Ovine papillomavirus type 3 virus-like particle-based tools for diagnosis and detection of infection.

The design of prophylactic and diagnostic tools specific to animal papillomaviruses is hampered by the difficulties of viral in vitro manipulation and by the scarce availability of dedicated biotechnological tools. This paper reports the production of Ovine Papillomavirus 3 (OaPV3)-based virus-like particles (OaPV3-VLPs) in the baculovirus system and their use to investigate host humoral immune response through the establishment of an indirect ELISA test., Polyclonal sera and monoclonal antibodies were generated against OaPV3-VLPs, and their isotype and reactivity were determined. Additionally, antibodies allowed OaPV3 detection in ovine squamous cell carcinoma (SCC) samples by immunohistochemistry. Results encourage the standardization of OaPV3-specific prophylactic and serological diagnostic tools, and open new perspectives for the study of host-viral interaction and SCC development.

Purification of an Acidic Polysaccharide with Anticoagulant Activity from the Marine Sponge Sarcotragus spinosulus.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

Skeletal calcification patterns of batoid, teleost, and mammalian models: Calcified cartilage versus bone matrix.

This study compares the skeletal calcification pattern of batoid Raja asterias with the endochondral ossification model of mammalians Homo sapiens and teleost Xiphias gladius. Skeletal mineralization serves to stiffen the mobile elements for locomotion. Histology, histochemistry, heat deproteination, scanning electron microscopy (SEM)/EDAX analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and Fourier transform infrared spectrometry (FTIR) have been applied in the study. H. sapiens and X. gladius bone specimens showed similar profiles, R. asterias calcified cartilage diverges for higher water release and more amorphous bioapatite. In endochondral ossification, fetal calcified cartilage is progressively replaced by bone matrix, while R. asterias calcified cartilage remains un-remodeled throughout the life span. Ca2+ and PO4 3- concentration in extracellular matrix is suggested to reach the critical salts precipitation point through H2 O recall from extracellular matrix into both chondroblasts or osteoblasts. Cartilage organic phase layout and incomplete mineralization allow interstitial fluids diffusion, chondrocytes survival, and growth in a calcified tissue lacking of a vascular and canalicular system. HIGHLIGHTS: Comparative physico-chemical characterization (TGA, DTG and DSC) testifies the mass loss due to water release, collagen and carbonate decomposition of the three tested matrices. R. asterias calcified cartilage water content is higher than that of H. sapiens and X. gladius, as shown by the respectively highest dehydration enthalpy values. Lower crystallinity degree of R. asterias calcified cartilage can be related to the higher amount of collagen in amorphous form than in bone matrix. These data can be discussed in terms of the mechanostat theory (Frost, 1966) or by organic/inorganic phase transformation in the course evolution from fin to limbs. Mineral analysis documented different charactersof R. asterias vs H. sapiens and X. gladius calcified matrix.

Collagenic architecture and morphotraits in a marine basal metazoan as a model for bioinspired applied research.

In some Porifera (Demospongiae: Keratosa), prototypes of the connective system are almost exclusively based on collagenic networks. We studied the topographic distribution, spatial layout, microtraits, and/or morphogenesis of these collagenic structures in Ircinia retidermata (Dictyoceratida: Irciniidae). Analyses were carried out on a clonal strain from sustainable experimental mariculture by using light and scanning electron microscopy. Histology revealed new insights on the widely diversified and complex hierarchical assemblage of collagenic structures. Key evolutionary novelties in the organization of sponge connective system were found out. The aquiferous canals are shaped as corrugate-like pipelines conferring plasticity to the water circulation system. Compact clusters of elongated cells are putatively involved in a nutrient transferring system. Knob-ended filaments are characterized by a banding pattern and micro-components. Ectosome and outer endosome districts are the active fibrogenetic areas, where exogenous material constitutes an axial condensation nucleus for the ensuing morphogenesis. The new data can be useful to understand not only the evolutionary novelties occurring in the target taxon but also the morpho-functional significance of its adaptive collagenic anatomical traits. In addition, data may give insights on both marine collagen sustainable applied researches along with evolutionary and phylogenetic analyses, thus highlighting sponges as a key renewable source for inspired biomaterials. Therefore, we also promote bioresources sustainable exploitation with the aim to provide new donors of marine collagen, thereby supporting conservation of wild populations/species.

