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Simple Summary: Enteric viruses are the main cause of gastrointestinal disease in dogs. Canine astrovirus (CaAstV) is an enteric virus that causes severe dehydration, diarrhea and vomiting, among other gastrointestinal symptoms. The present study is based on the design, standardization, and execution of a rapid and sensitive qPCR protocol using SYBR Green for the identification of CaAstV in dogs with gastroenteritis in Ecuador as a causative agent of gastrointestinal disease in co-infection with other viruses, such as canine parvovirus. The assay was shown to be repeatable, specific and sensitive based on the tests performed. The method was tested on 221 samples from dogs with gastroenteritis, of which 52.8% showed positivity for CaAstV. Sequencing of a segment of the ORF1b gene of the virus genome was performed to validate the presence of the virus, leading to the identification of two possible lineages of the virus circulating in Ecuador. This is the first report of CaAstV in the country. The present trial demonstrates the need for the application of molecular methods for the diagnosis and monitoring of emerging viruses in animals that could be influencing canine mortality due to gastrointestinal disease. Enteric viruses are responsible for a significant number of gastrointestinal illnesses in dogs globally. One of the main enteric viruses is the canine astrovirus (CaAstV), which causes diarrhea in dogs of various ages. It is linked to symptoms such as diarrhea, vomiting, depression and a significant mortality rate due to gastrointestinal disorders. It is a single-stranded positive RNA virus, with three open reading frames, ORF1a, ORF1b and ORF2, where the last one codes for the virus capsid protein and is the most variable and antigenic region of the virus. The aim of this work is to develop and standardize a quick detection method to enable the diagnosis of this etiological agent in dogs with gastroenteritis in Ecuador in order to provide prompt and suitable treatment. The assay was specific for amplification of the genome of CaAstV, as no amplification was shown for other canine enteric viruses (CPV-2, CCoV and CDV), sensitive by being able to detect up to one copy of viral genetic material, and repeatable with inter- and intra-assay coefficients of variation of less than 10% between assays. The standard curve showed an efficiency of 103.9%. For the validation of this method, 221 fecal samples from dogs affected with gastroenteritis of various ages from different provinces of Ecuador were used. From the RT-qPCR protocol, 119 samples were found positive for CaAstV, equivalent to 53.8% of the samples processed. CaAstV was detected in dogs where both the highest virus prevalence in the tested strains and the highest viral loads were seen in the younger canine groups up to 48 weeks; in addition, different strains of the virus were identified based on a sequenced fragment of ORF1b, demonstrating the first report of the presence of CaAstV circulating in the domestic canine population affected by gastroenteritis in Ecuador, which could be associated with the etiology and severity of enteric disease. [ABSTRACT FROM AUTHOR]

Fusarium graminearum is considered the main cause of Fusarium head blight (FHB) in Argentina. The development of this pathogen is dependent on weather conditions, and its occurrence results in economic losses associated with yield reduction, lower quality of grains, and frequent contamination with mycotoxins. The main inoculum source for FHB is F. graminearum perithecia in crop debris where the pathogen survives saprophytically. The adoption of conservationist tillage practices, which leave crop residues on the soil surface, impacts on the volume of inocula available which, in combination with the lack of FHB resistant wheat cultivars, increases the risk of disease development. Our objective was to analyze the long-term dynamics of F. graminearum inocula on different winter and summer crop residues. To do so, five winter cereal species (soft and durum wheat, barley, rye and tritordeum) were cultivated and harvested, and soybean was sown under no-till on the crop debris. The presence and viability of Fusarium graminearum propagules were later evaluated on the residues of the six crops in the 14 months following the harvest of soybean. A qPCR fungal quantification method was developed using previously available primers. The results obtained confirmed the presence and viability of the inoculum in all substrates throughout the time considered. Furthermore, a direct relationship was found between the amount of inoculum in the residues of the soybean sown under a zero-tillage system and the inoculum in the residues of the winter cereals previously sown. This research provides information that could help reduce the sources of F. graminearum inoculum and establish pre-harvest management strategies aimed at preventing mycotoxin contamination in the food chain. [ABSTRACT FROM AUTHOR]

The Kirsten rat sarcoma viral oncogene homolog, or KRAS gene is an oncogene that produces the small GTPase transductor protein known as KRAS. Because it may transmit outside signals to the cell nucleus, KRAS plays a role in controlling cell division. Mutations that activate the KRAS gene hinder the KRAS protein's capacity to transition between active and inactive states, which can cause cell transformation and heightened resistance to treatments that target epidermal growth factor receptors, such as chemotherapy and biological therapies. One of the leading causes of cancer-related death worldwide is colon adenocarcinoma. A mutation in KRAS causes your cells to proliferate uncontrollably, leading to the development of cancer. A wild-type gene is an ordinary (unmutated) gene. KRAS, an acronym for Kirsten rat sarcoma, is a gene that is altered in colorectal cancer quite commonly. Additionally connected to pancreatic and lung cancers are mutated forms of this gene. In this research work, we investigated the effect of Lactobacillus plantarum generated SCFA on the expression level of the KRAS gene. The level of KRAS gene expression was significantly lower, according to the data. With a rise in SCFA concentration, the fold change was lessened; at 100 µg/ml, the fold change value was less. [ABSTRACT FROM AUTHOR]

Early detection and prompt response are key factors in the eradication of huanglongbing (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of Candidatus Liberibacter asiaticus (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.

ObjectivesFusobacterium nucleatum is a gram-negative anaerobic bacillus associated with colorectal cancer (CRC). We aimed to determine the abundance of F. nucleatum and other CRC-associated bacteria using quantitative real-time polymerase chain reaction (qPCR) analysis to detect the possible correlations between tumor and normal tissues and the relationships between patients’ clinical characteristics, diet, and CRC-associated bacteria.MethodsA total of 249 biopsy samples of tumor and paired normal tissues were collected from patients with CRC. Biopsy samples were screened for detection of F. nucleatum using qPCR targeting nusG gene. Bacteroides fragilis, Escherichia coli, and Streptococcus gallolyticus were also detected in the samples using species-specific genes.ResultsThe frequencies of detection of F. nucleatum in the tumor and normal tissues of patients with CRC were 43.37 and 24.1%, respectively (P < 0.05). Statistical analysis using cycle threshold (Ct) values from qPCR data and clinical characteristics showed that tumor size, tumor location, and processed meat consumption were significantly correlated with the abundance of F. nucleatum (P < 0.05). The significance of the prevalence of B. fragilis and E. coli in tumor tissues was marginally higher than that in normal tissues (P < 0.1), and the consumption of processed/red meat affected the prevalence of these bacteria (P < 0.05).ConclusionsOur results showed an association between the presence of F. nucleatum in tumor tissues and CRC, indicating that F. nucleatum may be a potential marker for CRC diagnosis. F. nucleatum is enriched in CRC tissues and is associated with CRC development.

