Effects of diets with different phosporus availability on the intestinal microbiota of chickens and pigs

In the research works of the present thesis, 16S rRNA gene sequencing and metaproteomics were employed to investigate the gut microbiota of chickens and pigs kept at experimental diets with varying amount of calcium-phosphorus (CaP) and supplemented MP. This represents a valuable approach to investigate the bacterial specimens involved in the P absorption, allowing for a comprehensive understanding of how the intestinal bacteria adapt to a new diet and which metabolic routes are affected by changing levels of supplemented P and/or MP. Two major experimental trials were performed during the investigation. The first one was conducted on chickens operating a modulation in the dietary levels of Ca, P and MP. This trial highlighted a shift in the composition of the crop and ceca-associated microbial community depending on the composition of the diet fed. Also, investigated protein inventory revealed that the stress condition due to the reduced P availability is mirrored in the gastrointestinal tract (GIT)-associated microbiota. Marked differences were observed in the functions of the bacterial community in the case of P-available diets versus P-deficient ones. Protein repertoire of the first case draws a thriving microbial community focused on complex and anabolic functions. Contrariwise, the bacterial community in the case of P-lacking diets appears to deal with catabolic functions and stress response. The second trial was conducted on pigs and attempts to define the dynamics featuring the microbiota adaptation to a new challenging diet composed of different protein sources and varying levels of Ca and P. Statistical evidences reveal a stepwise adaptation of the fecal microbiota to the experimental diets fed. Both DNA-based approach and metaproteomics independently reveal three main adaptation phases: -before the feeding of the experimental trial (i.e. Zero), -the response of the microbial community to the challenging factor (i.e. MA) and, finally, - the newly achieved homeostatic balance (i.e. EQ). As observed in the first trial, feeding of the experimental diets impairs the overall fecal microbiota composition, stimulating the presence of phase-specific bacterial specimens and a characteristic relative abundance of the shared ones. Bacterial families responsible for the phase-specific architecture of the fecal microbiota are also active in the biochemical pathways driving the functional peculiarities of each adaptation phase. A deeper investigation of the identified protein repertoire revealed that the observed statistical differences among the adaptation phases are uniquely due to the Ca and P composition of the diets fed. None of the observed effects can be attributed to the diverse protein sources supplemented with the diets. Functional categorization of the identified protein inventory depicts three diverse functional assets of the microbial community. Specifically, prior the feeding of the experimental diets, bacteria are hypothesized to live under homeostatic condition, since they appear to be involved in complex and highly-specialized functions. Following the administration of the experimental diets microbial community changes its functional priority and reduce the expression of highly specialized functions to focus on more essential ones. Proteins involved in complex functions such as widening the substrates array and facing complex sugars tend to increase in abundance while the new homeostatic balance is achieved. Altogether, data from both trials provide useful information for future studies aimed to design effective breeding strategies finalized to reduce the P supplementation in the routinely breeding of livestock and maintain a balanced microbial activity in the animal GIT. Investigation of the dynamics of the porcine microbiota provides instructions on the minimal exposure time required from the intestinal microbiota to adapt to a new dietary composition. This is of fundamental importance for the design of future studies aimed to confirm and/or continue our results. Moreover, the anatomical and physiological similarities occurring between humans and pigs, make our findings of interest for future human nutritional studies, where the mechanisms and lasts of the microbiota adaptation process is still object of discussion.In den Forschungsprojekten der vorliegenden Arbeit wurden die 16S rRNA Gensequenzierung und Metaproteomik zur Untersuchung der Darmmikrobiota von Hühnern und Schweinen verwendet. Dabei wurde der Einfluss von Versuchsdiäten mit unterschiedlichen Mengen an Calcium-Phosphor (CaP) und ergänzten MP untersucht. Dies ist ein wertvoller Ansatz zur Identifizierung von Bakterienspezies, welche an der P-Absorption beteiligt