Diagnosis Of Canine Monocytic Ehrlichiosis Research Articles

Assessment of the effect of long-term serum storage for retrospective serologic diagnosis of canine monocytic ehrlichiosis (Ehrlichia canis)

There is currently sparse information on the possible effect of long-term storage of serum specimens for the retrospective serodiagnosis of canine monocytic ehrlichiosis (CME). The aim of this study was to assess the agreement between the original serologic outcome and the results of a repeat indirect fluorescent antibody (IFA) assay for the detection of IgG antibodies against E. canis. A secondary aim was to compare the diagnostic performance of two commercially available point-of-care (POC) immunochromatographic (IC) assays. Archived serum samples originally tested as positive (n=66) or negative (n=19) for E. canis IgG antibodies and kept frozen at −20°C for a median of 22 years, were retrospectively examined by IFA and by two POC IC assays. Cohen’s Kappa coefficient (0.748, p < 0.0001), indicated a substantial agreement between the original and repeat serologic testing results. An almost identical high sensitivity and moderate specificity were established for the two POC IC assays. Canine serum specimens on long-term storage may still be of value for seroepidemiologic surveys investigating the exposure to E. canis.

Occurrence and hematological aspects of dogs with Canine Monocytic Ehrlichiosis

Canine monocytic ehrlichiosis (CME) is an infectious disease caused by a gram-negative bacterium of the genus Ehrlichia spp., a monocyte parasite that leads to thrombocytopenia, anemia, hyporexia, apathy, pale mucous membranes, among others. The etiological agent is distributed worldwide, but there is a greater incidence and considerable prevalence in places with temperate climates due to the high presence of the tick vector Rhipicephalus sanguineus. In Brazil, the number of cases of this disease appears to have increased in many regions, due to several factors, but more studies are still needed regarding the prevalence in various regions. In view of the aforementioned points, this retrospective study aimed to address the findings of the clinical and laboratory evaluation of 43 medical records with the definitive diagnosis of canine monocytic ehrlichiosis, in naturally infected dogs, treated at HOVET - UFES, during the period of June 2017 to September 2023, in order to discuss and relate the findings found with current literature, as well as report the occurrence of this disease. For this, we used the clinical and laboratory records of dogs that showed positivity in the SNAP 4Dx Plus® rapid test and/or in blood smears, and presented Ehrlichia spp. morulae. in leukocytes. Simultaneously, clinical data, blood count findings and biochemical test results were prepared in figures, tables and organizational charts. Subsequently, with the information and results found, descriptive analysis of the data was used to demonstrate the occurrence through percentages. The most frequent clinical and laboratory changes were the presence of ticks, hyporexia, lymphadenomgelia, apathy, pale mucous membranes, thrombocytopenia, normocytic normochromic anemia, lymphopenia and hypoalbuminemia. Therefore, it is concluded that it is essential to report the occurrence of Ehrlichia sp. in dogs, as well as knowing the main clinical and laboratory changes mentioned so that it is possible to assist in the early identification of canine monocytic ehrlichiosis.

Tumor venéreo transmissível canino com remissão espontânea: estudo de caso com ênfase aos exames clínico e citopatológico para monitoramento da evolução tumoral

Abstract The canine transmissible venereal tumor (TVTC) is a neoplasm transmitted mainly through copulation and with a high incidence in stray dogs in Brazil. In the process of tumor evolution of TVTC, the progression, stationary and regression phases are recognized. The host immunity is related to the disease’s biological behavior, however, spontaneous regression observation in cases of naturally occurring TVTC is uncommon. A canine patient was attended, after beeing rescued from the street, due to an ulcerated mass in the external genitália and tick infestation. Cytopathological examination, which diagnosed TVTC, and laboratory tests that showed mild anemia and severe thrombocytopenia were performed. In view of the impossibility of carrying out other exams, it was made the presumptive diagnosis of canine monocytic ehrlichiosis (CME), and treatment was instituted. During follow-up it was observed quick improvement in clinical signs and laboratory changes, as well as a reduction in tumor mass. A new cytopathological evaluation was carried out, and was verified increase in mature lymphocytes and plasmocytes in the midst of the tumor cells, finding compatible with the stationary phase of the disease. From that moment on, it was decided to perform only clinical and cytopathological follow-up. In the following evaluations, continuous clinical remission and cytopathological findings compatible with those described in the regression phase were observed, until its complete remission. It is considered that the improvement in the general health of the patient after the treatment of CME is related to the spontaneous regression of TVTC, and that simultaneous performance of serial clinical and cytopathological exams may be feasible and useful for monitoring the stages of evolution of TVTC.

