Search Results (2,230)

Canine monocytic ehrlichiosis, caused by Ehrlichia canis, is an important tick-borne disease of dogs in tropical and subtropical regions. This cross-sectional study investigated the occurrence, associated risk factors, and molecular confirmation of E. canis infection among clinically suspected dogs in Andhra Pradesh, India, and evaluated the diagnostic performance of blood smear microscopy relative to PCR. Peripheral blood samples were collected from 442 clinically suspected dogs presented to 90 veterinary dispensaries across the state. Dogs were selected based on clinical features suggestive of ehrlichiosis, including inappetence, pale mucous membranes, tick infestation, fever, lymphadenopathy, weakness, and hemorrhagic signs. Samples were examined by Giemsa-stained blood smear microscopy and subsequently tested by PCR. Representative virB9 gene amplicons were sequenced and compared with reference sequences available in GenBank. Microscopy detected E. canis in 37/442 dogs (8.37%), whereas PCR detected infection in 51/442 dogs (11.54%). Logistic regression analysis indicated that female dogs, mongrels, kennel dogs, and dogs with tick infestation had significantly higher odds of infection, while dogs with a history of tick-control measures had lower odds. Sequence comparison and phylogenetic placement based on the partial virB9 fragment supported molecular confirmation of the detected E. canis isolates and showed similarity with previously reported Indian and international isolates. These findings provide regional evidence on the occurrence and epidemiology of canine ehrlichiosis in clinically suspected dogs and support PCR as a more reliable confirmatory method than microscopy. Full article

Dairy production in Indonesia relies on two contrasting calf-rearing systems: concentrate-driven commercial dairy systems (CDS) and smallholder farms (SH). As these systems differ in feeding practices, they may influence gut development in calves. This study examined how farm management affects the fecal microbiota and metabolites in healthy and diarrhea post-weaning calves. Fecal samples were collected from 11 and 14 calves from CDS and SH facilities, respectively. Gut bacterial communities were analyzed using 16S rRNA gene amplicon sequencing, and metabolites were characterized using untargeted gas chromatography–mass spectrometry (GC-MS). Microbial diversity, metabolite profiles, and correlation networks were compared between the farm types and diarrhea status. Our results showed that farm management strongly shaped microbial community structure and metabolic outputs, but did not alter microbial richness. Although diarrhea is typically associated with reduced microbial richness, it did not affect the microbial community structure, suggesting that it primarily impacted microbial function, particularly the metabolic environment. Correlation network analysis revealed stronger linkages between microbes and metabolites in SH calves, especially under healthy conditions. Overall, these findings indicate that dietary structure is a key determinant of fermentation stability, with CDS calves showing greater metabolic instability. In contrast, SH calves maintain a more resilient, fiber-driven functional state. Full article

Pear fruits host diverse microbial communities that influence postharvest quality, spontaneous fermentation, and susceptibility to microbial contamination. This study characterizes the fungal communities associated with naturally fallen overripe pears (Pyrus communis) using ITS2 amplicon sequencing combined with culture-dependent approaches. The fungal community exhibited low diversity and was dominated by Ascomycota (99%), primarily Saccharomycetes (91.8%), with Hanseniaspora, Aureobasidium, and Microcyclospora representing more than 90% of the total microbial community. Culture-dependent isolation confirmed Hanseniaspora uvarum as the dominant yeast species (~89%), followed by Metschnikowia spp. and Pichia spp. Pairwise co-culture assays, quantified using the Relative Interaction Index, demonstrated predominantly competitive interactions, with fast-growing H. uvarum exerting suppressive effects on slower-growing species. Among the isolated yeasts, Metschnikowia fructicola exhibited antibacterial activity against all tested bacteria Staphylococcus aureus, Listeria innocua and Salmonella typhimurium. The strongest antibacterial activity was exerted against the foodborne pathogen S. aureus. In a pear juice model system, co-cultivation with M. fructicola resulted in the elimination of S. aureus within four days, while yeast viability was maintained. These findings observe the fermentative yeasts distributed in overripe pears and demonstrate the potential of M. fructicola to inhibit bacterial growth under controlled conditions. The results provide a preliminary basis for further studies on fungal succession, yeast interactions, and the biocontrol potential of pear-associated yeasts. For broader ecological conclusions, larger-scale studies across locations, seasons, cultivars, and decay stages are required. Full article

Background: The aim of this study was to compare microbial diversity and compositional changes under digestive stress in yogurts produced using different culture strategies in a dynamic in vitro gastrointestinal system. Methods: Yogurts produced with probiotic starter cultures, standard yogurt cultures, and probiotic sachet supplementation were evaluated using a dynamic in vitro gastrointestinal system including mouth, stomach, and small intestinal phases under controlled pH, temperature, and digestion conditions. Microbial diversity and compositional changes before and after the in vitro gastrointestinal simulation were determined using a 16S rRNA amplicon-based Oxford Nanopore sequencing approach, and the resulting data were analyzed using bioinformatics, alpha-diversity, and beta-diversity metrics. Results: Probiotic sachet addition significantly increased microbial species richness, alpha diversity, and community balance compared to starter culture yogurts (Shannon and Inverse Simpson, p < 0.05). In vitro gastrointestinal system led to a reduction to a Firmicutes-dominant core microbiota in all samples; in contrast, beta diversity and PCoA analyses showed that the post-digestive microbial structure of sachet-supplemented yogurts was significantly different (PERMANOVA, p = 0.001). Conclusions: Consequently, it was demonstrated that adding probiotic sachets to yogurt increases the diversity and resilience of beneficial bacteria throughout the digestive tract, making the product a more robust and functional food for health. Full article

Background/Objectives: Museum collections constitute valuable material for investigating a wide range of histological processes. This results from the historical selection of unusual and advanced disease cases by museum curators, which are rarely encountered in contemporary clinical practice due to advances in medicine. Ancient DNA plays a crucial role in phylogenetic studies, as well as in analyses of population genetics. However, many commonly used DNA extraction techniques involve partial degradation of samples prior to DNA isolation. The use of non-destructive methods may enable the recovery of DNA appropriate for downstream analyses. Non-destructive methods of DNA extraction for research purposes are a recent development and facilitate genetic analyses of museum collections. ABO and Amel are examples of applications of the proposed method, although any set of primers can be used. ABO genotyping has been widely used in phylogenetic and population analyses. Methods: This study presents a non-destructive approach for PCR-based DNA extraction from preserved museum samples. Human tissue samples, filter materials used during preservation, and processed conservation fluids (after dilution and dialysis) were analyzed to determine ABO genotype and sex (based on Amelogenin). Results: The same replicable PCR profile (ABO blood group and sex determined by Amelogenin) was observed across all three sample types: tissues, filter papers, and conservation fluid. The use of a preservative solution is a new development, as it leaves the sample intact. However, this approach has a drawback: DNA diffuses into the preservative solution very slowly, and it takes several decades to reach a sufficient concentration. A major advantage of this approach is the ability to perform a PCR test without DNA preparation. Conclusions: Museum-derived samples represent a reliable source of DNA and can be effectively used in PCR-based analyses. The presented method works well with degraded DNA samples, combining an already established very short Amelogenin amplicon with PCR sequence-specific primers for ABO genotyping. Full article

Air-dried mutton is a traditional, culturally significant meat product, yet its spontaneous fermentation is inherently constrained by unstable microbial communities, leading to batch-to-batch quality inconsistency and potential food safety hazards. Elucidating whether composite starter cultures can modulate the microbiota and enhance product quality is therefore critical for standardized industrial processing. Herein, we investigated the effects of a defined starter culture (composed of Lactiplantibacillus plantarum and Pediococcus pentosaceus in a 2:1 ratio, with a total inoculum of 10⁸ CFU/g) on the quality and flavor of air-dried mutton, comparing inoculated samples (FJ) with naturally fermented controls (ZR). The fermentation was conducted at 30 °C and 95% relative humidity (RH) for 24 h, followed by air-drying at 4 °C for 21 d, with all assays performed in three biological and three technical replicates. Starter inoculation significantly reduced the pH, water activity (Aw), total volatile basic nitrogen (TVB-N), and thiobarbituric acid reactive substances (TBARS) values while improving sensory acceptability (p < 0.05). Amplicon sequencing analysis revealed a lactic acid bacteria (LAB)-dominated microbiota in FJ samples, with elevated abundances of Pediococcus and Lactobacillus and reduced abundance of Pseudomonas. The inoculated group also exhibited altered eicosapentaenoic acid content and a more diverse volatile flavor profile, with eight key aroma compounds positively correlating with LAB abundance. These findings demonstrate that composite starter inoculation improves physicochemical quality, stabilizes the microbial community, and enhances flavor in air-dried mutton. Further mechanistic validation and scale-up trials are required to confirm industrial applicability. Full article

Potato (Solanum tuberosum L.) is an important staple and food security crop to many communities in the world. However, potato production and quality is greatly constrained by bacterial wilt, a disease caused by a soil-borne pathogen, Ralstonia solanacearum. Ralstonia solanacearum can be managed through clean seed systems and therefore laboratory testing is a pre-requisite for seed certification to confirm the absence of the pathogen in potato seeds before planting. Molecular diagnostics is the gold standard for detection of R. solanacearum in potato seeds. However, the extraction of genomic DNA from R. solanacearum for molecular diagnostics is complex, tedious, lengthy and/or costly procedure. A simple, rapid and reliable DNA extraction protocol is required for use in routine molecular diagnosis of R. solanacearum, a high-risk quarantine pathogen. In this study, we developed a simple and rapid protocol for extracting genomic DNA from symptomatic and asymptomatic potato tubers infected with R. solanacearum and verified its efficiency for the detection and molecular characterization of the pathogen. The protocol was developed from the evaluation of distilled water, Tris-EDTA (TE) and Tris buffer as a base solution for tissue maceration. The DNA quantity and integrity was determined using the NanoDrop 2000C spectrophotometer and agarose gel electrophoresis, respectively. Both hot and cold solutions produced intact high molecular weight genomic DNA of sufficient yield and purity for molecular-based applications. The detection and determination of phylotypes of R. solanacearum, based on conventional and multiplex polymerase chain reaction (PCR), amplified the expected 280 and 372 bp amplicons, respectively, confirming that the quantity and quality of the extracted pathogen genomic DNA was sufficient for molecular diagnostic applications. The sequencing of the amplified products of the endoglucanase gene produced good quality sequences, which confirmed the R. solanacearum isolates to be members of phylotype II sequevar 1. This protocol is a simple, fast and reliable tool for the extraction of sufficient genomic DNA with high quality, directly from R. solancearum-infected potato tubers for PCR and sequencing applications. Its simplicity and throughput make it valuable for use in routine diagnostics and can be adopted by certification programs to ensure distribution of clean potato seeds to farmers. Full article

Tobacco cultivation leads to nicotine accumulation in soil, but how nicotine affects soil nitrification and ammonia-oxidizing microorganisms remains poorly understood. This study conducted a microcosm incubation using soil collected from a 10-year tobacco monoculture field. The soil was spiked with nicotine at 0, 10, or 100 mg kg⁻¹ (the two concentrations representing realistic root-zone levels and a worst-case residue hotspot, respectively) and incubated for 42 days under controlled conditions. Gross nitrification rates were measured by ¹⁵N isotope dilution, and the abundance (qPCR) and community composition (amplicon sequencing) of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were determined at multiple time points. Results showed nicotine at 10 mg kg⁻¹ slightly stimulated nitrification, whereas 100 mg kg⁻¹ caused a transient inhibition (day 1) followed by a sustained stimulation, with gross nitrification rates increased by up to 2-fold compared to the control. Nicotine explained 22% of the variation in bacterial community composition and significantly enriched Intrasporangiaceae and Bryobacter while suppressing Bradyrhizobium. AOB-amoA copy numbers increased within 3 days of nicotine addition and correlated strongly with nitrification rates, whereas AOA-amoA responded only after 6 weeks. Phylogenetic analysis showed that Nitrosospira (cluster Np 39-19) dominated the AOB community. We conclude that nicotine exerts concentration-dependent, biphasic effects on soil nitrification and that AOB, not AOA, drive the nitrification response to nicotine in this agricultural soil. Our findings highlight the potential of nicotine to shape nitrogen cycling in tobacco-cultivated ecosystems and call for field validation under long-term residue conditions. Full article

Background: Phenylketonuria (PKU) is a metabolic disorder requiring a strict low-phenylalanine diet. Oral health impairment, including bacteriome dysbiosis, is common in PKU, yet the mycobiome remains poorly defined. This pilot study aimed to characterise the salivary oral mycobiome of children with PKU compared with controls and to explore associations with dietary intake. Methods: Saliva samples from 18 children, including 8 patients with PKU and 10 age-matched controls, were profiled using internal transcribed spacer (ITS) amplicon sequencing. Alpha/beta diversity, taxonomic composition, diet–fungi correlations, discriminative taxa and LEfSe were analysed. Results: Alpha diversity did not differ significantly between groups after correction for multiple comparisons, although exploratory subgroup analyses suggested lower evenness in PKU children aged <10 years compared with older controls. Beta diversity differed by diagnosis (PERMANOVA: F = 1.7251, p = 0.0062) and in the age–diagnosis model (F = 1.8502, p = 0.0004). Taxonomic analyses identified nominal differences in several fungal taxa, including Candida (p = 0.011), Saccharomycetales_fam_Incertae_sedis (p = 0.011), Naganishia (p = 0.020), and Aspergillaceae (p = 0.036) in PKU samples; however, these findings should be interpreted as exploratory because many did not remain significant after FDR correction. Diet–mycobiome analyses identified selected FDR-supported associations, including an inverse relationship between phenylalanine intake and Naganishia in PKU. Conclusions: This pilot study suggests preliminary compositional differences in the oral mycobiome of children with PKU that may be related to dietary therapy and metabolic context. These exploratory findings require validation in larger cohorts with detailed oral health assessment and control of confounders. Full article

Soil microbiomes play a central role in nutrient cycling and ecosystem stability in forestry ecosystems. Here, we present an exploratory analysis of rhizosphere bacterial communities from eucalyptus plantations in two ecologically distinct Brazilian regions. Using 16S rRNA amplicon sequencing followed by ASV inference and phylogenetic reconstruction, we observed distinct taxonomic composition pattern between samples. Dominant phyla across both samples were Actinomycetota, Pseudomonadota, Acidobacteriota, and Bacillota, with Actinomycetota more abundant in TL (44.0%) than in ES (26.5%), and Acidobacteriota and Verrucomicrobiota more represented in ES. The family Streptomycetaceae and the genus Streptantibioticus were strongly enriched in ES (19.6% and 18.6%, respectively), whereas Solirubrobacteraceae, Pseudonocardiaceae, and Nocardiaceae were preferentially associated with TL. The Eldorado do Sul (ES) sample was characterized by higher observed richness and phylogenetic diversity, whereas Três Lagoas (TL) sample displayed relatively greater community evenness. Beta diversity metrics were consistent with high compositional dissimilarity between samples, with a limited fraction of ASVs forming a shared core microbiome. Despite this taxonomic variation, PICRUSt2-based predictions suggested a broadly conserved set of dominant metabolic pathways across samples. Predicted MetaCyc pathways were largely associated with central carbon metabolism, amino acid biosynthesis, and energy production. At the same time, variation in predicted metabolic profiles was observed between samples. The ES sample showed higher relative representation of pathways related to chitin degradation, purine metabolism, and nitrifier denitrification, whereas the TL sample displayed higher relative representation of pathways associated with alternative TCA variants, glyoxylate metabolism, menaquinol biosynthesis, and aromatic compound degradation. Overall, this exploratory analysis suggests that substantial taxonomic variation may coexist with a relatively conserved predicted functional framework across contrasting eucalyptus plantation systems. These observations should be interpreted as hypothesis-generating and highlight the need for future studies incorporating replicated sampling and direct functional measurements. Full article

