Hypervariable Region Research Articles

Maternal versus environmental contributions to the piglet pioneer microbiome.

The pioneer microbiome is the initial colonization and establishment of microorganisms within the neonate. The objective of this project was to quantify maternal and environmental contributions to the piglet's pioneer microbiome. Sterile swabs were used to collect samples from the gilt's rectum, the farrowing crate before and after gilts were moved in, the gilt's birth canal during farrowing, and the piglet's rectum on days 0 (prior to suckling), 3, and 10 post-farrowing and at weaning (21.6 ± 1.0 days post-farrowing). During farrowing, colostrum was collected from each gilt from a representative sample of teats into a single sterile collection cup. Bacterial DNA extraction and sequencing targeted the V4 hypervariable region of the 16S rRNA gene. The relative abundance of Lactobacillus in the piglet microbiome was lower on day 3 compared to day 0, 10, and at weaning (P < 0.05). For alpha diversity, piglet samples exhibited distinct clustering for bacterial richness by day (P < 0.01). Multiple regression analyses indicated that the birth canal explained 51.6% of the variation observed in the piglet day 0 microbiome (P < 0.0001) and 6.5% of the variation in the piglet day 10 microbiome (P = 0.013). The day 10 microbiome explained 58.6% of the variation observed in the piglet microbiome at weaning (P < 0.0001). Bacterial communities of the farrowing crate and colostrum did not impact the piglet microbiome for any day (P > 0.10). Results indicate that the piglet pioneer microbiome is largely influenced by the microbiome of the birth canal.

Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

On-farm study: cytokine profiles and vaginal microbiome of Bos indicus cattle before artificial insemination

Prior studies in beef cattle have shown shifts in the reproductive microbiome prior to artificial insemination (AI), yet few have characterized both the reproductive microbiome and immune responses prior to AI, particularly in purebred Bos indicus. Therefore, the aim of this study was to characterize the circulating cytokines and the vaginal microbiome of Bos indicus females prior to fixed-time artificial insemination (FTAI). Bos indicus females (n = 79) on four separate ranch operations within a 20-mile radius in East Texas were subjected to the 7-day CO-Synch + controlled intervaginal drug-releasing (CIDR) protocol beginning on day (d)-9 with FTAI on d0. Blood samples were collected on d-9 (CIDR IN), d-2 (CIDR OUT), and d0 (AI DAY) for cytokine concentration analyses using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per the manufacturer’s instructions. Sterile vaginal swabs were inserted past the vulva, rotated, and stored at -80°C for microbiome analysis. Bacterial community analyses targeted the V4 hypervariable region of the 16S rRNA gene. Pregnancy status was determined by transrectal ultrasonography approximately 60 days after FTAI for resulting open females (n = 45) and pregnant females (n = 34). Regardless of pregnancy status, the vaginal relative abundance of Firmicutes differed between CIDR IN, CIDR OUT, and AI DAY (63.74% vs. 28.31% vs. 60.86% ± 3.66%, respectively; P &amp;lt; 0.01). Genera with phylum Firmicutes including Ruminococcus, Clostridium, Blautia, Butyrvibrio, and Mogibacterium followed a similar trend (P &amp;lt; 0.05). Butyrvibrio tended to have greater relative abundance in the vaginal samples of Cows than Heifers (4.17% ± 0.75% vs. 3.26% ± 0.77%; P = 0.07). Concentrations of the interferon (IFN)γ (2005.98 ± 471.94 pg/mL vs. 1185.40 ± 482.65 pg/mL; P &amp;lt; 0.01), interleukin (IL)1F5 (153.89 ± 141.07 pg/mL vs. 627.30 ± 149.28 pg/mL; P &amp;lt; 0.01), and interferon gamma-induced protein (IP)10 (9363.26 ± 2929.83 pg/mL vs. 5905.53 ± 2983.60 pg/mL; P = 0.05) were greater in Cows than Heifers. There was a parity-by-status interaction for IP10, with Open Cows having the greatest concentration compared all other groups (P &amp;lt; 0.05). These results indicate differences in circulating cytokines and shifts in the vaginal microbiome for in Bos indicus cattle prior to FTAI.

