RESUMO
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.
Assuntos
Doenças do Cão , Ehrlichia canis , Ehrlichiose , Rickettsia , Animais , Cães , Brasil/epidemiologia , Ehrlichia canis/isolamento & purificação , Doenças do Cão/epidemiologia , Doenças do Cão/microbiologia , Doenças do Cão/parasitologia , Rickettsia/isolamento & purificação , Ehrlichiose/veterinária , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Masculino , Infecções por Rickettsia/veterinária , Infecções por Rickettsia/epidemiologia , Infecções por Rickettsia/microbiologia , Feminino , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Febre Maculosa das Montanhas Rochosas/veterinária , Febre Maculosa das Montanhas Rochosas/epidemiologia , Febre Maculosa das Montanhas Rochosas/microbiologia , PrevalênciaRESUMO
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.
RESUMO
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.
Assuntos
Anaplasmose , Coinfecção , Doenças do Cão , Ehrlichiose , Nocardia , Derrame Pleural , Síndrome do Desconforto Respiratório , Humanos , Cães , Feminino , Animais , Lactente , Ehrlichia canis/genética , Anaplasmose/microbiologia , Coinfecção/veterinária , Coinfecção/microbiologia , Ehrlichiose/veterinária , Ehrlichiose/microbiologia , RNA Ribossômico 16S/genética , Nocardia/genética , Derrame Pleural/veterinária , Doenças do Cão/microbiologiaRESUMO
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.
Assuntos
Babesia , Babesiose , Doenças do Cão , Ehrlichiose , Animais , Cães , Babesia/genética , Ehrlichia canis/genética , Colômbia/epidemiologia , Babesiose/diagnóstico , Ehrlichiose/epidemiologia , Ehrlichiose/veterinária , Genótipo , Doenças do Cão/diagnósticoRESUMO
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.
Assuntos
Doenças do Cão , Ehrlichiose , Animais , Cuba , Doenças do Cão/microbiologia , Cães , Ehrlichia , Ehrlichia canis/genética , Ehrlichiose/microbiologia , Ehrlichiose/veterinária , Genótipo , FilogeniaRESUMO
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.
Assuntos
Vetores Aracnídeos/parasitologia , Doenças do Cão/parasitologia , Ehrlichia canis/isolamento & purificação , Ehrlichiose/veterinária , Rhipicephalus sanguineus/parasitologia , Animais , Argentina , Doenças do Cão/epidemiologia , Doenças do Cão/transmissão , Cães , Ehrlichiose/epidemiologia , Ehrlichiose/parasitologia , Ehrlichiose/transmissão , Feminino , Monócitos/parasitologiaRESUMO
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.
RESUMO
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.
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.
Assuntos
Animais , Cães , Ehrlichia canis/citologia , Ehrlichia canis/genética , Reação em Cadeia da Polimerase/veterináriaRESUMO
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)
Assuntos
Animais , Cães , Ehrlichia canis/citologia , Ehrlichia canis/genética , Reação em Cadeia da Polimerase/veterináriaRESUMO
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 36â¯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 (nâ¯=â¯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; nâ¯=â¯21), Costa Rica (CR; nâ¯=â¯11), and Brazil (BR; nâ¯=â¯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 (USâ¯=â¯26), followed by the CR genogroup (CRâ¯=â¯19) and the BR genogroup (BRâ¯=â¯11). Notably, 26/53 trp36 PCR positive dogs had detectable antibodies to multiple E. canis genotypes (US/BR/CRâ¯=â¯8, BR/CRâ¯=â¯7, US/CRâ¯=â¯6 and US/BRâ¯=â¯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.
Assuntos
Doenças do Cão/epidemiologia , Ehrlichia canis/fisiologia , Ehrlichiose/veterinária , Variação Genética , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Colômbia/epidemiologia , DNA Bacteriano/análise , Doenças do Cão/microbiologia , Cães , Ehrlichia canis/classificação , Ehrlichia canis/genética , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Filogenia , Reação em Cadeia da Polimerase/veterinária , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análiseRESUMO
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.
