Development and validation of real-time PCR assays for the detection of Ehrlichia species and E. chaffeensis in clinical specimens.

Ehrlichiosis, caused by Gram-negative bacteria of the genus Ehrlichia, is considered an emerging infectious disease due to the increasing number of reported cases. Symptoms are non-specific and occur within 1 to 2 weeks following the bite of an infected tick. Confirmatory laboratory diagnostic methods vary in sensitivity and specimen requirements, which can lead to delayed diagnosis. PCR testing serves as an efficient approach to Ehrlichia confirmation in the acute stage of illness. Published assays have been effectively used to detect human ehrlichiosis at limit of detections ranging from 10 to 50 genomic copies (GC) of Ehrlichia DNA. With the discovery of new species capable of human infection, we wanted to develop assays that are sensitive and encompass a wide range of Ehrlichia. Here we developed and validated two sensitive and specific real-time PCR assays (PanE1 and PanE2) for the detection of Ehrlichia species, as well as two real-time PCR assays (ECh2 and ECh4) for the detection of Ehrlichia chaffeensis, specifically. The limit of detection was determined to be 10 GC per reaction with 100% confidence, and as little as 1 GC with lower efficiencies. Accuracy was assessed at 100% correlation. Specificity from exclusivity testing demonstrated that neither the Ehrlichia species assays (n = 60), nor the E. chaffeensis specific assays (n = 64) had cross reactivity with near neighbors or environmental bacteria. A positive predictive value of 100% and a negative predictive value of ≥93% was determined by evaluating banked clinical specimens from 62 patients with the assays. These real-time PCR assays are effective tools to detect human Ehrlichia species during the acute stage of illness. Early detection of Ehrlichia infection by these real-time PCR assays can facilitate diagnosis and treatment.

Multi-Locus Sequence Typing of Ehrlichia chaffeensis Reveals Extensive Genotypic Variation across the United States.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis, and its principal vector is the Amblyomma americanum tick. The most frequently identified cases of ehrlichiosis come from the southeastern and south central states of the United States. In this study, a molecular typing system was developed that allows for the genetic differentiation of E. chaffeensis isolates. This multi-locus typing system included sequencing and analyzing intergenic regions ECH0033-ECH0035 and ECH0217-ECH0218, plus, variable genes variable length PCR target, 28-kDa, 120-kDa, and hemE. We examined a total of 31 unique isolates from humans and white-tailed deer, and eight DNA samples extracted from infected A. americanum collected from multiple states. This is the largest evaluation of E. chaffeensis isolates and their genotypes. Our findings show that when sequences of all six loci were concatenated and compared, the 39 samples could be separated into 23 genotypes and further grouped into six phylogenetic clades. The data in this study show no clear pattern between the geographic alignment with the genetic differentiation between the strains. As a result, this poses a challenge to understanding the spread of E. chaffeensis in the United States. Interestingly, our findings indicate that multiple strains from distant geographic origins share the same mutations, which suggests that the strains are being moved from one site to another by their hosts or vectors. In addition, we are seeing a northward shift in the lone star tick distribution in the United States. Last, some data also suggest minimal genetic mutations have occurred over time among strains that are within geographical proximity.

Anaplasmataceae closely related to Ehrlichia chaffeensis and Neorickettsia helminthoeca from birds in Central Europe, Hungary.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

Two novel Ehrlichia strains detected in Amblyomma tigrinum ticks associated to dogs in peri-urban areas of Argentina.

