Evaluating the circulation of Ehrlichia canis and Rickettsia spp. in domestic dogs from a semiarid region in Brazil.

Tick-borne diseases are important for animal and human health, because they can cause death if not diagnosed and treated early. Canine monocytic ehrlichiosis (CME) can cause high morbidity in dog populations. Rocky Mountain Spotted Fever (RMSF) is among the most virulent infectious in humans; dogs are also susceptible to infection. The aims of this study were to evaluate the presence of Ehrlichia canis and Rickettsia spp. infections in domestic dogs, and to identify tick species parasitizing dogs among urban areas of two municipalities (Sobral and Alcântaras) in the Ceará State, Northeastern Brazil. A total of 208 domiciled dogs was sampled. After clinical evaluation, blood samples and ticks were collected and submitted to Real-Time Polymerase Chain Reaction (RT-PCR) targeting E. canis DNA. Serum samples were screened by Indirect Immunofluorescence Assays (IFA) for antibodies against different strains of Rickettsia spp. previously recognized in Brazil. The results of this study indicate the molecular detection of E. canis in the state of Ceará, Brazil, where the proportion of canine infection in Sobral (9.9%) was higher than in Alcântaras (5.6%). Rhipicephalus sanguineus sensu lato was the prevalent tick species infesting the dogs in both municipalities (43.5 and 53.3%, respectively). Our serological results indicate that dogs of the study area were at low risk of exposure to these tick-borne Rickettsia spp. of the spotted fever group. Our study offers epidemiological data of these diseases to better understanding Rickettsiales epidemic and enzootic cycles in the Brazilian semiarid region, improving prevention and control measures.

Molecular Detection and Characterization of Ehrlichia canis Isolates from Three Geographic Regions in Mexico: A Retrospective Study.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

First report of pediatric ehrlichiosis in Mexico.

BACKGROUND: Ehrlichia chaffeensis is responsible for most cases of human ehrlichiosis, an acute febrile tick-borne disease. This clinical entity is more commonly reported in adults from the United States. Therefore, it is of special interest to characterize this disease in children, given that very few cases in children have been reported outside of this country. CASE REPORT: We describe the case of a 15-year-old female from northeastern Mexico with a five-day history of myalgias, arthralgias, fever, abdominal pain, rash, and somnolence. The possibility of tick-borne disease was suspected considering that she lived with three tick-infested dogs that had recently died and a neighbor with similar symptoms who deteriorated rapidly and died a week earlier. Ehrlichia spp. was detected in blood samples by polymerase chain reaction. The patient completed a seven-day course of doxycycline and was discharged with complete resolution of symptoms. CONCLUSIONS: This case is the first report of ehrlichiosis in a pediatric patient in Mexico, illustrating the importance of considering tick-borne diseases as a differential diagnosis in patients with rash, fever, and altered level of consciousness. This initial clinical presentation may be indistinct from other conditions such as dengue, meningococcemia, and multisystem inflammatory syndrome in children (MIS-C), among others.

INTRODUCCIÓN: Ehrlichia chaffeensis es responsable de la mayoría de los casos de ehrlichiosis humana, una enfermedad febril aguda transmitida por garrapatas. Esta entidad clínica se reporta con mayor frecuencia en adultos de Estados Unidos. Por lo tanto, es de especial interés caracterizarla en niños, dado que se han reportado muy pocos casos en niños fuera de este país. CASO CLÍNICO: Se describe el caso de una paciente de sexo femenino de 15 años, originaria y residente del noreste de México con una historia de cinco días de mialgias, artralgias, fiebre, dolor abdominal, erupción cutánea y somnolencia. Se sospechó la posibilidad de una enfermedad transmitida por garrapatas considerando que convivió con tres perros infestados de garrapatas que habían muerto recientemente y una vecina con síntomas similares, quien se deterioró rápidamente y murió una semana antes. Ehrlichia spp. se detectó en una muestra sérica mediante reacción en cadena de la polimerasa. La paciente completó un curso de siete días de doxiciclina y fue dada de alta con resolución de los síntomas. CONCLUSIONES: Este caso es el primer reporte de ehrlichiosis en un paciente pediátrico en México que ilustra la importancia de considerar enfermedades transmitidas por garrapatas dentro del diagnóstico diferencial de pacientes con exantema, fiebre y alteración del estado de conciencia. Esta presentación clínica inicial puede ser indistinguible de otras entidades como dengue, meningococcemia y síndrome multisistémico inflamatorio, entre otras.