Genomic characterization of infectious bursal disease virus in Argentina provides evidence of the recent transcontinental spread of Chinese genotype A2dB1b

ABSTRACT The infectious bursal disease virus (IBDV) is a significant pathogen affecting the poultry industry worldwide. Its epidemiological history has been marked by the emergence of strains with different antigenic, pathogenic, and genetic features, some of which have shown notable spread potential. The A2dB1b genotype, also known as novel variant, has become widespread and gained increased relevance in IBDV epidemiology. This genotype was described in China in the 2010s and rapidly spread in Asia and Africa. The present study describes the circulation of the A2dB1b genotype in Argentina. Applying a next-generation sequencing approach, we obtained the complete coding sequence of 18 Argentine viruses. The high level of genomic homogeneity observed amongst these viruses, their monophyletic clustering in both partial and complete segments A and B derived phylogenies, and their close relatedness to some Chinese strains suggest that a unique transcontinental spread event from China to Argentina occurred recently. The apparent success of the A2dB1b genotype spreading throughout Asia, Africa, and South America may partially be due to specific amino acid characteristics. Novel residues in the hypervariable region of VP2 may help A2dB1b IBDVs evade the protection elicited by the applied commercial vaccines. Our findings underscore the importance of continuous characterization of field samples and evaluation of the control measures currently applied to fight against this specific IBDV genotype.

The bacterial and viral communities associated with onion bacterial bulb rot

Microbes occur in complex communities within plants as endophytes and establish a network of interactions that can influence plant health positively or negatively. Positive interactions may be synergistic and confer abiotic and biotic stress tolerance. The aim of this study was to identify bacteria and viruses present in storage onion bulbs that were symptomatic and asymptomatic for bacterial bulb rot, from crops grown in Georgia and Washington states; and to assess their potential role in the bulbs based on functions of bacterial and viral genes detected. DNA was extracted from nine asymptomatic bulbs and nine bulbs displaying symptoms of bacterial bulb rot and subjected to 16S rRNA amplicon sequencing and Metagenomic Assembled Genome (MAG) analysis. The Illumina platform was used to sequence the hypervariable region (V3-V4) of the 16S rRNA gene. The 16S rRNA amplicon profiling revealed the presence of numerous bacteria including potential onion pathogens in the genera Panteoa and Burkholderia. MAG assembly identified P. agglomerans. B. gladioli and B. cepacia, known bulb rot pathogens, including genes linked to fitness and those involved in the Type II and III secretion systems. Eighty-nine unique viral genomes were identified, of which 67 could be classified taxonomically. The bacterial and viral genomes differed significantly in asymptomatic versus symptomatic bulbs. Viral genomes showed evidence of auxiliary metabolic genes (AMGs), including genes involved in fitness and pathogenicity to bacterial hosts. The bulbs hosted endophytic bacteria and viruses, some of which were potentially beneficial, and others potentially pathogenic to onion or as hosts to bacteriophages.

Molecular epidemiology and strain diversity of circulating feline Calicivirus in Thai cats.

Feline calicivirus (FCV) is a significant viral pathogen causing upper respiratory tract and oral diseases in cats. The emergence of the virulent systemic FCV variant (VS-FCV) has raised global concern in the past decade. This study aims to explore the epidemiology, genetic characterization, and diversity of FCV strains circulating among Thai cats. Various sample types, including nasal, oral, and oropharyngeal swabs and fresh tissues, were collected from 184 cats across different regions of Thailand from 2016 to 2021. Using reverse transcription real-time polymerase chain reaction (RT-qPCR), FCV infection was investigated, with additional screening for feline herpesvirus-1 (FHV-1) by qPCR. The detection rates for FCV, FHV-1, and co-infection were 46.7, 65.8, and 31.5%, respectively. Significantly, the odds ratio (OR) revealed a strong association between the detection of a single FCV and the presence of gingivostomatitis lesions (OR: 7.15, 95% CI: 1.89-26.99, p = 0.004). In addition, FCV detection is notably less likely in vaccinated cats (OR: 0.22, 95% CI: 0.07-0.75, p = 0.015). Amino acid sequence analysis based on the VP1 major capsid protein gene of the 14 FCV-Thai (FCV-TH) strains revealed genetic diversity compared to the other 43 global strains (0 to 86.6%). Intriguingly, a vaccine-like FCV variant was detected in one cat. In summary, this study provides insights into the epidemiology and molecular characteristics of FCV diversity within the Thai cat population for the first time. The identification of unique physicochemical characteristics in the capsid hypervariable region of some FCV-TH strains challenges previous hypotheses. Therefore, further exploration of vaccine-like FCV variants is crucial for a comprehensive understanding and to improve viral prevention and control strategies.