sind, und ermöglicht es ein umfassendes Verständnis darüber zugewinnen, wie sich die Darmbakterien an eine neue Ernährung und an Stoffwechselwege, die von sich verändernden Mengen an ergänztem P und / oder MP beeinflusst werden, anpassen können. Im Rahmen dieser Arbeit wurden zwei große experimentelle Studien durchgeführt. Die erste Studie wurde an Hühnern durchgeführt und basierte auf der Verabreichung unterschiedlicher Mengen an Ca, P und MP in der Nahrung. Die Ergebnisse dieser Studie zeigten eine Nahrungs-abhängige Verschiebung in der Zusammensetzung der mikrobiellen Gemeinschaft in Kropf und Blinddarm. Außerdem zeigte das untersuchte Proteinrepertoire, dass der Stresszustand, aufgrund der reduzierten P-Verfügbarkeit, sich in der Mikrobiota im Gastrointestinaltrakt (GIT) widergespiegelt. Der Vergleich von Diäten mit verfügbarem P mit P-armen Diäten zeigte deutliche Unterschiede in den Funktionen der Bakteriengemeinschaft. Bei einer Diät mit verfügbarem P in der Nahrung, deutet die Analyse des Proteinrepertoires auf eine florierende mikrobielle Gemeinschaft hin, in der komplexe und anabole Funktionen vorherrschen. Im Gegensatz dazu scheinen bei Diäten mit P-Mangel katabole Funktionen und Stressreaktionen in der bakteriellen Gemeinschaft zu überwiegen. Die zweite Studie wurde an Schweinen durchgeführt und versuchte die Dynamik zu definieren, mit der sich die Mikrobiota an eine neue, herausfordernde, aus verschiedenen Proteinquellen und unterschiedlichen Mengen an Ca und P bestehende Nahrung, anpasst. Statistische Beweise zeigen eine stufenweise Anpassung der fäkalen Mikrobiota an die verabreichten Versuchsnahrung. Sowohl DNA- als auch metaproteomische Analysen, die getrennt voneinander durchgeführt wurden, zeigen drei Hauptanpassungsphasen, welche die folgenden experimentalen Adaptationsgruppen definieren: - vor der Verabreichung der Versuchsdiät (d.h. Null), - die Reaktion der mikrobiellen Gemeinschaft auf den herausfordernden Faktor (d.h. MA) und schließlich, - das Stadium des neu erreichten homöostatischen Gleichgewichts (d.h. EQ). Wie im ersten Versuch beobachtet wurde, beeinträchtigt die Fütterung der Versuchsnahrung die gesamte Zusammensetzung der fäkalen Mikrobiota, sie stimuliert das Vorhandensein von phasenspezifischen Bakterienarten und eine charakteristische relative Häufigkeit der phasenunspezifischen Bakterien. Bakterielle Familien, die für die phasenspezifische Struktur der fäkalen Mikrobiota verantwortlich sind, sind auch in den biochemischen Pfaden aktiv und sind im Wesentlichen für die funktionellen Besonderheiten jeder Anpassungsphase verantwortlich. Eine tiefere Untersuchung des identifizierten Proteinrepertoires ergab, dass die beobachteten statistischen Unterschiede zwischen den Adaptationsphasen eindeutig auf die Ca- und P-Gehalte der gefütterten Diäten zurückzuführen sind. Keine der beobachteten Effekten kann den verschiedenen gleichzeitig verabreichten Proteinquellen zugeschrieben werden. Die funktionelle Kategorisierung des identifizierten Proteinbestands zeigt drei verschiedene funktionelle Aspekte der mikrobiellen Gemeinschaft. Insbesondere wird angenommen, dass vor dem Füttern der experimentellen Diäten, die Bakterien unter homöostatischen Bedingungen leben, da sie in komplexe und hochspezialisierte Funktionen involviert zu sein scheinen. Nach der Verabreichung der experimentellen Diäten ändert die mikrobielle Gemeinschaft ihre funktionelle Priorität und reduziert die Ausübung hochspezialisierter Funktionen, um sich auf die wesentlichen zu konzentrieren. Während das neue homöostatische Gleichgewicht erreicht wird, nimmt die Menge an Proteinen, die an komplexen Funktionen beteiligt sind, wie z. B. die Verarbeitung einer großen Substratvielfalt sowie die Metabolisierung von komplexen Zuckern, zu. Zusammengefasst, liefern die aus beiden Experimente gewonnenen Daten nützliche Informationen für zukünftige Studien mit dem Ziel, effektive Zuchtstrategien zu entwickeln, um die P-Supplementierung bei der routinemäßigen Viehzucht zu reduzieren und eine ausgewogene mikrobielle Aktivität im Gastrointestinaltrakt des Tieres aufrechtzuerhalten. Die Untersuchung der Anpassungsdynamik der Schweinemikrobiota liefert genaue Informationen darüber, wie lange die intestinale Mikrobiota benötigt, um sich an eine neue Nahrungszusammensetzung anzupassen. Dies ist von zentraler Bedeutung für die Gestaltung zukünftiger experimenteller Studien, die darauf abzielen, unsere Ergebnisse zu bestätigen und/oder unsere Forschung fortzusetzen. Aufgrund der anatomischen und physiologischen Ähnlichkeiten, die zwischen Menschen und Schweinen auftreten, sind die erhaltenen Befunde auch für zukünftige Ernährungsstudien am Menschen von Interesse, bei denen die Mechanismen und die Dauer der Adaptationsprozesse der Mikrobiota noch untersucht werden

