82 results on '"Maria Filippa Addis"'
Search Results
2. Milk proteins as mastitis markers in dairy ruminants - a systematic review
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Anna Giagu, Martina Penati, Sara Traini, Simone Dore, and Maria Filippa Addis
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Serum Amyloid A Protein ,Goat Diseases ,Sheep ,Haptoglobins ,General Veterinary ,Goats ,Cattle Diseases ,Sheep Diseases ,Cell Count ,General Medicine ,Milk Proteins ,Mammary Glands, Animal ,Milk ,Animals ,Cattle ,Female ,Mastitis, Bovine ,Biomarkers - Abstract
Mastitis is one of the most impacting diseases in dairy farming, and its sensitive and specific detection is therefore of the greatest importance. The clinical evaluation of udder and mammary secretions is typically combined with the milk Somatic Cell Count (SCC) and often accompanied by its bacteriological culture to identify the causative microorganism. In a constant search for improvement, several non-enzymatic milk proteins, including milk amyloid A (M-SAA), haptoglobin (HP), cathelicidin (CATH), and lactoferrin (LF), have been investigated as alternative biomarkers of mastitis for their relationship with mammary gland inflammation, and immunoassay techniques have been developed for detection with varying degrees of success. To provide a general overview of their implementation in the different dairy species, we carried out a systematic review of the scientific literature using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. Our review question falls within the type “Diagnostic test accuracy questions” and aims at answering the diagnostic question: “Which are the diagnostic performances of mastitis protein biomarkers investigated by immunoassays in ruminant milk?”. Based on 13 keywords combined into 42 searches, 523 manuscripts were extracted from three scientific databases. Of these, 33 passed the duplicate removal, title, abstract, and full-text screening for conformity to the review question and document type: 78.8% investigated cows, 12.1% sheep, 9.1% goats, and 6.1% buffaloes (some included more than one dairy species). The most frequently mentioned protein was M-SAA (48.5%), followed by HP (27.3%), CATH (24.2%) and LF (21.2%). However, the large amount of heterogeneity among studies in terms of animal selection criteria (45.5%), index test (87.9%), and standard reference test (27.3%) resulted in a collection of data not amenable to meta-analysis, a common finding illustrating how important it is for case definitions and other criteria to be standardized between studies. Therefore, results are presented according to the SWiM (Synthesis Without Meta-analysis) guidelines. We summarize the main findings reported in the 33 selected articles for the different markers and report their results in form of comparative tables including sample selection criteria, marker values, and diagnostic performances, where available. Finally, we report the study limitations and bias assessment findings.
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- 2022
3. Mycoplasma species isolated from bovine milk collected from US dairy herds between 2016 and 2019
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M. Wieland, Paolo Moroni, R.D. Watters, B. Gross, Anja Sipka, P.D. Virkler, M.J. Zurakowski, Maria Filippa Addis, Gloria Gioia, Daryl V. Nydam, and Carlos Santisteban
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Mycoplasma bovis ,Veterinary medicine ,New York ,Mycoplasma species ,Biology ,medicine.disease_cause ,03 medical and health sciences ,Genetics ,medicine ,Animals ,Mycoplasma Infections ,Mastitis, Bovine ,030304 developmental biology ,Molecular identification ,0303 health sciences ,Dairy herds ,0402 animal and dairy science ,04 agricultural and veterinary sciences ,Mycoplasma ,medicine.disease ,biology.organism_classification ,Texas ,040201 dairy & animal science ,Mastitis ,Acholeplasma ,Milk ,Herd ,Cattle ,Female ,Animal Science and Zoology ,Food Science - Abstract
Determining the species of mycoplasma isolated from culture-positive milk samples is important for understanding the clinical significance of their detection. Between August 2016 and December 2019, 214,518 milk samples from 2,757 dairy herds were submitted to Quality Milk Production Services (QMPS) at Cornell University for mycoplasma culture. From these samples, 3,728 collected from 204 herds were culture positive. Based on the request of herd managers, owners, or veterinarians, 889 isolates from 98 herds were subjected to molecular identification by PCR and amplicon sequencing. The largest proportion of the identified isolates were from New York State (78.1%), while the others came from the eastern United States (17.8%), Texas (2.0%), and New Mexico (2.1%). As expected, Mycoplasma spp. were the most common (855 isolates, 96.2%) and Acholeplasma spp. accounted for the remainder (34 isolates, 3.8%). Mycoplasma bovis was the most prevalent Mycoplasma species (75.1%), followed by M. bovigenitalium (6.5%), M. canadense (5.9%), M. alkalescens (5%), M. arginini (1.7%), M. californicum (0.1%), and M. primatum (0.1%). A portion of the isolates were confirmed as Mycoplasma spp. other than M. bovis but were not identified at the species level (16 isolates, 1.8%) because further information was not requested by the manager, owner, or veterinarian. Mycoplasma bovis was the only species identified in 59 of the 98 herds. However, more than 1 Mycoplasma sp. was identified in 29 herds, suggesting that herd infection with 2 or more mycoplasmas is not uncommon. Moreover, a Mycoplasma sp. other than M. bovis was the only species identified in 8 herds. From the subset of 889 mycoplasma culture-positive isolates from 98 herds, we determined that over a third of the herds had either more than 1 Mycoplasma sp. or a Mycoplasma sp. other than M. bovis detected in their milk samples. In conclusion, we observed that M. bovis is the most common pathogenic Mycoplasma species found in mastitic milk, but other Mycoplasma species are not uncommon. Our results suggest that it is critical to test milk samples for mycoplasmas using diagnostic tests able to identify both the genus and the species.
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- 2021
4. Species identification by MALDI-TOF MS and gap PCR-RFLP of non-aureus Staphylococcus, Mammaliicoccus, and Streptococcus spp. associated with sheep and goat mastitis
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Nives Maria, Rosa, Martina, Penati, Sara, Fusar-Poli, Maria Filippa, Addis, and Sebastiana, Tola
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Goat Diseases ,Sheep ,Goats ,Staphylococcus ,Cattle Diseases ,Sheep Diseases ,Streptococcus ,Staphylococcal Infections ,Polymerase Chain Reaction ,Milk ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Animals ,Cattle ,Female ,Mastitis, Bovine ,Polymorphism, Restriction Fragment Length - Abstract
Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus.
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- 2022
5. Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci
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Elisa Azara, Carla Maria Longheu, Sonia Attene, Silvana Sanna, Marco Sale, Maria Filippa Addis, and Sebastiana Tola
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Enterotoxins ,Sheep ,Virulence ,General Veterinary ,Biofilms ,Staphylococcus ,Animals ,Humans ,Female ,General Medicine ,Adhesins, Bacterial - Abstract
Background In a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. Results Phenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans. Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected. Conclusions Considerable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.
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- 2022
6. Proteomic profiles and cytokeratin 13 as a potential biomarker of Ovis aries papillomavirus 3-positive and negative cutaneous squamous cell carcinomas
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Maria Filippa Addis, Carla Cacciotto, S Pirino, Salvatore Pisanu, Elisabetta Antuofermo, Tiziana Cubeddu, Veronica Vitiello, Alberto Alberti, Giovanni Pietro Burrai, and DIPARTIMENTO DI MEDICINA SPECIALISTICA, DIAGNOSTICA E SPERIMENTALE
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Skin Neoplasms ,Proteome ,Cell ,Reactome ,0403 veterinary science ,Tandem Mass Spectrometry ,Squamous cell carcinoma ,Viral ,Papillomaviridae ,Domestic ,Epithelial cell differentiation ,Chromatography ,Liquid ,0303 health sciences ,Tumor ,biology ,04 agricultural and veterinary sciences ,Papillomavirus ,medicine.anatomical_structure ,Carcinoma, Squamous Cell ,Biomarker (medicine) ,Immunohistochemistry ,Extracellular matrix organization ,040301 veterinary sciences ,Sheep Diseases ,03 medical and health sciences ,Cytokeratin ,Cytokeratin 13 ,Sheep ,Animals ,Biomarkers, Tumor ,Chromatography, Liquid ,DNA, Viral ,Keratin-13 ,Papillomavirus Infections ,Sheep, Domestic ,medicine ,neoplasms ,030304 developmental biology ,General Veterinary ,Carcinoma ,DNA ,biology.organism_classification ,stomatognathic diseases ,Squamous Cell ,Cancer research ,Biomarkers - Abstract
none 9 no Ovis aries papillomavirus 3 (OaPV3) is an epidermotropic PV reported in sheep cutaneous squamous cell carcinoma (SCC). The presence of OaPV3 DNA and its transcriptional activity in cutaneous SCC, as well as its in vitro transforming properties, suggest a viral etiology for this neoplasm. Nevertheless, the reactome associated with viral-host interaction is still unexplored. Here, we investigated and compared the proteomic profiles of OaPV3-positive SCCs, OaPV3-negative SCCs, and non-SCC samples by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, bioinformatics tools, and immunohistochemistry (IHC). OaPV3-positive SCCs (n = 3), OaPV3-negative SCCs (n = 3), and non-SCCs samples (n = 3) were subjected to a shotgun proteomic analysis workflow to assess protein abundance differences among the three sample classes. Proteins involved in epithelial cell differentiation, extracellular matrix organization, and apoptotic signaling showed different abundances in OaPV3-positive SCCs tissues (P ≤ 0.05) when compared to the other tissues. Cytokeratin 13 (CK 13) was among the most increased proteins in OaPV3-positive SCC and was validated by immunohistochemistry on 10 samples per class, confirming its potential as a biomarker of OaPV3 infection in SCC. Collectively, results provide a preliminary insight into the reactome associated with viral-host interaction and pave the way to the development of specific biomarkers for viral-induced sheep SCC. none Vitiello V.; Burrai G.P.; Pisanu S.; Cacciotto C.; Addis M.F.; Alberti A.; Antuofermo E.; Cubeddu T.; Pirino S. Vitiello V.; Burrai G.P.; Pisanu S.; Cacciotto C.; Addis M.F.; Alberti A.; Antuofermo E.; Cubeddu T.; Pirino S.
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- 2021
7. Peptidomic Changes in The Milk of Water Buffaloes (Bubalus Bubalis) With Intramammary Infection By Non-Aureus Staphylococci
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Gabriele Di Vuolo, Esterina De Carlo, Fabrizio Ceciliani, Francesco Maria Tangorra, Martina Penati, Renata Piccinini, Giovanna Cappelli, Elisa Maffioli, Gabriella Tedeschi, Valerio Bronzo, Maria Filippa Addis, Domenico Vecchio, and Mariangela Albertini
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Veterinary medicine ,Buffaloes ,biology ,Caseins ,food and beverages ,Cell Count ,Staphylococcal Infections ,biology.organism_classification ,Water Buffaloes ,Intramammary infection ,Tandem Mass Spectrometry ,Animals ,Humans ,Cattle ,Female ,Bubalus ,Mastitis, Bovine ,Chromatography, Liquid - Abstract
Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.
