Fecal Samples Research Articles

Landscape structure influences the eukaryome of a folivorous-frugivorous primate

ABSTRACT Eukaryotes are important components of primate gut communities. Despite their role in the diversity and structure of the gut ecosystem, microbiome research has focused on the prokaryotic component of the gut community. While gut bacteria are shaped by host phylogeny and diet, these factors are known to have negligible effects on eukaryotic diversity, which is expected to be modulated by the characteristics of the habitat. We assess the influence of landscape composition and configuration on the eukaryome of black and gold howler monkeys (Alouatta caraya). We collected fecal samples from 10 independent social groups inhabiting small forest fragments or orchards enriched with native tree species and applied an 18S rRNA gene fragment metabarcoding approach to describe their eukaryotic communities. We used generalized linear models to assess the power of landscape metrics in predicting the richness, diversity, evenness, and phylogenetic diversity of the eukaryome. Most communities were dominated by Ascomycota, and it is likely that many of the reads had an environmental origin. Forest cover affected eukaryome richness positively and patch density showed a positive relationship with the Simpson’s diversity and Pielou’s evenness indexes. Howler monkeys living in landscapes with lower habitat coverage had a lower eukaryotic alpha-diversity in the fecal microbiome.

Is Butyrate Concentration in the Equine Gastrointestinal Tract Altered During and After Surgery for Treatment of Large Colon Obstruction?

A major cause of morbidity and mortality in horses with large colon obstructive lesions is injury to the colonic mucosal barrier from ischemic injury. Since butyrate has been shown to play a critical role in the maintenance of a healthy mucosal barrier, it may play a role in the recovery process. This study’s objective was to determine whether the differences in butyrate concentrations existed between horses with surgical large colon obstructive lesions and healthy horses both during and after surgery. Eleven horses presenting with surgical colic lesions were enrolled; colonic samples were acquired during surgery, and fecal samples were obtained 36 h later. Colonic and fecal samples were also obtained from control groups. Samples were analyzed for butyrate, acetate, and propionate concentrations. There was no significant difference in butyrate content between surgical colonic or fecal samples and controls; however, an alteration in the proportion of SCFAs in relation to one another was noted. These changes in the individual SCFA levels were not statistically significant. The study findings demonstrated that there were no significant differences in butyrate proportions when comparing samples from horses with surgical colic lesions to healthy control horses.

Association analysis of gut microbiota with LDL-C metabolism and microbial pathogenicity in colorectal cancer patients

BackgroundColorectal cancer (CRC) is the most common gastrointestinal malignancy worldwide, with obesity-induced lipid metabolism disorders playing a crucial role in its progression. A complex connection exists between gut microbiota and the development of intestinal tumors through the microbiota metabolite pathway. Metabolic disorders frequently alter the gut microbiome, impairing immune and cellular functions and hastening cancer progression.MethodsThis study thoroughly examined the gut microbiota through 16S rRNA sequencing of fecal samples from 181 CRC patients, integrating preoperative Low-density lipoprotein cholesterol (LDL-C) levels and RNA sequencing data. The study includes a comparison of microbial diversity, differential microbiological analysis, exploration of the associations between microbiota, tumor microenvironment immune cells, and immune genes, enrichment analysis of potential biological functions of microbe-related host genes, and the prediction of LDL-C status through microorganisms.ResultsThe analysis revealed that differences in α and β diversity indices of intestinal microbiota in CRC patients were not statistically significant across different LDL-C metabolic states. Patients exhibited varying LDL-C metabolic conditions, leading to a bifurcation of their gut microbiota into two distinct clusters. Patients with LDL-C metabolic irregularities had higher concentrations of twelve gut microbiota, which were linked to various immune cells and immune-related genes, influencing tumor immunity. Under normal LDL-C metabolic conditions, the protective microorganism Anaerostipes_caccae was significantly negatively correlated with the GO Biological Process pathway involved in the negative regulation of the unfolded protein response in the endoplasmic reticulum. Both XGBoost and MLP models, developed using differential gut microbiota, could forecast LDL-C levels in CRC patients biologically.ConclusionsThe intestinal microbiota in CRC patients influences the LDL-C metabolic status. With elevated LDL-C levels, gut microbiota can regulate the function of immune cells and gene expression within the tumor microenvironment, affecting cancer-related pathways and promoting CRC progression. LDL-C and its associated gut microbiota could provide non-invasive markers for clinical evaluation and treatment of CRC patients.

