Search Results (287)

Feed supplementation strategies are essential for optimizing cattle productivity, and the incorporation of non-conventional feed resources may reduce both production costs and environmental impact. This study evaluated the effects of pelletized protein concentrates (including Acacia farnesiana, A. schaffneri, and Agave duranguensis bagasse) on rumen fermentation parameters, microbial communities, and gas emissions. Fistulated bullocks received the concentrate daily, and ruminal contents were collected and filtered before and after supplementation to assess in vitro gas and methane production, pH, and microbial composition using high-throughput sequencing of 16S rRNA and mcrA amplicons. In addition, in situ degradability was evaluated during and after the supplementation period. Supplementation led to a significant (p < 0.05) reduction in degradability parameters and methane production, along with a marked decrease in the abundance of Methanobrevibacter and an increase in succinate-producing taxa. These effects were attributed to the enhanced levels of non-fiber carbohydrates, hemicellulose, crude protein, and the presence of bioactive secondary metabolites and methanol. Rumen microbiota composition was consistent with previously described core communities, and mcrA-based sequencing proved to be a valuable tool for targeted methanogen detection. Overall, the inclusion of non-conventional ingredients in protein concentrates may improve ruminal fermentation efficiency and contribute to methane mitigation in ruminants, although further in vivo trials on a larger scale are recommended. Full article

Rift Valley fever (RVF) is a zoonotic disease caused by the Rift Valley fever virus (RVFV), affecting humans, livestock, and wild ruminants. This study aimed to characterize and assess the genetic diversity of RVFV strains circulating among livestock in Uganda. Blood samples were collected between January 2021 and May 2024 from apparently healthy cattle, goats, and sheep in four districts. The samples were first screened for RVFV antibodies using ELISA; antibody-positive samples were subsequently tested for viral RNA using reverse transcriptase quantitative PCR (RT-qPCR). The PCR-positive samples underwent targeted amplicon sequencing, and phylogenetic analyses of the small (S) and large (L) genome segments were conducted to determine viral lineages. Of the 833 ELISA-positive samples, 10 (all from cattle) tested positive for RVFV RNA using RT-qPCR. Consensus sequences were successfully generated for six S segments and one L genome segment. A phylogenetic analysis revealed that all sequences belonged to lineage C, showing close genetic similarity to RVFV strains previously identified in Uganda, Kenya, Sudan, Madagascar, and Saudi Arabia. Limited genetic diversity was observed at both the nucleotide and amino acid levels. The detection of RVFV in apparently healthy cattle suggests ongoing, low-level viral circulation in Uganda. These findings offer important insights for guiding RVF surveillance, control, and policymaking in the country. Full article

The long-term monocropping of red kidney beans in agricultural fields can lead to the occurrence of soil-borne diseases. Alterations in the composition of the soil microbial community are a primary cause of soil-borne diseases and a key factor in continuous cropping obstacles. Research exploring how different cultivation modes can modify the diversity and composition of the rhizosphere microbial community in red kidney beans, and thus mitigate the effects of continuous cropping obstacles, is ongoing. This study employed three cultivation modes: the continuous monocropping of red kidney beans, continuous monocropping of soybeans, and red kidney bean–soybean intercropping. To elucidate the composition and diversity of rhizosphere microbial communities, we conducted amplicon sequencing targeting the V3-V4 hypervariable regions of the bacterial 16S rRNA gene and the ITS1 region of fungal ribosomal DNA across distinct growth stages. The obtained sequencing data provide a robust basis for estimating soil microbial diversity. We observed that, under the intercropping mode, the composition of both bacteria and fungi more closely resembled that of soybean monocropping. The monocropping of red kidney beans increased the richness of rhizosphere bacteria and fungi and promoted the accumulation of pathogenic microorganisms. In contrast, intercropping cultivation and soybean monocropping favored the accumulation of beneficial bacteria such as Bacillus and Streptomyce, reduced pathogenic fungi including Alternaria and Mortierell, and exhibited less microbial variation across different growth stages. Compared to the monocropping of red kidney beans, these systems demonstrated more stable microbial structure and composition. The findings of this study will inform sustainable agricultural practices and soil management strategies. Full article

