Claudia Bezerra da Silva

Seroepidemiological aspects of Leishmania spp. in dogs in the Itaguai micro-region, Rio de Janeiro, Brazil.

This study evaluated factors associated with the frequency of Leishmania spp. antibodies in dogs residing in the Itaguai micro-region, State of Rio de Janeiro, Brazil. Blood samples were collected from 524 dogs. The serum samples were submitted to indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for Leishmania spp. The frequency of seropositive dogs was 28.24% (n = 148) in the micro-region, and among the three municipalities within that region, the highest frequency (p < 0.05) was observed in Seropedica (59.46%), followed by Itaguai (29.05%) and Mangaratiba (11.49%). Regarding factors associated with the host, mongrel dogs and those over the age of two presented higher frequency of antibodies to Leishmania spp. (p < 0.05). Concerning factors related to the environment and habits of the animal, dogs residing in rural areas (FR = 1.67, p = 0.0002), living outside the residence (FR = 1.42, p = 0.0197), with access to forest, streams and pastures (FR = 2.81, p = 0.0007), remaining loose (FR = 1.66, p = 0.0073), and those that had no shelter (FR = 2.16, p < 0.0001) were more likely to be seropositive. Canine leishmaniasis is a disease with high occurrence in the Itaguai micro-region, and aspects such as the definition of breed, age, habits and care by owners showed significant association in this micro-region.

A new quantitative PCR method for the detection of Anaplasma platys in dogs based on the citrate synthase gene

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.

Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs

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 70 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.

Molecular characterization of Theileria equi in horses from the state of Rio de Janeiro, Brazil

Theileria equi is one of the etiologic agents of the equine piroplasmosis. This infectious disease is transmitted by ticks and is a worldwide problem in the international horse movement. The 18S rRNA gene of T. equi is often used for genotyping and phylogenetic purpose. This study aimed to analyze the degree of the heterogeneity of the 18S rRNA gene of T. equi in horses from the state of Rio de Janeiro, Brazil. The complete T. equi 18S rRNA sequences were obtained from twenty naturally infected horses. The PCR amplicons were cloned and sequenced. The phylogenetic analyses were performed using a set of T. equi 18S rRNA sequences and other related organisms available in ARB-Silva database. There were twelve distinct T. equi 18S rRNA gene sequences circulating in horses in the state of Rio de Janeiro, Brazil. Monophyletic clades with 2% evolutionary divergence between clades and high bootstrap value were the support to divide T. equi sequences in three distinct clades. The sequences from this study grouped into clades I (70%, n=14/20) and II (30%, n=6/20). All of the T. equi sequences grouped within a node other than the theileriids. This study reported a clear division of two distinct genotypes of T. equi 18S rRNA sequences in state of Rio de Janeiro, Brazil, and it demonstrates that distinct isolates of T. equi can coexist in the same geographic region.

STATUS OF THE AMERICAN TEGUMENTARY LEISHMANIASIS IN THE STATE OF RIO DE JANEIRO , BRAZIL, FROM 2004 TO 2013

SUMMARY The aim of the present study was to analyze the status of the American Tegumentary Leishmaniasis (ATL) in the state of Rio de Janeiro, from 2004 to 2013, through its spatiotemporal distribution. We also described variables considered relevant to the epidemiology of the disease, such as the clinical form, gender, ethnic group, age group, and progression of disease. This is a descriptive study, which used notified secondary data from the Brazilian Information System of Notifiable Diseases (SINAN), Ministry of Health, Brazil, regarding confirmed diagnoses. To help the calculation of coefficients of detection and mortality, we used population data from the Brazilian Institute of Geography and Statistics (IBGE). We analyzed 1,470 cases of ATL with the predominance of the cutaneous clinical form (1,292/87.89%). The data has also revealed seven deaths, a predominance of males (922/62.72%), and a higher incidence of ATL in the white ethnic group (731/49.72%). We observed a high incidence of ATL in the group of 20 - 39 years old (477/32.44%). We concluded that there was a decrease in the number of ATL cases in the state of Rio de Janeiro, based on a coefficient of detection of 1.44/100.000 inhabitants in 2004 decreasing to 0.20/100.000 inhabitants in 2013. The localities with the highest occurrences of ATL were the metropolitan region (843 cases) and the municipality of Rio de Janeiro (740 cases). In 2005, the highest incidence of the disease was observed (351 cases) in the study. Among the variables selected to describe the epidemiology of the disease, the following categories: cutaneous clinical form, male patients, white ethnic group, and the age group of 20 - 39 years old were more affected than the others.