IntroductionThe gastrointestinal microbiota profoundly influences the health and productivity of animals. This study aimed to characterize microbial community structures of the mouth, gastrointestinal tract (GIT), and feces of cattle.MethodsSamples were collected from 18 Akaushi crossbred steers at harvest from multiple locations, including the oral cavity, rumen, abomasum, duodenum, jejunum, ileum, cecum, spiral colon, distal colon, and feces. These cattle were raised without exposure to antimicrobial drugs or hormone implants. Total microbial abundance was assessed using qPCR targeting the V3–V4 region of the 16S rRNA gene, and microbial community composition was evaluated through 16S rRNA gene sequencing.ResultsTotal microbial abundance was lesser in the small intestine than in other GIT regions (p ≤ 0.05). Additionally, microbial communities in the small intestine had lower richness and diversity than other regions (p ≤ 0.05). Microbial community compositions were measurably different along the GIT, with greater relatedness in adjacent GIT sections when progressing from oral to aboral locations. Firmicutes, Bacteroidota, and Actinobacteria were the dominant phyla in all samples. However, variations in composition were evident at lower taxonomic levels within these dominant phyla among samples from different regions. Genera previously associated with healthy gut microbiome communities were observed in low abundance across GIT regions. Taxa historically associated with liver abscesses (e.g., Fusobacterium and Trueperella) were detected in low abundance (≤0.02% relative abundance) throughout the GIT. In contrast, Bacteroides, which recently has been identified as a dominant feature in many liver abscesses, was observed in greater relative abundance (5.2% on average) in the hindgut.DiscussionThis study provides an in-depth evaluation of the GIT of harvest-ready Akaushi crossbred cattle of varying growth rates. Clear differences exist in the abundance and composition of microbial populations at different points of the GIT. Unfortunately, no single GIT location can adequately represent the microbial communities of the entire GIT, which has important implications for future research. Additionally, examining microbiome data only at the phylum level likely oversimplifies important complexities of the microbial community structures, and investigations of lower taxonomic ranks should be included.

ABSTRACT To prevent the spread of foodborne illnesses, the presence of pathogens in the food chain is monitored by government agencies and food producers. The culture-based methods currently employed are sensitive but time- and labor-intensive, leading to increasing interest in exploring culture-independent diagnostic tests (CIDTs) for pathogen detection. However, few studies quantify the relative sensitivity and reliability of these CIDTs compared to current approaches. To address this issue, we conducted a comparison of the limit of detection (LOD50) for Salmonella between a culture-based method and three CIDTs: qPCR (targeting invA and stn), metabarcode (16S) sequencing, and shotgun metagenomic sequencing. Samples of chicken feed and chicken caecal contents were spiked with S. serovar Enteritidis and subjected to culture- and DNA-based detection methods. To explore the impact of non-selective enrichment on LOD50, all samples underwent both immediate DNA extraction and overnight enrichment prior to gDNA extraction. In addition to this spike-in experiment, feed and caecal samples acquired from the field were tested with culturing, qPCR, and metabarcoding. In general, LOD50 was comparable between qPCR and shotgun sequencing methods. Overnight microbiological enrichment resulted in an improvement in LOD50 with up to a three-log decrease. However, Salmonella reads were detected in some unspiked feed samples, suggesting false-positive detection of Salmonella. In addition, the LOD50 in feeds was three logs lower than in caecal contents, underscoring the impact of background microbiota on Salmonella detection using all methods.IMPORTANCEThe appeal of culture-independent diagnostic tests (CIDTs) is increased speed with lowered cost, as well as the potential to detect multiple pathogen species in a single analysis and to monitor other areas of concern such as antimicrobial resistance genes or virulence factors. This study provides quantitative data on the sensitivity of CIDTs relative to current approaches, which is essential for determining the feasibility of implementing these methods in pathogen surveillance programs.

Human papilloma virus (HPV) infections vary in their oncogenic potential, and whether an infection progresses to cervical intraepithelial neoplasia (CIN) also depends on the immune response. Therefore, the aim of the present study was to explore biomarkers related to the immune system and cell proliferation, in combination with HPV classified as having high (HOP) or low oncogenic potential (LOP), that can possibly guide a more accurate identification of women following cervical cancer screening programmes in need for immediate follow-up with a biopsy. A next-generation sequencing transcriptomic immune profile analysis applied to 28 persistent CIN3 lesions and 14 normal biopsies identified four genes, the immune markers ARG1 and HLA-DQB2 and the tumour markers CDKN2A and KRT7, as possible markers for differentiating between CIN3 and normal tissue. To validate these findings, analysis of the relative gene expression of these markers by use of reverse transcriptase real-time quantitative polymerase chain reaction was performed in an independent cohort of 264 (82 normal, 64 CIN1, and 118 CIN2/CIN3) biopsies, and the data were combined with information on the HOP- or LOP-HPV identified in the biopsies. Statistical analysis was performed with receiver operating characteristic curves, reporting area under the curve (AUC) with 95% confidence intervals (CIs), and logistic regression. Statistically significantly higher median expression levels of CDKN2A (p < 0.001) and KRT7 (p = 0.045) and significantly lower expression of ARG1 (p = 0.012) were found in biopsies with HOP-HPV infections, with no difference detected for HLA-DQB2 (p = 0.82). Models using expression levels of CDKN2A (AUC, 0.91; 95% CI, 0.86–0.95), KRT7 (0.86, 0.81–0.91), or ARG1 (0.78, 0.70–0.85) together with HOP/LOP-HPV class were significantly better than HPV class alone (0.72, 0.66–0.79) in discriminating CIN2/3 versus CIN1 (p < 0.001, p < 0.001, and p = 0.014, respectively).