Mitochondrial DNA in Fornescis: Principles, Applications, and Limitations

Forensic science involves the scientific analysis and interpretation of evidence that is used in legal proceedings to establish guilt or innocence. The use of mitochondrial DNA (mtDNA) in forensics is crucial as it has several advantages over other types of DNA. For instance, mtDNA is more resistant to degradation than nuclear DNA, making it suitable for analysis even in small or degraded samples. Additionally, mtDNA is maternally inherited, and this makes it useful in tracing an individual's maternal lineage or identifying relationships between individuals across multiple generations. Another advantage of mtDNA analysis is its reliability and accuracy since it is less prone to errors or contamination. This is because mtDNA is found in the mitochondria, which are separate from the cell nucleus and are less likely to be affected by extraneous DNAor contamination. Overall, the use of mtDNA in forensic science has significantly improved the accuracy and reliability of criminal investigations, making it a vital tool in the criminal justice system. In mitochondrial DNA (mtDNA) analysis, the two regions of the mtDNA genome that are typically analyzed are hyper variable regions 1 and 2 (HV1 and HV2). HV1 and HV2 are non-coding regions of the mtDNA genome, meaning that they do not contain information for the production of proteins. Instead, they contain a high degree of variation in their nucleotide sequences, which makes them useful for identifying individuals and their maternal lineages. The interpretation of mtDNA analysis results from HV1 and HV2 involves comparing the nucleotide sequences obtained from the evidence sample to a reference database of mtDNA sequences. This comparison is done to identify any matches or differences between the sample and the reference sequences. If there are no differences or only a few, the sample is considered to be a match to the reference sequence. The more differences there are between the sample and the reference sequences, the less likely it is that the sample and reference sequences came from the same individual or maternal lineage.The interpretation of mtDNA analysis results from HV1 and HV2 can be used to provide information about the identity and maternal lineage of an individual. For example, if an evidence sample matches a reference sequence from a known maternal relative, this can be used to support the identification of the individual,

Novel indigenous probiotic isolated from the healthy Pacific white shrimp Litopenaeus vannamei intestine in differing stages based on metagenomic and screening approaches

The healthy intestinal microbiota of shrimp can be used as an indicator sustainable shrimp production. In this study, the integrated of metagenomic and screening probiotic approach from healthy Litopenaeus vannamei intestines in differing stages was studied to find novel indigenous probiotics. The microbiota from intestine of naupli, post larva (PL-10), juvenile (40 days), and adult (80 days) of Pacific white shrimp were characterized using a high-quality sequence of V3–V4 of 16S rRNA gene as the hypervariable region. The classifiable sequence number was detected in 54 phyla. Several core intestine bacteria, 35 of these 557 genera, have a prevalence >10 sequences across all samples. We found microbiota were different taxa in the difference stages, such as Proteobacteria, Firmicutes, and Bacteriodetes. The top 10 most abundant genera were Vibrio, Pseudoalteromonas, Spingomonas, Marinibacterium, Klebsiella, Alteromonas, Aestuaribacter, Shimia, Stenotrophomonas, and Ruegeria. Microbiota profiling based on a metagenomic approach was integrated with screening assessment for pathogenicity, antagonistic activity with Vibrio parahaemolyticus Vp5, antibiotic resistance, and digestive enzyme activities. As their assessment activity, several screened culturable bacteria were 19 of these 84 isolates. Three isolates with high activities (P < 0.05) found as novel indigenous probiotics were Shewanella algae A1, Shewanella algae A3, and Vibrio diabolicus UB3. Integrating metagenomic and screening methods was a new signature for the isolating novel indigenous probiotics in Pacific white shrimp.

Analyzing imbalance of short tandem repeats for pancreatic cancer detection.

