Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30 -Based PCR Assay

Abstract

Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30, to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30-based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30-based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.Canine monocytic ehrlichiosis (CME), sometimes known as tropical canine pancytopenia, is a cosmopolitan tick-borne disease of dogs that is primarily caused by the rickettsia Ehrlichia canis (10,(16)(17)(18)31). This parasite invades and develops in monocytes and macrophages of dogs, resulting in leukopenia, thrombocytopenia, fever, depression, and anorexia, and this species has been reported in association with at least one human case (26). Three other ehrlichial organisms, E. chaffeensis, the human granulocytic ehrlichiosis (HGE) agent, and Ehrlichia ewingii, are zoonotic in the United States (5, 9), and several reports suggest that these organisms also occur naturally in dogs and deer (3, 4, 6-8, 11, 12, 15). Ixodid ticks transmit most of the Ehrlichia spp. characterized to date and appear to be the biological vectors of E. chaffeensis, the HGE agent, and E. ewingii. E. canis and E. chaffeensis are closely related, based on 16S rRNA gene (16S ribosomal DNA [rDNA]) sequences and antigenic cross-reactivity (2). These observations indicate that tick transmission and canine infection studies with E. canis can serve as a model for further work with E. chaffeensis in dogs and humans and lead to better understanding of both pathogens.A sensitive, specific PCR assay would be useful for detection of Ehrlichia spp. that typically occur at low l...

Development of a p28-based PCR assay for Ehrlichia chaffeensis

“…The E. chaffeensis 91HE17 isolate was kindly provided by Dr. David Walker at the University of Texas Medical Branch at Galveston, and the E. chaffeensis St. Vincent isolate was purchased from ATCC (Manassas, VA). DNA was isolated from infected host cells by protein digestion (0.1 mg/ml proteinase K, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.45% (v/v) Tween-20, 0.45% (v/v) NP40) at 55 °C for 1 h) followed by phenol/chloroform extraction and ethanol precipitation as previously described for E. canis [13]. DNA from E. muris was provided as reported elsewhere [19].…”

“…More recently, single-step reverse transcription (RT)-PCR has also been utilized to amplify an outer membrane protein-1 (omp-1) paralog, p28-10, from pools of experimentally infected ticks [12], indicating that an outer membrane protein gene sequence could provide an even more sensitive PCR assay for this organism. Indeed, an assay based on p30, which is the E. canis homolog of p28, indicated that this assay was significantly more sensitive than the respective 16S rDNA-based assay for that organism [13]. This finding could be because species-specific nucleic acid sequences, such as those often encoding outer membrane proteins, can provide greater ranges of unique targets for design of primers for PCR assays [14,15].…”

“…Candidate primer sequences were identified with Prime software (Wisconsin Package V. 10.2; Genetic Computer Group [GCG], Madison, WI) and further evaluated with Pileup (GCG) as previously described with a few modifications [13]. Briefly, primer sets were identified from the p28 open reading frame (ORF) of the E. chaffeensis Arkansas isolate or from a consensus of p28 ORF sequences representing the Arkansas, 91HE17 and St. Vincent isolates.…”

“…This finding could be because species-specific nucleic acid sequences, such as those often encoding outer membrane proteins, can provide greater ranges of unique targets for design of primers for PCR assays [14,15]. Several such PCR assays have been used to directly detect other anaplasmal pathogens within both vertebrate and invertebrate hosts [13,16,17].…”

“…This paralog from the E. chaffeensis omp-1 multiple gene cluster was chosen because it has been characterized from several E. chaffeensis and E. canis isolates, allowing the design of primers predicted to be both common and restricted to the target species [13][14][15]. Several candidate primer sets were considered, resulting in a single-step PCR assay that was species-specific for E. chaffeensis, to amplify DNA from isolates representative of the three major p28 sequence clusters, and to be more sensitive than a conventional nested 16S rDNA-based PCR assay.…”