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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.

Radiospongilla cfr. philippinensis (Annandale, 1909) Figs 1 a, 2, 3, 4, 5; Tables 1, 2, 3 Material examined. Four specimens NTM ZOO 5052, NTM ZOO 5056, NTM ZOO 5063, NTM ZOO 5069. Dry shore of a billabong, Mamukala Wetlands, 12��38���S 132��35���E, 7 km East of the South Alligator River, South off the Arnhem Highway, Kakadu National Park (Fig. 1, site a), 10.vii.1998, R. Manconi leg. Some slides and stubs (DTRG-FW571a, b, c, d) are deposited in the authors��� collection. Additional material. NTM Z001431, Tom Lagoon, NT, 16��22���S 134��50���E, Latz leg., 3.i.1971, large clusters on roots (DTRG-FW706); NTM Z001435, unknown location, Stn n. FW-0016 (DTRG-FW707) Comparative material. Radiospongilla sceptroides, AM Z2837, neotype, Merrika River, Nadgee Faunal Reserve near Womboyne (37��15���S, 149��55���E), New South Wales, Australia, on large rock, 0.5 m depth, 6.iii.1958, leg. F. Hersey, det. Penney & Racek (DTRG-FW542). Spongilla philippinensis, BMNH 34.4.28.1 box 13.III. C, alcohol, leg. L.E. Cheesman, Lake Sentani, Dutch New Guinea (DTRG-FW382). Spongilla sceptroides Haswell, BMNH 86.8. 27.665, box 13.III. C, alcohol, Brisbane, Australia (DTRG-FW387), BMNH 86.8.27.658, box 5, dry, leg. von Lendenfeld, Kakalum River, Australia (DTRG-FW409). Spongilla cerebellata Bowerbank, 1863, schizotype, Bowerbank collection, leg. Brudley, Dominion of Nizam (DTRG- FW412). Description. Growth form encrusting (5���6 mm in diameter, 2���5 mm thick) to cushion-shaped (3.5 cm in diameter, 1 cm thick). Consistency extremely fragile (dry specimens are, at present, completely fragmented due to their extremely fragile consistency). Colour whitish in dry condition. Oscules scattered. Ectosomal skeleton with no special architecture. Choanosomal skeleton irregularly reticulate network, with ascending paucispicular primary fibres and vague secondary tracts. Spongin scanty. Megascleres acanthoxeas of two size classes straight to slightly bent, from stout [232���302 (266��17) x 6���13 (9.28��0.96) ��m] to slender [188���226 (208��13) x 2.9���5.8 (5.24��1.06) ��m] ornamented by large, straight to curved variably dense small spines except at the tips. Microscleres absent. Gemmules subspherical (600���750 ��m in diameter) single to grouped (2���3) scattered in the skeletal network at the sponge basal portion. Foramen in a more or less conical depression, simple, without collar, with brown, slender porus tube. Gemmular theca trilayered with gemmuloscleres in a single layer more or less radially embedded (NTM ZOO 5052, 5056, 5063, 5069) to in double layer both tangentially arranged at theca surface and radially embedded in it (NTM Z001431, NTM Z001435). Outer gemmular surface hispid due to the emerging distal apices of gemmuloscleres. Pneumatic layer with vague chambers of spongin to with welldeveloped polygonal chambers. Inner layer sublayered of compact spongin. Gemmuloscleres [122���174 (148��12) x 2.3���8.7 (5.25��2.26) ��m] straight to slightly curved of two types from slender acanthoxeas abruptly pointed with few small scattered spines except at the tips, to stout acanthostrongyles with large straight spines scattered along the axis, and curved spines densely clustered at the tips. Habitat. Several dry sponges were discovered along the shore of an almost dry billabong in shaded areas on the painted metallic piers of a bird-watching platform and on surrounding dry timbers and bushes between the platform and the path (Fig. 2). Data confirm the typical growth mode of this species on natural and artificial substrata (Racek 1969). Geographic distribution. The geographic range of R. philippinensis is from the Philippines to northern Australia (Tables 1���2). The present record in Kakadu National Park confirms the presence of this species in coastal tropical-subtropical areas of the north, western and southern Australia (Racek 1969). On the other hand R. streptasteriformis