Maguimithrax gen. nov. is erected to accommodate the extant species Damithrax spinosissimus, while Damithrax cf. pleuracanthus is recognized for the first time from the fossil record . Furthermore, two new species are described from the lower Miocene coral-associated limestones of Jamaica (Mithrax arawakum sp. nov. and Nemausa windsorae sp. nov.). Spurred by a recent revision of the subfamily, two known species from the same deposits are refigured and transferred to new genera: Mithrax donovani to Nemausa, and Mithrax unguis to Damithrax. The diverse assemblage of decapods from these coral-associated limestones underlines the importance of reefs for the abundance and diversity of decapods in deep time. Finally, we quantitatively show that these crabs possess allometric growth in that length/width ratios drop as specimens grow, a factor that is not always taken into account while describing and comparing among taxa.Spider crabs (Majoidea) are well-known from modern oceans and are also common in the western part of the Atlantic Ocean. When spider crabs appeared in the Western Atlantic in deep time, and when they became diverse, hinges on their fossil record. By reviewing their fossil record, we show that (1) spider crabs first appeared in the Western Atlantic in the Late Cretaceous, (2) they became common since the Miocene, and (3) most species and genera are found in the Caribbean region from the Miocene onwards. Furthermore, taxonomic work on some modern and fossil Mithracidae, a family that might have originated in the Western Atlantic, was conducted. Specifically,  Modern spider crabs (Majoidea) range in size from a few millimeters to more than a meter in carapace length . Long, sThe Mithracidae sensu Damithrax unguis  determined to the genus- and species-levels based on the literature and previously unreported material from the FLMNH Invertebrate Paleontology Collection. Possible majoids not determined to at least the genus-level ; fossil specimens in Invertebrate Paleontology (IP).http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:6049E531-ABA7-43EA-8308-EEB5029F667F. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Genera and species known today  are well-represented in the dataset  on fossiAll modern majoid families are represented in Although the pattern that most fossil majoid genera in the Western Atlantic are found in the Caribbean region  is consiMithrax arawakum sp. nov. and Nemausa windsorae sp. nov., erected below, add to the number of decapod species known from the lower Miocene limestones at the Duncans Quarry in Jamaica. The spider crabs Maguimithrax gen. nov.Etymology—Combination of part of the family name of Tobey Maguire, the actor in three Spider-Man movies , and Mithrax. Gender masculine.Type species—Maia spinosissimusSpecies included—Maguimithrax spinosissimus , 11447 (1♂), 11457 (1♀), 31157 , 11388 , all FLMNH IZ collection.Diagnosis—Carapace slightly longer than wide to about equally wide as long in large specimens (l/w ratio = ∼ 1.09–0.97) (09–0.97) , maximumRemarks—D. spinosissimus are close to Nemausa acuticornis and N. cornuta. There are indeed many similarities between Nemausa and D. spinosissimus including the spinose character of the carapace and appendages, a comparable third maxilliped  the third maxilliped endopod merus distomesial margin has a deep, angular excavation at the articulation with the palp in D. spinosissimus, whereas this merus exhibits no pronounced concavity in Mithrax  the propodus bears tubercles and spines in the examined specimens of D. spinosissimus, but it is smooth in Mithrax; and (4) molecular phylogenetics separates D. spinosissimus from Mithrax  the Mithrax .Damithrax. However, it should be noted that D. spinosissimus is much more spinose on the dorsal carapace than other species of Damithrax  are slightly longer than wide or about equally wide as long, unlike the diagnosis of the genus. Not surprisingly, the species plots as a sister taxon to all other modern Damithrax spp. , anterior two visible in dorsal view. Orbit complete, dorsal margin weakly armed behind strong pre-ocular tooth, eyestalk protected above by single blunt dorsal tooth or tubercle separated by closed fissure from two or three blunt post-ocular teeth or tubercles. Third maxilliped endopod merus distomesial margin deeply, angularly excavated at articulation with palp. Cheliped greater than or equal to carapace length; merus dorsal surface spinous, spines not laminar; carpus varied from smooth to rough; propodus smooth; dactylus with enlarged proximal tooth when mature, opposed margins of fingers otherwise crenulate. Pereiopods two to five (ambulatory legs) decreasing in size anterior to posterior; articles finely setose; merus dorsal surface bearing large tubercles and spines, ventral surface with one to six tubercles or spinules; carpus dorsal surface spinous; propodus without spination; dactylus strong, approximately half length of propodus, dactylar lock well developed .Stratigraphic horizon—lower Miocene, Montpelier Formation (uppermost unit) (st unit) .Material—Holotype: UF 106697; Paratypes: UF 73089, 73165, 103955, 106768, 106772, 111483; Topotypes: UF 112783–112785, 112795, 112942, 112946, 113010, 113011, 113117, 113586, 113587, 113675, 113677, 255051–255054. All internal molds of carapaces, some RTV silicone rubber casts of external molds of carapaces.Diagnosis—Pyriform carapace, l/w ratios vary from ∼0.90 for the largest specimens of ∼16 mm width, to ∼1.00 for small specimens. Short rostrum with two small spines downturned, slightly longer than axialmost inner orbital spine. Four usually single spines  on anterolateral margin excluding outer orbital spine. Forwardly directed shallow orbit with spines on the upper orbital margin: four upper orbital spines including outer orbital spine with center two converging; suborbital margin with three spines, axialmost one strongest. Smaller orbital spines less pronounced in small specimens. Tubercular gastric and branchial regions.Description—See Measurements— Remarks—Mithrax unguis based on early Miocene specimens from the Duncans Quarry, Trelawny Parish, Jamaica. The generic placement was reassessed here because of the revision of extant Mithracidae by Damithrax hispidus, as was also indicated by Damithrax, Mithrax unguis is transferred to Damithrax. The species cannot be retained in Mithrax because of the non-spinose character on the dorsal carapace not including the lateral margins. The species differs from D. hispidus, D. pleuracanthus, and D. tortugae in that the rostrum is sharp instead of blunt and that D. unguis seems to have sharper upper orbital spines. Moreover, the length/width ratios separate D. unguis from D. hispidus equal to length, and even length is greater than width for the smallest specimens. Therefore, providing a range of l/w ratios along with specimen sizes for diagnoses and descriptions seems even more useful.Stratigraphic and geographic range—lower Miocene, Jamaica.Damithrax cf. pleuracanthus .Stratigraphic horizon—late Pliocene–early Pleistocene, spoil (float).Material—Single carapace (UF 29057), cuticle.Diagnosis—See Description—Carapace pyriform, about as long as wide (l/w ratio = 1.01), maximum width at ∼61% of carapace length, weakly convex longitudinally and moderately so transversely. Rostrum with two forward projections, only bases preserved; with blunt triangular axial projection oriented downward and posteriorly, with rims. Orbits directed anterolaterally, about as wide as tall, deep, with seven spines around orbit: two spines on antennal segment of which the axialmost one is strongest, separated by a notch and then followed by weak spine more laterally; upper orbital margin with four spines including strong outer orbital spine and stronger axialmost spine; two weak spines in between. Circular antennal holes between axialmost suborbital spine and rostral spines. Anterolateral margin with four spines , third spine weakest, last spine at transition from antero- to posterolateral margin, oriented laterally. Posterolateral margin more rounded than anterolateral margin, with single small spine just posterior to previous spine. Posterior margin with convex protrusion axially, exhibiting row of tubercles and granules continuing onto posterolateral margin. Frontal region including epigastric region with double row of tubercles. Hepatic regions small, at lower level compared to gastric region, with single anterolateral spine. Protogastric regions bulbous, with major tubercle laterally and less pronounced tubercle axially. Mesogastric region with tubercle on process; base swollen, divided into three regions, central region oval. Uro- and/or protogastric region small, wider than long. Cardiac region pentagonal to triangular, with concave margins, about equally long as wide, tubercular. Branchial regions confluent. Intestinal region not delineated, with two strong tubercles. Cervical groove moderately deep, with two slits axially, V-shaped overall but rounded axially, bends more laterally near anterolateral margin. Shallow groove extends from cervical groove near base hepatic region to below outer orbital spine. Grooves around cardiac and uro- and/or metagastric regions. Dorsal carapace surface of cuticle with very small pits, armed with tubercles all over, more granules posteriorly; row of five tubercles in center of gastric region. Ventrolateral sides below anterolateral margins contain small spines. Of hardened parts: most of ventral surface, abdomen, and appendages lacking.Measurements—Excluding spines and rostrum: 13.9 mm long, 13.8 mm wide.Remarks—The specimen is very well-preserved and is ascribed to Damithrax because of the close similarity to extant species, notably Damithrax hispidus, D. pleuracanthus, and D. tortugae. These modern species were synonymized by D. pleuracanthus. Ontogenetic variability of tubercles on the dorsal carapace was also found for D. hispidus in that the largest specimen (75.4 mm carapace width) exhibits fewer tubercles compared to small specimens (<∼30 mm carapace width) . D. hispidus, D. pleuracanthus, and D. tortugae. The FLMNH IZ collection contained sufficient specimens of D. hispidus and D. pleuracanthus to verify identifications. Indeed, specimens of D. pleuracanthus contain more tubercles on the carpus, but ornamental differences were difficult to verify for the merus. While large specimens of D. hispidus (>∼35 mm carapace width) often contained two spines on the inner side of the merus, smaller specimens (<∼23 mm carapace width) often contained only a single tubercle, much like similar-sized specimens of D. pleuracanthus , the ascription is with some query. Nevertheless, this is the first record of this species in the fossil record. The results in D. hispidus .Type material—UF 112682 , UF 112941 .Type locality—FLMNH-IP XJ015: Duncans Quarry 01, Trelawny Parish, Jamaica .Type horizon—lower Miocene, Montpelier Formation (uppermost unit) (st unit) .Material—No material known other than type specimens.Diagnosis—Carapace pyriform, slightly longer than wide (l/w ratio = 1.03 for holotype). Short rostrum with two small spines downturned. Orbits directed forward, with at least four distinct spines around orbit: one long spine at angle of suborbital margin near rostral horns, other such spines not preserved; a slender and long outer orbital spine; a small central upper orbital spine; and a large projection on upper margin near rostral horns. Anterolateral margin with four strong spines , middle two with small spine at anterior base; last spine at transition from antero- to posterolateral margin, oriented laterally. Posterolateral margin more rounded than anterolateral margin, with single small spine just posterior to previous spine. Frontal region with two longitudinal rims connecting to rostral spines and tubercular epigastric regions. Cervical groove deep and wide, U-shaped. Branchiocardiac groove strongest around cardiac region, weaker more laterally. Dorsal carapace surface armed with tubercles, granules, and spines , not very densely so.Description—Carapace pyriform, slightly longer than wide (l/w ratio = 1.03 for holotype), maximum width at ∼65% of carapace length, weakly convex longitudinally and moderately so transversely. Short rostrum with two small spines downturned. Orbits directed forward, wider than tall, not very deep, at least four distinct spines around orbit: one long spine with a smaller spine axially at angle of suborbital margin near rostral horns, other such spines not preserved (may be broken); a slender and long outer orbital spine; a small central upper orbital spine; and a large projection on upper margin near rostral horns. Single small spine present below orbit. Anterolateral margin with four strong spines , middle two with small spine