Pleural effusion-related Nocardia otitidiscaviarum, Anaplasma platys and Ehrlichia canis coinfection in a dog.

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum.

Molecular characterization of Ehrlichia canis and Babesia vogeli reveals multiple genogroups associated with clinical traits in dogs from urban areas of Colombia.

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 - 18.0%) and 1.09% (2/185; CI 95,% -0.43 - 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors.

First report of pediatric ehrlichiosis in Mexico / Primer reporte de ehrlichiosis pediátrica en México

Abstract Background: Ehrlichia chaffeensis is responsible for most cases of human ehrlichiosis, an acute febrile tick-borne disease. This clinical entity is more commonly reported in adults from the United States. Therefore, it is of special interest to characterize this disease in children, given that very few cases in children have been reported outside of this country. Case report: We describe the case of a 15-year-old female from northeastern Mexico with a five-day history of myalgias, arthralgias, fever, abdominal pain, rash, and somnolence. The possibility of tick-borne disease was suspected considering that she lived with three tick-infested dogs that had recently died and a neighbor with similar symptoms who deteriorated rapidly and died a week earlier. Ehrlichia spp. was detected in blood samples by polymerase chain reaction. The patient completed a seven-day course of doxycycline and was discharged with complete resolution of symptoms. Conclusions: This case is the first report of ehrlichiosis in a pediatric patient in Mexico, illustrating the importance of considering tick-borne diseases as a differential diagnosis in patients with rash, fever, and altered level of consciousness. This initial clinical presentation may be indistinct from other conditions such as dengue, meningococcemia, and multisystem inflammatory syndrome in children (MIS-C), among others.

Resumen Introducción: Ehrlichia chaffeensis es responsable de la mayoría de los casos de ehrlichiosis humana, una enfermedad febril aguda transmitida por garrapatas. Esta entidad clínica se reporta con mayor frecuencia en adultos de Estados Unidos. Por lo tanto, es de especial interés caracterizarla en niños, dado que se han reportado muy pocos casos en niños fuera de este país. Caso clínico: Se describe el caso de una paciente de sexo femenino de 15 años, originaria y residente del noreste de México con una historia de cinco días de mialgias, artralgias, fiebre, dolor abdominal, erupción cutánea y somnolencia. Se sospechó la posibilidad de una enfermedad transmitida por garrapatas considerando que convivió con tres perros infestados de garrapatas que habían muerto recientemente y una vecina con síntomas similares, quien se deterioró rápidamente y murió una semana antes. Ehrlichia spp. se detectó en una muestra sérica mediante reacción en cadena de la polimerasa. La paciente completó un curso de siete días de doxiciclina y fue dada de alta con resolución de los síntomas. Conclusiones: Este caso es el primer reporte de ehrlichiosis en un paciente pediátrico en México que ilustra la importancia de considerar enfermedades transmitidas por garrapatas dentro del diagnóstico diferencial de pacientes con exantema, fiebre y alteración del estado de conciencia. Esta presentación clínica inicial puede ser indistinguible de otras entidades como dengue, meningococcemia y síndrome multisistémico inflamatorio, entre otras.

Novel Ehrlichia canis genogroup in dogs with canine ehrlichiosis in Cuba.

BACKGROUND: Canine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene. METHODS: Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis. RESULTS: Four of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the 'Cuba (CUB) genogroup'. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions. CONCLUSIONS: The results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence.

