Serological, Hematologic, and PCR Studies of Cattle in an Area of Switzerland in Which Tick-Borne Fever (Caused by Ehrlichia phagocytophila) Is Endemic

The purpose of this study was to examine the seasonal variations in seroprevalence to Ehrlichia phagocytophila in cattle pastured during the summer months in an area where tick-borne fever is endemic. The study was performed during a 1-year period from April 1996 to March 1997 and involved 34 cows, 22 pregnant heifers, and 14 calves. Blood samples, collected from all 70 cattle once a month, were used to determine serum immunoglobulin G titers by indirect immunofluorescence. In addition, blood smears were examined for Ehrlichia organisms, and PCR amplification was performed for the molecular detection of E. phagocytophila. Prior to the pasture period, the seroprevalence was 16%. Two weeks after the start of pasturing, it was 43%, after which it progressively increased and reached a maximum of 63% in September. The seroprevalence progressively decreased after the end of pasturing to a low of 23%. The variation in antibody titers was similar to that of seroprevalence. E. phagocytophila organisms were detected in blood smears of 7 animals and by nested PCR in 12. Only four cows, which were on the pastures of endemicity for the first time, had clinical signs of ehrlichiosis. This study demonstrated marked seasonal variations in seroprevalence and in serum titers of antibody to E. phagocytophila in cattle. The incidence of clinical signs of ehrlichiosis was increased in cattle grazing on the pastures of endemicity for the first time.     

Detection of Ehrlichia phagocytophila DNA in Ixodes ricinus Ticks from Areas in Switzerland Where Tick-Borne Fever Is Endemic

A total of 1,523 adult Ixodes ricinus ticks were collected from regions where bovine ehrlichiosis is endemic and were examined for Ehrlichia phagocytophila via PCR. Of the ticks from cattle with ehrlichiosis, the ticks from healthy cattle, and the free-living ticks, 26.5% (18 of 68), 4.4% (35 of 802), and 0.8% (5 of 653), respectively, were positive.     

Sequential Evaluation of Dogs Naturally Infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii

Historically, disease manifestations in dogs seroreactive to Ehrlichia canis antigens by indirect immunofluorescent antibody testing have been attributed to infection with either E. canis or Ehrlichia ewingii. A 1996 study by Dawson and colleagues provided PCR evidence that healthy dogs from southeastern Virginia could be naturally infected with Ehrlichia chaffeensis. This observation stimulated us to determine which Ehrlichia spp. infected sick dogs that were referred to our hospital from the same region. Based upon PCR amplification with species-specific primers, sick dogs seroreactive to E. canis antigens were determined to be infected with four Ehrlichia species: E. canis, E. chaffeensis, E. equi, and E. ewingii. Coinfection with three Ehrlichia species (E. canis, E. ewingii, and E. equi) was documented for one dog. An additional canine pathogen presumed to be tick transmitted, Bartonella vinsonii subsp. berkhoffii, was identified in 7 of 12 dogs. Importantly, our results indicate that in naturally infected dogs, E. chaffeensis can cause severe disease manifestations that are clinically and serologically indistinguishable from disease manifestations of E. canis or E. ewingii. In addition, our findings support the efficacy of doxycycline for treatment of E. canis, E. equi, and E. ewingii infections but indicate that, based upon the persistence of E. chaffeensis DNA for 1 year following treatment, E. chaffeensis infection in dogs may be more refractory to doxycycline treatment. Undetected coinfection with Bartonella may also complicate the evaluation of treatment efficacy while resulting in disease manifestations that mimic ehrlichiosis.     

Identification of a Granulocytic Ehrlichia Strain Isolated from a Horse in Switzerland and Comparison with Other Rickettsiae of the Ehrlichia phagocytophila Genogroup

This case report describes a 12-year-old Arabian mare with granulocytic ehrlichiosis. Clinical signs included fever, apathy, anorexia, icterus, limb edema, and reluctance to move. Examination of buffy coat smears revealed Ehrlichia organisms in neutrophils and eosinophils. A band of 1,428 bp was amplified from DNA of leukocytes via nested PCR and was identified as part of the Ehrlichia 16S rRNA gene. It differed from the gene sequences of Ehrlichia phagocytophila and E. equi at two and three positions, respectively. Interestingly, the nucleotide sequence of the 16S rRNA was 100% identical to that of the agent of human granulocytic ehrlichiosis.     

