RESUMO
The aim of this study was to detect Theileria equi (Laveran 1901) DNA in horses and ticks using real-time PCR and to list the factors associated with infection in animals located in the Seropedica and Petropolis municipalities of the state of Rio de Janeiro. We tested blood samples from 314 horses and samples from 300 ticks, including 191 Amblyomma cajennense, 104 Dermacentor nitens, and 5 Ixodida larvae. Factors inherent to the horse, the ownership, and animal management were obtained from an epidemiological questionnaire and were evaluated in association with the presence of T. equi DNA in the animals. Among the horses in the study, 81 % (n = 253/314) presented T. equi DNA, and the animals of the Seropedica municipality had the highest infection frequency (91 %, n = 128/141, p < 0.001). The factors that had significantly different infection frequencies by chi-squared or Fisher's exact tests (p < 0.2) were included in a logistic regression model using the R programming package. Work and walking activity (odds ratio [OR] = 5.7, CI = 2.3-14.4), reproductive activity (OR = 3.8, CI = 1.3-11.5), and tick infestation (OR = 2.6, CI = 1.1-6.2) were factors that favored the presence of T. equi DNA in the animals (p < 0.05). Among the tick samples, A. cajennense and D. nitens were the identified species. The presence of T. equi DNA was observed in 9.9 % (n = 19/191) of the A. cajennense samples and 3.8 % (n = 4/104) of the D. nitens samples. A multivariate analysis revealed that the presence of A. cajennense on the animals (OR = 4.1, CI = 1.8-9.1) was associated with the presence of T. equi DNA in the horses. In the studied municipalities, activities related to work, walking, and reproduction and the presence of ticks on the horses, particularly an intense infestation of A. cajennense, are factors that lead to infection with T. equi in the horses.
Assuntos
Vetores Aracnídeos/parasitologia , Doenças dos Cavalos/parasitologia , Epidemiologia Molecular , Theileria/genética , Theileriose/parasitologia , Carrapatos/parasitologia , Animais , Brasil/epidemiologia , DNA de Protozoário/análise , DNA de Protozoário/genética , Doenças dos Cavalos/epidemiologia , Cavalos , Reação em Cadeia da Polimerase em Tempo Real , Theileria/classificação , Theileriose/epidemiologia , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/parasitologia , Carrapatos/classificaçãoRESUMO
Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (â¼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.
Assuntos
Variação Genética , Doenças dos Cavalos/parasitologia , RNA Ribossômico 18S/genética , Theileria/genética , Theileriose/parasitologia , Animais , Brasil , Sequência Consenso , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças Endêmicas/veterinária , Doenças dos Cavalos/sangue , Cavalos , Funções Verossimilhança , RNA de Protozoário/sangue , RNA de Protozoário/genética , RNA Ribossômico 18S/sangue , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência/veterinária , Theileria/classificação , Theileriose/sangueRESUMO
Hemoparasitic diseases are prominent in domestic animals, particularly in Brazil, a tropical country with a wide range of vectors. This study investigated the epidemiology of Babesia vogeli in the whole blood of dogs from the southeastern region of Rio de Janeiro, Brazil. Whole blood samples from 390 dogs were screened for the presence of B. vogeli DNA by qPCR using the heat shock protein 70â¯kDa (hsp70) gene of B. vogeli. Characteristics related to the host and its environment were collected using a questionnaire. Bivariate analysis was used to evaluate each factor individually. A phi correlation test was used to verify collinearity. The variables with pâ¯<â¯.1 and a low or moderate correlation with the other variables were selected for the multivariate analysis. Multiple models were created, and the best logistic regression model was chosen using the Akaike Information Criterion (AIC). The final model was used to determine which variables were closely related to B. vogeli infections in dogs. Of the 390 dog blood samples, 15.66% were positive for B. vogeli. The variables cat contact, age, shelter, street or woods access, tick infestation and fur lengthwere included in the final model. Per the logistic regression analysis, three variables explained B. vogeli detection in dogs: age (odds ratio [OR]â¯=â¯2.12; p-value <.05; confidence interval [CI]: 1.13-3.96), tick infestation (ORâ¯=â¯2.08; p-value <.05; CI: 1.10-3.93) and shelter (ORâ¯=â¯2.22; p-value <.05; CI: 1.16-4.26). These variables were determined to be associated with B. vogeli detection in domiciled dogs in the southeastern region of Rio de Janeiro, Brazil. These data indicate that the age of the animal, the presence of ticks and the lack of shelter directly affect the epidemiology of B. vogeli.