at anterior base; last spine at transition from antero- to posterolateral margin, oriented laterally. Posterolateral margin with single small spine just posterior to previous spine. Posterior margin with convex protrusion axially, with row of granules adjacent to convexity. Frontal region with two longitudinal rims connecting to rostral spines and tubercular epigastric regions. Hepatic regions small, at lower level compared to gastric region, with single strong anterolateral spine. Protogastric regions bulbous, with major tubercle laterally and less pronounced one axially. Mesogastric region with tubercle on process; base swollen, divided into three regions. Uro- and/or protogastric region small, appears as a laterally elongated tubercle. Cardiac region pentagonal, about equally long as wide, tubercular. Branchial regions weakly divided; epi- and mesobranchial regions confluent, tubercular; metabranchial separated from others, with spines, tubercles, and granules. Intestinal region not delineated, with two strong tubercles. Cervical groove deep and wide, U-shaped, bends more laterally near anterolateral margin to continue on ventral carapace, where it bends forward. Short groove extends from cervical groove near base hepatic region to outer orbital spine. Branchiocardiac groove strongest around cardiac region, weaker more laterally, not expressed to very weak on ventral carapace. Dorsal carapace surface armed with tubercles, granules, and spines , not very densely so; row of five tubercles in center of gastric region. Of hardened parts: most of ventral surface, abdomen, cuticle, and appendages lacking.Measurements—Excluding spines and rostrum: 14.0 mm long, 13.6 mm wide (UF 112682); length not measurable, 13.0 mm wide (UF 112941).Remarks—The species appears to fit best in Mithrax because (1) the carapace is about equally long as wide (l/w ratio = 1.03); (2) the dorsal ornamentation has tubercles, granules, and spines ; and (3) the orbit is weakly produced and has two spines on the upper margin excluding the outer orbital spine.M. aculeatus, M. armatus, M. bellii, M. besnardi, and M. hemphilli exhibit a dense cover of granules (M. aculeatus), whereas granules are much less abundant in the new species. Additionally, M. besnardi has a higher number of spines on the upper orbital margin . For M. braziliensis, M. caboverdianus seems comparable (tubercles and spines with some interspersed granules) to the new species, the similar-sized holotype in Mithrax arawakum sp. nov.), but more specimens are needed to confirm this potential difference. Distinct rostral spines are missing in M. caboverdianus, but are present in Mithrax arawakum sp. nov. Additionally, the cardiac region in M. caboverdianus appears wider. The upper orbital margin contains more spines in M. clarionensis and the spines on the lateral margin are less prominent for a similar-sized specimen .Stratigraphic and geographic range—lower Miocene, Jamaica.NemausaType species—Pisa spinipesSpecies included—Nemausa acuticornis .Type locality—FLMNH-IP XJ015: Duncans Quarry 01, Trelawny Parish, Jamaica .Type horizon—lower Miocene, Montpelier Formation (uppermost unit) (st unit) .Material—No material known other than type specimen.Diagnosis—Length/width ratio pyriform carapace = 1.19; orbital margins with seven spines, one long spine at angle of suborbital margin near rostral horns and two additional, smaller spines on same margin; anterolateral margin of carapace with four strong spines, anteriormost two with small spine at anterior base; mesogastric region flattened, anterior part not defined.Description—Carapace pyriform, length/width ratio = 1.19, maximum width at 59% of carapace length, moderately convex longitudinally and transversely. Rostrum incompletely preserved, but with bases of two diverging spines. Orbits anterolaterally directed, wider than tall, deepest in most lateral part, seven spines around orbit: one long spine at angle of suborbital margin near rostral horns and two additional, smaller spines on same margin, separated by notch that marks boundary between antennal segment and rest of suborbital structure; one strong outer orbital spine with elongated base; three supraorbital spines, one closest to rostrum strongest. Anterolateral margin with four strong spines , anteriormost two with small spine at anterior base; last strong spine at transition from antero- to posterolateral margin, directed laterally. Posterolateral margin more rounded than anterolateral margin, with single spine just posterior to previous spine. Gastric and hepatic regions mostly undifferentiated; epigastric regions appear as tubercles; base of mesogastric region swollen, anterior part not defined; uro- and/or metagastric region small, wider than long, sandwiched between mesogastric and cardiac regions. Cardiac region hexagonal. Branchial and intestinal regions confluent. Cervical groove deepest axially; curves around base mesogastric region, then becomes shallower and bends transversely to intersect lateral margin between first and second anterolateral spines. Branchiocardiac groove only defines lateral parts of cardiac region, does not reach lateral margin. Dorsal carapace surface armed with larger and smaller tubercles; row of five pronounced tubercles in center of gastric region; other strong tubercles present on epigastric, branchial, and cardiac regions. Of hardened parts: ventral surface, abdomen, and appendages missing; rostral spines largely missing.Measurements—Excluding spines and rostrum: 27.7 mm long, 23.3 mm wide, and 14 mm tall (as preserved).Remarks—The anterolateral spines are about equally prominent on the cast and the internal mold. The bases of the rostral spines and many of the orbital spines are much better seen on the cast. This is not surprising given the delicate nature of spines, which have the tendency to break easily on the internal mold. Perhaps surprisingly, the small tubercles on the dorsal carapace are not as numerous on the cast, yet another example that ornamentation with and without the cuticle can differ  (N. sinensis.. (1.19) . FurtherN. spinipes are figured, with Nemausa spinipes has a better defined mesogastric region (Very few specimens of Nemausa donovani is different in that the mesogastric region is outlined entirely and a distinct elevation is seen in the center of the posterior part of this region, both unlike in the new species. This is unlikely to be related to ontogeny because the mesogastric features appear stable throughout ontogeny in a congeneric species (Nemausa (N. acuticornis, N. donovani and N. windsorae sp. nov. is much greater, supporting the hypothesis that these are two separate species. Furthermore, N. windsorae sp. nov. bears a denser ornamentation of tubercles, which may only in part be explained by ontogeny (N. acuticornis bears distinct tubercles on the branchial regions, whereas these regions are nearly smooth in N. donovani, unlike for N. windsorae sp. nov. species . Althougontogeny  because Stratigraphic and geographic range—lower Miocene, Jamaica.10.7717/peerj.1301/supp-1Table S1Database fossil majoids from the Western Atlantic.Click here for additional data file.10.7717/peerj.1301/supp-2Table S2Measurements modern and fossil mithracids from the Western Atlantic.Click here for additional data file.10.7717/peerj.1301/supp-3Table S3Damithrax hispidus from Florida.Data on Click here for additional data file.


Desmophyllum dianthus and colonial Lophelia pertusa.In recent years, several types of molecular markers and new microscale skeletal characters have shown potential as powerful tools for phylogenetic reconstructions and higher-level taxonomy of scleractinian corals. Nonetheless, discrimination of closely related taxa is still highly controversial in scleractinian coral research. Here we used newly sequenced complete mitochondrial genomes and 30 microsatellites to define the genetic divergence between two closely related azooxanthellate taxa of the family Caryophylliidae: solitary L. pertusa and D. dianthus were identical. Variability of the mitochondrial genomes of the two species is represented by only 12 non-synonymous out of 19 total nucleotide substitutions. Microsatellite sequence (37 loci) analysis of L. pertusa and D. dianthus showed genetic similarity is about 97 %. Our results also indicated that L. pertusa and D. dianthus show high skeletal plasticity in corallum shape and similarity in skeletal ontogeny, micromorphological  and microstructural characters .In the mitochondrial control region, an astonishing 99.8 % of nucleotides between Desmophyllum and the dendroid Lophelia appear to be significantly more similar to each other than other unambiguous coral genera analysed to date. This consequently leads to ascribe both taxa under the generic name Desmophyllum (priority by date of publication). Findings of this study demonstrate that coloniality may not be a robust taxonomic character in scleractinian corals.Molecularly and morphologically, the solitary The online version of this article (doi:10.1186/s12862-016-0654-8) contains supplementary material, which is available to authorized users. Mitogenomics, or the analysis of complete mitochondrial genomic data sets, is a powerful tool used in a wide range of organisms to improve phylogenetic estimations, reconstruct robust phylogenies and resolve long-standing phylogenetic uncertainties –6. SclerDesmophyllum dianthus  and Lophelia pertusa  are azooxanthellate scleractinian corals living in cold waters worldwide, with the exception of the polar seas  we propose that the solitary and the colonial species are ascribed herein to the genus Desmophyllum and are named Desmophyllum dianthus  and Desmophyllum pertusum , respectively. Below we modify the diagnosis of the genus Desmophyllum based on the information acquired in this study. We also provide a short description of the most important skeletal features of the species Desmophyllum pertusum . A comprehensive revision of the genus Desmophyllum, including fossil species, is currently in progress.The genus Order: Scleractinia[‘Robust’ Scleractinia Group]Family: Caryophylliidae Dana, 1846Desmophyllum Ehrenberg, 1834Genus: Diagnosis: Corallum solitary or colonial. Corallum of solitary species greatly variable in shape, trochoid with subcylindrical pedicel or cylindrical, ceratoid, scolecoid. Colonial corallum bushy in shape, often with anastomosing branches. Up to 6 cycles of septa non-hexamerally arranged. Pali absent. Columella poorly developed, visible only in early developmental stages. Septothecal wall in ontogenetically adult corallites commonly covered by thickening deposits.Type species: Madrepora dianthus Esper, 1794, pl. 69, Figures 1, 2, 3; subsequent designation Cairns, 1994, p. 76Desmophyllum striatum Cairns, 1979—for description see Cairns (1979:120), Desmophyllum quinarium Tenison-Woods, 1879—for description see Tenison-Woods (1879:18), Desmophyllum dianthus —for description see Cairns (1979:26), Desmophyllum pertusum Recent species included: Desmophyllum pertusum  comb. nov.Madrepora pertusa Linnaeus, 1758: 38, pl. 2, Figures 1, 2.Lophelia pertusa Zibrowius, 1980: 126, pl. 66 a-l (synonymy).—Cairns, 2000: 100–102 (synonymy).Lophelia prolifera Cairns, 1979: 125–127, pl. 24, Figures one, two, three, four, five (synonymy).Diagnosis: Corallum forming large bushy colonies. Corallite skeleton connected to the parental one at least in the early growth stage Fig. . CoralliRemarks: The species has been widely described, as belonging to the genus Lophelia , might be weak characters from a taxonomic point of view to maintain D. dianthus and D. pertusum as sympatric species. Detailed studies on the systematics of the genus Desmophyllum, currently on-going, might lead to synonymise these two species. Further research on biogeography and reproduction of the genus are needed to support (or not) this taxonomic distinction. If differences of environmental features and ontogenetic mechanism would not be statistically significant to support species differentiation, the comparison of molecular data performed in this study  would demonstrate that D. dianthus and D. pertusum are the same species. In such a case, following the rule of priority by date of publication of the International Code of Zoological Nomenclature (ICZN) [Art. 23.1], since the species D. dianthus  was described after D. pertusum , both species would be named as D. pertusum.Insufficient information about Hydra (Hydrozoa), Nematostella (sea anemone), and Acropora (scleractinian)  free of the parent before a third generation bud appears”  database [Accession Numbers KX000882-KX000894].The new taxon name and this article are registered in [ZooBank] registry with an identifier code [urn:lsid:zoobank.org:act:949C5CE4-4841-423 F-958D-A8D6A4E65E6B].


