Comparison of an indirect immunofluorescence assay, western blot analysis, and a commercially available ELISA for detection of Ehrlichia canis antibodies in canine sera

OBJECTIVE: To examine the correlation between results for an indirect immunofluorescence assay (IFA) that uses Ehrlichia canis antigen as a substrate (ie, E canis-IFA), 2 western blot (WB) analyses, and a commercially available ELISA in the detection of E canis antibody in dog sera. SAMPLE POPULATION: 54 canine serum samples that were reactive on E canis-IFA and 16 canine serum samples that were E canis-IFA nonreactive. PROCEDURE: Serum samples were evaluated by use of 2 WB analyses and a commercially available ELISA. Correlation between results of the 3 testing modalities (ie, IFA, WB analyses, and the ELISA) was examined by use of nonreactive (E canis-IFA reciprocal titer, < 20), low-titer (reciprocal titer, 80 to 160), medium-titer (reciprocal titer, 320 to 2,560), and high-titer (reciprocal titer, 5,120 to > 20,480) serum samples. RESULTS: For all serum samples in the nonreactive (n = 16), medium-titer (17), and high-titer (18) groups, correlation of results among IFA, WB analyses, and the commercially available ELISA was excellent. A poor correlation was found between IFA results and those of WB analyses and the ELISA for serum samples in the low-titer group (19), with only 4 of the 19 serum samples having positive results on both WB analyses and the commercially available ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: The discrepancy between E canis-IFA, WB analyses, and the commercially available ELISA results for the low-titer serum samples may be related to a high IFA sensitivity or, more likely, a lack of specificity associated with cross-reactivity among Ehrlichia spp.     

Detection of platelet-bound antibodies in beagle dogs after artificial infection with Ehrlichia canis

Six dogs were infected with Ehrlichia canis by intravenous injection of heavily infected DH82 cells. All dogs developed typical signs of canine monocytic ehrlichiosis. Using flow cytometric technology, platelet-bound IgG (PBIgG) were detected in 5 of the 6 dogs after experimental infection with E. canis over a period of 3-10 days post infection (PI). The first detection of PBIgG was made as early as day 3 PI in 2 out of 6 dogs, and on day 5 PI in 1 dog. On day 7 PI, PBIgG was detected in 2 dogs, and on day 10 PI in 3 out of 6 dogs. This is the first report documenting the presence of PBIgG following E. canis infection in dogs. This finding further supports the theory that the thrombocytopenia seen in canine monocytic ehrlichiosis has an immunological component and that exposure to an infectious agent, in this case the rickettsia E. canis, can trigger autoimmune mechanisms. Due to the heterogeneous appearance of PBIgG among the infected dogs it was concluded that other non-immunological mechanisms are probably also involved in the pathogenesis of the thrombocytopenia seen in canine monocytic ehrlichiosis.     

A modification of the indirect immunofluorescence test for detection of Ehrlichia phagocytophila antibodies.

A modified technique for production of antigen and performance of the test is described. A suspension of infected neutrophils was directly applied to multiwell slides. Multichannel pipettes may be used for dilution and application of sera. The modification increases the capacity both by production of the antigen and by performance of the test. This paper also gives a quantitative determination of the antibodies.     

Polymyositis associated with Ehrlichia canis infection in two dogs

Clinical, haematological, biochemical, electrophysiological and pathological features of two dogs infected with Ehrlichia canis and with concurrent signs of polymyositis are presented. Both dogs had a history of relatively acute onset, progressive tetraparesis, hyporeflexia and generalised muscle wasting. Skeletal muscles were atrophic and characterised histologically by plasmocytic, lymphocytic and immature lymph-oreticular cellular infiltrates with accompanying areas of necrosis. Histopathological similarities between ehrlichiosis and polymyositis are noted and a probable aetiological relationship is inferred.     

Efficacy of a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

Investigation of renal protein loss in dogs with acute experimentally induced Ehrlichia canis infection.