A panoramic view of the molecular epidemiology, evolution, and cross-species transmission of rosaviruses

Rosavirus is a newly discovered member of the family Picornaviridae that was initially detected in wild rodents and subsequently in children with diarrhoea. Nevertheless, there is a significant gap in our understanding of the geographical distribution, phylogenetic relationships, evolutionary patterns, and transmission of rosaviruses. To address these issues, we analysed 434 rodents and shrews from five different species that were collected in southern China. Using PCR screening of faecal samples, we detected rosaviruses in Norway rats (Rattus norvegicus) and identified two previously undocumented host species: tanezumi rats (Rattus tanezumi) and Asian house shrews (Suncus murinus). Rosaviruses were particularly common in these animals, with an overall prevalence rate of 32.49% (141/434). For genetic and evolutionary analyses, we selected six representative positive samples to amplify the complete genomes of rosaviruses. Bayesian phylogenetic analysis suggested that our sequences clustered within the genus Rosavirus, where genotype B sequences are the closest relatives. The elevated nonsynonymous-to-synonymous ratios observed in rosavirus B may be attributed to relaxed selection pressures driven by virus spillover events. On the basis of the available data, it is hypothesized that the genus Rosavirus may have originated from Norway rats around the year 1339. In summary, these findings provide valuable insights into the complex evolutionary history of rosaviruses and underscore the urgent need for ongoing surveillance of this virus.

Child health, nutrition and gut microbiota development during the first two years of life; study protocol of a prospective cohort study from the Khyber Pakhtunkhwa, Pakistan

Recent evidence suggests that the development of gut microbiota during infancy affects several metabolic, immune, and endocrine pathways in humans. An imbalance in gut microbiota diversity or function, also known as dysbiosis, not only affects early child growth and development, but is also linked with the development of chronic, non-communicable diseases in later life. The Child Health And Microbiome Development Study – Pakistan (CHAMP) study aimed to longitudinally assess gut microbiota development and associated factors (maternal, child, and demographic) during early childhood in populations residing in malnutrition-endemic communities in Pakistan. A prospective cohort of mother-infant pairs (n=70) will be recruited from District Swat, Pakistan, and followed for two years. Complete information about demographic characteristics, anti-natal and post-natal care, dietary intake, feeding practices, and child health will be collected at baseline and 3, 6, 12, 18, and 24 months. Anthropometric measurements (height, weight, mid-upper arm circumference, and head circumference), dry blood spots, and fecal samples were also collected. Ethical approval for the study was obtained from Khyber Medical University, Pakistan. The study is also registered on clincaltrial.gov (Ref no: NCT05793294). The study findings will help researchers understand gut microbiota development, associated factors, and their impact on longitudinal growth in infants during the first two years of life.

Alterations of Gut Microbiome in Patients with Colorectal Advanced Adenoma by Metagenomic Analyses.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide, mostly arising from adenomatous polyps. Mounting evidence has demonstrated that changes in the gut microbiome play key roles in CRC progression, while quite few studies focused on the altered microbiota architecture of advanced adenoma (AA), a crucial precancerous stage of CRC. Thus, we aimed to investigate the microbial profiles of AA patients. Fecal samples were collected from 26 AA patients and 26 age- and sex-matched normal controls (NC), and analyzed by shotgun metagenomic sequencing. Gut microbial dysbiosis was observed in AA patients with lower alpha diversity. Advanced adenoma was characterized by an increased Bacillota/Bacteroidota ratio and higher Pseudomonadota levels compared to normal individuals. Linear discriminant analysis effect size (LEfSe) analysis was performed and identified 14 microbiota with significantly different abundance levels between AA and NC groups. Functional analysis revealed that tryptophan metabolism was upregulated in AA. Correspondingly, the expressions of gut microbes implicated in tryptophan metabolism also changed, including Akkermansia muciniphila, Bacteroides ovatus, Clostridium sporogenes, and Limosilactobacillus reuteri. The microbial network suggested that AA exhibited decreased correlation complexity, with Escherichia coli and Enterobacteriaceae unclassified harboring the strongest connectivity. A diagnostic model consisting of 3 microbial species was established based on random forest, yielding an area under the curve (AUC) of 0.799. Our study profiled the alterations of the gut microbiome in AA patients, which may enrich the knowledge of microbial signatures along with colorectal tumorigenesis and provide promising biomarkers for AA diagnosis.