Culicoides imicola represents the main bluetongue virus (BTV) vector, other European Culicoides biting midges, possibly implicated in virus transmission, have been detected here. Understanding their distribution, seasonal abundance, and infection rates is necessary to predict disease incidence and spread across coastal and inland areas, and to define their role in virus overwintering.Bluetongue (BT) epidemics have affected the Mediterranean island of Sardinia since 2000. While Culicoides species were morphologically and molecularly identified. Infection rates in prevailing midge species captured in 2013 during a BTV-1 outbreak were determined using RT-qPCR based virus detection in insect body pools, supplemented by specific body region analyses. The seasonal infection prevalence in Culicoides samples collected in 2001 in a BTV-2 affected farm was also determined.Biting midge abundance was determined by light traps on selected farms representing diverse climatic conditions of Sardinia. Livestock-associated C. newsteadi species A and species B) prevailed among all biting midge species (47.7 %), followed by C. imicola (27.8 %) and the Obsoletus complex (C. obsoletus and C. scoticus) (17.6 %). Whilst Culicoides imicola was more abundant along the coast, the Newsteadi complex was frequently collected at higher altitude and the Obsoletus complex was notably associated to cattle farms. Culicoides pulicaris and C. punctatus abundance was found to be marginal in all farms. BTV was detected in parous female samples of all these species, and the full dissemination of the virus within the body of C. imicola, C. obsoletus, C. scoticus, and Newsteadi complex species was confirmed by analyses of thorax and head, containing salivary glands. Higher infection rates were associated with C. scoticus, C. newsteadi species A and species B, compared to C. imicola. The virus was detected in C. newsteadi species A and C. obsoletus in winter and spring, whereas it was mainly found in summer and autumn in C. imicola.The Newsteadi complex (Culicoides vectors, including C. imicola and the Newsteadi complex being the most important. The Newsteadi complex and other midge species can play an important role in internal areas and are likely to be directly involved in virus overwintering.In Sardinia, bluetongue virus is transmitted by multiple  Culicoides (Diptera: Ceratopogonidae) is characterized by a significant diversity of biting midges . Th. ThCulicC. imicola, C newsteadi species A and B, and C. scoticus parous females, that represented the majority of pools analyzed during this period. Culicoides obsoletus, C. pulicaris and C. punctatus, collected from April to May, were represented in only a few female pools analyzed, but were also found to be BTV-1 positive (Ct range: 25–30)  and 17 (10.4 %) carried a high (Ct range: 25–30) and a very high (Ct < 25) virus genome load, respectively. The higher loads were found in pools of C. scoticus and C. newsteadi species A, and in thorax of C. imicola and C. newsteadi species B  confirmed the full dissemination of BTV within the main midge vectors. Higher virus genome loads were found in head and thorax of  B Table .Table 4RCulicoides scoticus had the highest MIR value of 0.058, while the lowest rate was that for C. imicola (0.023). C. newsteadi species A and species B showed intermediate values (Table The minimum infection rates (MIRs), expressed per 1,000 parous females, has been calculated for the prevailing midge vectors for which a sufficient number of samples was available. Culicoides vectors, collected monthly, in Sassari-1 farm during a major BTV-2 epidemic in 2001. In this farm, C. newsteadi species A (95 % of the Newsteadi complex) was captured throughout the year, with higher abundance in spring, and represented the most prevalent species, followed by C. imicola, captured from April and reaching the population peak in September. The Obsoletus complex, showed a lower population density and included mainly C. obsoletus (85 %)  Fig. .Fig. 3CC. imicola and C. obsoletus. BTV-2 positive pools  of C. obsoletus and C. newsteadi species A were detected during the winter-spring months and sporadically in summer, whereas C. imicola pools showed virus positivity only from September to November  and C. scoticus, represented 17 % of the total Culicoides midges collected in Sardinia and was detected in all farms with a general prevalence of C. scoticus. The females of this complex were identified by morphometric measures of genitalia combined with molecular analyses. Although measurements of spermathecae of our samples were generally higher in comparison with samples reported in France [European species of livestock-associated Sardinia . The NewSardinia  and CorsSardinia . Both C.and Fig. . Culicoieratures . This iseratures , 56 and eratures , 58. Howeratures . Furtheral sites . The Obsn France  and othen France , these vC. obsoletus and C. scoticus are able to breed in a great variety of habitats, dung heaps and cow pats are emerging as their most important farm development substrates in Germany [Culicoides pulicaris and C. punctatus were generally collected in smaller amounts and a higher abundance of the first species was especially recorded in farms at higher altitudes.Higher abundance was associated with cow farms along the coast, probably due to the composted cow manure accumulating in cattle holding and spread on cropping lands and pastures. Although  Germany . CulicoiCulicoides imicola is a multivoltine species, having several generations per year regulated by temperature and other climatic factors [Culicoides vectors, including Obsoletus and Newsteadi complex species that produce 3–4 generations per year [ factors . In Sard factors , 62. Forper year , parous C. imicola, C. obsoletus, C. scoticus and C. newsteadi species A and B, support their vector competence during BTV-2 and BTV-1 epidemics in Sardinia. As a confirmation of the ability of the virus to replicate inside females of these species, meaningful high virus genome loads  were found in the thorax, where salivary glands are located, and exceptionally in the head. BTV infection prevalence was also observed in few parous female pools of C. obsoletus, C. pulicaris and C. punctatus, indicating their possible implication in virus transmission in Sardinia. This study confirmed the ability of different Culicoides species to harbor both BTV-1 and BTV-2 serotypes, but specific experiments are needed to assess possible differences in their susceptibility levels.The significant viral RNA titers detected by qRT-PCR in the body of parous females of C. imicola, C. obsoletus, and C. scoticus are recognized as proven vectors in Europe, as supported by repeated virus isolation from field-collected females [Culicoides newsteadi has been regarded as a potential vector since few intrathoracically-inoculated females from Israel sustained virus replication in the laboratory [C. newsteadi species A and B, supported the full BTV dissemination in field-collected parous females, which corroborates the results of other investigations in Italy [C. imicola was only sporadically collected. In addition, their winter-spring activity period makes C. newsteadi species A and B the main candidates for virus overwintering.Among BTV-positive midge species collected in this study,  females , 17 and  females –24. Culiboratory . The higin Italy . While tC. imicola, which is consistent with laboratory infection studies in England [The estimated prevalence of BTV-1 infection in field-collected parous females of most abundant vector species in our study was comparable to previous infection rate determinations in Sardinia and other Italian regions . Higher  England . However England .C. brevitarsis, in respect to the more efficient but less abundant C. fulvus [On the other hand, neither the field infection rate determination nor the evaluation of vector competence represent the actual potential of vector populations to transmit the disease among animals. This could more appropriately be estimated through the vector capacity, defined as the average number of infective bites per host per day . Vector . fulvus .C. imicola plays a main role in BTV transmission along coastal areas where this species is significantly abundant, as documented by extraordinary capture levels in certain farms where more than 200,000 midges per night were collected by a single trap. This multivoltine and thermophilic species was rarely captured during the first half of the year and peaked in summer-autumn in conjunction with BT epidemics. Both in the same areas and inland, an augmentative vector role can be attributed to the more abundant C. newsteadi species A and B that were found all over the year. An additional contribution to BT spread along the coast and inland may derive from populations of other vectors like C. obsoletus, C. scoticus and C. pulicaris, that can reach high local abundance, especially in cow farms.In Sardinia, Culicoides adult lifespan following the active virus transmission period; (iv) ongoing and slow/low-level cycle of infection and transmission between ruminants and midges over the interseasonal period [Culicoides vectors with high viral load are found in winter-spring in conjunction with low seroconvertion rate in animals in Sardinia, would support a continuous cycle of infection and transmission between ruminants and midge vectors.The BT epidemic waves in Sardinia normally lasted 2–3 years, and achieved an end as a consequence of natural animal immunization and vaccination campaigns. Infection continuity, over the years, revealed obvious virus overwintering. In this context, parous females of Newsteadi and Obsoletus complex species associated with high viral loads were collected during winter-spring in diverse areas where these species could be directly implied in virus overwintering , 33. Prol period –69. WhilCulicoides species, Bluetongue in Sardinia and probably in other Mediterranean regions, is likely to be transmitted by multiple vectors, which ensures virus spread in areas characterized by different environmental and climatic conditions. In addition, different species can be involved in virus overwintering, thus contributing to endemicity of this disease that almost continuously appeared during the last 15 years in Sardinia and the neighboring Mediterranean regions.Based on this study and on previous knowledge on distribution and abundance of s.l., sensu lato; s.str., sensu strictoasl, above sea level; BT, Bluetongue; BTV, bluetongue virus; COI, mithocondrial cytochrome oxidase c subunit I; ITS2 rDNA, ribosomal internal transcribed spacer 2; MIR, minimum infection rate; RT-qPCR, Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction;


Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P) or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG) levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect. Staphylococcus aureus in their nose ..StaphyloS. aureus bacteremia in S. aureus carriers, the risk of death due to bacteremia might be lower once carriers acquire bacteremia. An explanation for this has not yet been provided, although a role for the immune system has been proposed. More than 80% of health care-associated S. aureus infections are caused by an endogenous strain [S. aureus strain, carriers may have developed antibodies or cellular immune responses that protect against endogenous bacteremia-related death. Non-carriers may have developed humoral responses that protect against colonization more than against invasive disease. Several studies have been conducted comparing anti-staphylococcal antibody levels in carriers and non-carriers. Carriers show higher immunoglobulin G (IgG) levels than non-carriers against toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA) [S. aureus strain in the improvement of the course and outcome of S. aureus bacteremia. In this study, S. aureus carriers who developed endogenous S. aureus bacteremia showed a stronger and broader pre-bacteremia IgG response to their own invasive, endogenous S. aureus strain compared to non-carriers, who develop an exogenous S. aureus bacteremia. Recently, Montgomery et al. [S. aureus skin and soft tissue infection (SSTI) protects against secondary endogenous SSTI. This protection was mediated by antibody and interleukin (IL) 17A and inhibited by interferon (IFN) γ. Conclusions regarding the antigen-specificity of these antibodies were not drawn. Their observation suggests, in addition to a role of humoral immunity, a protective role of cellular immunity. Protection against exogenous infection was not studied.In spite of the higher risk of acquiring nosocomial s strain , 11. Thi A (SEA)  and the  A (SEA) . In cont A (SEA) , extrace A (SEA) . In addi A (SEA)  also sugy et al.  showed iS. aureus carriage and/or S. aureus exposure and the role of humoral and/or cellular immunity on the course and outcome of subsequent endogenous or exogenous S. aureus infection are difficult as both carriers and non-carriers harbor a diversity of anti-staphylococcal antibodies. In non-carriers, these antibodies may be induced by S. aureus carriage or (sub-)clinical infection in the past. Studies in mice initially free of S. aureus and anti-staphylococcal antibodies may provide further insight.In humans, conclusive studies on the exact influence of S. aureus bacteremia is influenced by prior S. aureus exposure and whether this is dependent on the S. aureus strain (endogenous or exogenous) causing the initial exposure. For this purpose, a mouse model of mild S. aureus skin infection once or twice was established. We focused in this study on humoral immunity only, by analyzing the pre-bacteremia IgG levels against a broad panel of 25 S. aureus antigens following skin infection, and we assessed whether improvement in the course of S. aureus bacteremia was associated with pre-bacteremia IgG levels.In the present study in mice, we investigated whether the course of S. aureus isolate and S. aureus strain 8325–4, a well-characterized laboratory strain  [S. aureus isolate P  is a clinical isolate recovered from blood of a septic patient and was previously described and analyzed by proteomics by Ziebandt et al.  [S. aureus, grown in Brain Heart Infusion broth  until OD560 ~ 1.0, were stored with 5% glycerol at -80°C. For infection, a suspension of staphylococci was defrosted and centrifuged for 10 minutes at 14,000 x g. The S. aureus pellet was resuspended in saline, and diluted to obtain the desired inoculum.Bacterial strains used were a clinical SA, ST8) . S. aure-type 1) . StaphylS. aureus-free animals were 11–13 weeks old on the day of infection, and were given food and water ad libitum. Before each experiment, one mouse per group was sacrificed to confirm the S. aureus-free status in terms of cultures of fresh fecal and nasal microbiota and the absence of anti-staphylococcal IgG levels in blood (see below). The animal experimental protocols adhered to the rules laid down in the Dutch Animal Experimentation Act and the EU Animal Directive 2010/63/EU, and the Institutional Animal Care and Use Committee of the Erasmus University Medical Centre Rotterdam approved the present protocols (permit number: EMC2415).Specified opportunistic pathogen-free (SOPF) female BALB/cBYJ mice were obtained from Charles River . These S. aureus skin infection in mice was adapted from Brown et al. [®, 0.5 mg/kg; Eurovet Animal Health, Bladel, the Netherlands), midazolam  and fentanyl . S. aureus isolate P (3–6 x 107 CFU) or S. aureus 8325–4 (5–10 x 107 CFU) was injected intradermally (50 μL) (n = 10 per group). For placebo skin infection, mice received saline. Anesthesia was antagonized to quickly awaken the mice using a mixture of atipamezole , flumazenil  and naloxon . Anesthetic and antagonistic agents were administered intraperitoneally, in a total volume of 175 and 250 μL, respectively. Mouse body weight was assessed three times a week. Two and five weeks after intradermal S. aureus inoculation, blood was withdrawn from the tail artery to determine anti-staphylococcal IgG levels in serum. Blood was collected in a Microvette® CB300 tube  and sera were prepared and stored at -80°C. At week five, mice were sacrificed by CO2 exposure. Presence of S. aureus in intestines and nasopharynx five weeks after intradermal S. aureus inoculation was determined by culturing fresh feces and nasopharyngeal lavage in phenol-red mannitol salt broth  at 35°C for 7 days. Nasopharyngeal lavage was performed by flushing the nares with 5 mL sterile phosphate buffered saline + 0.4% Tween 20 . When PHMB turned yellow, this was subcultured overnight at 35°C on Colombia III blood agar. S. aureus was identified based on colony morphology and Slidex Staph Plus agglutination testing . Multi-locus variable number of tandem repeat analysis (MLVA) of SIRU01, SIRU07, SIRU13 and SIRU15 [S. aureus strains were the same as the strain used for infection of mice.The method of induction of n et al. . In shorS. aureus isolate P into the tail vein. A S. aureus inoculum at the 50% lethal dose  or at the 100% lethal dose  was used for establishment bacteremia. Clinical signs of illness in each mouse were evaluated twice daily during the experiment to minimize suffering as described before [2 exposure. Euthanized mice were considered as deaths, as pilot experiments showed that mice with severe signs of illness die before the next time point. Animal survival rate over 14 days after infection was monitored. At day 28, S. aureus load in blood and organs of surviving animals was determined to investigate whether surviving mice next to survival also showed improved S. aureus clearance. The mice were sacrificed by CO2 exposure. A blood sample was taken via a transcutaneous cardiac puncture and collected in a vial containing Lithium Heparin . The lungs, spleen, liver, and kidneys were removed aseptically and homogenized  in 2 mL of saline for 10 seconds at 30,000 rpm at room temperature. Undiluted homogenate suspensions and blood as well as 10-fold serial dilutions in saline were plated onto Colombia III blood agar. After incubation overnight at 35°C, colonies were counted.Bacteremia was induced by inoculation of 100 μL of d before . Mice weS. aureus isolate P or with S. aureus 8325–4 (n = 15 per group) was induced 35 days before induction of bacteremia. Controls received intradermal inoculation of saline. In case of skin infection twice, three weeks after the first skin infection, a second skin infection using the same S. aureus strain was applied, near the inoculation site of the first skin infection. Bacteremia caused by S. aureus isolate P was always established at five weeks after the first skin infection, when mice were 16–18 weeks old (adults).Experimental set-up is shown in cis/trans isomerase (PrsA); hypothetical protein SA0104; serine-aspartate dipeptide repeat protein D (SdrD); staphylococcal superantigen-like proteins 1, 3, 5, and 10 ; and wall teichoic acid (WTA). Genes encoding these S. aureus antigens were all present in both S. aureus isolate P and S. aureus 8325–4.Serum IgG levels against the following antigens were semi-quantified: alpha toxin; clumping factors A and B (ClfA and ClfB); extracellular fibrinogen-binding protein (Efb); ESAT-6-like protein (EsxA) and CFP-10-like protein (EsxB); formyl peptide receptor-like1 inhibitory protein (FLIPr); fibronectin-binding protein A (FnbpA); immunodominant staphylococcal antigen A (IsaA); iron-responsive surface determinants A and H (IsdA and IsdH); lipase; leukocidins D and E (LukD and LukE); peptidoglycan hydrolase (LytM); endonuclease (Nuc); peptidoglycan (PG); a parvulin-type peptidyl-prolyl Alpha toxin, LukD, and LukE were prepared as described previously . ClfB, FIgG levels were semi-quantified simultaneously in multiplex assays using a bead-based flow cytometry technique . Methods have been described elsewhere –26. TestS. aureus isolate P or S. aureus 8325–4 (n = 10 per group), and from mice with placebo skin infection were analyzed. Sera from three non-infected mice were used as negative controls.Serum samples from mice with skin infection caused by U test was used to compare median differences in anti-staphylococcal IgG levels in different groups. The Wilcoxon Signed Rank test was used to compare anti-staphylococcal IgG levels in paired samples. In individual mice, high IgG levels against an antigen were not correlated with high IgG levels against other antigens, or vice versa. The Bonferroni correction was applied to correct for multiple testing. As a result, P-values < 0.002 were considered to be statistically significant. These statistical analyses were performed using the Statistical Package of Social Sciences version 17.0 for Windows .The Mann-Whitney S. aureus colonization status. The log rank test was used to determine statistical differences in animal survival rate between groups. Differences were considered statistically significant when 2-sided P-values were < 0.05. GraphPad Prism 5 for Windows  was used for these statistical analyses.The Fisher’s exact test was used to compare differences in S. aureus isolate P or S. aureus 8325–4 developed a scab (area of ~1.5 cm2) at the inoculation site within 1 week. Body weight loss was only minor  and mice appeared healthy. Five weeks after infection, when S. aureus bacteremia was induced, the skin had healed and body weight was restored. S. aureus cultures from the inoculation site were always negative at week 5 after skin infection. However, the intestines of mice with skin infection once or twice, caused by S. aureus isolate P or S. aureus 8325–4, were always culture positive for the infecting S. aureus strain. Nasopharynges were not always culture positive: S. aureus was cultured from mice after S. aureus isolate P skin infection once (3 of 10 mice) or twice (5 of 10 mice), and after S. aureus 8325–4 skin infection once (1 of 10 mice) or twice (4 of 10 mice). No significant differences in number of nasopharynx culture positive mice were observed.Mice infected with S. aureus isolate P skin infection once or twice , whereas the second skin infection with S. aureus 8325–4 resulted in a significant rise in IgG levels at week 5 against Efb, IsaA, and IsdA (P < 0.002).Serum anti-staphylococcal IgG levels against a broad panel of 25 staphylococcal antigens were assessed in mice with or twice  or with or twice  .Bacteremia was always induced by S. aureus bacteremia in mice with prior skin infection is shown in Fig S. aureus isolate P or S. aureus 8325–4, animal survival rate of bacteremic mice was not significantly improved compared to placebo skin infection.The course of LD50 S. aureus bacteremia in mice with prior skin infection is shown in Fig S. aureus isolate P. The time to death of bacteremic mice was increased after skin infection once (P = 0.033), and was further prolonged after the second skin infection (P = 0.001). Mouse survival at day 14 was increased from 0% to 20%. Skin infection once or twice with S. aureus 8325–4 had no effect on the course of bacteremia.The