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- 2021
8. Liver proteomics of gilthead sea bream (Sparus aurata) exposed to cold stress
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Riccardo Melis, Roberto Anedda, H. Slawski, Stefania Ghisaura, Maria Filippa Addis, Grazia Biosa, Sergio Uzzau, and Daniela Pagnozzi
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Fish Proteins ,Proteomics ,0106 biological sciences ,Physiology ,030310 physiology ,Carbohydrate metabolism ,010603 evolutionary biology ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Methionine ,Aquaculture ,Tandem Mass Spectrometry ,Protein biosynthesis ,Animals ,Food science ,Shotgun proteomics ,0303 health sciences ,Catabolism ,business.industry ,Cold-Shock Response ,Metabolism ,Sea Bream ,Liver ,chemistry ,General Agricultural and Biological Sciences ,business ,Metabolic Networks and Pathways ,Developmental Biology - Abstract
The gilthead sea bream (Sparus aurata, L.) is very sensitive to low temperatures, which induce fasting and reduced growth performances. There is a strong interest in understanding the impact of cold on fish metabolism to foster the development and optimization of specific aquaculture practices for the winter period. In this study, an 8 week feeding trial was carried out on gilthead sea bream juveniles reared in a Recirculated Aquaculture System (RAS) by applying a temperature ramp in two phases of four weeks each: a cooling phase from 18 °C to 11 °C and a cold maintenance phase at 11 °C. Liver protein profiles were evaluated with a shotgun proteomics workflow based on filter-aided sample preparation (FASP) and liquid chromatography-mass spectrometry (LC-ESI-Q-TOF MS/MS) followed by label-free differential analysis. Along the whole trial, sea breams underwent several changes in liver protein abundance. These occurred mostly during the cooling phase when catabolic processes were mainly observed, including protein and lipid degradation, together with a reduction in protein synthesis and amino acid metabolism. A decrease in protein mediators of oxidative stress protection was also seen. Liver protein profiles changed less during cold maintenance, but pathways such as the methionine cycle and sugar metabolism were significantly affected. These results provide novel insights on the dynamics and extent of the metabolic shift occurring in sea bream liver with decreasing water temperature, supporting future studies on temperature-adapted feed formulations. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011059.
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- 2019
9. Comparative profiling of biofilm-production, quorum sensing system and virulence genes in human and ovine non-aureus staphylococci
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Sebastiana Tola, Marco Sale, Sonia Attene, Maria Filippa Addis, Carla Longheu, Silvana Sanna, and Elisa Azara
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Quorum sensing ,Biofilm ,Virulence ,Profiling (information science) ,Computational biology ,Biology ,Gene - Abstract
Background: This study assessed the genetic characteristics shared by non-aureus staphylococci (NAS) responsible for human infections and those causing mastitis in dairy ewes. In a collaboration between animal and human health care professionals, we collected and identified 125 ovine and 70 human NAS isolates and compared them for biofilm production, presence of autolysins, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), pyrogenic toxins, and agr alleles regulating quorum-sensing systems. Ovine NAS included: S. epidermidis (57), S. chromogenes (29), S. haemolyticus (17), S. simulans (8), S. caprae (6), S. warneri (5), S. saprophyticus, S. intermedius, and S. muscae (1 each) while human NAS included: S. haemolyticus (28), S. epidermidis (26), S. hominis (4), S. lugdunensis (4), S. capitis (3), S. warneri (2), S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis (1 each).Results: Based on colony characteristics on Congo Red Agar, 4 (3.2%) ovine, and 49 (70%) human isolates produced biofilm. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA) and the bhp, aap, and embp genes coding biofilm accumulation proteins. PCR amplification of the genes coding for autolysins (atlE and aae), microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, sdrG and sdrF), enterotoxins (sea, seb, sec, sed, and see), and the toxic shock syndrome toxin (tsst), revealed that 40%, 39.2%, 47.2% and 52.8% of the sheep isolates carried atlE, aae, sdrF and sdrG, respectively, against 37.1%, 42.8%, 32.8%, and 60% of human isolates. Enterotoxins and tsst were not detected. Fifty-nine sheep isolates (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the accessory gene regulator (agr), responsible for the regulation of virulence factors: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans.Conclusions: This comparative study provided a detailed characterization of the putative virulence genes present in human and ovine NAS and indicated that the ability to form biofilms, observed mainly in human S. epidermidis, could be a major virulence factor facilitating colonization, infection, diffusion, and resistance.
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- 2021
10. Draft Genome Sequence of Acholeplasma laidlawii Isolated from the Conjunctiva of a Heifer with Infectious Bovine Keratoconjunctivitis
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Laura B. Goodman, Belinda Thompson, Paolo Moroni, Patrick K. Mitchell, Maria Filippa Addis, Gloria Gioia, and Erin L. Goodrich
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Whole genome sequencing ,Conjunctiva ,fungi ,Genome Sequences ,Biology ,biology.organism_classification ,Microbiology ,medicine.anatomical_structure ,Immunology and Microbiology (miscellaneous) ,Infectious bovine keratoconjunctivitis ,Acholeplasma laidlawii ,Genetics ,medicine ,bacteria ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Pathogen ,Sequence (medicine) - Abstract
Acholeplasma laidlawii can be isolated from cattle environments and different body sites of bovines. It is still under evaluation if A. laidlawii acts as a primary pathogen. Here, we present the whole-genome sequence of A. laidlawii isolated from the conjunctiva of a heifer with infectious bovine keratoconjunctivitis.
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- 2021
11. The untargeted lipidomic profile of quarter milk from dairy cows with subclinical intramammary infection by non-aureus staphylococci
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Matteo Audano, Mariangela Albertini, Fabrizio Ceciliani, Francesco Maria Tangorra, Maria Filippa Addis, Renata Piccinini, Donatella Caruso, Nico Mitro, M.H. Ghaffari, Cristina Lecchi, and Valerio Bronzo
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Staphylococcus ,Mammary gland ,Cattle Diseases ,Cell Count ,Biology ,Microbiology ,Transcriptome ,03 medical and health sciences ,Mammary Glands, Animal ,Genetics ,medicine ,Metabolome ,Animals ,Udder ,Mastitis, Bovine ,030304 developmental biology ,Subclinical infection ,0303 health sciences ,0402 animal and dairy science ,food and beverages ,04 agricultural and veterinary sciences ,Lipidome ,Staphylococcal Infections ,medicine.disease ,040201 dairy & animal science ,Mastitis ,medicine.anatomical_structure ,Milk ,Proteome ,Lipidomics ,Animal Science and Zoology ,Cattle ,Female ,Food Science - Abstract
This observational study determined the lipidome of cow milk during subclinical intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted approach. Among the pathogens causing bovine IMI, NAS have become the most frequently isolated bacteria from milk samples. Although the application of system biology approaches to mastitis has provided pivotal information by investigating the transcriptome, proteome, peptidome, and metabolome, the milk lipidome during mammary gland inflammation remains undisclosed. To cover this gap, we determined the milk lipidome of 17 dairy cows with IMI caused by NAS (NAS-IMI), and we compared the results with those of healthy quarter milk from 11 cows. The lipidome was determined following a liquid chromatography-quadrupole time-of-flight mass spectrometry approach. Sixteen subclasses of lipids were identified in both groups of animals. From 2,556 measured lipids, the abundance of 597 changed more than 10-fold in quarter milk with NAS-IMI compared with healthy quarters. The results demonstrate the influence of NAS-IMI on the milk lipidome, implying significant changes in lipid species belonging to the family of triacylglycerols and sphingomyelins, and contribute to the understanding of inflammatory processes in the bovine udder, highlighting potential novel biomarkers for improving mastitis diagnostics.
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- 2020
12. Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics
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Daniela Pagnozzi, Alberto Alberti, Carla Cacciotto, Maria Filippa Addis, Marco Pittau, Elisabetta Coradduzza, Cacciotto C., Addis M.F., Pagnozzi D., Coradduzza E., Pittau M., and Alberti A.
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Proteomics ,Proteome ,040301 veterinary sciences ,Mycoplasma agalactiae ,Immunology ,ved/biology.organism_classification_rank.species ,Bacterial Protein ,Context (language use) ,Biology ,Immunoproteomics ,Microbiology ,0403 veterinary science ,03 medical and health sciences ,Immune system ,Antigen ,Bacterial Proteins ,Conserved antigen ,Animals ,Contagious agalactia ,030304 developmental biology ,0303 health sciences ,Antigens, Bacterial ,Sheep ,General Veterinary ,Animal ,ved/biology ,Immunodominant Epitopes ,Immunoproteomic ,Proteomic ,04 agricultural and veterinary sciences ,Subcellular localization ,Immunodominant protein ,Antigens, Surface ,Immunodominant Epitope - Abstract
Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.
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- 2020
13. Identification of secreted and cellular antigens of Staphylococcus aureus causing dairy sheep mastitis and their potential for vaccine development
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Gavino Marogna, Elisa Azara, Maria Filippa Addis, Sebastiana Tola, and Carla Longheu
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Proteomics ,Staphylococcus aureus ,040301 veterinary sciences ,Immunology ,Bacterial Toxins ,Leukocidin ,Cattle Diseases ,Exotoxins ,Sheep Diseases ,Biology ,medicine.disease_cause ,Immunoproteomics ,Microbiology ,0403 veterinary science ,03 medical and health sciences ,Immune system ,Antigen ,Leukocidins ,Tandem Mass Spectrometry ,medicine ,Animals ,Sheep milk ,Mastitis, Bovine ,030304 developmental biology ,0303 health sciences ,Antigens, Bacterial ,Sheep ,General Veterinary ,Staphylococcal Vaccines ,04 agricultural and veterinary sciences ,Staphylococcal Infections ,medicine.disease ,Mastitis ,Immunity, Humoral ,Cattle ,Female ,Staphylococcus - Abstract
Staphylococcus aureus is the leading cause of clinical mastitis and is associated with persistent subclinical infections in ewes, significantly compromising the quality and quantity of milk productions. To date, vaccines intended for use in sheep have been mainly focused on biofilm production traits, but many S. aureus pathogenic isolates do not produce biofilm, including those circulating in Sardinia, one of the leading sheep milk producers in Europe. The aim of this work was to identify suitable immunodominant, alternative candidates to biofilm components for vaccine and diagnostic development. An immunoproteomics study was carried out by testing sera from naturally infected sheep with a prevalent S. aureus lineage against cellular and secreted antigens, followed by tandem mass spectrometry identification of the most prominent immunogens. Four cellular and three secreted S. aureus antigens elicited a strong humoral host immune response. The four cellular antigens were the housekeeping proteins pyruvate kinase, elongation Factor Tu, dihydrolipoyl dehydrogenase, and alpha-keto acid dehydrogenase. The three secreted antigens were the bifunctional autolysin (Atl) and the two components of the Panton-Valentine leukocidin, lukF-PV/lukM, demonstrating the carriage of prophage phiPV83 in a sheep isolate and the strong response of the sheep host against them. In consideration of the key role played by these secreted proteins in S. aureus replication and immune evasion, these antigens may represent suitable candidates for developing vaccines eliciting a more successful immunological protection in areas where non-biofilm forming Staphylococcus spp. are the most widespread intramammary pathogens.