MS2Lipid: A Lipid Subclass Prediction Program Using Machine Learning and Curated Tandem Mass Spectral Data

Background: Untargeted lipidomics using collision-induced dissociation-based tandem mass spectrometry (CID-MS/MS) is essential for biological and clinical applications. However, annotation confidence still relies on manual curation by analytical chemists, despite the development of various software tools for automatic spectral processing based on rule-based fragment annotations. Methods: In this study, we present a novel machine learning model, MS2Lipid, for the prediction of known lipid subclasses from MS/MS queries, providing an orthogonal approach to existing lipidomics software programs in determining the lipid subclass of ion features. We designed a new descriptor, MCH (mode of carbon and hydrogen), to increase the specificity of lipid subclass prediction in nominal mass resolution MS data. Results: The model, trained with 6760 and 6862 manually curated MS/MS spectra for the positive and negative ion modes, respectively, classified queries into one or several of 97 lipid subclasses, achieving an accuracy of 97.4% in the test set. The program was further validated using various datasets from different instruments and curators, with the average accuracy exceeding 87.2%. Using an integrated approach with molecular spectral networking, we demonstrated the utility of MS2Lipid by annotating microbiota-derived esterified bile acids, whose abundance was significantly increased in fecal samples of obese patients in a human cohort study. This suggests that the machine learning model provides an independent criterion for lipid subclass classification, enhancing the annotation of lipid metabolites within known lipid classes. Conclusions: MS2Lipid is a highly accurate machine learning model that enhances lipid subclass annotation from MS/MS data and provides an independent criterion.

Host genetics and gut microbiota influence lipid metabolism and inflammation: potential implications for ALS pathophysiology in SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons, with genetic and environmental factors contributing to its complex pathogenesis. Dysregulated immune responses and altered energetic metabolism are key features, with emerging evidence implicating the gut microbiota (GM) in disease progression. We investigated the interplay among genetic background, GM composition, metabolism, and immune response in two distinct ALS mouse models: 129Sv_G93A and C57Ola_G93A, representing rapid and slow disease progression, respectively.Using 16 S rRNA sequencing and fecal metabolite analysis, we characterized the GM composition and metabolite profiles in non-transgenic (Ntg) and SOD1G93A mutant mice of both strains. Our results revealed strain-specific differences in GM composition and functions, particularly in the abundance of taxa belonging to Erysipelotrichaceae and the levels of short and medium-chain fatty acids in fecal samples. The SOD1 mutation induces significant shifts in GM colonization in both strains, with C57Ola_G93A mice showing changes resembling those in 129 Sv mice, potentially affecting disease pathogenesis. ALS symptom progression does not significantly alter microbiota composition, suggesting stability.Additionally, we assessed systemic immunity and inflammatory responses revealing strain-specific differences in immune cell populations and cytokine levels.Our findings underscore the substantial influence of genetic background on GM composition, metabolism, and immune response in ALS mouse models. These strain-specific variations may contribute to differences in disease susceptibility and progression rates. Further elucidating the mechanisms underlying these interactions could offer novel insights into ALS pathogenesis and potential therapeutic targets.

Synbiotic Effects of Lacticaseibacillus paracasei K56 and Prebiotics on the Intestinal Microecology of Children with Obesity.

Lacticaseibacillus paracasei K56 (L. paracasei K56) is a probiotic with weight-loss effects. However, symbiosis research on the combined effects of Lacticaseibacillus paracasei K56 and prebiotics is lacking. Therefore, the aim of this study was to investigate the effects of L. paracasei K56, xylooligosaccharide (XOS), galactooligosaccharide (GOS), polyglucose (PG), and their synbiotic combinations (XOS + K56, GOS + K56, and PG + K56) on metabolism and gut composition in children with obesity, using an in vitro fermentation model. Fecal samples were collected from 14 children with obesity for in vitro fermentation, and the effects of the various treatments in gas production and short chain fatty acid synthesis (SCFAs) were assessed. Treatment with probiotics, prebiotics, and synbiotics regulated gut microbiota and metabolites in children with obesity. GOS and XOS had higher degradation rates than PG + K56 synbiotics in the gut microbiota of children with obesity. Moreover, treatment with XOS, GOS, and their synbiotic combinations, (XOS + K56) and (GOS + K56), significantly reduced the production of gas, propionic acid, and butyric acid compared with PG + K56 treatment. Treatments with GOS + K56 and XOS + K56 altered the composition of the gut microbiota, improved the abundance of Bifidobacteria and Lactobacilli, and reduced the abundance of Escherichia/Shigella. Overall, this study provides a theoretical foundation for the use of K56-based synbiotics.