RESUMO
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.
RESUMO
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)
Assuntos
Animais , Cães , Ehrlichia canis/isolamento & purificação , Anaplasma/isolamento & purificação , Anaplasmose/epidemiologia , Ehrlichiose/epidemiologia , Ehrlichiose/veterinária , Trombocitopenia/veterinária , Reação em Cadeia da Polimerase/veterináriaRESUMO
The aim of the present study was to evaluate the genetic diversity of Ehrlichia canis in naturally infected dogs from six mesoregions of Rio de Janeiro state. E. canis was diagnosed with a real-time polymerase chain reaction (qPCR) targeting a 93 base pair (bp) fragment of the 16S rDNA gene. To evaluate the genetic diversity of the parasite, we amplified a positive sample from each mesoregion by distinct conventional PCR assays with targets in the gp19 (414 bp), gp36 (814 bp), and p28 (843 bp) genes. A total of 267 samples were collected from dogs in Rio de Janeiro state. Among the samples analyzed, 42.3% (n = 113/267) were 16S rDNA-qPCR positive. When performing PCR for the gp19 and gp36 genes, 100% (n = 113/113) and 5.3% (n = 6/113) of the samples amplified fragments of 414 bp and 814 bp, respectively. The six PCR-positive samples for the gp36 gene also amplified the p28 gene fragment. The characterization based on the gp19 gene demonstrated that it is highly conserved. In protein analysis (TRP36), all samples showed a tandem repeat protein (TRP) that comprised 11 replicates. Seven high-entropy amino acid sites were distributed throughout the gp36 gene. Eleven high-entropy amino acid sites were found throughout the p28 gene. There is a positive selection pressure in both genes (p ≤ 0.05). Comparing and characterizing an organism are useful for providing important information about the host's immune response and identifying new antigenic targets, as well as essential characteristics for the development of vaccines and new diagnostic tools.
Assuntos
Doenças do Cão/microbiologia , Ehrlichia canis/genética , Ehrlichia canis/isolamento & purificação , Ehrlichiose/veterinária , Animais , Proteínas de Bactérias/genética , Brasil , Cães , Ehrlichia canis/classificação , Ehrlichiose/microbiologia , Variação Genética , FilogeniaRESUMO
ABSTRACT: 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.
RESUMO: 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.
RESUMO
This cross-sectional, observational, and descriptive study aims to investigate the epidemiology of Ehrlichia canis in healthy owned dogs from the Southeastern region of Rio de Janeiro, Brazil. Blood samples were collected from 390 households dogs. During the visits, an epidemiological questionnaire was filled out concerning the dogs' characteristics as well as the environments in which they lived. The variables were analyzed using a bivariate test, while the correlation analysis between the variables was performed via a phi test. The variables that had p-values lower than 0.2 in the bivariate analysis and had a low or moderate correlation were selected for the multivariate analysis. The model that had the lowest Akaike information criterion (AIC) value was retained. Among the 390 blood samples tested, 24.8% were considered positive for E. canis. The parsimonious logistic regression model presented an AIC value of 408.75 and showed three variables that favored the presence of E. canis DNA in the tested dogs: the animal's access to urban streets and neighborhoods (odds ratio [OR] = 1.91; p-value = 0.02; confidence interval [CI]: 1.14 - 3.18), tick infestation (OR = 2.01; p-value = 0.006; CI: 1.22 - 3.32), and poor hygienic conditions (OR = 2.19; p-value = 0.002; CI: 1.31 - 3.67). The model was considered well-calibrated based on the Hosmer-Lemeshow test (p = 0.39). According to the present study, dogs that have access to the street and neighborhood, are infested with ticks, and live under poor hygienic conditions are more likely to be infected with E. canis in the state of Rio de Janeiro, Brazil.