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- 2020
14. Proteomic datasets of uninfected and
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Salvatore, Pisanu, Carla, Cacciotto, Daniela, Pagnozzi, Sergio, Uzzau, Claudia, Pollera, Martina, Penati, Valerio, Bronzo, and Maria Filippa, Addis
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Proteomics ,Goat milk ,FASP ,Mass spectrometry ,NSAF ,food and beverages - Abstract
We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled “Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats”. After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243.
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- 2020
15. The value of the biomarkers cathelicidin, milk amyloid A, and haptoglobin to diagnose and classify clinical and subclinical mastitis
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A. Kossatz, Laurent Meriaux, S. Bertulat, Maria Filippa Addis, Giulia Maria Grazia Puggioni, L. Wollowski, and Wolfgang Heuwieser
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medicine.medical_specialty ,Cell Count ,Gastroenterology ,03 medical and health sciences ,Mammary Glands, Animal ,Cathelicidins ,Internal medicine ,Genetics ,Medicine ,Animals ,Serum amyloid A ,Udder ,Mastitis, Bovine ,030304 developmental biology ,Subclinical infection ,0303 health sciences ,Serum Amyloid A Protein ,biology ,Haptoglobins ,business.industry ,Haptoglobin ,0402 animal and dairy science ,food and beverages ,04 agricultural and veterinary sciences ,medicine.disease ,040201 dairy & animal science ,Mastitis ,medicine.anatomical_structure ,Milk ,biology.protein ,Biomarker (medicine) ,Animal Science and Zoology ,Cattle ,Female ,business ,Somatic cell count ,Biomarkers ,Food Science ,California mastitis test ,Antimicrobial Cationic Peptides - Abstract
Timely and objective diagnosis and classification of mastitis is crucial to ensure adequate management and therapeutic decisions. Analyzing specific biomarkers in milk could be advantageous compared with subjective or semiquantitative criteria, such as palpation of the udder in clinical mastitis cases or evaluation of somatic cell count using cow side tests (e.g., California Mastitis Test) in subclinical mastitis quarters. The objective of this study was to investigate the diagnostic value of 3 biomarkers; cathelicidin, milk amyloid A, and haptoglobin for the diagnosis of subclinical and clinical mastitis. Furthermore, the suitability of these biomarkers to differentiate between mild, moderate, and severe clinical mastitis and the influence of different pathogens on biomarker levels was tested. A total of 67 healthy cows, 119 cows with subclinical mastitis, and 212 cows with clinical mastitis were enrolled in the study. Although cathelicidin, haptoglobin, and milk amyloid A were measured in all samples from healthy cows and those with subclinical mastitis, haptoglobin, and cathelicidin results were only available from 121 out of 212 cows with clinical mastitis. Milk amyloid A was measured in all samples. In cows with clinical mastitis, the mastitic quarter and a second healthy quarter serving as a healthy in-cow control quarter were sampled. It was possible to differentiate between healthy quarters, quarters with subclinical mastitis, and quarters with clinical mastitis using all 3 biomarkers. Concerning cathelicidin, thresholds were 0.000 [sensitivity (Se) = 0.83, specificity (Sp) = 0.97] and 0.053 (Se = 0.98, Sp = 0.99) for normalized optical density at 450 nm (NOD450) for differentiating between healthy quarters and quarters with subclinical or clinical mastitis, respectively. Thresholds of 1.28 µg/mL (Se = 0.65, Sp = 0.76) and 1.81 µg/mL (Se = 0.77, Sp = 0.83) for milk amyloid A and 3.65 µg/mL (Se = 0.92, Sp = 0.94) and 5.40 µg/mL mL (Se = 0.96, Sp = 0.99) for haptoglobin were calculated, respectively. Healthy in-cow control quarters from cows with CM showed elevated milk amyloid A and haptoglobin levels compared with healthy quarters from healthy cows. Only the level of milk amyloid A was higher in severe clinical mastitis cases compared with mild ones. In contrast to clinical mastitis, cathelicidin and haptoglobin in subclinical mastitis quarters were significantly influenced by different bacteriological results. The measurement of cathelicidin, milk amyloid A, and haptoglobin in milk proved to be a reliable method to detect quarters with subclinical or clinical mastitis.
- Published
- 2020
16. Influence of subclinical mastitis and intramammary infection by coagulase-negative staphylococci on the cow milk peptidome
- Author
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Gabriella Tedeschi, Mariangela Albertini, Maria Filippa Addis, Valentina Zamarian, Valerio Bronzo, Francesco Maria Tangorra, Renata Piccinini, Elisa Maffioli, and Fabrizio Ceciliani
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0301 basic medicine ,Coagulase ,Biophysics ,Mastitis ,Biology ,Biochemistry ,Cow milk ,03 medical and health sciences ,Tandem Mass Spectrometry ,Casein ,medicine ,Animals ,Humans ,Mastitis, Bovine ,Subclinical infection ,030102 biochemistry & molecular biology ,food and beverages ,Gel electrophoresis of proteins ,Staphylococcal Infections ,medicine.disease ,030104 developmental biology ,Milk ,Immunology ,Herd ,Cattle ,Female ,Somatic cell count - Abstract
Coagulase-negative staphylococci (CNS) are the most prevalent microorganisms isolated from cow milk and are associated with subclinical mastitis and persistent increases in the bulk milk somatic cell count (BMSCC) of low BMSCC herds. By combining peptide enrichment, LC-ESI-MS/MS, and statistical analysis, we investigated the influence of subclinical mastitis and CNS infection on the milk peptidome. Quarter milk samples from clinically healthy Holstein cows were subjected to bacteriological culture (BC) and somatic cell counting (SCC) for two consecutive samplings and 28 (including 11 negatives and 17 positives) were selected for peptidomic analysis. The study identified 1363 different endogenous peptides and highlighted a significant increase of peptides in CNS-positive milk, mainly represented by casein fragments. Milk peptidome changes increased with the SCC, as also demonstrated by protein electrophoresis and densitometry. Peptides significantly different in CNS or CONTROL samples were identified and characterized. Our results indicate that subclinical mastitis by CNS can induce significant changes in the milk peptidome, opening the way to future studies for the identification of a biomarker panel as well as for the understanding of their consequences for the technological and sensorial characteristics of cow milk and dairy products. SIGNIFICANCE: This is the first investigation on the impact of subclinical CNS mastitis on the bovine milk peptidome. The peptide enrichment strategy combined with a highly sensitive MS/MS analysis enabled the compilation of a very large peptide dataset for healthy and mastitic milk. The comparison of CNS and Control samples, also considering SCC classes, highlighted several peptides with potential for understanding milk protein and peptide dynamics in subclinical mastitis, with possible implications for its detection.
- Published
- 2020
17. Diversity and functions of the sheep faecal microbiota: a multi-omic characterization
- Author
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Maria Filippa Addis, Alessandro Tanca, Marcello Abbondio, Cristina Fraumene, Massimo Deligios, Antonio Palomba, Sergio Uzzau, Daniela Pagnozzi, and Valeria Manghina
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DNA, Bacterial ,0301 basic medicine ,Carbohydrate transport ,Firmicutes ,Microbial metabolism ,Bioengineering ,Gut flora ,DNA, Ribosomal ,Applied Microbiology and Biotechnology ,Biochemistry ,Bacterial genetics ,Microbiology ,Feces ,03 medical and health sciences ,fluids and secretions ,RNA, Ribosomal, 16S ,Animals ,Cluster Analysis ,Intestine, Large ,Phylogeny ,Research Articles ,Genetics ,Sheep ,Bacteria ,biology ,Phylum ,Sequence Analysis, DNA ,biology.organism_classification ,Archaea ,Gastrointestinal Microbiome ,DNA, Archaeal ,030104 developmental biology ,Metaproteomics ,Euryarchaeota ,Metabolic Networks and Pathways ,Research Article ,Biotechnology - Abstract
Summary Little is currently known on the microbial populations colonizing the sheep large intestine, despite their expected key role in host metabolism, physiology and immunity. This study reports the first characterization of the sheep faecal microbiota composition and functions, obtained through the application of a multi‐omic strategy. An optimized protocol was first devised for DNA extraction and amplification from sheep stool samples. Then, 16S rDNA sequencing, shotgun metagenomics and shotgun metaproteomics were applied to unravel taxonomy, genetic potential and actively expressed functions and pathways respectively. Under a taxonomic perspective, the sheep faecal microbiota appeared globally comparable to that of other ruminants, with Firmicutes being the main phylum. In functional terms, we detected 2097 gene and 441 protein families, finding that the sheep faecal microbiota was primarily involved in catabolism. We investigated carbohydrate transport and degradation activities and identified phylum‐specific pathways, such as methanogenesis for Euryarchaeota and acetogenesis for Firmicutes. Furthermore, our approach enabled the identification of proteins expressed by the eukaryotic component of the microbiota. Taken together, these findings unveil structure and role of the distal gut microbiota in sheep, and open the way to further studies aimed at elucidating its connections with management and dietary variables in sheep farming.