Immunophenotypical and pathological changes in dogs experimentally infected with Ehrlichia canis / Alterações imunofenotípicas e patológicas em cães experimentalmente infectados por Ehrlichia canis

Canine monocytic ehrlichiosis (CME) is one of the most important tick-borne diseases worldwide, with multisystemic presentations. Immune dysregulation has been proposed as the primary mechanism involved in its pathogenesis and in tissue injury in dogs with CME. Experimental infection of German Shepherd dogs in the present study demonstrated that CME caused marked pathological changes in their lymph nodes and spleen, and also gave rise to mononuclear infiltration in organs and tissues. Immunophenotyping of cells in lymph nodes, spleen and injured tissues highlighted differences in lymphocyte subsets, local expression of immunoglobulin subclasses and MHCII molecules between infected and control dogs. These findings suggest that the immunophenotypic and immunopathological changes in dogs with acute experimental CME are related to Th1 bias and compartmentalized immune response.(AU)

A erliquiose monocítica canina (EMC) é uma das doenças veiculadas por carrapatos com apresentações multisistêmicas mais relevantes em todo o mundo. A desregulação do sistema imune vem sendo proposta como o principal mecanismo envolvido na patogênese e lesão de tecidos em cães com EMC. A infecção experimental de pastores alemães nesta pesquisa evidenciou marcadas alterações patológicas em linfonodos, baço e também infiltração mononuclear em órgãos e tecidos. A imunofenotipagem de células em linfonodos, baço e tecidos lesados destacou diferenças em subconjuntos de linfócitos, expressão local de subclasses de imunoglobulinas e de moléculas MHCII entre cães infectados e controle. Esses achados sugerem que um viés Th1 e uma resposta imune compartimentalizada estão relacionados às alterações imunofenotípicas e imunopatológicas em cães com EMC experimental aguda.(AU)

Epidemiological link between canine monocytic ehrlichiosis caused by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto in Argentina.

In this work, we analyze data that support an epidemiological link between cases of canine monocytic ehrlichiosis (CME) by Ehrlichia canis and the presence of Rhipicephalus sanguineus sensu stricto as vector in an endemic area for this tick in Argentina. In a blood sample of a 1-year-old toy poodle with CME compatible clinical signs, which showed CME typical morulae in monocytes in Giemsa-stained blood smear, DNA of E. canis was detected by PCR. Further, DNA of E. canis was also detected in a female of R. sanguineus s.s. collected on the infected dog. Rhipicephalus sanguineus s.s. is the only member of the R. sanguineus group that prevails in the study area. The results of this study suggest that R. sanguineus s.s. may play a more important role in the transmission of E. canis than it was assumed so far. The epidemiological link between CME cases and R. sanguineus s.s. as vector in temperate areas of Argentina described in this work contrast previous studies which found that R. sanguineus sensu lato "tropical lineage" (which is absent in the study area) is competent to transmit E. canis but not R. sanguineus s.s.

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs / Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães

This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each techniques performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R²) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.(AU)

Este estudo tem como objetivo descrever um novo método de detecção de uma reação em cadeia da polimerase quantitativa em tempo real (qPCR) visando o gene da proteína da membrana externa de 28 kDa (p28), bem como comparar este método com um PCR convencional (cPCR), que visa o mesmo gene, a fim de avaliar o desempenho da técnica desenhada neste estudo na detecção de Ehrlichia canis (E. canis). As concentrações ideais de oligonucleotídeos foram alcançadas e a sensibilidade analítica e a especificidade do qPCR foram determinadas. Um total de 218 amostras de sangue total de cães foram coletadas convencionalmente para este estudo. O DNA foi extraído de cada amostra. Posteriormente, as amostras foram testadas por um cPCR estabelecido e o novo qPCR para comparar os desempenhos entre cada técnica. A curva padrão revelou 100% de eficiência e coeficiente de determinação (R²) equivalente a 99,8%. Dentre as amostras examinadas por qPCR, 24,31% foram consideradas positivas, percentual significativamente maior do que as detectadas por cPCR (15,13%). A técnica qPCR atingiu uma sensibilidade maior do que a cPCR na detecção de E. canis. A qPCR padronizada neste estudo é um método eficiente para a confirmação do diagnóstico de erliquiose monocítica canina (EMC) e pode fornecer o monitoramento de níveis de parasitemia ao longo do tratamento da doença.(AU)