Protective effect of ginseng extract and total ginsenosides on hematopoietic stem cell damage by inhibiting cell apoptosis and regulating the intestinal microflora.

Ginseng may improve the myelosuppression and intestinal microbiota disorder induced by cyclophosphamide (CY); however, the effect of ginseng components on hematopoietic stem cell (HSC) damage remains largely unexplored. The present study aimed to assess the protective effect of ginseng extract (GE), total ginsenosides (TG) and total polysaccharides (TP) from ginseng on the intestinal microflora and HSCs of model mice. In the present study, a mouse model of HSC damage induced by CY was constructed, intestinal microflora of fecal samples were sequenced using the 16S ribosomal RNA (rRNA) sequencing techniques, the differentially expressed genes (DEGs) of HSCs were analyzed using high‑throughput RNA‑sequencing, cell apoptosis and erythroid differentiation were detected using flow cytometry and the blood cell parameters were analyzed using a hematology analyzer. Analysis of the 16S rRNA in fecal samples showed that GE, TG and TP improved an imbalanced intestinal microflora, where the relative abundance of Lactobacillus intestinalis had a positive correlation with ginsenosides content. Specifically, TP significantly increased the expression of low‑abundance microflora. Transcriptomic analysis results revealed 2,250, 3,432 and 261 DEGs in the GE, TG and TP groups compared with those in the Model group, respectively. In the expression analysis of DEGs, both TG and GE were found to markedly increase the expression levels of Klf4, Hhex, Pbx1, Kmt2a, Mecom, Zc3h12a, Zbtb16, Lilr4b, Flt3 and Klf13. Furthermore, TG inhibited the apoptosis of HSCs by increasing the expression levels of Bcl2 and Mcl1, whilst decreasing the expression of Bax. By contrast, GE inhibited the apoptosis of HSCs by reducing the expression of Bax and Bad. Regarding erythroid differentiation and blood cell parameters, GE was found to significantly increase the expression of TER‑119. In addition, GE and TG improved all blood cell parameters, including the count of white blood cells, neutrophils (NEUT), lymphocytes (LYMPH), red blood cells (RBC), hemoglobin (HGB) and reticulocyte and platelets (PLT), whereas TP could only improve the counts of LYMPH, RBC, HGB and PLT. The improvement effect of GE and TG on WBC, NEUT and Ret was superior to TP. In conclusion, TG may protect the hematopoiesis function of HSCs in a CY‑induced mouse model of HSC damage, followed by GE. However, TP did not appear to improve HSC damage. Ginsenosides may therefore be considered essential ingredients in GE when protecting HSCs against damage. GE and TG exerted their protective effects on HSCs by inhibiting the apoptosis of HSCs whilst improving the imbalance of intestinal microflora.

The grey wolf (Canis lupus) as a host of Echinococcus multilocularis, E. granulosus s.l. and other helminths – a new zoonotic threat in Poland

Abstract Introduction The aim of this study was to estimate the occurrence of Echinococcus spp. and other helminth infections in grey wolves in south-eastern Poland. Material and Methods Overall, 74 samples of wolf faeces were examined with a multiplex PCR and a system of real-time quantitative PCR methods to detect and identify Echinococcus spp. The faeces were additionally examined microscopically. Also, 20 samples of wolf intestines were examined with a sedimentation and counting technique (SCT). Results Echinococcus multilocularis DNA was detected in 6.8% and E. granulosus s.l. (identified as E. ortleppi) in 4.1% of faeces samples. Taenia spp. DNA was found in 43.2% and Mesocestoides in 4.1%. Examination of the intestines by SCT showed E. multilocularis worms in 10%, E. granulosus s.l. (E. ortleppi) in10%, Taenia spp. in 100%, hookworms in 30%, Alaria alata in 20%, Mesocestoides sp. in 10%, Trichuris vulpis in 15%, Molineus sp. in 5% and Euryhelmis sp. in 5%. By coproscopy, Capillariidae eggs were found in 59% of faeces samples. Genetic analysis of E. multilocularis worms showed the presence of two European haplotypes previously described in Poland in red foxes and pigs. Sequences of nad1 obtained from E. ortleppi worms shared full identity with a sequence from a human case in Poland. Conclusion The study showed the presence of E. multilocularis in wolves for the first time in Poland and confirmed our earlier observations on E. ortleppi. This double threat from Echinococcus in this wolf population should be taken into account when assessing the epidemiological risk. The study enriched the knowledge of other helminths found in wolves, also those (Euryhelmis) that were recorded for the first time in this species.

