RESUMEN

Ehrlichia canis is a common tick-borne intracellular pathogen causing canine monocytic ehrlichiosis (CME) in dogs worldwide. The aims of this study were to investigate the genetic diversity and antigenicity of E. canis based on the p28 and trp36 genes in dogs in Thailand. The E. canis p28 and trp36 genes were amplified by the polymerase chain reaction (PCR) and cloned for sequencing and bioinformatic analyses. 36% (44/120) of dog blood samples were positive for E. canis DNA consisting of p28 (31%, 14/44) and trp36 (69%, 30/44) genes with 792 and 882 bp of PCR products size, respectively. The E. canis TRP36 from all Thailand sequences exhibited encoded nine amino acids (TEDSVSAPA) with 11 copies of tandem repeats along the sequences. The phylogenetic trees of E. canis, using the p28 and trp36 genes, exhibited that the Thailand isolates fell into two clades and one clade with similarity ranging from 55.95 to 100% and 100%, respectively. The results of diversity analysis revealed 10 and 20 haplotypes of the p28 and trp 36 genes, respectively. The entropy analysis of the p28 and trp36 nucleic acid sequences showed 442 and 1321 high entropy peaks respectively, whereas those of the P28 and TRP36 amino acid sequences showed 477 and 388 high entropy peaks, respectively. For B-cell epitopes analysis, the conserved amino acid of P28 and TRP36 sequences has been also demonstrated. Therefore, the results could be utilized to improve the understanding of phylogenetic relationship, genetic diversity and antigenicity of E. canis Thailand isolates.

Asunto(s)

RESUMEN

Ehrlichia minasensis is a new pathogenic bacterial species that infects cattle, and Borrelia theileri causes bovine borreliosis. We detected E. minasensis and B. theileri DNA in cattle from southwestern Colombia by using PCR. E. minasensis and B. theileri should be considered potential etiologies of febrile syndrome in cattle from Colombia.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Ehrlichia canis is among the most prevalent tick-borne pathogens infecting dogs worldwide, being primarily vectored by brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.). The genetic variability of E. canis has been assessed by analysis of different genes (e.g., disulfide bond formation protein gene, glycoprotein 19, tandem repeat protein 36 - TRP36) in the Americas, Africa, Asia, and in a single dog sample from Europe (i.e., Spain). This study was aimed to assess the variations in the TRP36 gene of E. canis detected in naturally infected canids and R. sanguineus s.l. ticks from different countries in Asia and Europe. DNA samples from dogs (n = 644), foxes (n = 146), and R. sanguineus s.l. ticks (n = 658) from Austria, Italy, Iran, Pakistan, India, Indonesia, Malaysia, the Philippines, Singapore, Thailand, Vietnam, and Taiwan were included in this study. Ehrlichia canis 16S rRNA positive samples (n = 115 from the previous studies; n = 14 from Austria in this study) were selected for molecular examination by analyses of TRP36 gene. Out of 129 E. canis 16S rRNA positive samples from dogs (n = 88), foxes (n = 7), and R. sanguineus s.l. ticks (n = 34), the TRP36 gene was successfully amplified from 52. The phylogenetic analysis of the TRP36 pre-repeat, tandem repeat, and post repeat regions showed that most samples were genetically close to the United States genogroup, whereas two samples from Austria and one from Pakistan clustered within the Taiwan genogroup. TRP36 sequences from all samples presented a high conserved nucleotide sequence in the tandem repeat region (from 6 to 20 copies), encoding for nine amino acids (i.e., TEDSVSAPA). Our results confirm the US genogroup as the most frequent group in dogs and ticks tested herein, whereas the Taiwan genogroup was present in a lower frequency. Besides, this study described for the first time the US genogroup in red foxes, thus revealing that these canids share identical strains with domestic dogs and R. sanguineus s.l. ticks.

