Antigenic analysis of four species of the genus Ehrlichia by use of protein immunoblot

The antigenic profile of Ehrlichia canis, E risticii, E sennetsu, and E equi was investigated by the use of protein (western) immunoblot technique. Results of analysis of serum from acutely and chronically infected animals indicated that the 4 Ehrlichia species share a unique 25-kD polypeptide in addition to other peptides. Immune sera from dogs inoculated with E canis recognized a wide range of E canis polypeptide antigens, as determined by western blot analysis. A larger number of E sennetsu polypeptides were detected when homologous antiserum and antiserum to E equi were used. The latter antiserum did not recognize antigens of E canis or E risticii. Antisera to E canis, E risticii, and E sennetsu detected E equi antigens. Data indicate that a 25-kD protein is a common antigen among the species of the genus Ehrlichia and that the ascending order of abundance of immunodominant determinants in the 4 species of Ehrlichia studied would be: E risticii----E equi----E sennetsu----E canis. Implications of these findings for diagnosis of ehrlichial infections and prophylaxis are evident.     

Molecular detection and characterization of Ehrlichia canis from dogs in Turkey

Seroprevalence of Ehrlichia canis antibodies among dogs in Turkey were previously reported, however, the ehrlichial organism has never been characterized in this region. The current study examined dogs from Ankara with febrile illness for E. canis infection with E. canis-specific PCR. Three of the 12 blood specimens from dogs showing clinical signs compatible with canine ehrlichiosis were found to be positive by PCR using E. canis-specific primers. E. canis detected in one of the blood specimens was designated as Kutahya strain. The representative E. canis strain was characterized by 16S rRNA gene sequencing and Western blot analysis of the plasma sample from the dog infected with E. canis. The 16S rRNA sequence (1,388 bp) of the E. canis Kutahya was identical to that of Ehrlichia ovina from a sheep in Turkey and Venezuelan Dog Ehrlichia (VDE) and was closely related (99.9%) to that of type strain of E. canis, Oklahoma. The plasma of the dog infected with E. canis Kutahya was analyzed by Western blotting using the purified E. canis Oklahoma strain as antigen. The reactive antibody profiles of the dog infected with E. canis Kutahya was found to be similar to those of dogs infected with E. canis Oklahoma and VDE, suggesting the antigenic similarities among these strains. The findings in this study would help for a better understanding of epidemiology of canine ehrlichiosis. This is the first report of molecular detection and characterization of an ehrlichial agent in Turkey.     

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.     

Significance of serological testing for ehrlichial diseases in dogs with special emphasis on the diagnosis of canine monocytic ehrlichiosis caused by Ehrlichia canis

Dogs are susceptible to a number of ehrlichial diseases. Among them, canine monocytic ehrlichiosis is an important and potentially fatal disease of dogs caused by the rickettsia Ehrlichia canis. Diagnosis of the disease relies heavily on the detection of antibodies and is usually carried out using the indirect immunofluoresence antibody (IFA) test. The IFA test may be confounded by cross-reactivities between a number of the canine ehrlichial pathogens. This article presents a review of the ehrlichial diseases affecting dogs with reference to their immune responses, host specificities, cross-reactivites and diagnosis. Diagnostic means such as Western immunblot, dot-blot and PCR are discussed. The use of the IFA test as a diagnostic means for E. canis is presented along with its potential pitfalls. The review emphasizes that the disease process, cross-reactivites with other ehrlichial species, multiple tick-borne infections and persistent IFA antibody titers post-treatment, should all be considered when interpreting E. canis serological results.     

Detection of ehrlichial infection by PCR in dogs from Yamaguchi and Okinawa Prefectures, Japan

Species-specific nested polymerase chain reaction (PCR) was used to detect the presence of possible canine ehrlichial agents (Ehrlichia canis, E. chaffeensis, E. ewingii, E. equi and E. platys) and monocytic ehrlichial agents found in Japan (E. muris and a recently discovered Ehrlichia species detected from Ixodes ovatus) in blood samples from dogs in Yamaguchi and Okinawa Prefecture, Japan. Partial sequence of E. platys was detected from 1 of 67 dogs (1.5%) tested from Yamaguchi Prefecture and 24 out of 87 (27.6%) in the subtropical Okinawa Prefecture. Dogs in Okinawa and Miyako Islands had a higher positive rate (69.2 and 45.0%, respectively) than Ishigaki Island (11.1%). Another dog in Yamaguchi Prefecture had a positive PCR reaction to the Ehrlichia sp. detected from I. ovatus. No other Ehrlichia were found in these samples.     

