks.


   Acknowledgments
 
We thank Ms. Chuanmin Cheng for technical help in growing Ehrlichia cultures, Drs. Patricia Payne and Michael Dryden for help in establishing contacts with clinicians and veterinary practitioners for sharing clinical samples, and Dr. J. Stephen Dumler for providing A. phagocytophilum DNA.


   Footnotes
 
Address reprint requests to Roman R. Ganta, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., Manhattan, KS 66506. E-mail: rganta{at}vet.k-state.edu

Supported by the Morris Animal Foundation (grant D01CA-91) and the National Institutes of Health (grants AI50785, AI55052, and RR17686).

This manuscript is published as contribution No. 04-319-J of the Kansas Agricultural Experiment Station, Manhattan.

Accepted for publication December 17, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR: Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001, 51:2145-2165[Abstract]
  2. Dawson JE, Anderson BE, Fishbein DB, Sanchez CY, Goldsmith CY, Wilson KH, Duntley CW: Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J Clin Microbiol 1991, 29:2741-2745[Abstract/Free Full Text]
  3. Maeda K, Markowitz N, Hawley RC, Ristic M, Cox D, McDade JE: Human infection with Ehrlichia canis, a leukocytic rickettsia. N Engl J Med 1987, 316:853-856[Medline]
  4. Chen SM, Dumler JS, Bakken JS, Walker DH: Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol 1994, 32:589-595[Abstract/Free Full Text]
  5. Buller RS, Arens M, Hmiel SP, Paddock CD, Sumner JW, Rikhisa Y, Unver A, Gaudreault-Keener M, Manian FA, Liddell AM, Schmulewitz N, Storch GA: Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis. N Engl J Med 1999, 341:148-155[Abstract/Free Full Text]
  6. Breitschwerdt EB, Hegarty BC, Hancock SI: Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin Microbiol 1998, 36:2645-2651[Abstract/Free Full Text]
  7. French TW, Harvey JW: Canine infectious cyclic thrombocytopenia (Ehrlichia platys infection in dogs). Woldehiwet Z Ristic M eds. Rickettsial and Chlamydial Diseases of Domestic Animals. 1993:pp 195-208 Pergamon Press Oxford
  8. Murphy GL, Ewing SA, Whitworth LC, Fox JC, Kocan AA: A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E ewingii in dogs and ticks from Oklahoma. Vet Parasitol 1998, 79:325-339[Medline]
  9. Paddock CD, Childs JE: Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin Microbiol Rev 2003, 16:37-64[Abstract/Free Full Text]
  10. Perez M, Rikihisa Y, Wen B: Ehrlichia canis-like agent isolated from a man in Venezuela: antigenic and genetic characterization. J Clin Microbiol 1996, 34:2133-2139[Abstract]
  11. Kordick SK, Breitschwerdt EB, Hegarty BC, Southwick KL, Colitz CM, Hancock SI, Bradley JM, Rumbough R, Mcpherson JT, MacCormack JN: Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 1999, 37:2631-2638[Abstract/Free Full Text]
  12. Hoskins JD, Breitschwerdt EB, Gaunt SD, French TW, Burgdorfer W: Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group rickettsiae in Louisiana dogs. J Vet Intern Med 1988, 2:55-59[Medline]
  13. Hua P, Yuhai M, Shide T, Yang S, Bohai W, Xiangrui C: Canine ehrlichiosis caused simultaneously by Ehrlichia canis and Ehrlichia platys. Microbiol Immunol 2000, 44:737-739[Medline]
  14. Chang YF, Novosel V, Chang CF, Kim JB, Shin SJ, Lein DH: Detection of human granulocytic ehrlichiosis agent and Borrelia burgdorferi in ticks by polymerase chain reaction. J Vet Diagn Invest 1998, 10:56-59[Abstract/Free Full Text]
  15. Sexton DJ, Corey GR, Carpenter C, Kong LQ, Gandhi T, Breitschwerdt E, Hegarty B, Chen SM, Feng HM, Yu XJ, Olano J, Walker DH, Dumler SJ: Dual infection with Ehrlichia chaffeensis and a spotted fever group rickettsia: a case report. Emerg Infect Dis 1998, 4:311-316[Medline]
  16. Paddock CD, Folk SM, Shore GM, Machado LJ, Huycke MM, Slater LN, Liddell AM, Buller RS, Storch GA, Monson TP, Rimland D, Sumner JW, Singleton J, Bloch KC, Tang YW, Standaert SM, Childs JE: Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus. Clin Infect Dis 2001, 33:1586-1594[Medline]
  17. Meinkoth JH, Ewing SA, Cowell RL, Dawson JE, Warner CK, Mathew JS, Bowles M, Thiessen AE, Panciera RJ, Fox C: Morphologic and molecular evidence of a dual species ehrlichial infection in a dog presenting with inflammatory central nervous system disease. J Vet Intern Med 1998, 12:389-393[Medline]
  18. Codner EC, Farris-Smith LL: Characterization of the sub-clinical phase of ehrlichiosis in dogs. J Am Vet Med Assoc 1986, 189:47-50[Medline]
  19. Dumler JS, Sutker WL, Walker DH: Persistent infection with Ehrlichia chaffeensis. Clin Infect Dis 1993, 17:903-905[Medline]
  20. Egenvall A, Lilliehook I, Bjoersdorff A, Engvall EO, Karlstam E, Artursson K, Heldtander M, Gunnarsson A: Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. Vet Rec 2000, 146:186-190[Abstract/Free Full Text]
  21. Kieser ST, Eriks IS, Palmer GH: Cyclic Rickettsemia during persistent Anaplasma marginale infection of cattle. Infect Immun 1990, 58:1117-1119[Abstract/Free Full Text]
  22. Chen SM, Popov VL, Feng HM, Walker DH: Analysis and ultrastructural localization of Ehrlichia chaffeensis proteins with monoclonal antibodies. Am J Trop Med Hyg 1996, 54:405-412