Fecal and Sputum Microbiota and Treatment Response in Patients with Mycobacterium abscessus Pulmonary Disease.

The microbiota is a potential source of biomarkers for clinical outcomes in chronic respiratory conditions, but its role in Mycobacterium abscessus pulmonary disease (PD) remains unexplored. We aimed to identify microbial signatures in fecal and sputum microbiotas associated with treatment response in patients with M. abscessus PD. We conducted a cohort study prospectively enrolling patients undergoing antibiotic treatment for M. abscessus PD. Fecal and sputum samples were collected before, and 2 weeks and 6 months after, treatment initiation. 16S rRNA amplicon sequencing approach was used to characterize the microbiotas. After categorizing patients as early treatment responders and non-responders based on sputum culture results at 2 weeks, we elucidated the differences in their diversity and composition of microbiotas. Among 32 patients enrolled, 27 patients (median 66 years, 85.2% women, 48.1% with subspecies abscessus) were included for microbiota analysis. Fifteen patients (55.6%) achieved negative conversion at 2 weeks, which was maintained in 14 (93.3%) after 6 months. Alpha-diversity of the fecal microbiota after 2 weeks of treatment decreased significantly in responders, but not in non-responders (P=0.029). Increased abundance of Eubacterium hallii group in baseline fecal microbiota was indicative of unresponsiveness to antibiotics, whereas an increase in Enterococcus in fecal microbiota at week 2 was linked with favorable response. Increased abundance of Burkholderia-Caballeronia-Paraburkholderia and Porphyromonas in baseline, and decreased abundance of Rothia in week 2 sputum microbiota were also associated with good treatment response. In M. abscessus PD, changes in microbial diversity and compositional signatures vary with treatment response.

The gut microbiome is associated with susceptibility to febrile malaria in Malian children

Malaria is a major public health problem, but many of the factors underlying the pathogenesis of this disease are not well understood, including protection from the development of febrile symptoms, which is observed in individuals residing in areas with moderate-to-high transmission by early adolescence. Here, we demonstrate that susceptibility to febrile malaria following Plasmodium falciparum infection is associated with the composition of the gut microbiome prior to the malaria season in 10-year-old Malian children, but not in younger children. Gnotobiotic mice colonized with the fecal samples of malaria-susceptible children were shown to have a significantly higher parasite burden following Plasmodium infection compared to gnotobiotic mice colonized with the fecal samples of malaria-resistant children. The fecal microbiome of the susceptible children was determined to be enriched for bacteria associated with inflammation, mucin degradation and gut permeability, and to have increased levels of nitric oxide-derived DNA adducts and lower levels of mucus phospholipids compared to the resistant children. Overall, these results indicate that the composition of the gut microbiome is associated with the prospective risk of febrile malaria in Malian children and suggest that modulation of the gut microbiome could decrease malaria morbidity in endemic areas.

The role of gut microbiota in a generalist, golden snub-nosed monkey, adaptation to geographical diet change

Changes in diet causing ecological stress pose a significant challenge to animal survival. In response, the gut microbiota, a crucial part of the host’s digestive system, exhibits patterns of change reflective of alterations in the host’s food component. The impact of temporal dietary shifts on gut microbiota has been elucidated through multidimensional modeling of both food component and macronutrient intake. However, the broad distribution of wild generalist and the intricate complexity of their food component hinder our capacity to ascertain the degree to which their gut microbiota assist in adapting to spatial dietary variations. We examined variation in patterns of the gut microbial community according to changes in diet and in a colobine monkey with a regional variable diet, the golden snub-nosed monkey (Rhinopithecus roxellana). Specifically, we analyse the interactions between variation in food component, macronutrient intake and the gut microbial community. We compared monkeys from four populations by quantifying food component and macronutrient intake, and by sequencing 16S rRNA and the microbial macro-genomes from the faecal samples of 44 individuals. We found significant differences in the diets and gut microbial compositions, in nutrient space and macronutrient intake among some populations. Variations in gut microbiota composition across distinct populations mirror the disparities in macronutrient intake, with a notable emphasis on carbohydrate. Geographical differences in the diet among of golden snub-nosed monkey populations will result in macronutrient intake variation, with corresponding differences in macronutrient intake driving regional differences in the compositions and abundances of gut microbiota. Importantly, the gut microbiota associated with core digestive functions does not vary, with the non-core gut microbiota fluctuating in response to variation in macronutrient intake. This characteristic may enable species heavily reliant on gut microbiota for digestion to adapt to diet changes. Our results further the understanding of the roles gut microbiota play in the formation of host dietary niches.