Treatment of Mycobacterium tuberculosis requires multi-drug regimens, and resistance to any individual antibiotic can compromise outcomes. For slow-growing organisms like M. tuberculosis, rapid detection of resistance-conferring mutations enables timely initiation of effective therapy. Conversely, confirming wild-type status in resistance-associated genes supports confidence in standard regimens. We developed an amplicon-based next generation sequencing (amplicon tNGS) assay on the Illumina platform targeting eight genes linked to resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and fluoroquinolones. Sequencing results were analyzed using a custom bioinformatics pipeline. Forty-seven samples were used for assay development, and 37 additional samples underwent post-implementation clinical validation. Compared to whole genome sequencing (WGS), amplicon tNGS demonstrated 97.7% sensitivity, 98.9% specificity, and 98.7% overall accuracy for variant detection in targeted regions. Resistance prediction showed 79.3% concordance with WGS; discrepancies were primarily due to mutations outside of target regions. Among post-implementation samples, 27/37 passed quality metrics for all targets, with 95.7% concordance between amplicon tNGS results and final susceptibility results. This assay is now in use in our laboratory and offers significantly faster turnaround than both WGS and phenotypic methods on cultured isolates, enabling more rapid, informed treatment decisions for tuberculosis patients. Full article

Avian metapneumovirus subtype B (aMPV/B) is an economically significant pathogen in poultry, causing respiratory and reproductive disorders. In this study, 167 clinical samples were collected from commercial poultry farms across Thailand to investigate the prevalence, genetic diversity, and evolutionary dynamics of aMPV/B. Nested RT-PCR targeting the G gene revealed a positivity rate of 34.13% (57/167). Phylogenetic and Median-joining network analyses of sequenced amplicons identified two distinct Thai lineages: one genetically similar to vaccine strains and another of unknown origin. Divergence time analysis using a Bayesian framework estimated the time to the most recent common ancestor (tMRCA) of these lineages around 2006, with further sub-lineage diversification occurring around 2009 and 2016. These findings suggest that the circulating Thai aMPV/B strains likely stem from limited introduction events followed by local evolution. Lineage-specific amino acid substitutions within the G gene were identified, which may affect antigenic properties and immune recognition. This study highlights the molecular heterogeneity and ongoing diversification of aMPV/B in Thailand and underscores the need for sustained genomic surveillance and regionally tailored vaccination strategies. Full article

Evolving technologies available to clinical laboratories and laboratory-related updates to clinical guidelines both drive the need for clinical laboratories to keep their test menu updated and in line with current technological and clinical developments. Our laboratory has developed a targeted Illumina-based amplicon next-generation sequencing (NGS) assay to interrogate the hsp65 and erm(41) genes of Mycobacterium spp. for the purposes of providing species-level ± subspecies-level identification of Mycobacterium spp. organisms in clinical samples and genotypic predictions for inducible macrolide resistance (in the case of M. abscessus complex members). The developed assay demonstrated 100% sensitivity and specificity for M. tuberculosis and M. abscessus complex cultured organisms, 98% ID overall concordance relative to the available reference identification, and a nearly 60% “rescue” rate for primary samples that could not be identified using our previous method. There was 94.6% concordance between genotypic and phenotypic results for inducible macrolide resistance. The developed assay was successfully implemented in our clinical laboratory and has been accredited for clinical use. Full article