Leptospirosis is a neglected zoonotic disease that is endemic in tropical regions, including Ecuador. It is caused by spirochetes of the genus Leptospira, which can infect humans through animal reservoirs such as rats and dogs, or through contact with contaminated water or soil. In March 2023, public health authorities declared a concerning outbreak of leptospirosis in Durán Cantón, located in the Coastal region of Ecuador. For the first time in the country, a multidisciplinary approach involving physicians and veterinarians was implemented for the surveillance and management of this leptospirosis outbreak. A total of 335 samples were collected, including suspected human cases, household contacts, household dogs, synanthropic rats, and water samples within the area of human cases. Samples were processed by qPCR targeting lipL32, secY, and rrs fragment genes and characterized further for Sanger sequencing. Overall, 26.2 % of human samples, 43.8 % of dog samples, 38.5 % of rat samples, and 39.4 % of water samples tested positive for Leptospira. Further, phylogenetic analysis shows that human, dog, and rat sequences are clustered within the pathogenic subclade P1, within the branch of L. kirschneri and L. interrogans. This study is the first of its kind in Ecuador, where an ongoing outbreak of leptospirosis was managed in real-time by using molecular diagnosis and not serological tools, and where the epidemiological surveillance was done following a One Health approach. This experience should inspire public and animal health authorities in Ecuador to promote a national One Health surveillance and control program for zoonotic diseases.

ABSTRACT: Growing broiler chickens of the Cobb500 strain were used to determine the effects on intestinal microbiota composition of a protein-free (PF) diet as compared to a diet based in casein (CAS) as the only protein source. CAS was formulated to contain the same amount of protein (190 g kg−1) as a commercial Maize-soy diet which was used as a practical reference. The ileal AA flow (g kg−1 dry matter intake) was significantly higher (P < 0.001) than PF in birds fed protein containing diets (CAS or Maize-soy). Taken as a whole (discriminant and ANOSIM analysis), the intestinal (ileal and caecal contents and ileal tissue) microbiota composition of PF and CAS were significantly (P < 0.001) different from Maize-soy and not different from each other in some cases. RT-qPCR and sequencing analysis of the ileal and caecal microbiota revealed significant (P < 0.05) differences in a number of bacterial groups between broilers fed PF, CAS or Maize-soy diets. The main result was that the lack of protein in the intestinal medium of PF birds resulted in a drop of Lactobacillus spp. counts (on average, 43 in PF vs 1,734 in the Maize-soy diet) and increased Enterobacteriaceae (on average, 419 in PF vs 172 in the Maize-soy diet) and other potentially pathogenic bacterial groups (in both intestinal contents and tissue). Thus, the lack of protein in the intestinal medium of PF birds resulted in a microbiota composition compatible with a pro-inflammatory state, and this effect was somewhat less marked in birds fed CAS. The results reported here suggest that the adverse effects on microbiota composition in broilers fed CAS were less marked than in those fed PF, which would be in line with a preferential use of a highly digestible protein containing diet to determine endogenous AA excretion instead of a PF diet.

We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12–0.21% and 0.62–1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

The identification of novel therapeutic agents using nanomedicine is critical in the fight against diabetes mellitus (DM). Cholic acid (CA) has shown potential in diabetes management, but its effectiveness is limited by poor solubility and stability. To overcome these challenges, CA was encapsulated in chitosan nanoparticles (CACNPs). The CACNPs had a negative zeta potential (ZP) of −13.6 ± 5.81 mV, which is an indication of good stability and potential for enhanced uptake by diseased cells. The average particle size (PS) measured by dynamic light scattering (DLS) was 169.8 ± 84.3 nm. The polydispersity index (PDI) was 0.220, indicating uniform particle size distribution. The drug loading capacity (DL%) of the CACNPs was 60.96 ± 0.9 %, whereas, the entrapment efficiency (EE%) was 69.19 ± 1.02 %. The MTT assay on 3 T3-L1 cells revealed a concentration-dependent effect on cell viability, with an IC50 value of 766.0 ± 0.09 µg/ml. Furthermore, CACNPs demonstrated dose-dependent enhancement of glucose uptake in differentiated adipocytes, while at 500 µg/ml, they inhibited adipocyte differentiation, suggesting a potential role in adipogenesis inhibition. Quantitative PCR (qPCR) indicated positive modulation of glucose metabolism-related genes (PI3K, GLUT4, PPARg) upon treatment with CACNPs. These findings suggest that CACNPs could serve as a novel inhibitor of adipocyte differentiation and may influence key pathways in glucose metabolism, making them promising candidates for the management of DM.

Black tea is highly favored by consumers worldwide, with enzymatic reactions being recognized as a pivotal factor influencing tea quality. The role of microorganisms in shaping the composition of black tea has emerged as a focus of research due to their involvement in enzyme catalysis and metabolic processes. In this study, full-length amplicon sequencing combined with qPCR more accurately reflected microbial profile, and Pantoea, Pseudomonas, Paucibacter, and Cladosporium were identified as the main microbial genera. Moreover, by comprehensively analyzing color, aroma, and taste components over time in black tea samples, correlations were established between the dominant genus and various quality factors. Notably, peroxidase activity levels, total soluble sugar content, and tea pigments concentration exhibited significant associations with the dominant genus. Consequently, this microbiological perspective facilitated the exploration of driving factors for improving black tea quality while establishing a theoretical foundation for quality control in industrial production.

