Ct with a macrophage-stimulatory activity [38]. Also, the Vpma and Avg families of variable lipoproteins provide M. agalactiae a mechanism by which it potentially avoids opsonization, phagocytosis, macrophage killing, and antibodies neutralization [39]. Membrane nucleases play a key role for the survival of mycoplasmas into their hosts [40]. Since most mycoplasma species lost essential components of the biochemical machinery in which ribose phosphate, selected amino acids, CO2, and NH3 are combined in successive reactions to form nucleotides (de novo pathway), these microorganisms optimized the use of free bases and nucleosides released from the breakdown of the host nucleic acids by converting them back into nucleotides (salvage pathway, 3, 4]. Under this scenario, membrane nucleases and transport systems for the uptake of nucleotide precursors play not only a crucial functional role in promoting mycoplasmas survival but also contribute to pathogenicity as virulence factors [4,11,14,34,40,41]. Indeed, it has been demonstrated that severalM. agalactiae SNaseFigure 5. MAG_5040 antigenic properties. (A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10 polyacrylamide 15900046 gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes GSK -3203591 designated MW. doi:10.1371/journal.pone.0057775.gnucleases are implicated in mycoplasma-mediated host cell cytotoxicity. The M. pneumoniae nuclease Mpn133 induces apoptosis-like death of A549 mammalian cells, after binding and internalization [6,13]. Also, the Ca2+ and Mg2+ ion-dependent endonucleases produced by M. hyorhinis and M. penetrans induce apoptotic changes in epithelial cells, and trigger apoptosis in cultured lymphocytes, respectively [11,14].The surface expressed MAG_5040 protein of M. agalactiae was found to contain a TNASE_3 thermonuclease domain profile. Recombinant GST-MAG_5040 nuclease activity tested on different nucleic acid substrates showed that MAG_5040 is a sugarnonspecific exonuclease that preferentially cleaves nucleic acid residues from double and single strand DNA. Slightly smaller exonuclease activity was observed against RNA. RecombinantM. agalactiae SNaseFigure 6. MAG_5040 western blotting reactivity with sera collected from naturally infected hosts, and indirect order Pleuromutilin detection of homologs in selected mycoplasma species. 200 ng of cleaved rMAG_5040 were run in single well in 10 polyacrylamide gels and probed with sera collected alternatively from Piedmont goats 
naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) wit.Ct with a macrophage-stimulatory activity [38]. Also, the Vpma and Avg families of variable lipoproteins provide M. agalactiae a mechanism by which it potentially avoids opsonization, phagocytosis, macrophage killing, and antibodies neutralization [39]. Membrane nucleases play a key role for the survival of mycoplasmas into their hosts [40]. Since most mycoplasma species lost essential components of the biochemical machinery in which ribose phosphate, selected amino acids, CO2, and NH3 are combined in successive reactions to form nucleotides (de novo pathway), these microorganisms optimized the use of free bases and nucleosides released from the breakdown of the host nucleic acids by converting them back into nucleotides (salvage 
pathway, 3, 4]. Under this scenario, membrane nucleases and transport systems for the uptake of nucleotide precursors play not only a crucial functional role in promoting mycoplasmas survival but also contribute to pathogenicity as virulence factors [4,11,14,34,40,41]. Indeed, it has been demonstrated that severalM. agalactiae SNaseFigure 5. MAG_5040 antigenic properties. (A) Sensitivity of western blotting based on recombinant MAG_5040. Decreasing amounts of cleaved rMAG_5040 (250 to 6 ng) were run in a 10 polyacrylamide 15900046 gel and tested against a pool of high titre M. agalactiae naturally infected sheep sera. (B) Reactivity of rMAG_5040 (upper panel) and total M. agalactiae protein lysate (bottom panel) with sera collected from a sheep selected as representative of the longitudinal study. Sera were collected every two weeks for 9 months (numbers in lanes indicates selected sampling occasions, from T0 to T18). Neg indicates a pool of negative sera used as control in western blotting. Protein ladders (Magic Marker XP, Invitrogen) were loaded in lanes designated MW. doi:10.1371/journal.pone.0057775.gnucleases are implicated in mycoplasma-mediated host cell cytotoxicity. The M. pneumoniae nuclease Mpn133 induces apoptosis-like death of A549 mammalian cells, after binding and internalization [6,13]. Also, the Ca2+ and Mg2+ ion-dependent endonucleases produced by M. hyorhinis and M. penetrans induce apoptotic changes in epithelial cells, and trigger apoptosis in cultured lymphocytes, respectively [11,14].The surface expressed MAG_5040 protein of M. agalactiae was found to contain a TNASE_3 thermonuclease domain profile. Recombinant GST-MAG_5040 nuclease activity tested on different nucleic acid substrates showed that MAG_5040 is a sugarnonspecific exonuclease that preferentially cleaves nucleic acid residues from double and single strand DNA. Slightly smaller exonuclease activity was observed against RNA. RecombinantM. agalactiae SNaseFigure 6. MAG_5040 western blotting reactivity with sera collected from naturally infected hosts, and indirect detection of homologs in selected mycoplasma species. 200 ng of cleaved rMAG_5040 were run in single well in 10 polyacrylamide gels and probed with sera collected alternatively from Piedmont goats naturally infected with M. agalactiae (A) or from sheep selected from an outbreak of contagious agalactia occurred in Sicily (B). In A, odd lanes (1 to 15) identify sera collected from 8 different goats. Even numbers (2 to 16) indicate sera taken from the same goats at two weeks distance. In B lanes 1 to 10 were probed with sera collected from 10 sheep. Neg in all panels designates lanes in which a pool of negative sera was loaded. (C) Reactivity of of cleaved rMAG_5040 (200 ng) wit.