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

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 na?ve dogs.     

Molecular and antigenic comparison of Ehrlichia canis isolates from dogs, ticks, and a human in Venezuela

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 using E. 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. canis Oklahoma. 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.     

Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay

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.     

Sequence and expression analysis of virB9 of the type IV secretion system of Ehrlichia canis strains in ticks, dogs, and cultured cells

Ehrlichia canis virB9 was cloned and expressed. The sequences of virB9 from six geographic locations were identical. virB9 was transcribed by E. canis in dogs, ticks, and cell culture. Infected dogs had antibodies to recombinant VirB9, indicating that VirB9 was produced by E. canis in dogs and was antigenic.     

Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans.

Ehrlichia chaffeensis, E. canis, and E. ewingii are genetically closely related, as determined by 16S rRNA gene base sequence comparison, but they exhibit biologic differences. E. chaffeensis is the etiologic agent of human ehrlichiosis. E. canis and E. ewingii cause two distinctly different forms of canine ehrlichiosis and infect different types of leukocytes, monocytes and granulocytes, respectively. E. chaffeensis can also infect dogs. In the study, Western immunoblot analysis of sera from dogs inoculated with E. chaffeensis, E. canis, or E. ewingii was performed to determine antigenic specificity and the intensities of the reactions to purified E. chaffeensis and E. canis antigens. At 2 to 3 weeks postexposure, antisera from four dogs inoculated with E. chaffeensis reacted with 64-, 47-, 31-, and 29-kDa proteins of E. chaffeensis but reacted poorly with E. canis antigen. In contrast, at 2 to 3 weeks postexposure, antisera from four E. canis-inoculated dogs reacted strongly with the 30-kDa major antigen of E. canis but reacted poorly with proteins from E. chaffeensis. At 4 weeks postexposure, the sera from three E. ewingii-inoculated dogs showed weak binding to 64- and 47-kDa proteins of both E. chaffeensis and E. canis. Convalescent-phase sera from human ehrlichiosis patients and sera from dogs chronically infected with E. ewingii strongly reacted with similar sets of proteins of E. chaffeensis and E. canis with similar intensities. However, sera from dogs chronically infected with E. canis reacted more strongly with a greater number of E. canis proteins than with E. chaffeensis proteins. The protein specificity described in the report suggests that dogs with E. canis infections can be distinguished from E. chaffeensis-infected animals by Western immunoblot analysis with both E. canis and E. chaffeensis antigens.     

Expression of members of the 28-kilodalton major outer membrane protein family of Ehrlichia chaffeensis during persistent infection

The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.     

Molecular detection of Ehrlichia canis,Anaplasma bovis,Anaplasma platys,Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.     

Structural analysis of chlamydial major outer membrane proteins.

The primary structure and surface exposure of the major outer membrane protein (MOMP) isolated from 14C intrinsically or 125I extrinsically radiolabeled Chlamydia trachomatis serotypes D/UW-3, G/UW-57, H/UW-4, I/UW-12, and L2/434 and the Chlamydia psittaci meningopneumonitis strain were analyzed by two different peptide-mapping techniques. Radiolabeled proteins were digested with either Staphylococcus aureus V8 protease, the patterns of peptide fragments produced being displayed by sodium dodecyl sulfate gel electrophoresis, or alpha-chymotrypsin, the peptides being analyzed after separation by high-voltage electrophoresis and thin-layer chromatography. The comparative structural data obtained from these two different techniques were remarkably similar. From these data, the following points could be made. (i) MOMPs are structurally heterogeneous between members of chlamydial species; the C. psittaci MOMP was clearly distinct from each of the C. trachomatis MOMPs. (ii) Considerable structural homology occurs among MOMPs from different C. trachomatis serotypes; however, distinct differences in the primary structure of each C. trachomatis MOMP were evident. (iii) These observed differences were most obvious in peptide maps of MOMPs isolated from chlamydiae that had been surface labeled by lactoperoxidase-mediated radioiodination. The surface-exposed portions of the MOMPs from serotypes L2 and D were very similar. In contrast, those from serotypes G, H, and I were quite different. These structural data are in agreement with the serospecificities described for these proteins.     

Antigenic characterization of ehrlichiae: protein immunoblotting of Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii.