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs / Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs' whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique's performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

RESUMO: Este estudo tem como objetivo descrever um novo método de detecção de uma reação em cadeia da polimerase quantitativa em tempo real (qPCR) visando o gene da proteína da membrana externa de 28 kDa (p28), bem como comparar este método com um PCR convencional (cPCR), que visa o mesmo gene, a fim de avaliar o desempenho da técnica desenhada neste estudo na detecção de Ehrlichia canis (E. canis). As concentrações ideais de oligonucleotídeos foram alcançadas e a sensibilidade analítica e a especificidade do qPCR foram determinadas. Um total de 218 amostras de sangue total de cães foram coletadas convencionalmente para este estudo. O DNA foi extraído de cada amostra. Posteriormente, as amostras foram testadas por um cPCR estabelecido e o novo qPCR para comparar os desempenhos entre cada técnica. A curva padrão revelou 100% de eficiência e coeficiente de determinação (R2) equivalente a 99,8%. Dentre as amostras examinadas por qPCR, 24,31% foram consideradas positivas, percentual significativamente maior do que as detectadas por cPCR (15,13%). A técnica qPCR atingiu uma sensibilidade maior do que a cPCR na detecção de E. canis. A qPCR padronizada neste estudo é um método eficiente para a confirmação do diagnóstico de erliquiose monocítica canina (EMC) e pode fornecer o monitoramento de níveis de parasitemia ao longo do tratamento da doença.

New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs / Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães

This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R²) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

Este estudo tem como objetivo descrever um novo método de detecção de uma reação em cadeia da polimerase quantitativa em tempo real (qPCR) visando o gene da proteína da membrana externa de 28 kDa (p28), bem como comparar este método com um PCR convencional (cPCR), que visa o mesmo gene, a fim de avaliar o desempenho da técnica desenhada neste estudo na detecção de Ehrlichia canis (E. canis). As concentrações ideais de oligonucleotídeos foram alcançadas e a sensibilidade analítica e a especificidade do qPCR foram determinadas. Um total de 218 amostras de sangue total de cães foram coletadas convencionalmente para este estudo. O DNA foi extraído de cada amostra. Posteriormente, as amostras foram testadas por um cPCR estabelecido e o novo qPCR para comparar os desempenhos entre cada técnica. A curva padrão revelou 100% de eficiência e coeficiente de determinação (R²) equivalente a 99,8%. Dentre as amostras examinadas por qPCR, 24,31% foram consideradas positivas, percentual significativamente maior do que as detectadas por cPCR (15,13%). A técnica qPCR atingiu uma sensibilidade maior do que a cPCR na detecção de E. canis. A qPCR padronizada neste estudo é um método eficiente para a confirmação do diagnóstico de erliquiose monocítica canina (EMC) e pode fornecer o monitoramento de níveis de parasitemia ao longo do tratamento da doença.

Human Monocytic Ehrlichiosis, Mexico City, Mexico.

Little information is available about human infections by the members of the genus Ehrlichia in Mexico. Only 2 species, Ehrlichia canis and E. chaffensis, are known to cause disease in this country. We report a fatal case of human monocytic ehrlichiosis in Mexico City in a man who was homeless.

Ehrlichia canis TRP36 diversity in naturally infected-dogs from an urban area of Colombia.