  23. Reddy GR, Sulsona CR, Barbet AF, Mahan SM, Burridge MJ, Alleman AR: Molecular characterization of a 28 kDa surface antigen gene family of the tribe Ehrlichiae. Biochem Biophys Res Commun 1998, 247:636-643[Medline]
  24. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. 1982 Cold Spring Harbor Laboratory Cold Spring Harbor
  25. Devereux J: Genetics computer group sequence analysis software package, version 6.1. Nucleic Acids Res 1984, 12:387-395
  26. Stefano JE, Genovese L, An Q, Lu L, McCarty J, Du Y, Stefano K, Burg JL, King W, Lane DJ: Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase. Mol Cell Probes 1997, 11:407-426[Medline]
  27. Mackay IM: Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 2004, 10:190-212[Medline]
  28. Szollosi J, Damjanovich S, Matyus L: Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research. Cytometry 1998, 34:159-179[Medline]
  29. Waner T, Harrus S, Jongejan F, Bark H, Keysary A, Cornelissen AW: Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis. Vet Parasitol 2001, 95:1-15[Medline]
  30. Felek S, Unver A, Stich RW, Rikihisa Y: Sensitive detection of Ehrlichia chaffeensis in cell culture, blood, and tick specimens by reverse transcription-PCR. J Clin Microbiol 2003, 39:460-463
  31. Lane RS, Foley JE, Eisen L, Lennette ET, Peot MA: Acarologic risk of exposure to emerging tick-borne bacterial pathogens in a semi-rural community in northern California. Vector Borne Zoonotic Dis 2001, 1:197-210[Medline]
  32. Warner CK, Dawson JE: Genus and species level identification of Ehrlichia species by PCR and sequencing. Persing DH eds. PCR protocols for emerging infectious diseases. 1996:pp 100-105 ASM Press Washington DC
  33. Goodman RA, Hawkins EC, Olby NJ, Grindem CB, Hegarty B, Breitschwerdt EB: Molecular identification of Ehrlichia ewingii infection in dogs: 15 cases (1997–2001). J Am Vet Med Assoc 2003, 222:1102-1107[Medline]
  34. McBride JW, Corstvet RE, Gaunt SD, Chinsangaram J, Akita GY, Osburn BI: PCR detection of acute Ehrlichia canis infection in dogs. J Vet Diagn Invest 1996, 8:441-447[Abstract/Free Full Text]
  35. Stich RW, Rikihisa Y, Ewing SA, Needham GR, Grover DL, Jittapalapong S: Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay. J Clin Microbiol 2002, 40:540-546[Abstract/Free Full Text]
  36. Engvall EO, Pettersson B, Persson M, Artursson K, Johansson KE: A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J Clin Microbiol 1996, 34:2170-2174[Abstract]
  37. Chang WL, Pan MJ: Specific amplification of Ehrlichia platys DNA from blood specimens by two-step PCR. J Clin Microbiol 1996, 34:3142-3146[Abstract]
  38. Arens MQ, Liddell AM, Buening G, Gaudreault-Keener M, Sumner JW, Comer JA, Buller RS, Storch GA: Detection of Ehrlichia spp. in the blood of wild white-tailed deer in Missouri by PCR assay and serologic analysis. J Clin Microbiol 2003, 41:1263-1265[Abstract/Free Full Text]
  39. Dumler JS: Molecular methods for ehrlichiosis and Lyme disease. Clin Lab Med 2003, 23:867-884[Medline]
  40. Sparagano OA, de VA, Paoletti B, Camma C, de Vos AP, Otranto D, Giangaspero A: Molecular detection of Anaplasma platys in dogs using polymerase chain reaction and reverse line blot hybridization. J Vet Diagn Invest 2003, 15:527-534[Abstract/Free Full Text]
  41. Bekker CP, de Vos S, Taoufik A, Sparagano OA, Jongejan F: Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization. Vet Microbiol 2002, 89:223-238[Medline]
  42. Pusterla N, Huder JB, Leutenegger CM, Braun U, Madigan JE, Lutz H: Quantitative real-time PCR for detection of members of the Ehrlichia phagocytophila genogroup in host animals and Ixodes ricinus ticks. J Clin Microbiol 1999, 37:1329-1331[Abstract/Free Full Text]
  43. Loftis AD, Massung RF, Levin ML: Quantitative real-time PCR assay for detection of Ehrlichia chaffeensis. J Clin Microbiol 2003, 41:3870-3872[Abstract/Free Full Text]
  44. Brouqui P, Salvo E, Dumler JS, Raoult D: Diagnosis of granulocytic ehrlichiosis in humans by immunofluorescence assay. Clin Diagn Lab Immunol 2001, 8:199-202[Abstract/Free Full Text]
  45. Dawson J, Fishbein D, Eng T, Redus M, Greene N: Diagnosis of human ehrlichiosis with the indirect fluorescent antibody test: kinetics and specificity. Ann NY Acad Sci 1990, 590:308[Medline]
  46. French TW, Harvey JW: Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test. Am J Vet Res 1983, 44:2407-2411[Medline]
  47. Belanger M, Sorenson HL, France MK, Bowie MV, Barbet AF, Breitschwerdt EB, Alleman AR: Comparison of serological detection methods for diagnosis of Ehrlichia canis infections in dogs. J Clin Microbiol 2002, 40:3506-3508[Abstract/Free Full Text]
  48. Reddy GR, Sulsona CR, Harrison RH, Mahan SM, Burridge MJ, Barbet AF: Sequence heterogeneity of the major antigenic protein 1 genes from Cowdria ruminantium isolates from different geographical areas. Clin Diag Lab Immunol 1996, 3:417-422[Abstract]
  49. Uilenberg G, Barre N, Camus E, Burridge MJ, Garris GI: Heartwater in the Caribbean. Prev Vet Med 1984, 2:255-267