e16359 Background: Short tandem repeats (STRs), alternatively known as microsatellites, encompass repeat units of 1 to 6 base pairs (bp) in the genome and function as regulators of gene expression, impacting plasticity and complex traits. STRs are prone to hypermutation with alterations correlated to pathogenicity in multiple Mendelian and acquired disorders. STR variant classes include insertions, mobile element insertions (MEIs), deletions, and multi-allelic copy number variants (mCNVs). These can lead to repeat expansions, allelic imbalance (AI), and microsatellite instability (MSI) observed in diseases such as Huntington's disease, hereditary ataxia, and multiple cancers. In pancreatic cancer, AI and associated loss-of-heterozygosity (LOH) at tumor suppressor loci flanked by hypervariable STR regions correspond to a pathogenic phenotype with a risk of aggressive disease. Consequently, it is critical to develop accurate and robust methods to precisely identify and genotype pancreatic samples with varying specimen quality in a clinical setting to aid in diagnosis. Methods: Capillary electrophoresis (CE) and Sanger sequencing have been used to analyze STRs. The availability of newer tools such as 2nd generation short-read (NGS) and 3rd generation long-read (TGS) sequencers could allow for high throughput scaling, however, informatics challenges associated with analyzing repetitive regions remain. Here we developed amplification and enrichment methods for targeted analysis for detection by CE, NGS and TGS. Results: Amplification and enrichment strategies for detecting 17 short tandem repeat regions that play a role in pancreatic carcinogenesis were developed. Methods were optimized for detecting STR alterations in cell-free nucleic acids isolated from endoscope-guided pancreatic fine needle aspirate biopsies. Furthermore, informatics tools were developed to analyze short-read and long-read sequencing data to accurately analyze repeat regions. Conclusions: Here we compared 3 platforms (CE, NGS, and TGS) and found that CE remains the gold standard for analyzing AI/LOH with data comparable to long-read TGS, while short-read NGS is not always amenable to accurate analysis dependent on the region of interest. Additionally, we found that artifacts of amplification persist, irrespective of the 3 platforms analyzed.

Fowl Aviadenovirus (FAdV-11) as the causative agent of a vertical outbreak of inclusion body hepatitis in commercial broiler breeders in Brazil

Inclusion body hepatitis (IBH), caused by Fowl Aviadenovirus (FAdV), is a significant disease affecting chickens worldwide. FAdV-11 associated with IBH was identified in 9-day-old commercial broiler breeders in Brazil. The chicks exhibited a sudden increase in daily mortality, accompanied by nonspecific clinical signs, in one house on the farm. The cumulative mortality rate reached 11.13 % by 15 days of age. Macroscopically, the liver was markedly enlarged and pale red with a distinct lobular pattern. Histologically, the liver showed mild to severe multifocal coalescent vacuolar degeneration and necrosis, with hepatocyte nuclei diffusely enlarged and containing large basophilic inclusion bodies. Additionally, the heart exhibited a diffuse, moderate infiltration of lymphocytes and plasma cells, with focal thrombosis in the epicardium. FAdV was detected in liver samples using specific primers for the hexon gene (52 K-fw and 52 K-rv) in a polymerase chain reaction (PCR) assay. Sequencing of the hypervariable region of this gene (Hex L1) for phylogenetic analysis revealed FAdV-D species serotype 11. To our knowledge, this is the first report of FAdV-D serotype 11 as the causative agent of a vertical outbreak of inclusion body hepatitis in broiler breeders in Brazil.

Ex-vivo investigation of human salivary microbial growth with lysogeny broth for translational research–A pilot study

Background/purposeSalivary microbiome has become a surrogate indicator of oral disease due to its collective reservoirs and convenience in sampling. However, failed clinical trials often lead to wastes of resources, indicating a need for preclinical models. In this pilot study, we aimed to compare the salivary microbiome by metagenomics analysis before and after lysogeny broth culture for prospective translational studies. Materials and methodsThe study cohort included seven patients with severe periodontitis (Stage III/IV, Grade C), from whom unstimulated saliva was collected. The salivary microbiome was sequenced over the 16S rRNA gene V3–V4 hypervariable regions at baseline and after 6 hours of lysogeny broth culture. ResultsThe results revealed changes in salivary microbiome and reduced bacterial diversity after culture, mainly due to the expansion of genera Neisseria (Median (Mdn) 15.95% to 37.52%, P < 0.05), Rothia (Mdn 10.21% to 16.32%, P < 0.05), and Haemophilus (Mdn 5.88% to 13.25%, P < 0.05). Periodontitis-related pathogens such as phyla Bacteroidetes, Fusobacteria and Spirochaetes were identified, while genera Porphyromonas, Parvimonas, Peptostreptococcus, and Campylobacter showed a decrease after lysogeny broth culture. Caries-related pathogens, including genera Veillonella, Leptotrichia, and species Haemophilus parainfluenzae and Streptococcus salivarius, were also detected. ConclusionThis pilot study revealed that periodontitis- and caries-related bacteria could be identified in the saliva at baseline and after 6 hours ex-vivo culture with lysogeny broth. Our findings also suggested that lysogeny broth favored the growth of specific genera and may serve as a reference to monitor short-term modulation of these bacteria in salivary microbiome.