Metabolomic profiling of human feces and plasma from extrauterine growth restriction infants.

Extrauterine growth restriction (EUGR) affects a substantial proportion of preterm infants and may influence both short-term complications and long-term sequelae. While many preterm infants with EUGR are secondary to small for gestational age (SGA) or very low birth weight (VLBW), a subset of EUGR infants do not exhibit these conditions. The purpose of this study is to investigate the metabolic profiles and biomarkers of EUGR infants in the absence of SGA and VLBW. A total of 100 feces (n = 50) and plasma samples (n = 50) were collected from participants categorized as either EUGR (EUGR group) or non-EUGR (NonEUGR group) in the absence of SGA and VLBW. Metabolites were characterized via UPLC-MS/MS using the Discovery HD4® platform. Data normalization, partial least squares discriminant analysis (PLSDA), and KEGG enrichment analysis of metabolite profiles were performed using the MetaboAnalyst 6.0. The clinical characteristics of preterm infants differed significantly between the EUGR and NonEUGR groups at discharge, including length of stay, weight Z-score, weight, height Z-score, height, head circumference, and fat-free mass. The PLSDA model exhibited clustering within groups and separation between groups. A total of 58 and 71 differential metabolites were identified in feces and plasma samples, respectively. They were involved in pathways such as caffeine, galactose, glutathione, cysteine, and methionine metabolisms. In the feces sample, 1-palmitoyl-galactosylglycerol exhibited a significant negative correlation with the growth characteristics of preterm infants, while 1-palmitoyl-2-palmitoleoyl-GPC displayed the opposite pattern. In plasma samples, androsterone glucuronide displayed a significant positive correlation with the growth characteristics of preterm infants, whereas 2-methoxyhydroquinone sulfate generated an opposite pattern. Moreover, 2-oleoylglycerol and sphinganine-1-phosphate exhibited the highest area under the curve in feces and plasma samples, respectively, according to diagnostic ROC curves. Preterm infants with EUGR, in the absence of SGA and VLBW, exhibit specific clinical characteristics and metabolomic profiles. Sphinganine-1-phosphate and 2-oleoylglycerol may hold promise as diagnostic markers for EUGR in the absence of SGA and VLBW. The objective of this study is to identify the differential metabolites in preterm infants with extrauterine growth restriction (EUGR) in the absence of small for gestational age (SGA) or very low birth weight (VLBW). Preterm infants with EUGR without SGA and VLBW exhibit specific clinical characteristics and metabolomic profiles. Sphinganine-1-phosphate and 2-oleoylglycerol emerged as potential diagnostic biomarkers for EUGR. This study enhances our understanding of the metabolomic profile in preterm infants with EUGR without SGA or VLBW. Our findings will offer valuable evidence for improving nutritional management and shedding light on the associated pathophysiological mechanisms of EUGR.

Rapid production of recombinant rotaviruses by overexpression of NSP2 and NSP5 genes with modified nucleotide sequences.

