Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens' ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In the present study, the expression of genes involved in immune signaling pathways of R. microplus adults' guts when challenged with a high or low parasitic load of T. equi was evaluated. This research demonstrates divergences in the immune gene expression pattern linked to T. equi infection in R. microplus since the Toll, IMD, and JNK signaling pathways were transcriptionally repressed in the guts of adult ticks infected with T. equi. Moreover, the results showed that different infectious doses of T. equi induce differential gene expression of key components of immune signaling cascades in R. microplus gut, suggesting a link between the intensity of infection and the activation of tick immunity response. The present study adds knowledge to elucidate the gut immune signaling response of R. microplus to T. equi infection. In addition, the generated data can serve as a basis for further investigations to develop strategies for controlling and preventing equine piroplasmosis.

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.

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.