Rheumatoid arthritis (RA) is a predominant inflammatory arthritis in human. The function of HLA-B27 gene in other types of arthritis has been studied, however, its function in RA is unclear. This study investigated the relative expression of HLA-B27 gene in RA patients compared to normal control and assesses its suitability as a biomarker for early detection of RA. Bioinformatics analysis was used to determine the profile of HLA-B27 gene in different human tumors to demonstrate the possible involvement of HLA-B27 in both RA and cancer; and also, to discover its functional association with other human genes. Samples of human blood from RA patients and healthy individuals were collected, and RNA extraction, cDNA synthesis and qPCR were carried out to detect relative expression of HLA-B27. ROC analysis was undertaken to investigate HLA-B27 diagnostic performance; GENT2 platform was used to compare HLA-B27 expression levels in different human tumors, and gene-gene interaction network was generated using GeneMANIA to identify correlation of HLA-B27 with other human genes. The qPCR analysis demonstrated an increase in the HLA-B27 expression by 1.65 fold in RA compared to normal control. ROC analysis indicated that HLA-B27 expression could efficiently differentiate RA from normal, supporting its potential use as diagnostic molecular biomarkers. The GENT2 revealed that HLA-B27 expression levels vary across different tumor types, most notably in heart tissue. The gene-gene interaction network revealed that KIR3DL1, KIR3DS1, LILRB1, B2M and LILRA1 were the leading genes showing the highest correlations with the HLA-B27. Our results indicate that HLA-B27 gene is involved in the RA pathogenesis and it can be used as a molecular biomarker for the diagnosis of RA. Our findings could lead to the discovery of novel diagnostic, preventive and therapeutic strategies.

Little information is presently available regarding the frequency of the silent shedders of respiratory viruses in healthy sport horses and their impact on environmental contamination. Therefore, the aim of this study was to investigate the detection frequency of selected respiratory pathogens in nasal secretions and environmental stall samples of sport horses attending a multi-week equestrian event during the summer months. Six out of fifteen tents were randomly selected for the study with approximately 20 horse/stall pairs being sampled on a weekly basis. Following weekly collection for a total of 11 weeks, all samples were tested for the presence of common respiratory pathogens (EIV, EHV-1, EHV-4, ERAV, ERBV, and Streptococcus equi ss equi (S. equi)) using qPCR. A total of 19/682 nasal swabs (2.8%) and 28/1288 environmental stall sponges (2.2%) tested qPCR-positive for common respiratory pathogens. ERBV was the most common respiratory virus (17 nasal swabs, 28 stall sponges) detected, followed by EHV-4 (1 nasal swab) and S. equi (1 nasal swab). EIV, EHV-1, EHV-4 and ERAV were not detected in any of the study horses or stalls. Only one horse and one stall tested qPCR-positive for ERBV on two consecutive weeks. All the other qPCR-positive sample results were related to individual time points. Furthermore, only one horse/stall pair tested qPCR-positive for ERBV at a single time point. The study results showed that in a selected population of sport horses attending a multi-week equestrian event in the summer, the frequency of the shedding of respiratory viruses was low and primarily restricted to ERBV with little evidence of active transmission and environmental contamination.

Human adenovirus (HAdV) F40/41 is an important pathogen in pediatric acute gastroenteritis cases. However, the diversity of study designs and diagnostic methods often leads to misinterpretations of their impact. Our study explored the genetic diversity of HAdV-F40/41 in Brazil using a specific qPCR assay for HAdV species F, combined with a phylogenetic analysis of the partial hexon and fiber genes. Our results demonstrated that HAdV-F41 strains predominated and exhibited higher diversity than HAdV-F40 strains. Based on the hexon gene, Brazilian HAdV-F41 strains were grouped into two genome type clusters (GTC), further divided into subclusters, with most strains clusteringto GTC2. The partial shaft region of the fiber gene exhibited higher conservation among HAdV-F41. The specific qPCR assay for HAdV species F identified HAdV-F in an additional 31.5% (34/108) of previously uncharacterized HAdV-positive samples detected using a non-specific HAdV qPCR assay. Both assays strongly correlated in detecting HAdV-F, and the specific qPCR assay for enteric types can enhance HAdV surveillance, especially when sequencing is not possible. Our study provides novel insights regarding the genetic diversity of HAdV-F species in Brazil.

Quantitative PCR is a molecular technique for DNA quantification that depends on reaction efficiency and the Ct value (“cycle threshold”). However, the results are dependent on laboratory conditions and mathematical approaches. Thus, the data of 16 genes from Pseudomonas aeruginosa strain AG1 were generated using qPCR to assess the effect of DNA concentration and three mathematical methods (a standard curve and two individual-curve-based approaches called exponential and sigmoidal models) on efficiency and DNA quantification. Differences in efficiency were revealed depending on the mathematical method used; the values were 100% in three out of the four standard curves, but estimations of the expected fold change in DNA serial dilutions were not achieved, indicating the possible overestimation of efficiency. Moreover, when efficiency was compared to DNA concentration, a decreasing trend in efficiency as DNA concentration increased in the reaction was observed in most cases, which is probably related to PCR inhibitors. For all 16 genes at a single DNA concentration, the efficiencies for the exponential model were found in the range of 1.5–2.79 (50–79%), and for the sigmoidal approach, the range was 1.52–1.75 (52–75%), with similar impact on normalized expression values, as indicated by the genes for standard curves. Jointly, DNA concentration and mathematical model choice were demonstrated to impact the estimation of reaction efficiency and, subsequently, DNA quantification when using qPCR.

Objectives: The study involving 20 SARS CoV patients found that elevated microRNA levels, specifically miR-23a overexpression and miR-195-F1gene downregulation, could serve as potential diagnostic factors for COVID-19 infections, potentially aiding in early detection. Materials and Methods: 20 patients suffered from coronavirus (SARS CoV) (Patient group) and ten samples of healthy persons (control group). The blood sample was collected from the patients who attended Al-Zafaraniyah General Hospital, Fatima-Alzahra Hospital, and Al-Yarmook Teaching Hospital in Baghdad City. Nasopharyngeal and oropharyngeal swabs for each patient were taken simultaneously and added immediately into viral transport media (VTM). The microRNAs in the serum of coronavirus (SARS CoV patients were miRNA concentration upon conversion to cDNA is necessary to analyze and compute regulatory element levels of one or more genes—all procedures, including data analysis, qPCR amplification, and total RNA purification. Results: The miR-23a-3p levels were markedly elevated, and serum levels of the miR-195-F1 gene were shown to be substantially downregulated in COVID-19 individuals. Conclusions: The study reveals that COVID-19 patients exhibit increased expression levels of miR-23a-3p and miR-195-F1gene, suggesting it as a potential diagnostic indicator for COVID-19 diagnosis and treatment monitoring.