Assuntos
Babesia/genética , Babesiose/epidemiologia , Doenças do Cão/epidemiologia , Infestações por Carrapato/parasitologia , Fatores Etários , Animais , Babesia/isolamento & purificação , Brasil/epidemiologia , DNA de Protozoário/genética , Doenças do Cão/parasitologia , Cães/parasitologia , Feminino , Proteínas de Choque Térmico HSP70/genética , Modelos Logísticos , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Infestações por Carrapato/epidemiologia , Carrapatos/parasitologiaRESUMO
A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70â¯kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.
Assuntos
Babesia/isolamento & purificação , Babesiose/sangue , Doenças do Cão/diagnóstico , Proteínas de Choque Térmico HSP70/genética , Filogenia , RNA Ribossômico 18S/genética , Animais , Babesia/química , Babesia/genética , Babesiose/diagnóstico , Babesiose/epidemiologia , Babesiose/parasitologia , Brasil/epidemiologia , Primers do DNA/genética , DNA de Protozoário/genética , Doenças do Cão/epidemiologia , Doenças do Cão/parasitologia , Cães , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Anaplasma platys is an obligate intracellular bacterium that primarily affects dogs, but it can also infect humans. Our study aimed to standardize a quantitative real-time (q)PCR method using the citrate synthase gene (gltA) as a specific target for A. platys detection in naturally infected dogs. Primers (gltA84F and gltA84R) and probe (PLATYSp) were designed to amplify an 84-bp fragment based on the gltA gene sequences of A. platys available in GenBank. A total of 186 dog blood samples originating from the Brazilian state of Rio de Janeiro were tested by qPCR. Additionally, the same samples were tested by cytology and a nested (n)PCR that targeted the 16S ribosomal DNA to determine the performance of our qPCR method compared to these existing techniques. Among the samples tested with qPCR, 17.2% were considered positive, significantly more than detected by nPCR (14.0%). Under optical microscopy, inclusions were observed in platelets of 25.3% of the samples, and among these samples, only 33.9% were identified as positive for A. platys using qPCR. The qPCR technique proved to be more specific than cytology and to have superior sensitivity to nPCR for detecting A. platys in dogs. The development of this new qPCR method contributes to the advancement of research involving A. platys Furthermore, it can be used to quantify the presence of this bacterium to evaluate the treatment of infected animals, or even as a more sensitive and specific tool for situations indicating possible clinical disease but with negative cytology.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/diagnóstico , Doenças do Cão/diagnóstico , Anaplasma/genética , Anaplasma/metabolismo , Anaplasmose/microbiologia , Animais , Brasil , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Primers do DNA , Doenças do Cão/microbiologia , Cães , Valor Preditivo dos Testes , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Destruxins (DTXs) are cyclic depsipeptides produced by many Metarhizium isolates that have long been assumed to contribute to virulence of these entomopathogenic fungi. We evaluated the virulence of 20 Metarhizium isolates against insect larvae and measured the concentration of DTXs A, B, and E produced by these same isolates in submerged (shaken) cultures. Eight of the isolates (ARSEF 324, 724, 760, 1448, 1882, 1883, 3479, and 3918) did not produce DTXs A, B, or E during the five days of submerged culture. DTXs were first detected in culture medium at 2-3 days in submerged culture. Galleria mellonella and Tenebrio molitor showed considerable variation in their susceptibility to the Metarhizium isolates. The concentration of DTXs produced in vitro did not correlate with percent or speed of insect kill. We established endophytic associations of M. robertsii and M. acridum isolates in Vigna unguiculata (cowpeas) and Cucumis sativus (cucumber) plants. DTXs were detected in cowpeas colonized by M. robertsii ARSEF 2575 12 days after fungal inoculation, but DTXs were not detected in cucumber. This is the first instance of DTXs detected in plants endophytically colonized by M. robertsii. This finding has implications for new approaches to fungus-based biological control of pest arthropods.
Assuntos
Depsipeptídeos/metabolismo , Fabaceae/microbiologia , Proteínas Fúngicas/metabolismo , Insetos/microbiologia , Metarhizium/fisiologia , Micotoxinas/metabolismo , Animais , Depsipeptídeos/análise , Fabaceae/química , Fabaceae/fisiologia , Proteínas Fúngicas/análise , Micotoxinas/análiseRESUMO
BACKGROUND: Anaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil. METHODS: We tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum. RESULTS: Among the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs. CONCLUSIONS: This is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.