Assuntos
Infecções Assintomáticas/epidemiologia , Doenças do Cão/epidemiologia , Ehrlichia canis/isolamento & purificação , Ehrlichiose/veterinária , Infestações por Carrapato/veterinária , Animais , Proteínas da Membrana Bacteriana Externa/análise , Brasil/epidemiologia , Estudos Transversais , Doenças do Cão/microbiologia , Cães , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Feminino , Masculino , Análise Multivariada , Reação em Cadeia da Polimerase/veterinária , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/parasitologiaRESUMO
Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p < 0.001). The number of dogs positive for A. platys was larger in Winter (p < 0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p < 0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.(AU)
As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p < 0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p < 0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p < 0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.(AU)
Assuntos
Animais , Cães , Anaplasma/isolamento & purificação , Ehrlichia canis/isolamento & purificação , Babesia/isolamento & purificação , Epidemiologia Molecular , Métodos Epidemiológicos/veterinária , Ehrlichiose/veterinária , Babesiose/epidemiologia , Ehrlichiose/epidemiologia , Reação em Cadeia da Polimerase/veterináriaRESUMO
Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p<0.001). The number of dogs positive for A. platys was larger in Winter (p<0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p<0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.(AU)
As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p<0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p<0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p<0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.(AU)
Assuntos
Animais , Cães , Anaplasma/isolamento & purificação , Babesia/isolamento & purificação , Ehrlichia canis/isolamento & purificação , Epidemiologia Molecular , Babesiose/epidemiologia , Ehrlichiose/epidemiologia , Ehrlichiose/veterinária , Métodos Epidemiológicos/veterinária , Reação em Cadeia da Polimerase/veterináriaRESUMO
ABSTRACT: Hemoparasitic infections are tick-borne diseases, which affect animals and humans. Considering the importance of canine hemoparasitic infections in veterinary clinics, this study aimed to determine the occurrence of Anaplasma platys, Ehrlichia canis and Babesia vogeli in blood samples from 182 dogs not domiciled in the city of Pato Branco, southwestern region of Paraná State, Brazil, using polymerase chain reaction (PCR). The prevalence of A. platys and B. vogeli was 32.9% and 10.9% respectively, and A. platys infection prevailed (p 0.001). The number of dogs positive for A. platys was larger in Winter (p 0.05). All blood samples were negative for E. canis. In the dogs, infestation by Amblyomma cajennense predominated over that by Rhipicephalus sanguineus (p 0.001); but there was no significant association between PCR and the variables presence of ticks, sex and age. Dogs infected by A. platys and B. vogeli showed thrombocytopenia, lymphopenia and leukocytosis; but there was no correlation between such hematological changes and infection by hemoparasites. This appears to be the first molecular study that demonstrates the existence of A. platys and B. vogeli in dogs from the southwestern region of Paraná.
RESUMO: As hemoparasitoses são enfermidades transmitidas por carrapatos que afetam os animais e os humanos. Considerando a importância das hemoparasitoses caninas na clínica médica veterinária, este estudo objetivou determinar a ocorrência de Anaplasma platys, Ehrlichia canis e Babesia vogeli em amostras de sangue de 182 cães não domiciliados do município de Pato Branco, região sudoeste paranaense, Brasil, utilizando a reação em cadeia da polimerase (PCR). A prevalência de A. platys e B. vogeli foi de 32,9% e 10.9%, respectivamente, predominando a infecção por A. platys (p 0,001). Constatou-se um maior número de cães positivos para A. platys no período do inverno (p 0.05). Todas as amostras de sangue foram negativas para E. canis. Nos cães, a infestação por Amblyomma cajennense prevaleceu sobre a infestação por Rhipicephalus sanguineus (p 0,001), mas não foi observada associação significativa entre a PCR e as variáveis presença de carrapatos, sexo e idade. Cães infectados por A. platys e B. vogeli apresentaram trombocitopenia, linfopenia e leucocitose, porém não houve correlação destas alterações hematológicas com a infecção pelos hemoparasitas. Este é o primeiro estudo molecular que demonstra a existência de A. platys e de B. vogeli em cães da região sudoeste paranaense.