Evaluation of granulocytic ehrlichiosis in dogs of Missouri, including serologic status to Ehrlichia canis, Ehrlichia equi and Borrelia burgdorferi.

Canine granulocytic ehrlichiosis was diagnosed in 37 dogs by finding ehrlichial morulae in 0.1 to 26.2% of their blood neutrophils and eosinophils. All 37 dogs had clinical signs of arthritis or muscular stiffness. Titer to Ehrlichia canis was determined in sera from 31 of the 37 dogs; 25 dogs had titer ranging from 1:20 to 1:5,120. In the other 6 dogs, titer to E canis was less than 1:10. The most common hematologic abnormality in these dogs, other than rickettsiemia, was thrombocytopenia. Granulocytes infected with ehrlichial organisms were not found in another 10 dogs that had clinical signs of arthritis or muscular stiffness. Of these 10 dogs, 3 had titer to E canis ranging from 1:40 to 1:320. Titer in the other 7 dogs was less than 1:10. Ehrlichial morulae were not found in the granulocytes of 18 healthy dogs. Of these 18 dogs, 9 had titer to E canis ranging from 1:20 to 1:5,120. Titer in the other 9 dogs was less than 1:10 Titer to Borrelia burgdorferi was determined in dogs with granulocytic ehrlichiosis, arthritic dogs without detected rickettsiemia, and in healthy dogs. Low titer determined by 2 laboratories was considered to be nonspecific reaction in all 3 groups of dogs and, thus, did not indicate that the arthritic disorders were attributable to canine borreliosis.     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

Granulocytic Ehrlichiosis in Dogs from North Carolina and Virginia

Medical records of 3 dogs from North Carolina and 3 dogs from Virginia with ehrlichial morulae in circulating neutrophils were studied retrospectively. Two clinically distinct disease syndromes, including chronic, moderate to severe anemia (n = 3) and polyarthritis (n = 2) were associated with canine granulocytic ehrlichiosis (CGE) in these dogs. One dog was clinically healthy, and abnormalities were not detected during physical examination. Clinical signs were nonspecific and included fever, lethargy, anorexia, vomiting, and diarrhea. The most frequent laboratory abnormalities were normocytic normochromic nonregenerative anemia, moderate thrombocytopenia with large platelets, lymphopenia, and eosinopenia. Considerable variability was found in the serologic responses to Ehrlichia equi, Ehrlichia canis , and Ehrlichia chaffeensis antigens among the 5 dogs for which stored sera were available for indirect fluorescent antibody testing. Polymerase chain reaction amplification and sequencing of portions of the 16S rRNA gene from blood (collected in ethylenediaminetetraacetic acid) of 1 severely anemic dog (dog 3) and 1 polyarthritic dog (dog 4) resulted in DNA sequences nearly identical to the GenBank accessions for Ehrlichia ewingii. The DNA sequence from a 3rd dog (dog 5) was most similar to that of E. canis. Serologic or molecular results support the possibility of E. ewingii, E. equi , and E. canis coinfection or serologic cross-reactivity among canine granulocytic and monocytic Ehrlichia species in dogs from North Carolina and Virginia. Variability in response to tetracycline or doxycycline treatment was noted in these dogs, with more rapid resolution of signs in dogs with polyarthritis. We report the 1st cases of CGE in dogs from North Carolina and Virginia, including recognition of CGE in a healthy dog.     

