Detection and molecular characterization of Avipoxvirus in Culex spp. (Culicidae) captured in domestic areas in Rio de Janeiro, Brazil.

Avian pox is a highly contagious poultry disease that causes significant economic losses. Mosquitoes belonging to the genus Culex (Diptera: Culicidae) have a fundamental role in disseminating Avipoxvirus (Poxviridae). This study proposes investigating the presence of Avipoxvirus (APV) DNA in Culex spp. from Rio de Janeiro to determine its frequency and perform a phylogenetic analysis based on the core like the 4b protein (p4b) gene. The detection of APVs was conducted individually on four hundred Culex spp. mosquitoes. A total of 12.23% (47/384) of the Culex spp. were positive in the PCR. Sequencing the p4b gene revealed that this study's sequences displayed 98.8-99% identity with Fowlpoxvirus (FWPW) sequences available in GenBank. In the phylogenetic analysis, these APVs were clustered in the A1 subclade together with FWPW sequences from several countries. The evolutionary distance of the p4b gene was 0.61 ± 0.21% in rural areas and 0.38 ± 0.16% in peri-urban areas. The current investigation is the first study to report the detection of APVs in field-caught mosquitoes. Moreover, a high frequency of APV DNA was observed in Culex spp. captured in domestic areas, where backyard poultry is present. This data demonstrates the importance of implementing control measures for Culex spp. to mitigate the transmission of APVs in backyard poultry in Rio de Janeiro.

Robust comparative performance of genomic DNA extraction methods from non-engorged phlebotomine sandflies.

The present study is a comparative analysis of DNeasy Blood & Tissue Qiagen® kit (Qiagen®, Hilden, Alemanha), salting out, HotShot and phenol-chloroform protocols to extract DNA from sandflies. In addition, a comparative test using sandflies with and without eyes evaluated the potential inhibitory effect in the cPCR. An inhibition test was performed using an exogenous DNA added to the qPCR. The genomic DNA quality of each sample was evaluated by cPCR based on the cytochrome c oxidase subunit I (cox1) gene. The DNA extraction protocols showed the following percentage of amplification: HotShot (91.6% [55/60]), salting out (71.6% [43/60]), phenol-chloroform (95% [57/60]) and kit DNeasy Blood & Tissue Qiagen® (73.3% [44/60]). The phenol-chloroform method achieved a significantly higher frequency of cox1 gene amplification. The pigment present in the phlebotomine's eyes seems to inhibit cPCR reactions since the frequency of amplification of the cox1 gene increased in the sandflies without eyes (p < 0.0001). The HotShot method showed the highest inhibitory potential. These manual extraction techniques can be an inexpensive and effective alternative to study vector-pathogen interactions.

A cross-sectional study of Leishmania spp. in draft horses from the Distrito Federal, Brazil: Seroprevalence, spatial distribution, and associated factors.

Equine leishmaniasis is a neglected tropical disease caused by the protozoan of the Leishmania genus, and it has been reported in several countries around the world, especially Brazil. Therefore, the present investigation aims to conduct a cross-sectional study to determine the prevalence, spatial distribution, and associated factors with seropositivity for Leishmania spp. in draft horses from the Distrito Federal, Brazil. The serological survey was conducted on 411 animals, employing the Indirect Immunofluorescence Test (IFA) and Enzyme-Linked Immunosorbent Assay (ELISA). The Kappa (κ) and gross agreement indexes evaluated the Leishmania spp. seropositivity by IFA and ELISA test. The statistical analysis was performed using the Chi-square test and logistic regression. The spatial analysis showed the areas with the highest number of seropositive and the Moran autocorrelation analyses between the spatial distribution and the epidemiological model's explanatory variables. A 27.01 % co-positivity was observed with a κ index of 52.64 %. The final model considered the variables: access to water bodies (p-value = 0.008, Odds Ratio (OR) = 2.26, Confidence Interval (CI) = 1.24-4.13), the absence of the use of ectoparasiticide (p-value = 0.008, OR = 1.93 CI = 1.18-3.15) and traveling animal (p-value = 0.059, OR = 1.54, CI = 0.98-2.41). The Kernel map showed hot areas with a high concentration of nine positive animals per area and some lighter areas ranging from five to seven positive animals per area where control measures should be performed. The Moran autocorrelation analysis was significant for the variables: traveling animal (Moran's I = 0.540 and pseudo-p-value = 0.001) and the absence of use ectoparasiticide (Moran's I = 0.259 and pseudo-p-value = 0.005). The current study exposes a high seroprevalence of Leishmania spp. in horses in the Distrito Federal, Brazil. Moreover, it proposes that traveling animal, the access to water bodies and the absence of the use of ectoparasiticide are significantly associated with seropositivity for Leishmania spp. in draft horses, which may contribute to the implementation of prophylactic and controls measures where leishmaniasis is already stalled.

Spatial distribution and molecular epidemiology of Babesia vogeli in household dogs from municipalities with different altitude gradients in the state of Rio de Janeiro, Brazil.

We performed a cross-sectional epidemiological study with 456 household dogs from urban and rural areas in two different regions situated at different altitudes in the state of Rio de Janeiro. The PCR technique using 18S rRNA as target revealed prevalence of 7.9% of dogs positive for piroplasmids. These samples were sequenced, and all the sequences were 99.9% to 100% similar to Babesia vogeli sequences from other countries. The spatial distribution of positive cases was analysed using kernel interpolation in the QGIS software, and the spatial correlation indicators among positive dogs, altitude, and presence of ticks were obtained by calculating the local Moran index using the GeoDa software. The spatial correlation between positive cases and altitude was clear based on both visual and statistical observations. Logistic regression applying the Wald method with a cutoff point of 0.1 revealed that dogs from a region with altitude <600 m had a 2.29-fold chance of B. vogeli infection (OR = 2.29; p-value = 0.04; CI: 1.03-5.07), while the rainy season was 2.45 times more associated with B. vogeli infection (OR = 2.45; p-value = 0.01; CI: 1.20-5.01), and dogs infested with Rhipicephalus sanguineus sensu lato had a 2.47 times higher chance of being infected (OR = 2.47; p-value = 0.02; CI: 1.13-5.38). Entropy analysis of the alignment between B. vogeli 18S rRNA (> 1.600 bp) sequences revealed that the most variable region corresponds to the hypervariable V4 region. Genetic homogeneity was observed among the B. vogeli 18S rRNA sequences, with distance values ranging from 0 to 0.007 and a mean value of 0.001. The evolutionary distance (0.003) was greater between the sequences from the municipalities of Barra do Pirai (low altitude) and Teresopolis (high altitude). This study expands the molecular epidemiologic knowledge of B. vogeli and shows points of variability in the B. vogeli 18S rRNA. The results indicate the potential use of spatial analysis tools to improve screening for positive cases, enabling more in-depth studies to strengthen understanding of tick infection prevention in dogs.

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

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

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