Proteomic profiles and cytokeratin 13 as a potential biomarker of Ovis aries papillomavirus 3-positive and negative cutaneous squamous cell carcinomas.

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.

Identification of Mycobacterium avium subsp. paratuberculosis (MAP) in Sheep Milk, a Zoonotic Problem.

Johne's disease (JD) is a life-threatening gastrointestinal disease affecting ruminants, which causes crucial economical losses globally. This ailment is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious intracellular pathogen that belongs to the Mycobacteriaceae family. This acid-fast, hard-to-detect bacterium can resist milk pasteurization and be conveyed to dairy product consumers. Many studies have emphasized the zoonotic nature of MAP, suggesting an association between MAP and some gastroenteric conditions such as Crohn's disease in humans. This underlines the importance of utilizing efficient pasteurization alongside a state-of-the-art diagnostic system in order to minimize the possible ways this pathogen can be conveyed to humans. Until now, no confirmatory MAP screening technique has been developed that can reveal the stages of JD in infected animals. This is partially due to the lack of an efficient gold-standard reference method that can properly evaluate the performance of diagnostic assays. Therefore, the following research aimed to compare the merits of qPCR and ELISA assessments of milk for the detection of MAP in a total of 201 Sardinian unpasteurized sheep milk samples including 73 bulk tank milk (BTM) and 128 individual samples from a MAP-infected flock (MIF) applying various reference models. Accordingly, milk qPCR and ELISA assessments, together and individually, were used as reference models in the herd-level study, while serum ELISA and fecal PCR were similarly (together and in isolation) considered as the gold standards in the individual-level diagnosis. This study showed that the type of gold-standard test affects the sensitivity and specificity of milk qPCR and ELISA significantly. At the individual level in the MAP-infected flock, serum ELISA in isolation and together with fecal PCR were recognized as the best references; however, the best correlation was seen between milk and serum ELISA (p < 0.0001). Regarding the detection of MAP in BTM, qPCR IS900 was recognized as the most sensitive and specific diagnostic test (p < 0.0001) for monitoring the MAP shedders and animals with clinically developed symptoms within herds, under the condition that both milk qPCR and milk ELISA tests formed a binary reference model. The BTM analyses (qPCR and ELISA) revealed that MAP positivity has a seasonal pattern. This hypothesis was proven through a longitudinal study on 14 sheep herds.

MHO_0730 as a Surface-Exposed Calcium-Dependent Nuclease of Mycoplasma hominis Promoting Neutrophil Extracellular Trap Formation and Escape.

Mycoplasma lipoproteins play a relevant role in pathogenicity and directly interact with the host immune system. Among human mycoplasmas, Mycoplasma hominis is described as a commensal bacterium that can be associated with a number of genital and extragenital conditions. Mechanisms of M. hominis pathogenicity are still largely obscure, and only a limited number of proteins have been associated with virulence. The current study focused on investigating the role of MHO_0730 as a virulence factor and demonstrated that MHO_0730 is a surface lipoprotein, potentially expressed in vivo during natural infection, acting both as a nuclease with its amino acidic portion and as a potent inducer of Neutrophil extracellular trapsosis with its N-terminal lipid moiety. Evidence for M. hominis neutrophil extracellular trap escape is also presented. Results highlight the relevance of MHO_0730 in promoting infection and modulation and evasion of innate immunity and provide additional knowledge on M. hominis virulence and survival in the host.