Ehrlichia canis is the etiologic agent of a highly prevalent tick-borne disease, canine monocytic ehrlichiosis (CME). Four defined E. canis genotypes based on the trp36 gene sequences have been reported, three of them identified in North or South America. The diversity of E. canis has been investigated using genetic and serologic approaches based on distinct 36 kDa tandem repeat protein (trp36) gene sequences that have been reported. The main objectives of this study were to determine the prevalence of E. canis infection in dogs from Medellín, Colombia by PCR and determine the E. canis diversity using molecular and serologic approaches. Blood was collected from dogs (n = 300) with clinical signs of CME for PCR detection of E. canis 16S rRNA, dsb and trp36 DNA. Phylogenetic analysis of trp36 gene sequences was performed using MEGA. A serological evaluation was performed using immunofluorescence microscopy and ELISA with species-specific peptides from E. canis TRP19 and TRP36 (3 genotypes) and E. chaffeensis (TRP32). E. canis DNA (16S rRNA and/or dsb) was detected in 18 % (53/300) of dogs by PCR amplification. The trp36 gene was amplified and sequenced from 35/53 16S rRNA/dsb PCR positive samples revealing three genotypes: United States (US; n = 21), Costa Rica (CR; n = 11), and Brazil (BR; n = 3). Most dogs (33/35) with detectable trp36 DNA had anti-E. canis TRP19 and TRP36 peptide antibodies that corresponded to the genotype detected by PCR. Dogs that had antibodies to the TRP19 peptide (82/300; 38 %), also had antibodies to one or more genotype-specific TRP36 peptides. Based on TRP36 serology, the dogs exhibited highest frequency of infection with the US genogroup (US = 26), followed by the CR genogroup (CR = 19) and the BR genogroup (BR = 11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CR = 8, BR/CR = 7, US/CR = 6 and US/BR = 5) suggesting coinfection or multiple sequential infections with different genotypes. Colombian dogs did not have antibodies to E. chaffeensis as determined by a TRP32 species-specific ELISA. Our results demonstrate the presence of three previously defined genotypes in North and South America in Colombian dogs (US, BR, CR). These results also demonstrate that TRP19 and TRP36 serology can provide valuable information regarding E. canis exposure and the potential genotype(s) involved in infection.

Ehrlichiosis monocítica canina en la provincia de Entre Ríos, Argentina: confirmación molecular de casos en la ciudad de Concordia / Canine monocytic ehrlichiosis in Entre Ríos province, Argentina: molecular confirmation of cases in Concordia city

Resumen El objetivo del siguiente trabajo fue confirmar la presencia de Ehrlichia canis por diagnóstico molecular, a partir de muestras de perros con diagnósticos presuntivos, oriundos de la ciudad de Concordia, provincia de Entre Ríos. ADN fue extraído de 14 muestras de sangre de perros con diagnóstico presuntivo de EMC. Para la detección de ADN deE. canis se amplificó un fragmento del gen dsb, específico de este género, y su confirmación específica se realizó mediante secuenciación. Doce de las 14 muestras mostraron bandas del tamaño esperado. Las secuencias obtenidas mostraron un 98-100% de identidad con secuencias registradas en GenBank para E. canis. Además, se amplificó un fragmento del gen ARNr 16S mitocondrial de garrapatas obtenidas de un perro positivo. Las secuencias demostraron que corresponden a R. sanguineus sensu stricto (linaje templado). En este estudio se confirma la presencia de E. canis en la ciudad de Concordia, Entre Ríos, resultando en una alerta para los médicos veterinarios de la región, a fin de incentivar estrategias de prevención de la enfermedad y control del vector.

Abstract The aim of this work was to confirm the presence of Ehrlichia canis in blood samples of dogs from Concordia, Entre Ríos Province, Argentina. DNA was extracted in 14 blood samples of dogs with presumptive diagnosis of CME. A PCR protocol targeting dsb gene, specific for genus Ehrlichia was carried out for detection and species confirmation was carried out by sequencing. Twelve out of 14 samples shown bands of the expected size. The obtained sequences revealed 98-100% identity with E. canis sequences registered in GenBank. Moreover, ticks were retrieved from an E. canis positive dog and a fragment of mitochondrial ARNr 16S gene was amplified. The obtained sequences were identified as R. sanguineus sensu stricto (temperate linage). This work confirmed the presence of E. canis in Concordia city, Entre Ríos province resulting in an alert for veterinary clinicians in this region aiming to encourage prevention strategies of this disease and vector control.