- Published
- 2017
18. Evaluation of a bovine cathelicidin ELISA for detecting mastitis in the dairy buffalo: Comparison with milk somatic cell count and bacteriological culture
- Author
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Sergio Uzzau, Paolo Moroni, Vittorio Tedde, Giulia Maria Grazia Puggioni, Maria Filippa Addis, Jacopo Guccione, Paolo Ciaramella, Claudia Pollera, Valerio Bronzo, Puggioni, Giulia Maria Grazia, Tedde, Vittorio, Uzzau, Sergio, Guccione, Jacopo, Ciaramella, Paolo, Pollera, Claudia, Moroni, Paolo, Bronzo, Valerio, and Addis, Maria Filippa
- Subjects
Veterinary medicine ,Staphylococcus aureus ,Buffaloes ,040301 veterinary sciences ,medicine.medical_treatment ,Cell Count ,Enzyme-Linked Immunosorbent Assay ,Mastitis ,medicine.disease_cause ,Sensitivity and Specificity ,Cathelicidin ,0403 veterinary science ,03 medical and health sciences ,Cathelicidins ,medicine ,Animals ,Subclinical mastitis ,030304 developmental biology ,0303 health sciences ,Subclinical mastitis Water buffalo milk Cathelicidin ELISA Somatic cell count Bacteriological culture ,General Veterinary ,biology ,Diagnostic Tests, Routine ,04 agricultural and veterinary sciences ,Staphylococcal Infections ,biology.organism_classification ,medicine.disease ,stomatognathic diseases ,Milk ,Herd ,Cattle ,Female ,Bubalus ,Somatic cell count ,True positive rate ,Antimicrobial Cationic Peptides - Abstract
A recently developed bovine cathelicidin (CATH) ELISA was evaluated in the dairy buffalo (Bubalus bubalis) by testing 618 quarter milk samples from a herd with subclinical mastitis cases. Somatic cell count (SCC) and bacteriological culture (BC) were carried out on the same samples for comparison. Out of 618 quarters, 258 (41.75%) were positive to CATH, 289 (46.76%) had SCC > 200,000 cells/mL, and 457 (73.95%) were positive to BC. The most prevalent microorganism was Staphylococcus aureus (SAU, 35.76% of all quarters), followed by non-aureus staphylococci (NAS, 22.17% of all quarters). Clinical mastitis quarters were only 7 (1.13%). CATH levels were significantly higher in clinical quarters and in high SCC, BC-positive quarters than in healthy, low SCC, BC-negative quarters. The highest median values were observed for SAU and the lowest for NAS. Differences among microorganism classes were generally more significant for SCC than for CATH. Test char acteristics of the CATH ELISA, evaluated by considering as true positives all BC-positive quarters with SCC > 200,000 cells/mL (N = 242), and as true negatives all sterile quarters with SCC < 200,000 cells/mL (N = 44), were as follows: sensitivity 57.85%, specificity 84.09%, positive predictive value 95.24%, negative predictive value 26.62%, accuracy 61.89%. Therefore, the bovine CATH ELISA showed a fair sensitivity and a good specificity in detecting water buffalo mastitis.
- Published
- 2019
19. Milk cathelicidin and somatic cell counts in dairy goats along the course of lactation
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Sergio Uzzau, Valerio Bronzo, Giulia Maria Grazia Puggioni, Antonio Casula, Paolo Moroni, Claudia Pollera, Giulio Curone, Vittorio Tedde, and Maria Filippa Addis
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Microbiological culture ,040301 veterinary sciences ,medicine.medical_treatment ,Mammary gland ,Biology ,medicine.disease_cause ,Cathelicidin ,0403 veterinary science ,Animal science ,Cathelicidins ,Lactation ,medicine ,Animals ,Streptococcus ,Goats ,0402 animal and dairy science ,04 agricultural and veterinary sciences ,General Medicine ,medicine.disease ,040201 dairy & animal science ,Mastitis ,medicine.anatomical_structure ,Milk ,Herd ,Animal Science and Zoology ,Female ,Somatic cell count ,Food Science ,Antimicrobial Cationic Peptides - Abstract
This research communication reports the evaluation of cathelicidin in dairy goat milk for its relationship with the somatic cell count (SCC) and microbial culture results. Considering the limited performances of SCC for mastitis monitoring in goats, there is interest in evaluating alternative diagnostic tools. Cathelicidin is an antimicrobial protein involved in innate immunity of the mammary gland. In this work, half-udder milk was sampled bimonthly from a herd of 37 Alpine goats along an entire lactation and tested with the cathelicidin ELISA together with SCC and bacterial culture. Cathelicidin and SCC showed a strong correlation (r = 0.72; n = 360 milk samples). This was highest in mid-lactation (r = 0.83) and lowest in late lactation (r = 0.61), and was higher in primiparous (0.80, n = 130) than in multiparous goats (0.71, n = 230). Both markers increased with stage of lactation, but cathelicidin increased significantly less than SCC. In addition, peak level in late lactation was lower for cathelicidin (5.05-fold increase) than for SCC (7.64-fold increase). Twenty-one (5.8%) samples were positive to bacteriological culture, 20 for coagulase-negative staphylococci and one for Streptococcus spp.; 18 of them were positive to the cathelicidin ELISA (85.71% sensitivity). Sensitivity of SCC >500 000 and of SCC >1 000 000 cells/ml was lower (71.43 and 23.81%, respectively). Therefore, the high correlation of cathelicidin with SCC during the entire lactation, along with its lower increase in late lactation and good sensitivity in detecting intramammary infection (IMI), indicate a potential for monitoring subclinical mastitis in dairy goats. However, based on this preliminary assessment, specificity should be improved (40.41% for cathelicidin vs. 54.57 and 67.85% for SCC >500 000 and >1 000 000 cells/ml, respectively). Therefore, the application of cathelicidin for detecting goat IMI will require further investigation and optimization, especially concerning the definition of diagnostic thresholds.
- Published
- 2019
20. Proteomic changes occurring along gonad maturation in the edible sea urchin Paracentrotus lividus
- Author
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Maria Filippa Addis, Grazia Biosa, Sergio Uzzau, Maura Baroli, Stefania Ghisaura, Daniela Pagnozzi, Tonina Roggio, Roberto Anedda, and Barbara Loi
- Subjects
0301 basic medicine ,Gonad ,Biophysics ,Zoology ,Sexing ,Biochemistry ,Paracentrotus lividus ,03 medical and health sciences ,Aquaculture ,biology.animal ,medicine ,Animals ,Shotgun proteomics ,Sea urchin ,biology ,business.industry ,Ecology ,04 agricultural and veterinary sciences ,biology.organism_classification ,030104 developmental biology ,medicine.anatomical_structure ,Sea Urchins ,Proteome ,Paracentrotus ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Gamete ,business - Abstract
The reproductive stage of Paracentrotus lividus strongly influences product quality that, in turn, impacts significantly on the market price. Large, compact and sweet gonads are preferred, and sensory attributes are positively related to the ratio of nutritive phagocytes to gametes. Gonads at advanced maturation stages, although larger, have less desirable attributes, being more watery and bitter especially in females. Therefore, the best compromise among size, texture, and taste needs to be reached. In this study, wild P. lividus were collected along coastal Sardinia, and gonads in the recovery, pre-mature, mature, and spent stages were analyzed by gel-based and by shotgun proteomics. A detailed characterization of the proteome changes occurring in gonads of both sexes along maturation was achieved, and significant modifications were seen in numerous proteins involved in nutrient accumulation in nutritive phagocytes, as well as in gamete biology and maturation. Adding to an improved understanding of the P. lividus reproductive cycle in its natural environment, the results described in this work may form the basis for defining novel protein markers and procedures for an easier sexing and staging, and for monitoring sea urchin gonad maturation cycles in aquaculture plants. Mass spectrometry data are deposited in ProteomeXchange ( PXD004200 ). Significance The sensory quality of P. lividus gonads is strongly influenced by the reproductive cycle, with significant changes in flavor, texture, and size. A better knowledge of the protein profiles, patterns, and markers associated with gonad sex and maturation stage can have useful implications for understanding and monitoring these changes. One of these is the ability to identify protein profiles specifically associated with a given stage and, in perspective, to identify maturation and sex markers. The comprehensive proteomic evaluation achieved in this work was made possible by the application of combined gel-based and shotgun approaches. As a result, this study generated the largest proteomic dataset available in the literature for P. lividus , as well as a general picture of protein abundance changes occurring along maturation.
- Published
- 2016
21. Mycoplasmalipoproteins are major determinants of neutrophil extracellular trap formation
- Author
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Stefano Rocca, Tania Carta, Vittorio Tedde, Carla Cacciotto, Tiziana Cubeddu, Bernardo Chessa, Antonio Anfossi, Alberto Alberti, Gessica Tore, Marco Pittau, and Maria Filippa Addis
- Subjects
0301 basic medicine ,Innate immune system ,ved/biology ,Mycoplasma agalactiae ,Immunology ,ved/biology.organism_classification_rank.species ,Antimicrobial peptides ,Lipopeptide ,Inflammation ,Neutrophil extracellular traps ,Mycoplasma ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,TLR2 ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Virology ,medicine ,medicine.symptom - Abstract
Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.
- Published
- 2016
22. Proteomic analysis ofRhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast
- Author
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Daniela Pagnozzi, Alessandro Tanca, Grazia Biosa, Marcello Abbondio, Maria Filippa Addis, Ilaria Mannazzu, Sergio Uzzau, Raffaela Cutzu, and Sara Landolfo
- Subjects
0301 basic medicine ,Fungal protein ,biology ,business.industry ,030106 microbiology ,Bioengineering ,Computational biology ,Rhodotorula ,biology.organism_classification ,Proteomics ,Applied Microbiology and Biotechnology ,Biochemistry ,Rhodotorula mucilaginosa ,Yeast ,Biotechnology ,03 medical and health sciences ,Genetics ,Database search engine ,Identification (biology) ,Shotgun proteomics ,business - Abstract
Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.
- Published
- 2016
23. Influence of seasonal and environmental patterns on the lipid content and fatty acid profiles in gonads of the edible sea urchin Paracentrotus lividus from Sardinia
- Author
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Maria Filippa Addis, Ivan Guala, Tonina Roggio, Maura Baroli, Sergio Uzzau, Riccardo Melis, Silvia Siliani, Roberto Anedda, Roberta Sanna, and Barbara Loi
- Subjects
0106 biological sciences ,endocrine system ,Zoology ,Aquatic Science ,Oceanography ,01 natural sciences ,Paracentrotus lividus ,Cyclical trend ,biology.animal ,Mediterranean Sea ,Animals ,Gonads ,Sea urchin ,Ecosystem ,chemistry.chemical_classification ,photoperiodism ,biology ,Ecology ,010604 marine biology & hydrobiology ,Fatty Acids ,Sampling (statistics) ,Fatty acid ,04 agricultural and veterinary sciences ,General Medicine ,Lipid Metabolism ,biology.organism_classification ,Lipids ,Pollution ,Sea surface temperature ,Italy ,chemistry ,Lipid content ,Paracentrotus ,040102 fisheries ,0401 agriculture, forestry, and fisheries ,Seasons - Abstract
The influence of seasonal and environmental patterns on the lipid fraction of Paracentrotus lividus gonads was investigated. For this purpose, sea urchins were collected monthly over a year from two Sardinian coastal areas. Total lipids in gonads follow an annual cyclical trend, described by a sine wave curve, that it is more influenced by season than by growing area. The lowest lipid content in gonads corresponds to a high percentage of mature reproductive stages (i.e. winter season), independently of sampling area. A variation in total lipid content follows a change in photoperiod, while it is related to sea surface temperature. Multivariate analysis on fatty acid profiles of gonads, detected by gas chromatography, clusters the collected specimens mainly according to the sampling area, secondly according to the sites within the same sampling area and finally according to season.