Reverse genetics systems for rotaviruses (RV) facilitate the generation of genetically engineered RVs by transfection of 11 plasmids encoding 11 genomic viral RNA segments. In addition to viral genome expression, overexpression of NSP2 and NSP5 has been used to increase the rescue efficiency of recombinant RVs. Here, we showed that the overexpression of nucleotide sequence-modified NSP2 and NSP5 enabled the rapid and efficient production of recombinant RVs. Using improved reverse genetics, we established a reverse genetics system for human and bovine RV clinical isolates, as well as laboratory strains of bovine RV (NCDV and UK) and porcine RV (Gottfried). In addition, we rescued low-replicating recombinant RVs carrying a mutant NSP4 lacking the double-layered particle-binding domain, which was deficient in the efficient production of mature virions. These advancements in reverse genetics enabled the generation of molecular clones of RV clinical isolates and recombinant RVs harboring critical amino acid mutations, offering a versatile platform for investigating RV biology and pathogenesis.IMPORTANCERecombinant rotavirus (RV) synthesis via reverse genetics relies on both the viral propagation capacity and the efficiency of the experimental system. Since the establishment of our reverse genetics system, several enhancements have been implemented to augment the rescue efficiency. Nevertheless, challenges persist in generating RV clinical strains and recombinant viruses with low replication capacities. Notably, this improved reverse genetics system successfully facilitated the establishment of molecular clones of human and bovine RV clinical isolates. Fecal samples from patients with RV typically harbor quasi-species or, occasionally, multiple genotypes of RV. In the present study, we performed the genetic sequencing of clinical viral strains during the early propagation stages in cultured cells. Subsequently, infectious viruses were synthesized, allowing the characterization of circulating viruses in nature. This approach provides valuable insights into the genetic diversity and dynamics of RV populations and contributes to a more comprehensive understanding of viral pathogenesis and evolution.

Urban Pigeons as Reservoirs of Critical Pathogens: Improved protocol for sequencing pigeon faeces in disease monitoring

The spread of pathogens by animals is a serious issue around the world that causes a severe threat to human health. Feral pigeons (Columba livia forma domestica) that live in urban areas are zoonotic carriers of various pathogens that can be transmitted to humans by faecal contamination. This study aimed to detect the presence of bacterial, viral, and specifically fungal pathogens in pigeon faeces based on the World Health Organization's (WHO) priority pathogen list published in 2022. Fresh faecal samples were collected at Uppsala, Central Station, and Svandammen, the pigeons' most relevant gathering spots and feeding sites. Genomic DNA was directly extracted from these samples, and ScilifeLab performed High throughput sequencing through Oxford Nanopore Technologies(ONT) (PromethION). Metagenomic analysis revealed that most of the critically prioritized viral and bacterial pathogens listed by WHO were present in pigeon faeces. Regarding fungal pathogens, which were the main objective of this study, samples from both studied locations contained all critical, high and, medium-important fungal pathogens published in the WHO list, such as Aspergillus fumigatus, Candida albicans, Candida auris, Cryptococcus neoformans, Nakaseomyces glabratus, Candida tropicalis, and Cryptococcus gattii. These fungal pathogens pose the risk of invasive fungal diseases and severe infections in low-immunity individuals and vulnerable populations. The findings indicate the importance of conducting further research to comprehensively understand potential exposure to feral pigeons. Furthermore, keeping pigeons away from sensitive areas, such as hospitals, and implementing measures to control pigeon populations can significantly decrease the spread of pathogens.

Identification of a novel GP60 subtype family of Cryptosporidium wrairi from capybaras (Hydrochoerus hydrochaeris) inhabiting urban areas in the state of Mato Grosso do Sul, Brazil

Capybaras (Hydrochoerus hydrochaeris) are hosts for several parasites of public health importance, including Cryptosporidium spp. Therefore, this study aimed to perform the molecular characterization of Cryptosporidium spp. in fecal samples from capybaras inhabiting urban areas. We analyzed 401 fecal samples from capybaras in two municipalities of the state of Mato Grosso do Sul, Brazil. Fecal samples were purified using centrifugal sedimentation with ethyl acetate. They were then screened for Cryptosporidium spp. by malachite green negative staining and a nested PCR protocol targeting the 18S rRNA gene. Samples positive by microscopy or PCR were examined by PCR protocols targeting the actin, HSP-70, and GP60 genes. Amplicons from all PCR protocols were subjected to genetic sequencing. Microscopic screening and 18S rRNA gene-targeted PCR identified 0.25 % (1/401) and 0.5 % (2/401) of samples, respectively, as positive for Cryptosporidium spp. The genetic sequences of the 18S rRNA, HSP-70, actin, and GP60 genes showed genetic similarity to Cryptosporidium wrairi sequences of 99.07 %, 99.69 %, 99.57 %, and 91.51 %, respectively. Genetic sequencing and phylogenetic analyses identified the novel GP60 subtype family VIIbA13 of C. wrairi. In conclusion, we report in this study a low prevalence of the novel GP60 subtype family VIIbA13 of C. wrairi in free-living capybaras from urban areas of the state of Mato Grosso do Sul, Brazil. We propose that capybaras act as a novel host for C. wrairi.