Experimental Transmission of Ehrlichia equi to Horses through Naturally Infected Ticks (Ixodes pacificus) from Northern California

We report the experimental transmission of Ehrlichia equi from naturally infected Ixodes pacificus ticks to horses. Three weeks after exposure to ticks, two of three horses developed clinical signs compatible with E. equi infection, while one horse remained asymptomatic. 16S rRNA gene PCR of blood leukocyte lysates was positive for all horses at various time points; two horses seroconverted. The 16S rRNA gene sequences amplified from tick-exposed horses showed more than 99% homology to corresponding fragments of the 16S rRNA genes of E. equi, Ehrlichia phagocytophila, and the human granulocytic ehrlichiosis agent.     

Amplification of Ehrlichial DNA from Dogs 34 Months after Infection with Ehrlichia canis

In order to determine whether dogs in the subclinical phase of canine monocytic ehrlichiosis (CME) are carriers of Ehrlichia canis and to determine the significance of persistent indirect immunofluorescent anti-E. canis antibody titers during this phase, PCR was performed with blood, bone marrow, and splenic aspirates collected 34 months postinoculation from six clinically healthy beagle dogs experimentally infected with E. canis. At least one of the three samples (spleen, bone marrow, and blood) from four of the six dogs was PCR positive. The spleens of all four of these dogs were PCR positive, and the bone marrow and blood of two of the four dogs were PCR positive. Indirect immunofluorescent-antibody titers increased progressively during the first 5 months postinfection, remained high for an additional period of more than 11 months, and declined thereafter, suggesting that the dogs were recovering from the disease. Five of the dogs remained seropositive 34 months postinfection. The data obtained in this study demonstrate for the first time that clinically healthy dogs in the subclinical phase of CME are carriers of the rickettsia. It was shown that dogs can harbor E. canis for years without developing the chronic clinical disease and that dogs can eliminate the parasite and recover from CME without medical treatment. Our findings suggest that the spleen is the organ most likely to harbor E. canis parasites during the subclinical phase and the last organ to accommodate the parasite before elimination. It was concluded that PCR of DNA extracted from splenic aspirates is a reliable method for determining the carrier state of CME.     

Human Exposure to a Granulocytic Ehrlichia and Other Tick-Borne Agents in Connecticut

Indirect fluorescent-antibody (IFA) staining methods with Ehrlichia equi (MRK or BDS strains) and Western blot analyses containing a human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain) were used to confirm probable human cases of infection in Connecticut during 1995 and 1996. Also included were other tests for Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis (HME), Babesia microti, and Borrelia burgdorferi. Thirty-three (8.8%) of 375 patients who had fever accompanied by marked leukopenia or thrombocytopenia were serologically confirmed as having HGE. Western blot analyses of a subset of positive sera confirmed the results of the IFA staining methods for 15 (78.9%) of 19 seropositive specimens obtained from different persons. There was frequent detection of antibodies to a 44-kDa protein of the HGE agent. Serologic testing also revealed possible cases of Lyme borreliosis (n = 142), babesiosis (n = 41), and HME (n = 21). Forty-seven (26.1%) of 180 patients had antibodies to two or more tick-borne agents. Therefore, when one of these diseases is clinically suspected or diagnosed, clinicians should consider the possibility of other current or past tick-borne infections.     

Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis

Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.     

Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection.

Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.     

Therapeutic Effect of Doxycycline in Experimental Subclinical Canine Monocytic Ehrlichiosis: Evaluation of a 6-Week Course

The efficacy of doxycycline treatment (10 mg/kg of body weight every 24 h for 42 days) in eliminating Ehrlichia canis from four subclinically infected dogs was evaluated. One dog remained PCR positive, suggesting that 6 weeks of doxycycline treatment may not be sufficient to clear E. canis parasites from all subclinically infected dogs. Serology (indirect immunofluorescent antibody assay) was shown to be unreliable in assessing recovery from the carrier state, as anti-E. canis antibodies persisted after elimination of the parasite. Our findings suggest that an increase in the platelet count may be an important indicator for dogs that recover from subclinical ehrlichiosis.     