The measurements and tests of the samples conducted in the laboratories of the College of Agriculture included isolating bio-fertilizers and testing the efficiency of isolates that fix atmospheric nitrogen and solubilize phosphorous compounds. Bacteria were isolated and identified from the rhizosphere soils of different plants collected from various agricultural areas. A total of 74 bacterial isolates were obtained based on the phenotypic characteristics of the developing colonies, as well as biochemical and microscopic traits. The results of isolation and identification showed that among the 74 bacterial isolates, there were 15 isolates of A. chroococcum, 13 of Az. lipoferum, 13 of B. megaterium, 10 of P. putida, 10 of Actinomycetes, and nitrifying bacteria (Nitrosomonas and Nitrobacter) with 7 and 6 isolates respectively. Bacillus isolates demonstrated the ability to dissolve phosphate compounds with a dissolution diameter ranging between 6-11 mm. The results of the polymerase chain reaction (qPCR) examination confirmed the diagnostic results using the quantitative polymerase interaction technique after extracting DNA from bacterial isolates from the soil. The first group of 10 isolates of P. putida showed, when the gene (GltA F, GltAR) was amplified, resulting bundles with a molecular size of 288 base pairs for P. putida bacteria, confirming the diagnosis using molecular, biological, microscopic, and phenotypic tests. This match confirms the accuracy of the classification of P. putida bacteria. The second group of 10 isolates of P. putida showed, when the gene (PR o DF, PRp o DR) was amplified, resulting bundles with a molecular size of 478 base pairs for B. megaterium, confirming its classification. The third group of 10 isolates of bacteria showed, upon gene amplification (N i f HF, N i f HR), resulting bundles with a molecular size of 371 base pairs for A. chroococcum bacteria, confirming their identification. The fourth group of 14 isolates showed, when the gene (16sAZ F and 16sAZ R) was amplified, resulting bundles with a molecular size of 646 base pairs for Az. lipoferum bacteria, confirming its classification. The fifth group did not show any type of nitrogen-fixing bacteria or phosphate solubilizers after amplification, based on the Sr DNA 71 genotype sequence.

Abstract Background Digital dermatitis (DD) is a contagious bovine foot disease causing reduced animal welfare and negative economic consequences for the farmer. Treponema spp. are the most important causative agents. Studies indicate that trimming equipment can transfer DD-associated treponemes between cows. The aim of this observational study in 22 DD-positive Norwegian dairy herds was to investigate the risk of transferring Treponema spp. with trimming equipment and chutes after claw trimming, and after washing and disinfection. Swabs from the trimming equipment and chutes were collected from nine different locations, at five different time points. Bacterial DNA was extracted from 647 swabs and analysed by qPCR for Treponema spp. In addition, 172 swabs taken immediately after trimming, were analysed by a multiplex qPCR targeting T. phagedenis, T. pedis and T. medium/vincentii. Biopsy sampling from DD lesions was performed on cows in the same herds during trimming. Altogether 109 biopsies were analysed by FISH for confirmation of the DD diagnosis and identification of Treponema phylotypes (PTs). Results High numbers of Treponema spp. were detected from all nine locations on the trimming equipment and chutes immediately after trimming, and T. phagedenis was detected on two or more locations in all but two herds, 1 and 19. There was a decline in the amount of Treponema spp. after washing and disinfection. The belly belt, the cuff, and the footrest on the chute had the highest proportion of positive samples after disinfection. The belly belt had the highest copy numbers of all nine locations (median = 7.9, max = 545.1). No Treponema spp. was detected on the hoof knives after disinfection. Treponema phagedenis, T. pedis, and Treponema phylotype 3 (T. refringens) were detected by FISH analysis of the biopsies. Treponema phagedenis was detected in biopsies from all herds except 1 and 19. Conclusion This study shows that DD-associated Treponema spp. were present on the trimming equipment and chutes after trimming cows in DD-positive herds. Washing and disinfection reduced the load of Treponema spp. However, large differences in Treponema spp. between different locations were documented. High copy numbers on the grinder and the chute after disinfection, indicates that sufficient cleaning and disinfection of these locations is difficult, and that passive transfer of DD-associated treponemes (viable or not) is possible.

Abstract Background There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. Results In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. Conclusions The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.

The monk seal is the most endangered pinniped in the world and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness and records are biased towards occasional, mostly coastal encounters. Nowadays, molecular techniques allow us to detect and quantify minute amounts of DNA traces released into the environment (eDNA) by any organism. A species-specific molecular assay is now available for detecting the recent presence of the monk seal in the water column through the analysis of sea-water samples collected from the sea surface. The project “Spot the Monk” uses this non-invasive detection tool to monitor monk seal occurrence in Mediterranean waters by means of eDNA analysis. The simplicity in the acquisition of samples together with the need to collect samples in multiple points simultaneously made the project well suited to the involvement of the general public. Up to today, about 350 samples have been collected and analysed in the central-western Mediterranean by researchers and a multifarious range of citizen scientists - from recreational sailing organisations, both amateur and competitive sportsmen, to fishermen. This work announces the launch of an open-source Observatory (https://www.spot-the-monk-observatory.com/) where the project outcomes are publicly accessible as soon as they are produced. Embracing the principles of Open Science, we believe that such an approach can contribute to filling the knowledge gap about the distribution of this charismatic species in our seas, providing, at the same time, a proof of concept on how data collected by a variety of actors can be returned to the scientific and non-scientific communities in an innovative format for immediate consultation.

