H. pylori infection in recent years. First-line therapies are facing a challenge because of increasing treatment failure due to elevated antibiotics resistance. Several new treatment strategies that recently emerged to overcome antibiotic resistance have been surveyed. Alternative first-line therapies include bismuth-containing quadruple therapy, sequential therapy, concomitant therapy, and hybrid therapy. Levofloxacin-based therapy shows impressive efficacy but might be employed as rescue treatment due to rapidly raising resistance. Rifabutin-based therapy is also regarded as a rescue therapy. Several factors including antibiotics resistance, patient compliance, and CYP 2C19 genotypes could influence the outcome. Clinicians should use antibiotics according to local reports. It is recommended that triple therapy should not be used in areas with high clarithromycin resistance or dual clarithromycin and metronidazole resistance.This paper reviews the literature about first-line therapies for  Helicobacter pylori (H. pylori) is the most important aspect of managing H. pylori-related gastrointestinal diseases. In the past decade, the Maastricht III Consensus Report has recommended that proton pump inhibitor- (PPI-) clarithromycin-amoxicillin or metronidazole treatment is the first choice for H. pylori infection [Eradicating nfection . Althougnfection , 3, mostnfection –7. Accornfection –10. The nfection . The facnfection , 13. BesHelicobacter pylori meeting 2012 in Singapore: (1) in areas with low clarithromycin resistance rates, standard triple therapy should be the primary choice, while bismuth-containing quadruple, sequential therapy, and concomitant therapy could be alternative first-line therapies and (2) in areas with high clarithromycin resistance, regimens including bismuth-containing quadruple, sequential therapy, and concomitance should be the better choice for first-line regimens. This paper will introduce recent novel and acceptable regimens as the first-line therapies of H. pylori and the factors influencing eradication.In order to overcome the challenge of decreasing eradication rates, many novel first-line therapies have been developed. According to guidelines of the Maastricht III, the minimal acceptable eradication level recommended is an 80% eradication rate (by intention-to-treat (ITT) analysis) . Graham Triple therapies are still the most commonly used first-line therapy in the world despite decreasing efficacy . Clarith However, prolonged duration of standard triple therapy might be a good method to overcome the challenge of resistance. A systemic review showed that the distribution of clarithromycin-resistant strains ranged from 49% (Spain) to 1% (Netherlands) worldwide . One AmeH.  pylori Infection [H. pylori infection. Three studies with this combination given for 10 days have demonstrated eradication  rates more than 90% [The Maastricht III Consensus Report  and the nfection  both recthan 90% –23. One  However, there is no agreement with the duration of bismuth-containing quadruple therapy now. Ten or fourteen days are often used durations in these regimens . FurtherH. pylori eradication was 1.21 (95% CI 1.17–1.25). This meta-analysis showed a trend preferring sequential therapy to triple therapy. Sequential therapies also demonstrated better eradication rates than standard triple therapy for clarithromycin-resistant strains  [A 10-day sequential therapy consists of a 5-day dual therapy with a PPI  and amoxicillin  followed by a 5-day triple therapy with a PPI , clarithromycin  and metronidazole . This novel therapy shows an impressive eradication rate above 90% –28. The However, there is significant heterogeneity observed between results from Asia and Italy. One study in Asia compared the sequential therapy with standard triple therapy and found that the two methods did not have significantly different eradication rates  . This su This regimen containing four-drug regimen: a PPI , clarithromycin , amoxicillin , and metronidazole . All drugs are given during the course of concomitant therapy . A meta-H. pylori with dual resistance (metronidazole and clarithromycin). They demonstrated hybrid therapy with high eradication rates: 97%  and 99% . This study also surveyed the efficacy in the treatment of H. pylori with dual resistance. It also showed encouraging results. Tests on the efficacy of this new regimen is needed with further studies. Hsu et al.  presenteLevofloxacin could be used as an alternative agent for clarithromycin in either a standard triple, quadruple, or sequential regimens. The use of levofloxacin in first-line therapy has also been surveyed. The eradication rates of levofloxacin-based triple therapy ranged from 72% to 96% . The var The optimal dose of levofloxacin is another interesting point. The commonly used dosage of levofloxacin was 500 mg daily in many studies in Asia . StudiesH. pylori. Primary resistance to levofloxacin