Primer reporte de un caso de ehrlichiosis monocítica canina en la provincia de Santa Fe, Argentina

La ehrlichiosis monocitica canina (EMC) es una enfermedad causada por la bacteria Ehrlichia canis, de distribución mundial, alta mortalidad en caninos doméstico y síntomas inespecíficos, lo que dificulta su diagnóstico clínico. Ehrlichia canis es transmitida por la garrapata Rhipicephalus sanguineus sensu lato a un hospedador, en Argentina se reconocen dos linajes (tropical y sensu stricto) de dicha especie. El objetivo del presente trabajo es reportar el primer caso confirmado de EMC por E. canis en un canino de la ciudad de Rafaela, Santa Fe, área endémica de R. sanguineus s.s. El 18/02/2019 llegó a la consulta privada un canino con síntomas inespecíficos como hipertermia tarde/noche, depresión, letargia, aplasia, inapetencia y pérdida de peso; más antecedente de parasitismo por garrapatas. La mascota presentaba anemia leve (4.730.000/mm³), enzimas hepáticas aumentadas (AST/ASA/GOT=72 U/l) y esplenomegalia. El test serológico in vitro y PCR para amplificar ADN E. canis fueron positivos, por lo que la mascota recibe tratamiento para EMC con doxiciclina 10 mg/kg/día durante 30 días, antiinflamatorios durante 5 días y protector hepático. A los 60 días de iniciado el tratamiento la mascota recuperó su peso normal y a los 120 días se realiza PCR como monitoreo de la eficacia del tratamiento dando resultado negativo.

Canine monocytic ehrlichiosis (CME) is a disease caused by Ehrlichia canis bacteria. It has a globally distributed and cause high mortality in domestic canines with nonspecific symptoms, which makes clinical diagnosis difficult. Eherlichia canis is transmitted to a host by the tick Rhipicephalus sanguineus sensu lato. In Argentina two lineages of this species are recognized (tropical and sensu stricto). The objective of this work is to report the first confirmed case of CME by E. canis in endemic area of R. sanguineus s.s from Rafaela, Santa Fe. On February 2, 2019 a canine arrived at the private clinic with non-specific symptoms such as late / night hyperthermia, depression, lethargy, aplasia, loss of appetite and weight loss, as well as, antecedent of tick parasitism. The pet had mild anemia (4,730,000/ m³), increased liver enzymes (AST/ASA/GOT = 72 U/l) and splenomegaly. The in vitro serological test and PCR to amplify E. canis DNA were positive, so the dog received treatment for CME with doxycycline 10 mg/kg/day for 30 days, anti-inflammatory for 5 days and liver protector. After 60 days of starting the treatment, the animal regained its normal weight and after 120 days the PCR have given negative result, checking the effectiveness of the treatment.

Molecular detection of Ehrlichia canis and Anaplasma platys in dogs from municipality of Belém, State of Pará, Brazil / Detecção molecular de Ehrlichia canis e Anaplasma platys em cães do município de Belém, Estado do Pará, Brasil

The occurrence of diseases transmitted by ticks in dogs is very frequent in Brazil, among these diseases we can highlight the ehrlichiosis and anaplasmosis, which are caused by Ehrlichia canis and Anaplasma platys, respectively. The objective of this study was to survey the occurrence of these pathogens in blood samples from domiciled and stray dogs from the city of Belém, Pará. Two hundred and seventy six dogs were sampled for convenience, and the DNA extracted from the blood of these animals was submitted to nested-PCR for research of E. canis and A. platys. E. canis DNA was detected in 39.4% (109/276) and A. platys DNA in 23.1% (64/276) of the samples, there was a statistically significant difference between the frequency of these agents (P<0.0001), and there was coinfection in 13.4% (37/276) of animals. The frequency of detection of these parasites was higher in stray dogs than in those domiciled for both E. canis (OR=2.84) and A. platys (OR=10.5). Considering the results, it was possible to conclude that E. canis and A. platys are present in the studied population, with stray dogs being more affected by these parasites.(AU)