Chronic intramammary infection by Listeria monocytogenes in a clinically healthy goat - a case report.

BACKGROUND: Listeria monocytogenes is a ubiquitous Gram-positive bacterium responsible for a severe foodborne disease in humans, and contaminated dairy products can be an important source of infection. Typically, infected dairy ruminants show clinical manifestations including encephalitis, septicemia, abortion, and diarrhea, but may also become asymptomatic carriers and shed L. monocytogenes in the feces acting as an important source of viable bacteria. Isolation from individual goat milk has been documented very rarely, and chronic, asymptomatic intramammary infection by L. monocytogenes with continuous milk shedding of viable bacteria has never been described in this dairy species. CASE PRESENTATION: At the routine controls, cheese and bulk milk were positive for L. monocytogenes in a herd of 200 lactating Alpine goats, but none showed clinical signs of listeriosis. Individual milk was subjected to bacterial culture and a clinically healthy goat was identified as affected by a chronic intramammary infection (IMI) by L. monocytogenes. The goat had never shown clinical signs of mastitis or other diseases. Her right half-udder milk was positive to L. monocytogenes in two consecutive samples collected one week apart, as demonstrated by bacterial culture and molecular analysis. Mammary tissues collected after culling were also positive to L. monocytogenes by culture. Histological examination highlighted a chronic interstitial mastitis with leukocyte infiltration, atrophy of the alveoli and presence of corpora amylacea. Immunohistochemistry (IHC) and immunofluorescence (IF) confirmed the presence of high numbers of bacteria in the lumen of mammary alveoli, with intracellular bacteria mainly located in macrophages, but also present in neutrophils and epithelial cells. After culling of the positive goat, bulk tank milk tested negative to L. monocytogenes at the following controls. CONCLUSION: This study demonstrates that L. monocytogenes can establish a chronic, subclinical IMI in goats with high numbers of bacteria shed in milk, representing a source of contamination for the herd and its dairy products. This underscores the importance of frequently monitoring all dairy herds that sell directly milk and/or fresh cheese and indicates that a chronic L. monocytogenes IMI should also be considered as source of bacteria when bulk tank milk contamination is detected in a dairy goat farm.

Transforming properties of ovine papillomaviruses E6 and E7 oncogenes.

An increasing number of studies suggest that cutaneous papillomaviruses (PVs) might be involved in skin carcinogenesis. However, only a few animal PVs have been investigated regard to their transformation properties. Here, we investigate and compare the oncogenic potential of 2 ovine Delta and Dyokappa PVs, isolated from ovine skin lesions, in vitro and ex vivo. We demonstrate that both OaPV4 (Delta) and OaPV3 (Dyokappa) E6 and E7 immortalize primary sheep keratinocytes and efficiently deregulate pRb pathway, although they seem unable to alter p53 activity. Moreover, OaPV3 and OaPV4-E6E7 expressing cells show different shape, doubling time, and clonogenic activities, providing evidence for a stronger transforming potential of OaPV3 respect to OaPV4. Also, similarly to high-risk mucosal and cutaneous PVs, the OaPV3-E7 protein, constantly expressed in sheep squamous cell carcinomas, binds pRb with higher affinity compared to the E7 encoded by OaPV4, a virus associated to fibropapilloma. Finally, we found that OaPV3 and OaPV4-E6E7 determine upregulation of the pro-proliferative proteins cyclin A and cdk1 in both human and ovine primary keratinocytes. Collectively, results provide evidence for implication of ovine PVs in cutaneous proliferative lesions and skin cancer progression, and indicate sheep as a possible animal model for the study of cutaneous lesions and malignancies.

Characterization of paucibacillary ileal lesions in sheep with subclinical active infection by Mycobacterium avium subsp. paratuberculosis.