Stanisic, 1979 is apparently endemic to the Northern Territory (Tables 1���2) (Stanisic 1979) whereas R. pedderensis Osborne, Forteath & Stanisic, 2008 is endemic to Tasmania (Osborne et al. 2008). The doubtful presence in Australia of R. crateriformis (Potts, 1882) is discussed by Racek (1969). The biogeographic pattern of the speciose Radiospongilla (18 species) is in the Australasian, Afrotropical, Neotropical, Nearctic, Oriental, and Palaearctic regions (Fig. 5) (Penney & Racek 1968; Manconi & Pronzato 2002, 2007). Remarks. In his synopsis on Australian freshwater sponges Racek (1969) clarified the taxonomic status of Radiospongilla philippinensis (previously a junior synonym of R. sceptroides) as a valid species (Penney & Racek 1968). On the basis of comparative analysis of specimens from Kakadu National Park bearing gemmules with a single layer of radial gemmuloscleres are here ascribed to R. cfr. philippinensis (Table 3) because they diverge from descriptions of Philippine and other Australian material (Annandale 1909ab). Also additional material from the NTM bearing gemmules with two layers of gemmuloscleres i.e. tangentially and radially arranged in the gemmular theca is here ascribed to R. cfr. philippinensis (Table 3). Radiospongilla Radiospongilla philippinensis cfr. philippinensis Gemmuloscleres acanthostrongyles acanthostrongyles acanthostrongyles acanthostrongyles ��m 80���122 x 31 105���180 x 4���5 acanthoxeas acanthoxeas 102���149 x 4.5���7 122���174 x 2.3���8.7 Gemmular tangential spicules in irregularly radial irregularly radial theca single layer spicules in single tangential and radial spicules in spicules in single layer double layer layer References *Annandale 1909ab Racek 1969 Present paper Present paper # Gee, 1931b GENUS Pectispongilla ANNANDALE , 1909, Published as part of Manconi, R., Cubeddu, T. & Pronzato, R., 2016, Australian freshwater sponges with a new species of Pectispongilla (Porifera: Demospongiae: Spongillida), pp. 61-76 in Zootaxa 4196 (1) on pages 65-69, DOI: 10.11646/zootaxa.4196.1.3, http://zenodo.org/record/167679, {"references":["Racek, A. A. (1969) The freshwater sponges of Australia (Porifera: Spongillidae). Australian Journal of Marine and Freshwater Research, 20, 267 - 310.","Stanisic, J. (1979) Freshwater sponges from the Northern Territory (Porifera: Spongillidae). Proceedings of the Linnean Society of North South Wales, 103 (2), 123 - 130.","Osborn, A. W., Forteath, G. N. R. & Stanisic, J. (2008) A new species of freshwater sponge (Porifera: Spongillidae) of the genus Radiospongilla from Lake Pedder in Tasmania. Papers and Proceedings of the Royal Society of Tasmania, 142 (2), 39 - 44.","Penney, J. T. & Racek, A. A. (1968) Comprehensive revision of a world-wide collection of freshwater sponges (Porifera: Spongillidae). United States National Museum Bulletin, 272, 1 - 184.","Manconi, R. & Pronzato, R. (2002) Spongillina n. subord. Lubomirskiidae, Malawispongiidae n. fam., Metaniidae, Metschnikowiidae, Palaeospongillidae, Potamolepidae, Spongillidae. In: J. Hooper & R. W. M. van Soest (Eds.), Vol. 1. Systema Porifera. A guide to the classification of sponges. Kluwer Academic / Plenum Publisher: New York, pp. 921 - 1019. http: // dx. doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 97","Manconi, R. & Pronzato, R. (2007) Gemmules as a key structure for the adaptive radiation of freshwater sponges: a morphofunctional and biogeographical study. In: Custodio M. R., Lobo-Hajdu G., Hajdu, E. & Muricy, G. (Eds.), Porifera research: biodiversity, innovation and sustainability. Serie Livros. Museu Nacional: Rio de Janeiro, pp. 61 - 77.","Gee, N. G. (1931 b) Freshwater sponges of the Philippine Islands. The Philippine Journal of Science, 46 (1), 61 - 74."]}