Serum cardiac troponin I concentration in dogs with ehrlichiosis

Background: Ehrlichiosis is a multisystemic disease with the potential to cause cardiomyocyte injury in naturally infected dogs.
Hypothesis: Myocardial injury occurs in dogs infected with Ehrlichia canis .
Animals: One-hundred and ninety-four dogs from Brazil with clinical and laboratory abnormalities indicative of ehrlichiosis. Sixteen healthy dogs served as controls.
Methods: Electrocardiogram, echocardiogram, noninvasive blood pressure measurement, and serum cardiac troponin I (cTnI) concentrations were evaluated. Serologic assays and PCR determined the exposure and infection status for E. canis, Anaplasma spp., Babesia canis vogeli, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia chaffeensis, Ehrlichia ewingii, Leishmania chagasi , and spotted-fever group Rickettsia . Dogs were assigned to groups according to PCR status: E. canis infected, infected with other vector-borne organisms, sick dogs lacking PCR evidence for infection, and healthy controls.
Results: E. canis -infected dogs had higher serum cTnI concentrations than controls (median: 0.04 ng/dL; range 0.04–9.12 ng/dL; control median: 0.04 ng/dL; range: 0.04–0.10 ng/dL; P = .012), and acute E. canis infection was associated with myocardial injury (odds ratio [OR]: 2.67, confidence interval [CI] 95%: 1.12–6.40, P = .027). Severity of anemia was correlated with increased risk of cardiomyocyte damage ( r = 0.84, P < .001). Dogs with clinical signs of systemic inflammatory response syndrome (SIRS) were at higher risk for myocardial injury than were other sick dogs (OR: 2.55, CI 95%: 1.31–4.95, P = .005).
Conclusions and Clinical Importance: Acute infection with E. canis is a risk factor for myocardial injury in naturally infected Brazilian dogs. Severity of anemia and SIRS might contribute to the pathophysiology of myocardial damage.     

Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand

Forty-nine dogs from Thailand were evaluated for serologic evidence of exposure or polymerase chain reaction (PCR) evidence of infection with vectorborne pathogens, including Ehrlichia sp. (Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, and Ehrlichia risticii), Bartonella vinsonii subsp. berkhoffi (Bvb), spotted fever group (SFG) rickettsiae (Rickettsia rickettsii), Typhus group (TG) rickettsiae (Rickettsia canada, Rickettsia prowazekii, and Rickettsia typhi), and Babesia sp. (Babesia canis and Babesia gibsonii). All study dogs had at least 1 of 3 entry criteria: fever, anemia, or thrombocytopenia. By immunofluorescence antibody (IFA) testing, seroreactivity was most prevalent to E chaffeensis (74%) and E canis (71%) antigens, followed by E equi (58%), Bvb (38%), E risticii (38%), R prowazekii (24%), B canis (20%), R rickettsii (12%), R canada (4%), and B gibsonii (4%) antigens. There was 100% concordance between E canis IFA and Western blot immunoassay (WI) for 35 of 35 samples; 2 samples were IFA and WI reactive only to E equi antigens. By PCR amplification, 10 dogs were found to be infected with E canis, 5 with Ehrlichia platys, and 3 with B canis. Sequencing of PCR products was undertaken to compare Ehrlichia strains from Thailand to strains originating from the United States. Partial DNA sequence analysis confirmed infection with E canis and E platys, with identical 16S rRNA sequence alignment to E canis (U26740) and to E platys (M83801), as reported in GenBank. Partial E canis P28.1 and P28.2 amino acid sequences from Thai dogs were divergent from analogous sequences derived from North American E canis (AF082744) strains, suggesting that the Thai dogs were infected with a geographically distinct strain of E canis compared to North American strains. The results of this study indicate that dogs in Thailand have substantial exposure to vectorborne diseases and that coinfection with these pathogens may be common.     

Experimental transmission of granulocytic ehrlichial organisms in dogs

Canine granulocytic ehrlichial organisms were transmitted from an infected dog from Missouri to two male, 10-month-old dogs by an intravenous injection of whole blood. Physical or behavioral abnormalities were not detected during the 98 days of evaluation other than a mild pyrexia from Day 18 to 20. Ehrlichial morulae were found in blood granulocytes of Dog 1 from Day 13 to 44 and of Dog 2 from Day 14 to 34 with the peak rickettsemia occurring on Day 16 for both dogs. By Day 21 after inoculaiuon, both dogs had positive titers to Ehrlichia canis. The highest titers for both dogs were found 63 days after inoculation, after which the titers decreased. Most of the hematologic abnormalities (i.e., neutropenia, lymphocytosis, thrombocytopenia) and fever occurred between 18 and 24 days after inoculation. The pathologic bases of these abnormalities were not investigated but their concurrent presence suggested an association with the dogs' immunologic responses to the granulocytic ehrlichial agent. Results from the study indicated that the canine granulocytic ehrlichial agent of Missouri may produce subclinical infections and suggested that dogs may be able to clear the organism without antimicrobial therapy.     