In recent years a febrile illness apparently associated with tick bite in patients in the United States has been attributed to infection by an Ehrlichia species. This implication is based on serologic responses to E. canis, morphologic demonstration of ehrlichiae in clinical materials, and a single isolate distinct from E. canis which was obtained from a human patient by the Centers for Disease Control. Little is known about the antigens of the ehrlichiae. This report expands the breadth of available knowledge concerning the antigenic components and serologic responses to component antigens of E. canis, E. sennetsu, and E. risticii. Protein immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using density gradient-purified ehrlichiae and homologous antisera demonstrated reproducible and characteristic antigens within each species (for E. sennetsu, 91, 64, 54, 44, 36, 34, 28, 25, and 24 kDa; for E. risticii, 70, 52, 48, 44, 35, 28, 24, 23, and 20 kDa; for E. canis, 110, 64, 52, 42, 33, 28, 24, 23, and 20 kDa). When antisera were reacted with heterologous antigens, cross-reactivity among these species was virtually restricted to the 70-kDa antigen. Furthermore, when serum samples obtained from 10 patients who were convalescing from ehrlichiosis were tested against each antigen, only three serum samples had any reactivities, and these serum samples reacted with only a few of the antigenic bands. These results documented the molecular sizes of electrophoretically separated antigens of the three Ehrlichia species, confirm their serologic relationships, and support the novel nature of the agent(s) of human ehrlichiosis in the United States.     

Reisolation of Ehrlichia canis from blood and tissues of dogs after doxycycline treatment.

We present evidence that supports the carrier status of dogs experimentally infected with Ehrlichia canis after treatment with doxycycline. Canine ehrlichiosis was induced in five dogs by intravenous inoculation with E. canis-infected DH82 cells. All animals developed mild clinical signs of transient fever, body weight loss, thrombocytopenia, and increased gamma globulin levels in plasma. An indirect fluorescent-antibody test (IFA) revealed that all dogs had seroconverted (titer, 5,120) by day 10 postinoculation (p.i.). E. canis was reisolated from blood samples collected at intervals throughout the 2-month period p.i. Doxycycline was administered orally once daily at 10 mg/kg of body weight per day for 1 week starting at 2 months p.i. Following treatment, gamma globulin levels in plasma were decreased. At necropsy on days 54 to 59 after the start of treatment, spleen, liver, kidney, and lymph nodes were collected for E. canis culture and histopathologic examination. Although the dogs did not show significant clinical signs during or after treatment with the antibiotic, E. canis was reisolated from the blood and tissue samples of three of five dogs. A 16-fold reduction in IFA titer was noted in two dogs which were negative for E. canis reisolation at day 49 after the start of treatment, whereas a zero- to fourfold reduction in IFA titer was seen in the remaining three dogs. Western immunoblot reactions to higher-molecular-size E. canis antigens in the sera of two dogs which were negative for E. canis on blood culture decreased, whereas they remained continuously high or only transiently decreased for the duration of the study for antigens in the sera of three dogs from which E. canis was reisolated. Histopathologically, prominent plasmacytosis in the kidney cortex was present in three dogs from which E. canis was reisolated, whereas the kidney cortices of two dogs had moderate to minor plasmacytosis. These findings pose questions regarding the efficacy, dosage and duration of doxycycline treatment in dogs with E. canis infection. In addition, it was shown that IFA and Western immunoblotting may aid in assessing the efficacy of antibiotic therapy when definitive reisolation procedures are not readily available.     

Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil

The current study evaluated the prevalence of Ehrlichia canis Donatien and Lestoquard in domestic dogs, Canis familiaris L., and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) ticks from different areas of Brazil. In Monte Negro County (state of Rond?nia, Brazilian western Amazon), the indirect immunofluorescence assay detected E. canis-reactive antibodies (titer > or = 40) in 58/153 (37.9%) and 40/161 (24.8%) dogs from the urban and rural areas, respectively. These values were significantly different between the two areas. Ticks from a household in the urban area of Monte Negro, and from households in three other localities (162-165 adult ticks per household) in the state of S?o Paulo (SP) were tested by polymerase chain reaction (PCR) targeting an erlichial dsb gene fragment. The prevalence of infected ticks (given as minimal infection rate) was 2.3, 6.2, and 3.7% for populations 1 (Monte Negro), 2 (Jundiaí, SP), and 3 (S?o Paulo I, SP), respectively, which were statistically similar. In contrast, no infected tick was detected in population 4 (S?o Paulo II, SP). DNA sequences were determined for some of the PCR products generated from ticks and dogs from populations 1-3, being all identical to each other and to available sequences of E. canis in GenBank. These results reinforce previous records of E. canis-infecting dogs in Brazil. Natural infection of R. sanguineus ticks by E. canis is reported for the first time in Brazil, where this tick is the commonest species infesting dogs.     

C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis.