Pectispongilla gagudjuensis n. sp. Manconi & Pronzato Figs 1 b, 6, 7, 8, 9; Tables 1, 2, 4 Material. Type material NTM ZOO 2024, NTM ZOO 2680, NTM ZOO 4338, NTM ZOO 4339, NTM ZOO 4340 from a small unnamed dry creek, 12��43���S 132��46���E (Fig. 1, site b) along the Kakadu Highway, North of Malabanjbanjdju, Kakadu National Park, Alligator River Region, Northern Territory, 11.vii.1998, leg. Luca Pronzato. Some slides and stubs (DTRG-FW568 a, b, c, d, e) are deposited in the authors��� collection. Comparative material. Pectispongilla botryoides NTM Z001405 (DTRG-FW646) and part of the latter as AM Z3504 (DTRG-FW701), Tanami Gorge, 19��58���S 129��39���E, Camel Waterhole, Northern Territory, 25.v.1970, leg. P. Latz, S. Parker & D. Howe, det. A.A. Racek; AM Z2905, Manchester Lake, Queensland, 6.ix.61, det. A.A. Racek (DTRG-FW700). P. aurea var. subspinosa , syntype, BMNH 14.11.24.34 (ex ZEV 3790/7), Kochi (ex- Cochin), Ernakulam, Kerala, SW India (DTRG-FW401); USNM 34578, Penney collection 90124, schizosyntype, dry, Kochi (ex-Cochin), Ernakulam, Kerala, SW India (DTRG-FW553); ZMB 7981 from BMNH 14.11.24.34, alcohol, Kochi (ex-Cochin), Ernakulam, Kerala, SW India (DTRG-FW515). Etymology. The specific epithet refers to the native word Gagudju (from which Kakadu) derived from the name of one of the ca. thirty old native languages of the flood plain area in the Arnhem Land. Diagnosis. Pectispongilla gagudjuensis n. sp. is characterised by two types of short skeletal megascleres (acanthostrongyles and acanthoxeas), absence of microscleres, free gemmules, mature botryoidal gemmuloscleres with disto-lateral apices as irregular concavities grouped to form a botryum, and growth form as a small hollow cup in dry condition. Description. Growth form encrusting as minute almost flat cushions (max. 1 cm in diameter) scattered and strictly adhering to substrata by basal spongin plate. Surface smooth. Consistency hard and fragile. Colour white. Oscul e apical, single, central, large (300���350 ��m in diameter). Ectosomal skeleton compact and dense arrangement of spicules more or less tangential. Choanosomal skeleton vaguely reticulate network of spicules joined by scanty spongin. Spongin scanty except for basal spongin plate and gemmular theca. Megascleres of two types. Dominant acanthostrongyles [90���168 (135��19) x 8���13 (10��4) ��m] almost straight to notably bent (boomerang-like shaped), spiny by few scattered short spines sometimes more dense at the tips. Acanthoxeas (mucronate acanthostrongyles?) [130���205 (163��19) x 5���12 (8.5��1.9) ��m] less frequent, gradually to abruptly pointed, with short spines from scattered to more dense at the tips. Microscleres absent. Gemmules free (up to 8 for each specimen), small (200���350 ��m in diameter), subspherical after rehydration with gemmuloscleres more or less radially embedded. Foramen single with a short tube and a well developed collar. Gemmular theca thick, trilayered. Outer layer with a variable amount of compact spongin, as a honeycomb-like surface due to the partial emergence of distal botryoidal apices of gemmuloscleres. Pneumatic layer as an irregular network of anastomosing thin spongin fibres. Inner layer of sublayered compact spongin. Gemmuloscleres botryoidal (mature) to pseudo-botryoidal (immature) [26���46 (36��4) x 2���3 (2.9��0.29) ��m] with smooth, straight to slightly bent shafts and a range of variable shape at the convex side of each tip according with the age of the spicules. Immature gemmuloscleres with disto-lateral arrangement of simple small spines in rows (pseudo-botryum); botryum-like tips in growing spicules with a progressive increase of siliceous webs joining spines one to each other; true botryoidal tips with a cluster of well developed rounded concavities in more aged gemmuloscleres. Remarks. A comparative analysis of Pectispongilla gagudjuensis n. sp. showed that it matches only in part diagnostic traits of the other species of the genus (Table 4). The skeletal megascleres are shorter than the other species of the genus and the gemmules are the largest of the genus. Microscleres are absent in P. gagudjuensis n. sp., whereas microscleres of P. aurea Annandale, 1909 and P. subspinosa Annandale, 1912 are smooth to microspined oxeas (Penney & Racek 1968, p. 78���79) and those of P. stellifera Annandale, 1915 range from microspined oxeas to subspherical tubercled spherasters (Penney & Racek 1968, p. 79). Compared with the Australian P. botryoides Haswell, (1882) and the other species of the genus the gemmuloscleres of the new species have the shorter shafts. Moreover the gemmuloscleres of P. gagudjuensis are characterized by displaying the entire range of the various tip morphs (Fig. 8) hitherto recorded in the other four species of Pectispongilla i.e. from small spines in rows (immature gemmuloscleres) up to well developed botryoidal-like apices (mature spicules). The peculiar body architecture deeply diverges from Pectispongilla species and all other Spongillida i.e. reduced in dry condition to an almost hollow cup with megascleres to form the body wall and containing only free gemmules and a few spicular tracts. This morpho-functional trait has never been hitherto described for the family Spongillidae and is comparable only to the Baikalian Swartschewskia papyracea (Dybowsky, 1880) in dry condition. This body architecture, closely adhering to the substratum by a thin spongin basal plate, indicates its functional role as a protective device for gemmules during the long, harsh dry season. Its morpho-functional role is comparable to that performed by the gemmular cages of megascleres enveloping the gemmular theca found in other genera of Spongillida. P. gagudjuensis however diverges in the depth of the gemmular cage architecture described for other genera (e.g. Corvospongilla Annandale, 1911; Heterorotula Penney & Racek, 1968; Uruguayella Bonetto & Ezcurra De Drago, 1969; Pachyrotula Volkmer-Ribeiro & R��tzler, 1997). In synthesis, exclusive traits of P. gagudjuensis n. sp. in comparison with those of the other Pectispongilla species are as follows: i) single apical oscular aperture, ii) presence of free gemmules in the hollow internal space of the dry body, iii) megascleres of two types i.e. acanthoxeas and dominant acanthostrongyles, iv) short length of megascleres (ca. half of those of other species), v) largest gemmules of the genus, vi) shorter shafts of gemmuloscleres in the genus (see Annandale 1915 and Penney & Racek 1968). The diagnosis of the genus is here emended adding the presence of spiny strongyles among megascleres. Genus Pectispongilla Habitat. Lotic, temporary creek. Several dry, scattered small specimens under pebbles, cobbles, and boulders, from 1 to 6 per substratum. Sponges were absent from the surveyed horizontal rocky bed and timbers. Encrusting bryozoans, with statoblasts, on the same substrata but not strictly associated to the sponges, were also collected and preserved together with the sponges. Geographic distribution. P. gagudjuensis n. sp. is known only from the type locality. The disjunct Oriental, Australasian, and far east Palaearctic biogeographic pattern of Pectispongilla (5 species) seems to indicate Gondwanan origins and is restricted to the south-western Indian sub-region and Australia with an enclave in Korea and Japan (Annandale 1911, 1915; Penney & Racek 1968; Racek 1969; Manconi & Pronzato 2002, 2007, 2015) (Fig. 9). P. botryoides Haswell, 1882 is reported exclusively from Australia (Tables 1���2). Two species P. aurea Annandale, 1909 and P. stellifera Annandale, 1915 are endemic to restricted areas in the SW-Indian subregion, while P. subspinosa Annandale, 1911 is known from SW-India, Japan, and Korea (Tables 1���2)., Published as part of Manconi, R., Cubeddu, T. & Pronzato, R., 2016, Australian freshwater sponges with a new species of Pectispongilla (Porifera: Demospongiae: Spongillida), pp. 61-76 in Zootaxa 4196 (1) on pages 69-74, DOI: 10.11646/zootaxa.4196.1.3, http://zenodo.org/record/167679, {"references":["Penney, J. T. & Racek, A. A. (1968) Comprehensive revision of a world-wide collection of freshwater sponges (Porifera: Spongillidae). United States National Museum Bulletin, 272, 1 - 184.","Annandale, N. (1915) Notes on freshwater sponges