Ahmet Unver

Ehrlichia ewingii, a Newly Recognized Agent of Human Ehrlichiosis

BACKGROUND Human ehrlichiosis is a recently recognized tick-borne infection. Four species infect humans: Ehrlichia chaffeensis, E. sennetsu, E. canis, and the agent of human granulocytic ehrlichiosis. METHODS We tested peripheral-blood leukocytes from 413 patients with possible ehrlichiosis by broad-range and species-specific polymerase-chain-reaction (PCR) assays for ehrlichia. The species present were identified by species-specific PCR assays and nucleotide sequencing of the gene encoding ehrlichia 16S ribosomal RNA. Western blot analysis was used to study serologic responses. RESULTS In four patients, ehrlichia DNA was detected in leukocytes by a broad-range PCR assay, but not by assays specific for E. chaffeensis or the agent of human granulocytic ehrlichiosis. The nucleotide sequences of these PCR products matched that of E. ewingii, an agent previously reported as a cause of granulocytic ehrlichiosis in dogs. These four patients, all from Missouri, presented between May and August 1996, 1997, or 1998 with fever, headache, and thrombocytopenia, with or without leukopenia. All had been exposed to ticks, and three were receiving immunosuppressive therapy. Serum samples obtained from three of these patients during convalescence contained antibodies that reacted with E. chaffeensis and E. canis antigens in a pattern different from that of humans with E. chaffeensis infection but similar to that of a dog experimentally infected with E. ewingii. Morulae were identified in neutrophils from two patients. All four patients were successfully treated with doxycycline. CONCLUSIONS These findings provide evidence of E. ewingii infection in humans. The associated disease may be clinically indistinguishable from infection caused by E. chaffeensis or the agent of human granulocytic ehrlichiosis.

Molecular and Antigenic Comparison of Ehrlichia canis Isolates from Dogs, Ticks, and a Human in Venezuela

ABSTRACT We previously culture isolated a strain of Ehrlichia canis, the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with E. canis in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis usingE. canis-specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four Rhipicephalus sanguineus ticks each were positive. An ehrlichial agent (Venezuelan dog Ehrlichia [VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human Ehrlichia isolate (VHE) and was closely related (99.9%) to that of E. canisOklahoma. The 5′ (333-bp) and 3′ (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional E. canis-positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and E. canis Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human E. canis infection and that R. sanguineus, which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of E. canis directly from naturally infected ticks.

Analysis of Transcriptionally Active Gene Clusters of Major Outer Membrane Protein Multigene Family in Ehrlichia canis and E. chaffeensis

ABSTRACT Ehrlichia canis and E. chaffeensis are tick-borne obligatory intramonocytic ehrlichiae that cause febrile systemic illness in humans and dogs, respectively. The current study analyzed the pleomorphic multigene family encoding approximately 30-kDa major outer membrane proteins (OMPs) of E. canis andE. chaffeensis. Upstream from secA and downstream of hypothetical transcriptional regulator, 22 paralogs of the omp gene family were found to be tandemly arranged except for one or two genes with opposite orientations in a 28- and a 27-kb locus in the E. canis and E. chaffeensisgenomes, respectively. Each locus consisted of three highly repetitive regions with four nonrepetitive intervening regions. E. canis, in addition, had a 6.9-kb locus which contained a repeat of three tandem paralogs in the 28-kb locus. These total 47 paralogous and orthologous genes encoded OMPs of approximately 30 to 35 kDa consisting of several hypervariable regions alternating with conserved regions. In the 5′-end half of the 27-kb locus or the 28-kb locus of each Ehrlichia species, 14 paralogs were linked by short intergenic spaces ranging from −8 bp (overlapped) to 27 bp, and 8 remaining paralogs in the 3′-end half were connected by longer intergenic spaces ranging from 213 to 632 bp. All 22 paralogs, five unknown genes, and secA in the omp cluster inE. canis were transcriptionally active in the monocyte culture, and the paralogs with short intergenic spaces were cotranscribed with their adjacent genes, including the respective intergenic spaces at both the 5′ and the 3′ sides. Althoughomp genes are diverse, our results suggest that the gene organization of the clusters and the gene locus are conserved between two species of Ehrlichia to maintain a unique transcriptional mechanism for adaptation to environmental changes common to them.

The omp-1 major outer membrane multigene family of Ehrlichia chaffeensis is differentially expressed in canine and tick hosts.

ABSTRACT Sixteen of 22 omp-1 paralogs encoding 28-kDa-range immunodominant outer membrane proteins of Ehrlichia chaffeensis were transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed by E. chaffeensis in three developmental stages of Amblyomma americanum ticks before or after E. chaffeensis transmission to naïve dogs.

Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan.

Abstract: In the present study, three canine blood samples from Japan, that were suspected to be ehrlichia positive were examined. After sequencing the 16S rRNA genes, each dog was found to be infected either with Ehrlichia canis (Kagoshima 1), Anaplasma platys (Okinawa 1), or Wolbachia sp. (Okinawa 2). Phylogenic analysis was performed on these sequences. The nearly entire 16S rRNA sequence of Kagoshima 1 was found to be most similar to the sequences from Oklahoma and Venezuela E. canis strains (1 base pair difference out of 1,387, 99.9% sequence identity). The 16S rRNA gene sequence of Okinawa 1 showed the closest DNA identity to the French strain of A. platys (1 base deletion out of 1,385 bp, 99.6% sequence identity). The 16S rRNA gene sequence of Okinawa 2 illustrated the closest DNA identity to that of a Wolbachia sp. from Dirofilaria immitis (98.9% sequence similarity). These data imply a low diversity within E. canis strains and within A. platys strains, including those strains reported in this study. This is also the first demonstration of Wolbachia DNA in dog blood, suggesting the involvement of Wolbachia sp. in canine febrile illnesses.

