Dissecting the impact of Anaplasma phagocytophilum infection on functional networks and community stability of the tick microbiome.

CONTEXT: Pathogens can manipulate microbial interactions to ensure survival, potentially altering the functional patterns and microbiome assembly. The present study investigates how Anaplasma phagocytophilum infection affects the functional diversity, composition, and assembly of the Ixodes scapularis microbiome, with a focus on high central pathways-those characterized by elevated values in centrality metrics such as eigenvector, betweenness, and degree measures, in the microbial community. METHODS: Using previously published data from nymphs' gut V4 region's amplicons of bacterial 16S rRNA, we predicted the functional diversity and composition in control and A. phagocytophilum-infected ticks and inferred co-occurrence networks of taxa and ubiquitous pathways in each condition to associate the high central pathways to the microbial community assembly. RESULTS: Although no differences were observed concerning pathways richness and diversity, there was a significant impact on taxa and functional assembly when ubiquitous pathways in each condition were filtered. Moreover, a notable shift was observed in the microbiome's high central functions. Specifically, pathways related to the degradation of nucleosides and nucleotides emerged as the most central functions in response to A. phagocytophilum infection. This finding suggests a reconfiguration of functional relationships within the microbial community, potentially influenced by the pathogen's limited metabolic capacity. This limitation implies that the tick microbiome may provide additional metabolic resources to support the pathogen's functional needs. CONCLUSIONS: Understanding the metabolic interactions within the tick microbiome can enhance our knowledge of pathogen colonization mechanisms and uncover new disease control and prevention strategies. For example, certain pathways that were more abundant or highly central during infection may represent potential targets for microbiota-based vaccines.

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.

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.

Molecular investigation of Babesia caballi in horses from the state of Rio de Janeiro, Brazil: Epidemiological aspects associated with the infection.

The epidemiological aspects of Babesia caballi infection were evaluated in 516 horse samples from Rio de Janeiro, Brazil. The presence and infestation level of ticks on horses, breed conditions, and animal management were evaluated on each farm through an epidemiological questionnaire. The gene that codes for rhoptry-associated protein-1 (RAP-1) of B. caballi was amplified by nested PCR (nPCR). Among the horses sampled, 17.2% (n = 89/516) presented B. caballi DNA. The characterized samples showed 99-100% similarity with other isolates of B. caballi based on the RAP-1 gene, available in GenBank. In the final logistic regression model, the variables associated with B. caballi infection in horses were as follows: age below two years (OR = 3.33; IC = 1.7-6.5), farms located in low altitudes (OR = 3.52; IC = 1.7-7.3) and Dermacentor nitens infestation (OR = 1.91; IC = 1.1-3.4). Furthermore, a high level of D. nitens infestation in horses was also a factor associated with positivity for B. caballi (OR = 2.11; IC = 1.25-3.54). In summary, young horses bred in low altitude regions characterized with high temperatures, and infested by D. nitens, mainly with a higher level of infestation, are more likely to be infected by B. caballi. This epidemiological study provides statical evidence that the D. nitens tick play a role as the biological vector of B. caballi in the studied region.

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.

Cross-sectional study of Brucella spp. using real-time PCR from bovine whole blood in Colombia.

A cross-sectional study was conducted in Colombia to recover Brucella spp. DNA from bovine whole-blood samples through probe-based real-time PCR (qPCR). By an SNP-based assay, vaccine strains were differentiated from field strains. The associated factors were evaluated using logistical regression models. A total of 656 random cows from 40 herds were selected and analyzed using serology and PCR. The qPCR assay detected 9.5% (n = 62/656; 95% CI: 7.3, 12.0) of the animals with Brucella-DNA presence, while the serological test detected a 6.6% (n = 43/656; CI: 4.8, 8.7). 62.5% (n = 25/40; 95% CI: 45.8, 77.3) of positive cases were detected at the herd-level by the qPCR, while only 27.5% (n = 11/40; 95% CI: 14.6, 43.9) were detected by the serological test. All positive samples were identified as field Brucella strains employing the SNP-based assay. In the final regression model at the animal-level, five variables were associated with Brucella-DNA presence: the use of bulls for mating recorded history of reproductive problems, pregnant cows, parlor milking, and cows belonging to farms ≤200 m from the main road. At the herd-level, two variables were associated with Brucella-DNA presence: recorded history of reproductive problems and the use of bulls for mating. Given the fluctuant brucellosis prevalence in endemic areas, updated epidemiological studies are necessary to evaluate the disease dynamic and if established prevention and control measures have been effective or need to be adjusted. The increase in the prevalence of brucellosis in animal reservoirs creates an important risk of transmission in humans.

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.

Author Correction: Detection of Neorickettsia risticii, the agent of Potomac horse fever, in horses from Rio de Janeiro, Brazil.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Detection of Neorickettsia risticii, the agent of Potomac horse fever, in horses from Rio de Janeiro, Brazil.

This study aims to report the presence of Neorickettsia risticii DNA in blood samples from naturally infected horses in Rio de Janeiro, provide clinicopathological findings related to the infection, and report the phylogenetic diversity of the 16S rDNA of N. risticii in order to evaluate its heterogeneity. Real-time quantitative polymerase chain reaction (qPCR) was performed to investigate the presence of N. risticii in samples collected from horses (n = 187). Five positive samples were found in the molecular screening. Hypoalbuminemia and high levels of creatine kinase and lactate dehydrogenase were the predominant findings in the biochemical analysis. The sequences were similar to those of N. risticii. Phylogenetic analysis revealed genotype segregation based on the geographical distribution in the N. risticii sequence clade. Dendrograms constructed with five hypervariable regions revealed that V4 distinguished Neorickettsia at the species level and produced a phylogeny that best represented the phylogeny obtained with the complete 16S rDNA sequence. This is the first report of N. risticii DNA in the blood of Brazilian horses based on sequences deposited in GenBank. Further studies are necessary to clarify the epidemiological chain of this vector-borne parasite in order to determine and establish appropriate preventive measures in the equine trading market.

Molecular characterization of Ehrlichia canis from naturally infected dogs from the state of Rio de Janeiro.

The aim of the present study was to evaluate the genetic diversity of Ehrlichia canis in naturally infected dogs from six mesoregions of Rio de Janeiro state. E. canis was diagnosed with a real-time polymerase chain reaction (qPCR) targeting a 93 base pair (bp) fragment of the 16S rDNA gene. To evaluate the genetic diversity of the parasite, we amplified a positive sample from each mesoregion by distinct conventional PCR assays with targets in the gp19 (414 bp), gp36 (814 bp), and p28 (843 bp) genes. A total of 267 samples were collected from dogs in Rio de Janeiro state. Among the samples analyzed, 42.3% (n = 113/267) were 16S rDNA-qPCR positive. When performing PCR for the gp19 and gp36 genes, 100% (n = 113/113) and 5.3% (n = 6/113) of the samples amplified fragments of 414 bp and 814 bp, respectively. The six PCR-positive samples for the gp36 gene also amplified the p28 gene fragment. The characterization based on the gp19 gene demonstrated that it is highly conserved. In protein analysis (TRP36), all samples showed a tandem repeat protein (TRP) that comprised 11 replicates. Seven high-entropy amino acid sites were distributed throughout the gp36 gene. Eleven high-entropy amino acid sites were found throughout the p28 gene. There is a positive selection pressure in both genes (p ≤ 0.05). Comparing and characterizing an organism are useful for providing important information about the host's immune response and identifying new antigenic targets, as well as essential characteristics for the development of vaccines and new diagnostic tools.

Molecular evidence of Anaplasma phagocytophilum and Theileria equi coinfection in horses from Rio de Janeiro, Brazil.

The present study aims to determine the frequencies of Theileria equi and Anaplasma phagocytophilum antibodies among horses from the state of Rio de Janeiro, Brazil, and to detect the presence of DNA of these pathogens through molecular methods. A total of 98 serum samples of horses from the municipality of Seropedica were tested by indirect immunofluorescence antibody (IFA) to detect anti-A. phagocytophilum and anti-T. equi IgG antibodies. In addition, quantitative real-time PCR (qPCR) was used to detect these pathogens in the DNA extracted from the whole blood and buffy coat of horses. Bivariate analysis and odds ratio were performed to verify the possible association between positivity and characteristics related to the horses. As evaluated by IFA and qPCR, the frequency of animals that tested positive for T. equi was 89.8% (n = 88/98) and 91.8% (n = 90/98), whereas A. phagocytophilum was 17.4% (n = 17/98) and 1.0% (n = 1/98), respectively. Serological evidence of exposure to A. phagocytophilum and T. equi was observed in 16.3% (n = 16/98) of the horses; however, exposure was confirmed by qPCR in only 1.0% (n = 1/98). No statistical association was found in the bivariate and odds ratio analysis. This is the first study reporting the molecular detection of A. phagocytophilum DNA in horses from the state of Rio de Janeiro, and also the coinfection of A. phagocytophilum and T. equi in a horse from Brazil confirmed by molecular methods. Equine granulocytic anaplasmosis is circulating in Brazilian horses, together with T. equi, and should be included in the differential diagnosis of tick-borne diseases.

Epidemiology of Ehrlichia canis in healthy dogs from the Southeastern region of the state of Rio de Janeiro, Brazil.

This cross-sectional, observational, and descriptive study aims to investigate the epidemiology of Ehrlichia canis in healthy owned dogs from the Southeastern region of Rio de Janeiro, Brazil. Blood samples were collected from 390 households dogs. During the visits, an epidemiological questionnaire was filled out concerning the dogs' characteristics as well as the environments in which they lived. The variables were analyzed using a bivariate test, while the correlation analysis between the variables was performed via a phi test. The variables that had p-values lower than 0.2 in the bivariate analysis and had a low or moderate correlation were selected for the multivariate analysis. The model that had the lowest Akaike information criterion (AIC) value was retained. Among the 390 blood samples tested, 24.8% were considered positive for E. canis. The parsimonious logistic regression model presented an AIC value of 408.75 and showed three variables that favored the presence of E. canis DNA in the tested dogs: the animal's access to urban streets and neighborhoods (odds ratio [OR] = 1.91; p-value = 0.02; confidence interval [CI]: 1.14 - 3.18), tick infestation (OR = 2.01; p-value = 0.006; CI: 1.22 - 3.32), and poor hygienic conditions (OR = 2.19; p-value = 0.002; CI: 1.31 - 3.67). The model was considered well-calibrated based on the Hosmer-Lemeshow test (p = 0.39). According to the present study, dogs that have access to the street and neighborhood, are infested with ticks, and live under poor hygienic conditions are more likely to be infected with E. canis in the state of Rio de Janeiro, Brazil.