The aim of this work was to describe two novel strains of Ehrlichia associated to Amblyomma tigrinum from Argentina. Molecular detection of agents belonging to the family Anaplasmataceae was performed targeting three different loci: 16S rRNA gene, dsb gene and a fragment of groESL heat shock operon. The results have shown that two different strains of Ehrlichia sp. associated to A. tigrinum are circulating in peri-urban areas of Argentina. The Ehrlichia strain detected in ticks from San Luis Province, named as Ehrlichia sp. strain San Luis, is closely related to the Ehrlichia chaffeensis. The novel Ehrlichia strain detected in Córdoba Province, named as Ehrlichia sp. strain Córdoba, is phylogenetically related to three Ehrlichia strains from Brazil, two of them isolated from wild carnivorous and the third one isolated from horse. Even though Ehrlichia sp. strain Córdoba was clustered with the three Ehrlichia strains from Brazil, the genetic similarity was too low to consider them as the same taxonomic entity. Blood samples of dogs were positive to Anaplasma platys. The association of these two novel strains with A. tigrinum has epidemiological relevance because adult stages of this tick species are common parasite of dogs in rural and peri-urban areas and they are aggressive to humans. The presence of these two novel Ehrlichia strains implies a potential epidemiological risk in Argentina because the species of the genus Ehrlichia are known to be pathogenic to both domestic mammals and humans.

Fatal Monocytic Ehrlichiosis in Woman, Mexico, 2013.

Human monocytic ehrlichiosis is a febrile illness caused by Ehrlichia chaffeensis, an intracellular bacterium transmitted by ticks. In Mexico, a case of E. chaffeensis infection in an immunocompetent 31-year-old woman without recognized tick bite was fatal. This diagnosis should be considered for patients with fever, leukopenia, thrombocytopenia, and elevated liver enzyme levels.

Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets.

During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets for the design of alternative therapeutics against intracellular pathogens. The collection of putative effectors of E. chaffeensis described in our paper could serve as a roadmap for future studies of the function and evolution of effectors.

Detection of bacterial agents in Amblyomma americanum (Acari: Ixodidae) from Georgia, USA, and the use of a multiplex assay to differentiate Ehrlichia chaffeensis and Ehrlichia ewingii.

Amblyomma americanum, the lone star tick, is the most common and most aggressive human biting tick in the Southeastern United States. It is known to transmit the agents of human ehrlichioses, Ehrlichia chaffeensis and Ehrlichia ewingii. In addition, it carries agents of unspecified pathogenicity to humans, including Rickettsia amblyommii, Borrelia lonestari, and the newly emerging Panola Mountain Ehrlichia (PME). Surveillance of these ticks for recognized or emerging pathogens is necessary for assessing the risk of human infection. From 2005 to 2009, we surveyed A. americanum ticks from four locations in the state of Georgia. Ticks (1,183 adults, 2,954 nymphs, and 99 larval batches) were tested using a multiplex real-time polymerase chain reaction (PCR) assay designed to detect and discriminate DNA from Rickettsia spp., E. chaffeensis, and E. ewingii. This assay was capable of detecting as few as 10 gene copies of the aforementioned agents. Ticks were also tested for PME and B. lonestari by nested PCR. The prevalence of infection ranged from 0 to 2.5% for E. chaffeensis, 0 to 3.9% for E. ewingii, 0 to 2.2% for PME, 17 to 83.1% for R. amblyommii, and 0 to 3.1% for B. lonestari. There were 46 (4.1%) individual adults positive for two agents, and two females that were each positive for three agents. Two larval batches were positive for both B. lonestari and R. amblyommii, indicating the potential for transovarial transmission of both agents from a single female. Although infrequent in occurrence, the dynamics of coinfections in individual ticks should be explored further, given the potential implications for differential diagnosis and severity of human illness.

Myocarditis after trimethoprim/sulfamethoxazole treatment for ehrlichiosis.

The manifestations of human monocytic ehrlichiosis range from a mild febrile syndrome to a severe multisystem illness. Myocardial involvement is uncommon. We report a woman, 78 years of age, who was treated with trimethoprim/sulfamethoxazole after a tick bite, in whom myocarditis was subsequently diagnosed. She recovered completely after doxycycline therapy.

Ehrlichia chaffeensis TRP120 binds a G+C-rich motif in host cell DNA and exhibits eukaryotic transcriptional activator function.

