Towards facilitated interpretation of shotgun metagenomics long-read sequencing data analyzed with KMA for the detection of bacterial pathogens and their antimicrobial resistance genes.

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k-mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU.

Genomic and Antimicrobial Surveillance of Campylobacter Population in Italian Poultry.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni. ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni, while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli. Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli, the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

Metagenomic Characterization and Volatile Compounds Determination in Rumen from Saanen Goat Kids Fed Olive Leaves.

The accumulation and disposal of by-products deriving from the agro-food industry represents a problem both from an economic and environmental point of view. The use of these matrices in zootechnical nutrition could represent a feasible solution. The aim of the study was to examine the effect of a diet containing olive leaves (OL), a by-product of the olive industry, on the ruminal microbial community of Saanen goat kids and on volatile organic compounds (VOCs) produced during the digestion. Twenty goat kids were randomly divided into two groups of ten goat kids each. The control group (CTR) was fed with a standard diet, while the experimental group (OL+) received a custom-formulated diet containing 10 % OL on a dry matter (DM) basis. After 30 days of trial, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the rumen microbiota; furthermore, rumen VOCs were also characterized by solid-phase microextraction coupled with gas chromatography-mass spectrometry. The Shannon's alpha index was not significantly different between the two groups, on the contrary, Bray-Curtis (p < 0.01) and Jaccard (p < 0.01) distances evidenced that feed affected microbial community. Eleven genera were influenced by OL supplementation, with a significant increase (p < 0.05) in Paludibacter, Fibrobacter, Sphaerochaeta Christensenella, Rikenella, Oligosphaera, Candidatus Endomicrobium, Anaerovorax, and Atopobium was observed, while the percentages of Bacteroides and Selenomonas were reduced (p < 0.05). Differences were also observed between the two groups at the family level (p < 0.004). Fibrobacteriaceae, Christensenellaceae, Coriobacteriaceae, Oligosphaeraceae, Candidatus Endomicrobium, and Planctomycetaceae were significantly higher (p < 0.05) in goat kids fed OL diet compared to CTR, while the levels of other identified families, Succinivibrionaceae and Bifidobacteriaceae, were opposite (p < 0.05). Finally, results showed that the main phyla in both groups were Bacteroidetes and Firmicutes; however, no significant differences in the relative abundance of any phyla were observed between the two groups. In addition to what has been reported, the analysis of VOCs at the rumen level showed the ability of the OL integration to induce an increase in hexanoic acid and a parallel decrease in decanal. Furthermore, only in OL+ samples there was the accumulation of α-terpineol to which a wide range of interesting biological properties is attributed. The presence of VOCs associated with health status suggests a favorable role of OL in preserving and improving animal welfare.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

Investigating the cecal microbiota in broiler poultry farms and its potential relationships with animal welfare.

The present study assessed the modulation of cecal microbiota and correlations with Campylobacter colonization and animal welfare status. For these purposes, we conducted a cross sectional study of the cecal microbiota from 187 broilers reared in 13 batches from 10 poultry farms by performing 16S rRNA sequencing (regions V3-4). The welfare of each batch was assessed using a simplified Welfare Quality® protocol, scoring higher in organic batches, compared to both antibiotic-free and conventional batches. The bioinformatics analyses were conducted in QIIME 2 and a linear discriminant analysis determined the association between microbiota and animals with different Campylobacter carriage status and welfare levels. In the microbiota from the subjects negative for Campylobacter or with high welfare scores, Bacteroidetes was the predominant phylum with the genus Megamonas significantly increased in abundance. A greater abundance of Parabacteroides, Phascolarctobacterium, Helicobacter in poultry negative for Campylobacter was also found at the genus level. Animals with the lowest welfare scores showed an increased abundance of Proteobacteria. The results suggested a different microbial composition and diversity in the analyzed groups.

Neutralization of SARS-CoV-2 Variants by Serum from BNT162b2 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.

A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May-June 2018.

Introduction. In May-June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy.Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures.Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak.Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was 'caciotta' cheese (odds ratio 2.40, 95 % confidence interval 1.10-5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection.Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.

Genotyping and Antibiotic Resistance Traits in Campylobacter jejuni and coli From Pigs and Wild Boars in Italy.