The unique microbial communities present on fruit surfaces significantly influence the fermentation process and product quality of artisanal cider production, constituting a microbial terroir analogous to that recognized in viticulture. In this study, we investigated the microbial composition and diversity associated with the apple varietals (Empire, Golden Delicious, and Idared) cultivated by two different orchard producers in the Hudson River Valley of New York. Using 16S rRNA and ITS amplicon sequencing, we identified distinct bacterial and fungal communities that varied significantly according to the apple varietal and orchard location. Notably, the orchard was the dominant factor shaping both the bacterial and fungal communities on the apples’ surfaces, with the varietal differences also playing a significant, albeit secondary, role. For example, we found that the bacterial genera Acidophilim sp. and 1174-901-12 sp., as well as the fungus Sporobolmyces patagonicus, were important markers of the orchard in which the apples were cultivated. These microbial signatures persisted into the early stages of cider fermentation, suggesting their potential influence on the cider quality and flavor profile. Our findings underscore the critical importance of the microbial terroir in cider production, and suggest that targeted management practices can leverage regional microbial diversity to enhance the distinctiveness and marketability of artisanal cider products. Full article

Potato early dying disease complex (PED) leads to premature senescence and rapid decline in potato plants. Unlike potato wilt caused solely by Verticillium species, PED symptoms are more severe due to the synergistic effects of multiple pathogens, including root-lesion nematodes, fungi such as Colletotrichum and Fusarium, and soft-rot bacteria. To investigate the microbiome responsible for PED, soil and stem samples from healthy-looking and symptomatic plants were analyzed using amplicon-targeted next-generation sequencing (Illumina MiSeq and PacBio technologies). Samples were collected from four locations in New Brunswick, Canada from fields previously rotated with barley or oat. Comparative analysis of the bacterial, fungal, and eukaryotic diversity in soil samples showed minimal differences, with only bacterial alpha diversity influenced by the plant health status. Verticillium dahliae was abundant in all soil samples, and its abundance was significantly higher in the stems of diseased plants. Additional fungal species implicated in PED, including Plectosphaerella cucumerina, Colletotrichum coccodes, Botrytis sp., and Alternaria alternata, were also identified in the stems. This study highlights the complex, plant-associated microbial interactions underlying PED and provides a foundation for microbiome-informed disease management strategies. Full article

Androgenetic alopecia (AGA) is the most common form of patterned hair loss, exhibiting gender-specific clinical features. Recent studies highlight the importance of the skin microbiome in maintaining skin health, but the relationship between the hair follicle microbiome and hair loss, particularly AGA, remains understudied. Hair follicle layer samples were collected directly from the crown region of female pattern hair loss (FPHL), male pattern hair loss (MPHL), and healthy adult women (control) groups. Microbial DNA was extracted and analyzed using Illumina 16S rRNA V3–V4 gene amplicon sequencing. Alpha-diversity and beta-diversity analyses and taxonomic and functional profiling were conducted through relative abundance, LEfSe, and PICRUSt2 analyses. The alpha-diversity analysis showed a significant decrease in microbial richness in the hair loss groups. Unweighted UniFrac-based beta-diversity analysis revealed significant clustering between the control group and the FPHL group. Taxonomic profiling and LEfSe analysis identified differences in microbial composition and biomarkers. PICRUSt2 analysis further revealed altered pathways related to porphyrin metabolism, fatty acid biosynthesis, and steroid hormone metabolism. Additionally, differences in microbiome composition and potential functions were found between the FPHL and MPHL groups. This study provides comprehensive insights into the hair follicle microbiome, revealing unique microbial patterns and functional alterations associated with FPHL. Understanding these microbiome characteristics may contribute to targeted approaches for addressing AGA. Further research is warranted. Full article