Assuntos
Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/isolamento & purificação , Doenças Transmissíveis Emergentes/epidemiologia , Ehrlichiose/veterinária , Ixodidae/microbiologia , Zoonoses/microbiologia , Animais , Brasil/epidemiologia , Doenças do Cão/epidemiologia , Doenças do Cão/microbiologia , Cães , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Masculino , Epidemiologia Molecular , Razão de Chances , Filogenia , RNA Bacteriano , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Zoonoses/epidemiologiaRESUMO
Equine neorickettsiosis (EN), also known as Potomac Horse Fever, is a non-contagious disease caused by the bacterium Neorickettsia risticii of the Anaplasmataceae family. The objectives of this study were to detect the presence of anti-N. risticii antibodies by the indirect immunofluorescence assay (IFA) and of its DNA by qPCR in equids at high and low altitude regions in the State of Rio de Janeiro, Brazil, and to identify factors associated with seropositive equids by multiple logistic regression analysis. The frequency of anti-N. risticii antibodies was 16.05% (n=113/704). The animal age and breeding region were the factors that influenced the seropositivity rate for N. risticii in the equids (p<0.05). Equids from the lowland region had higher seropositivity (p<0.05; OR=5.87) compared to those of the mountain region. The presence of snails on the farm was a factor associated with this result (p<0.05; OR=2.88). In the lowland region, age of the animal and site of breeding were protective factors for the detection of antibodies anti-N. risticii in equids, with lower frequency of seropositivity in younger animals (p<0.05; OR=0.06) and in animals raised in dry areas (p<0.05; OR=0.22). The presence of the target DNA of N. risticii by qPCR was not observed in any of the samples tested. The existence of seropositive equids for N. risticii demonstrates a possible circulation of this agent in the studied area, and that the age related characteristics and equids breeding region are important factors regarding seropositivity in the State of Rio de Janeiro.(AU)
A Neorickettisiose equina (NE), também conhecida como Febre do Cavalo de Potomac, é uma doença não contagiosa causada pela bactéria Neorickettsia risticii da família Anaplasmataceae. Os objetivos deste estudo foram detectar a presença de anticorpos anti-N. risticii através da reação de Imunofluorescência Indireta (RIFI) e do DNA dessa bactéria através da qPCR em equídeos de regiões de alta e baixa altitude no Estado do Rio de Janeiro, Brasil; e identificar os fatores associados com a soropositividade dos equídeos através da análise de regressão logística múltipla. A frequência de anticorpos anti-N. risticii foi de 16,05% (n=113/704). Observou-se que a idade e a região de criação foram os fatores que influenciaram a taxa de soropositividade para N. risticii nos equídeos (p<0,05). Equídeos da região de baixada apresentaram maior soropositividade (p<0,05; OR=5,87) quando comparado aos criados em região de montanha. A presença de caramujos na propriedade foi um fator associado a este resultado (p<0,05; OR=2,88). Na região de baixada, animais mais jovens (p<0,05; OR=0,06), criados em áreas secas (p<0,05; OR=0,22) demonstraram serem fatores de proteção na detecção de anticorpos anti-N. risticii. Não foi observada a presença do DNA-alvo de N. risticii através da qPCR em nenhuma das amostras testadas. A existência de equídeos soropositivos para N. risticii demonstra a possível circulação desse agente na área estudada, e as características inerentes a idade e a região de criação dos equídeos são fatores importantes relacionados à soropositividade no estado do Rio de Janeiro.(AU)
Assuntos
Animais , Infecções por Anaplasmataceae/epidemiologia , Infecções por Anaplasmataceae/veterinária , Fatores Epidemiológicos , Cavalos , Neorickettsia risticii/isolamento & purificação , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Modelos Logísticos , Reação em Cadeia da Polimerase/veterinária , Testes Sorológicos/veterináriaRESUMO
Anaplasma phagocytophilum was detected in dogs from Brazil in the municipalities of Seropédica and Itaguaí, Rio de Janeiro state, by real-time polymerase chain reaction (PCR) using SYBR Green to detect the amplification. Of 253 samples, 18 (7.11%) were positive, with a threshold cycle (Ct) ranging from 31 to 35 cycles. The PCR product from a positive sample was cloned and sequenced. The sequence obtained demonstrated 100% identity with other A. phagocytophilum sequences published in the GenBank database. The analytical sensitivity of RT-PCR using SYBR Green system was able to detect 3 plasmid copies when defined numbers of plasmid copies containing 122 base pairs from the msp2 gene were used. The assay was considered specific when DNA from bacteria (Anaplasma platys, Anaplasma marginale, Ehrlichia canis, Neorickettsia risticii, Rickettsia rickettsii) closely related to A. phagocytophilum was placed in the reaction. These results demonstrate that the canine granulocytic anaplasmosis agent is present in regions in which dogs could be a source of infection for tick vectors. The current study reports the detection of A. phagocytophilum, a zoonotic agent responsible for Human granulocytic anaplasmosis, in Brazilian dogs.