Identification and antimicrobial susceptibility profiles of Campylobacter isolated from camel at municipal abattoirs in eastern Ethiopia

Campylobacteriosis is an infectious zoonotic disease caused by the genus Campylobacter. The disease is transmitted from animals to humans predominantly through the consumption of contaminated food and water. However, the lack of information on the status of Campylobacter makes it difficult to quantify the role of camel meat in the dissemination of the pathogen. A cross-sectional study was conducted from June 2022 to August 2023 to investigate the occurrence of Campylobacter and associated risk factors and to determine the antimicrobial susceptibility profiles of Campylobacter species from camels slaughtered at municipal abattoirs in the towns of Harar, Babile, Jigjiga and Dire Dawa in eastern Ethiopia. A total of 324 (146 carcass swabs, 146 camel feces and 32 abattoir environmental swab samples) were collected and analyzed using TaqMan real-time PCR and culture techniques following standard procedures. In addition, antimicrobial susceptibility tests were performed using the disk diffusion method for eight antimicrobial agents according to the Clinical Laboratory Standards Institute. The overall prevalence of Campylobacter was 7.7%. Campylobacter was more frequently detected from carcasses and surface contact environmental swabs. We isolated Campylobacter at the genus level from approximately half of the PCR-positive samples, representing 54.2% (13 out of 24) of the total. The isolation levels of C. jejuni and C. coli were also 5.56% and 2.2%, respectively, which varied significantly (p = 0.037) based on sample type and site. The odds of occurrence of C. jejuni in samples collected from abattoir environments was 7.52 times greater than those in carcass and fecal samples. We detected resistance to chloramphenicol (78.6%), followed by amoxicillin (71.4%). However, 93%, 78.6%, and 71.4% of the isolates were susceptible to ceftriaxone, ciprofloxacin, and nalidixic acid, respectively. Multidrug resistance (MDR) was detected in 60% of the isolates. Of these MDR isolates, 9 (75%) were C. jejuni and 3 (25%) were C. coli. This study revealed that a considerable proportion of multidrug-resistant Campylobacter species circulate in both camel meat and abattoir environments. This indicates possible carcass cross-contamination by Campylobacter during slaughtering that can pose a threat to humans and limit therapeutic options, which could be prevented by applying good hygienic practices at abattoirs. Therefore, abattoir workers need to be aware of abattoir hygienic standard operating procedures. Regular coordinated actions should be implemented for the rational use of veterinary and medical drugs at the national level, together with training and awareness of hygienic practices.

A growing-finishing diet formulated to reduce the soybean meal does not compromise the growth performance, health, behaviour and gut health of Italian heavy pigs

Soy contributes to the environmental impact of Italian pork production. This study aimed to evaluate the effect of a reduced soybean meal (SBM) and crude protein (CP) finishing diet on growth performance, health, behaviour and gut health of Italian heavy pigs. 1920 pigs (35.6 kg body weight; (BW)) balanced by sex and BW were assigned to the control diet (CO), or the treated diet (TRT) formulated by reducing SBM by 31%, 67% and 69% (replaced by pea and sunflower meal) in 3 feeding phases, respectively, and 1.2% CP in the third phase. 251 pigs were individually weighed at d11, d94 and d181. Feed intake (FI), behavioural indices and air gases at pen-level were monitored monthly. Faecal samples (20/pigs/group) for microbiota, ammonia and volatile fatty acids (VFAs) and hair for stress biomarkers were collected. Diet did not affect final BW, faecal ammonia, cortisol and dehydroepiandrosterone. From d11–d94, the CO group had higher gain to feed (G:F) (p = .007), favourable faecal VFAs profile and a lower environmental ammonia (p < .0001). From d10–d181, the TRT diet increased the ADG (p = .04) and G:F (p = .01), reduced FI (p < .0001) and the lesions score index at d102 (p = .03) and promoted Methanobrevibacter (d94; padj. = 0.013) and Clostridium sensu stricto (d181; padj. = 0.001). Overall, the TRT diet combined with the stress of the transport and acclimatisation to the farm may limit the growth of pigs in the initial period, but it can increase their growth in the long term. Concluding, replacing 56% of SBM with sustainable alternatives seems promising for heavy pig.