A ocorrência de doenças transmitidas por carrapatos em cães é muito frequente no Brasil, dentre estas enfermidades podemos destacar a erliquiose e a anaplasmose, que são causadas por Ehrlichia canis e Anaplasma platys, respectivamente. O objetivo deste trabalho foi fazer um levantamento da ocorrência destes patógenos em amostras de sangue de cães domiciliados e errantes do município de Belém, Pará. Foram amostrados 276 cães por conveniência, sendo que o DNA extraído do sangue desses animais foi submetido à nested-PCR para a pesquisa de E. canis e A. platys. O DNA de E. canis foi detectado em 39,4% (109/276), e o DNA de A. platys em 23,1% (64/276) dos cães amostrados. Houve diferença estatisticamente significante entre a frequência desses agentes (p<0,0001), pois foi encontrada coinfecção entre os agentes em 13,4% (37/276) dos animais. A frequência de detecção desses parasitos foi maior em cães errantes do que nos domiciliados tanto para E. canis (OR=2,84) quanto para A. platys (OR=10,5). Diante dos resultados, foi possível concluir que E. canis e A. platys estão presentes na população canina estudada, sendo os cães errantes mais acometidos por esses parasitos.(AU)

Canine monocytic ehrlichiosis in Buenos Aires, Argentina: comparison of serological and molecular assays / Ehrlichiose monocítica canina em Buenos Aires: comparação de testes serológicos e moleculares

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.(AU)

A ehrlichiose monocítica canina (CME) é uma doença infecciosa transmitida pelo carrapato Rhipicephalus sanguineus sensu lato com distribuição mundial causada por Ehrlichia canis, que pode produzir uma doença grave se não foi diagnosticada e tratada precocemente. A confirmação da doença é feita diretamente pela detecção do DNA fazendo a reação em cadeia da polimerase (PCR) ou indiretamente por métodos sorológicos. O objetivo deste estudo foi comparar o método sorológico baseado na imunocromatografia e a técnica de PCR para o diagnóstico de E. canis em cães suspeitos da Cidade de Buenos Aires e da região sul da Grande Buenos Aires. As amostras de sangue de 20 cães clinicamente saudáveis (Grupo Controle) e de 80 cães com suspeita clínica de CME (Grupo 1-4) foram avaliadas em paralelo. O diagnóstico serológico foi feito pelo teste imunocromatográfico SPEED® EHRLI (BVT, França). Para a detecção molecular, foi utilizada uma PCR de triagem para amplificar um fragmento de 345 pb do gene que codifica a subunidade 16S do rRNA da família Anaplasmataceae. As amostras positivas depois foram processadas pela PCR aninhada específica para E. canis. No Grupo Controle, a presença de E. canis não foi detectada por PCR ou anticorpos específicos com o teste imunocromatográfico. No grupo em que a sorologia foi solicitada inicialmente (1 e 2), a concordância entre os testes foi baixo (kappa: 0,200) enquanto que no grupo onde o teste inicialmente solicitado foi a PCR, a concordância entre os testes era adequado (kappa: 0,650). A concordância entre os testes avaliados na população total estudada foi moderada (kappa: 0,496). Em conclusão, os resultados do nosso estudo sugerem que o uso de testes serológicos rápidos inicialmente, juntamente com a confirmação subsequente por PCR, permitirá melhorar o diagnóstico de CME.(AU)

Canine monocytic ehrlichiosis in Buenos Aires, Argentina: comparison of serological and molecular assays / Ehrlichiose monocítica canina em Buenos Aires: comparação de testes serológicos e moleculares

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.(AU)