- Published
- 2016
24. The Role of Innate Immune Response and Microbiome in Resilience of Dairy Cattle to Disease: The Mastitis Model
- Author
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Valerio Bronzo, Bianca Castiglioni, Federica Riva, Erminio Trevisi, Giulio Curone, Maria Filippa Addis, Massimo Amadori, Paolo Moroni, V. Lopreiato, and Paola Cremonesi
- Subjects
lcsh:Veterinary medicine ,Innate immune system ,General Veterinary ,Settore AGR/19 - ZOOTECNICA SPECIALE ,Dairy cattle diseases ,Metabolic stress ,Microbiome ,Review ,Disease ,Biology ,medicine.disease ,Mastitis ,Immune system ,lcsh:Zoology ,Immunology ,medicine ,lcsh:SF600-1100 ,Animal Science and Zoology ,lcsh:QL1-991 ,Epigenetics ,Adaptation ,Dairy cattle - Abstract
Simple Summary A major concern for the development of livestock activities is represented by the gradual reduction of antibiotic usage in farm animals, which may disturb the fragile balance between animal health and production. Therefore, it is necessary to maintain the immunocompetence of farm animals within the structure of this new trend toward reduced drug usage. High-yielding dairy cattle often experience more disease prevalence associated with short life expectancy and reduced environmental fitness. These signs of immunosuppression can be linked to metabolic changes observed around calving, which confirms the crucial link between immunity and milk production levels. The immunocompetence of these animals should be re-appraised and new disease control strategies should be based on creating a more efficient immune system. This review summarizes the dairy cow’s metabolic response to stress and what role the innate immune system and microbiome play. The review also discusses how new approaches to animal health based on specific intervention at dry-off and in the first weeks after calving are needed as the relevant stressors are pivotal to disease occurrence. Abstract Animal health is affected by many factors such as metabolic stress, the immune system, and epidemiological features that interconnect. The immune system has evolved along with the phylogenetic evolution as a highly refined sensing and response system, poised to react against diverse infectious and non-infectious stressors for better survival and adaptation. It is now known that high genetic merit for milk yield is correlated with a defective control of the inflammatory response, underlying the occurrence of several production diseases. This is evident in the mastitis model where high-yielding dairy cows show high disease prevalence of the mammary gland with reduced effectiveness of the innate immune system and poor control over the inflammatory response to microbial agents. There is growing evidence of epigenetic effects on innate immunity genes underlying the response to common microbial agents. The aforementioned agents, along with other non-infectious stressors, can give rise to abnormal activation of the innate immune system, underlying serious disease conditions, and affecting milk yield. Furthermore, the microbiome also plays a role in shaping immune functions and disease resistance as a whole. Accordingly, proper modulation of the microbiome can be pivotal to successful disease control strategies. These strategies can benefit from a fundamental re-appraisal of native cattle breeds as models of disease resistance based on successful coping of both infectious and non-infectious stressors.
- Published
- 2020
25. Liver proteome dataset of Sparus aurata exposed to low temperatures
- Author
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Grazia Biosa, Daniela Pagnozzi, Stefania Ghisaura, Roberto Anedda, Sergio Uzzau, Riccardo Melis, H. Slavski, and Maria Filippa Addis
- Subjects
Low temperature exposure ,Biology ,lcsh:Computer applications to medicine. Medical informatics ,Proteomics ,Tandem mass spectrometry ,Differential analysis ,03 medical and health sciences ,0302 clinical medicine ,Biochemistry, Genetics and Molecular Biology ,Shotgun proteomics ,Maintenance phase ,Research article ,Food science ,lcsh:Science (General) ,Cold stress ,Liver proteins ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Gilthead sea bream ,Proteome ,lcsh:R858-859.7 ,Liver proteomic dataset ,030217 neurology & neurosurgery ,lcsh:Q1-390 - Abstract
We report the proteomic dataset of livers from Sparus aurata exposed to low temperature during growth. Gilthead sea bream juveniles were reared in Recirculating Aquaculture Systems (RAS) and exposed to a temperature ramp made of two phases of four weeks each: a Cooling phase from 18 °C (t0) to 11 °C (t1) and a Cold Maintenance phase at 11 °C (t1-t2) in a 8 week feeding trial. At the end of the experiment, sea bream livers were collected and analyzed with a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry, peptide identification carried out using Sequest-HT as search engine within the Proteome Discoverer informatic platform, and label-free differential analysis.The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011059 (Vizcaíno et al., 2016; Deutsch et al., 2017; Perez-Riverol et al., 2016). The dataset described here is also related to the research article entitled “Liver proteomics of gilthead sea bream (Sparus aurata) exposed to cold stress” (Ghisaura et al., 2019). Keywords: Cold stress, Gilthead sea bream, Low temperature exposure, Liver proteins, Shotgun proteomics, Liver proteomic dataset
- Published
- 2019
26. Differences in the peptide profile of raw and pasteurised ovine milk cheese and implications for its bioactive potential
- Author
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Daniela Pagnozzi, Tonina Roggio, Maria Filippa Addis, A. Pirisi, M. Pes, Salvatore Pisanu, and Sergio Uzzau
- Subjects
chemistry.chemical_classification ,medicine.diagnostic_test ,Proteolysis ,Proteolytic enzymes ,Pasteurization ,Ripening ,Biological activity ,Peptide ,Biology ,Applied Microbiology and Biotechnology ,law.invention ,Enzyme ,chemistry ,Biochemistry ,law ,Casein ,medicine ,Food science ,Food Science - Abstract
Cheese produced using raw ovine milk (R) or pasteurised ovine milk (P) was subjected to peptide extraction, characterisation by mass spectrometry and bioinformatic analysis with the aim of assessing the impact of milk pasteurisation on the final peptide profile. In total, 187 peptides arising from β-casein, αS1-casein, and αS2-casein were identified. Upon label-free quantitation, 58 peptides were found to be significantly different in the two preparations; 38 were more abundant in R and 20 were more abundant in P. Of these, 27 were unique to R and 10 were unique to P. Bioinformatic analysis by EnzymePredictor provided insights into the influence of milk pasteurisation on susceptibility of cheese proteins to proteolytic enzymes during ripening. Finally, BIOPEP analysis predicted a biological activity for 37 of the 187 identified sequences (20%), with a significantly higher abundance of peptides with immunomodulating and ACE-inhibitor properties in R cheeses.
- Published
- 2015
27. Mastitis, milk quality and yield
- Author
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F.L. Welcome, Maria Filippa Addis, and Paolo Moroni
- Subjects
Animal science ,Yield (finance) ,media_common.quotation_subject ,medicine ,Quality (business) ,medicine.disease ,Mathematics ,media_common ,Mastitis - Published
- 2017
28. Cathelicidin production and release by mammary epithelial cells during infectious mastitis
- Author
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Alberto Alberti, Simone Dore, Vittorio Tedde, Carla Cacciotto, Tiziana Cubeddu, Salvatore Pisanu, Maria Filippa Addis, Marco Pittau, Agnese E. Cannas, Stefano Rocca, Sergio Uzzau, Cubeddu T., Cacciotto C., Pisanu S., Tedde V., Alberti A., Pittau M., Dore S., Cannas A., Uzzau S., Rocca S., and Addis M.F.
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Mycoplasma agalactiae ,Immunology ,Antimicrobial peptides ,ved/biology.organism_classification_rank.species ,Blotting, Western ,Sheep Diseases ,Mammary epithelial cell ,Enzyme-Linked Immunosorbent Assay ,Mastitis ,Cathelicidin ,Microbiology ,03 medical and health sciences ,Immunomicroscopy ,Mammary Glands, Animal ,Cathelicidins ,medicine ,Animals ,Mycoplasma Infections ,In Situ Hybridization, Fluorescence ,Streptococcus uberis ,Mastiti ,Sheep ,General Veterinary ,biology ,ved/biology ,Degranulation ,Epithelial Cells ,Neutrophil extracellular traps ,Staphylococcal Infections ,biology.organism_classification ,medicine.disease ,Milk ,030104 developmental biology ,lipids (amino acids, peptides, and proteins) ,Female ,Antimicrobial peptide ,Antimicrobial Cationic Peptides - Abstract
Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers.
- Published
- 2017
29. Effect of whey concentration on protein recovery in fresh ovine ricotta cheese
- Author
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S. Furesi, Giovanni Falchi, Maria Filippa Addis, M. Pes, Myriam Fiori, Enrico Salvatore, A. Pirisi, Tonina Roggio, and Daniela Pagnozzi
- Subjects
Proteomics ,Whey protein ,Hot Temperature ,Food Handling ,Ultrafiltration ,Fraction (chemistry) ,Lactoglobulins ,Protein aggregation ,Cheese ,Tandem Mass Spectrometry ,Genetics ,Animals ,Food science ,Sheep ,Chromatography ,Chemistry ,Hydrogen-Ion Concentration ,Milk Proteins ,Dietary Fats ,Ricotta cheese ,Whey Proteins ,Yield (chemistry) ,Lactalbumin ,Electrophoresis, Polyacrylamide Gel ,Animal Science and Zoology ,Composition (visual arts) ,Rennet ,Dietary Proteins ,Chymosin ,Chromatography, Liquid ,Food Science - Abstract
Ricotta cheese, particularly the ovine type, is a typical Italian dairy product obtained by heat-coagulation of the proteins in whey. The aim of this work was to investigate the influence of whey protein concentration, obtained by ultrafiltration, on yield of fresh ovine ricotta cheese. Ricotta cheeses were obtained by thermocoagulation of mixtures with protein content of 1.56, 3.10, 4.16, and 7.09g/100g from the mixing of skim whey and ultrafiltered skim whey. A fat-to-protein ratio of 1.1 (wt/wt) was obtained for all mixtures by adding fresh cream. The initial mixtures, as well as the final ricotta cheeses, were analyzed for their composition and by SDS-PAGE. Protein bands were quantified by QuantityOne software (Bio-Rad, Hercules, CA) and identified by liquid chromatography-tandem mass spectrometry. Significant differences in the composition of the ricotta cheese were observed depending on protein concentration. Particularly, ricotta cheese resulting from the mixture containing 7.09g/100g of protein presented higher moisture (72.88±1.50g/100g) and protein (10.18±0.45g/100g) contents than that prepared from the mixture with 1.56g/100g of protein (69.52±1.75 and 6.70±0.85g/100g, respectively), and fat content was lower in this sample (12.20±1.60g/100g) compared with the other treatments, with mean values between 15.72 and 20.50g/100g. Each protein fraction presented a different behavior during thermocoagulation. In particular, the recovery of β-lactoglobulin and α-lactalbumin in the cheese increased as their content increased in the mixtures. It was concluded that concentrating ovine rennet whey improved the extent of heat-induced protein aggregation during the thermal coagulation process. This resulted in a better recovery of each protein fraction in the product, and in a consequent increase of ricotta cheese yield.