Asunto(s)

RESUMEN

Tandem repeat proteins (TRPs) are major immunoreactive proteins of Ehrlichia canis, which have been used in the serological diagnosis of different genotypes of the microorganism. TRP19 is preserved among different E. canis isolates expressed on both reticulate and dense-core cells and observed in the extracellular matrix or associated with the morula membrane. TRP36 is differentially expressed only on the surface of the dense-core form of the bacterium and exhibits more divergence among isolates. The aim of this study was to evaluate the distribution of the American (USTRP36), Brazilian (BrTRP36) and Costa Rican (CRTRP36) genotypes of E. canis in Brazil, using ELISA assays. Serum samples of 814 dogs from 49 municipalities from all over Brazil were analyzed. Our results showed that 33.9% of the samples were reactive to the USTRP36 genotype and 32.6% to the BrTRP36 genotype. The two genotypes appeared to occur equally throughout Brazil, although the frequency of seropositivity was lower in the south than in the country's other regions. Dogs that reacted to at least one of the synthetic peptides (TRP19 and TRP36) were 456 (56%). A few dogs (n = 5; 0.6%) reactive to the E. canis TRP36 genotype (CRTRP36) were also detected in the northeast and southern regions. We concluded that the American and Brazilian genotypes of E. canis are distributed evenly in Brazil, especially in the tropical region, while the temperate region in the south presented the lowest prevalence rates. This study offers the first report of dogs seropositive for the Costa Rican genotype in Brazil.

RESUMEN

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

Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil.

Asunto(s)

RESUMEN

Ehrlichia minasensis, a recently described Ehrlichia species that is the most closely related to, but clearly distinct from, Ehrlichia canis, has been circulating in not only bovines, cervids, and dogs but also several tick species from Canada, Brazil, France, Pakistan, Ethiopia, and Israel. However, there are no reports of E. minasensis in China. The purpose of this study was to explore whether E. minasensis is present naturally in ticks in China. Through PCR targeting of the genus-conserved dsb gene, E. minasensis DNA was detected in Haemaphysalis hystricis ticks removed from free-ranging sheep in Hainan Province, South China in 2017. The partial sequence of the dsb, 16S rRNA, and groEL genes demonstrated that the Hainan strain shared 99% identity with the dsb gene of E. minasensis strain UFMG-EV (GenBank: JX629808), with the 16S rRNA of E. minasensis isolate E-2650 (MH500005) and with the groEL gene of E. minasensis strain UFMG-EV (JX629806), respectively. Moreover, sequence analysis of the major immunogenic tandem repeat protein (trp36) revealed that the Hainan strain harbored a unique tandem repeat sequence (APEAAPVSAPEAAPVSAPVS) and a C-terminal region that differed from those of other known E. minasensis strains. Additionally, phylogenetic analysis based on the entire amino acid sequence of trp36 revealed that the Hainan strain was closely related to a recently described E. minasensis strain from Brazil, of which the sister clade contained different strains of E. canis. The discovery of this novel Hainan strain in H. hystricis ticks represents the first known natural presence of E. minasensis in South China, highlighting the need for its constant surveillance.

RESUMEN

Canine monocytic ehrlichiosis, an important tick-borne disease caused by Ehrlichia canis, is cosmopolitan but particularly prevalent in tropical and subtropical regions. In Turkey, the genetic diversity of E. canis remains undefined. The aim of this study was to characterize E. canis in naturally infected dogs from Turkey by sequencing and phylogenetic analysis of the Tandem Repeat Protein 36 (TRP36) encoded by the trp36 gene. A total of 167 archived blood samples randomly collected from municipal shelter dogs in three distinct geographic regions were analyzed for E. canis. Only ten samples (5.98%) were found positive by PCR assays target regions of the trp36 and 16S rRNA genes. Sequence analysis of Turkish E. canis TRP36 revealed five Tanden Repeat sequences (TRs) resulting to three TR genotypes: i) the previously reported US genotype composed exclusively from TRs of "TEDSVSAPA" sequence (14 or 8 TRs), ii) the previously Brazilian genotype composed exclusively from TRs of ASVVPEAE sequence (13 TRs), and iii) a novel genotype. In addition, phylogenetic analysis based on the entire sequences of TRP36 revealed that these genotypes correspond to four distinct genogroups (US genogroups I and II, Brazilian genogroup and Costa Rica-Turkey genogroup), all containing Turkish genotypes amongst other geographically distant E. canisgenotypes.