Canonical correlative analyses among an enzyme-linked immunosorbent assay using synthetic peptides, an indirect fluorescent antibody test, and hematologic measurements in dogs infected with Ehrlichia canis.

Immunoreactive tandem repeat proteins (TRPs) in amino acid sequences were identified and employed in the serologic diagnosis of canine monocytic ehrlichiosis (CME). This study evaluated using TRP19 and TRP36 synthetic protein antigens with enzyme-linked immunosorbent assays (ELISAs) and compared the results with an indirect fluorescent antibody test (IFAT) to diagnose CME in the serum of dogs with suspected CME. The sera of 243 dogs that exhibited clinical and hematologic signs suggestive of CME had IFATs performed. An ELISA with synthetic TRP19 and TRP36 antigens from two Ecanis genotypes (USTRP36 and BrTRP36) that circulate in Brazil were also performed. Canonical correlations and chi-square tests were evaluated on the results of these tests. Among the 243 dogs, 179 (73.6%) were reactive at an IFAT≥40, 167 (68.72%) reacted at the cut-off ≥160, and 149 (61.31%) reacted at ≥640. The ELISA revealed that 172 (70.8%) dogs reacted to TRP19 peptide, 163 (67.1%) reacted to USTRP36, and 114 (46.9%) reacted to BrTRP36. The canonical correlation analyses showed that the TRP19 ELISA was the most effective diagnostic method. Serum total protein levels showed strong positive correlations with the USTRP36 peptide. Qualitative analyses revealed an association between the TRP19 peptide and the presence of anemia, thrombocytopenia, and hyperproteinemia. The IFAT results at titers of ≥160 and ≥640 were associated with thrombocytopenia. The results of this study indicated that the TRP19 peptide was an excellent antigen and that the IFAT was diagnostically important in confirming the diagnosis of CME in serum samples.

Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests.

Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.

Canine monocytic ehrlichiosis in Buenos Aires, Argentina: comparison of serological and molecular assays

ABSTRACT: Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the bacterium Ehrlichia canis and transmitted by Rhipicephalus sanguineus sensu lato, a tick with worldwide distribution. When not diagnosed and treated early, disease can be severe. Currently, the disease is confirmed by serological or molecular assays. The objective of this study was to compare a serological assay based on immunochromatography (SPEED® EHRLI immunochromatographic test; BVT, France) and a molecular assay (a screening PCR followed by a nested PCR specific for E. canis) for the diagnosis of E. canis in suspected dogs from Buenos Aires city and southern Greater Buenos Aires, Argentina. Blood samples from 20 clinically healthy dogs (Control Group) and from 80 sick dogs suspected of having CME (Groups 1 to 4) were tested in parallel. Neither the immunochromatographic test nor the PCR assay was able to detect the presence of E. canis in the Control Group. In the group which had been previously tested by serology, the agreement between the tests was low (kappa: 0.200), whereas in the group which had been previously tested by PCR, the concordance between the tests was adequate (kappa: 0.650). The concordance between the tests evaluated in the total population studied was moderate (kappa: 0.496). The results of our study suggest that the use of rapid serological tests as a first approach, together with subsequent confirmation by PCR, will improve the diagnosis of CME.

Serological and molecular diagnosis of Ehrlichia canis and associated risk factors in dogs domiciled in western Cuba.