course of LD100 S. aureus was never cultured from blood, lungs, spleen, and liver at day 28. In 17–60% of surviving mice, cultures from kidneys were S. aureus positive, ranging from 1 x 101–2 x 107 CFU per two kidneys, but differences in number of S. aureus positive kidneys between the mice that survived moderate bacteremia were not observedIn mice that survived LD50 bacteremia Fig  and 2B oS. aureus bacteremia was associated with increased levels of anti-staphylococcal IgG induced by S. aureus skin infection. To this aim, in mice receiving skin infection twice caused by S. aureus isolate P or S. aureus 8325–4 (n = 10 per group), serum IgG levels against a broad panel of 25 staphylococcal antigens at week 5 after skin infection, the day of induction of S. aureus isolate P bacteremia were compared. IgG levels are shown in S. aureus isolate P skin infected mice, IgG levels against alpha toxin, Efb, EsxA, EsxB, IsaA, IsdA, IsdH, lipase, LukD, LukE, LytM, Nuc, PG, PrsA, and SA0104 were significantly higher than in mice with placebo skin infection. In S. aureus 8325–4 skin infected mice, IgG levels against Efb, EsxA, EsxB, IsaA, IsdA, IsdH, lipase, LukD, LukE, LytM, Nuc, PG, PrsA, SA0104, SSL1 and SSL10 were significantly higher than in mice with placebo skin infection. Comparing both IgG profiles, IgG levels against Efb, LukD, LukE, Nuc, and WTA were significantly higher in S. aureus isolate P skin infected mice than in S. aureus 8325–4 skin infected mice.We assessed whether improvement of the course of S. aureus skin infection improved the course of subsequent endogenous S. aureus bacteremia. The use of S. aureus-free mice enabled us to draw conclusions in this respect. In humans, conclusive studies on the exact influence of S. aureus exposure on the outcome of S. aureus bacteremia remain difficult, as both carriers and non-carriers have been exposed to S. aureus [S. aureus bacteremia compared to non-carriers, hypothesizing that anti-staphylococcal antibody levels or components of the cellular immune system that are increased in carriers, induced by prior S. aureus exposure, may play a role in protection against death due to S. aureus bacteremia. Our observations in the present study in mice are in line with the suggestions made by Wertheim et al.In the present study, we showed for the first time that mild . aureus –15. Wert. aureus  already S. aureus skin infection caused by either S. aureus isolate P, a clinical sepsis isolate [S. aureus 8325–4, a frequently used sequenced strain [S. aureus isolate P. Skin infection led to S. aureus colonization in intestines and nasopharynges, and with respect to the humoral immune response, anti-staphylococcal IgG in blood was found. The skin infection described above was mild and transient, simulating a mild S. aureus infection in humans. We showed that prior S. aureus skin infection improved the course of LD100 S. aureus isolate P bacteremia provided that bacteremia was caused by the endogenous S. aureus strain. This effect was not observed in exogenous bacteremia caused by S. aureus isolate P when skin infection was induced by S. aureus 8325–4. While a single prior skin infection resulted in delayed time to death and improved animal survival in our endogenous LD100 S. aureus bacteremia model, in endogenous LD50 S. aureus bacteremia prior skin infection did not. This lack of a protective effect in LD50 S. aureus bacteremia may be related to the relatively small window to detect significant differences in this respect. Whereas in the present study bacteremia was always caused by S. aureus isolate P, in future experiments it would be informative to study the effects of skin infection on the course and outcome of subsequent S. aureus 8325–4 bacteremia. S. aureus 8325–4 showed a very steep inoculum-response curve in the mouse model of bacteremia, and as a consequence unfortunately, we were not able to realize reproducible infection models with LD50 and LD100 inocula.We established local  isolate , or S. ad strain . SubsequS. aureus bacteremia. Also in the model of endogenous LD50 S. aureus bacteremia, mortality was reduced, although this difference was only borderline significant (P = 0.052), indicating a trend in improvement of survival rate in skin infected mice with subsequent endogenous bacteremia. Interestingly, following the second skin infection, pre-bacteremia serum IgG levels were elevated as well. As improved survival rates were associated with strong increases in pre-bacteremia anti-staphylococcal IgG levels, this suggests that IgG resulting from prior mild skin infection may potentially act protective against antigens determining the outcome of S. aureus bacteremia. Future studies including successful adoptive transfer of antibody purified from serum of skin infected mice to naive mice prior to S. aureus bacteremia are necessary to confirm a protective role of pre-bacteremia IgG.We also demonstrated that a second skin infection following the first skin infection further enhanced the survival rate in mice with endogenous LD100 S. aureus isolate P bacteremia. Next to IgG against these S. aureus antigens, pre-bacteremia IgG against Efb, LukD, and LukE may also contribute in this respect. These IgG levels were elevated in mice with S. aureus isolate P skin infection twice and endogenous bacteremia compared to mice with S. aureus 8325–4 skin infection twice and exogenous bacteremia.Regarding the pre-bacteremia IgG levels, most striking were the IgG levels against IsaA, Nuc, PrsA, and WTA, which were further elevated following the second skin infection. These IgG levels may contribute to the delayed time to death and improved animal survival in mice with S. aureus isolate P skin infection improved the course of subsequent endogenous S. aureus bacteremia in mice are in line with those obtained in a recently published experimental study in mice by Montgomery et al. [S. aureus SF8300 (USA300) skin infection (SSTI) on a secondary SSTI caused by the same S. aureus strain. It was shown that skin infection protected against the subsequent skin infection. Protection against exogenous SSTI was not studied. They demonstrated that this protection was mediated by antibody and IL-17A and inhibited by IFN-γ as shown by antibody transfer to naive mice and neutralization of IL-17A or IFN-γ prior to infection, respectively. While in their study IgG levels against Hla and IsdB were assessed, the present study in mice included IgG levels against a broad panel of 25 S. aureus antigens. Future studies in mice on the effect of immunization targeting IsaA, Nuc, PrsA, WTA, Efb, LukD and LukE prior to the induction of S. aureus bacteremia may shed further light on the protective role of these anti-staphylococcal IgGs in bacteremia. In addition to these antigens, antibodies against S. aureus antigens not included in the present study may also be associated with protection against S. aureus bacteremia. Further insight may be obtained from adoptive antibody transfer studies.Our observation that y et al. . They stS. aureus bacteremia, as the cellular immune response has a role in S. aureus infections [S. aureus bacteremia-related mortality, conclusions cannot be drawn from the present study which was focused on the humoral immune response prior to S. aureus bacteremia. However Montgomery et al. [S. aureus bacteremia model used in the present study.Next to the potential role of pre-bacteremia anti-staphylococcal antibodies, components of cellular immunity are expected to contribute as well to the improvement of the animal survival rate in mice with endogenous fections , 27, 28.y et al.  showed tS. aureus in the intestines and nasopharynx following S. aureus skin infection may also contribute to the improvement of the animal survival rate. As differences in number of intestine or nasopharynx culture positive mice were not observed between the groups, no conclusions can be drawn on the immunological relevance of colonization of these sites. As quantitative cultures from the intestinal and nasopharyngeal flora were not performed, definite conclusions on the role of intestinal and nasopharyngeal colonization cannot be drawn, but this merits further investigation.In addition to pre-bacteremia immune responses, the consequences of exposure to S. aureus isolate P skin infection prior to S. aureus bacteremia improved the outcome of endogenous invasive infection. Improved animal survival rates may be associated with the elevated levels of anti-staphylococcal IgGs against specified antigens. The observation that anti-S. aureus IgG may contribute to improvement of the course and outcome of endogenous S. aureus bacteremia opens new perspectives to investigate the protective capacity of active and passive immunization as a non-antibiotic-based treatment regimen in patients with S. aureus bacteremia. Further studies using other S. aureus strains are needed to generalize and support our conclusion that S. aureus skin infection improves the course of subsequent endogenous S. aureus bacteremia. In this respect, also the role of cellular immunity should be further investigated.In conclusion, we show that


Helicobacter pylori is a gram-negative urease-producing bacterium causally linked with gastritis, peptic ulcer disease and gastric adenocarcinoma. Infection is more frequent and acquired at an earlier age in developing countries compared to European populations. The incidence of Helicobacter pylori infection in dyspeptic Ghanaian patients was 75.4%. However, epidemiological factors associated with infection vary across populations.Helicobacter pylori infection was confirmed by rapid-urease examination of antral biopsies at upper Gastro-intestinal endoscopy.This study used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the Korle-Bu Teaching Hospital, Accra between 2010 and 2012. The study questionnaire elicited their epidemiological clinical characteristics. H. pylori-infection was even across most age – groups, ranging from 69.2% (61 – 70) years to 80% (21 – 30) years. Helicobacter pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence with rural areas having the highest prevalence. The unemployed and patients in farming had relatively high Helicobacter pylori infection rates of 92.3% and 91.7% respectively.The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5% were females. The age distribution of Helicobacter pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use suggests likely re-crudescence or re-infection from multiple sources in a developing country. Socio-cultural factors such as residential class and farming may be facilitating factors for its continued prevalence. Helicobacter pylori is a spiral-shaped gram-negative urease-producing bacterium [H. Pylori has been demonstrated worldwide and in individuals of all ages with conservative estimates suggesting 50% of the world′s population is affected [H. Pylori is rarely found before age 10 but increases to 10% in those between 18 and 30 years of age and to 50% in those older than age 60 [H. pylori infection in Ghanaian patients with dyspeptic symptoms referred for upper gastrointestinal endoscopy at the Korle-Bu Teaching Hospital (KBTH) has previously been found to be 75.4% [H. Pylori is related to socioeconomic status and living conditions in early life. Overcrowded conditions associated with childhood poverty lead to increased transmission and higher prevalence rates [H. pylori have been investigated in humans and chronic excessive salt intake has been shown to enhance H. pylori