Background/Objectives: Traditionally, Sanger sequencing was used to characterize reference materials and confirm discordant allele calls from different STR typing kits at the National Institute of Standards and Technology (NIST). Sequencing can also identify genomic variations within polymerase chain reaction (PCR) amplicons containing STRs, particularly variants that result in null alleles and alleles that do not migrate within allele sizing bins provided by kit manufacturers. Methods: Sanger methods are low-throughput, time- and labor-intensive, and require additional procedures for analysis of heterozygous alleles. To address these limitations, a quicker, more straightforward protocol that uses next-generation sequencing (NGS) was developed. Results: This research provides the criteria used to individually sequence thirty-five autosomal STR loci, with PCR primer locations chosen to increase amplicon length and maximize the likelihood of detecting variants in the flanking region. The list of targeted sequences, associated primers, and chromosomal coordinates is also included. Conclusions: By applying NGS technology to forensic samples containing variant alleles, additional information can be obtained about their molecular basis, and this information can be published and shared across the forensic community. The development of this protocol can increase awareness and encourage the integration of NGS technology into forensic laboratories to improve forensic DNA typing for human identification. Full article

Cold-temperate forest ecosystems in northeastern China have experienced significant changes due to long-term anthropogenic disturbances. To elucidate how different natural forest stands influence the structure, function, and assembly processes of soil microbial communities, we performed 16S rRNA and ITS amplicon sequencing (n = 3 plots per stand) across three natural stands in Northeast China: a 10-year-old natural poplar forest (A1), a 20-year-old natural larch mixed forest (A2), and a 40-year-old natural spruce-pine mixed forest (A3). The results demonstrated that significant variations in aboveground vegetation were accompanied by significant differences in soil physicochemical properties. Specifically, soil organic carbon (SOC) and total nitrogen (TN) were significantly lower in A1 compared to the coniferous stands (p < 0.05). While α-diversity remained stable across stands (p > 0.05), β-diversity revealed distinct community structures (PERMANOVA, p < 0.05). Neutral Community Model (NCM) analyses indicated a shift in bacterial assembly from a stronger stochastic influence in A1 (R² = 0.431) to a more prominent deterministic influence in A3 (R² = 0.188). Because stand age and canopy composition co-vary in our sampling design, we cannot fully disentangle their individual contributions. This comparative observational study reveals distinct soil microbial community structures across the three natural forest types. The transition across forest stands was associated with shifts in dominant microbial taxa and a tendency toward stronger deterministic assembly processes in coniferous stands. These patterns are consistent with differences in litter quality and soil physicochemical properties, providing observational evidence that may inform sustainable management of cold-temperate forests. Full article