Asunto(s)

RESUMEN

The tick-borne pathogens Ehrlichia canis and Anaplasma platys are the causative agents of canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT). Although molecular evidence of E. canis has been shown, phylogenetic analysis of this pathogen has not been performed and A. platys has not been identified in Mexico, where the tick vector Rhipicephalus sanguineus sensu lato (s.l.) is common. The aim of this research was to screen, identify and characterize E. canis and A. platys by PCR and phylogenetic analysis in dogs from La Comarca Lagunera, a region formed by three municipalities, Torreon, Gomez-Palacio and Lerdo, in the Northern states of Coahuila and Durango, Mexico. Blood samples and five engorged R. sanguineus s.l. ticks per animal were collected from 43 females and 57 male dogs presented to veterinary clinics or lived in the dog shelter from La Comarca Lagunera. All the sampled dogs were apparently healthy and PCR for Anaplasma 16S rRNA, Ehrlichia 16S rRNA, and E. canis trp36 were performed. PCR products were sequenced and used for phylogenetic analysis. PCR products were successfully amplified in 31% of the samples using primers for Anaplasma 16S rRNA, while 10% and 4% amplified products using primers for Ehrlichia 16S rRNA and E. canis trp36 respectively. Subsequent sequencing and phylogenetic analyses of these products showed that three samples corresponded to A. platys and four to E. canis. Based on the analysis of trp36 we confirmed that the E. canis strains isolated from Mexico belong to a conservative clade of E. canis and are closely related to strains from USA. In conclusion, this is the first molecular identification of A. platys and the first molecular characterization and phylogenetic study of both A. platys and E. canis in dogs in Mexico.

Asunto(s)

RESUMEN

The aim of this study was to characterize Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro, Brazil. In addition, all the clinical and hematological findings observed in these dogs were reported. PCR targeting the 16S rRNA gene was used for diagnostic purposes, and the TRP19 and TRP36 genes were sequenced to evaluate the genetic diversity. Fifteen samples were positive for E. canis. The polymerase chain reaction for the TRP19 gene resulted in 11 amplicons (11/15), which were cloned into the pGEM-T easy vector for sequencing. The complete sequence of TRP19 gene was compared to those in the GenBank, revealing high identicalness. Phylogenetic analysis on the TRP36 gene sequences demonstrated two distinct strains from two dogs, named 56C and 70C. The 56C strain was grouped with the strain Cuiaba 16, which is a hybrid strain formed by Brazilian and US genogroups; and the 70C strain was grouped with other strains of the US genogroup, thus suggesting that there are at least two genogroups of E. canis in Rio de Janeiro (US and Brazilian). Those animals, in which the 70C and 56C strains were isolated, showed distinct clinical and hematological manifestations of 1the disease. The appearance of different genotypes may express new phenotypes, thus resulting in different forms of presentation of the disease and making its diagnosis more complex.

O objetivo deste estudo foi caracterizar as cepas de Ehrlichia canis em cães naturalmente infectados no Rio de Janeiro, Brasil. Além disso, os achados clínicos e hematológicos observados nos cães foram relatados. O gene 16S rRNA foi utilizado como alvo da PCR para fins diagnósticos, e os genes TRP19 e TRP36 para avaliar a diversidade genética. Quinze amostras foram positivas para E. canis. PCR para o gene TRP19 produziu 11 amplicons (11/15) que foram clonados no pGEM-T easy vector para sequenciamento. A comparação das sequências completas do gene TRP19 com outras sequências depositadas no GenBank revelou uma alta identidade. Duas amostras (56C e 70C) após o ensaio da PCR, tendo como alvo o gene TRP36, geraram sequências, e a análise filogenética mostrou que a cepa 56C foi agrupada com a cepa Cuiabá 16, que é uma cepa híbrida, formada pelo genogrupo Brasileiro e o genogrupo US; e a cepa 70C agrupou com as outras cepas do genogrupo US, sugerindo a existência de pelo menos dois genogrupos de E. canis no Rio de Janeiro (US e Brasileiro). Esses animais apresentaram manifestações clínicas e hematológicas distintas, e diferentes genótipos podem expressar novos fenótipos, resultando em diferentes formas de apresentação da doença e fazendo com que o diagnóstico seja mais complexo.

Asunto(s)