colonization in mice and in humans [H. pylori infection in a West African country with a known high prevalence of the condition.acterium . It is tacterium , 3. It iacterium . It has acterium . H. Pylon humans . This stH. pylori eradication treatment or proton-pump inhibitor-use two weeks preceding endoscopic analysis were excluded from the study. Consenting patients were subsequently recruited into the study and administered the study questionnaire which elicited patients’ demographics, household, environmental characteristics, associated symptoms and relevant background history. This included occupation, alcohol intake, dietary preferences and herbal remedies. Household structure was defined as follows: detached (a single house in an enclosed compound); semi-detached ; compound structure (more than two separate houses enclosed in the same compound). Urban residences were categorized into class 1 – 3 based on the Accra Metropolitan Assembly (AMA) classification of Accra with tax imposition rates of Ghana Cedi (GHC) 100, GHC 60 and GHC 40 respectively. These reflect relative wealth and housing density in the Metropolis with class 1 corresponding to the most affluent and class 3 the most densely populated regions. Rural regions were geographical areas in Ghana not within an urban area and outside cities and towns.Ethical approval was granted by the Protocol and Ethical Review Committee of the University of Ghana Medical School, College of Health Sciences, Accra, Ghana. This study was conducted in accordance with the Helsinki Declaration. It used a cross-sectional design to consecutively sample dyspeptic patients at the Endoscopy Unit of the KBTH, Accra between April, 2010 and August, 2012. Korle-Bu Teaching Hospital has approximately 2,500 beds and is the main tertiary referral center in the capital, Accra serving the majority of the southern half of Ghana. The Endoscopy Unit runs two four-hour sessions per day on four days of the week. Approximately five patients attend each endoscopy session for upper gastro-intestinal (GI) endoscopy. Sampling was conducted during three such sessions per week. All patients were taken through the explanatory statement of the project before endoscopy. Patients with prior H. pylori infection was confirmed by rapid-urease Campylobacter-like-organism (CLO) examination of antral biopsies at upper GI endoscopy . Data was analyzed using the SPSS 16 Program. Categorical data was expressed as proportions and presented in tables. Chi-square was used to demonstrate the differences between observed variables with a p-value of <0.05 used to indicate statistical significance. Logistic regression analysis was used to demonstrate the relative risk of H. pylori infection in patients with specific predictor variables.H. pylori obtained by immediate CLO-testing of gastric antral biopsies was 74.8%. The age distribution of H. pylori-CLO-positive cases was even across most age - groups as illustrated by H. pylori prevalence, 80.0% (n = 24) while age group (61–70) had the lowest, 69.2% (n = 27). There was no statistically significant difference in H. pylori prevalence between age groups, (p = 0.957). H. pylori prevalence including number in household, type of household and residential location. There was a statistically significant difference between residential classes in relation to H. pylori prevalence (p = 0.046) with rural areas having the highest prevalence . However, H pylori prevalence decreased across areas mapping to the three residential classes in accordance with increasing affluence  of patients living in households with 1 – 5 members. By contrast, there was no statistical difference in H. pylori prevalence between household structural types; detached, semi-detached or compound house-structure, (p = 0.469).The sample population of dyspeptic patients attending the Endoscopy Unit for upper GI endoscopy yielded 242 patients of which 47.5% were females, 52.5% males. The overall prevalence of ffluence . ConsistH. pylori cases by occupation; p = 0.107; the unemployed and patients involved in farming/agriculture had relatively high H. pylori infection rates of 92.3% and 91.7% respectively. Potential associations were also sought between consumption of alcohol, herbal remedies, dietary preference  and H. pylori infection prevalence. However, none achieved statistical significance, H. pylori infection with specific predictor variables after logistic regression analysis. Residential class was the most significant risk factor for the presence of H. pylori infection following multi-variate analysis . Household size  and structure  were however potential risk factors for H. pylori infection.H. pylori infection in this study was 74.8% comparable to previous studies in Ghana [H. pylori prevalence demonstrated supports the notion that the rate of acquisition of infection is predominantly highest in early life [H. pylori following successful bacterial cure is not uncommon in developing countries, occurring in approximately ∼ 12% of individuals shown to have been originally cleared of this bacterium. By contrast, in developed countries∼1% such individuals are subsequently re-infected. Additionally, when re-infection occurs this has most commonly been shown to represent recrudescence of the original bacterial strain [The prevalence of in Ghana  and otherly life . Indeed,rly life . Re-infel strain , 14, whil strain . Re-infel strain , 14.H. pylori infection. However, all nine patients with a household size of over 30 members had H. pylori infection in comparison to 67.9% of patients living in households with only 1 – 5 individuals. A study has shown that in UK adults, aged 50-- 59 years, sibling number was independently associated with prevalence of H. pylori infection [H. pylori infection [H. pylori among contacts and household members. For example, a child may have an enhanced susceptibility to H. pylori through increased exposure and a possible lowered immune response [Rising numbers of household membership and household structure were not significantly associated with nfection . Similarnfection , 18. Speresponse .H. pylori prevalence in Black and Hispanic people was also inversely related to the social class during childhood. In support of the notion that childhood environment was critical, this relationship remained even after adjusting for the present social class and age of study participants [H. pylori prevalence in developed countries [H. pylori in vomitus, human faeces and unclean water, all potentially serving as sources of infection of vulnerable residents in over-crowded and densely populated accommodations [In this study, occupation was used as a surrogate marker of socio-economic status of patients. Those who were classified as unemployed or students had a relatively high infection prevalence of 96.2%. Given the frequent correlation between employment status and wealth, these data would appear to be consistent with the finding that children from low-income families have significantly higher infection rates than those from high-income families . The H. icipants . More afountries  as well ountries . This maodations .H. pylori strains and an increased risk of infection with exposure to sheep has also been reported in a number of studies [Helicobacter species also strongly produce urease, the CLO test would not be able to differentiate H. Pylori from other species such as H. Heilmannii[Helicobacter-like organisms (GHLOs), commonly noted in dogs and cats, often infect patients who own pets suggesting a zoonotic link. However, they are associated with approximately 0.08-1% of gastritis in humans [H. Pylori from material other than gastric tissue which has made identification of portals of entry and exit problematic [H. pylori in abattoir workers [H. pylori from the inflamed gastric lining of commercially reared animals [H. pylori[H. pylori transmission from infected animals who have close human contact.Farming and agriculture was associated with an infection prevalence of 91.7% suggesting possible zoonotic transmission. In Ghana, poultry, cows, sheep and goats are some of the common animals frequently reared in farming communities. Many animals such as cats, monkeys have been successfully infected with  studies , 22. Sinilmannii. The larn humans . Laboratblematic . Reports workers . These r workers . Thus, t animals , and the. pylori, continuH. pylori is endemic in Ghana but the persistently high prevalence across age groups despite significant community anti-microbial use and abuse [H. pylori in Ghana.nd abuse , suggest


Bacillus sp. strain GZT, a 2,4,6-tribromophenol (TBP)-degrading bacterium previously isolated from an electronic waste-dismantling region. The draft genome sequence is 5.18 Mb and has a G+C content of 35.1%. This is the first genome report of a brominated flame retardant-degrading strain. Here, we report the draft genome sequence of  Bacillus sp. strain GZT, capable of efficient biodegradation of TBP, was isolated from a creek near electronic waste-dismantling workshops using TBP as the sole carbon and energy source , a widely used brominated flame retardant, is released into the environment, resulting in deleterious effects on the environment and human beings, triggering effective techniques for its removal , 2. Amony source . The gendenovo version 2.04 , per the manufacturer’s instructions, and quantified by use of a NanoDrop 2000. The genome sequencing was performed using an Illumina MiSeq system . A total of 2,196,615 paired-end reads, with an average insert size of 400 bp, were generated by sequencing, which yielded approximately 208-fold depth of coverage. The reads obtained were assembled using SOAPion 2.04 . The finBacillus cereus AND1407  and B. cereus ISP3191 .The draft genome of GZT was analyzed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) . Genome B. cereus and Bacillus thuringiensis. However, which dehalogenase in GZT is directly related to the TBP degradation is still unknown and needs further identification. The genome information and annotation reported here are valuable for future research aimed at better understanding the molecular principles of TBP degradation.Six dehalogenase subunits were found in the genome of GZT, including the previously confirmed haloacid-degrading gene cluster in Bacillus sp. GZT has been deposited at DDBJ/EMBL/GenBank under the accession no. LVVJ00000000. The version described in this paper is the first version, LVVJ01000001.This draft genome sequence of


Mycoplasma mycoides subsp. mycoides is generally considered one of most pathogenic Mycoplasma species, and it is the etiological agent of contagious bovine pleuropneumonia (CBPP). Here, we present the annotated genome sequence of M. mycoides subsp. mycoides Italian strain 57/13, isolated in 1992 during CBPP outbreaks in Italy. Mycoplasma mycoides subsp. mycoides is the etiological agent of contagious bovine pleuropneumonia (CBPP) (a (CBPP) , which ia (CBPP) .M. mycoides subsp. mycoides strain 57/13 that was isolated from the lung of a cow showing CBPP pathological lesions in 1992, during a CBPP outbreak in northern Italy. The strain was identified by PCR as M. mycoides subsp. mycoides , the M. mycoides subsp. mycoides strain 57/13 showed an overall identity of 99% and one major insertion. Genome organization highlighted a high degree of overlapped genes. As with other European strains, this isolate lacks the gtsC and lppB genes (The pB genes .CP010267.The whole-genome assembly has been deposited at GenBank under accession no.