Abstract Background The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. Methods Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. Results The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95–100% similarity to the isolates from the gene bank. Conclusion The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

Abstract Background Intramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows’ udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available. Results Among the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens. Conclusions The effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable – especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi–positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilt in plants. The Verticillium wilt disease severity assay on plants caused by V. dahliae mainly includes two methods, one is a plant disease grade classification based on disease severity statistics [namely the disease index (DI)], and the other is the V. dahliae biomass quantification in plants (namely the fungal biomass). In this study, the relationships of pathogenicity with the DI, pathogenicity with the biomass, and the correlation analysis of the DI and relative fungal biomass were analysed. The results showed that pathogenicity assessment of V. dahliae strains using the DI method was able to give an intuitive reflection of the pathogenic ability for defoliating and non-defoliating strains; moreover, the method of quantitative PCR for fungal biomass also had high repeatability and stability. As a whole, the correlation coefficient between the DI and fungal biomass values of 28 strains was 0.728, indicating that the two data sets were highly correlated; however, the correlation coefficients of the defoliating strains and non-defoliating strains were only 0.5384 and 0.4547, respectively. In conclusion, the correlation coefficient between the DI and the fungal biomass presented high consistency, which could provide some meaningful exploration for the more accurate pathogenicity identification of V. dahliae.

Background: The effectiveness and reliability of diagnostic tests that detect DNA sequences largely hinge on the quality of the used primers and probes. This importance is especially evident when considering the specific sample being analyzed, as it affects the molecular background and potential for cross-reactivity, ultimately determining the test’s performance. Methods: Predicting primers based on the consensus sequence of the target has multiple advantages, including high specificity, diagnostic reliability, broad applicability, and long-term validity. Automated curation of the input sequences ensures high-quality primers and probes. Results: Here, we present a use case for developing a set of consensus primers and probes to identify antibiotic resistance and virulence genes in Staphylococcus (S.) aureus using the ConsensusPrime pipeline. Extensive qPCR experiments with several S. aureus strains confirm the exceptional quality of the primers designed using the pipeline. Conclusions: By improving the quality of the input sequences and using the consensus sequence as a basis, the ConsensusPrime pipeline pipeline ensures high-quality primers and probes, which should be the basis of molecular assays.

Abstract Background Pathogenic infections pose a significant threat to global health, affecting millions of people every year and presenting substantial challenges to healthcare systems worldwide. Efficient and timely testing plays a critical role in disease control and transmission prevention. Group testing is a well-established method for reducing the number of tests needed to screen large populations when the disease prevalence is low. However, it does not fully utilize the quantitative information provided by qPCR methods, nor is it able to accommodate a wide range of pathogen loads. Results To address these issues, we introduce a novel adaptive semi-quantitative group testing (SQGT) scheme to efficiently screen populations via two-stage qPCR testing. The SQGT method quantizes cycle threshold (Ct) values into multiple bins, leveraging the information from the first stage of screening to improve the detection sensitivity. Dynamic Ct threshold adjustments mitigate dilution effects and enhance test accuracy. Comparisons with traditional binary outcome GT methods show that SQGT reduces the number of tests by 24% on the only complete real-world qPCR group testing dataset from Israel, while maintaining a negligible false negative rate. Conclusion In conclusion, our adaptive SQGT approach, utilizing qPCR data and dynamic threshold adjustments, offers a promising solution for efficient population screening. With a reduction in the number of tests and minimal false negatives, SQGT holds potential to enhance disease control and testing strategies on a global scale.

Peng Wu,1,&ast; Xiaoyong Wang,2,&ast; Min Yin,1,&ast; Wenjie Zhu,3,&ast; Zheng Chen,1 Yang Zhang,1 Ziyu Jiang,4 Longqing Shi,5 Qiang Zhu1 1Children’s Hospital of Nanjing Medical University, Nanjing, People’s Republic of China; 2The People’s Hospital of Rugao, Nantong, People’s Republic of China; 3Kangda College of Nanjing Medical University, Nanjing, People’s Republic of China; 4Department of Oncology, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, People’s Republic of China; 5Department of Hepatobiliary and Pancreatic Surgery, Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Longqing Shi, Department of Hepatobiliary and Pancreatic Surgery, The Third Affiliated Hospital of Soochow University, Jiangsu, People’s Republic of China, Email 20204135017@stu.suda.edu.cn Qiang Zhu, Children’s Hospital of Nanjing Medical University, Nanjing, People’s Republic of China, Email zhu20081023@yeah.netBackground: Liver cancer remains to be one of the leading causes of cancer worldwide. The treatment options face several challenges and nanomaterials have proven to improve the bioavailability of several drug candidates and their applications in nanomedicine. Specifically, chitosan nanoparticles (CNPs) are extremely biodegradable, pose enhanced biocompatibility and are considered safe for use in medicine.Methods: CNPs were synthesized by ionic gelation, loaded with rutin (rCNPs) and characterized by ultraviolet–visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The rCNPs were tested for their cytotoxic effects on human hepatoma Hep3B cells, and experiments were conducted to determine the mechanism of such effects. Further, the biocompatibility of the rCNPs was tested on L929 fibroblasts, and their hemocompatibility was determined.Results: Initially, UV–vis and FTIR analyses indicated the possible loading of rutin on rCNPs. Further, the rutin load was quantitatively measured using Ultra-Performance Liquid Chromatography (UPLC) and the concentration was 88 μg/mL for 0.22 micron filtered rCNPs. The drug loading capacity (LC%) of the rCNPs was observed to be 13.29 ± 0.68%, and encapsulation efficiency (EE%) was 19.55 ± 1.01%. The drug release was pH-responsive as 88.58% of the drug was released after 24 hrs at the lysosomal pH 5.5, whereas 91.44% of the drug was released at physiological pH 7.4 after 102 hrs. The cytotoxic effects were prominent in 0.22 micron filtered samples of 5 mg/mL rutin precursor. The particle size for the rCNPs at this concentration was 144.1 nm and the polydispersity index (PDI) was 0.244, which is deemed to be ideal for tumor targeting. A zeta potential (ζ-potential) value of 16.4 mV indicated rCNPs with good stability. The IC50 value for the cytotoxic effects of rCNPs on human hepatoma Hep3B cells was 9.7 ± 0.19 μg/mL of rutin load. In addition, the increased production of reactive oxygen species (ROS) and changes in mitochondrial membrane potential (MMP) were observed. Gene expression studies indicated that the mechanism for cytotoxic effects of rCNPs on Hep3B cells was due to the activation of Unc-51-like autophagy-activating kinase (ULK1) mediated autophagy and nuclear factor kappa B (NF-κB) signaling besides inhibiting the epithelial–mesenchymal Transition (EMT). In addition, the rCNPs were less toxic on NCTC clone 929 (L929) fibroblasts in comparison to the Hep3B cells and possessed excellent hemocompatibility (less than 2% of hemolysis).Conclusion: The synthesized rCNPs were pH-responsive and possessed the physicochemical properties suitable for tumor targeting. The particles were effectively cytotoxic on Hep3B cells in comparison to normal cells and possessed excellent hemocompatibility. The very low hemolytic profile of rCNPs indicates that the drug could be administered intravenously for cancer therapy.Keywords: rCNPs, Hep3B, ROS, qPCR, mechanism