Paratuberculosis (PTB) or Johne's disease is a contagious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Ovine PTB is less understood than bovine PTB, especially concerning paucibacillary infection and its evolution into clinical disease. We combined shotgun proteomics, histopathology and immunohistochemistry for the characterization of ileal tissues collected from seven asymptomatic sheep negative to serum ELISA, positive to feces and tissue MAP IS900 and F57 PCR, histologically classified as paucibacillary, actively infected, together with 3 MAP-free controls (K). Following shotgun proteomics with label-free quantitation and differential analysis, 96 proteins were significantly changed in PTB vs K, and were mostly involved in immune defense processes and in the macrophage-MAP interaction. Principal component analysis (PCA) of protein abundances highlighted two PTB sample clusters, PTB1 and PTB2, indicating a dichotomy in their proteomic profiles. This was in line with the PCA of histopathology data and was related to features of type 2 (PTB1) and type 3a (PTB2) lesions, respectively. PTB2 proteomes differed more than PTB1 proteomes from K: 43 proteins changed significantly only in PTB2 and 11 only in PTB1. The differential proteins cathelicidin, haptoglobin, S100A8 and S100A9 were evaluated by immunohistochemistry. K tissues were negative to cathelicidin and haptoglobin and sparsely positive to S100A8 and S100A9. PTB tissues were positive to all four proteins, with significantly more cells in PTB2 than in PTB1. In conclusion, we described several pathways altered in paucibacillary PTB, highlighted some proteomic differences among paucibacillary PTB cases, and identified potential markers for disease understanding, staging, and detection.

Liver Injury, Endotoxemia, and Their Relationship to Intestinal Microbiota Composition in Alcohol-Preferring Rats.

BACKGROUND: There is strong evidence that alcoholism leads to dysbiosis in both humans and animals. However, it is unclear how changes in the intestinal microbiota (IM) relate to ethanol (EtOH)-induced disruption of gut-liver homeostasis. We investigated this issue using selectively bred Sardinian alcohol-preferring (sP) rats, a validated animal model of excessive EtOH consumption. METHODS: Independent groups of male adult sP rats were exposed to the standard, home-cage 2-bottle "EtOH (10% v/v) versus water" choice regimen with unlimited access for 24 h/d (Group Et) for 3 (T1), 6 (T2), and 12 (T3) consecutive months. Control groups (Group Ct) were composed of matched-age EtOH-naïve sP rats. We obtained samples from each rat at the end of each experimental time, and we used blood and colon tissues for intestinal barrier integrity and/or liver pathology assessments and used stool samples for IM analysis with 16S ribosomal RNA gene sequencing. RESULTS: Rats in Group Et developed hepatic steatosis and elevated serum transaminases and endotoxin/lipopolysaccharide (LPS) levels but no other liver pathological changes (i.e., necrosis/inflammation) or systemic inflammation. While we did not find any apparent alteration of the intestinal colonic mucosa, we found that rats in Group Et exhibited significant changes in IM composition compared to the rats in Group Ct. These changes were sustained throughout T1, T2, and T3. In particular, Ruminococcus, Coprococcus, and Streptococcus were the differentially abundant microbial genera at T3. The KEGG Ortholog profile revealed that IM functional modules, such as biosynthesis, transport, and export of LPS, were also enriched in Group Et rats at T3. CONCLUSIONS: We showed that chronic, voluntary EtOH consumption induced liver injury and endotoxemia together with dysbiotic changes in sP rats. This work sets the stage for improving our knowledge of the prevention and treatment of EtOH-related diseases.

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

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.

Ovis aries Papillomavirus 3 in Ovine Cutaneous Squamous Cell Carcinoma.