Unraveling the adipose tissue proteome of transition cows through severe negative energy balance

Fat mobilization in high-yielding dairy cows during early lactation occurs to overcome negative energy balance (NEB), caused by insufficient feed intake and the concomitant increased nutritional requirements. For this reason, adipose tissue represents an essential organ for healthy and performant lactation. However, only a few data are known about adipose tissue proteome and its metabolic status during peripartum. The aim of this study was to analyze the differential proteomics profiles of subcutaneous adipose tissue belonging to cows with different NEB scores (low NEB and severe NEB). Both groups were analyzed at three different time points (one month before calving, one and sixteen weeks after calving) that were related to different levels and rates of adipose tissue mobilization. The dataset highlighted the differential expression of the same four key proteins (annexin A2, actin-related protein 10, glyceraldehyde-3-phosphate dehydrogenase, and fatty acid-binding protein) involved in lipid metabolism during all time points and of other 22 proteins typical of the other comparisons among remaining time points. The obtained dataset suggested that the individual variability in adipose tissue metabolism/mobilization/energy availability could be linked to the different outcomes in levels of energy balance and related physical complications among dairy cows during peripartum

Assessing the pathogenicity of BRCA1/2 variants of unknown significance: Relevance and challenges for breast cancer precision medicine

IntroductionBreast cancer (BC) is the leading cause of cancer-related death in women worldwide. Pathogenic variants in BRCA1 and BRCA2 genes account for approximately 50% of all hereditary BC, with 60-80% of patients characterized by Triple Negative Breast Cancer (TNBC) at an early stage phenotype. The identification of a pathogenic BRCA1/2 variant has important and expanding roles in risk-reducing surgeries, treatment planning, and familial surveillance. Otherwise, finding unclassified Variants of Unknown Significance (VUS) limits the clinical utility of the molecular test, leading to an “imprecise medicine”.MethodsWe reported the explanatory example of the BRCA1 c.5057A>C, p.(His1686Pro) VUS identified in a patient with TNBC. We integrated data from family history and clinic-pathological evaluations, genetic analyses, and bioinformatics in silico investigations to evaluate the VUS classification.ResultsOur evaluation posed evidences for the pathogenicity significance of the investigated VUS: 1) association of the BRCA1 variant to cancer-affected members of the family; 2) absence of another high-risk mutation; 3) multiple indirect evidences derived from gene and protein structural analysis.DiscussionIn line with the ongoing efforts to uncertain variants classification, we speculated about the relevance of an in-depth assessment of pathogenicity of BRCA1/2 VUS for a personalized management of patients with BC. We underlined that the efficient integration of clinical data with the widest number of supporting molecular evidences should be adopted for the proper management of patients, with the final aim of effectively guide the best prognostic and therapeutic paths

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p < 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p < 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p < 0.0001) or urgent (20.4% vs. 38.5%; p < 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p < 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Immunoinformatic-Based Prediction of Candidate Epitopes for the Diagnosis and Control of Paratuberculosis (Johne’s Disease)