This article has been cited by other articles:


Home page
 J. Mol. Diagn.Home page
J. Dong, J. P. Olano, J. W. McBride, and D. H. Walker
Emerging Pathogens: Challenges and Successes of Molecular Diagnostics
J. Mol. Diagn., May 1, 2008; 10(3): 185 - 197.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
A. Luce-Fedrow, T. Von Ohlen, D. Boyle, R. R. Ganta, and S. K. Chapes
Use of Drosophila S2 Cells as a Model for Studying Ehrlichia chaffeensis Infections
Appl. Envir. Microbiol., March 15, 2008; 74(6): 1886 - 1891.
[Abstract] [Full Text] [PDF]

Home page
 CVIHome page
A. M. Cardenas, C. K. Doyle, X. Zhang, K. Nethery, R. E. Corstvet, D. H. Walker, and J. W. McBride
Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis of Ehrlichia canis Infection
Clin. Vaccine Immunol., February 1, 2007; 14(2): 123 - 128.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
R. R. Ganta, C. Cheng, E. C. Miller, B. L. McGuire, L. Peddireddi, K. R. Sirigireddy, and S. K. Chapes
Differential Clearance and Immune Responses to Tick Cell-Derived versus Macrophage Culture-Derived Ehrlichia chaffeensis in Mice
Infect. Immun., January 1, 2007; 75(1): 135 - 145.
[Abstract] [Full Text] [PDF]

Home page
 Ann. N. Y. Acad. Sci.Home page
K. R SIRIGIREDDY, D. C MOCK, and R. R GANTA
Multiplex Detection of Ehrlichia and Anaplasma Pathogens in Vertebrate and Tick Hosts by Real-Time RT-PCR
Ann. N.Y. Acad. Sci., October 1, 2006; 1078(1): 552 - 556.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sirigireddy, K. R.
Right arrow Articles by Ganta, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sirigireddy, K. R.
Right arrow Articles by Ganta, R. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS