BACKGROUNDShiga toxin-producing Escherichia coli (STEC) can cause illnesses ranging from self-limiting diarrhoea to severe manifestations such as haemolytic-uraemic syndrome (HUS). In 2023, an increase in notified STEC cases was observed in the German federal state of Lower Saxony and nationwide.AIMWe aimed to investigate possible reasons for the observed increase.METHODSWe analysed data on notified STEC cases at federal and state level. All available STEC isolates from Lower Saxony from 2023 were whole genome sequenced. We sent a survey on detection and identification methods to 25 clinical microbiology laboratories in Lower Saxony.RESULTSIn 2023, a statistically significant increase in notified STEC cases in all ages was seen in Lower Saxony and nationwide when compared with case numbers in 2022 and the median of 2015–2019 (p < 0.01). The highest increase was observed in people aged 60–69 years: 110 cases were notified in Lower Saxony in 2023 (median 2015–2019: 26) and 471 cases nationwide (median 2015–2019: 182). No overall increase was seen in disease severity or in the number of HUS cases. No larger genetic clusters or outbreaks were identified in Lower Saxony. The survey among the 17 responding laboratories in Lower Saxony revealed an increased use of multiplex PCR assays for gastrointestinal pathogens, introduced mainly in 2023.CONCLUSIONThe increase in notified STEC cases was probably associated with the implementation of multiplex PCR assays for the analysis of gastrointestinal specimens. Our findings highlight the need to monitor diagnostic practices when assessing and evaluating surveillance data.

The diagnosis of upper respiratory infections is commonly made using nucleic acid amplification technologies (NAAT). The impact of asymptomatic carriage is not often appreciated. We aimed to detect respiratory pathogens present in tonsil tissues from children without acute infection. Remnant tonsil tissues were obtained from children undergoing tonsillectomy procedures. Nucleic acids of respiratory pathogens were detected using laboratory-developed PCR assays. The most frequently detected virus was Enterovirus, followed by Adenovirus and Rhinovirus. Among 522 enrolled children, 41.0% had at least one pathogen detected, and co-detections with more than one pathogen were found in 5.6%. Younger children were more likely to have a virus detected, and co-detection was more frequent. Results of standard of care testing for children with suspected respiratory infection during the same study period were also analyzed. Our data demonstrate that a wide array of pathogens can be detected in the tonsil tissues from children absent of acute respiratory symptoms. It emphasizes the need for careful interpretation of test results, particularly in the era of widespread multiplex NAAT usage.IMPORTANCEThe diagnosis of upper respiratory infections is now commonly made using nucleic acid amplification technologies (NAATs). One potential limitation of NAATs is the increased detection of the nucleic acids of pathogens in healthy individuals in the absence of symptoms. The findings from this study highlight the critical importance of understanding asymptomatic carriage of respiratory pathogens, particularly in pediatric populations. The detection of pathogens in asymptomatic individuals, especially younger children, calls for cautious interpretation of test results to avoid misdiagnosis and unnecessary treatment. It emphasizes the need for careful interpretation of test results, particularly in the era of widespread multiplex NAAT usage.

Hantaviruses are zoonotic, tri-segmented, negative-sense RNA viruses and a significant public health threat. Viral pathogenesis varies between host species, with rodent reservoir infection being asymptomatic and human infection resulting in severe, immune-mediated disease. Viral pathogenesis is highly dependent on virus replication efficiency since it affects the virus’s ability to evade detection and determines the magnitude of the host immune response. However, the molecular replication kinetics for hantaviruses remain poorly defined. Therefore, we developed a sense- and segment-specific quantitative real-time PCR assay and an SYBR-based RT-qPCR assay, allowing us to quantify both negative-sense genome levels and total viral RNA synthesis of the small (S), medium (M), and large (L) segments of Seoul virus (SEOV). We then measured total viral RNA and genome accumulation in reservoir rat endothelial cells (RLMVEC), non-reservoir human endothelial cells (HUVEC-C), and Vero E6 epithelial cells. We also measured the ratio of each segment released into the culture supernatant, approximating the relative packaging efficiency. We found that, while the magnitude of viral RNA differed, RNA replication kinetics were largely similar between reservoir and non-reservoir endothelial cells. However, replication and release kinetics differed between infection of endothelial and Vero cells. We also found that the S, M, and L segments were not equally abundant during viral infection or release but instead followed a trend of M>L>S. Overall, this study validates two RT-qPCR assays to measure SEOV RNA, details the accumulation and release of each viral segment and demonstrates the impact of host cell type on hantavirus replication.

The Booroola fecundity (FecB) gene, a mutation in the bone morphogenetic protein receptor-1B (BMPR1B), is known to increase ovulation rate and litter size in sheep. Efficient and accurate, large-scale detection of this single nucleotide polymorphism (SNP) is critical for marker assisted introgression/selection programs for genetic improvement of prolificacy. This study aimed to develop and validate a robust, cost-effective, and high-throughput genotyping assay for detecting the FecB mutation across diverse sheep populations globally. A competitive allele-specific PCR (KASP) assay targeting the A746G nucleotide substitution in BMPR1B gene was designed and tested on a diverse panel of 224 individuals across 22 sheep breeds and validated against direct sequencing. The KASP genotyping results showed 100% concordance with sequence data, with clear fluorescence-based discrimination of homozygous wild-type Fec++ (AA), heterozygous FecB+ (AG), and homozygous mutant FecBB (GG) genotypes. The assay was compatible across three real-time PCR platforms, BioRad CFX96, Roche LightCycler 480 II, and ABI QuantStudio 6 demonstrating reproducible genotyping and cross-platform interoperability. Subsequently, the KASP assay was applied to a global screening of 1471 sheep from 47 breeds in 14 countries for further validation of the assay. The mutation was not observed in the sheep samples investigated from Africa, Europe, Latin America, and West Asia, whereas it was polymorphic or fixed in certain populations from South (India and Bangladesh) and Southeast Asia (Indonesia). Notably, native Bangladeshi sheep exhibited high frequencies of the FecB allele, with some populations (e.g., Bangladesh Central) nearing fixation, reflecting possible selection for high prolificacy. In conclusion, the validated KASP assay offers a powerful, scalable tool for the rapid genotyping of the FecB mutation, enabling efficient screening and incorporation of prolificacy traits in sheep breeding programs worldwide.

Prevalence and genetic diversity of Theileria and Anaplasma species infecting cattle in Paraguay.

Establishment and application of a SYBR Green-based absolute real-time quantitative PCR assay for chilli yellow ringspot virus.

Influence of virus analytical methods on the estimation of virus reductions by ultrafiltration.

ROS-induced allosteric regulation of NikR coordinates HP0910-mediated OMP2 methylation to modulate H. pylori biofilm dynamics and therapeutic targeting.

A high-sensitivity qPCR method for detecting residual Vero cell DNA in rabies vaccine production.

Echinococcosis, a serious zoonotic parasitic disease caused by tapeworms of the genus Echinococcus, is clinically characterized by a long latent period of up to 10years. An accurate diagnosis is critical for the efficient management and treatment of patients. The aim of this study was to identify robust diagnostic signatures for echinococcosis. Using co-immunoprecipitation and RNA sequencing (RNA-seq), we comparatively profiled the Argonaute 2-binding microRNAs (abmiRNAs) in hepatocytes of Echinococcus multilocularis-infected mice. Using receiver operating characteristic curve (ROC) analysis, we established quantitative PCR (qPCR) assays based on circulating liver-specific abmiRNAs and their combinations, and further validated these assays using blinded serum samples from infected mice and patients. Three abmiRNAs were identified as being predominantly expressed in the liver: miR-192-5p, miR-122-5p and miR-21a-5p. While these three abmiRNAs are upregulated in liver cancer and hepatitis virus infections, we found that all circulating abmiRNAs were significantly and gradually downregulated as the Emultilocularis infection progressed; however, they were rapidly upregulated following anthelmintic treatment. The qPCR assays targeting these circulating abmiRNAs and their combinations showed high sensitivity and specificity. Individual circulating abmiRNAs and their combinations accurately distinguished infections in both mice (n = 50) and humans (n = 117), with the combination of miR-192-5p and miR-122-5p being particularly effective in distinguishing infections. This combination was also sensitive to anthelmintic treatment. These results suggest that circulating miR-192-5p and miR-122-5p are serum signatures for the diagnosis and prognostic management of echinococcosis.