Next-Generation Sequencing of the Human Aqueous Humour Microbiome.

The microbiome of the ocular surface has been characterised, but only limited information is available on a possible silent intraocular microbial colonisation in normal eyes. Therefore, we performed next-generation sequencing (NGS) of 16S rDNA genes in the aqueous humour. The aqueous humour was sampled from three patients during cataract surgery. Air swabs, conjunctival swabs from patients as well as from healthy donors served as controls. Following DNA extraction, the V3 and V4 hypervariable regions of the 16S rDNA gene were amplified and sequenced followed by denoising. The resulting Amplicon Sequence Variants were matched to a subset of the Ribosomal Database Project 16S database. The deduced bacterial community was then statistically analysed. The DNA content in all samples was low (0-1.49 ng/µL) but sufficient for analysis. The main phyla in the samples were Acinetobacteria (48%), Proteobacteria (26%), Firmicutes (14%), Acidobacteria (8%), and Bacteroidetes (2%). Patients' conjunctival control samples and anterior chamber fluid showed similar patterns of bacterial species containing many waterborne species. Non-disinfected samples showed a different bacterial spectrum than the air swab samples. The data confirm the existence of an ocular surface microbiome. Meanwhile, a distinct intraocular microbiome was not discernible from the background, suggesting the absence of an intraocular microbiome in normal eyes.

Molecular determinants of Ras-mTORC2 signaling

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC’s specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC’s membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC’s own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

Identifying SNP threshold from P2 sequences for investigating norovirus transmission

Noroviruses are a group of non-enveloped single-stranded positive-sense RNA virus belonging to Caliciviridae family. They can be transmitted by the fecal-oral route from contaminated food and water and cause mainly acute gastroenteritis. Outbreaks of norovirus infections could be difficult to detect and investigate. In this study, we developed a simple threshold detection approach based on variations of the P2 domain of the capsid protein. We obtained sequences from the norovirus hypervariable P2 region using Sanger sequencing, including 582 pairs of epidemiologically-related strains from 35 norovirus outbreaks and 6402 pairs of epidemiologically-unrelated strains during the four epidemic seasons. Genetic distances were calculated and a threshold was performed by adopting ROC (Receiver Operating Characteristic) curve which identified transmission clusters in all tested outbreaks with 80 % sensitivity. In average, nucleotide diversity between outbreaks was 67.5 times greater than the diversity within outbreaks. Simple and accurate thresholds for detecting norovirus transmissions of three genotypes obtained here streamlines molecular investigation of norovirus outbreaks, thus enabling rapid and efficient responses for the control of norovirus.

Effects of Scallop Mantle Toxin on Intestinal Microflora and Intestinal Barrier Function in Mice.

Previous studies have shown that feeding mice with food containing mantle tissue from Japanese scallops results in aggravated liver and kidney damage, ultimately resulting in mortality within weeks. The aim of this study is to evaluate the toxicity of scallop mantle in China's coastal areas and explore the impact of scallop mantle toxins (SMT) on intestinal barrier integrity and gut microbiota in mice. The Illumina MiSeq sequencing of V3-V4 hypervariable regions of 16S ribosomal RNA was employed to study the alterations in gut microbiota in the feces of SMT mice. The results showed that intestinal flora abundance and diversity in the SMT group were decreased. Compared with the control group, significant increases were observed in serum indexes related to liver, intestine, inflammation, and kidney functions among SMT-exposed mice. Accompanied by varying degrees of tissue damage observed within these organs, the beneficial bacteria of Muribaculaceae and Marinifilaceae significantly reduced, while the harmful bacteria of Enterobacteriaceae and Helicobacter were significantly increased. Taken together, this article elucidates the inflammation and glucose metabolism disorder caused by scallop mantle toxin in mice from the angle of gut microbiota and metabolism. SMT can destroy the equilibrium of intestinal flora and damage the intestinal mucosal barrier, which leads to glucose metabolism disorder and intestinal dysfunction and may ultimately bring about systemic toxicity.

Endophytic bacterial diversity in the latex-bearing caulosphere of Hevea brasiliensis Müll. Arg.