The present study investigated the genomic constitution and antimicrobial resistance (AMR) of 238 Campylobacter from pigs and wild boars in Italy between 2012 and 2019. Campylobacter strains were genotyped using multilocus sequence typing (MLST) and whole genome MLST (wgMLST), screened for antimicrobial resistance genes, and tested for phenotypic susceptibility to six different antibiotics. C. coli was detected in 98.31% and 91.66% of pigs and wild boars, while C. jejuni was isolated in the remaining cases. MLST assigned 73 STs and 13 STs in pigs and wild boars, respectively, including 44 novel STs. The predominant ST in pigs was ST-854 (12.36%), followed by ST-9264 (6.18%). ST-1055 and ST-1417 were predominant in wild boars (30% and 13.33%, respectively). The minimum spanning tree using 1,121 global MLST profiles showed specific Italian clusters and a clear separation between pig and wild boar profiles. The wgMLST confirmed the MLST clustering and revealed a high genetic diversity within C. coli population in Italy. Minimum inhibitory concentrations (MIC) of six antibiotics revealed higher resistance in pigs to ciprofloxacin, nalidixic acid, streptomycin and tetracycline, compared to wild boar. In contrast, most strains were susceptible to gentamicin. Worrying levels of multidrug resistance (MDR) were observed mostly in pig isolates. Molecular screening of AMR mechanisms revealed the predominance of gyrA T86I substitution among fluoroquinolone- and quinolone-resistant isolates, and the 23S rRNA A2075G mutation among macrolide-resistant isolates. Other resistance determinants were observed: (i) tet(O) gene was present among tetracycline-resistant isolates; (ii) rpsL and aph(3')-III genes conferring resistance to aminoglycosides, were identified only in streptomycin or gentamicin-resistant pig isolates; (iii) cmeA, cmeB, cmeC, cmeR genes responsible of pump efflux mechanisms, were observed in almost all the strains; (iv) OXA-61, encoding ß-lactamase, was found in the half of the strains. Genotypic and phenotypic AMR profiling was fairly correlated for quinolones/fluoroquinolones. Campylobacter infection is common also in wild boar populations in Italy, suggesting that wild boars could be a reservoir of resistant and multi-resistant Campylobacter species, which may be of public health concern. The present study adds to our knowledge on the epidemiological and ecological traits of this pathogen in domesticated and wild swine.

Genomic Comparison of Salmonella Enteritidis Strains Isolated from Laying Hens and Humans in the Abruzzi Region during 2018.

Salmonellosis is a major cause of bacterial foodborne infection. Since 2016, an increased number of cases of gastroenteritis caused by Salmonella enterica serovar Enteritidis linked to eggs produced in Poland has been reported in Europe. In Italy, S. Enteritidis is one of the three most commonly reported serotypes, associated mainly with the consumption of contaminated eggs and derived products. In our work, we analysed 61 strains of S. Enteritidis obtained from humans and farms in the Abruzzi region, Italy, in 2018. We used Multiple-Loci Variable-Number Tandem Repeat (VNTR) analysis (MLVA)-based typing and Whole-Genome Sequencing (WGS) tools to identify closely related strains and perform cluster analysis. We found two clusters of genetically similar strains. The first one was present in the local farms and isolated from human cases and had single-linkage distance of no more than two core genes and less than five Single-Nucleotide Polymorphisms (SNPs). The second cluster contained strains isolated from humans and from a dessert (tiramisù) sample that shared identical core genome and were assigned the same SNP address. Cluster 2 isolates were found to be genetically similar to an S. Enteritidis strain from a multi-country outbreak linked to Polish eggs.

Molecular Characterization and Antimicrobial Susceptibility of C. jejuni Isolates from Italian Wild Bird Populations.

Poultry is considered a major reservoir of human campylobacteriosis. It also been reported that not only poultry, but also wild birds, are capable of carrying C. jejuni, thus demonstrating to be a risk of spreading the bacteria in the environment. To gain insight into the population structure and investigate the antimicrobial resistance genotypes and phenotypes, we analyzed a collection of 135 C. jejuni from 15 species of wild birds in Italy. MLST revealed the presence of 41 sequence types (STs) and 13 clonal complexes (CCs). ST-179 complex and the generalist ST-45 complex were the most prevalent. Core genome MLST revealed that C. jejuni from ST-45 complex clustered according to the bird species, unlike the ST-179 complex which featured 3 different species in the same cluster. Overall we found a moderate prevalence of resistance to tetracycline (12.5%), ciprofloxacin and nalidixic acid (10%). The novel ST isolated from one pigeon showed resistance to all the antibiotics tested. The ST-179 complex (33.3%) was identified with significantly higher nalidixic acid resistance relative to other tested STs. Nine AMR genes (tet(O), cmeA, cmeB, cmeC, cmeR, aad, blaOXA-61, blaOXA-184 and erm(B)) and 23S rRNA and gyrA-associated point mutations were also described, indicating a concordance level between genotypic and phenotypic resistance of 23.3%, 23.4% and of 37.5% for streptomycin, tetracycline and quinolones/fluoroquinolones, respectively. We recommend that particular attention should be given to wild birds as key sentinel animals for the ecosystem contamination surveillance.

Tendon Biomimetic Electrospun PLGA Fleeces Induce an Early Epithelial-Mesenchymal Transition and Tenogenic Differentiation on Amniotic Epithelial Stem Cells.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers' axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

Correction: Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

[This corrects the article DOI: 10.1371/journal.pone.0223804.].

Antimicrobial resistance genotypes and phenotypes of Campylobacter jejuni isolated in Italy from humans, birds from wild and urban habitats, and poultry.

Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA-184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations.

Amniotic epithelial stem cell biocompatibility for electrospun poly(lactide-co-glycolide), poly(ε-caprolactone), poly(lactic acid) scaffolds.