GAS1 affects proliferation, apoptosis, and steroid hormone levels by regulating mitochondrial functions and the PI3K/AKT pathway in bovine granulosa cells.

Comparative analysis of reovirus replication efficiency in HEK293T, L929, and huh-7 cell lines: implications for reovirus propagation in oncolytic therapy.

Detection of cross-reactivity of antibodies to the N proteins of feline morbillivirus and canine distemper virus in Japanese cat plasma samples.

Direct detection and rapid speciation and subspeciation of Mycobacterium avium complex using five-target multiplex PCR and clinical correlations in HIV-positive patients in Tehran, Iran.

Four new piperidine amide alkaloids from Piper longum fruits and their anti-inflammatory activities.

Development of an endpoint PCR assay for monkeypox virus clade differentiation

The interactive responses of cattle genetics and the rumen microbiome (G×M) govern variations in the feed efficiency and methane emissions, which subsequently impact cattle productivity and their environmental footprint. Modulation of the rumen microbiome can be done through dietary supplementation, such as with the antimicrobial ionophore monensin, which offers a pathway to favorably alter metabolic outcomes. However, the limited data on breed-specific microbiome shifts in response to dietary changes restrict the understanding of the impacts of G×M on fermentation and nutrient utilization. The objective of the study was to determine the effect of a monensin-fed diet on the ruminal microbiome and the short-chain fatty acid (SCFA) profile of temperate and tropically adapted cattle breeds. A total of 10 steers each of the Angus, Brahman, and F1 (Angus × Brahman) breed types were fed forage ± a monensin ionophore supplement. Ruminal fluid samples were collected during four 21-day periods (one equilibrium and three treatments). At the conclusion of each period, the SCFAs were analyzed via gas chromatography. The microbiome profiles were analyzed through DNA extraction, quantitative PCR (qPCR) assays, and sequencing to evaluate the G×M interactions. SCFA analysis showed a decrease in the acetate/propionate ratio ( p = 0.001) across all breed types under monensin treatment. However, breed type variations were evident, as the total SCFA concentrations were lower only in the Brahman steers that consumed monensin. The qPCR assays indicated significantly lower ruminal methanogen contents ( mcrA gene; p &amp;lt; 0.01) and a reduced methanogen/prokaryote ratio (MPR; p &amp;lt; 0.001) in monensin-fed steers compared with the control. A treatment-by-breed interaction was observed for the fungi/prokaryote ratio (FBR; p = 0.003), with only F1 steers on the monensin diet showing a lower FBR than those on the control diet. The permutational analysis of variance (PERMANOVA) and beta diversity analyses demonstrated significant differences in the ruminal microbiome structure between the control and the monensin-treated groups for both prokaryotic and fungal communities. Several amplicon sequence variants (ASVs) within the genera Faecalimonas , Streptococcus , and Prevotella showed variable abundance among breeds in response to monensin treatment, confirming the influence of (G×M) interactions on the microbiome structure. This study established the potential of dietary supplementation with an antimicrobial ionophore (monensin) to modulate the rumen microbiome structure, alter the metabolic profiles, and reduce methanogens while emphasizing the need for breed-specific dietary strategies due to the influence of G×M interactions.

Bacteria of the genus Mannheimia are major pathogens of respiratory diseases in ruminants and pose a significant threat to the global ruminant industry. However, the biological characteristics and pathogenic mechanisms of Mannheimia glucosida remain unclear. In this study, we isolated five strains of M. glucosida, which specifically hydrolyzed esculin, from sheep with respiratory disease in China. All five strains of M. glucosida were found to encode the adhesion-related gene adh and the anti-phagocytosis-related gene plpD, as determined by a virulence gene assay. Moreover, all M. glucosida isolates were resistant to streptomycin. Phylogenetic analysis based on 16S rRNA, infB, and sodA genes showed that the sodA gene could be a valuable indication for the analysis of bacterial genetic evolution in the genus Mannheimia. By mouse modeling, M. glucosida D251 was further found to cause multiorgan damage with an LD50 of 1.35 × 106 CFU. Meanwhile, by combining whole genome sequencing with bioinformatic analysis, we found that the D251 genome encodes a large number of virulence and drug resistance genes. Finally, we established a highly sensitive and specific PCR assay for M. glucosida. Collectively, these results indicate that M. glucosida may be an important pathogen in respiratory disease in sheep in China and provides a theoretical basis for the clinical diagnosis and treatment of this disease.