ABSTRACT: The source of infection of digital dermatitis (DD), an infectious lameness condition, is still uncertain. In this cross-sectional study, we aimed to identify potential reservoirs of DD bacteria in dairy cattle body sites with different stages of the disease and farm environments. We collected skin swabs from 85 dairy cows from 5 herds, 3 with and 2 without DD, from foot, hock, and udder cleft skin (with lesions or not), saliva, urine, and feces. We also obtained environmental samples. Real-time quantitative PCR targeted Treponema phagedenis, Treponema medium, Treponema pedis, Porphyromonas levii, Bacteroides pyogenes, Fusobacterium necrophorum, and Fusobacterium mortiferum. Digital dermatitis-associated Treponema spp. were exclusively detected in DD-affected herds in DD-foot and other skin lesions, healthy skin, saliva, and environmental samples. In contrast, the non-Treponema spp. were found in samples from both DD-negative and affected herds. As expected, DD lesions had higher bacterial loads than healthy skin. Interestingly, similar counts were observed in udder cleft lesions, indicating a potential opportunistic behavior on compromised skin. None of the targeted species were detected in fecal samples, but P. levii, B. pyogenes, and F. necrophorum were detected in urine. All 7 species were detected in saliva, although in low quantities. No associations were observed between the presence of each bacterial species in DD lesions and urine; however, there was an association between the presence of DD-Treponema spp. in lesions and saliva, hock, and udder skin. Feces and urine do not seem to be a DD bacteria primary source, but saliva and other skin lesions may play a role. Longitudinal studies would improve our understanding of DD-associated bacteria's transient or persistent presence in these sites. Investigating the sources of DD-associated bacteria will guide future interventions to minimize bacterial shedding and transmission, ultimately more effectively reducing bacterial load, transmission, and sources of infection in dairy herds.

Background: Bacterial identification can be done using various testing techniques. Molecular techniques are often used to research dangerous diseases, an approach using genetic information on the pathogenic agent. The enterohemorrhagic invasive species Escherichia coli 0157:H7 was identified from the feces of working horses on the island of Sumbawa. Another advance in molecular technology is genome amplification with qPCR which is the gold standard for detecting E. coli. Aim: This study aims to detect and identify the invasive species Escherichia coli 0157:H7 using the gene encoding chuA with the qPCR method sourced from horse feces. Methods: Fresh fecal samples from horses on Sumbawa Island were isolated and identified, then continued with molecular examination using the gene encoding chuA using the qPCR method. Results: qPCR testing in this study showed that 6 sample isolates that were positive for Escherichia coli 0157:H7 were detected for the presence of the chuA gene, which is a gene coding for an invasive species of E. coli bacteria. The highest to lowest Cq values and Tm from the qPCR results of the sample isolates were 15.98 (4KJ), 14.90 (19KG), 14.6 (3KJ), 13.77 (20KG), 12.56 (5KGB), 12.20 (6KJ). Tm values are 86.7 (4KJ), 86.69 (3KJ), 86.56 (5KGB), 85.88 (20KGB), 85.81 (19KG), 85.74 (6KJ). Conclusion: Validation, standardization of the development, and modification of qPCR technology must be carried out to harmonize testing throughout to avoid wrong interpretation of the test results so that the determination of actions to eradicate and control diseases originating from animals in the field does not occur. [Open Vet J 2024; 14(4.000): 1051-1058]

Botrytis cinerea is a well-known pathogen that can be challenging to control in crops, such as wine grapes. To adapt to the increasing problems of climate change and strain resistance, it is important to find new methods to detect Botrytis cinerea and differentiate strains. These methods include strain differentiation and classification by simple sequence repeats (SSRs) and early detection of the fungus by qPCR. Various strains were analysed using SSR markers and either agarose gel electrophoresis or capillary sequencing via PCR. A sensitive qPCR method was refined to achieve an early detection method for the pathogen. The results demonstrate promising ways to distinguish between strains using both agarose gel electrophoresis and capillary sequencing as well as to detect infection before it becomes visible on grapes. This can be used to further understand and analyse different Botrytis cinerea strain characteristics such as laccase activity, regional or annual effects. The early detection method can be used to better prepare growers for an impending infection so that targeted efforts can be made.

Abstract Objective Breast cancer is the leading cause of cancer incidence and mortality among Indonesian women. A comprehensive investigation is required to enhance the early detection of this disease. Mitochondrial DNA copy number (mtDNA-CN) and relative telomere length (RTL) have been proposed as potential biomarkers for several cancer risks, as they are linked through oxidative stress mechanisms. We conducted a case–control study to examine peripheral blood mtDNA-CN and RTL patterns in Indonesian breast cancer patients (n = 175) and healthy individuals (n = 181). The relative ratios of mtDNA-CN and RTL were determined using quantitative real-time PCR (qPCR). Results Median values of mtDNA-CN and RTL were 1.62 and 0.70 in healthy subjects and 1.79 and 0.73 in breast cancer patients, respectively. We found a positive association between peripheral blood mtDNA-CN and RTL (p

Abstract The diagnostic assays currently used to detect Shigella spp. (Shigella) and enterotoxigenic Escherichia coli (ETEC) are complex or elaborate which make them difficult to apply in resource poor settings where these diseases are endemic. The simple and rapid nucleic acid amplification-based assay "Rapid LAMP-based Diagnostic Test (RLDT)" was evaluated to detect Shigella spp (Shigella) and enterotoxigenic Escherichia coli (ETEC) and determine the epidemiology of these pathogens in Kolkata, India. Stool samples (n = 405) from children under five years old with diarrhea seeking care at the hospitals were tested, and 85(21%) and 68(17%) by RLDT, 91(23%) and 58(14%) by quantitative PCR (qPCR) and 35(9%) and 15(4%) by culture, were positive for Shigella and ETEC, respectively. The RLDT showed almost perfect agreement with qPCR, Kappa 0.96 and 0.89; sensitivity 93% and 98%; specificity 100% and 97% for Shigella and ETEC, respectively. While RLDT detected additional 12% Shigella and 13% ETEC than culture, all culture positives for Shigella and ETEC except one each were also positive by the RLDT, sensitivity 97% and 93% respectively. RLDT is a simple, sensitive, and rapid assay that could be implemented with minimum training in the endemic regions to strengthen the disease surveillance system and rapid outbreak detection.