RESUMO
Ehrlichiosis is a worldwide illness, endemic in tropical and subtropical countries where seroprevalence can reach up to 33% as it is the case in México and Israel. However, the low sensitivity of the serological tests used for diagnosis, 67% in tests with IFI, has led to a required searching for new alternatives to diagnose the disease quickly and effectively. PCR is one of these techniques that could provide high sensitivity and specificity. The target of this study was to implement the PCR test for the diagnosis of Ehrlichia spp., on blood samples from animals with suspected illness from veterinary clinics in the city of Medellin. 90 samples were taken, 33 samples from animals suspected of having symptoms and 57 from healthy animals as probable negatives. DNA from the Madrid strain was used as a positive control. The PCR was performed using as an example the protocol suggested by Aguirre et al (2004). EEC and ECB primers reported by Dawson et al (2004) were used (aquí el propio texto en español no está claro). In this study the 500pb band was amplified, corresponding to the 16s rRNA of Ehrlichia spp., in 11 samples of the animals with suspected illness, obtaining a presentation rate of 33.3%, confirming the presence of bacteria in the animals of the environment, and achieving the implementation of PCR for Ehrlichia spp. as a diagnostic tool in the city.
La Ehrlichiosis es una enfermedad de distribución mundial, es endémica en los países tropicales y subtropicales en donde la seroprevalencia puede llegar a ser hasta del 33%, como en México e Israel. La baja sensibilidad de las pruebas utilizadas para el diagnóstico, 67% en las pruebas con IFI, han llevado a la necesidad de buscar nuevas alternativas que permitan diagnosticar la enfermedad de manera rápida y eficaz. La PCR es una de estas técnicas que podría ofrecer una alta sensibilidad y especificidad. El objetivo de este trabajo fue implementar la prueba de PCR, para el diagnóstico de Ehrlichia spp., en muestras de sangre de caninos sospechosos provenientes de consultorios veterinarios de la ciudad de Medellín. Se tomaron 90 muestras, 33 de animales sospechosos de ehrlichiosis por sintomatología, y 57 de animales sanos como probables negativos. Se utilizó como control positivo el ADN de la cepa Madrid. La PCR fue realizada utilizando como ejemplo el protocolo sugerido por Aguirre et al. (2004). Se utilizaron los primers EEC y ECB reportados por Dawson et al. (1994). En este estudio se logró amplificar la banda de 500 pb correspondientes al gen 16s ARNr de Ehrlichia spp., en 11 muestras de los animales sospechosos, obteniendo una frecuencia de presentación del 33,3%, confirmando la presencia de la bacteria en los animales de nuestro medio, y logrando la implementación de PCR para Ehrlichia spp. como herramienta diagnóstica en nuestra ciudad.
A ehrlichiose é uma doença em todo o mundo, é endêmica em países tropicais e subtropicais, onde a soroprevalência pode atingir até 33%, como no México e Israel. A baixa sensibilidade dos testes utilizados para o diagnóstico, 67% nos testes de IFI, levaram à necessidade de buscar novas formas de diagnosticar a doença de forma rápida e eficaz. PCR é uma técnica de tal forma que poderia fornecer alta sensibilidade e especificidade. O objetivo deste trabalho foi implementar o teste de PCR para o diagnóstico de Ehrlichia spp., Em amostras de sangue de cães suspeitos de clínicas veterinárias da cidade de Medellín. Demorou 90 amostras, 33 amostras de suspeitos que apresentavam sintomas de erliquiose e 57 animais saudáveis como negativo provável. Foi utilizado como controle positivo de ADN a partir da estirpe de Madrid. A PCR foi padronizada utilizando o exemplo do protocolo proposto por Aguiar et al. (2004). Utilizou-se primers BCE CES e relatado por Dawson et al. (1994). Neste estudo foram amplificados de banda de 500 pb para o gene 16S rRNA do gênero Ehrlichia em 11 amostras de animais suspeitos, a obtenção de uma taxa de apresentação de 33,3%, o que confirma a presença de bactérias em animais nosso meio ambiente e obter a implementação de PCR por Ehrlichia spp. como uma ferramenta de diagnóstico em nossa cidade.