Transcriptional Analysis of p30 Major Outer Membrane Multigene Family of Ehrlichia canis in Dogs, Ticks, and Cell Culture at Different Temperatures

ABSTRACT Ehrlichia canis, an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis. E. canisimmunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs andRhipicephalussanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs in E.canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups ofR. sanguineus ticks (adult males and females and nymphs) were separately infected with E.canis by feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower inE. canis cultivated in dog monocyte cell line DH82 at 25°C than in E. caniscultivated at 37°C. Analysis of differential expression ofp30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E. canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

Sensitive Detection of Ehrlichia chaffeensis in Cell Culture, Blood, and Tick Specimens by Reverse Transcription-PCR

ABSTRACT Ehrlichia chaffeensis is an obligatory intracellular bacterium of monocytes and macrophages and the etiologic agent of human monocytic ehrlichiosis, an emerging zoonosis. The Lone Star tick (Amblyomma americanum) has been implicated as the primary vector of E. chaffeensis. The present study examined the sensitivity of the nested reverse transcription (RT)-PCR based on the 16S rRNA gene relative to that of the nested PCR for detection ofE. chaffeensis in infected DH82 cells, experimentally infected dog peripheral blood mononuclear cells, or experimentally infected A. americanum tick samples. The RT-PCR was found to be approximately 100 times more sensitive than the PCR for detection of E. chaffeensis regardless of the nature of the specimens. Thus, this RT-PCR is useful for detection of E. chaffeensis when a high sensitivity is required. Positive results by RT-PCR also imply the presence of viable pathogens. This is the first demonstration of RNA of E. chaffeensis in infected blood and acquisition-fed male, nymphal, and larval A. americanum ticks.

Prevalence and molecular analysis of Anaplasma platys in dogs in Lara, Venezuela

Blood specimens from clinically normal military dogs and their trainers, in Lara, Venezuela were screened for Anaplasma platys, A. phagocytophilum, or Ehrlichia ewingii using 16S rRNA PCR tests. Sixteen percent (7/43) of dog specimens were positive by A. platys PCR test followed by sequencing of the PCR products, and all human blood specimens [25] were negative. All specimens from these dogs and humans were PCR negative for E. ewingii or A. phagocytophilum. Twelve Rhipicephalus sanguineus ticks removed from these dogs were negative for A. platys by reverse transcription PCR test. Almost the entire 16S rRNA gene (1,364 bp) and groESL operon (1,646 bp) sequences of A. platys isolated from a dog were determined, revealing that both sequences were closely related to the sequences of an A. platys strain detected in R. sanguineus ticks from the Democratic Republic of Congo.

Western Blot Analysis of Sera Reactive to Human Monocytic Ehrlichiosis and Human Granulocytic Ehrlichiosis Agents

ABSTRACT Laboratory diagnosis of human ehrlichioses is routinely made by an indirect immunofluorescence assay (IFA) using cultured ehrlichia-infected whole cells as antigen. Concern has been raised that incorrect diagnoses of human monocytic ehrlichiosis (HME) or human granulocytic ehrlichiosis (HGE) may be made on the basis of serologic cross-reactivity between Ehrlichia chaffeensis and the agent of HGE. The present study examined whether two recombinant major outer membrane proteins, rP30 and rP44, that were previously shown to be sensitive and specific serodiagnostic antigens for HME and HGE, respectively, could be used to discriminate IFA dually reacting sera. Thirteen dually IFA-reactive sera, three sera that were IFA positive only with E. chaffeensis, and three sera that were IFA positive only with the HGE agent were examined by Western immunoblot analysis using purified whole organisms and recombinant proteins as antigens. All 16 E. chaffeensis IFA-positive sera reacted with rP30. However, none of these sera reacted with rP44, regardless of IFA reactivity with the HGE agent. The three HGE-agent-only IFA-positive sera reacted only with rP44, not with rP30. Western immunoblotting using purified E. chaffeensis and the HGE agent as antigens suggested that heat shock and other proteins, but not major outer membrane proteins, cross-react between the two organisms. Therefore, Western immunoblot analysis using rP44 and rP30 may be useful in discriminating dually HME and HGE IFA-reactive sera.

Cytokine Gene Expression by Peripheral Blood Leukocytes in Dogs Experimentally Infected with a New Virulent Strain of Ehrlichia canis

Abstract:  Ehrlichia canis (E. canis) is a lipopolysaccharide (LPS)‐deficient obligatory intracellular bacterium that causes canine monocytic ehrlichiosis, a chronic febrile disease accompanied with hematological abnormality. This study analyzed temporal expression levels of IL‐1β, IL‐2, IL‐6, IL‐8, IFN‐γ, and TNF‐α mRNA by peripheral blood leukocytes from dogs experimentally infected with a new virulent strain of E. canis by using real‐time RT‐PCR. Relative levels of IL‐1β and IL‐8 transcripts normalized by the β‐actin transcript levels, were significantly upregulated, whereas those of TNF‐α and IFN‐γ transcripts were only weakly upregulated in all three infected dogs, starting from 2 days up to 52 days post inoculation. The expressions of IL‐2 and IL‐6 genes were extremely low compared with the positive control (ConA‐stimulated canine peripheral blood leukocytes). This study showed that E. canis can induce chronic expression of a subset of proinflammatory cytokine genes: balance, timing, and duration of these cytokine generations may contribute to the progression of canine ehrlichiosis.