Abstract Background Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. Methods Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. Results Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. Conclusions The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered. Graphical Abstract

Lentils (Lens culinaris) cannot withstand prolonged flooding. This project aimed to study molecular and physiological responses in L. culinaris under flood stress to understand this plant's key mechanisms for flood tolerance. Six- to eight-week-old lentil seedlings were flooded in a growth chamber for four days. On the fourth day, molecular and physiological experiments were conducted on the flooded and control lentil plants. In addition, a morphological study of the stems and leaves using a scanning electron microscope and volatile compound studies (serving as potential biomarkers for flood stress) using GC–MS were conducted. Under flooded conditions in most of the lentil plants, the expressions of the following genes were upregulated: acetyl-CoA carboxylase carboxyltransferase beta subunit (accD), aldolase, and petD-cytochrome b6/f complex subunit IV (PetD). On the other hand, chloroplast copper/zinc superoxide dismutase (SD) and PBA flash photosystem II D1 precursor (psbA) constituted two downregulated genes among most of the plants. Additionally, higher levels of absorbance for soluble sugar, surface roughness, and surface wax deposition were observed in lentils under flooded conditions. Higher wax accumulation leads to higher leaf hydrophobicity, which is a flood-related adaptation. On the contrary, chlorophyll content, catalase activity, and volatile emissions were reduced among flooded lentils, probably due to cell death and reduced photosynthesis. Oxidative damage occurs when cells’ catalase activity is low, meaning cells are unable to detoxify reactive oxygen species. In conclusion, flooding can cause a notable difference in lentils at anatomical, physiological, and molecular levels.

Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus-Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity of these primers to estimate the relative abundance of each genotype within individual nodules and after serial transfers to new hosts. For the moderate and non-beneficial genotypes, qPCR allowed us to differentiate genotypes that are phenotypically indistinguishable and investigate host control with suboptimal symbionts. We consistently found the wild type increasing in the proportion of the population, but our data suggest a potential reproductive trade-off between the moderate and non-beneficial genotypes. The multi-generation framework we used, coupled with qPCR, can easily be scaled up to track dozens of M. japonicum mutants simultaneously. Moreover, these mutants can be used to explore M. japonicum genotype abundance in the presence of a complex soil community.

A voluntary upper respiratory biosurveillance program in the USA received 9740 nasal swab submissions during the years 2008-2021 from 333 veterinarians and veterinary clinics. The nasal swabs were submitted for qPCR testing for six common upper respiratory pathogens:equine influenza virus (EIV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV). Additional testing was performed for equine gamma herpesvirus-2 (EHV-2) and equine gamma herpesvirus-5 (EHV-5) and the results are reported. Basic frequency statistics and multivariate logistic regression models were utilized to determine the associations between risk factors and EIV positivity. The EIV qPCR-positivity rate was 9.9%. Equids less than 9 years of age with a recent history of travel and seasonal occurrence in winter and spring were the most common population that were qPCR positive for EIV. This ongoing biosurveillance program emphasizes the need for molecular testing for pathogen identification, which is critical for decisions associated with therapeutics and biosecurity intervention for health management and vaccine evaluations and development.

Daqu is a typical multi-enzyme and multi-microbes fermentation starter of traditional Chinese liquor. The culture-independent molecular method has become a general method for the study of the Daqu microbial community. To determine the most suitable DNA extraction method of Daqu samples, the effects of different DNA extraction methods on Daqu genomic DNA extraction and amplicon libraries were evaluated. In this study, four DNA extraction methods were selected for the extraction of microbial DNA from two kinds of Daqu. Combined with DNA extraction quality, qPCR quantitative results, Illumina Miseq sequencing results of bacterial 16S rRNA gene, and fungal ITS gene amplicon library, the effects of different extraction methods of Daqu genomic DNA were evaluated. The comprehensive analysis of DNA yield and microbial biomass showed that the DNA yield of the SDS-based extraction method was higher than that of the kit extraction method, and the DNA yield of the in-situ SDS-based extraction method was as high as 5.46 x 104 ng/g, but the DNA purity was low. The qPCR quantitative results of the SDS-based extraction method were 101 - 6 orders of magnitude higher than those of the kit extraction method, and the microbial biomass of the washing-pretreatment plus SDS-based extraction method was the highest, which was 101-3 orders of magnitude higher than that of the in-situ SDS-based extraction method. Whereas the microbial biomass of the DNA extraction kit was the lowest, with only 102-4of the Shannon diversity index among amplicon libraries showed that different DNA extraction methods had significant differences among fungal ITS amplicon libraries, but no significant differences among bacterial 16S rDNA amplicon libraries. The Shannon index of the fungal amplicon library of the washing-pretreated samples were higher than that of the in-situ treated samples. By comparing the operational taxonomic units (OTU) with significant differences among the amplicon libraries, it could be seen that most of the OTUs in the bacterial 16S rDNA amplicon library had differences in abundance, but most of them could be detected in different DNA extraction methods. However, among the fungal ITS amplicon libraries, most OTUs showed a similar trend between different DNA extraction methods, indicating that the DNA extraction method had a significant effect on the extraction of some fungal microbial genomes, and the washing-pretreatment was beneficial to the extraction of fungal genomic DNA. Compared with the four DNA extraction methods, the washing-pretreatment plus SDS-based extraction method was the most suitable for the quantitative and community structure analysis of Daqu microorganisms. [ABSTRACT FROM AUTHOR]