of the genus Pectispongilla and its allies. Records of the Indian Museum, 11, 171 - 178.","Haswell, W. A. (1882) On Australian freshwater sponges. Proceedings of the Linnean Society of North South Wales, 7, 208 - 210.","Annandale, N. (1911) Freshwater sponges, hydroids and polyzoa. Porifera. In: A. E. Shipley (Ed.), Fauna of British India, Including Ceylon and Burma. Taylor & Francis: London, pp. 27 - 126, 241 - 245","Racek, A. A. (1969) The freshwater sponges of Australia (Porifera: Spongillidae). Australian Journal of Marine and Freshwater Research, 20, 267 - 310.","Manconi, R. & Pronzato, R. (2002) Spongillina n. subord. Lubomirskiidae, Malawispongiidae n. fam., Metaniidae, Metschnikowiidae, Palaeospongillidae, Potamolepidae, Spongillidae. In: J. Hooper & R. W. M. van Soest (Eds.), Vol. 1. Systema Porifera. A guide to the classification of sponges. Kluwer Academic / Plenum Publisher: New York, pp. 921 - 1019. http: // dx. doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 97","Manconi, R. & Pronzato, R. (2007) Gemmules as a key structure for the adaptive radiation of freshwater sponges: a morphofunctional and biogeographical study. In: Custodio M. R., Lobo-Hajdu G., Hajdu, E. & Muricy, G. (Eds.), Porifera research: biodiversity, innovation and sustainability. Serie Livros. Museu Nacional: Rio de Janeiro, pp. 61 - 77."]}

In the rat brown fat peroxisomes - thermogenetic organules - an peroxisomal enzyme activities undergo remarkable changes during the adaptation to cold of the animals (see 3). In this paper was show that changes of peroxisomal enzyme activities occur also in liver and kidney during cold-adaptation. Catalase, L-hydroxyacid oxidase, uricase and D-aminoacid oxidase (DAO) were assayed as in (6). During cold-adaptation, the activity of the former three enzymes (Table 2) increases with the weight of the organs (Table 1) whereas that of DAO exhibits a much larger increase (Table 3). Results are discussed with regard to the contribution of the liver to non-shivering thermogenesis.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13567-018-0612-0) contains supplementary material, which is available to authorized users.

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.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0196-x) contains supplementary material, which is available to authorized users.

Objectives: To characterize clinically and genetically a family with autosomal dominant lateral temporal epilepsy (ADLTE) negative to LGI1 exon sequencing test. Methods: All participants were personally interviewed and underwent neurologic examination. Most affected subjects underwent EEG and neuroradiologic examinations (CT/MRI). Available family members were genotyped with the HumanOmni1-Quad v1.0 single nucleotide polymorphism (SNP) array beadchip and copy number variations (CNVs) were analyzed in each subject. LGI1 gene dosage was performed by real-time quantitative PCR (qPCR). Results: The family had 8 affected members (2 deceased) over 3 generations. All of them showed GTC seizures, with focal onset in 6 and unknown onset in 2. Four patients had focal seizures with auditory features. EEG showed only minor sharp abnormalities in 3 patients and MRI was unremarkable in all the patients examined. Three family members presented major depression and anxiety symptoms. Routine LGI1 exon sequencing revealed no point mutation. High-density SNP array CNV analysis identified a genomic microdeletion about 81 kb in size encompassing the first 4 exons of LGI1 in all available affected members and in 2 nonaffected carriers, which was confirmed by qPCR analysis. Conclusions: This is the first microdeletion affecting LGI1 identified in ADLTE. Families with ADLTE in which no point mutations are revealed by direct exon sequencing should be screened for possible genomic deletion mutations by CNV analysis or other appropriate methods. Overall, CNV analysis of multiplex families may be useful for identifying microdeletions in novel disease genes.