Squamous cell carcinoma (SCC) is a common malignancy affecting humans and other animals. Papillomaviruses (PVs) are frequently reported as causal agents of cutaneous benign and malignant epithelial lesions in different animal species, but only few studies have investigated their role in ovine SCC. In this study, we explore the possible involvement of the Ovine aries PVs (OaPV1, OaPV2, OaPV3) in cutaneous SCC using an integrated histological and molecular approach. Forty cutaneous SCCs from different anatomical locations of Sardinian sheep and 40 matched non-SCC samples were evaluated histologically and by polymerase chain reaction (PCR) to assess the presence of ovine PVs. In addition, DNA in situ hybridization (ISH) and reverse transcription-polymerase chain reaction (RT-PCR) were carried out to evaluate the cellular localization and viral transcriptional activity, respectively. OaPV3 DNA was detected in 26 of 40 (65%) SCCs and in 12 of 40 (30%) non-SCC samples using PCR. OaPV1 and OaPV2 were not detected. OaPV3 viral DNA was observed by ISH in malignant epithelial squamous cells of 18 of 40 (45%) SCCs. In addition, the viral transcriptional activity was identified in 24 of 40 (60%) SCCs by RT-PCR. Notably, a higher viral positivity was observed in SCCs compared with non-SCC samples. The considerable infection rate of OaPV3 in the most common skin tumor of the sheep suggests that PV could represent a key factor in the onset of ovine SCC.

Impact of pe_pgrs33 Gene Polymorphisms on Mycobacterium tuberculosis Infection and Pathogenesis.

PE_PGRS33 is a surface-exposed protein of Mycobacterium tuberculosis (Mtb) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the pe_pgrs33 gene of Mtb clinical isolates and evaluate their impact on protein functions. We sequenced pe_pgrs33 in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the Mtb complex (MTBC). Overall, an association between pe_pgrs33 alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on pe_pgrs33 against deleterious SNPs. Among a total of 19 pe_pgrs33 alleles identified in this study, 5 were cloned and used to complement the pe_pgrs33 knock-out mutant strain of Mtb H37Rv (MtbΔ33) to assess the functional impact of the respective polymorphisms in in vitro infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of Mtb H37Rv, impairing the cell entry capacity of Mtb, but neither its intracellular replication rate nor its immunomodulatory properties. In vivo studies performed in the murine model of tuberculosis (TB) demonstrated that the MtbΔ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly, MtbΔ33 showed an enhanced virulence during the chronic steps of infection compared to Mtb H37Rv. Similarly, the complementation of MtbΔ33 with a frameshift allele also resulted in a Mtb strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of Mtb.

A first immunohistochemistry study of transketolase and transketolase-like 1 expression in canine hyperplastic and neoplastic mammary lesions.

BACKGROUND: Canine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the 'Warburg effect', the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells. RESULTS: TKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05). CONCLUSIONS: This study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

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.

Paratuberculosis in sheep: Histochemical, immunohistochemical and in situ hybridization evidence of in utero and milk transmission.

To investigate in utero and milk transmission of Mycobacterium avium subsp. paratuberculosis (MAP), tissues from thirteen pregnant sheep, naturally infected and serologically positive to MAP, were examined by means of histochemistry, immunohistochemistry and in situ hybridization. Soon after parturition, ewes were euthanized and tissues samples were collected and prepared. The offspring (18 lambs) were divided into three groups to investigate different routes of MAP transmission. Lambs were sacrificed at three months old and the tissue samples collected, formalin-fixed and paraffin embedded. Hematoxylin and eosin and Ziehl-Neelsen staining methods were performed on fixed tissues for general examination and for detection of acid-fast bacteria. Additionally, immunohistochemical and in situ hybridization techniques were used to detect MAP antigen and MAP DNA respectively. This study of a flock of MAP-infected sheep indicates both in utero and milk transmission of MAP from dams to their offspring. Importantly, this study detected the presence of MAP in the mammary gland and mammary lymph nodes of adult ewes therefore indicating a significant route for the potential exposure to humans from this bacterial infection.

Neutrophil extracellular traps in sheep mastitis.

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.

Evaluating the impact of different sequence databases on metaproteome analysis: insights from a lab-assembled microbial mixture.

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge. This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.