Rubber trees are a commercial cash crop, and the milky latex or polyisoprene they produce is the natural source of rubber. Little is known about the bacterial populations found in active zone of latex-bearing caulosphere. We employed a tailored cloud microbial bioinformatic approach for the identification and potential hypothetical ecological roles of an uncultured endophytic hidden bacterial community in the active zone of the latex-bearing caulosphere of Hevea brasiliensis. Small pieces of slivers were collected from healthy plant from the village: Belonia, South Tripura, rubber plantation in Northeastern India. These uncultured bacteria were identified using the V3-V4 hypervariable amplicon region of the 16S rDNA gene. A total of 209,586 contigs have been generated. EasyMAP Version 1.0, a cloud-based microbial bioinformatics tool with an integrated QIIME2 pipeline, was used to analyze contigs. We detected 15 phyla and 91 OTUs (operational taxonomic units). Proteobacteria (73.5%) was the most enriched phylum, followed by Firmicutes (13.8%), Bacteroidetes (5.2%), and Actinobacteria (3.2%). Ammonia oxidizers, sulfate reducers, dehalogenation, chitin degradation, nitrite reducers, and aromatic hydrocarbon degraders were the most prevalent functional categories in the active zones of caulosphere. Furthermore, Gammaproteobacteria (49.2%) and Erwinia (29.19%) were the most abundant classes and genera of endophytic bacterial communities. Thus, the presence of a substantial amount of phosphate-solubilizing Gammaproteobacteria (PSB) may stimulate growth, increase plant resilience, suppress disease, and aid in the rubber and sugar breakdown. This is the first report of microbial endophytes associated with Hevea caulosphere.

Shifts in uterine microbiome associated with pregnancy outcomes at first insemination and clinical cure in dairy cows with metritis

Objectives were to assess differences in uterine microbiome associated with clinical cure and pregnancy outcomes in dairy cows treated for metritis. Cows with metritis (reddish-brownish, watery, and fetid vaginal discharge) were paired with cows without metritis based on parity and days postpartum. Uterine contents were collected through transcervical lavage at diagnosis, five days later following antimicrobial therapy (day 5), and at 40 days postpartum. Uterine microbiome was assessed by sequencing the V4 hypervariable region of the 16S rRNA gene. Although alpha-diversity based on Chao1, Shannon, and inverse Simpson indexes at diagnosis did not differ between cows with and without metritis, disease was associated with differences in beta-diversity. Prevalence of Porphyromonas, Bacteroides, and Veillonella was greater in cows with metritis. Streptococcus, Sphingomonas, and Ureaplasma were more prevalent in cows without metritis. Differences in beta-diversity between cows with and without metritis persisted on day 5. Uterine microbiome was not associated with clinical cure. Richness and alpha-diversity, but not beta-diversity, of uterine microbiome 40 days postpartum were associated with metritis and pregnancy. No relationship between uterine microbiome and pregnancy outcomes was observed. Results indicate that factors other than changes in intrauterine bacterial community underlie fertility loss and clinical cure in cows with metritis.

Subcuticular and biofilm microbiomes in Holothuria tubulosa and their potential for denitrification

Holothurians, as benthic invertebrates inhabiting marine ecosystems, have a crucial function in that they actively process organic detritus in the sediments. Previous works have provided evidence of the capability of holothurians to reduce nitrate and ammonium concentrations in aquaculture tanks. However, the mechanisms underlying this nitrogen decrease still need to be elucidated and might be related to bacterial symbionts in the holothurians. Here we characterize the community of bacterial symbionts in the biofilm and subcuticle of Holothuria tubulosa and explore the presence of nitrification and denitrification genes. To characterize these bacterial symbionts, we extracted DNA and amplified the V3-V4 hypervariable region of the 16S rRNA gene. We obtained a notable contribution of Bacteroidota, Alphaproteobacteria (mostly Rhodobacterales), and Gammaproteobacteria (mostly Pseudomonadales) both within the biofilm and subcuticle of H. tubulosa. Subsequently, we tested the presence of specific genes encoding enzymes involved in nitrification (i.e. archaeal amoA and bacterial amoA) and denitrification (i.e. nirS and nosZ). Our results confirm the presence of denitrification genes in the holothurian biofilms. These findings indicate that the holothurians house a diverse community of bacterial symbionts, which includes species with the potential for nitrogen removal. Therefore, holothurian holobionts may play a multifaceted ecological role, both processing organic detritus and reducing nitrogen levels in coastal areas. These roles could be extended to sustainable aquaculture, making them valuable ecosystem engineers with significant implications for ecosystem and aquaculture health.