Brewers’ spent grain (BSG), which accounts for approximately 85% of the by-products generated during beer production, is a valuable source of dietary fibre, proteins and antioxidant compounds. This study aimed to characterise the chemical composition, techno-functional properties, antioxidant capacity and potential prebiotic effect of BSG flour as a sustainable functional ingredient. Dietary fibre composition, mineral content, and extractable and non-extractable (poly)phenol fractions were determined. The prebiotic potential of BSG flour was evaluated using an in vitro fermentation model with human faeces. Microbial metabolic activity was assessed through the production of short-chain fatty acids (SCFAs), lactate and ammonium, alongside changes in antioxidant capacity during fermentation, while microbiota composition was analysed by 16S rRNA amplicon sequencing. BSG flour showed high levels of insoluble fibre, mainly hemicellulose and arabinoxylans, as well as proteins and non-extractable (poly)phenols, particularly hydroxycinnamic acid derivatives. In vitro fermentation led to a significant increase in SCFA production, particularly acetate and propionate, indicating active degradation of fibre polysaccharides. These metabolic changes were accompanied by enhanced antioxidant capacity and shifts in microbiota composition, including an increased relative abundance of Bifidobacterium species. Overall, this study suggests that BSG flour could be used as a novel ingredient for the development of dietary-fibre-rich foods with potential gut health benefits. Full article

Genome-edited (GE) crops are reaching consumers faster than the analytical infrastructure designed to monitor them. Unlike transgenic crops, most GE products carry only small sequence changes and no foreign DNA, making conventional element-based polymerase chain reaction (PCR) screening, which has underpinned genetically modified organism (GMO) enforcement for two decades, largely ineffective. This review critically evaluates detection technologies not by listing them sequentially, but by comparing their performance against a shared set of enforcement-relevant criteria: sensitivity at regulatory thresholds, allele discrimination capacity, prior target knowledge requirement, and validation maturity. Building on detection/discrimination distinctions already present in ENGL guidance documents and the DETECT project, we formalize a two-axis framework separating detectability (technically achievable for most known targets in defined seed or DNA mixtures at or near the 0.1% MRPL) from identifiability (rarely achievable without developer disclosure), with detection as a necessary precondition for identification, and apply it product by product to each commercialized GE crop for which public molecular data are available. The sulfonylurea-tolerant canola (SU Canola) case, in which analytical specificity is established but forensic event specificity is contested, and the German DETECT project are examined as contrasting case studies of analytical success and attribution failure, extracting generalizable lessons for the field. A technology comparison table, a product-specific feasibility matrix, and a tiered enforcement workflow are provided as practical tools. We conclude with five research priorities for closing the detection–identification gap across near-term, mid-term and longer-term horizons. Full article

Genetic screening of monogenic traits is important for improving genetic health and increasing favorable milk protein in dairy cattle. This study developed and applied an amplification refractory mutation system PCR (ARMS-PCR) assay to simultaneously screen 21 causal variants underlying 18 monogenic traits in Holstein cattle, including 13 recessive genetic defects, 2 milk protein loci, and 3 morphological loci. The assay was designed as a unified multiplex PCR-capillary electrophoresis workflow, enabling clear detection of allele-specific products distinguished by fragment size and fluorescent color. Sanger sequencing validation of newly incorporated loci supported the accuracy of the assay. A total of 1656 cows from 12 commercial farms were genotyped using the multiplex ARMS-PCR panel, and amplicons were analyzed by capillary electrophoresis. Carriers were detected for all genetic defects except DUMPS, with carrier frequencies ranging from 0.12% to 6.64%. The highest frequencies were observed for HH5 (6.64%) and MWS (6.58%), whereas HH3, HH1, and HCD showed intermediate frequencies of 1.81% to 3.08%; all the remaining defects were below 1%. Overall, 22.10% of sampled cows carried at least 1 defect allele, including 20.47% carrying 1 defect, 1.51% carrying 2, and 0.12% carrying 3. For milk protein loci, the desirable β-casein A2A2 and κ-casein BB genotypes occurred at frequencies of 45.83% and 14.13%, respectively, and the favorable A2A2/BB combination was present in 6.22% of sampled cows. Dominant red, recessive red, and polled alleles were rare. These results indicate that multiplex fluorescent ARMS-PCR can serve as a practical targeted screening tool for the simultaneous management of known deleterious alleles and selection of favorable monogenic variants in dairy cattle breeding. Full article