ranges between 8% and 31% in different countries or regions [One critical point should be remembered that quinolone resistance is raised rapidly in eradication of  regions –43. Inap regions .H. pylori [Rifabutin is an antituberculous agent and it is also effective for eradicating . pylori . The opt. pylori . AnotherH. pylori. Resistance rates are remarkably variable in different geographic areas and therefore it is necessary to select the drugs according to local resistance patterns [H. pylori clarithromycin resistance rates was mainly resulted from the long-term use of clarithromycin for respiratory tract infections [Antibiotic resistance is the most serious problem in eradicating patterns . Clarithfections . A systefections . A meta-fections . Metroni The resistance to metronidazole is between 30 and 40% , 49, alt Resistance against amoxicillin is usually low around the world, so its resistance does not influence the use in treatment regimens.H. pylori in Europe (2008-2009) is around 14.1% (4.0–28.0%) [Resistance to levofloxacin has increased rapidly in recent years and the worldwide resistance rate is around 16.2% (95% CI 14.4–18%). In Taiwan, about five-fold increase in levofloxacin resistance was observed in primary resistance  . Average0–28.0%) . ResistaH. pylori include (1) PPIs make acid-labile antibiotics more stable by increasing gastric pH value, especially clarithromycin, thereby increasing concentration and H. pylori sensitivity to antibiotics (2) PPIs may alter transport of antibiotics from plasma to gastric juice, increase luminal concentrations and elevating the success rate of eradication [H. pylori eradication rates were noted in many kinds of PPIs [The polymorphisms of CYP2C19 lead some patients to metabolize PPI more rapidly than others. Patients are divided into three phenotypes: extensive (EM), intermediate (IM), and poor (PM) metabolizers. Ethnic differences in the frequencies of CYP2C19 gene polymorphism are well known. Asian people have a higher proportion of poor metabolizers (20 versus 5%) compared to whites , 51. Thedication . CYP2C19 of PPIs , 54. How of PPIs . The effect of increasing dose is unclear. One study in China demonstrated that increasing the dosage of omeprazole (20 to 40 mg) would improve the efficacy of eradication , but othH. pylori, so it is necessary to find alternative methods to improve eradication rate and compliance in first-line therapy. So many studies have tried new treatment approaches by using probiotics. Several studies have previously reported that certain probiotics exhibit inhibitory activity against H. pylori in vitro and in vivo [H. pylori eradication therapy because they could increase compliance by increasing tolerability and preventing side effects [H. pylori include production of inhibitory substance, host immune modulation or competition for adhesion [It is difficult to develop new effective antibiotics to eradicate  in vivo , 59. Ear in vivo –62. So p effects –66. The adhesion , 67, 68.adhesion , 70.Saccharomyces boulardii and Lactobacillus supp. are the most common probiotics used in clinical trials. Several meta-analysis studies showed that standard triple therapy accompanied with the Saccharomyces boulardii or Lactobacillus supp. could increase eradication rates and decrease therapy-related side effects, especially diarrhea and taste disturbance [ In previous studies, turbance –74. H. pylori eradication. In summary, the exactly mechanism of probiotics is largely unknown and further research is greatly needed. The restoration of the normal flora in the intestine might be important in patients receiving triple therapy for H. pylori infected patients allergic to penicillin, the previously recommended first-line treatment with omeprazole-clarithromycin-metronidazole has low efficacy for curing the infection. So other regimens which include bismuth-containing, non-bismuth-conatining quadruple therapies or levofloxacin-based triple therapy should be taken into consideration. These regimens showed better and acceptable eradication outcomes [Drug allergy to penicillin is also an important factor influencing regimen chosen. In outcomes . So it iH. pylori eradication therapy. One meta-analysis of 5538 patients in 2006 revealed that the summary OR for eradication failure among smokers relative to nonsmokers was 1.95 . It also showed a better eradication rate of 8.4%  in nonsmokers [ Smoking might cause failure of H. pylori infection are facing a challenge because of increasing treatment failure. The paper reviews several new treatment strategies with the intention to overcome antibiotic resistance (H. pylori infection is a common and serious infection, and we should prescribe the first-line regimens more carefully and empirically. Clinicians should use antibiotics according to local reports. First-line therapies of sistance . Alterna