- Published
- 2014
30. Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis
- Author
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Maria Filippa Addis, Carla Cacciotto, Vittorio Tedde, Clara Locatelli, Giulia Maria Grazia Puggioni, Sergio Uzzau, Paolo Moroni, Daniela Pagnozzi, Valerio Bronzo, Antonio Casula, Giulio Curone, Addis M.F., Bronzo V., Puggioni G.M.G., Cacciotto C., Tedde V., Pagnozzi D., Locatelli C., Casula A., Curone G., Uzzau S., and Moroni P.
- Subjects
0301 basic medicine ,medicine.drug_class ,medicine.medical_treatment ,Staphylococcus ,Antibiotics ,Cell Count ,Biology ,medicine.disease_cause ,Serratia ,Cathelicidin ,Microbiology ,03 medical and health sciences ,Disease severity ,Cathelicidins ,cathelicidin ,Genetics ,medicine ,clinical mastiti ,Animals ,Pathogen ,Mastitis, Bovine ,intramammary infection ,dairy cow ,0402 animal and dairy science ,04 agricultural and veterinary sciences ,medicine.disease ,biology.organism_classification ,040201 dairy & animal science ,Mastitis ,Anti-Bacterial Agents ,030104 developmental biology ,Milk ,Streptococcus agalactiae ,Immunology ,Animal Science and Zoology ,lipids (amino acids, peptides, and proteins) ,ELISA ,Cattle ,Female ,Somatic cell count ,Food Science ,Antimicrobial Cationic Peptides - Abstract
The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC
- Published
- 2016
31. Production and Release of Antimicrobial and Immune Defense Proteins by Mammary Epithelial Cells following Streptococcus uberis Infection of Sheep
- Author
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Carla Cacciotto, Maria Filippa Addis, Salvatore Pisanu, Daniela Pagnozzi, Stefano Rocca, Sergio Uzzau, Franca Campesi, Gavino Marogna, Tiziana Cubeddu, Giuseppe Martino Schianchi, Addis M.F., Pisanu S., Marogna G., Cubeddu T., Pagnozzi D., Cacciotto C., Campesi F., Schianchi G., Rocca S., and Uzzau S.
- Subjects
Proteomics ,medicine.medical_treatment ,Immunology ,Mammary gland ,Inflammation ,Microbiology ,Cathelicidin ,Mammary Glands, Animal ,Immune system ,Anti-Infective Agents ,Cathelicidins ,Streptococcal Infections ,medicine ,Animals ,Lactation ,Mastitis, antimicrobial peptides, proteomics ,Udder ,Glycoproteins ,Streptococcus uberis ,Host Response and Inflammation ,Sheep ,Innate immune system ,biology ,Streptococcus ,Epithelial Cells ,Lipid Droplets ,biology.organism_classification ,Milk ,Infectious Diseases ,medicine.anatomical_structure ,Parasitology ,Glycolipids ,Calprotectin ,medicine.symptom ,Leukocyte L1 Antigen Complex ,Antimicrobial Cationic Peptides - Abstract
Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis . The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.
- Published
- 2013
32. Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome
- Author
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Grazia Biosa, Daniela Pagnozzi, Sergio Uzzau, Maria Filippa Addis, and Alessandro Tanca
- Subjects
Proteomics ,Proteome ,Detergents ,Biology ,medicine.disease_cause ,Orbitrap ,Biochemistry ,Specimen Handling ,Workflow ,law.invention ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,law ,Protein purification ,Escherichia coli ,medicine ,Protein precipitation ,Sample preparation ,Molecular Biology ,Chromatography ,Escherichia coli Proteins ,Membrane Proteins ,Ammonium bicarbonate ,chemistry ,Membrane protein ,Chromatography, Liquid - Abstract
This work presents a comparative evaluation of several detergent-based sample preparation workflows for the MS-based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest- and SDS-based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in-solution digestion (SC), protein precipitation followed by in-solution digestion in ammonium bicarbonate or urea buffer, filter-aided sample preparation (FASP), and 1DE separation followed by in-gel digestion. On the whole, about 1000 proteins were identified upon LC-MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented.
- Published
- 2013
33. Proteomic characterization of hepatitis C eradication: Enzyme switch in the healing liver
- Author
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Daniela Pagnozzi, P. Nieddu, Maria Pina Dore, Maria Stella Mura, Sergio Uzzau, Andrea Soddu, Alessandro Tanca, Paolo Cossu-Rocca, Maria Filippa Addis, Giordano Madeddu, Sergio Babudieri, and Giovannino Massarelli
- Subjects
Adult ,Proteomics ,Proteome ,medicine.medical_treatment ,Alpha interferon ,Interferon alpha-2 ,Carbohydrate metabolism ,Pharmacology ,Biology ,Antiviral Agents ,Polyethylene Glycols ,Diabetes Complications ,Insulin resistance ,Downregulation and upregulation ,Virology ,Ribavirin ,medicine ,Humans ,Insulin ,Interferon-alpha ,Lipid metabolism ,Hepatitis C ,medicine.disease ,Recombinant Proteins ,Alcoholism ,Metabolic pathway ,Infectious Diseases ,Liver ,Immunology ,Female - Abstract
Lipid pathway impairment, decrease in the antioxidant pool and downregulation in amino-acid metabolism are just some of the metabolic variations attributed to chronic HCV infection. All of them have been studied separately, mainly in animal models. Thanks to proteomic analysis we managed to describe (for the fist time to the best of our knowledge), in vivo and in humans, the metabolic alterations caused by HCV, and the recovery of the same alterations during HCV treatment. We performed proteomic analysis on liver specimens of a 28-year-old woman affected by hepatitis C genotype 1a, alcoholism and diabetes mellitus type 1, before and after antiviral treatment with pegylated interferon alpha 2b and ribavirin. The subject, thanks to a patient-tailored therapy, reached Sustained Virological Response. Throughout the treatment period the patient was monitored with subsequent biochemical, clinical and psychological examinations. The data obtained by the patient's close monitoring suggest a direct interaction between insulin resistance and an active HCV genotype 1 infection, with a leading role played by the infection, and not by insulin resistance, as demonstrated by the sharp fall of the insulin units needed per day during treatment. The proteomic analysis showed that after therapy, a downregulation of enzymes involved in amino acid metabolism, glycolysis/gluconeogenesis and alcohol catabolism takes place, the latter probably due to cessation of alcohol abuse. On the contrary, the metabolic pathways linked to metabolism of the reactive oxygen species were upregulated after therapy. Finally, a significant alteration in the pathway regulated by peroxisome proliferator-activated receptor alpha (PPARA), a major regulator of lipid metabolism in the liver, was reported. These "real time" data confirm in vivo, in humans, that during HCV infection, the pathways related to fatty acids, glucose metabolism and free radical scavenging are inhibited. The same enzyme deficit is completely recovered after HCV eradication.
- Published
- 2013
34. High throughput genomic and proteomic technologies in the fight against infectious diseases
- Author
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Sergio Uzzau, Maria Filippa Addis, Massimo Deligios, and Alessandro Tanca
- Subjects
Proteomics ,education.field_of_study ,Emerging technologies ,business.industry ,Population ,Reverse vaccinology ,Genomics ,General Medicine ,Computational biology ,Biology ,Proteogenomics ,Communicable Diseases ,Microbiology ,Biotechnology ,Infectious Diseases ,Virology ,Communicable Disease Control ,Molecular targets ,Humans ,Parasitology ,Identification (biology) ,education ,business - Abstract
New technologies have shown significant promise in the fight against infectious diseases, with the discovery of novel molecular targets for in vitro diagnostics and the improved design of vaccines. In developing countries, especially in areas of neglected diseases and resources-poor settings, a number of technological innovations are further needed, such as the integration of old and new biomarkers in suitable analysis platforms, the simplification of existing analysis systems, and the improvement of sample preservation and management. However, in these areas, identification of new biomarkers for infectious diseases is still a core issue in the diagnostic quest. Similarly, new technologies will allow scientists to design vaccines with improved immunogenicity, efficacy and safety in the local area, according to the circulating pathogenic strains and the genetic background of the population to be immunized. In this work we review the current omics-based technologies and their potential for accelerating the development of next generation vaccines and the identification of biomarkers suitable for point-of-care (POC) diagnostic applications.
- Published
- 2013
35. Characterization of size and composition of milk fat globules from Sarda and Saanen dairy goats
- Author
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Maria Filippa Addis, Anna Maria Roggio, G. Leo, Alessandro Tanca, Salvatore Pisanu, Tonina Roggio, M. Piccinini, Daniela Pagnozzi, Sergio Uzzau, and Gavino Marogna
- Subjects
Animal science ,Food Animals ,biology ,Biochemistry ,Homogeneous ,Milk fat ,Animal Science and Zoology ,Sarda ,Composition (visual arts) ,Protein composition ,biology.organism_classification ,Relative species abundance ,Breed - Abstract
The small size of goat milk fat globules (MFGs) is one of the factors contributing to the higher digestibility of goat milk compared to other milks. In this study, size, protein composition and lipid distribution of MFGs were evaluated comparatively in a popular dairy breed, Saanen, and in a minor breed, Sarda. MFGs were found to be significantly smaller in Sarda compared to Saanen goats, with average diameters of 2.73 ± 0.15 μm and 3.63 ± 0.27 μm, respectively. Raman spectroscopy revealed differences in the lipid profiles of differently sized MFGs within each breed, with MFGs of the same size class having comparable profiles between breeds. Proteomic characterization by SDS-PAGE followed by tandem mass spectrometry (GeLC–MS/MS) and label-free differential quantification highlighted significant differences in expression levels of MFG proteins from the two breeds, with a higher abundance of cytoplasmic proteins in Sarda MFGs and of membrane proteins in Saanen MFGs. Moreover, differences in the relative abundance of several major MFG proteins were observed for the two breeds. In conclusion, this study demonstrates the existence of breed-dependent differences in the lipid and protein makeup of goat MFGs, likely related to their different size distribution. This highlights once again the importance of investigating biodiversity in autochthonous and neglected breeds, which often possess valuable attributes that might be lost as a consequence of the widespread diffusion of highly productive, but more homogeneous, dairy breeds.
- Published
- 2013
36. Proteomic dataset of Paracentrotus lividus gonads of different sexes and at different maturation stages
- Author
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Tonina Roggio, Grazia Biosa, Maura Baroli, Daniela Pagnozzi, Stefania Ghisaura, Roberto Anedda, Sergio Uzzau, Maria Filippa Addis, and Barbara Loi
- Subjects
0301 basic medicine ,Multidisciplinary ,Gonad ,030102 biochemistry & molecular biology ,biology ,Zoology ,biology.organism_classification ,Bioinformatics ,lcsh:Computer applications to medicine. Medical informatics ,Differential analysis ,Paracentrotus lividus ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,biology.animal ,Proteome ,medicine ,lcsh:R858-859.7 ,Research article ,Protein identification ,Shotgun proteomics ,lcsh:Science (General) ,Sea urchin ,lcsh:Q1-390 ,Data Article - Abstract
We report the proteomic dataset of gonads from wild Paracentrotus lividus related to the research article entitled "Proteomic changes occurring along gonad maturation in the edible sea urchin Paracentrotus lividus" [1]. Gonads of three individuals per sex in the recovery, pre-mature, mature, and spent stages were analyzed using a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry, protein identification carried out using Sequest-HT as the search engine within the Proteome Discoverer informatics platform, and label-free differential analysis. The dataset has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD004200.
- Published
- 2016
37. Proteomic changes in the ileum of sheep infected with Mycobacterium avium subspecies paratuberculosis
- Author
-
Maria Filippa Addis, Stefano Rocca, Sergio Uzzau, Salvatore Pisanu, and Tiziana Cubeddu
- Subjects
0301 basic medicine ,Proteomics ,Proteome ,040301 veterinary sciences ,Phagosome acidification ,Paratuberculosis ,Gene Expression ,Sheep Diseases ,Microbiology ,0403 veterinary science ,03 medical and health sciences ,Ileum ,medicine ,Animals ,KEGG ,Shotgun proteomics ,Pathogen ,Sheep ,General Veterinary ,biology ,04 agricultural and veterinary sciences ,medicine.disease ,biology.organism_classification ,Mycobacterium avium subspecies paratuberculosis ,Mycobacterium avium subsp. paratuberculosis ,030104 developmental biology ,Animal Science and Zoology ,Female - Abstract
Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, β-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.
- Published
- 2016
38. Characterization of sheep milk fat globule proteins by two-dimensional polyacrylamide gel electrophoresis/mass spectrometry and generation of a reference map
- Author
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Grazia Biosa, Maria Filippa Addis, Giovanni Falchi, Stefania Ghisaura, Alessandro Tanca, Tonina Roggio, Daniela Pagnozzi, Salvatore Pisanu, and Sergio Uzzau
- Subjects
Chromatography ,food.ingredient ,Vesicle ,food and beverages ,Biology ,Mass spectrometry ,Applied Microbiology and Biotechnology ,medicine.anatomical_structure ,food ,Biochemistry ,Lactation ,Skimmed milk ,medicine ,Reference map ,Globules of fat ,Sheep milk ,Polyacrylamide gel electrophoresis ,Food Science - Abstract
Milk fat globules (MFGs) are fat droplets released in milk as vesicles with a tripartite membrane structure. MGF proteins (MFGPs) are involved in many metabolic processes, and are recently gaining growing attention. Sheep MFGPs were extracted and characterized by two-dimensional polyacrylamide gel electrophoresis/mass spectrometry (2-DE/MS), and a reference map was established. Specifically, 29 MFGPs were successfully identified by tandem MS from 61 spots, localized in the reference map, and characterized for gene ontology. For comparison, skim milk and whey were also subjected to 2-DE/MS, achieving identification of all the major sheep milk proteins and their localization in reference maps. Reproducibility of MFGP maps was then assessed; no significant variations were detectable when comparing samples from different healthy animals at the same stage of lactation. These results provide the first 2-DE reference map of sheep MFGPs, opening the way to studies on their expression in different physiological and pathological conditions.
- Published
- 2012
39. Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation
- Author
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Valli De Re, Salvatore Pisanu, Sergio Uzzau, Daniela Pagnozzi, Vincenzo Canzonieri, Marica Garziera, Maria Paola Simula, Alessandro Tanca, Maria Filippa Addis, Renato Cannizzaro, and Grazia Biosa
- Subjects
Bouin's Fixative ,Clinical Biochemistry ,Proteome ,Computational biology ,Biology ,Proteomics ,Bioinformatics ,Biochemistry ,Analysis method ,Paraffin embedded tissue ,Analytical Chemistry - Abstract
Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.
- Published
- 2012
40. Proteomic analysis of Rhodotorula mucilaginosa: dealing with the issues of a non-conventional yeast
- Author
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Maria Filippa, Addis, Alessandro, Tanca, Sara, Landolfo, Marcello, Abbondio, Raffaela, Cutzu, Grazia, Biosa, Daniela, Pagnozzi, Sergio, Uzzau, and Ilaria, Mannazzu
- Subjects
Fungal Proteins ,Proteomics ,Gene Ontology ,Sequence Analysis, Protein ,Tandem Mass Spectrometry ,Rhodotorula ,Electrophoresis, Gel, Two-Dimensional ,Databases, Protein ,Carotenoids ,Biotechnology - Abstract
Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John WileySons, Ltd.
- Published
- 2015
41. Comparison of blood serum peptide enrichment methods by Tricine SDS-PAGE and mass spectrometry
- Author
-
Maria Filippa Addis, Tonina Roggio, Sergio Uzzau, Grazia Biosa, Daniela Pagnozzi, Salvatore Pisanu, and Alessandro Tanca
- Subjects
Serum ,chemistry.chemical_classification ,Tricine ,Chromatography ,Glycine ,Biophysics ,Ultrafiltration ,Peptide ,Fractionation ,Chemical Fractionation ,Mass spectrometry ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Ultrafiltration (renal) ,Blood serum ,chemistry ,Electrophoresis, Polyacrylamide Gel ,Biomarker discovery ,Peptides ,Polyacrylamide gel electrophoresis ,Biomarkers - Abstract
Characterisation of blood serum peptides can provide valuable information on physiological and pathological processes. However, the analysis of raw serum samples by MS results in the identification of a limited number of peptides. In order to improve sensitivity, many peptide enrichment methods have been proposed during the last ten years. Here, we present a comparison of fractionation methods aimed to simplify analysis of small proteins and peptides in blood serum, one of the most promising sources of putative biomarkers. Specifically, three methods based on ultrafiltration, differential precipitation, and peptide ligand libraries (ProteoMiner) were evaluated for the enrichment of peptides and low molecular weight proteins, as demonstrated by Tricine SDS-PAGE and subsequent LC-MS/MS (GeLC-MS/MS). As a result, differential solubilisation (DS) allowed the identification of the highest number of peptides. Moreover, the DS method enabled also the quantitative comparison of samples, producing fundamental information in biomarker discovery approaches.
- Published
- 2011
42. Impact of fixation time on GeLC–MS/MS proteomic profiling of formalin-fixed, paraffin-embedded tissues
- Author
-
Grazia Biosa, Giovanni Falchi, Maria Filippa Addis, Alessandro Tanca, Stefano Rocca, Sergio Uzzau, Daniela Pagnozzi, and Gisella Foddai
- Subjects
Proteomics ,Paraffin Embedding ,Time Factors ,Chromatography ,Spectral counting ,Formalin fixed paraffin embedded ,Proteomic Profiling ,Biophysics ,Ms analysis ,Proteins ,Fixation time ,Biology ,Bioinformatics ,Biochemistry ,Fixatives ,Dogs ,Tandem Mass Spectrometry ,Formaldehyde ,Protein purification ,Animals ,Biomarker discovery ,Fixation (histology) - Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissue banks represent an invaluable resource for biomarker discovery. Recently, the combination of full-length protein extraction, GeLC-MS/MS analysis, and spectral counting quantification has been successfully applied to mine proteomic information from these tissues. However, several sources of variability affect these samples; among these, the duration of the fixation process is one of the most important and most easily controllable ones. To assess its influence on quality of GeLC-MS/MS data, the impact of fixation time on efficiency of full-length protein extraction efficiency and on quality of label-free quantitative data was evaluated. As a result, although proteins were successfully extracted from FFPE liver samples fixed for up to eight days, fixation time appeared to negatively influence both protein extraction yield and GeLC-MS/MS quantitative proteomic data. Particularly, MS identification efficiency decreased with increasing fixation times. Moreover, amino acid modifications putatively induced by formaldehyde were detected and characterized. These results demonstrate that proteomic information can be achieved also from tissue samples fixed for relatively long times, but suggest that variations in fixation time need to be carefully taken into account when performing proteomic biomarker discovery studies on fixed tissue archives.
- Published
- 2011
43. Proteomic analysis of muscle tissue from gilthead sea bream (Sparus aurata, L.) farmed in offshore floating cages
- Author
-
Vittorio Tedde, Maria Filippa Addis, Maria Cristina Porcu, Roberto Cappuccinelli, Tonina Roggio, Elia Bonaglini, Sergio Uzzau, and Daniela Pagnozzi
- Subjects
Muscle tissue ,Sparidae ,biology ,business.industry ,Parvalbumins ,Fish farming ,Zoology ,Aquatic animal ,Aquatic Science ,biology.organism_classification ,Fishery ,medicine.anatomical_structure ,Aquaculture ,Proteome ,medicine ,Mariculture ,business - Abstract
Characterization of the muscle tissue proteome is key to many aspects of fish aquaculture, encompassing physiology, growth, food safety, seafood authentication and quality, traceability and shelf-life. In this study, a 2D-PAGE-MS study was performed on gilthead sea bream (Sparus aurata, L.) muscle tissue along the production cycle in four offshore floating cage plants and two repopulation lagoons located in different areas of Sardinia, Italy. The aim of this study was to accomplish systematic characterization of the gilthead sea bream muscle proteome, and to gather data about its variability in physiological conditions occurring in both farmed and wild fish. In general, a relatively stable protein expression pattern was observed in farmed sea bream muscle compared to other more dynamic tissue proteomes, such as liver. However, several statistically significant variations in abundance of some proteins and their isoforms were detected, related to growth and environmental factors. Among these, parvalbumins, troponins, and Wap65 showed variations according to fish length and water temperature. Interestingly, the ratio of structural proteins versus glycolytic enzymes was also observed to change during the production cycle, showing an increase with fish length. In order to assess whether the farming conditions were able to induce alterations in the muscle proteome, farmed and wild fish were subjected to a differential proteomics analysis. The data gathered in this study indicate that the protein expression profile of muscle tissue is comparable in wild and maricultured gilthead sea breams of commercial size, supporting the view that farming in offshore floating cages might favor proper muscle tissue development, and therefore enable the production of higher quality fish. In conclusion, this work describes the detailed characterization of the sea bream muscle proteome, and provides a number of insights on its size and environment-related variability.