In Mexico, hospital wastewater (HWW) is a source of chemical and microbiological contamination, and it is released into the municipal sewage system without prior treatment. This water may contain pathogenic bacteria and antimicrobial resistance genes, which represent a risk to Public Health and the environment. So far, there are no studies that analyse this problem comprehensively, relating bacterial population structures, chemical contaminants, and seasonality. The aim of this work was to seasonally characterise the bacterial communities of HWW, including clinically relevant bacteria and resistance genes in Hospital Juárez de México (HJM), and to evaluate the impact of physicochemical factors on their composition. A one-year observational, cross-sectional study was conducted at five HWW discharge points of HJM. Fourteen physicochemical parameters were determined by using standard methodologies, and statistical differences between discharges and seasons were evaluated. Bacterial communities were analysed by targeted amplicon sequencing of the V3-V4 region of the 16S rRNA gene. In addition, the presence of eight antimicrobial resistance genes of local epidemiological importance was assessed. Data were analysed using alpha and beta diversity indices, principal component analysis, and multivariate statistical tests. HWW showed high taxonomic diversity, with Proteobacteria, Firmicutes, and Bacteroidetes standing out. Clinically relevant bacteria were identified in 73.3% of the analyses, with Enterobacter and Escherichia-Shigella predominating. Total and dissolved solids, temperature, nitrate, and pH significantly influenced the bacterial composition of HWW. Seven out of the eight genes evaluated were identified, with bla_KPC, bla_OXA-40, and mcr-1 being the most frequent, showing significant seasonal differences. This study underlines the microbiological and chemical complexity of HWW, highlighting the impact of clinically relevant bacteria and antimicrobial resistance genes on Public Health. The findings emphasise the need to implement hospital waste management programmes and ideally specific treatment plants to minimise the associated risks and protect the environment and human health. Full article

Biosecurity is vital to Australia’s efforts to prevent and respond to pests and diseases. Here, we report on testing suspected illegal goods (SIGs) as part of an active Australian biosecurity response in Sydney. The Australian Government, Department of Agriculture, Fisheries and Forestry detected and secured consignments containing tuber products of unknown biosecurity risk and origin. Swab samples were collected from vacuum-sealed yam products, organic packing material (background negative controls), and field negative controls to assess possible cross-contamination from the storage facility. DNA from all samples was analysed using high-throughput metabarcoding targeting the Internal Transcribed Spacer 2 (ITS2) and the chloroplast trnL (UAA) P6 Loop gene regions by two independent teams in Australia. A plant community profile comprising Australian native species and other non-native established species would support the notion of produce being harvested and/or packaged domestically, while their absence would suggest foreign production. Of the 5,764,942 total reads produced, the bioinformatic analysis generated 5,181,530 amplicon sequencing variants employed for species identification. Twenty plant taxa were identified via ITS2 and 15 via trnL, corresponding to worldwide distributed plants, non-native species established in Australia, or species not recorded in Australia. No Australian endemic species were detected. The absence of common Australian native plants, combined with the presence of species not known to occur in Australia, provided strong evidence that the suspect tuber products were illegally imported. Full article

Malaria remains a significant public health challenge, particularly in endemic regions. The extensive genetic diversity of Plasmodium falciparum (Pf) complicates outbreak prediction and transmission control. One of its most polymorphic markers, merozoite surface protein 2 (MSP2), presents a potential target for molecular surveillance. This cross-sectional study, conducted at King Faisal Hospital Rwanda (KFHR) from October 2021 to June 2023, assessed MSP2’s utility in malaria prediction. PfMSP2 was sequenced, and selected amplicons were cloned, expressed in bacteria, and purified. These antigens were tested against sera from malaria patients and geographically diverse healthy individuals, with complementary surveys contextualizing serological findings. Of the 75 processed monoallelic clinical isolates, 3D7 strains predominated over FC27. Three MSP2-derived biomarkers were produced, eliciting significantly low IgG responses in malaria patients and Belgian controls, but a complex pattern emerged in healthy individuals, with significant differences between Rwandan and Cameroonian samples. IgG3 was the predominant subclass in individuals with high IgG responses. Notably, Rwandan individuals with weak humoral responses to the tested antigens but also other with high responses experienced malaria episodes in the subsequent year. These findings highlight MSP2 polymorphism as a valuable tool for malaria surveillance and outbreak prediction. Integrating genotyping and serology could enable precise, community-specific malaria risk assessments, strengthening control strategies. Full article