Ocular signs of canine monocytic ehrlichiosis: a retrospective study in dogs from Barcelona, Spain

Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by the rickettsia Ehrlichia canis. Ocular lesions are a common feature of the disease and can be present in all stages. The purpose of this retrospective study was to determine the prevalence, type and response to treatment of ocular lesions associated with monocytic ehrlichiosis in 46 affected dogs presented to the Autonomous University of Barcelona-Veterinary Teaching Hospital (UAB-VTH) from January 2000 to December 2002. Dogs were included in the study only if they had a positive serologic test for E. canis and information about the clinical outcome was available. Eighteen breeds were represented, with the German Shepherd dog (n = 6) being the most common. There were 25 intact and three castrated males, and 16 intact and two neutered females. Twenty dogs (43.4%) were between 5 and 10 years old. Seventeen dogs (37% of all cases of monocytic ehrlichiosis diagnosed during the study period) had ocular signs, and 11 dogs (64.7% of the ocular cases) had only ocular lesions without apparent systemic signs. Exudative retinal detachment was the most common ocular manifestation; other prevalent findings included anterior exudative uveitis and optic neuritis. Five of the 17 cases with ocular lesions (29.4%) had ocular bleeding disorders (hyphema or retinal hemorrhages). All the dogs with ocular disease presented with bilateral signs. Dogs with posterior segment disease had titers against E. canis that were > or = 1 : 320, while lower titers were noted in dogs with anterior exudative uveitis. Two dogs presented with chronic autoimmune panuveitis after ehrlichiosis treatment. Canine ehrlichiosis should be considered in the differential diagnosis of exudative retinal detachment and anterior uveal inflammatory lesions.     

Concurrent babesiosis and ehrlichiosis in the dog: blood smear examination supplemented by the indirect fluorescent antibody test, using Cowdria ruminantium as antigen

Giemsa-stained, peripheral blood smears of 67 dogs, showing clinical signs typical of babesiosis or reminiscent of concurrent babesiosis and ehrlichiosis, were examined for the presence of Babesia canis and Ehrlichia canis. Since Cowdria ruminantium cross-reacts with Ehrlichia, the sera of these dogs were also subjected to the indirect fluorescent antibody (IFA) test in which C. ruminantium was used as antigen. Fifty-five per cent of these dogs had mixed infections of B. canis and E. canis, as judged by blood smear examination and serology. The serum of 32% of these dogs with mixed infections reacted positively in the IFA test. Six out of 9 dogs, the blood smears of which were negative for both B. canis and E. canis, were serologically positive for E. canis. Furthermore, sero-conversion from a negative in the initial serum sample to titres of up to 1:160 in a subsequent sample was recorded in 9 out of 13 dogs with suspected mixed infection on blood smear.     

Transmission of Ehrlichia canis to dogs by ticks (Rhipicephalus sanguineus).

Two strains of Rhipicephalus sanguineus acquired Ehrlichia canis by feeding as either larvae or nymphs on acutely infected dogs and, in subsequent instars, transmitted the agent to normal dogs. Three strains of R sanguineus transmitted E canis as adults after their larval and nymphal stages fed on infected dogs. More than 400 adult female ticks were fed on infected dogs as larvae or nymphs or both, but none transmitted E canis transovarially.     

Experimental Ehrlichia canis infection in the dog does not cause immunosuppression

A carrier state develops in some Ehrlichia canis-infected dogs due to ineffective host defenses. The subsequent development of immune-mediated diseases or opportunistic infections in chronic ehrlichiosis suggests dysregulation of immunity; however, the immunobiology of this infection has not been well characterized. In this study, eight dogs were infected with E. canis, and changes in seroreactivity, serum immunoglobulin (Ig) concentrations, peripheral blood T cell subsets, lymphocyte blastogenesis (LBT), and lymphokine-activated killer (LAK) activity were evaluated over 4 months. Infection, which was documented by seroconversion, polymerase chain reaction, and blood culture, caused self-limiting fever and thrombocytopenia. Infected dogs developed an anti-E. canis antibody response but were not immune to re-infection. Serum IgM, IgG, and IgA concentrations were unaffected by E. canis. The percentage of circulating CD4(+) T cells was similar in uninfected and infected dogs at all points. Infected dogs developed a CD8(+) lymphocytosis 6 weeks after inoculation that subsequently subsided, despite organism persistence. Functional defects of cell-mediated immunity, measured as suppression of LAK activity or mitogen-driven LBT, were not observed. These results suggest that immune responses are not grossly impaired in young dogs during the first several months following experimental E. canis infection.     