#279 The impact of synbiotics on the uremic toxins, inflammation, and gut microbiome of CKD patients—randomized, double-blind, placebo-controlled trial

Abstract Background and Aims The microbiome represents a complex system of microorganisms coexisting within the human body. The intestinal tract is the most densely populated region and is essential in preserving health and developing diseases such as chronic kidney disease (CKD). The relationship between the gut microbiome and the kidney is complex. CKD can significantly affect the composition of the gut microbiome, which, through the fermentation process, participates in the production of over 90 different uremic toxins with the potential to aggravate systemic inflammation, oxidative stress, and decrease renal function. Indoxyl-sulfate (IS), p-cresyl-sulfate (PCS), and trimethylamine-N-oxide (TMAO) are the most closely studied. Supplementation with pre-, pro-, or synbiotics could reduce the production and improve the intestinal clearance of uremic toxins. Assessing the efficacy and safety of this potential strategy has been the aim of our randomized, double-blind, placebo-controlled study. Method A total of 84 patients, aged over 18 years, with an estimated glomerular filtration rate (eGFR) between 15 and 45 ml/min, were randomized into two groups, the one treated with synbiotic—64 billion colonies of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium lactis with 6.4 g of inulin in the form of capsules, and a control group treated with an identical number and appearance of capsules but with a total of 6.8 g of bioinert maltodextrin, over a period of 12 weeks. Strict control of the diet and the use of antibiotics and other probiotics was carried out. The study examined the effect of therapy on the serum level of the uremic toxins IS, PCS, and TMAO, the composition of the stool microbiome, the serum level of interleukin 6 (IL6), high-sensitivity C-reactive protein (hs-CRP), eGFR, albuminuria, degree of liver steatosis according to ultrasound elastomeric measurement, occurrence of gastrointestinal symptoms and gut transition time. The level of uremic toxins was determined using ultra-performance liquid chromatography. To determine the composition of the intestinal microbiome, we used 16s rRNA amplicon sequencing targeting the V3-V4 hypervariable region. Results The synbiotic treatment significantly reduced the levels of IS (−11% vs. 1%, p = 0.033) and PCS (−6% vs. 0%, p = 0.011) in the intervention arm. We also observed that treatment with the synbiotic significantly reduced hsCRP levels (ΔhsCRP 0 vs −29%, p = 0.03). Synbiotic treatment greatly enriched the intestinal microbiome with the genera Lactobacillus, Bifidobacterium, and Faecalibacterium by 81%, 55%, and 40%, respectively, with a reduction in the genera Ruminococcus and Enterobacterium by 35% and 40%. The use of synbiotics led to a significant reduction of liver steatosis according to the dynamics of the attenuation coefficient—ATT (ΔATT% −0.6 vs −2.3%, p = 0.33), with a decrease in the serum alanine aminotransferase—ALT (ΔALT −19% vs −28%, p = 0.035). A statistically significant shortening of the gut transition time—GTT was observed in the intervention arm (ΔGTT −5 h vs −10 h, p = 0.031), paralleled by a reduction in the intensity and prevalence of constipation symptoms. No side effects were observed. Conclusion Synbiotics could be an effective, safe, and affordable therapy that could be used to modify the intestinal microbiome of CKD patients and contribute to the reduction of the level of uremic toxins, inflammation, degree of fatty liver, shorten gut transit time and reduce the symptoms of constipation.

Variability and Number of Circulating Complementary Sex Determiner (Csd) Alleles in a Breeding Population of Italian Honeybees under Controlled Mating.

In Apis mellifera, csd is the primary gene involved in sex determination: haploid hemizygous eggs develop as drones, while females develop from eggs heterozygous for the csd gene. If diploid eggs are homozygous for the csd gene, diploid drones will develop, but will be eaten by worker bees before they are born. Therefore, high csd allelic diversity is a priority for colony survival and breeding. This study aims to investigate the variability of the hypervariable region (HVR) of the csd gene in bees sampled in an apiary under a selection scheme. To this end, an existing dataset of 100 whole-genome sequences was analyzed with a validated pipeline based on de novo assembly of sequences within the HVR region. In total, 102 allelic sequences were reconstructed and translated into amino acid sequences. Among these, 47 different alleles were identified, 44 of which had previously been observed, while 3 are novel alleles. The results show a high variability in the csd region in this breeding population of honeybees.