Three biodegradable thermoplastic polymers, poly(ε-caprolactone) (PCL), poly(l-lactide-co-d,l-lactide) (PLA) and poly(l-lactide-co-glycolide) (PLGA), have been used to produce nonwovens scaffolds with uniform micrometer fibres. Scaffolds' physical and morphological characterization was performed by X-ray diffraction, Scanning Electron Microscopy and Contact-Angle test. Morphological investigations revealed that all produced fibres were randomly orientated with interconnected pores ranging between 5 and 12µm in diameter. An average fibre diameter of 1.5, 0.75 and 1.2µm was found for PCL, PLA and PLGA, respectively. Moreover, experiments were designed to verify whether the fabricated electrospun substrates were biocompatible for ovine amniotic epithelial stem cells (oAECs) under in vitro conditions. Cell adhesion, survival, spatial organization on fibres, proliferation index, and DNA quantification after 48h culture, showed an enhanced adhesion and proliferation, especially for PLGA scaffolds. The favourable interaction between oAECs and the fibrous scaffolds was attributed to the greatly improved porosity and pore size distribution of the electrospun scaffolds. In addition, AECs can be considered ideal for tissue engineering especially when using biocompatible and opportunely produced scaffolds.

M1 and M2 macrophage recruitment during tendon regeneration induced by amniotic epithelial cell allotransplantation in ovine.

Recently, we have demonstrated that ovine amniotic epithelial cells (oAECs) allotransplanted into experimentally induced tendon lesions are able to stimulate tissue regeneration also by reducing leukocyte infiltration. Amongst leukocytes, macrophages (Mφ) M1 and M2 phenotype cells are known to mediate inflammatory and repairing processes, respectively. In this research it was investigated if, during tendon regeneration induced by AECs allotransplantation, M1Mφ and M2Mφ phenotype cells are recruited and differently distributed within the lesion site. Ovine AECs treated and untreated (Ctr) tendons were explanted at 7, 14, and 28 days and tissue microarchitecture was analyzed together with the distribution and quantification of leukocytes (CD45 positive), Mφ (CD68 pan positive), and M1Mφ (CD86, and IL12b) and M2Mφ (CD206, YM1 and IL10) phenotype related markers. In oAEC transplanted tendons CD45 and CD68 positive cells were always reduced in the lesion site. At day 14, oAEC treated tendons began to recover their microarchitecture, contextually a reduction of M1Mφ markers, mainly distributed close to oAECs, and an increase of M2Mφ markers was evidenced. CD206 positive cells were distributed near the regenerating areas. At day 28 oAECs treated tendons acquired a healthy-like structure with a reduction of M2Mφ. Differently, Ctr tendons maintained a disorganized morphology throughout the experimental time and constantly showed high values of M1Mφ markers. These findings indicate that M2Mφ recruitment could be correlated to tendon regeneration induced by oAECs allotransplantation. Moreover, these results demonstrate oAECs immunomodulatory role also in vivo and support novel insights into their allogeneic use underlying the resolution of tendon fibrosis.

Cellular and molecular maturation in fetal and adult ovine calcaneal tendons.

Processes of development during fetal life profoundly transform tendons from a plastic tissue into a highly differentiated structure, characterised by a very low ability to regenerate after injury in adulthood. Sheep tendon is frequently used as a translational model to investigate cell-based regenerative approaches. However, in contrast to other species, analytical and comparative baseline studies on the normal developmental maturation of sheep tendons from fetal through to adult life are not currently available. Thus, a detailed morphological and biochemical study was designed to characterise tissue maturation during mid- (2 months of pregnancy: 14 cm of length) and late fetal (4 months: 40 cm of length) life, through to adulthood. The results confirm that ovine tendon morphology undergoes profound transformations during this period. Endotenon was more developed in fetal tendons than in adult tissues, and its cell phenotype changed through tendon maturation. Indeed, groups of large rounded cells laying on smaller and more compacted ones expressing osteocalcin, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) were identified exclusively in fetal mid-stage tissues, and not in late fetal or adult tendons. VEGF, NGF as well as blood vessels and nerve fibers showed decreased expression during tendon development. Moreover, the endotenon of mid- and late fetuses contained identifiable cells that expressed several pluripotent stem cell markers [Telomerase Reverse Transcriptase (TERT), SRY Determining Region Y Box-2 (SOX2), Nanog Homeobox (NANOG) and Octamer Binding Transcription Factor-4A (OCT-4A)]. These cells were not identifiable in adult specimens. Ovine tendon development was also accompanied by morphological modifications to cell nuclei, and a progressive decrease in cellularity, proliferation index and expression of connexins 43 and 32. Tendon maturation was similarly characterised by modulation of several other gene expression profiles, including Collagen type I, Collagen type III, Scleraxis B, Tenomodulin, Trombospondin 4 and Osteocalcin. These gene profiles underwent a dramatic reduction in adult tissues. Transforming growth factor-ß~1 expression (involved in collagen synthesis) underwent a similar decrease. In conclusion, these morphological studies carried out on sheep tendons at different stages of development and aging offer normal structural and molecular baseline data to allow accurate evaluation of data from subsequent interventional studies investigating tendon healing and regeneration in ovine experimental models.