Serologic Evidence of a Natural Infection of White-Tailed Deer with the Agent of Human Granulocytic Ehrlichiosis in Wisconsin and Maryland

White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.     

Serological evidence of human granulocytic ehrlichiosis in Switzerland

To investigate whether human granulocytic ehrlichiosis (HGE) is prevalent in Switzerland, 1515 human serum samples from individuals with different risks for tick exposure were tested for antibodies toEhrlichia phagocytophila, a surrogate marker of the agent of HGE. The distribution of titres showed marked differences between sera of individuals with no or low risk for tick exposure and those with a high risk. The results of serological testing provided evidence of HGE in Switzerland as well as evidence of two types of coinfections: those with the agent of HGE andBorrelia burgdorferi, and those with the agent of HGE and the central European tickborne encephalitis virus.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State.

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

Detection of Antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis Antigens in Sera of Korean Patients by Western Immunoblotting and Indirect Immunofluorescence Assays

Two hundred seventy one serum samples from South Korean patients were tested to detect antibodies against Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) and Ehrlichia chaffeensis (the human monocytic ehrlichiosis agent) by indirect fluorescent-antibody assay (IFA) and the Western blot assay. These sera were collected from patients with symptoms of high fever. The rate of seropositivity for Orientia tsutsugamushi was 50.9% by IFA at the Public Health & Environmental Research Institute and National Institute of Health in South Korea. By IFA, 30 (11.1%) and 39 (14.4%) of the serum samples reacted with A. phagocytophilum and E. chaffeensis antigens, respectively. By the Western blot assays, 24 (8.9%) and 29 (10.7%) of the serum samples reacted with purified A. phagocytophilum and E. chaffeensis protein antigens, respectively. This report strengthens other evidence regarding the presence of A. phagocytophilum and E. chaffeensis infections in humans in South Korea.     

Serological evidence of infection with Ehrlichia spp. in red foxes (Vulpes vulpes) in Switzerland

Serum samples from 1,550 red foxes in Switzerland were tested for antibodies to the agents of canine granulocytic and monocytic ehrlichiosis by an indirect immunofluorescent technique. Forty-four (2.8%) of the samples were positive for Ehrlichia phagocytophila, which is an antigen marker for granulocytic ehrlichiosis. In contrast, none of the samples had antibodies specific to Ehrlichia canis, the agent of monocytic ehrlichiosis.     

Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.     

Immunodiagnosis of Human Granulocytic Ehrlichiosis by Using Culture-Derived Human Isolates

Human granulocytic ehrlichiosis (HGE) is an emerging infection caused by an Ehrlichia species closely related to Ehrlichia equi and Ehrlichia phagocytophila. Recent advances in the isolation and cultivation of this organism have allowed us to develop an immunofluorescence assay (IFA), enzyme immunoassay (EIA), and Western immunoblotting (WB) using HL-60 cell culture-derived human isolates. Antibody was detected in sera from culture-confirmed HGE patients by IFA and EIA, and these samples were reactive when analyzed by immunoblot analysis. HGE patient sera had high antibody titers and did not react with uninfected HL-60 cells. When IFA, EIA, and WB were used to analyze sera from healthy donors or those with a range of other disorders, including infections caused by Ehrlichia chaffeensis, Rickettsia rickettsii, and Coxiella burnetti, no significant cross-reactivity could be detected by EIA or immunoblot analysis with the exception of two of four serum samples from R. rickettsii-infected patients that were reactive by IFA only. Sera from HGE patients did not significantly cross-react in serologic tests for Borrelia burgdorferi. Using sera from patients previously enrolled in two clinical trials of treatment for early Lyme disease, we evaluated a two-step approach for estimation of the seroprevalence of antibodies reactive with the etiologic agent of HGE. On the basis of the immunoblot assay results for sera from culture-confirmed HGE patients, WB was used to confirm the specificity of the antibody detected by EIA and IFA. EIA was found to be superior to IFA in the ability to detect WB-confirmed antibodies to the HGE agent. When EIA and WB were used, 56 (19.9%) patients with early Lyme disease (n = 281) had either specific immunoglobulin M (IgM) or IgG antibodies; 38 patients (13.5%) had IgM only, 6 (2.1%) had IgG only, and 12 (4.3%) had both IgM and IgG. Therefore, Lyme disease patients are at high potential risk for exposure to Ehrlichia. Analysis by immunoblotting of serial samples from persons with culture-confirmed HGE or patients with Lyme disease and antibodies to the agent of HGE revealed a reproducible pattern of the immune response to specific antigens. These samples confirmed the importance of the 42- to 45-kDa antigens as early, persistent, and specific markers of HGE infection. Other significant immunogenic proteins appear at 20, 21, 28, 30, and 60 kDa. Use of the two-test method of screening by EIA and confirming the specificity by WB appears to offer a sound approach to the clinical immunodiagnosis of HGE.     