Ehrlichia canis is a rickettsia transmitted by the tick, Rhipicephalus sanguineus, and is the causative agent of canine monocytic ehrlichiosis (CME). In Cuba, the first diagnosis of CME was made in 2001, but few studies have since investigated this disease locally. The objective of this study was to estimate the prevalence of E. canis in dogs domiciled in four municipalities within the western region of Cuba and determine the associated risk factors. Blood was drawn from 378 selected dogs living in four municipalities in two provinces of western Cuba. From the total number of samples, 206 plasma samples were selected to perform an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against E. canis. Using the original 378 samples of extracted DNA, a nested polymerase chain reaction (nPCR) was performed to amplify a specific fragment of the 16S rRNA gene of E. canis. Analysis of the 206 plasma samples revealed a total of 162 animals that were seropositive for E. canis (78.64%) with a density index between 109.5 and 970.7. In contrast, 179 samples were positive based on the nPCR assay (47.35%). As well, there was a high concordance (kappa = 0.7), calculated through the Kappa index, between the animals found to be positive based on nPCR and those determined based on ELISA. The analysis of risk factors showed that residing in the municipality of Boyeros in addition to having a history of infestation by ticks increases the probability of having a positive result based on nPCR.

Diagnóstico de la ehrlichiosis monocítica canina: una revisión actualizada

&lt;p&gt;La ehrlichiosis monocítica canina (EMC) es causada por la infección de una rickettsia denominada Ehrlichia canis; este microorganismo se reproduce solamente en el citoplasma de los monocitos sanguíneos y macrófagos tisulares. La EMC ha sido reportada en todo el mundo, pero las frecuencias más altas se reportan en las regiones tropicales y subtropicales. La EMC no siempre es reconocida clínicamente, pues es difícil de diagnosticar debido a que los signos clínicos son variables, a que se presenta en diferentes fases y a la existencia de coinfecciones con otros patógenos transmitidos por garrapatas y otros agentes infecciosos. Las manifestaciones clínicas y las técnicas de diagnóstico tradicionales, como el diagnóstico hematológico y la demostración típica de mórulas de E. canis en el citoplasma de monocitos en frotis sanguíneos, se han utilizado para el diagnóstico de la EMC; sin embargo, las pruebas de diagnóstico por PCR y la serología mediante el uso de antígenos específicos son las pruebas más sensibles y específicas para el diagnóstico de la enfermedad. Este artículo presenta una revisión actualizada sobre el diagnóstico de la infección por E. canis en perros.&lt;/p&gt;

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast

Canine monocytic ehrlichiosis caused by Ehrlichia canis is distributed globally, but its prevalence in Africa is poorly known. In the study reported herein, 27% of Rhipicephalus sanguineus ticks collected from watchdogs in Abidjan, Ivory Coast, were positive for E. canis using quantitative real-time PCR. A new molecular strategy is proposed that can be used not only for epidemiological study, but also for the diagnosis of canine monocytic ehrlichiosis. Our findings show for the first time the presence of E. canis using molecular tools in the Ivory Coast, providing direct evidence for the presence of this pathogen.

Serum Acute Phase Proteins as Clinical Phase Indicators and Outcome Predictors in Naturally Occurring Canine Monocytic Ehrlichiosis

Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick-borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). Fifty-six dogs naturally infected with E. canis and 7 clinically healthy control dogs. C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis-specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.

Diagnosis of canine monocytic ehrlichiosis: Development of advanced techniques to combat a global disease

Diagnosis of canine monocytic ehrlichiosis: Development of advanced techniques to combat a global disease

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates ( P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs ( P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.

Comparison of a Clinic-Based ELISA Test Kit with the Immunofluorescence Test for the Assay of Ehrlichia Canis Antibodies in Dogs

The "gold standard" for the detection of antibodies to Ehrlichia canis, the cause of canine monocytic ehrlichiosis (CME), is the indirect immunofluorescence antibody (IFA) test. The IFA test however is generally available only in selected laboratories and requires extensive equipment and trained personnel. A double-blind study was conducted to compare the ability of an in-clinic standardized enzyme-linked immunosorbent assay (ELISA) test kit to measure E. canis IgG antibodies in dogs compared with the standard IFA technique. A good correlation was found between the 2 techniques (r2 = 0.8793; P < 0.0001). Evidence for the sensitivity of the ELISA technique for the early detection of E. canis IgG antibodies was demonstrated by comparing the appearance of E. canis antibody titers by the IFA and ELISA techniques after artificial infection of 2 sets of dogs. In both experimental infections, both tests were equally sensitive for the early detection of IgG antibodies against E. canis, and the results correlated well with the appearance of fever and clinical signs. Proposed application of the in-clinic ELISA test is to aid in the diagnosis of CME.