Ehrlichia chaffeensis is an obligately intracellular bacterium that modulates host cell gene transcription in the mononuclear phagocyte, but the host gene targets and mechanisms involved in transcriptional modulation are not well-defined. In this study, we identified a novel tandem repeat DNA-binding domain in the E. chaffeensis 120-kDa tandem repeat protein (TRP120) that directly binds host cell DNA. TRP120 was observed by immunofluorescent microscopy in the nucleus of E. chaffeensis-infected host cells and was detected in nuclear extracts by Western immunoblotting with TRP120-specific antibody. The TRP120 binding sites and associated host cell target genes were identified using high-throughput deep sequencing (Illumina) of immunoprecipitated DNA (chromatin immunoprecipitation and high-throughput DNA sequencing). Multiple em motif elicitation (MEME) analysis of the most highly enriched TRP120-bound sequences revealed a G+C-rich DNA motif, and recombinant TRP120 specifically bound synthetic oligonucleotides containing the motif. TRP120 target gene binding sites were mapped most frequently to intersecting regions (intron/exon; 49%) but were also identified in upstream regulatory regions (25%) and downstream locations (26%). Genes targeted by TRP120 were most frequently associated with transcriptional regulation, signal transduction, and apoptosis. TRP120 targeted inflammatory chemokine genes, CCL2, CCL20, and CXCL11, which were strongly upregulated during E. chaffeensis infection and were also upregulated by direct transfection with recombinant TRP120. This study reveals that TRP120 is a novel DNA-binding protein that is involved in a host gene transcriptional regulation strategy.

Ehrlichia chaffeensis infection of sika deer, Japan.

To determine whether Ehrlichia chaffeensis exists in Japan, we used PCR to examine blood from sika deer in Nara, Japan. Of 117 deer, 36 (31%) were infected with E. chaffeensis. The E. chaffeensis 16S rRNA base and GroEL amino acid sequences from Japan were most closely related to those of E. chaffeensis Arkansas.

Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela.

BACKGROUND: Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease. OBJECTIVE: The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques. METHODS: Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications. RESULTS: The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs. CONCLUSIONS: This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.

Virulence potential of Ehrlichia chaffeensis strains of distinct genome sequences.

Human monocytic ehrlichiosis, one of the most frequent life-threatening tick-borne zoonoses, is caused by Ehrlichia chaffeensis that lacks endotoxin and peptidoglycan. While sequence polymorphisms in several genes in E. chaffeensis strains have been reported, global genomic divergence and biological differences among strains are unknown. The objectives of the present study were to compare the genome sequences of strains of E. chaffeensis and to examine the virulence potentials of the strains with defined genome sequences. Genomic DNA was extracted from purified E. chaffeensis strains Wakulla and Liberty, and comparative genome hybridization was performed using a densely tiled microarray of 147,027 chromosome positions of the E. chaffeensis strain Arkansas genome. The results revealed that 4,663 and 5,325 positions in the chromosomes of strains Wakulla and Liberty, respectively, were different from those in the chromosome of strain Arkansas, including three common major polymorphic chromosomal regions. Of various functional categories, the differences were most concentrated in genes predicted to encode cell envelope proteins. Of all the open reading frames (ORFs), 21 omp-1 (p28 gene) paralogs, nine genes encoding hypothetical proteins, two genes encoding ankyrin repeat proteins, and hemE contained the most differences. Several highly polymorphic ORFs were confirmed by sequencing. When the E. chaffeensis strains were inoculated into severe combined immunodeficiency mice, the order of the severity of clinical signs and the bacterial burden detected in mice was Wakulla > Liberty > Arkansas. Severe diffuse inflammation and granulomatous inflammation were evident in the livers of mice infected with strains Wakulla and Arkansas, respectively, but not in the livers of mice infected with strain Liberty. These results revealed distinct virulence phenotypes of E. chaffeensis strains with defined genome sequences.

Restriction and expansion of Ehrlichia strain diversity.