- Published
- 2010
44. Identification and characterization of novel Mycoplasma spp. belonging to the hominis group from griffon vultures
- Author
-
Alberto Alberti, Michael Lierz, Bernardo Chessa, Carla Cacciotto, Laura Carcangiu, Fiammetta Berlinguer, Marco Pittau, Maria Filippa Addis, Marco Muzzeddu, Elisabetta Coradduzza, Roberta Lecis, Lecis R., Chessa B., Cacciotto C., Addis M.F., Coradduzza E., Berlinguer F., Muzzeddu M., Lierz M., Carcangiu L., Pittau M., and Alberti A.
- Subjects
Mycoplasma hominis ,Mycoplasma synoviae ,medicine.disease_cause ,Microbiology ,Mycoplasma ,Phylogenetics ,RNA, Ribosomal, 16S ,medicine ,Animals ,Mycoplasma Infections ,Respiratory Tract Infections ,Falconiformes ,Phylogeny ,Hominis group ,IGS 16S/23S ,General Veterinary ,biology ,Bird Diseases ,16SrDNA ,Ribosomal RNA ,biology.organism_classification ,16S ribosomal RNA ,Commensalism ,Birds of prey ,Gyps fulvus - Abstract
Mycoplasmas are commensals and pathogens of various avian species, and are also regularly found in birds of prey, although their significance to birds' health remains unclear. Here we describe two novel Mycoplasma isolated from the upper respiratory tract of four Eurasian griffon vultures (Gyps fulvus) housed in a wildlife recovery centre in Sardinia (Italy). By sequencing the 16S rRNA gene and the entire 16S/23S intergenic spacer region, the new strains were classified within the Mycoplasma taxonomy at the group and cluster levels, showing that the two isolates fall into the Mycoplasma synoviae and Mycoplasma hominis clusters of the hominis group, respectively. We combined molecular tools and immunoblotting methods in order to further characterize these isolates, and antigenic analyses overall confirmed the molecular findings. Different levels of pathogenicity and prevalence of these strains might have different implications for the conservation and reintroduction of vultures. © 2010 Elsevier Ltd.
- Published
- 2010
45. The Sarda Sheep Host Fecal Proteome
- Author
-
Maria Filippa Addis, Alessandro Tanca, Antonio Palomba, Sergio Uzzau, and Daniela Pagnozzi
- Subjects
0301 basic medicine ,Proteome ,Proteolysis ,Shotgun ,Context (language use) ,Computational biology ,Gut flora ,Biochemistry ,Feces ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Bacterial Proteins ,Sequence Analysis, Protein ,medicine ,Animals ,Intestinal Mucosa ,Shotgun proteomics ,Molecular Biology ,Sheep ,biology ,medicine.diagnostic_test ,biology.organism_classification ,030104 developmental biology ,030220 oncology & carcinogenesis - Abstract
The first characterization of the sheep fecal microbiota was recently reported, as obtained by using a multi meta-omic approach. Here, the mass spectra generated by single-run LC/high-resolution MS in the context of that study were reanalyzed using a host-specific database, in order to gain insights for the first time into the host fecal proteome of healthy Sarda sheep. On the whole, 5349 non-redundant tryptic peptide sequences were identified, belonging to 1046 different proteins. The "core" fecal proteome (common to all animals) comprised 431 proteins, mainly related to biological processes as immune response and proteolysis. Proteins involved in the immune/inflammatory response and peptidases were specifically investigated. This dataset provides novel insights into the repertoire of proteins secreted in the sheep intestinal lumen, and constitutes the basis for future shotgun and targeted proteomics studies aimed at monitoring changes in the sheep fecal proteome in response to production variables, infectious/inflammatory states, and variations in the gut microbiota. Data are available via ProteomeXchange with identifier PXD006145.
- Published
- 2018
46. Stress relaxation behaviour and structural changes of muscle tissues from Gilthead Sea Bream (Sparus aurata L.) following high pressure treatment
- Author
-
Nicola Secchi, Vittorio Tedde, Roberto Cappuccinelli, Luca Pretti, Tonina Roggio, Maria Cristina Porcu, Maria Filippa Addis, Giuseppe Stara, and Marco Campus
- Subjects
Muscle tissue ,Food storage ,Anatomy ,Biology ,medicine.anatomical_structure ,High pressure ,medicine ,Stress relaxation ,Water holding capacity ,Desmin ,Food science ,Elasticity (economics) ,Stress relaxation test ,Food Science - Abstract
Sea Bream muscle tissue was subjected to different high pressure treatments, and rheological changes were monitored during storage by means of the stress relaxation test. The best fit was obtained by application of the three term Maxwell exponential model, followed by the Nussinovich model. The application of 300 and 400 MPa pressures appeared to enable preservation of elasticity and stiffness of fish muscle during storage, compared to untreated samples. On the contrary, samples treated at 200 MPa underwent a decrease in elasticity during storage. The water holding capacity of dorsal muscle was also assessed, and it was found to decrease with increasing pressures. Immunoblot studies performed on the main structural proteins revealed that a pronounced time-dependent degradation of desmin, observed in untreated samples, could be prevented by treatment at 400 MPa. Taken together, our results strongly suggest that high pressure treatments inactivate degrading enzymes acting on proteins that are related to tissue integrity preservation, texture quality, and water holding capacity.
- Published
- 2010
47. 2-D PAGE and MS analysis of proteins from formalin-fixed, paraffin-embedded tissues
- Author
-
Stefano Rocca, Sergio Uzzau, Daniela Pagnozzi, Alessandro Tanca, and Maria Filippa Addis
- Subjects
Silver Staining ,Formalin fixed paraffin embedded ,Immunoblotting ,Muscle Proteins ,Biology ,Proteomics ,Biochemistry ,Mass Spectrometry ,Specimen Handling ,Silver stain ,Formaldehyde ,Skeletal Muscle Tissue ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Isoelectric Point ,Frozen tissue ,Muscle, Skeletal ,Molecular Biology ,Western immunoblotting ,Paraffin Embedding ,Sheep ,2 d page ,Hydrolysis ,Ms analysis ,Proteins ,Molecular biology - Abstract
In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin-fixed, paraffin-embedded (FFPE) tissues. However, 2-D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2-D PAGE separation and MS identification of full-length proteins extracted from FFPE skeletal muscle tissue. The 2-D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2-D maps of proteins from FFPE tissue following standard mass-compatible silver staining. Protein spots from both FFPE and frozen tissue 2-D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2-D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh-frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full-length proteins from FFPE tissues might be suitable to 2-D PAGE-MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological-fixed tissues.
- Published
- 2009
48. Characterisation of Mycoplasma capricolum P60 surface lipoprotein and its evaluation in a recombinant ELISA
- Author
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Ennio Bandino, Tiziana Cubeddu, Richard Thiéry, Pascale Mercier, Sergio Rosati, Alberto Alberti, Marco Pittau, Margherita Profiti, Maria Filippa Addis, Patrizia Robino, Bernardo Chessa, and Alessandro Mannelli
- Subjects
Basic membrane proteins ,P60 ,Lipoproteins ,Recombinant Fusion Proteins ,Mycoplasma agalactiae ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Enzyme-Linked Immunosorbent Assay ,Mycoplasmataceae ,Sensitivity and Specificity ,Microbiology ,Mycoplasma capricolum ,law.invention ,Bacterial Proteins ,Antigen ,law ,Animals ,Point Mutation ,Mycoplasma capricolum subspecies capricolum ,Cloning, Molecular ,Pleuropneumonia, Contagious ,Contagious agalactia ,Goat Diseases ,Base Sequence ,General Veterinary ,biology ,ved/biology ,Goats ,food and beverages ,General Medicine ,ELISA ,biology.organism_classification ,Antibodies, Bacterial ,Membrane protein ,Recombinant DNA ,Mollicutes ,Rabbits ,Bacteria - Abstract
This paper reports the identification and characterisation of a 60 kDa surface protein antigen (P60) of Mycoplasma capricolum subspecies capricolum (Mcc), and describes its diagnostic application. Genomic localization and presence in P60 of conserved functional domains suggested a structural and functional relationship with the immunodominant antigen P48 of Mycoplasma agalactiae, a basic membrane protein. A rP60-ELISA was developed, and it resulted in a high specificity for Mcc infections after evaluation with 125 goat sera. The comparison with an existent ELISA based on whole Mcc cell lysates showed that the two assays have comparable sensitivities, but the rP60-ELISA has the significant advantage of a greater specificity. Results indicate that P60 is a potential marker of Mcc infection, especially useful in areas where the presence of M. capricolum subspecies capripenumoniae is also reported.
- Published
- 2008
49. Draft Genome Sequence of Rhodotorula mucilaginosa, an Emergent Opportunistic Pathogen
- Author
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Cristina Fraumene, Alessandro Tanca, Marcello Abbondio, Ilaria Mannazzu, Massimo Deligios, Sergio Uzzau, and Maria Filippa Addis
- Subjects
Whole genome sequencing ,Opportunistic pathogen ,Eukaryotes ,parasitic diseases ,Genetics ,Biology ,Molecular Biology ,Genome ,Yeast ,Rhodotorula mucilaginosa ,Microbiology - Abstract
Rhodotorula mucilaginosa , a yeast with valuable biotechnological features, has also been recorded as an emergent opportunistic pathogen that might cause disease in both immunocompetent and immunocompromised individuals. Here, we report the draft genome sequence of R. mucilaginosa strain C2.5t1, which was isolated from cacao seeds in Cameroon.
- Published
- 2015
50. Full-length protein extraction protocols and gel-based downstream applications in formalin-fixed tissue proteomics
- Author
-
Alessandro, Tanca, Sergio, Uzzau, and Maria Filippa, Addis
- Subjects
Proteomics ,Two-Dimensional Difference Gel Electrophoresis ,Tandem Mass Spectrometry ,Blotting, Western ,Proteins ,Electrophoresis, Gel, Two-Dimensional ,Chromatography, Liquid - Abstract
Archival formalin-fixed, paraffin-embedded (FFPE) tissue repositories and their associated clinical information can represent a valuable resource for tissue proteomics. In order to make these tissues available for protein biomarker discovery and validation studies, dedicated sample preparation procedures overcoming the intermolecular cross-links introduced by formalin need to be implemented. This chapter describes a full-length protein extraction protocol optimized for downstream gel-based proteomics applications. Using the procedures detailed here, SDS-PAGE, western immunoblotting, GeLC-MS/MS, 2D-PAGE, and 2D-DIGE can be carried out on FFPE tissues. Technical tips, critical aspects, and drawbacks of the method are presented and discussed.
- Published
- 2015
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