Amblyomma cajennense are responsible for severe impact on livestock and public health. In the present work, we present the isolation and molecular identification of a group of culturable bacteria associated with A. cajennense eggs from females sampled in distinct geographical sites in southeastern Brazil. Additional comparative analysis of the culturable bacteria from Anocentor nitens, Rhipicephalus sanguineus and Ixodes scapularis tick eggs were also performed. 16S rRNA gene sequence analyses identified 17 different bacterial types identified as Serratia marcescens, Stenotrophomonas maltophilia, Pseudomonas fluorescens, Enterobacter spp., Micrococcus luteus, Ochrobactrum anthropi, Bacillus cereus and Staphylococcus spp., distributed in 12 phylogroups. Staphylococcus spp., especially S. sciuri, was the most prevalent bacteria associated with A. cajennense eggs, occurring in 65% of the samples and also frequently observed infecting A. nitens eggs. S. maltophilia, S. marcescens and B. cereus occurred infecting eggs derived from specific sampling sites, but in all cases rising almost as pure cultures from infected A. cajennense eggs. The potential role of these bacterial associations is discussed and they possibly represent new targets for biological control strategies of ticks and tick borne diseases.Ticks represent a large group of pathogen vectors that blood feed on a diversity of hosts. In the Americas, the Ixodidae ticks  Amblyomma cajennense are responsible for the transmission and environmental maintenance of pathogens with public health relevance, including reports in southeastern Brazil A. cajennense can transmit several pathogens to a variety of animals and is considered the main vector of Rickettsia rickettsii, the etiological agent of Rocky Mountain Spotted Fever , Argasidae (soft ticks), and the Nuttalliellidae (restricted to southern Africa)  collected in the U.S., part of Europe and Australia . Sampled ticks were maintained in laboratory until oviposition. Field collected samples of A. nitens and R. sanguineus actively feeding on horses and dogs were also obtained for comparative analyses. I. scapularis tick samples were also analyzed and obtained from U.S. colonies maintained at the Vector-Host Laboratory  (3 in a growth chamber at 26 °C and under a photoperiod of 14 h: 10 h (light: dark) until oviposition. The obtained egg masses were washed in ethanol 70% (v/v), air-dried and homogenized in 500 μL of sterile PBS  with sterile mortar and pestle. Each egg mass, from a single female tick, was split in two to four sub-samples to generate independent egg/PBS homogenates which were individually plated for bacterial isolation.In this work, actively feeding adult females of on, CDC) . Tick saBacteria were isolated by direct streaking of 50 μL of the egg/PBS suspensions on LB (Luria-Bertani medium) agar plates, without any enrichment step to prevent competitive selection to occur. Replicas were incubated for 24 h at 30 °C and 37 °C. Bacterial colonies with the same morphology were purified in triplicates and directly inoculated into liquid LB medium for subsequent DNA extraction and storage at −80 °C. Bacterial genomic DNA extraction was routinely performed using the DNeasy Blood and Tissue Kit  procedure and eluted in 100 μL of TE buffer. DNA integrity was analyzed by agarose gel electrophoresis (data not shown).The eubacterial 16S rRNA universal primers 27f, 338f, 907r and 1492r A. cajennense from 4 sampling sites located in the states of Rio de Janeiro and Minas Gerais, Brazil, and 26 comparative samples of egg masses from A. nitens, R. sanguineus and I. scapularis species. DNA sequence BLAST analyses grouped all isolates into a total of 17 bacterial types  belonging to eight bacterial genera (Firmicutes (Bacilli) , Gamma-proteobacteria , Alpha-proteobacteria  and Actinobacteria  after 16S rRNA gene sequence analyses , which occurred in tick eggs sampled in different states of Brazil, both from Cayenne ticks sampled in the cities of Rio do Ouro (Rio de Janeiro state) and Pouso Alto (Minas Gerais state), and from A. nitens obtained in Seropedica (Rio de Janeiro state). Staphylococcus spp. were also abundantly detected in a recent metagenomic assessment of bacteria in R. microplus, but S. sciuri was only found associated to adult ticks of this species  and Stenotrophomonas maltophilia (Clade D) were the only Gamma-Proteobacteria isolated from A. cajennense egg samples and observed coinfecting the eggs from females collected in the Três Rios site. In addition, members of Stenotrophomonas sp. (Clade D) and phylogenetically close to the Cayenne tick isolates, were also observed in association with I. scapularis in the U.S.S. sciuri is in fact considered common colonizers of dogs, cats, found in the skin of cattle and other animals (S. sciuri (Clade H), S. maltophilia (Clade D), S. marcescens (Clade B), P. fluorescens (Clade C), M. luteus (Clade F), S. aureus (Clade L), B. cereus/B. thuringiensis (Clade G) S. marcensces and B. cereus are also known to combat insects and nematodes, organisms that could represent predators of tick eggs (S. sciuri and S. maltophilia are also known as multidrug resistant bacteria (A. cajennense egg samples, as a consequence of their potential resistance to competing microorganisms or to other antimicrobial components on eggs surface or in the surrounding environment (Ticks can uptake and carry bacteria from their environment or from the skin surface of hosts and some of these bacteria are able to survive and replicate in ticks . S. sciu animals . This suB. cereus/B. thuringiensis. The Clade B S. marcensces also seems to be harmful to ticks, since it was already described that the wax from the eggs of Amblyomma hebraeum has antibiotic activity for their protection specifically against this bacterium (Amblyomma variegatum also present specific substances against S. marcensces (Some bacterial species isolated are also known to be pathogenic to arthropod hosts, as the Clade G rcensces  and ableS. sciuri, are potential candidates for future paratransgenesis strategies, once they were easily isolated from tick eggs and reported infecting both nymph and adult ixodidae ticks of different species, including R. microplus, Ixodes holocyclus, I. ricinus, Dermacentor reticulatus, Haemaphysalis concina and Amblyomma fimbriatum (It is important to mention that the bacteria presented here were cultured and isolated, not only described at the DNA sequence level. These bacteria, specially


Toxin production by pathogenic microorganisms likely serves to (i) protect against phagocytosis by predatory cells, (ii) aid in penetrating tissue barriers, (iii) promote nutrient release, or (iv) alter cellular architecture and metabolism in ways that facilitate the establishment of a niche for colonization and replication. Useful enzymatic targets for some bacterial toxins may be similar between prokaryotic and eukaryotic environments, necessitating a need for eukaryotic-specific cofactors to regulate toxin activity. Host cell—derived factors may impart localization properties to an effector, induce folding events, provide a platform for the inhibition of cellular processes or support greater substrate promiscuity . The aimP. aeruginosa. Monoubiquitin, ubiquitin polymers, and ubiquitylated proteins are capable of activating ExoU [P. asymbiotica, B. thailandensis, and P. fluorescens [Many bacterial toxins contain catalytic domains with homology to plant patatins, which are lipid acyl hydrolases found in potato tubers. Cofactor activation of phospholipase activity is best characterized for ExoU, a lipid membrane—hydrolyzing protein encoded by the opportunistic pathogen ing ExoU . Bioinfoing ExoU . Functioorescens .R. typhus encodes ExoU homologs, Pat1 and Pat2 [R. prowazekii, the etiologic agent of typhus, displays enhanced enzymatic activity in the presence of bSOD1 , whiciquitin, ). Ricketar entry , phagosoar entry .L. pneumophila displays a Rab5-dependent PLA1 activity [Salmonella, is activated by GTP-RhoA [Y. enterocolitica homolog to SseJ, YspM, may have a similar function [VipD lipase from activity  that tarGTP-RhoA  likely fGTP-RhoA . The Y. function .P. aeruginosa was one of the first identified bacterial ADP-ribosyltransferases requiring a cofactor for activation [A. hydrophila [V. parahaemolyticus [Most cofactor-stimulated transferases catalyze the transfer of an ADP-ribose moiety from nicotinamide adenine dinucleotide to a target protein. Exoenzyme S from tivation . Memberstivation . Homologdrophila  and V. polyticus .P. aeruginosa, ExoT, is similar (76% identity) to ExoS and requires 14-3-3 proteins as cofactors, but has limited target recognition and is not overtly cytotoxic [A. hydrophila and A. salmonicida encode enzymes similar to ExoT/S termed AexT [Another enzyme from ytotoxic . CrkI anytotoxic . ADP-ribmed AexT .Yersinia that inhibits immune responses through acetylation of several residues in the kinases involved in the MAPK and NFκB pathways [P. syringae secretes an IP6-activated acetyltransferase, HopZ1, which interferes with plant cell cytoskeletal networks and downstream cell wall—mediated immune responses by acetylating microtubules [S. typhimurium AvrA suggests that stimulation of acetyltransferase activity in this family of enzymes likely involves allosteric conformational changes [V. cholerae RTX toxin [Clostridium difficile toxin A [Bacterial effectors with acetyltransferase activity include YopJ, a type III secreted enzyme from pathways . Acetyltpathways ). P. syrotubules . The bin changes . StructuTX toxin  and Clos toxin A  suggest V. cholerae, LT-I and LT-II from E. coli, are the most studied enzymes of this family to date; each are highly homologous in sequence and structure. Cholera toxin A1 subunit was shown to fold on host cell lipid rafts [s), locking it into the GTP-bound form. This stimulates adenylyl cyclase activity and generates supraphysiological amounts of cAMP, deregulating the chloride balance in the intestinal lumen to cause diarrhea.ADP-ribosylation factors (ARFs) have been identified as stimulators of type I and type II heat labile toxin (LT) activity from enteric bacteria. Cholera toxin from id rafts  before iid rafts . ActivatB. pertussis. These enzymes contain structurally similar components but differ in affinity and binding mechanism for their eukaryotic activator, calmodulin (CaM). Edema factor has three domains: a protective antigen-binding domain, the adenylyl cyclase core domain, and an alpha-helical domain. The helical