Ehrlichia are tick-borne gram negative, obligately intracellular bacteria. The 16S rRNA gene DNA sequences are highly conserved among strains of each Ehrlichia species. The 28-kDa/Map-1 outer membrane protein genes are highly diversified among strains of Ehrlichia chaffeensis and E. ruminantium, but are highly conserved among E. canis isolates. The diversity of the immunodominant proteins of E. chaffeensis and E. ruminantium in contrast with the conservation of the immunodominant proteins of E. canis suggests that E. chaffeensis and E. ruminantium face more host immune pressure than E. canis or that E. chaffeensis and E. ruminantium evolved earlier than E. canis and have diverged.

A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii.

A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCyclertrade mark instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.

Evidence of tick-borne organisms in mule deer (Odocoileus hemionus) from the western United States.

Free-ranging mule deer (MD; Odocoileus hemionus) from Arizona and California were tested for evidence of infection with several tick-borne pathogens, including species of Ehrlichia, Anaplasma, Babesia, and Borrelia. Of 125 mule deer tested from Arizona, 29 (23%) and 11 (9%) had antibodies reactive to E. chaffeensis and A. phagocytophilum by indirect immunofluorescent antibody testing, respectively; none of the six MD tested from California were seropositive. Using a commercial competitive ELISA kit, antibodies reactive to Anaplasma spp. were detected in 19 (15%) MD from Arizona and four of six (67%) MD from California. Polymerase chain reaction (PCR) testing for tick-borne pathogens was conducted on blood samples from 29 MD from Arizona and 11 MD from California. Twenty-two of 29 (75.9%) MD from Arizona had PCR evidence of infection with at least one tick-borne pathogen. We detected an Anaplasma sp. in 19 of 29 (65.5%) MD and a Babesia sp. in 10 of 29 (34%) MD. Sequencing of these amplicons indicated that the Anaplasma sp. was the same that had previously been detected in MD from California and the Babesia sp. was similar to one previously detected in a reindeer (Rangifer tarandus tarandus) from California. All of the California MD had evidence of infection with a tick-borne pathogen. Two different species of Anaplasma spp. were detected in MD from California, eight of of 11 MD were infected with an Anaplasma sp., and three of 11 MD were infected with A. ovis. This is the first report of a mule deer naturally infected with A. ovis. Ten of 11 MD from California were infected with a Babesia-like organism previously associated with human disease, and a single MD was PCR positive for Borrelia coriaceae, which has been associated with epizootic bovine abortion. Together, these data suggest that MD in northern Arizona and eastern California are exposed to several pathogens of human and veterinary importance.

Ehrlichia chaffeensis: a prototypical emerging pathogen.

Ehrlichia chaffeensis is an obligately intracellular, tick-transmitted bacterium that is maintained in nature in a cycle involving at least one and perhaps several vertebrate reservoir hosts. The moderate to severe disease caused by E. chaffeensis in humans, first identified in 1986 and reported for more than 1,000 patients through 2000, represents a prototypical "emerging infection." Knowledge of the biology and natural history of E. chaffeensis, and of the epidemiology, clinical features, and laboratory diagnosis of the zoonotic disease it causes (commonly referred to as human monocytic ehrlichiosis [HME]) has expanded considerably in the period since its discovery. In this review, we summarize briefly the current understanding of the microbiology, pathogenesis, and clinical manifestations associated with this pathogen but focus primarily on discussing various ecological factors responsible for the recent recognition of this important and potentially life-threatening tick-borne disease. Perhaps the most pivotal element in the emergence of HME has been the staggering increases in white-tailed deer populations in the eastern United States during the 20th century. This animal serves as a keystone host for all life stages of the principal tick vector (Amblyomma americanum) and is perhaps the most important vertebrate reservoir host for E. chaffeensis. The contributions of other components, including expansion of susceptible human populations, growth and broadening geographical distributions of other potential reservoir species and A. americanum, and improvements in confirmatory diagnostic methods, are also explored.

Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet.

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.