BackgroundAedes aegypti, the principal vector of dengue and other arboviruses, is widely distributed in Pakistan, yet its population genetics and endosymbiont status remain poorly characterized. This study aimed to investigate the genetic structure, haplotype diversity, and phylogeographic patterns of Ae. aegypti in dengue-endemic regions of Pakistan, and to screen for natural Wolbachia infections to provide baseline data for surveillance and vector control.MethodsOvitrap collections were conducted in 2021 across the provinces of Punjab (Bakkar) and Khyber Pakhtunkhwa (Charsadda, DI Khan, Kohat, and two sites within Peshawar: Hayat Abad and Tarnab). Following the morphological identification of adult Ae. aegypti, we extracted genomic DNA from confirmed specimens to amplify and sequence a 658-bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene. Phylogenetic analyses, haplotype network construction, and population differentiation statistics were performed. Additionally, 300 field-caught adult mosquitoes were screened for Wolbachia using validated conventional and quantitative PCR assays targeting the Wolbachia surface protein (wsp) gene.ResultsPhylogenetic analysis of 166 COI sequences (92 from Pakistan) revealed a monophyletic Ae. aegypti clade with 99.65–100% sequence identity, with Pakistani isolates clustering with those from Saudi Arabia, Iran, and India. In total, 13 global haplotypes were identified, with Hap_3 dominating (53%) and shared across regions. Within Pakistan, eight haplotypes were detected, including region-specific variants, yielding high overall diversity (Hd 0.69; π = 0.007). District-level analysis showed that DI Khan and Bakkar had the highest haplotype diversity (Hd 0.73 and 0.71) but low nucleotide diversity (π = 0.005–0.006), whereas Kohat exhibited no haplotype diversity. Population structure was higher in Pakistan (FST 0.26; Nm 0.7) than globally (FST 0.17; Nm 1.19), consistent with low gene flow among Pakistani populations. No natural Wolbachia infections were detected in Ae. aegypti.ConclusionsAedes aegypti in Pakistan belong to a globally monophyletic lineage and show moderate mitochondrial diversity with higher population structure than the global population. The lack of detected Wolbachia infections suggests that natural strains are either absent or occur at very low prevalence. These findings provide a baseline for surveillance and support integrating Wolbachia-based biocontrol alongside conventional interventions in Pakistan.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-025-07134-x.

Fusobacterium nucleatum (Fn) is increasingly recognized as a cancer-associated bacterium, yet reliable quantification in human specimens is challenging due to low bacterial burden and abundant host DNA. We analyzed 145 Fusobacterium genomes to design primers targeting conserved regions of the fadA adhesin gene and developed a duplex quantitative real-time PCR (qPCR) assay for simultaneous detection of fadA and a human PGT as an internal control. Analytical sensitivity, specificity, precision, and reproducibility were evaluated using serially diluted Fn DNA, spike-in experiments with human DNA, and cross-platform/operator validation. Clinical performance was assessed in colorectal cancer patient tissues, including fresh tissue (n = 24) and formalin-fixed paraffin-embedded (FFPE) samples (n = 22), using 16S rRNA-based methods as references. The assay successfully detected all four major Fn subspecies (nucleatum, animalis, polymorphum, and vincentii). The limit of detection was ≤0.1 pg, with no interference between duplex targets. Spike-in experiments demonstrated consistent target detection in human-DNA-rich samples, with strong linearity (R2 = 0.998) across dilutions. High precision (coefficient of variations &lt; 5%) was observed across intra-day, inter-day, inter-instrument, and inter-operator evaluations. In fresh tissues, the assay yielded 86% sensitivity, 94% specificity, and 92% accuracy. Using the FFPE samples, the assay achieved 91% sensitivity and 100% specificity, confirming robust classification in both clinical samples. This duplex qPCR assay enables broad detection of Fn with high analytical performance in both fresh and FFPE tissues. Its simplicity, reproducibility, and compatibility with pathology workflows support deployment in multi-center studies and downstream applications in diagnostic studies and prognostic modeling.