Serological survey of Ehrlichia and Anaplasma infection of feral raccoons (Procyon lotor) in Kanagawa Prefecture, Japan

Numbers of feral raccoon; the possible reservoir animal of Ehrlichia and Anaplasma, are increasing in Japan. Thus serological methods were utilized to examine Ehrlichia and Anaplasma infection in raccoons from Kanagawa Prefecture, Japan. By using an indirect immunofluorescence assay, among 187 feral raccoons examined, 1 (0.5%) serologically reacted with Ehrlichia canis, 3 (1.6%) with Ehrlichia chaffeensis and 1 (0.5%) with Anaplasma phagocytophilum with the titers of 1:40 or more. Although screening PCR for Ehrlichia and Anaplasma species failed to detect the presence of ehrlichial DNA in serum samples, results of the serological tests suggested that the feral raccoons might be infected with some species of Ehrlichia and Anaplasma.     

Detection of the new Ehrlichia species closely related to Ehrlichia ewingii from Haemaphysalis longicornis in Yonaguni Island, Okinawa, Japan

We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3-98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0-94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.     

Cyclic CD8+ lymphopenia in dogs experimentally infected with Bartonella vinsonii subsp. berkhoffii

Until recently, it was presumed that Bartonella vinsonii only infected voles, a species of North American rodents. In April of 1993, however, our laboratory isolated a novel subspecies of B. vinsonii (B. vinsonii subsp. berkhoffii) from the blood of a dog diagnosed with vegetative valvular endocarditis. Subsequently, based on a seroepidemiologic survey of dogs from North Carolina and Virginia presenting for a variety of medical problems, we found evidence supporting a potentially important association between B. vinsonii and Ehrlichia canis co-infection in dogs. In the following study, eight dogs were infected with B. vinsonii: four specific pathogen free dogs and four dogs that had previously been infected with E. canis. Flow cytometric analysis of peripheral blood lymphocytes revealed a cyclic elevation of the CD4/CD8 T-cell ratio that correlated with cyclic CD8+ lymphopenia in all dogs infected with B. vinsonii, regardless of prior exposure to E. canis.     

Failure of imidocarb dipropionate to clear experimentally induced Ehrlichia canis infection in dogs

The recommended treatment for canine ehrlichiosis is tetracycline or its analog doxycycline, although recent reports have documented ineffective clearing of Erchlichia canis after doxycycline administration. Imidocarb dipropionate is used as an alternative treatment to tetracycline or is used in conjunction with doxycycline. The effectiveness of imidocarb dipropionate in clearing Ehrlichia species from the blood and tissues of dogs with E. canis infection has not been thoroughly evaluated. Fifteen dogs were experimentally infected with E. canis. Ten dogs were treated with imidocarb dipropionate (6.6 mg/kg, IM, 2 injections given 2 weeks apart). Five infected control dogs were not treated. Blood samples from all 15 dogs were E. canis DNA positive by PCR assay by 3 weeks after inoculation (PI), and E. canis antibodies were detected by IFA assay by 1 week PI. Blood platelet counts in all dogs were below the reference interval by 4 weeks PI. E. canis DNA was detected in bone marrow and splenic aspirates by PCR assay 4 weeks PI but not before infection. Bone marrow aspirates were E. canis DNA positive by PCR assay in 14/15 dogs, and splenic aspirates were E. canis DNA positive by PCR assay in 13/15 dogs. Blood samples from all treated and control dogs remained positive for E. canis DNA by PCR assay, and platelet counts remained below preinoculation values 13 weeks PI (6 weeks after 2nd treatment). As administered in this study, imidocarb dipropionate did not clear experimental E. canis infection in dogs.     

Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings

Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10-14-day incubation period, infected dogs developed pyrexia up to 41 degrees C for 6-8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs.