Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors.in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis  and Ph. papatasi . LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental  The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain  exhibited lower infection intensity compared to BH49 and FV1 in both vectors.L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host. Intraspecific variation in the LPG of  Leishmania major is one of the most important species causing cutaneous leishmaniasis (CL) in the Old World. It is widely distributed from Sub-Saharan Africa to the Indian subcontinent South-Central Asia. This species is transmitted by the bite of the sand flies, Phlebotomus papatasi and Phlebotomus duboscqi, depending on the region (L. major and were named L. major-like (e region . In sevejor-like .L. major-like strains were named BH49 (Goiás) and BH121 (Minas Gerais). By the time they were isolated, specific epidemiological studies were not available to incriminate the vector(s) and wild host(s); however, a lot of information was available on regarding the vectors and hosts of Leishmania braziliensis  and 5-ASKH by isoenzyme, RAPD and SSR-PCR analysis . The L. iliensis . Althougiliensis . To asceiliensis . The latL. major . These r PpGalec . HoweverL. major, their glycobiology and infectivity to New and Old World vectors remain unknown. Here, we qualitatively evaluated the presence of galactose/arabinose in the LPGs of L. major-like strains (BH49 and BH121). We additionally evaluated their interactions with vectors Lutzomyia longipalpis (permissive) and Ph. papatasi (restrictive).Although these strains were genetically similar to Old World Parasites and LPG purification - World Health Reference strains of L. major (MHOM/IL/1980/Friedlin), Leishmania infantum (MCAN/BR/89/Ba-262), and L. major-like strains BH49 (MHOM/BR/1971/BH49) and BH121 (MHOM/BR/1971/BH121) were used. L. major-like strains were isolated from human CL cases in Brazil: BH49 in the State of Goiás and BH121 in the State of Minas Gerais. Frozen stocks of strains passaged in mice were characterised and typed, as previously reported  were extracted in solvent E , as previously described , equilibrated with the same buffer. LPG was eluted in solvent E, dried, and resuspended in sterile, LPS-free distilled water .reported . Promastescribed . The solImmunoblotting - Purified LPGs (10 μg) from different strains were transferred to nitrocellulose paper; the membrane was blocked in 5% milk in PBS and probed with the following antibodies : WIC 79.3, which recognises terminal Gal sequences that branch off the repeat units, and 3F12, which recognises terminal AraGal  , 1994. Fat units . After tat units .Sand fly colonies, experimental infections, and morphometry - Colonies of Lu. longipalpis  and Ph. papatasi  were maintained in the insectary at the Department of Parasitology, Charles University in Prague under standard conditions (6 promastigotes/mL. Reference strain of L. major (MHOM/IL/1980/Friedlin) was used as the control since it is the most used model for sand fly infections. Sand-flies were infected with the strains in two parallel sets of experiments using all sand-fly Leishmania combinations. Engorged females were separated, maintained in the same conditions as the colony, and dissected on days 1, 5, and 8 post-infection (PI). Dissected guts were divided into several parts: the abdominal midgut (AMG), thoracic midgut (TMG), stomodeal valve (SV), cardia, and endoperithrophic space. They were examined under a microscope to determine the localisation and intensity of Leishmania infections. Parasite loads were graded as light (< 100 parasites/gut), moderate (100-1000 parasites/gut), or heavy (> 1000 parasites/gut), as described by Leishmania, midgut smears fixed with methanol and stained with Giemsa were examined under a light microscope with an oil-immersion objective and photographed with an Olympus D70 camera. Body length, body width, and flagellar length of 120 randomly selected promastigotes from three females/smears were measured for each sand fly species and time interval using Image-J software. The morphological forms were distinguished, according to the criteria described by 2) tests using SPSS statistics 23 software.nditions . Sand flLPGs from L. major-like strains display intraspecific polymorphisms - For preliminary qualitative analysis, immunoblotting (western-blot and dot-blot) techniques were performed using standard mAbs specific for the repeat unit epitopes of Leishmania spp. By western-blot, LPGs purified from L. major-like strains were differentially recognised by antibody WIC 79.3, specific for terminal β-galactosyl residues. LPGs from FV1 and BH49 strains were strongly recognised by this antibody. However, the LPG from BH121 reacted very poorly with the antibody  reacted with 3F12. Since the LPGs from L. major-like strains exhibited different levels of glycosylation in their side-chains, they were subjected to a dot-blot assay using CA7AE that recognizes unbranched repeat units. Consistent with these observations, LPGs from L. major-like strains and the positive L. infantum control (Ba262) were recognised by this antibody. Moreover, a very clear cline was observed where the lower galactosylated LPGs were more reactive for CA7AE. The pattern could be seen more clearly in the dot-blot compared to western-blot. As expected, the LPG from L. major (FV1) reacted poorly with this antibody  and Ph. papatasi (PP) sand flies (Ph. papatasi (p < 0.05) (Ph. papatasi (p < 0.05) (nd flies . The int < 0.05) . Interes < 0.05) . Similar < 0.05) . Colonis < 0.05) . All exp < 0.05) . For exa < 0.05) . Additio < 0.05) . Similar < 0.05) .L. major-like strains have been isolated in several countries in Latin America, despite the fact that this species in commonly found in Old World regions. Several aspects of their epidemiology regarding vectors and reservoirs are scarce. Only two studies have addressed some biological, biochemical, and molecular parameters using L. major-like strains  has β-1,3 galactosyl side-chains (Ph. papatasi (L. major (FV1), BH49 exhibited β-galactosyl residues in their LPGs. An interesting feature of the BH121 strain was its absent or low galactosylation level, based on WIC 79.3 recognition. Although both strains did not show arabinosyl residues in their LPGs, these sugars are not important for attachment to the sand fly midgut and may be absent in other L. major strains, such as LV39  (L. major (Ph. papatasi). In permissive vectors such as Lu. longipalpis and Lu. migonei, intraspecies variations in the LPGs of L. infantum and L. amazonensis did not affect interaction (Ph. papatasi). This vector is only susceptible to Old World L. major and L. turanica (L. major-like strains bearing variations in their LPG galactosylation levels differently infected both vectors. Previous reports have shown that Lu. longipalpis is a permissive vector able to sustain infection with different Leishmania species including L. major (reviewed by Lu. longipalpis and Ph. papatasi. Although both strains were able to infect both vectors and accomplish metacyclogenesis, the intensity of this infection was significantly lower in the BH121 strain. This is the first evidence that Leishmania strains bearing intraspecies polymorphisms in their LPG exhibited variations during the interaction with restrictive and permissive sand-flies. One of the hypotheses is that the presence of a smaller number of galactose residues in the LPG could adversely affect parasite attachment to the midgut. Since both L. major-like strains were able to survive and sustain infection in Ph. papatasi, a restrictive vector, this is a strong indication that galactosylation may be one of the factors involved. Besides, since Ph. papatasi is not found in Brazil, those strains may have used an alternative vector in the New World. Although Lu. longipalpis has urbanized in many cities in Latin America (revised by L. major-like strains. Although Lu. longipalpis is a proven vector for L. infantum, the transmission of L. major-like strains by this vector and other vectors has not been observed yet. More importantly, other CL vectors in those geographical regions such as Lu. whitmani, Lu. migonei and Lu. intermedia should also be investigated. This work highlights the importance of returning to the field to address these questions.In order to determine whether such variations could have an impact during development within the invertebrate host, sand flies were infected with the strains om mouse , it is aeraction , 2017. WL. major-like strains were able to successfully infect Lu. longipalpis and Ph. papatasi. The observed differences, among other factors, seemed to be controlled by variations in the galactose residues in the LPG. However, there are still many uncertainties regarding the origin and introduction of L. major-like strains in the Americas. Like L. infantum, this could have occurred after the introduction of infected dogs (reviewed by L. major-like strains in the Americas remain unknown. After more than 40 years, the regions from where these strains were isolated were modified by anthropic action, and the eco epidemiology of leishmaniasis may have changed completely. This reinforces the need for novel epidemiological studies in those areas; we must also search for new L. major-like strains, vectors, and hosts.In conclusion,


This case report describes a 75-year-old male patient presenting with persistent cough.His chest X-ray showed a right-sided paratracheal opacification. A thoracic CT  was subsThe lusorian artery is also known as an aberrant right subclavian artery. It is ananatomical variant in which the brachiocephalic artery is absent. Thus, from the aorticarch there are 4 originating arteries: the right and left common carotid arteries andthe right and left subclavian arteries. The lusorian artery prevalence varies from 0.16to 0.8%, depending on the country. The anomaly is clinically asymptomatic in more than90% of the cases. Most symptoms arise in individuals at advanced ages, probably relatedto atherosclerotic phenomena. The most frequent symptoms are dysphagia, dyspnea,retro-sternal pain, coughing and weight loss. The lusorian artery and the right-sidedaortic arch coexist in 9.2% of the cases.


Staphylococcus pseudintermedius is the most prevalent inhabitant of the skin and mucosa of dogs and cats, and also the major bacterial pathogen causing canine skin and ear infections [S. intermedius and S. delphini [S. pseudintermedius can be isolated from the nares, mouth, anus, groin and forehead of healthy dogs and cats, as well as from dogs and cats with inflammatory skin disease [S. pseudintermedius (MRSP) [fections . It was fections , then indelphini . S. pseu disease ,5. In res (MRSP) ,7, especs (MRSP) . Infectis (MRSP) ,12. Furts (MRSP)  and subss (MRSP) , showingS. pseudintermedius is due to several potential virulence factors, such as cell wall-anchored proteins (CWA), exoenzymes and exotoxins [S. aureus than in S. pseudintermedius, although it has been shown that most of them have similar features [Staphylococcal Protein A (SpA), a 40–60 kDa protein belonging to the most prevalent group of CWA proteins, i.e. microbial surface component recognizing adhesive matrix molecules (MSCRAMM). In S. aureus, SpA comprises five repeated domains , each of them binding with high affinity the Fc region of immunoglobulin (Ig) G and the Fab region of Ig of the variable heavy VH3 subclass. Protein A is expressed by virtually all clinical S. aureus isolates [SpA binding to Fab and cross-linking of IgM promotes B cell superantigen activity [SpA cross-linking of B cell receptors triggers proliferation and apoptotic collapse of the expanded lymphocyte populations [SpAKKAA) enabled infected Guinea pigs to elicit a protective antibody response against S.aureus infection [The successful fitness of xotoxins ; these vfeatures . This isisolates  and it iisolates . Proteinisolates , whereasactivity . Notablyulations . Along wulations , recentlnfection .S. aureus and S. pseudintermedius protein A in order to understand if S. pseudintermedius protein A could be a candidate for vaccine development. To date, little is known about S. pseudintermedius SpA and the above authors have filled a gap. In fact, a putative spa gene was initially described by Moodley et al. in 2009 [S. pseudintermedius ED99 [S. pseudintermedius surface (sps) genes (from spsA to spsR), two genes had an orthologous conformation with S. aureus spa encoding protein A, namely spsP and spsQ [spsP gene encodes a protein that consists of 377 amino acids; it has a predicted N-terminal sequence of 33 amino acids, followed by a repeat region consisting of three IgG-binding domains. The C-terminal region has a predicted X-region which shares 63% sequence similarity to the X-region of S. aureus. On the other hand, the spsQ gene encodes a protein that consists of 462 amino acids, with a predicted terminal sequence of 33 amino acids, followed by a repeat region consisting of four IgG-binding domains. The C-terminal region has a predicted X-region which includes a 77 amino acid-long repeat sequence (Xr) and a constant region (Xc) with 70% similarity to the X-region of S. aureus [spsQ gene was always present in clinical isolates of S. pseudintermedius, while spsP gene was harbored less frequently [S. pseudintermedius isolates associated with different STs and demonstrated that spsQ gene was present in all the isolates, as already observed [In light of these considerations, the study conducted by Balachandran et al . has inv in 2009 ; subsequius ED99  revealedand spsQ . The sps. aureus . Riley eequently . In thisobserved , but itsS. pseudintermedius virulence are scarce and continuous efforts are necessary to better understand S. pseudintermedius pathogenicity. During the last decade, the relationship between dogs and their owners has changed dramatically. As a result, transmission of microorganisms between humans and their dogs is increasing, as observed for the MDR ST71, which has been isolated at high frequency both in animals and humans [S. pseudintermedius resides on the skin or mucosal surfaces of dogs and might easily be transmitted to pet owners either by direct contact or by sharing the same environment in the household [These results represent additional information on the worldwide success of these two lineages that are nowadays a considerable challenge for the treatment of MRSP infections. In general, data about d humans ,14. S. pousehold ,30. The ousehold .S. pseudintermedius and well speculated in S. aureus were addressed in this study. In fact, the authors demonstrated that S. pseudintermedius protein A is a CWA protein able to bind canine IgG primarily via its Fc region, it is recognized by the anti-protein A raised against S. aureus protein A, can be secreted during log phase, and its block makes S. pseudintermedius more susceptible to phagocytosis [S. pseudintermedius SpA is a virulence factor that allows the bacterium to evade the host immune response; the primarily interaction with the Fc fraction of IgG hinders phagocytosis, because bacteria coated with IgG in an inappropriate conformation which becomes not recognizable by the Fc receptors on polymorphonuclear cells [S. pseudintermedius isolates expressing protein A are able to escape from opsonization and phagocytosis, leading to persistent infections. These results were also confirmed by the phagocytosis assays performed by the authors.Other aspects of protein A not yet clarified in ocytosis . S. pseuar cells . This meSpA in S. aureus. Infections caused by S. aureus do not generate protective immunity in either humans and or animals [SpA [SpAKKAA, in which 20 amino acid residues essential for its association with Ig Fc and Fab were replaced, elicited antibodies that neutralize SpA [SpAKKAA – derived polyclonal antibodies promoted OPK of staphylococci, suppressed staphylococcal B cell superantigen activity and promoted humoral immune responses against a wide spectrum of antigens [SpAKKAA monoclonal antibodies (MAb). SpAKKAA MAb-mediated neutralization of SpA, promoted OPK of S. aureus and the development of antibody responses against many different antigens [S. aureus lacking protein A (Δspa), or expressing the nontoxigenic variant, displayed defects in abscess formation and could not suppress the adaptive immune responses of infected animals [On the other hands, several progresses have been made on immunological aspects of  animals . Vaccineals [SpA . To datelize SpA . The SpAantigens . Moreoveantigens . It has antigens . Recentl animals .S. pseudintermedius infections; moreover it is still necessary to determine if there are toxic effects of S. pseudintermedius protein A on B cells. The anti-protein A antibody used in this study was raised against S. aureus protein A [S. pseudintermedius should include antigenic targets rapresentative of a wide variety of strains and STs.These preliminary results underline the importance of the study of Balachandran et al . which protein A , suggest


Burkholderia sp. strain BE12, isolated from a French agricultural soil, possesses antifungal activity against a set of phytopathogenic fungi and has friendly interactions with grapevine. Here, we present the draft genome sequence of BE12, along with genes related to plant growth-promoting traits and siderophores that this strain contains, supporting its plant growth and antifungal activities. Burkholderia comprises more than 90 species that are distributed in a wide range of environments, including water, soil, plants, animals, and humans  traits, including siderophore production, phosphate solubilization, and the production of phytohormones. An in vivo assay showed that Burkholderia sp. BE12 caused a significant increase in growth parameters of grapevine (Vitis vinifera L.) and was able to reduce the development of gray mold, caused by B. cinerea, on grapevine plantlets (unpublished data).This strain also has been screened and characterized, http://www.microbesng.uk) using Illumina MiSeq and HiSeq 2500 technology platforms, with 2 to 250-bp paired-end reads. The closest existing reference genome was determined using Kraken (http://bio-bwa.sourceforge.net) to assess data quality. The reads were assembled by de novo assembly using SPAdes (http://cab.spbu.ru/software/spades/). The draft genome of 7,472,757 bp includes 97 contigs with a GC content of 66.94% and an N50 contig size of 180,386 bp. Gene function prediction was performed by the Rapid Annotations using Subsystems Technology (RAST) server (http://rast.nmpdr.org)  and sequenced at MicrobesNG   followedpdr.org) , resultiIn silico analysis using antiSMASH  deaminase, which potentially plays a role in promoting plant growth (Burkholderia sp. BE12 to successfully promote plant growth and protect plants from disease.ntiSMASH  and RASTntiSMASH . FurtherntiSMASH . The annt growth . In addiPREY00000000. The version described in this paper is the first version, PREY01000000.This whole-genome shotgun project has been deposited in GenBank under the accession no.


AbstractIssoca  is described. Issocaassanga sp. n. presents the diagnostic features of the genus, with the exception of the relative position of the subneural parenchymal muscle layer with the cephalic retractor muscle, which are overlapped in the type species of the genus but are intersected in the new species. Rather than a polymorphic character, the relative position of these muscle layers might reflect the polyphyletic status of the genus.A new species of the genus  Geoplaninae . The number of known species of this group exceeds 290 species . The genus Issoca Froehlich, 1955 (Geoplaninae) comprises five species, namely I.spatulata , I.rezendei , I.jandaia Froehlich, 1955, I.piranga Froehlich, 1955, and I.potyra Froehlich, 1958. The most notable morphological feature of Issoca is a cephalic glandulo-muscular organ. This organ consists of a cephalic retractor muscle derived from the ventral longitudinal cutaneous musculature, and a set of adhesive glands piercing the ventral surface of the cephalic region , the cephalic retractor muscle in Issoca is typically circular in cross-section and is traversed by muscle fibers of the parenchymal subneural musculature . Yet the internal organs of I.spatulata remain unstudied.Most Neotropical land planarians are grouped in the subfamily c region . In contculature . The retIssoca was briefly discussed by Geoplaninae. In that work, the genera Issoca (represented by three species), Luteostriata Carbayo, 2010 , and Supramontana Carbayo & Leal-Zanchet, 2003 (1 species), constituted the so-called clade LIS . Figures of sagittal and horizontal views are orientated with anterior to the left. The type material is deposited at the Museu de Zoologia da Universidade de São Paulo (MZUSP).The specimens were collected in the Atlantic Forest in the States of Espírito Santo and Rio de Janeiro, Brazil. We conducted searches on soil litter and trails during the day and night, with the light of a flashlight. Geographic coordinates of collecting sites were recorded either with Garmin eTrex GPS device (Espírito Santo) or with Googlemaps (Rio de Janeiro). Specimens were photographed, then killed with boiling water and subsequently fixed in 10% formalin. In the laboratory, the specimens were cut into pieces, dehydrated in a graded ethanol series, cleared in clove oil, then infiltrated and embedded in Paraplast® Tissue Embedding Medium. Tissue blocks were sectioned at 7 μm intervals using a microtome and affixed the sections with albumin-glycerol (1:1) onto glass slides placed on a hot plate, and stained them according to af annular-shaped fold of male atriumcb cerebral ganglionco common glandular ovovitelline ductdc parenchymal layer of deccussate fibresdi diagonal parenchymal muscledv dorso-ventral parenchymal musclee eyeej ejaculatory ductep esophagusf foldfa female atriumfd female genital canalg gonoporegl glandsgs ventral glandular surface of the cephalic regioni intestineln normal longitudinal cutaneous musclesm musclema male genital atriummc common muscle coatmo moutho ovaryov ovovitelline ductpb penis bulbpo pharyngeal pouchpp penis papillapv prostatic vesicler cephalic retractor musclesb subintestinal transverse musclessc subcutaneous nerve netsd sperm ductse necks of secretory cellssg shell glandssk sunken longitudinal cutaneous musclessn subneural transverse musclessp supraintestinal transverse musclest testisvi vitellariavn ventral nerve plateTaxon classificationAnimaliaORDOFAMILIAsp. n.http://zoobank.org/FD7F2CF1-B799-4215-B75C-80E16FA90B1DIssoca sp. 1; Type material. Parque Estadual do Desengano , Santa Maria Madalena, Rio de Janeiro State, Brazil: Holotype F4085 (MZUSP PL. 1085): J. Pedroni et al. col., 13 August 2009: sagittal sections of copulatory apparatus on 28 slides. Paratype F4057 (MZUSP PL. 1082): J. Pedroni et al. col., 12 August 2009: sagittal sections of copulatory apparatus on 29 slides.Reserva Biológica Augusto Ruschi , Santa Teresa, Espírito Santo State, Brazil: Paratype F2158 (MZUSP PL 1020): F. Carbayo et al. col., 26 March 2008: fixed in 80% ethanol. Paratype F2250 (MZUSP PL. 1023): F. Carbayo et al. col., 24 May 2008: fixed in 80% ethanol. Paratype F2266 (MZUSP PL. 1024): F. Carbayo et al. col., 24 May 2008: fixed in 80% ethanol. Paratype F2274 (MZUSP PL. 1025): F. Carbayo et al. col., 24 May 2008: sagittal sections of copulatory apparatus on 33 slides; transverse sections of cephalic region on 7 slides; horizontal sections of portion containing ovaries on 22 slides; sagittal sections of pharynx region on 34 slides; transverse sections of pre-pharyngeal region on 8 slides. Paratype F2309 (MZUSP PL. 1032): F. Carbayo et al. col., 24 May 2008: fixed in 80% ethanol. Paratype F2394 (MZUSP PL. 1037): F. Carbayo et al. col., 27 May 2008: sagittal sections of copulatory apparatus on 18 slides; sagittal sections of pharynx region on 20 slides; transverse sections of cephalic region on 4 slides; horizontal sections of portion containing ovaries on 8 slides; and transverse sections of pre-pharyngeal region on 6 slides. Paratype F2470 (MZUSP PL. 1042): F. Carbayo et al. col., 29 May 2008: transverse sections of cephalic region on 14 slides; horizontal sections of portion containing ovaries on 48 slides; sagittal sections of a portion posterior to ovaries on 23 slides; sections immediately before pre-pharyngeal region on 33 slides; transverse sections of pre-pharyngeal region on 16 slides; sagittal sections of pharynx region on 41 slides; sagittal sections of PageBreakcopulatory apparatus on 61 slides. Paratype F2507 (MZUSP PL. 1045): F. Carbayo et al. col., 29 May 2008: preserved in 80% ethanol.Parque Estadual do Desengano, Santa Maria Madalena, Rio de Janeiro State, Brazil.Issoca up to 97 mm in length. Widest dorsal pigment bands with 33–40% of body width. A few cutaneous muscle bundles in-sunk in the pre-pharyngeal region. Cephalic retractor muscle and the sub-neural parenchymal muscle not intersecting with each other. Copulatory apparatus relatively long. Prostatic vesicle extrabulbar, proximally dilated, and non-anastomosed. Ejaculatory duct thin, with its opening at the tip of the penis papilla; this papilla is conical with dorsal insertion posterior to the ventral one. Female atrium spacious with some lateral folds. Common glandular duct almost as long as the female atrium. Common muscle coat envelops male and female atria.Species of The body is elongated with nearly parallel margins Fig. . Its cepThe dorsal color consists of a cream-colored , a transverse supraintestinal layer , and a transverse subintestinal layer . Additionally, there is a subneural layer  of transverse muscles. Dorso-ventrally oriented parenchymal fibers are abundant in the pre-pharyngeal region.The three usual parenchymal muscle layers are present throughout the body: a dorsal layer of diagonal decussate fibers  encircles the male atrium.PageBreakits lumen. One of these folds is continuous with a fold coming from the male atrium , both partially intermingled. The female atrium receives gland cells producing erythrophilic granules. The male atrium is 1.2 times longer than the female. The common muscle coat is well-developed, and wraps the male and female atria.The female atrium is lined with a columnar epithelium, which is lacunar in aspect in some parts. In the anterior portion of the female atrium, the simple columnar epithelium is 25 μm high; whereas the posterior portion is lined by a pseudostratified columnar epithelium Fig. . The musassanga, meaning dense, thick (The specific epithet refers to the Tupi (indigenous Brazilian tribe) name e, thick . It refeParque Estadual do Desengano  and Reserva Biológica Augusto Ruschi , Brazil.I.assanga allows one to readily distinguish it from the remaining species of the genus by being considerably larger (97 mm in length after fixation) than the largest previously described species, i.e. I.jandaia, which is 50 mm in length alive, and 32 mm after fixation. Although the chromatic pattern of all species of the genus is striped, the stripes are as wide as 33–40% of the body width only in I.assanga sp. n. The widest colored stripe in other species of the genus is found in I.potyra, with a pair or paramedian bands with 17% of the body width each  or >30% , the copulatory apparatus in I.rezendei is relatively compact, the prostatic vesicle is intrabulbar, the penis papilla is vertical, and the female atrium is absent , differ from the new species in the following details. In contrast to I.assanga sp. n., the ejaculatory duct of I.jandaia opens into a cavity inside a penis papilla-like fold of the male atrium, the common glandular duct is relatively long, and the female atrium is intensely folded. The penis papilla of Issocapiranga occupies only the anterior half of the male atrium, and the muscle coat enveloping the male atrium is separated from that wrapping the female one  the polyphyly of Issoca has been highlighted  in I.potyra “is similar to that of the other species of the genus”. However, his diagrammatic reconstruction of the organ shows the cephalic retractor muscle underneath the subneural muscle layer, not intersected. Moreover, the retractor in I.rezendei diminishes by means of separating its fibers towards the body margins and the back see Fig. . Descripdaia see . Froehlithe back , whereas


Candida albicans, a ubiquitous polymorphic fungus that is part of the normal microbiota of the gastrointestinal and reproductive tracts of healthy individuals. In order to persistently colonize the oropharynx, C. albicans must adhere to the epithelial cell lining of the oral mucosa while avoiding being killed by host antimicrobial factors. OPC develops when local host defenses are weakened, permitting the fungus to invade and damage oral epithelial cells. The epithelial cells respond to fungal infection by secreting antimicrobial peptides that directly kill the fungus and by releasing pro-inflammatory cytokines that recruit neutrophils to the focus of infection, where they can kill C. albicans and limit the extent of epithelial cell damage [Oropharyngeal candidiasis (OPC) occurs in a diverse group of patients. Risk factors for OPC include the use of dentures, corticosteroid inhalers, cigarettes, broad-spectrum antibiotics, and immunosuppressive and chemotherapeutic agents. Patients with HIV, diabetes, and iatrogenic or autoimmune-induced dry mouth are also at substantial risk for OPC. This infection is caused primarily by l damage ,2. In thC. albicans can invade into oral epithelial cells by two distinct mechanisms, induced endocytosis and active penetration  and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9) pathways. Activation of these signaling pathways requires C. albicans hyphal formation and expression of the Als3 invasin [Although OPC is a superficial fungal infection, it is characterized by invasion of the epithelial cell lining of the oropharynx . C. albietration . During the Als3  and Ssa1the Als3  invasinsthe Als3  Fig 1)..C. albicthe Als3 . Additio invasin . The relC. albicans hyphae physically push their way into the epithelial cell  family. These proteases degrade E-cadherin and other inter-epithelial cell junctional proteins, enabling the organism to penetrate between epithelial cells [C. albicans infection also stimulates the activity of epithelial cell calpain, a cysteine protease that degrades E-cadherin. Moreover, calpain activity is dramatically enhanced in epithelial cells that are co-infected with C. albicans and Streptococcus oralis [al cells  Fig 1)..C. albics oralis .C. albicans invades epithelial cells, it damages them. Epithelial cells in turn respond to C. albicans by secreting proinflammatory cytokines and antimicrobial peptides. Recently, it was discovered that C. albicans-induced epithelial cell damage is mainly caused by candidalysin, a toxin released by the fungus  that is activated by yeast-phase C. albicans is currently unknown.Although the presence of candidalysin is required to induce a maximal epithelial cell response to  minutes . In addi minutes . Finally minutes . These dC. albicans. Different HDPs have different mechanisms of action, and their targets include the fungal cell membrane and mitochondria [C. albicans, clear the infection within 2–3 days. Mice that are deficient in either IL-17 receptor A (IL-17RA) or IL-17RC fail to up-regulate HDPs in the oral epithelium in response to C. albicans infection and are highly susceptible to OPC [Defb3 (encoding β-defensin 3) in the oral mucosa and increased susceptibility to OPC. This increased susceptibility is phenocopied by deletion of Defb3 [C. albicans, especially the production of HDPs, contributes to the host defense against OPC.Host defense peptides (HDPs) represent one of the first lines of defense against invading microbes. Epithelial cells release multiple types of HDPs, including β-defensins and cathelicidins, that either kill or inhibit the growth of chondria . HDPs thchondria . The impchondria . By conte to OPC . Recentlof Defb3 . CollectC. albicans colonizes the oral mucosa of healthy individuals and causes OPC in patients, it must be able to withstand HDPs. The fungus has evolved multiple different mechanisms that enable it to evade the deleterious effects of HDPs and persist in the oral cavity [C. albicans also expresses the Flu1 efflux pump that reduces intracellular HDP levels [When l cavity . These ml cavity ,20,21  pathway enables C. albicans to resist histatin 5 and human β-defensins 2 and 3 [Exposure to HDPs induces a stress response in  2 and 3 ,24. The  2 and 3 ,26. Conv 2 and 3 .C. albicans is frequently isolated from the oral cavity in conjunction with other microbial pathogens. In a study of AIDS patients with OPC [Candida. Among the subjects with multispecies OPC, C. albicans plus C. glabrata (68.2%) was the most common co-infection, followed by C. albicans plus C. tropicalis (15.0%) and C. albicans plus C. glabrata plus C. tropicalis (8.4%). These epidemiological data suggest that C. glabrata has unique characteristics that facilitate co-infection with C. albicans.with OPC , 107 outC. glabrata is unable to invade reconstituted human oral epithelium in vitro and causes minimal epithelial cell damage. A potential explanation for this finding is that C. glabrata grows only as yeast in vivo and does not form hyphae at foci of infection. However, when oral epithelial cells are infected with a mixture of C. glabrata and C. albicans, the C. glabrata cells bind to C. albicans hyphae and are carried deeper into the epithelium, where they cause extensive epithelial cell damage (C. glabrata alone is not able to cause disease in a mouse model of OPC. However, when mice are inoculated with a mixture of C. albicans and C. glabrata, they develop more severe disease than mice infected with C. albicans alone [C. albicans hyphae. Multiple C. glabrata adhesins, including Epa8, Epa19, Awp2, Awp7, and CAGL0F00181, mediate adherence to C. albicans hyphae; whether they interact specifically with C. albicans Als1 and/or Als3 remains to be determined [C. glabrata and C. albicans in patients with OPC. Although C. tropicalis can occasionally cause OPC in conjunction with C. albicans, the mechanistic basis for this co-infection is currently unknown.Silva et al.  reportedl damage . It was ns alone . This sytermined . CollectC. albicans can also augment infection with S. aureus. When mice are immunosuppressed with cortisone acetate and then orally inoculated with S. aureus, the bacteria persist in the oral cavity in relatively low numbers and do not cause detectable disease [C. albicans are orally inoculated with S. aureus, the bacteria adhere to the fungal hyphae, which transport the bacteria along with them as they invade into the superficial oral epithelium. Not only does the presence of C. albicans result in higher levels of S. aureus in the oral cavity but it also enables S. aureus to invade the oral mucosa and cause a fatal hematogenously disseminated infection. This pathologic interaction between S. aureus and C. albicans is dependent on Als3; even though an als3Δ/Δ null mutant is still able to cause OPC, S. aureus co-infection does not result in a disseminated staphylococcal infection [S. aureus with C. albicans at other anatomic sites, such as the gastrointestinal tract or peritoneal cavity [S. aureus bacteremia.Co-infection with  disease . Howevernfection . Althougl cavity , might fC. albicans is also known to interact with common oral commensal bacteria. In vitro studies demonstrate that Streptococcus gordonii and C. albicans co-adhere during mixed-species biofilm formation [S. gordonii cell wall-anchored proteins SspA and SspB and the C. albicans surface proteins Als3, Eap1, and Hwp1. The presence of S. gordonii enhances C. albicans hyphal development and increases biofilm mass [C. albicans virulence during OPC is currently unknown. Co-infection with Streptococcus mutans also increases C. albicans biofilm formation in vitro. However, Galleria mellonella larvae infected with both organisms have prolonged survival compared to larvae infected with C. albicans alone. This reduction in virulence is likely due to inhibition of fungal hyphal formation by the bacteria [Streptococcus oralis both augments C. albicans biofilm formation in vitro and increases virulence during experimental OPC in mice, as manifested by increased size of the oral lesions and greater fungal dissemination to distal organs [C. albicans with the oral microbiota has the potential to significantly influence the pathogenesis of OPC.ormation . This inilm mass ,34,35; wbacteria . By contl organs ,38. ThesC. albicans has evolved multiple strategies to breach the epithelial cell barrier, withstand HDPs, and cause a superficial infection. In mouse models of infection, other microbial pathogens can interact with C. albicans and alter virulence. This pathogenic interaction is undoubtedly relevant to human infection in the case of C. glabrata and streptococci because these organisms are commonly isolated in conjunction with C. albicans. Whether mucosal infection with C. albicans predisposes patients to develop invasive staphylococcal infections is currently unknown but is clearly worthy of investigation.The oral epithelium is a critical component of the host defense against OPC. However,


Helicobacter pylori colonizes the human stomach and induces inflammation, and in some cases persistent infection can result in gastric cancer. Attachment to the gastric mucosa is the first step in establishing bacterial colonization, and outer membrane proteins (OMPs) play a pivotal role in binding to human cells. Some OMP interaction molecules are known in H. pylori, and their associated host cell responses have been gradually clarified. Many studies have demonstrated that OMPs are essential to CagA translocation into gastric cells via the Type IV secretion system of H. pylori. This review summarizes the mechanisms through which H. pylori utilizes OMPs to colonize the human stomach and how OMPs cooperate with the Type IV secretion system. Acinetobacter baumannii, OMPs are essential for establishing infection, and an interaction network with β-lactamase regulates antibiotic inactivation . Tr. Tr30].  stomach . In addi. pylori . Recentl of mice . In addioipA mutant resulted in the reduced production of IL-8 in a gastric epithelial cell line [H. pylori adhesion to host cells and does not influence the production of IL-8 in gastric cells [hopM/N, babA/B/C, and sabA/B), whereas it was reported that the OipA locus was duplicated, referred to as OipA-2 [H. pylori hspEAsia and hspAmerind strains, but not in hpEurope or hspWAfrica strains, based on multi-locus sequence typing analysis.OipA was initially identified as a protein that induces a pro-inflammatory response based on the fact that an ell line . In contic cells ,36. Whetic cells ,35,36,37s OipA-2 . OipA-2 H. pylori strain. Akanuma and colleagues reported that the oipA-mutant H. pylori TN2 strain does not infect Mongolian gerbils [oipA-mutant H. pylori 7.13 strain infected Mongolian gerbils; however, oipA mutant strain-infected animals did not develop gastric cancer, whereas 27% of those infected with the wild-type strain did develop this condition [H. pylori strain and controversial. Therefore, it is difficult to demonstrate the relationships between OipA status and molecular processes as well as clinical outcomes.The role of OipA as an adhesion molecule is suggested by in vitro experiments using gastric epithelial cells ,35,39. H gerbils . In contondition . Thus, tcag PAI, vacA-s1 type, and vacA-m1 type [H. pylori strains; however, OipA-related signaling has been reported. OipA is suggested to induce the remodeling of actin stress fibers by activating the phosphorylation of focal adhesion kinase (FAK); this leads to the activation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) [oipA mutant H. pylori could not activate EGFR-related signaling involving phosphatidylinositol 3-kinase (PI3K) [Based on epidemiological studies, functional OipA correlates with other virulence factors such as -m1 type ,42,43,44(Erk1/2) . OipA-mee (PI3K) , phosphoe (PI3K) . Recentle (PI3K) .H. pylori. Therefore, OipA might cooperate with other virulence factors during the pathogenesis of H. pylori.It is difficult to demonstrate the relationships between OipA status and clinical outcomes. However, it is clear that OipA status is associated with the presence of the other virulence factors in cagA-positive and vacA-s1 type strains than in cagA-negative and vacA-s2 strains [H. pylori strains [vacA and cagA genotypes. HopQ is a paralog of the Hop family that includes BabA and SabA . HopQ is strains . Further strains . This incagPAI-encoded co-factor of the T4SS. Belogolva et al. suggested that HopQ is essential for CagA translocation, and that deletion of HopQ reduces T4SS-dependent activation of signaling  [HopQ was reported as a non-duction) . Furtherduction) . Recentlduction) ,54. ThisH. pylori expresses many different virulence factors. The function of each virulence factor has gradually been revealed. In regard to membrane proteins, the binding partners of BabA, SabA, and HopQ were identified, and subsequently BabA and HopQ were suggested to enhance CagA translocation into gastric cells via the T4SS. In addition, many epidemiological studies have demonstrated that OMPs correlate with other virulence factors and clinical outcomes. Therefore, these proteins might have a dynamic function in cooperating with the T4SS and other factors. However, less is known about the interplay between membrane proteins and the membrane region of H. pylori. Recently, it is clear that outer membrane vesicles (OMVs) have a unique function. OMVs are involved with periplasmic and outer membrane proteins, peptideglycanes, lipopiolysaccharides, DNA, RNA, and enzymes. OMVs are also involved in a mechanism to transfer virulence factors and maintain bacteria communities. Thus, OMPs at MOVs might play an important role in communicating with bacteria. Studies that focus on local or temporal interactions among virulence factors and the other molecules are required for a deeper understanding of the dynamic pathogenesis and communication of H. pylori.H. pylori utilize OMPs to colonize the human stomach. This indicates that OMPs might be a vaccine candidate. In fact, many reports suggested that OMPs might be a candidate as a vaccine based on in vitro experiments. However, there has been no report that shows that OMPs are useful as target molecules for vaccines in animal models. An animal model is essential to demonstrate the efficacy of a vaccine. Thus, this problem needs to be overcome to produce a vaccine for H. pylori infection.Many studies demonstrated that


Helicobacter pylori (H. pylori) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes , and also the epitope-rich regions of urease B subunit (UreB158–251 and UreB321–385) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes . In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4+ T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori. These results indic ate that a multivalent epitope-based vaccine including Th and B cell epitopes from various H. pylori antigens could be a promising candidate against H. pylori infection.Epitope-based vaccine is a promising strategy for therapeutic vaccination against  Helicobacter pylori (H. pylori) is a helix-shaped bacterium that infects more than half of the world's population , H. pylori adhesin A (HpaA) and neutrophil-activating protein (NAP), have been proved to be the excellent candidates for their ability to induce protective immune responses against H. pylori infection  which is composed of two subunits, UreA and UreB. Urease can hydrolyze urea to ammonia and carbon dioxide, thereby neutralizing gastric acid and facilitating H. pylori colonization  and the Dna K/J  chaperones  is commonly lost during colonization of mice , indicating the Mongolian gerbil model seems more suitable for H. pylori infection , NAP, HSP60, and HpaA, which are involved in the adhesion and virulence of H. pylori to gastric mucosa. Immunological properties of CWAE vaccine were evaluated in BALB/c mouse model, and its therapeutic effect was analyzed in Mongolian gerbils, in which H. pylori infection is similar to that in humans.In this study, our aim is to develop a multivalent epitope-based vaccine, CWAE against + T cell epitopes binding to human leukocyte antigen (HLA) class II molecules were screened from UreB by online T Cell Epitope Prediction Tools . The predicted CD4+ T cell epitopes with percentile rank ≤ 1 are considered high affinity and used as the candidate epitopes for vaccine design (Supplementary Data Sheet http://tools.iedb.org/main/bcell/). An output threshold of 0.9 was used  for identification of linear B cell epitopes. This threshold was chosen to provide a high level of certainty for predicted B-cell epitopes from UreB. The predicted B cell epitopes are as shown in Supplementary Data Sheet http://www.iedb.org/home_v3.php), as shown in Supplementary Data Sheet 158–251 and UreB321–385) and the other four B or Th cell epitopes .A protein A sepharose column (BioVision) was used to purify mouse IgG in the antiserum. H. pylori colonization in the stomachs. Briefly, the stomach was dissected into two tissue fragments along the lesser curvature. One fragment was homogenized in 2 ml of PBS by using a tissue homogenizer. Serial 10-fold dilutions of the homogenate were plated on H. pylori selective plate (QingDao Hopebio Technology) supplemented with 7% goat blood, trimethoprim (5 μg/ml), polymixin B (5 μg/ml), and vancomycin (10 μg/ml) under microaerobic conditions. After 4–6 days culture, colonies were counted and the number of Colony-Forming Units (CFU) per stomach was calculated. The degree of H. pylori colonization in the mouse stomach was also measured by rapid urease test. Briefly, the antral portion of the stomach was immediately immersed in 500 μl of sodium phosphate buffer containing 500 mM urea, 0.02 % phenol red, and 0.1 mM DTT. The stomach sample was incubated at 37°C for 3 h. The supernatant of specimens was used for quantification at 550 nm (A550).Two weeks after the final therapeutic vaccination, mice were killed for determination of the H. pylori infection by immunohistochemical (IHC) staining using polyclonal anti-H. pylori antibody  and polymer-HRP based detection system (BioGenex).One strip of stomach tissue was cut out and fixed with formalin. Then, the stomach tissue was embedded in paraffin and stained with hematoxylin and eosin (HE). For evaluation of gastritis, the slides were “blinded” and the extent of gastritis was graded as follows: 0, none; 1, a few leukocytes scattered in the deep mucosa; 2, moderate numbers of leukocytes in the deep to mid mucosa and occasional neutrophils in the gastric glands (microabscesses); 3, dense infiltrates in the deep to mid mucosa, a few microabscesses; and 4, dense, diffuse infiltrates throughout the lamina propria and into the submucosa, frequent microabscesses. The stomach tissue sections were also assessed for the presence of 5 cells/well) with synthetic peptides of the Th epitopes , NAP, Urease or H. pylori lysates (5 μg/ml) in plates at 37°C for 72 h. Then, 10 μl of the CCK-8 solution (Dojindo Molecular Technologies Inc) was added into plates for 4 h. The results were expressed as stimulation indices (SI), defined as the index of lymphocyte proliferation according to formula: SI = the absorbance 450 value of stimulated cultures/the absorbance 450 value of negative control cultures. To determine cytokine production, culture supernatants from splenic lymphocytes stimulated by H. pylori lysates for 72 h were collected to assay for IL-4, IFN-γ, and IL-17 by using ELISA kits  according to the manufacturer's instructions.Splenic lymphocytes were isolated with lymphocyte separation medium (Dakewe Biotechnology Company) and cultured .All data were analyzed with the GraphPad Prism 5 software and expressed as mean ± SD. Statistical significance was tested using Student's H. pylori urease composed of molecular adjuvants CTB and tandem copies of urease B and Th cell epitopes was constructed in previous study  against Urease (Ure), NAP, HSP60, and HpaA was constructed. The structure diagram of multivalent epitope vaccine CWAE is shown in Figure 158–251 and UreB321–385 in CWAE were obtained by using online T Cell Epitope Prediction Tools and B Cell Epitope Prediction Tools. The epitope-rich region UreB158–251 was found to contain four known Th or B cell epitopes [UreB158–172 and UreB181–195 /pETCWAE. Based on the SDS-PAGE, the content of CWAE in the inclusion bodies was about 60% of the total protein  was largely expressed in inclusion body of y Figure . SimilarP Figure . The adjP Figure , but theH. pylori urease, UreA, UreB, HSP60, HpaA, and NAP) were measured by ELISA. CWAE and CTB-UE could induce similar levels of antibodies specific to H. pylori urease, UreA, or UreB  in the CWAE vaccine were synthesized and tested. The results indicated that both CWAE and CTB-UE vaccine were capable of generating similar levels of antibodies directed specifically against the epitope peptides UreA183–203  were orally immunized with CWAE, urease or PBS. The H. pylori colonization in the stomach was analyzed by quantitative culture. The result showed that oral therapeutic immunization with CWAE, CTB-UE, or urease dramatically decreased the H. pylori loads in the stomachs of H. pylori-infected Mongolian gerbils compared with those using oral therapeutic immunization with PBS  and biological toxicity. For example, the active H. pylori urease comprising two subunits, UreA  and UreB , has a molecular weight of about 96 KD and induces damage to epithelial cells  in protection against H. pylori has been widely accepted  and mucosal adjuvant (CTB) within the vaccine, aluminum hydroxide adjuvant, and peptide components of CWAE. Furthermore, the lymphocyte proliferation results showed that splenic lymphocytes from Mongolian gerbils immunized with CWAE proliferated significantly after stimulation with NAP, Urease, and H. pylori lysates, indicating the CWAE can also induce lymphocyte responses against various H. pylori antigen. Interestingly, CWAE and Urease vaccinations elicited comparable production of IFN-γ, IL-4, and IL-17, while CWAE had significantly higher protective efficacy compared to URE vaccine, suggested that many factors, not only Urease, may be involved in H. pylori infection in vivo, and multi-target intervention in the process of H. pylori infection is critical for eradicating H. pylori colonization. The CWAE vaccine composed of HLA restricted CD4+ T (Th) cell epitopes could induce Th cell responses against H. pylori in Mongolian gerbils, implying the predicted epitope-rich regions of UreB may also contain some Mongolian gerbil Th cell epitopes. Besides, the CWAE vaccine also contains a lot of B cell epitopes, which can induce epitope-specific antibodies against H. pylori. Therefore, the immune protection of CWAE vaccine was correlated with high levels of Th cell responses and epitope-specific IgG, IgA, or sIgA antibodies.A critical role of CD4H. pylori was designed and constructed in this study. Oral immunization with CWAE significantly reduced H. pylori colonization in H. pylori-infected Mongolian gerbils, which may be related to mixed Th cell responses and epitope-specific antibodies against various H. pylori antigens. Given the complex nature of H. pylori infection, the CWAE vaccine will face many challenges on the road to a human vaccine. We will further evaluate the therapeutic effect of CWAE in other animal models and investigate other oral immune delivery systems, such as polysaccharide microsphere and food grade lactic acid bacteria, to help CWAE to effectively override or bypass enzymatic degradation in the human gastrointestinal tract. Furthermore, clinical trials of the therapeutic multivalent epitope-based vaccine are expected in the future. This multivalent epitope-based vaccine may be a promising vaccine candidate that may help to control H. pylori infection.In conclusion, a multivalent epitope-based vaccine CWAE against Conceived and designed the experiments: LG, KL, and GX. Performed the experiments: FT, RY, HY, XG, and HL. Analyzed the data: LG and FT. Contributed reagents/materials/analysis tools: JW. Wrote the manuscript: LG, YZ, and RY.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. Persistent Helicobacter pylori infection may induce several upper gastrointestinal diseases. Two major virulence factors of H. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution of vacA and cag-pathogenicity island (cag-PAI) alleles varies in H. pylori isolated from patients in different geographic regions. Aim. To assess the association between mixed infection of H. pylori clinical isolates from Taiwanese patients and the severity of gastrointestinal diseases. Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes. Results. The prevalence of mixed infections of all H. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P < 0.05). Conclusions. H. pylori mixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development. Helicobacter pylori is a gram-negative, spiral shaped microaerophilic bacterium that colonizes the human gastric mucosa throughout life  urea breath test and bacterial culture was performed on biopsies before therapy [From January 2011 to December 2014, a total of 70 patients withrollment . H. pylo therapy . Among t therapy . All the H. pylori isolates were cultured from the biopsies specimen and identified by biochemical reactions [ H. pylori were diagnosed with positive reaction in catalase, urease, and oxidase tests. The bacterial isolates were routinely cultured on Brucella agar plates  with appropriate antimicrobial agents as described previously [Two biopsied specimens of each patient were taken: one specimen from antrum (lesser curvature side) and another from low body (greater curvature side).eactions . H. pyloeviously . cagA, cagE, cagT, cagM, and vacA s- or m-regions among the 6 isolates isolated from one host.After obtaining positive cultures from the biopsies, 6 isolated colonies from a single culture plate were examined for the genotypes using polymerase chain reaction (PCR) approach as described previously , 30, 32.P value of less than 0.05 was considered significant.The relationship of between-group comparisons was performed using the Chi-square test with Fisher's exact test. A  H. pylori-positive status  were enrolled in this study. From each patient, the biopsies were taken from the antrum and body of stomach. We obtained 6 colonies from pooled isolates in one culture plate, for a total of 420 isolates, to study the H. pylori genes. cag-PAI status and the s-region or m-region of vacA in H. pylori were assessed by PCR using unique primers. Mixed infection was defined as distinct expression of cagA, cagE, cagT, cagM, and vacA s- or m-regions among the 6 isolates in one host. The PCR distribution of positive/negative results for individual genes was shown in n = 300) and mixed infection (n = 120) were positive for cagA. There were no major differences of the gene distributions between the two groups.From January 2011 to December 2014, 70 patients diagnosed with upper gastrointestinal diseases and H. pylori clinical isolates from patients and disease severity. As shown in  H. pylori mixed infections, two patients (22.2%) with chronic gastritis, 10 patients (47.6%) with duodenal ulcer, 4 patients (18.2%) with gastric ulcer, and 4 patients (22.2%) with gastric carcinoma, had mixed infections. Additionally, patients with duodenal ulcer showed a higher prevalence of H. pylori mixed infection compared to that in other gastrointestinal diseases (P < 0.05). In all studied subjects, mixed infections of H. pylori strains were found in 20 (28.6%) patients.We then analyzed the association between mixed infections inH. pylori mixed infections were defined as having more than one allele of either the s-region or m-region of vacA or both iceA1 and iceA2 genotypes [ cagA, cagE, cagT, cagM, and vacA s- or m-regions. We evaluated a large number of colonies (n = 420) isolated from 70 patients. Moreover, 11 pairs of primers, including those for the genes cag-PAI and vacA s- or m-regions, were used. Therefore, we accurately and sensitively analyzed H. pylori mixed infections.enotypes –24. The enotypes , 26–28. enotypes . In this H. pylori-infected samples isolated from patients who lived in southern Taiwan [ H. pylori mixed infections was 28.6% (20/70) in patients who were residents of northern Taiwan. This discrepancy in the prevalence of mixed infections in Chinese populations may be explained by the different analysis strategies and the fact that enrolled subjects were from different geographic regions [Mixed genotypes were found in 24% of patients who were Chinese residents of Hong Kong . Anothern Taiwan . In this regions , 35. H. pylori survival in the harsh environment of the stomach where disease progression occurs [A high percentage of subjects (77%) carrying a mix of metronidazole-susceptible/resistant strains have been reported . Mixed in occurs , 40. H. pylori mixed infections was high in residents living in northern Taiwan and that the rates differed from those in other Chinese populations from other geographic regions. The mixed infections in H. pylori with high genetic diversity may promote bacterial adaptation to the stomach and contribute to disease development.In conclusion, our study reported that the prevalence of


AbstractCheraxwarsamsonicussp. n., endemic to the Warsamson River drainage, in the western part of the Kepala Burung (Vogelkop) peninsula, West Papua, Indonesia, is described, figured and compared with its closely related species, Cheraxmisolicus Holthuis, 1949. The new species may be easily distinguished from C.misolicus by the shape of the rostrum, absence of setae on the rostrum, the shape of the chelae, the presence of 3-4 cervical spines and by using sequence divergence, which is substantial for considering C.warsamsonicussp. n. to be a new species. The new species is collected and exported for ornamental purposes and its commercial name in the pet trade is Cherax “irian jaya”, Cherax “pink coral”, or Cherax “hoa creek“. Due to similar colouration it is often confused with the recently described Cheraxpulcher Lukhaup, 2015.A new species,  Cherax “irian jaya” and Cherax “hoa creek” , Papua New Guinea and Australia, their exact provenances could not be ascertained, with dealers claiming they came from Ajamaru (West Papua) and other places in the area that could not be confirmed. Also species have been mixed at the places of exporters in Sorong and Jakarta. Therefore in January 2016 we visited the Sorong Regency and South Sorong Regency to clarify the distribution of some of the species present in the pet trade. In the present contribution, this species is described as new to science and establish that it is in fact native to the Warsamson River Drainage, Sorong Regency of the Kepala Burung (Vogelkop) Peninsula West Papua, Indonesia. Cheraxwarsamsonicus sp. n., is genetically and morphologically most similar to Cheraxmisolicus Holthuis, 1949 endemic to the Island of Misool, one of four major islands in the Raja Ampat Islands in West Papua, Indonesia and two other undescribed species from Sorong and South Sorong Recency.The crayfishes of the island of New Guinea were extensively studied by a creek” . These hCheraxmisolicus and Cheraxwarsamsonicus sp. n. may be easily distinguished using sequence divergence, by colouration and pattern of live individuals, by the shape of the chelae, the shape of rostrum, and presence of dense setae on the rostrum in C.misolicus which is absent in the new species.Cheraxwarsamsonicus sp. n. as well as three other species were collected from streams in the southwestern part of the Kepala Burung peninsula in February 2016. In addition, sequence from seven species of Cherax and from two other parastacid genera used as outgroup were downloaded from GenBank (see Table Samples of PCR) was used to amplify two mitochondrial gene fragments, a ~535 bp region of the 16S ribosomal RNA gene (16S) using primers 1471 and 1472 (COI) using primers LCO1490 and HCO2198  or 50 °C (16S) for 60 s, and 72 °C for 1 min (COI) or 90 s (16S), with a final elongation step of 5 min at 72 °C. The same primers were used in PCR and sequencing. PCR products were sent to Macrogen Europe for purification and cycle sequencing of both strands of each gene.COI) and with MAFFT (16S) using the G-INS-i strategy suitable for thorough alignments of sequences with global homology (COI) and 543 bp (16S), respectively. To determine the best substitution model for Bayesian inference analyses (see below), hierarchical likelihood ratio tests were carried out with jModelTest (PageBreaksequence sets (24 models tested). Based on the Akaike Information Criterion and the Bayesian Inference Criterion, the GTR + I + G (COI) and the GTR + G (16S) models were chosen. The two sequence datasets were subsequently analysed both separately and combined.Sequences were aligned by eye (homology . The resodelTest  on both MP) using the heuristic search algorithm as implemented in PAUP* (ML) analyses were conducted with RAxML (BI) was employed to infer phylogeny by using MrBayes 3.2.2  using the different models for the two genes in the BI analyses. All new sequences have been deposited in GenBank, see Table The combined dataset was subjected to a partitioned analysis , among roots along banks of a unnamed creek draining into Warsamson River, north of Sorong City, 0°49'16.62"S 131°23'3.34"E, West Papua, Indonesia. coll. Chris Lukhaup, Irianto Wahid and unnamed local guide January 20 2016. Allotype: female (MZB Cru 4530), same data as holotype. Paratypes: (MZB Cru 4531), same data as holotype.Carapace surface smooth with four small spiniform tubercles posterior to cervical groove on lateral carapace. Eyes large, pigmented. Cornea slightly broader than eyestalk. Rostrum lanceolate in shape with excavated margins. Rostral margins with three prominent teeth. Rostral carinae prominent. Postorbital ridges prominent with one acute tubercle at anterior terminus. Uncalcified patch on lateral margin of chelae of adult male white, translucent. Propodal cutting edge with row of small granules and one large tubercle. Chelipeds blue and white with white joints. Fingers blue in distal third black with hooked tips. Other walking legs blue-gray. Pleon black with pinkish-red pattern. Lateral pleura lighter becoming greyish green.Figs –5.Body and eyes pigmented. Eyes not reduced. Body subovate, slightly compressed laterally. Pleon narrower then cephaPageBreaklothorax (width 16.7 mm and 17.5 mm respectively). Rostrum .Ventrolateral parts smooth with scattered pits; anterior margin strongly produced, rounded upper margin directed inward.Scaphocerite . Left chelae . Few scattered short setae present in posterior ventral part of fixed finger. Cutting edge of fixed finger with row of rather small granular teeth at posterior half and one at middle of anterior part.Dorsal surface of carpus (11.77 mm) smooth and pitted, with slight excavation in middle part and with well-developed acute and hooked spiniform tubercle in middle of dorsolateral inner margin. Ventral carpal surface margins slightly elevated, non-setose and with fovea; inner margin with one acute spiniform tubercle oriented in angle of approx. 45°; outer margin smooth with one spiniform tubercle oriented almost anteriorly.Merus (19.2 mm) laterally depressed in basal part; surface slightly pitted; one prominent spine at anterior part at dorsal surface. Row of 12-13 small granules on inner ventrolateral margin, four prominent spines, one at midlength other in middle of anterior part, third on distal ventrolateral outer margin, fourth on distal ventrolateral inner margin.Ischium (10.8 mm) smooth with small spine and three granules at midlength of ventrolateral inner margin.Second pereopod reaching anteriorly to approximately middle of scaphocerite. Finger as long as palm (5.6 mm), of same height. Short setae present on dactyl and fixed finger, getting denser anteriorly. Cutting edge of fixed finger and carpus with row of short setae. Carpus, smooth, not pitted, slightly longer than palm. Merus (12.7 mm) 1.7 times longer than carpus (7.2 mm). Ischium (6.2 mm) half as long as merus.Third pereopod overreaching second by almost length of finger of second pereopods. Fingers shorter than palm.Fourth pereopod reaching distal margin of scaphocerite. Dactylus with corneous tip. Short scattered setae present. Propodus more than twice as long as dactylus, nearly 1.5 times as long as carpus; somewhat flattened, carrying many stiff setae on lower margin. Merus just slightly longer than propodus.Fifth pereopod similar to fourth, slightly shorter.Dorsal surface of pleon smooth, with scattered pits; abdominal segments with short setae present on caudal margins.Telson with posterolateral spines, dense short setae present in posterior third. Posterior margins setose. Uropodal protopod with distal spine on mesial lobe. Exopod of uropod with transverse row of posteriorly directed diminutive spines ending in one PageBreakmore prominent spine, posteriorly directed on outer margin of mesial lobe. Terminal half of exopod with small tubercles and short hairs, slightly corrugated. Endopod of uropod smooth. Short scattered hairs present on posterior third of dorsal exopod. Postrolateral spine on outer margin present. Second spine on medial dorsal surface present, directed posteriorly.Fig. . Chela oSize. The biggest male examined has a carapace length of 48.7 mm, and a total length of 109 mm (n = 4) ,the holotype male has a total length of 92,8 mm the other males have a total length of 73mm and 96 mm; the female has a carapace length of 31.8 mm and a total length of 73 mm (n = 1).Colour. The living animals  of 1.9-2.1 %, respectively, supporting the morphology-based description of C.warsamsonicus as a new species.MZB Cru 4529), allotype (MZB Cru 4530) and paratypes (MZB Cru 4531) are deposited at the Museum Zoologicum Bogoriense (= Bidang Zoologi) Reseach Centre for Biology (=Pusat Penelitian Biologi), Indonesian Institute of Sciences (= LIPI), Jalan Raya Jakarta-Bogor Km 46 Cibinong 16911, Indonesia.The holotype  from southwest Australia. The other group contains species that have the rostral and sometimes the median carina well developed and referred to as the Astaconephrops group with Astaconephropsalbertisii as the type. Newly described species have been placed into one or the other of the two subgenera . Cherax into two subgenera, as conceived by Holthuis and subsequent authors dealing with New Guinea crayfish has to be reconsidered. Based on Munasinghe et al. (2004), Cheraxwarsamsonicus sp. n. belongs to the northern species group lineage consisting of 22 species:ubgenera , b, AustC.albertisii C.boesemani Lukhaup & Pekny, 2008C.boschmai Holthuis, 1949C.buitendijkae Holthuis, 1949C.communis Holthuis, 1949C.divergens Holthuis, 1950C.gherardii Patoka, Bláha & Kouba, 2015C.holthuisi Lukhaup & Pekny, 2006C.lorentziaruanus C.lorentzilorentzi C.longipes Holthuis, 1949C.misolicus Holthuis, 1949C.murido Holthuis, 1949C.monticola Holthuis, 1950C.minor Holthuis, 1996C.peknyi Lukhaup & Herbert, 2008C.pallidus Holthuis, 1949C.papuanus Holthuis, 1949C.paniaicus Holthuis, 1949C.pulcher Lukhaup, 2015C.solus Holthuis, 1949C.snowden Lukhaup, Panteleit & Schrimpf, 2015C.warsamsonicus, is most similar to C.misolicus, a species that is endemic to Misool Island, one of four major islands in the Raja Ampat Islands in West Papua, Indonesia.In comparison to all species of the northern group the new species, Cheraxwarsamsonicus sp. n. differs from C.misolicus in the following characters: shape of the chelae,  peninsula. The creeks from where these crayfish have been collected are shallow (20–60 cm) with a moderate flow, the water is clear, and have a pH of approximately 6.5. In most of the parts no water plants are present. The substrate of the creek is gravel or sand and soil cks Fig. . CrayfisCherax “irian jaya”, Cherax “pink coral”, and sometimes it is sold also as Cheraxpulcher. Therefore we propose the name Warsamson River Crayfish as a common name for the new species.The common name of the new species in the pet trade is


Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA–DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.Although the genus  Clavibacter was originally proposed by Davis et al. [Clavibacter michiganensis, Clavibacter iranicum, Clavibacter rathayi, Clavibacter toxicus, Clavibacter tritici and Clavibacter xyli. Subsequently, the grass-specific pathogens, C. iranicum, C. rathayi, C. toxicus and C. tritici, were reclassified into the genus Rathayibacter on the basis of DNA–DNA hybridization and their unique menaquinone structures [C. xyli were placed in the genus, Leifsonia [Clavibacter consists of only one species, C. michiganensis, which is subdivided into seven subspecies of plant pathogenic bacteria with narrow host specificities and two subspecies with close association with tomato and pepper seeds. Five of the subspecies comprise well-known pathogens, namely, C. michiganensissubsp.michiganensis , C. michiganensissubsp.sepedonicus , C. michiganensissubsp.insidiosus , C. michiganensissubsp.nebraskensis , and C. michiganensissubsp.tessellarius . More importantly, the first three subspecies are quarantine or regulated pathogens of important agricultural crops in many countries. Recently, C. michiganensissubsp.phaseoli was described as the causal agent of bacterial leaf yellowing on bean [C. michiganensissubsp.capsici (Cmc) as the causal agent of bacterial canker on pepper [C. michiganensissubsp.californiensis and C. michiganensissubsp.chilensis were named to include bacterial isolates from tomato and pepper seeds produced in California and Chile, respectively [C. michiganensissubsp.capsici with available genome sequence data  sequencing at Genome Quebec . The assembled sequences were compared with published sequences of C. michiganensissubsp.michiganensis and subsp. insidiosus, and other clavibacter sequences in GenBank (Clavibacter michiganensis were included in this study. The genome sequences generated in this study were deposited in Genbank with accession numbers of MZMQ00000000 (Cmt ATCC 33566), MZMM00000000 (Cms CFIA-Cs3N), MZMN00000000 (Cms CFIA-CsR14), MZMO00000000 (Cmi LMG 3663) and MZMP00000000 (Cmm LMG 7333).To better define the taxonomic position of the subspecies of  GenBank . Currentblast-based ANI calculation method described by Goris et al. [Average nucleotide identity (ANI) values of whole genomes represent the degree of identity/similarity between homologous regions shared by two genomes and has emerged as a powerful genome-based criterion for establishing species identity amongst genetically related micro-organisms . The apps et al. . ANI wasClavibacter were generally below the 96 % cutoff value for species delineation suggested by Richter and Rosselló-Móra [Clavibacter michiganensis [The ANI values among the subspecies of lló-Móra . ANI valganensis , were wein silico replacement for the wet-lab based DNA–DNA hybridization. In this study dDDH values were calculated using GGDC 2.0 server  by means of genome-to-genome sequence comparison and pairwise dDDH values were estimated using the GGDC calculator [Clavibacter subspecies into distinct clades with high degree of congruency with genomospecies allocation  or digital DNA–DNA hybridization (dDDH)  measureslculator . Consistlocation . The dDDlocation , below tlocation .acnA, gapA, lcdA, mdh, mtlD, pgi and proA .Multi-locus sequence analysis (MLSA) based on concatenated segments of housekeeping genes is used in phylogenetic studies to resolve taxonomic relationships among closely related species . MLSA waand proA . Strainsp values . The grop values . Of the C. michiganensis subspecies does not undermine classification based on phenotypic characterization of this group of plant pathogenic bacteria but rather supports their classification as individual species which are easily differentiated by classical bacteriological methods as previously reported [C. michiganensis subspecies is highly host-specific and in culture can also be readily differentiated by colony pigmentation on many commonly used growth media and substrate utilization  and multi-locus phylogenetic analysis presented in this paper and previously reported phenotypic characteristics, we propose that the bacteria presently classified as Clavibacter michiganensis subsp. capsici Oh et al. 2016, Clavibacter michiganensis subsp. nebraskensis (Vidaver and Mandel 1974) Davis et al. 1984, Clavibacter michiganensis subsp. insidiosus (McCulloch 1925) Davis et al. 1984, Clavibacter michiganensis subsp. sepedonicus (Spieckermann and Kotthoff 1914) Davis et al. 1984, and Clavibacter michiganensis subsp. tessellarius (Carlson and Vidaver 1982) Davis et al. 1984 be reclassified as Clavibacter capsici sp. nov., comb. nov.Clavibacter nebraskensis,  comb. nov., Clavibacter insidiosus comb. nov., Clavibacter sepedonicus comb. nov., and Clavibacter tessellarius sp. nov., comb. nov., respectively. The original type strains of the subspecies become type strains for each of the new species and species descriptions remain the same as for the former descriptions of corresponding subspecies [Traditional classification of plant pathogens faces critical challenges in the genome era as sequence data become routinely accessible through next-generation sequencing methods. The growing number of sequenced genomes of plant pathogens provides a rich source of information for new approaches to resolve complex taxonomic questions. In this study, the draft genomes of three type strains of Clavibacter capsici .Clavibacter michiganensissubsp.capsici Oh et al. 2016.Basonym: Clavibacter michiganensissubsp.capsici given by Oh et al. [The species description is unchanged from its description as h et al. .T (=KACC 18448T=LMG 29047T). The type strain was originally isolated from pepper showing bacterial canker disease in Anyang, Republic of Korea.The type strain is PF008Clavibacter insidiosus .Corynebacterium insidiosum (McCulloch 1925) Jensen 1934, Corynebacterium michiganensesubsp.insidiosum (McCulloch 1925) Carlson and Vidaver 1982, Clavibacter michiganensissubspinsidiosus (McCulloch 1925) Davis et al. 1984.Basonym: Medicago sativa L.). DNA G+C content of the type strain is 72.7 %. The type strain is LMG 3663T (=ATCC 10253T=NCPPB1109T).Gram-stain-positive, non-spore forming, aerobic bacterium without flagella. Produces yellowish colonies with blue granules on common laboratory growth media. Grows on TTC but not CNS medium. It does not liquefy gelatin nor produces levan. It does not produce acid from either sorbitol or mannitol. It utilizes glycerol but not acetate or succinate; it hydrolyses aesculin, and has α-mannosidase activity but no alkaline phosphatase activity. It causes bacterial wilt disease of alfalfa (lucerne) .Corynebacterium nebraskense Vidaver and Mandel 1974, Corynebacterium michiganensesubsp.nebraskense (Vidaver and Mandel 1974) Carlson and Vidaver 1982, Clavibacter michiganensissubsp.nebraskensis (Vidaver and Mandel 1974) Davis et al. 1984.Basonym: Zea mays L.) DNA G+C content of the type strain is 73.0 %. The type strain is NCPPB 2581T (=ATCC 27794T=LMG 3700T).Gram-stain-positive, non-spore forming, aerobic bacterium without flagella. Produces yellow to orange colonies on common laboratory growth media. It grows on CNS but does not grow on TTC medium. It does not liquefy gelatin but it does produce levan. It produces acid from sorbitol but it does not produce acid from mannitol. It utilizes acetate, glycerol and succinate. It hydrolyses aesculin, it has alkaline phosphatase activity, but it does not have α-mannosidase activity. It causes leaf freckles and a wilt disease of maize .Corynebacterium sepedonicum (Spieckermann and Kotthoff 1914) Skaptason and Burkholder 1942, Corynebacterium michiganensesubsp.sepedonicum (Spieckermann and Kotthoff 1914) Carlson and Vidaver 1982, Clavibacter michiganensissubsp.sepedonicus (Spieckermann and Kotthoff 1914) Davis et al. 1984.Basonym: Clavibacter species in producing acid from both sorbitol and mannitol. It utilizes acetate and succinate but not glycerol; it hydrolyses aesculin; alkaline phosphatase activity is weak, and α-mannosidase activity is lacking. It causes bacterial ring rot disease of potato (Solanum tuberosum L). DNA G+C content of the type strain is 72.4 %. The type strain is ATCC 33113T (=LMG 2889T=NCPPB 2137T).Gram-stain-positive, non-spore forming, aerobic bacterium without flagella. Produces white mucoid colonies at an optimum growth temperature of 20–23 °C. It does not grow on CNS or TTC media. It does not liquefy gelatin nor produces levan. It differs from the other Clavibacter tessellarius (tes.sel.la′ri.us. L. masc. n. tessellarius a mosaic stone maker).Clavibactermichiganensesubsp.tessellarius, Clavibacter michiganensissubsp.tessellarius (Carlson and Vidaver 1982) Davis et al. 1984.Basonym: Clavibacter michiganensissubsp.tessellarius given by Carlson and Vidaver, 1982 [The species description is unchanged from its description as er, 1982 .T (=NCPPB 3664T=LMG 7294T).The type strain is ATCC 33566Clavibacter, but it also provides a practical solution for plant pathologists and policy makers dealing with quarantine and regulated plant pathogens. C. michiganensis, C. sepedonicus and C. insidiosus are quarantine or regulated pathogens of important agricultural crops in many countries, while C. capsici is a newly described plant pathogen for which the range of distribution and risk to agriculture need to be assessed. The revised classification, and accordingly a simpler nomenclature, uncomplicates regulatory documents and more accurately reflects biological reality.This new taxonomy not only resolves the long-standing problem of having only a single species within the well-established genus, Clavibacter michiganensis subspecies, pathogens of important agricultural crops’. It is quoted here ‘the study also assessed the taxonomic position of the subspecies based on 16S rRNA and genome-based DNA homology and concludes that there is ample evidence to elevate some of the subspecies to species-level’.While this manuscript was under review, one of the co-authors  of this


Caenorhabditis elegans, has been shaped by interactions with environmental microbes, which for nematodes are both sources of food and agents of disease. As a result, C. elegans has evolved protective host responses coordinated through multiple pathways, which are required for host survival during microbial infection. Following exposure to a pathogen, putative immune effectors are transcriptionally up-regulated, which has led to an extensive search for the mechanisms underlying pathogen recognition in this simple metazoan host. Biological rationale for the existence of inducible immune defenses has come from the recognition that physiologic [The evolution of bacterivorous nematodes, such as siologic ,2 or abesiologic  activatisiologic –6, but asiologic –9. A relC. elegans, this appears to have created selection pressure to evolve mechanisms that monitor overall translation capacity as a means to detect pathogen infection. Characterization of these mechanisms by three separate groups working in parallel provided the first demonstration of surveillance immunity in nematodes [Pseudomonas aeruginosa and cripples elongation of the growing peptide strand by ribosylating host elongation factor 2. Exposure of C. elegans to P. aeruginosa interrupts translation in intestinal epithelial cells [C. elegans [C. elegans, which is controlled by a neuroendocrine axis involving serotonergic and c-Jun N-terminal kinase (JNK) signaling [A multitude of bacteria produce toxins that interrupt host mRNA translation. In the case of ematodes –9. One sal cells . This, i elegans ,8,10. Th elegans –9. Impor elegans . InductiC. elegans caused mitochondrial stress in the laboratory strain of C. elegans, which highlights the selection pressure faced by free-living nematodes [mt) [mt, is normally taken efficiently into the mitochondria and degraded, but under conditions of mitochondrial stress, the uptake of ATFS-1 into mitochondria is compromised, freeing cytosolic ATFS-1 to traffic to the nucleus, where it induces mitochondrial stress-response proteins [P. aeruginosa infection and causes the transcriptional induction of putative antibacterial immune effectors, which are required for C. elegans to resist infection by P. aeruginosa [C. elegans to detect pathogen invasion.Disruption of mitochondrial homeostasis often accompanies bacterial infection, which occurs at least in part through the direct effects of pathogen-encoded toxins that poison mitochondrial function . Interesematodes . Indeed,ematodes ,12–14. Druginosa . In addiruginosa . All togC. elegans. Disruption of the ubiquitin proteasome system, which targets proteins for degradation, leads to immune effector activation [Intriguingly, several other examples of surveillance immunity have been described in tivation ,15. In ativation . Likewistivation ,9.C. elegans has come from the recognition that host-derived signals of cellular damage are potent activators of immune responses. In mammals, these immune response elicitors are called damage-associated molecular patterns (DAMPs). A tyrosine derivative, hydroxyphenyllactic acid (HPLA), accumulates in C. elegans following infection with Drechmeria coniospora, a fungal pathogen that first attacks the extracellular cuticle of nematodes and evokes a potent antifungal immune response in the epidermis. HPLA is recognized by the epidermal G protein-coupled receptor DCAR-1 to activate immune defenses, which is required to resist killing by D. coniospora [C. elegans epidermis [Another key insight into the mechanisms of pathogen detection in niospora . The indpidermis . Disruptpidermis . Thus, iC. elegans possess a suite of inducible genes, including cytochrome P450s and glutathione-s-transferases, that metabolize toxins [An extension of the immune surveillance hypothesis involves the host response to chemical toxins, which often poison the same cellular processes as pathogen-encoded effectors. As in mammals, e toxins . Interese toxins –9. In ade toxins ,21. Moree toxins . The up-


Vaccinium corymbosum (VC). In contrast, the wild species V. arboreum (VA) is able to tolerate a wider range of soil pH. To assess the molecular mechanisms involved in near neutral pH stress response, plants from pH-sensitive VC (tetraploid) and pH-tolerant VA (diploid) were grown at near neutral pH 6.5 and at the preferred pH of 4.5.Blueberries are one of the few horticultural crops adapted to grow in acidic soils. Neutral to basic soil pH is detrimental to all commonly cultivated blueberry species, including V. corymbosum, transforming ~55% of the reads to gene counts. A quasi-likelihood F test identified differential expression due to pH stress in 337 and 4867 genes in VA and VC, respectively. Both species shared regulation of genes involved in nutrient homeostasis and cell wall metabolism. VA and VC exhibited differential regulation of signaling pathways related to abiotic/biotic stress, cellulose and lignin biosynthesis, and nutrient uptake.Transcriptome sequencing of root RNA was performed for 4 biological replications per species x pH level interaction, for a total of 16 samples. Reads were mapped to the reference genome from diploid The specific responses in VA likely facilitate tolerance to higher soil pH. In contrast, response in VC, despite affecting a greater number of genes, is not effective overcoming the stress induced by pH. Further inspection of those genes with differential expression that are specific in VA may provide insight on the mechanisms towards tolerance.The online version of this article (doi:10.1186/s12864-017-3967-0) contains supplementary material, which is available to authorized users. Vaccinium is an economically important genus within the Ericaceae family that includes blueberries (Vaccinium spp. subgenus Vaccinium) and cranberries (Vaccinium spp. subgenus Oxycoccus). V. corymbosum  is a high-value fruit crop that exhibits strict soil adaptation to acidic soils with high organic matter content , how VA responds to pH stress [VA (6.5–4.5)] and what genes are driving the differences in response to stress between the species [VC (6.5–4.5) - VA(6.5–4.5)]. Despite VC having 14 times more genes responding to pH stress, they only overlapped with 40% of the genes regulated by VA, most of which were regulated in the same direction Fig. . In view.5–4.5], p-value in ascending order. logFC, log-abundance ratio as fold change; logCPM, log-average concentration in counts per million reads, normalized by library size; PValue, exact p-value for differential expression using the NB model; FDR, the p-value adjusted for multiple testing; uniprot_id, evalue and description belong to best matches against plant TrEMBL protein database. Genes without annotation are not shown here  and diploid VA (B) blueberry species grown at pH 6.5 compared to growth at pH 4.5 in hydroponic medium, and differential response of VC compared to VA response (C). Genes are ordered by Soil pH is a major component of soil chemistry. Many essential chemical elements exhibit high solubility in the soil matrix at near neutral pH. However, blueberries and their relatives are adapted to acidic soils –3, 62, w+ ions, these species exhibited downregulated transcription of a nitrate excretion transporter (CUFF.28540) that is coupled with H+ excretion activity in Arabidopsis grown at acidic pH [The plants also regulated detoxification processes in response to the modification in soil ion composition at higher pH. Thus, they upregulated the expression of a drug efflux transporter of the MATE family (CUFF.2426) similar to a member that improves tolerance to biotic stress in Arabidopsis . In accocidic pH . Detoxifcidic pH . Additiocidic pH .Another focus in the transcriptional response to growth at higher pH in VA and VC is the cell wall. Primary cell walls are composed of cellulose microfibrils and structural proteins . SecondaVaccinium spp. when exposed to higher pH, [Higher pH also affected other physiological processes. For example, VC and VA shared downregulation of genes involved in the biosynthesis of signaling molecules in response to stress and salicylic acid , ethylene  or jasmonic acid . Upregulated metabolic enzymes also responding to ethylene were 2-oxoglutarate-dependent dioxygenases , which are related to iron-deficiency response in Arabidopsis . Additiogher pH, . Finallygher pH,  was alsoAltogether, these results indicate the similar transcriptional responses of VA and VC to growth at pH 6.5, including detoxification mechanisms, modification of the cell wall lignin component and signaling pathways involved in cell growth. We discuss the contrasting transcriptional responses between these two species next.When visualized in an MDS plot, the global gene expression patterns of VC roots separated samples between pH 4.5 and pH 6.5 on the second dimension, while the samples of VA clustered together. Previous field observations suggested that VA exhibits tolerance to higher pH soils , but thiVaccinium spp. has been previously documented [In this experiment, there were transcriptional responses related to nutrient uptake capacity. VA upregulated the expression of amino acid  and olicumented , these fcumented , with upcumented  was upreFurther downregulation was observed in genes related to the primary and secondary cell wall construction and repair in VA grown at higher pH, in addition to those previously described. For example, VA downregulated a boron efflux transporter (CUFF.48824), required for crosslinking of the pectic polysaccharide rhamnogalacturonan II (RG-II), as well as a xylanase (CUFF.51472) and a bifunctional α-L-arabinofuranosidase/β-D-xylosidase CUFF.53455), wall-modifying enzymes that act on hemicellulose at the cell wall. Finally, genes that encode proteins involved in monolignol production (CUFF.12829) and polymerization , and in suberin biosynthesis (gene.g8711.t1), were also downregulated. The modifications performed by VA to the cell wall machinery are less drastic in comparison to VC in view of the greater number of genes that VC regulates and VA doesn’t, coding proteins that modulate cell wall extensibility , cell wall plasticity  and lig455, wallSensitive to Proton Rhizotoxicity 1 (STOP1) were downregulated in VA (CUFF.54376) and VC (CUFF.15468 and CUFF.29512). Previous research showed that STOP1 is upregulated under low pH stress and participates in H+ and Al tolerance [STOP1 has also been associated with mechanisms for stabilizing the cell wall [STOP1, an H+ efflux transporter and multiple genes targeting the cell wall being downregulated at higher pH in both species. Intriguingly, while Al-responsive genes were not DE in VA, Al-activated malate transporters (CUFF.11109 and CUFF.20376) were upregulated in VC at higher pH. The pleiotropic effects of STOP1 suggest the activation of different responses in VA and VC by their respective genes that affect their tolerance capacity to higher pH. In addition to RNA-Seq, further validation by gene sequencing and qPCR will provide additional resources in future experiments to elucidate the role of STOP1.A small set of transcription factors were specifically DE in the roots of VA in comparison to VC. Some downregulated genes were involved in producing the Casparian band , cell wall formation (CUFF.1106) and ROS-mediated responses , while upregulated genes included a negative regulator of resistance genes , an ethylene signaling mediator  and a calmodulin-binding transcription activator (CUFF.56809). Additional transcription factors that were DE involved organ development  and stress response . Downregulation of a negative regulator of defense response with MYB30 ubiquitination activity (CUFF.39430) was also observed. Interestingly, different genes that encode homologs to Arabidopsis olerance . STOP1 hell wall . These f2+-dependent signaling were DE . Signaling cascades induce transcriptional changes that affect a vast number of physiological processes, some of which were already discussed. Additionally, DE is observed in genes associated with senescence , detoxification , components for endosomal trafficking , carbon transport (CUFF.51281) and lipid transport .DE of genes involved in hormone-mediated responses was also observed at higher pH in VA. Upregulated genes were involved in biosynthesis of ethylene  and jasmonic acid  and also gibberellin deactivation (CUFF.45537), whereas downregulated genes were involved in brassinosteroid biosynthesis (CUFF.18539), jasmonic acid metabolism (CUFF.14932) and cytokinin deactivation (CUFF.39626). Also, three hormone transporters of the ABC family were downregulated . Not only signaling was mediated through hormones, but also genes involved in CaVaccinium spp. at higher pH. In this scenario, VA and VC exhibit large transcriptional differences in relation to the cell wall and secondary metabolism, which participate in the mechanical and chemical protection of cells. The less extensive response to disease and healthy growth of VA at higher pH would be an indication of a better protection against pathogens than VC. Moreover, transcriptional responses to disease pressure might also play a role in the dissimilar soil adaptations of these species.Finally, a few genes related to disease resistance were either upregulated  or downregulated  in VA, in contrast with the response in VC that involved a much larger number of genes  or stressful higher pH (pH 6.5). While both VA and VC exhibited DE of nutrition- and detoxification-related genes in response to growth at higher pH, VA exhibited a wider transcriptional response towards Cu homeostasis suggesting a response to Cu scarcity. Both VA and VC exhibited downregulation of multiple genes involved in primary and secondary cell wall biosynthetic machinery at higher pH. However, the additional changes triggered by VC suggest a reduced adaptability of the cell wall in comparison to VA. Moreover, VA and VC downregulated different genes similar to the transcription factor Additional file 1: Figure S1.Hydroponic setup. (JPEG 2413 kb)Additional file 2: Figure S2.MA-plot of normalized counts. (TIFF 710 kb)Additional file 3: Table S1.p < 0.05 after Benjamini & Hochberg false discovery rate (FDR) correction. (XLSX 1020 kb)Lists of genes with differential expression in blueberry under pH stress. Significant genes in VC (A), VA (B) and their different response (C). Based on the quasi-likelihood F test. Additional file 4: Table S2.Arabidopsis thaliana sequences in NCBI. Arabidopsis descriptions include, when available, enzyme and pathway information from KEGG and AraCyc [Sequence similarity-based annotation of blueberry gene models. Annotations obtained from proteins in the TrEMBL database for plants and d AraCyc .  and VC (B) were subjected to singular enrichment analysis in AgriGO. The table contains output from the analysis performed using the combination AgriGO and REVIGO tools. GO terms with FDR < 0.5 after Yekutieli multi-test in AgriGO were used on REVIGO and searched for genes with that specific annotation. (XLSX 184 kb)Additional file 6: Table S4.Arabidopsis protein homolog, log fold change (FC) in V. arboreum and logFC in V. corymbosum. (XLSX 17 kb)Summary of discussed genes. Collection of genes present in discussion organized by function. The columns represent category/gene ID, description of best UniProt protein homolog, description of best