To elucidate whether acute-phase protein responses occur in dogs infected with Ehrlichia canis, C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels were serially measured in the plasma of five dogs experimentally inoculated with E. canis and 10 sham-inoculated or noninoculated control dogs. The CRP concentration was measured by a canine-specific capture enzyme-linked immunosorbent assay, and the AAG concentration was measured by a canine-specific radial immunodiffusion method. In all E. canis-inoculated dogs, a 3.3- to 6.5-fold increase in the plasma CRP concentration and a 1.9- to 8.6-fold increase in the plasma AAG concentration over the preinoculation level occurred at days 4 to 6 postexposure. Despite the persistence of E. canis and high antibody titers, both CRP and AAG concentrations gradually declined to preexposure levels by day 34 postexposure. E. canis-infected dogs had mild and transient clinical signs which resolved without treatment by day 14 postexposure. The CRP and AAG concentrations in control inoculated or nontreated dogs remained within the normal range throughout the experimental period. Of 12 dogs naturally infected with E. canis, 75% had greater than 50 micrograms of CRP per ml and 83% had greater than 500 micrograms of AAG per ml. All of these 12 dogs had chronic and severe clinical signs of canine ehrlichiosis. Thus, elevations in the levels of acute-phase proteins occur in both acute and chronic canine ehrlichiosis. Determination of CRP and AAG concentrations may help in assessing the severity of inflammatory damage in dogs with E. canis infections.     

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.     

Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains

The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.Ehrlichia canis is a globally distributed, tick-transmitted, obligately intracellular bacterium that is the primary etiological agent of canine monocytic ehrlichiosis and has been identified as being the cause of human ehrlichiosis in patients from Venezuela (38, 39). Rickettsiosis in dogs caused by E. canis was first reported in 1935 in Algeria and was later reported in southern India and other parts of Africa in the 1940s (9, 31). Subsequently, E. canis was relatively unrecognized until it was associated with outbreaks of canine tropical pancytopenia in Singapore and Malaysia from 1963 to 1968 (51) and was identified as being the cause of an epizootic of canine tropical pancytopenia in U.S. military dogs stationed in Vietnam in late 1968 (17, 36). E. canis infections have since been well documented in the United States, Israel, Brazil, and Vietnam (1, 3, 12, 16, 20-22, 36, 49), and serologic and/or molecular evidence of infection in temperate regions where Rhipicephalus sanguineus is commonly found, including Central and South America, the Caribbean, parts of Africa, southern Europe, and southeast Asia, has also been reported (2, 5-8, 15, 18, 19, 23, 32, 33, 41, 42, 44, 50).The development of globally useful serologically and molecularly based diagnostics as well as effective vaccines for canine monocytic ehrlichiosis is dependent on an understanding of the genetic diversity of E. canis, particularly with respect to major immunoreactive proteins. Molecular characterization of evolutionarily conserved genes such as 16S rRNA has provided little information on strain diversity and suggests a high level of conservation (39, 40, 43, 47, 48). Similarly, the immunoreactive major outer membrane proteins p28 and p30 in U.S. and Venezuelan strains of E. canis appear to be highly conserved (13, 29, 30, 46), an observation that was extended to characterized E. canis strains from six human patients from Venezuela (38). Other genes such as the thio-oxidoreductase gene (dsb) and gltA were also found to be conserved in geographically dispersed strains (23, 32).The genome of E. canis has been sequenced, and a small group of acidic tandem repeat- and ankyrin repeat-containing proteins associated with host-pathogen interactions were identified (24). Several of these proteins are considered major immunoreactive proteins and have been well studied, including gp200, gp140, gp36, and gp19 (11, 25, 26, 28, 53). E. canis gp36 is an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (11). Examination of the gp36 gene in U.S., Brazilian, and Cameroonian strains of E. canis identified variations in the numbers of tandem repeats and nucleic acid changes that resulted in four amino acid substitutions (10). However, the diversities of other major immunoreactive E. canis proteins in globally dispersed strains are not known. A homogeneous pattern of proteins reacting with E. canis dog sera from the United States, France, Israel, and the Virgin Islands by immunoblotting was previously reported (14). However, differences in protein reactivity were noted with sera collected from dogs from Italy and Zimbabwe, suggesting the potential for diversity in the antigenic composition of E. canis strains in these countries (14).The objective of this study was to determine the genetic and antigenic diversities of proteins subject to immune pressure in globally dispersed strains of E. canis. Four major immunoreactive protein genes (gp200, gp140, gp36, and gp19) were sequenced from each strain, and immunoblotting profiles for E. canis whole-cell lysates were compared. Strains from the United States and Brazil exhibited homogeneous immunoblotting patterns compared to that of the strain from Israel. Sequencing of four major immunoreactive protein genes demonstrated that U.S. and Brazilian strains were highly similar and that strains from Israel were the more divergent.     

Zoonotic Anaplasma phagocytophilum,Ehrlichia canis,Dirofilaria immitis,Borrelia burgdorferi,and spotted fever group rickettsiae (SFGR) in different types of dogs

Parasitology Research - Dogs can carry and share zoonotic pathogens with humans. This problem is understudied in different parts of the world, including Jordan. This study determined the prevalence...