Urinary protein-to-creatinine ratios and serum albumin concentrations were measured in 8 adult male dogs experimentally inoculated with Ehrlichia canis. Urinary protein concentration increased significantly, but transiently, during the acute phase of infection. Urinary protein-to-creatinine ratios were highest (mean, 8.6) during the third and fourth weeks after infection, and decreased to less than 0.5 by 6 weeks after infection. Correspondingly, albumin concentration decreased significantly during the acute phase. Serum albumin concentrations were lowest (mean, 2.1 g/dl) the fourth week after infection and increased to greater than 3.0 g/dl by 11 weeks after infection. There was an inverse linear correlation between urinary protein-to-creatinine ratio and serum albumin concentration. The magnitude of proteinuria and its inverse relationship with serum albumin concentration suggested that hypoalbuminemia associated with acute E canis infection may be attributable primarily to increased renal loss of protein, rather than decreased hepatic synthesis as previously suggested. Another dog was subsequently inoculated with E canis from 1 of the experimentally infected dogs and a renal biopsy was performed during peak proteinuria (urinary protein-to-creatinine ratio = 22 and serum albumin = 1.1 g/dl). Immunofluorescent staining revealed mild to moderate deposits of anti-canine IgM, and to a lesser extent, anti-canine IgG and complement factor C3 in the glomerular tufts and mesangium. Ultrastructural evaluation revealed distortion and fusion of podocyte foot processes and increased microvilli on podocytes. These morphologic changes were consistent with transient glomerular leakage of protein of a magnitude that would significantly contribute to hypoalbuminemia during acute E canis infection. An underlying immunologic mechanism was suggested by positive glomerular immunofluorescence and previously described histologic findings.     

Serosurvey of Ehrlichia canis and Hepatozoon canis infection in dogs in Yamaguchi Prefecture, Japan

Antibodies to Ehrlichia canis and Hepatozoon canis in dogs at the Animal Hospital in Yamaguchi University were surveyed and potential risk factors for both pathogens were evaluated. Among 430 dogs examined, 20 (4.7%) and 18 (4.2%) dogs showed positive findings for E. canis and H. canis, respectively. Neither, sex nor age was associated with the seropositivity of either pathogen, but the positive rate in dogs kept outside was slightly higher than that in dogs kept inside for both pathogens. A higher seropositive reaction to E. canis and H. canis was observed in dogs that lived in certain cities and towns. Beagles, golden retrievers and pointers had higher seropositivity than other breeds in E. canis, whereas shibas, akitas, beagles, pointers and mongrels had higher positive rates than other breeds in H. canis.     

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.     

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.     

Presence of immune-complexes, and absence of antinuclear antibodies, in sera of dogs naturally and experimentally infected with Ehrlichia canis

Antinuclear antibodies (ANA), immunoglobulin G (IgG) concentrations and circulating immune-complexes (CIC) were measured, over a period of 3 years, in 6 dogs experimentally infected with Ehrlichia canis, and in 10 dogs naturally infected with the rickettsia. No ANA were detected in any of the samples tested. The IgG concentrations were shown to be higher in the infected dogs when compared to the control dogs. CIC were detected in 2 of 10 naturally and 2 of 6 experimentally infected dogs, during both the acute and the subclinical phases of the disease. The results of this study suggest that ANA do not play a role in the pathogenesis of CME. It is however suggested that some manifestations in canine ehrlichiosis are immune-complex mediated.     

Detection of Ehrlichia canis by polymerase chain reaction in dogs from Portugal

Antibodies against Ehrlichia canis, the cause of canine monocytic ehrlichiosis, have been reported previously in clinically ill and stray dogs from Portugal. In this study, the 16S rRNA gene of E. canis was detected by the polymerase chain reaction (PCR) in 12/55 (22%) of dogs with suspected tick-borne disease in the Algarve region in Portugal.     

Antibodies reactive with Bartonella henselae and Ehrlichia canis in dogs from the communal lands of Zimbabwe

The prevalences of antibodies against Bartonella henselae and Ehrlichia canis were determined in sera from 228 dogs in 5 communal lands of Zimbabwe, areas where traditional subsistence agro-pastoralism is practised. The sera were collected from apparently healthy dogs during routine rabies vaccination programmes and tested with indirect fluorescent antibody assays using B. henselae (Houston-I) and E. canis (Oklahoma) as antigens. We found reactive antibodies (> or =1:80) against B. henselae in 14% of the dogs tested. Seropositive animals were found in Bikita (41%; 17/42), Omay (13%; 6/48), Chinamora (5%; 2/38) and Matusadona (15%; 7/48). No seropositive dogs were found in Chiredzi (0%; 0/52). Antibodies reactive with E. canis (> or =1:80) were found in 34% of the dogs tested, from Bikita (88%; 37/42), Chiredzi (31%; 16/52), Omay (17%; 8/48), Chinamora (26%; 10/38) and Matusadona (15%; 7/48). Our survey shows dogs in the communal lands of Zimbabwe are frequently exposed to E. canis and B. henselae or closely related species. Further studies are indicated to determine the pathogenicity of the organisms infecting these dogs and their clinical significance.     

Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from the North of Portugal

Tick-transmitted rickettsial pathogens belonging to the Ehrlichia and Anaplasma genera can infect dogs and humans. In this study, four dogs from the North of Portugal, in which an ehrlichial disease was suspected clinically, were tested by molecular methods. After DNA extraction from blood on filter paper, a 345 bp fragment of the Ehrlichia/Anaplasma 16S rRNA gene was amplified by the polymerase chain reaction (PCR). Sequence analysis of PCR products revealed one dog infected with Ehrlichia canis and three with Anaplasma platys. One of these latter animals was co-infected with Babesia canis subspecies vogeli. This is the first report of the genetic characterisation of both A. platys and E. canis in naturally infected dogs from the North of Portugal.     

Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.     

Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia

Ehrlichiosis and babesiosis are tick-borne diseases, caused mainly by Ehrlichia canis and Babesia canis, respectively, with a worldwide occurrence in dogs, whose main vector is the brown-dog tick, Rhipicephalus sanguineus. The present work aimed to detect the presence of E. canis and Babesia sp. in 91 dog blood samples in Colombia, by molecular and serological techniques. We also performed sequence alignment to indicate the identity of the parasite species infecting these animals. The present work shows the first molecular detection of E. canis and B. vogeli in dogs from Colombia. Immunoglobulin-G (IgG) antibodies to E. canis and Babesia vogeli were found in 75 (82.4%) and 47 (51.6%) sampled dogs, respectively. Thirty-seven (40.6%) and 5 (5.5%) dogs were positive in PCR for E. canis and Babesia sp., respectively. After sequencing, amplicons showed 99% of identity with isolates of E. canis and B. vogeli. The phylogenetic trees based on 16S rRNA-Anaplasmataceae sequences and 18S rRNA-piroplasmid sequences supported the identity of the found E. canis and B. vogeli DNAs, respectively. The present work shows the first molecular detection of E. canis and B. vogeli in dogs in Colombia.     

Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island

Sixty-eight dogs from four African countries and Reunion Island were tested for antibodies against Ehrlichia canis. Twenty-six dogs (50%) in Tunisia, Senegal and Chad were found positive using the indirect fluorescence antibody test. Dogs from both the Central African Republic and Reunion Island were all negative. Thus, this preliminary report confirms the presence of E. canis in Africa. Larger studies will be necessary to evaluate the current epidemiologic situation of canine ehrlichiosis in these countries.     

Accuracy of a point-of-care ELISA test kit for predicting the presence of protective canine parvovirus and canine distemper virus antibody concentrations in dogs

Canine parvovirus (CPV) and canine distemper virus (CDV) are highly infectious and often fatal diseases with worldwide distributions, and are important population management considerations in animal shelters. A point-of-care ELISA test kit is available to detect serum antibodies to CPV and CDV, and presumptively to predict protective status. The aim of this study was to determine the diagnostic accuracy of the test compared to CPV hemagglutination inhibition titers and CDV serum neutralization titers determined by a reference laboratory, using sera collected from dogs housed at animal shelters. The ELISA test was used under both field and laboratory conditions and duplicate specimens were processed using an extra wash step. The test kit yielded accurate results (CPV: sensitivity 92.3%, specificity 93.5%; CDV: sensitivity 75.7%, specificity 91.8%) under field conditions. CDV sensitivity was improved by performing the test under laboratory conditions and using an optical density (OD) meter (laboratory performed 94.0%; OD 88.1%). Point-of-care ELISA testing for serum CPV and CDV antibody titers was demonstrated to be a useful tool for determining antibody status when making decisions regarding the need for CPV and/or CDV vaccination and also in animal shelters for population management.