Predominance of Ehrlichia ewingii in Missouri dogs

To investigate the species distribution of Ehrlichia present in Missouri dogs, we tested 78 dogs suspected of having acute ehrlichiosis and 10 healthy dogs. Blood from each dog was screened with a broad-range 16S rRNA gene PCR assay that detects known pathogenic species of Ehrlichia and ANAPLASMA: The species was determined by using species-specific PCR assays and nucleotide sequencing. Ehrlichia antibody testing was performed by using an indirect immunofluorescence assay with Ehrlichia chaffeensis as the antigenic substrate. The broad-range assay detected Ehrlichia or Anaplasma DNA in 20 (26%) of the symptomatic dogs and 2 (20%) of the asymptomatic dogs. E. ewingii accounted for 20 (91%), and E. chaffeensis accounted for 1 (5%) of the positives. Anaplasma phagocytophilum DNA was detected in one dog, and the sequences of regions of the 16S rRNA gene and the groESL operon amplified from the blood of this dog matched the published sequences of this organism. Antibodies reactive with E. chaffeensis were detected in 14 (67%) of the 21 PCR-positive dogs and in 12 (19%) of the 64 PCR-negative dogs. Combining the results of PCR and serology indicated that 33 (39%) of 85 evaluable dogs had evidence of past or current Ehrlichia infection. We conclude that E. ewingii is the predominant etiologic agent of canine ehrlichiosis in the areas of Missouri included in this survey. E. canis, a widely recognized agent of canine ehrlichiosis, was not detected in any animal. The finding of E. ewingii in asymptomatic dogs suggests that dogs could be a reservoir for this Ehrlichia species.     

Isolation and Characterization of Two European Strains of Ehrlichia phagocytophila of Equine Origin

We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.     

Serological evidence of canine monocytic ehrlichiosis in Iran

Ehrlichia canis has a worldwide geographic distribution, but little is known about the occurrence of Canine monocytic ehrlichiosis (CME) in Iran. The current study was carried out to evaluate the seroprevalence and factors associated with a positive antibody response to E. canis in dogs from Kerman city, southeast of Iran. One hundred and twenty-three privately owned dogs were chosen from apparently healthy animals referred to veterinary hospital for health check or vaccination. All dogs were subjected to physical, hematological, and biochemical examinations and serological tests. Indirect immunofluorescence antibody test (IFA) and rapid immunochromatography assay (ICA) used to detect antibodies against E. canis in all sera. The overall seroprevalence of CME was 14.63% which was determined as 13.8% and 10.6% using IFA and ICA, respectively. Hyperglobulinemia (p = 0.001), age (p = 0.005), and elevated alkaline phosphatase (ALP) level (p = 0.02) showed a statistical relationship with seropositivity. Hematological abnormalities did not differ significantly between seronegative and seropositive dogs except for the group with high IFA titer. In blood smears from three infected dogs (16.66%), typical morulae of E. canis were observed in monocytes. All three cases were seropositive for E. canis and displayed obvious hyperglobulinemia, thrombocytopenia, leukopenia, anemia, and high ALP level. There was no sex and breed predilection among seropositive dogs. This is the first report that describes serological evidences of canine monocytic ehrlichiosis in southeast of Iran. Additional molecular studies are necessary to confirm E. canis infection and to identify the local strains of the organism.