Molecular detection and characterization of Anaplasma platys in dogs and ticks in Cuba

Canine cyclic thrombocytopenia, an infectious disease caused by Anaplasma platys is a worldwide dog health problem. This study aimed to detect and characterize A. platys deoxyribonucleic acid (DNA) in dogs and ticks from Cuba using molecular methods. The study was conducted in four cities of Cuba (Habana del Este, Boyeros, Cotorro and San José de las Lajas). Blood samples were collected from 100 dogs in these cities. The animals were inspected for the detection of tick infestation and specimens were collected. Genomic DNA was extracted from dog blood and ticks using a commercial kit. Genomic DNA samples from blood and ticks were tested by a nested polymerase chain reaction (nPCR) to amplify 678 base pairs (bp) from the 16S ribosomal DNA (rDNA) of A. platys. Positive samples in nPCR were also subjected to PCR to amplify a fragment of 580bp from the citrate synthase (gltA) gene and the products were sequenced. Only Rhipicephalus sanguineus sensu lato (s.l.) was found on dogs, and 10.20% (n=5/49) of these ticks plus sixteen percent (16.0%, n=16/100) of dogs were considered positive for A. platys by nPCR targeting the 16S rDNA gene. All analyzed gltA and 16S rDNA sequences showed a 99-100% identity with sequences of A. platys reported in around the world. Phylogenetic analysis showed two defined clusters for the 16S rDNA gene and three defined clusters for the gltA gene. Based on the gltA gene, the deduced amino acid sequence showed two mutations at positions 88 and 168 compared with the sequence DQ525687 (GenBank ID from Italian sample), used as a reference in the alignment. A preliminary study on the epidemiological aspects associated with infection by A. platys showed no statistical association with the variables studied (p>0.05). This is the first evidence of the presence of A. platys in dogs and ticks in Cuba. Further studies are needed to evaluate the epidemiological aspects of A. platys infection in Cuban dogs.

Molecular investigation of Neorickettsia risticii in trematodes and snails in a region with serological evidence of this agent in horses, state of Rio de Janeiro

No Brasil, estudos apontam a circulacao de Neorickettsia risticii em equinos, contudo nao estao claros quais os possiveis vetores intermediarios dessa bacteria no pais. O objetivo do presente estudo foi analisar a presenca de N. risticii, utilizando-se tecnicas moleculares, em caramujos e estagios larvais de trematodeos em propriedades rurais de uma regiao com historico de equinos sororreativos para essa bacteria, no Rio de Janeiro, Brasil. Uma amostragem por conveniencia foi utilizada na regiao de estudo. Os caramujos coletados foram expostos a luz incandescente (60W) durante duas-quatro horas para a investigacao de trematodeos nas formas larvais. A extracao de acido desoxirribonucleico (DNA) foi realizada em tecidos de caramujos e trematodeos. A tecnica de PCR em tempo real (qPCR) foi utilizada para investigar a presenca de um fragmento do gene 16S rRNA de N. risticii. Foram coletados 410 especimes de caramujos de 11 propriedades com criacoes de equinos, sendo identificadas as seguintes especies: Melanoides tuberculata, Pomacea sp., Biomphalaria tenagophila, Physa acuta, Drepanotrema anatinum e Biomphalaria straminea. Apenas 3,17% (n=13/410) dos caramujos identificados estavam infectados por trematodeos. As cercarias obtidas desses caramujos foram classificadas em Megalourous cercariae, Pleurolophocercus cercariae e Furcocercous cercariae. Nao foi observada a amplificacao do DNA-alvo de N. risticii, por meio da qPCR, em nenhuma das amostras de caramujos e trematodeos testadas. Com base nesses dados, a transmissao de N. risticii por trematodeos que utilizam as especies de caramujos nessa regiao parece nao ocorrer ou ocorre a taxas muito reduzidas. Portanto, novos estudos sao necessarios para elucidar quais especies de hospedeiros invertebrados se infectam por essa bacteria e potencialmente participam da cadeia de transmissao da neorickettsiose equina no estado do Rio de Janeiro, Brasil.