domain interacts with the core domain to maintain an unstructured activation loop. The N-terminal domain of CaM interacts with the EF helical domain, causing a conformational switch between the helical and core domains [Cofactor-stimulated nucleotide cyclases include the adenylyl cyclases edema factor (EF) of anthrax toxin and CyaA of  domains . The C-t domains .In contrast to soluble EF, CyaA localizes to the cytosolic interface at the plasma membrane. The CaM binding domain involves completely different sequences with an affinity that is roughly 100-fold stronger as compared to EF. N- and C-terminal domains of CaM participate in binding, stabilization, and activation of CyaA. The C-terminal domain stabilizes a catalytic loop, and the N-terminal domain is postulated to bind a β-hairpin motif of CyaA to promote substrate (ATP) binding within the catalytic chamber .P. aeruginosa [Exoenzyme Y is a type III-secreted, cofactor-stimulated adenylyl cyclase from ruginosa . Functioruginosa . The cofruginosa . Sensitiruginosa ,33. The ruginosa .Yersinia species inject actin-stimulated effector kinases into host cells [st cells . YopO orst cells .Shigella species, but also present in strains of Yersinia and enterohemorrhagic E. coli, is OspG [Another kinase characterized from  is OspG –39. OspG is OspG . Ubiquit is OspG  and UbcH is OspG . At leasV. parahaemolyticus has recently been shown to secrete an IP6-activated enzyme, VPA1380, with homology to the cysteine protease domains of multifunctional-autoprocessing RTX (MARTX) domains from Vibrio and large clostridial toxins A and B [P. syringae secretes a protease, AvrRpt2, which is homologous to the staphopain cysteine proteases that are activated through interactions with cyclophilins [ A and B . The mecophilins . AvrRpt2ophilins . Analysiophilins .Numerous secreted bacterial enzymes interact with host cell factors to ensure enzymatic activation in the correct environment, and in some cases, trafficking to a specific location within the host. Common properties of the activators are that they are ubiquitously distributed throughout the eukaryotic kingdom, are present in high concentration, and play roles in cell signaling. The prevailing theme of a high cellular concentration may facilitate the required conditions for toxins to establish a cross kingdom binding partner as they evolved . In this


Xylella fastidiosa, is a widely established and problematic disease of landscape ornamentals in Washington D.C. A multi-locus sequence typing analysis was performed using 10 housekeeping loci for X. fastidiosa strains in order to better understand the epidemiology of leaf scorch disease in this municipal environment. Samples were collected from 7 different tree species located throughout the District of Columbia, consisting of 101 samples of symptomatic and asymptomatic foliage from 84 different trees. Five strains of the bacteria were identified. Consistent with prior data, these strains were host specific, with only one strain associated with members of the red oak family, one strain associated with American elm, one strain associated with American sycamore, and two strains associated with mulberry. Strains found for asymptomatic foliage were the same as strains from the symptomatic foliage on individual trees. Cross transmission of the strains was not observed at sites with multiple species of infected trees within an approx. 25 m radius of one another. X. fastidiosa strain specificity observed for each genus of tree suggests a highly specialized host-pathogen relationship.Bacterial leaf scorch, associated with the bacterial pathogen  Xylella fastidiosa [X. fastidiosa include the Pierce’s disease of grapevine [X. fastidiosa causes a chronic leaf-scorching disease often referred to as bacterial leaf scorch (BLS) [stidiosa  is a bacrapevine , citrus rapevine , almond rapevine , and phorapevine . In addirapevine –11. In uch (BLS) . Leaf scX. fastidiosa is responsible for the decline of mature oak (Quercus spp.), elm (Ulmus spp.), sycamore (Platanus spp.), maple (Acer spp.), and red mulberry (Morus rubra) [Q. rubra), pin oak , and scarlet oak (Q. coccinea) [X. fastidiosa are also prevalent in Washington D.C. (the District), where a significant association was found between crown dieback and BLS infection on several common street trees including red oak, pin oak, American sycamore  and American elm (U. americana) [X. fastidiosa. Consequently, BLS was declared an urban forest disease of concern in the 2010 Forest Action Plan for the District (http://www.forestactionplans.org/states/district-columbia).s rubra) –15. In Noccinea) . Diseaseericana) . DespiteX. fastidiosa [X. fastidiosa subsp. fastidiosa [X. fastidiosa subsp. multiplex [X. fastidiosa subsp. pauca [X. fastidiosa subsp. sandyi [X. fastidiosa subsp. tashke [X. fastidiosa subsp. morus [Since the initial description of stidiosa , four sustidiosa , ii) X. ultiplex , iii) X.p. pauca , iv) X. . sandyi , while v. tashke  and iv) p. morus  have beep. morus , while tp. morus ,20. Diffp. morus ,19,21,22p. morus .X. fastidiosa. For example, X. fastidiosa subsp. fastidiosa is known for causing Pierce’s disease (PD) of grapevine and subsp. multiplex is known for causing almond leaf scorch. Both subspecies are capable of inducing almond leaf scorch [multiplex does not induce disease of grapevine [fastidiosa clade and the subsp. multiplex clade, all PD strains were confined to the subsp. fastidiosa clade [X. fastidiosa strains from citrus plants were not capable of sustainable colonization of coffee plants and strains isolated from coffee plants failed to develop symptoms in citrus [fastidiosa and subspecies sandyi [In several agricultural systems, the dynamics of host specificity for particular subspecific clonal complexes was demonstrated with multi-locus sequence typing (MLST), a method widely used for genetic typing and detecting recombination of f scorch , althougrapevine ,25. Thissa clade . Using in citrus . The strn citrus –30. Simis sandyi , two subs sandyi .X. fastidiosa responsible for bacterial leaf scorch in the District of Columbia [X. fastidiosa subsp. multiplex was found in association with decline of oak, sycamore, and elm. Moreover, white mulberry was found associated with subsp. sandyi and a subsp. multiplex strain different from the subsp. multiplex strain found on oak, sycamore, and elm. Some of this information agrees with previous findings for oak and sycamore [X. fastidiosa subsp. morus [multiplex [multiplex [X. fastidiosa at the subspecies level [Our previous survey identified several subspecies of Columbia . X. fastsycamore ,23. Howep. morus , and thaultiplex , or at les level . FurtherX. fastidiosa in urban environments, a MLST scheme for X. fastidiosa was used to genetically characterize X. fastidiosa among urban trees in the District. Our objective was to determine the strain diversity of X. fastidiosa infecting various tree species and elucidate any major clonal complexes that demonstrate host specificity.In order to better understand the epidemiology of leaf scorch disease associated with X. fastidiosa were selected throughout the District (X. fastidiosa based on the results of a survey that used an enzyme-linked immunosorbent assay (ELISA) and PCR [Quercus rubra), 29 pin oaks , 2 scarlet oaks (Quercus coccinea), 1 willow oak (Quercus phellos), 17 American elms (Ulmus americana), 11 American sycamores , and 4 white mulberries   . Of the X. fastidiosa in the District , KA representing synonymous mutations and KS representing non-synonymous mutations [Total DNA was extracted from petiole samples collected from infected trees as previously described . Ten houDistrict . These putations ,26,32.X. fastidiosa , 1.5 μl (10 μM solution) of each primer set designed for ten housekeeping loci of stidiosa , and 7.5fastidiosa) and ALS (subsp. multiplex) are mixed together, the strain added at a higher concentration is identified during a quantitative PCR melt analysis, and that dual peaks do not occur [Once there was visual confirmation of the target length gene product, the PCR products were cleaned using the EXOSAP-IT PCR purification kit . When necessary, gel extractions of target length gene product were performed using the gel extraction kit Nucleospin . The cleaned PCR products were sequenced with an ABI 3730XL sequencer  by MC Lab . Caution should be taken when collecting sequence data from DNA that is directly extracted from infected plant tissue without the isolation of the query organism. Non-specific amplification was an issue for several American elm DNA extracts and gel extraction of the target gene product was necessary. Furthermore, when using a Sanger based approach for sequencing (such as the one used for this study); the dominant strain may mask multiple co-occurring strains in an individual sample during base calling. It has been shown that when samples with DNA from PD , and compared to the pubmlst.org sequence types, denoted as (ST). A distance tree was created with all ten loci using the Geneious tree building option in Geneious [10 were run with 5 reference sequences in order to determine relatedness of strains from the District with strains representative of pre-defined subspecies. The reference strains included in the analysis were; Temecula 1 [fastidiosa; M12 [multiplex; MUL0034 [morus, and 9a5c [pauca which was used as an outgroup.All ten loci were concatenated for each bacterial sequence type in this analysis, denoted as , associated with members of the red oak family; red oak, pin oak, scarlet oak, and willow oak; ST10- 2 (ST-8) associated with American sycamore, ST10-3 (ST-41) associated with American elm, and ST10-4 and ST10-5 (both a derivative of ST-29) associated with mulberry Click here for additional data file.S1 Table* denotes that at least two trees of the same species were sampled at the site. ** denotes that an infected tree of a different genus was within an approx. 25 m radius.(PDF)Click here for additional data file.S2 Table* denotes a newly designed primer for this analysis.(PDF)Click here for additional data file.S3 Table* denotes an outlying allelic profile from the consensus host specific sequence types.  Parenthesis indicate the allele numbers found on pubmlst.org after resequencing with additional primers.(PDF)Click here for additional data file.S4 Tablemultiplex) and Temecula1 (subsp. fastidiosa) were also included for comparison.Reference strains M12 (subsp. (PDF)Click here for additional data file.S5 Table(XLS)Click here for additional data file.