The Study of Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) Circulation and Vectors at the Municipal Parks and Zoobotanical Foundation of Belo Horizonte, Minas Gerais, Brazil (FPMZB-BH).

Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) are Orbiviruses primarily transmitted by their biological vector, Culicoides spp. Latreille, 1809 (Diptera: Ceratopogonidae). These viruses can infect a diverse range of vertebrate hosts, leading to disease outbreaks in domestic and wild ruminants worldwide. This study, conducted at the Belo Horizonte Municipal Parks and Zoobotany Foundation (FPMZB-BH), Minas Gerais, Brazil, focused on Orbivirus and its vectors. Collections of Culicoides spp. were carried out at the FPMZB-BH from 9 December 2021 to 18 November 2022. A higher prevalence of these insects was observed during the summer months, especially in February. Factors such as elevated temperatures, high humidity, fecal accumulation, and proximity to large animals, like camels and elephants, were associated with increased Culicoides capture. Among the identified Culicoides spp. species, Culicoides insignis Lutz, 1913, constituted 75%, and Culicoides pusillus Lutz, 1913, 6% of the collected midges, both described as competent vectors for Orbivirus transmission. Additionally, a previously unreported species in Minas Gerais, Culicoides debilipalpis Lutz, 1913, was identified, also suspected of being a transmitter of these Orbiviruses. The feeding preferences of some Culicoides species were analyzed, revealing that C. insignis feeds on deer, Red deer (Cervus elaphus) and European fallow deer (Dama dama). Different Culicoides spp. were also identified feeding on humans, raising concerns about the potential transmission of arboviruses at the site. In parallel, 72 serum samples from 14 susceptible species, including various Cervids, collected between 2012 and 2022 from the FPMZB-BH serum bank, underwent Agar Gel Immunodiffusion (AGID) testing for BTV and EHDV. The results showed 75% seropositivity for BTV and 19% for EHDV. Post-testing analysis revealed variations in antibody presence against BTV in a tapir and a fallow deer and against EHDV in a gemsbok across different years. These studies confirm the presence of BTV and EHDV vectors, along with potential virus circulation in the zoo. Consequently, implementing control measures is essential to prevent susceptible species from becoming infected and developing clinical diseases.

Root Canal Microbiome Associated With Asymptomatic Apical Periodontitis as Determined by High-Throughput Sequencing.

INTRODUCTION: Advanced DNA sequencing technology allows more detailed analysis and description of the endodontic microbiome. This study used the MiSeq high-throughput sequencing platform (Illumina, San Diego, CA) to describe the endodontic microbiome of teeth with primary asymptomatic apical periodontitis with no sinus tract. METHODS: Root canal samples from 25 patients were prepared for DNA sequencing analysis. Bacterial diversity of the microbiome was identified and compared between cases and according to the size of the related apical periodontitis lesions. Statistical analyses of the operational taxonomic unit distribution was performed using principal component analysis with the Bray-Curtis distance and a principal coordinate analysis, 2-way permutational multivariate analysis of variance. The chi-square or Fisher exact test was used to evaluate the prevalence of different operational taxonomic units related to small and large apical periodontitis lesions. RESULTS: Although there was a very high bacterial diversity in the microbiome of teeth with asymptomatic apical periodontitis, 4 phyla dominated the microbiome: Firmicutes (27%), Bacteroidetes (21%), Proteobacteria (21%), and Actinobacteria (12%). There was high variability in species composition between root canal samples with no common species pattern for the cases. Large lesions showed a higher number of species but did not significantly differ from small lesions in bacterial diversity indexes. Bacteroidaceae [G-1] bacterium HMT 272, a previously uncultivated but still unnamed and uncharacterized taxon, was the most prevalent and abundant phylotype. CONCLUSIONS: High-throughput sequencing technology confirmed the complexity of the endodontic microbiome and revealed that microbial heterogeneity is a feature between cases. This indicates that various microbial combinations of the endodontic microbiome are able to illicit periapical inflammatory diseases.

Swine influenza A virus subtypes circulating in Brazilian commercial pig herds from 2012 to 2019.

The swine influenza A virus (SIAV) subtypes/lineages H1N1pdm09, H3N2, H1N2, and H1N1 of seasonal human origin are widespread in Brazilian swine herds. A monovalent inactivated H1N1pdm09 vaccine was licensed in Brazil in 2014. However, there are concerns about its efficacy due to the limited vaccine cross-protection against heterologous viruses and the potential for exacerbated reactions against vaccine strains. Thus, monitoring SIAVs subtypes/lineages that are circulating in the Brazilian swine population is important, by applying a fast and efficient diagnostic test in herd field samples. A RT-PCR assay was developed, using primers specific for HA subtyping of Brazilian SIAV, and was used to evaluate the occurrence of subtypes from samples collected between 2012 and 2019. From 167 field samples positive for influenza A, 117 were subtyped by nested RT-PCR assay. A higher occurrence of H1N1pdm was observed from 2012 to 2015, H3N2 in 2017, and H1hu in 2017 to 2019. A hemagglutination inhibition test was performed in serum samples received from 2017 to 2019, confirming these data. The molecular data highlights the importance of H1hu and H3N2 detection since there are no vaccines available for the subtypes/lineages and raises an alert of H1hu for its potential to infect humans. Serological data suggest a cyclical profile of occurrence between the H3N2 and H1N1pdm over time. Monitoring SIAVs circulating in Brazilian swine herds is necessary, which provides the relevant information for field veterinarians to apply effective control measures on the properties.

Study of molecular diagnosis and viremia of bluetongue virus in sheep and cattle.

Bluetongue virus (BTV) is an RNA virus that infects cattle and sheep. The objective of this study was to compare two real-time PCRs for the detection of BTV and to monitor Orbivirus viremia in sheep and cattle for 6 months. The PCR results showed the occurrence of infected animals throughout the experiment without records of clinical signs. The number of positive animals reduced during the experiment, but some animals were positive for BTV RNA during the entire experiment. The performance of the two RT-qPCRs for BTV detection techniques used in this work revealed a kappa index of 0.71 for cattle and 0.75 for sheep.

Development and validation of rt-qpcr for vesicular stomatitis virus detection (Alagoas vesiculovirus).

Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement. The threshold cycle values obtained in triplicate from each sample were evaluated by considering the variations between days, analysts and equipment in an analysis of variance aimed at determining the variances of repeatability and reproducibility. The results showed that RT-qPCRs had excellent sensitivity and specificity in the detection of RNA of the Alagoas vesiculovirus. The validation parameters showed low coefficients of variation and were equivalent to those found in other validation studies, indicating that the tests presented excellent repeatability and reproducibility.