In this study, 172 Single-Nucleotide Polymorphisms (SNPs) (94 identity-informative SNPs, 56 ancestry-informative SNPs, and 22 phenotypic-informative SNPs) included in the ForenSeq™ DNA Signature Prep kit/DNA Primer Mix B (Verogen) were used for genotyping DNA samples from a population of twenty-one unrelated subjects, native to Northeast Italy. SNP sequencing was performed with the MiSeq FGx™ Forensic Genomics System (Illumina-Verogen), and data were analyzed using the Universal Analysis Software (UAS) v1.2. Raw data underwent further examination with STRait Razor v3 (SRv3) to compare the target SNPs’ genotype calls made with UAS and to identify the presence of microhaplotypes (MHs) due to SNPs associated with the same target SNP’s amplicon. The allele (haplotype) frequencies, Hardy–Weinberg equilibrium, linkage disequilibrium, number of effective alleles (A_e), and relevant forensic statistic parameters were calculated. Among the 172 SNPs evaluated, 45 unique microhaplotypes were found, comprising a novel sequence variant never previously described. The presence of MHs resulted in an 8.00% rise in the typologies of unique sequences, leading to changes in A_e. Notably, for 12 out of the 94 iiSNPs, the values of A_e exceeded 2.00, which is generally associated with a higher expected heterozygosity and increased power of discrimination. Full article

Babesia odocoilei is an emerging zoonotic protozoan parasite primarily associated with cervids, with growing recognition among non-cervid hosts and in terms of potential public health implications. While this species has been documented in North America and parts of Europe, data on its presence in Romania remain scarce. This study aimed to investigate the presence of Babesia spp. in ticks collected from Romania, providing new information on the existing species and their distribution, as well as their potential epidemiologic significance. A total of 41 Ixodidae ticks were collected from 184 wild boars across six counties from Western and Central Romania. Ticks were identified using morphological assessments, and DNA was extracted from the samples using a standardized protocol. The presence of Babesia spp. was assessed using real-time PCR with primers and a Taq Man probe targeting 116 bp fragments of 18S rRNA genes. Molecular analysis was used to detect Babesia spp. DNA from a single tick sample (1/41, 2.43%), identified as Dermacentor marginatus, from Timiș County. The resulting amplicons were sequenced and compared with reference sequences in GenBank for species confirmation. This finding represents the first molecular identification of B. odocoilei in questing ticks from Romania. The expanding host range and geographic distribution of B. odocoilei raise concerns regarding its zoonotic potential. The presence of this pathogen in Dermacentor marginatus ticks suggests a broader vector competence than previously recognized, and future research should focus on host reservoirs, vector competence, and potential zoonotic transmission, with an emphasis on public health implications, including potential implications for veterinary diagnostics, vector control policies, and public health awareness regarding emerging tick-borne pathogens. Full article

The increasing incidence of tick-borne diseases in Europe necessitates the development of accurate and high-throughput molecular tools for detecting pathogens in tick populations. In this study, we present a novel flaB gene-based profiling method for the detection and identification of Borrelia and Borreliella species in Ixodes ricinus ticks, combining newly designed primers with next-generation sequencing (NGS). The method was evaluated alongside conventional nested PCR targeting the flaB gene, as well as microbial profiling based on the V4 region of the rrs gene, using tick DNA extracted from 1088 specimens pooled into 94 samples. Our results demonstrate that the flaB gene-based profiling approach was the highest-performing out of the three methods, detecting Borreliaceae DNA in 83 DNA pools, compared to 58 and 56 pools using nested PCR and V4 rrs profiling, respectively. A total of 23 distinct flaB sequence variants were identified, corresponding to five Borreliaceae species: Borreliella afzelii, Bl. garinii, Bl. valaisiana, Bl. burgdorferi, and Borrelia miyamotoi. Additionally, the method enabled putative strain-level discrimination within species. Our results highlight the value of flaB gene-based profiling as a robust tool for ecological and epidemiological studies of Borreliaceae diversity in ticks. Full article