Paratuberculosis is an infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of Crohn’s disease patients.Since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. However, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. The prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.In the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of 10 MAP proteins. These proteins were chosen according to a previously published immunoproteomics approach. For each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both B-cells and/or class I MHC antigens. The retrieved peptide sequences were analyzed for their specificity and cross-reactivity. The final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations

Antimicrobial Resistance in Veterinary Medicine and Public Health

Animal productions, and populations, have been rapidly expanding over the last decades, forcing the ever-closer coexistence of human beings and domestic animals on our yet “narrow” planet [...

Proteomic analysis of fresh and liquid-stored boar spermatozoa

In this study comparative proteomics was used to define changes in the expression of the spermatozoa proteins during liquid storage. Semen from eight boars was analyzed on the day of collection and after liquid preservation at 15–17 °C for three days. Sperm parameters (concentration, motility, morphology, vitality) and percentage of non-capacitated and acrosomal-reacted spermatozoa were determined. Sperm proteins were extracted and separated by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteomic profiles were computationally compared to highlight differentially expressed protein spots that were, in turn, identified by mass spectrometry. The intensities of four spots were significantly different between fresh and liquid stored sperm. Namely: ATP citrate lyase, chaperonin containing T-complex polypeptide 1 (TCP1) subunit ε and probable phospholipid-transporting ATP-ase were over-expressed in liquid stored sperm, whereas cytosolic non-specific dipeptidase was over-expressed in fresh sperm. These differentially expressed proteins could be used as plausible biomarkers for the evaluation of boar semen quality and spermatozoa survival after liquid storage and could help to address problems associated with sperm preservation

News in livestock research — use of Omics-technologies to study the microbiota in the gastrointestinal tract of farm animals

Technical progress in the field of next-generation sequencing, mass spectrometry and bioinformatics facilitates the study of highly complex biological samples such as taxonomic and functional characterization of microbial communities that virtually colonize all present ecological niches. Compared to the structural information obtained by metagenomic analyses, metaproteomic approaches provide, in addition, functional data about the investigated microbiota. In general, integration of the main Omics-technologies (genomics, transcriptomics, proteomics and metabolomics) in live science promises highly detailed information about the specific research object and helps to understand molecular changes in response to internal and external environmental factors. The microbial communities settled in the mammalian gastrointestinal tract are essential for the host metabolism and have a major impact on its physiology and health. The microbiotas of livestock like chicken, pig and ruminants are becoming a focus of interest for veterinaries, animal nutritionists and microbiologists. While pig is more often used as an animal model for human-related studies, the rumen microbiota harbors a diversity of enzymes converting complex carbohydrates into monomers which bears high potential for biotechnological applications. This review will provide a general overview about the recent Omics-based research of the microbiota in livestock including its major findings. Differences concerning the results of pre-Omics-approaches in livestock as well as the perspectives of this relatively new Omics-platform will be highlighted

Analysis of the Bacterial and Host Proteins along and across the Porcine Gastrointestinal Tract

Pigs are among the most important farm animals worldwide and research to optimize their feed efficiency and improve their welfare is still in progress. The porcine intestinal microbiome is so far mainly known from sequencing-based studies. Digesta and mucosa samples from five different porcine gastrointestinal tract sections were analyzed by metaproteomics to obtain a deeper insight into the functions of bacterial groups with concomitant analyses of host proteins. Firmicutes (Prevotellaceae) dominated mucosa and digesta samples, followed by Bacteroidetes. Actinobacteria and Proteobacteria were much higher in abundance in mucosa compared to digesta samples. Functional profiling reveals the presence of core functions shared between digesta and mucosa samples. Protein abundances of energy production and conversion were higher in mucosa samples, whereas in digesta samples more proteins were involved in lipid transport and metabolism; short-chain fatty acids production were detected. Differences were also highlighted between sections, with the small intestine appearing more involved in carbohydrate transport and metabolism than the large intestine. Thus, this study produced the first functional analyses of the porcine GIT biology, discussing the findings in relation to expected bacterial and host functions