A ehrlichiose monocítica canina (CME) é uma doença infecciosa transmitida pelo carrapato Rhipicephalus sanguineus sensu lato com distribuição mundial causada por Ehrlichia canis, que pode produzir uma doença grave se não foi diagnosticada e tratada precocemente. A confirmação da doença é feita diretamente pela detecção do DNA fazendo a reação em cadeia da polimerase (PCR) ou indiretamente por métodos sorológicos. O objetivo deste estudo foi comparar o método sorológico baseado na imunocromatografia e a técnica de PCR para o diagnóstico de E. canis em cães suspeitos da Cidade de Buenos Aires e da região sul da Grande Buenos Aires. As amostras de sangue de 20 cães clinicamente saudáveis (Grupo Controle) e de 80 cães com suspeita clínica de CME (Grupo 1-4) foram avaliadas em paralelo. O diagnóstico serológico foi feito pelo teste imunocromatográfico SPEED® EHRLI (BVT, França). Para a detecção molecular, foi utilizada uma PCR de triagem para amplificar um fragmento de 345 pb do gene que codifica a subunidade 16S do rRNA da família Anaplasmataceae. As amostras positivas depois foram processadas pela PCR aninhada específica para E. canis. No Grupo Controle, a presença de E. canis não foi detectada por PCR ou anticorpos específicos com o teste imunocromatográfico. No grupo em que a sorologia foi solicitada inicialmente (1 e 2), a concordância entre os testes foi baixo (kappa: 0,200) enquanto que no grupo onde o teste inicialmente solicitado foi a PCR, a concordância entre os testes era adequado (kappa: 0,650). A concordância entre os testes avaliados na população total estudada foi moderada (kappa: 0,496). Em conclusão, os resultados do nosso estudo sugerem que o uso de testes serológicos rápidos inicialmente, juntamente com a confirmação subsequente por PCR, permitirá melhorar o diagnóstico de CME.(AU)

Kidney disease in natural infection by Ehrlichia canis in dogs / Doença renal na infecção natural por Ehrlichia canis em cães

Canine monocytic ehrlichiosis, caused by the intracellular bacterium Ehrlichia canis, can affect different organs, including the kidneys, in different stages of infection, and kidney involvement is considered one of the main causes of death related to the disease. This study aimed to investigate the occurrence of kidney disease in dogs naturally infected with E. canis and to correlate antibody levels with the severity of renal disease. Serum concentrations of urea, creatinine, and proteins (albumin and globulin), along with urine concentration, urine gamma-glutamyl transferase, and urine protein levels, were evaluated in 60 dogs with E. canis infection diagnosed by polymerase chain reaction. The detection of anti-E. canis antibodies was also performed for each dog. Of the 60 dogs with E. canis infection, 73.33% presented anti-E. canis antibodies. Laboratory abnormalities consistent with renal disease were observed in 33 (55%) infected dogs, and of these, 43.3% were in stage I chronic kidney disease. A positive correlation was observed between antibody levels and total plasma protein (p = 0.0332) and serum globulin (p = 0.0057) levels. In this study, renal disease was observed on routine laboratory testing in 55% of dogs with monocytic ehrlichiosis; however, there was no correlation between the stage of renal disease and the antibody titer against E. canis.(AU)

A Erliquiose monocítica canina, causada pela bactéria intracelular Ehrlichia canis, pode acometer diferentes órgãos inclusive os rins, nas distintas fases da infecção, sendo considerada uma das principais causas de óbito relacionadas a essa doença. Este trabalho teve por objetivo investigar a ocorrência de doença renal em cães naturalmente infectados por E. canis correlacionando à gravidade da doença renal. Sessenta cães com infecção por E. canis diagnosticados pela reação em cadeia pela polimerase (PCR) foram avaliados a concentração sérica de ureia e creatinina, proteínas (albumina e globulinas), urinálise, gamaglutamil transferase urinária e proteinúria. Paralelamente foi pesquisado a presença de anticorpos anti-E. canis pelo ensaio imunoenzimático (dot ELISA). Dos 60 cães com infecção por E. canis, 73,33% apresentaram anticorpos anti- E. canis, enquanto, 33 (55%) cães apresentaram achados laboratoriais condizentes com doença renal, e destes 43,3% dos cães encontravam-se no estágio I da doença renal. Correlação positiva foi observada entre os níveis de anticorpos, globulina sérica (p=0,0057) e proteínas plasmáticas totais (p=0,0332). Neste estudo, a doença renal foi observada em 55% dos cães com erliquiose monocítica, utilizando exames laboratoriais empregados na rotina clínica, sem correlação com o estadiamento da doença renal, apesar dos altos títulos de anticorpos contra E. canis.(AU)