Flavobacterium columnare (F. columnare) is the causative agent of columnaris disease. This bacterium affects both cultured and wild freshwater fish including many susceptible commercially important fish species. F. columnare infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely remains unclear, compromising the further development of efficient curative and preventive measures to combat this disease. Besides elaborating on the agent and the disease it causes, this review aims to summarize these pathogenesis data emphasizing the areas meriting further investigation. The agent1.1 History and taxonomy1.1 Morphology and biochemical characteristics1.1 Epidemiology2. The disease2.1 Clinical signs, histopathology, ultrastructural features and haematology2.1 Diagnosis3. Pathogenesis3.1 Colonization3.1 Exotoxins, bacteriocins and endotoxins3.1 Interaction with the fish immune system4. Importance of environmental factors5. Experimental trials/pathogenicity tests6. Control6.1 Preventive measures6.1 Curative approach7. Conclusion8. Competing interests9. Authors’ contributions10. Acknowledgements11. ReferencesFlavobacterium columnare (F. columnare), the causative agent of columnaris disease, belongs to the family Flavobacteriaceae[Bacillus columnaris and the disease elicited columnaris disease. Over the decades the taxonomic status of the pathogen has changed several times since the pioneering work of Davis [Onchorhynchus nerka) [Myxobacteria are long, thin Gram-negative rods that are motile on agar media by a creeping or flexing motion. They have a life cycle composed of vegetative cells, microcysts (resting cells), and fruiting bodies, or only vegetative cells and microcysts [Chondrococcus columnaris[Cytophagaceae as Cytophaga columnaris after isolating a pathogenic bacterium resembling Chondrococcus columnaris morphologically, but not producing microcysts [Cytophagaceae and the genus Flexibacter, assigning it as Flexibacter columnaris[Flavobacterium columnare, based on DNA-rRNA hybridization data and protein and fatty acid profiles [F. columnare cluster was subdivided in three genomovars based on differences in 16S rRNA sequences, restriction fragment length polymorphism (RFLP) and DNA-DNA-hybridization [teriaceae-3. Columteriaceae. Althougteriaceae. Upon exof Davis . In 1944s nerka) . A dilutcrocysts . Ordal aolumnaris. Garnjobcrocysts . Bernardolumnaris. FinallyF. columnare are summarized in Table F. columnare, the reader is referred to Bernardet and Bowman [The morphological and biochemical characteristics of d Bowman .F. columnare is distributed worldwide in fresh water sources and may infect many different wild and cultured freshwater fish species, such as (but not limited to) carp, channel catfish, goldfish, eel, perch, salmonids and tilapia [Ictalurus punctatus) industry in the United States, F. columnare is the second most prevalent bacterium, after Edwardsiella ictaluri, to cause disease and mortality [F. columnare infection can release up to 5 × 103 colony forming units/mL/h of viable bacteria into tankwater [ tilapia ,12-17. T tilapia . In the ortality ,19, withortality . This orortality . Fish maortality ,23. Fujiankwater . The gilankwater .F. columnare to survive for extended periods in water. Survival was demonstrated to be influenced by physical and chemical characteristics of the surrounding water. Fijan indicated that F. columnare can survive up to 16 days at 25°C in hard, alkaline water with a high organic load [3, especially when acid or with a low organic content, does not provide a favorable environment for the organism [F. columnare in water [F. columnare in static, sterile river water was directly related to temperature, with a higher survival percentage at lower temperatures [F. columnare is also capable of surviving in sterile river mud [F. columnare longer than sterile river water. In this case, however, the percentage survival of F. columnare seeded into mud seems to be higher at 25°C than at 5°C. Temperatures below 5°C are even detrimental to F. columnare cells in mud. F. columnare also grows well on particulate fish feed [F. columnare can change from a virulent to a less virulent form with an altered colony morphology, probably to save energy [F. columnare strains at fish farms originate from environmental waters and that the farm environment and practices may select for virulent strains that cause outbreaks in the farm [Several studies have indicated the potential for in water . Ross aneratures . The baceratures . F. coluish feed . When sue energy . It has the farm ,30.F. columnare causes acute to chronic infections and typically affects the gills, the skin and fins. The clinical manifestation of columnaris disease amongst others is dependent on the virulence of the eliciting strain. In a study of Rucker et al., the strains of low virulence induced slow progressive infections at water temperatures above 21°C and caused massive tissue damage before death occurred [Salmo salar) in 12 to 24 h at 20°C. Ordinarily, these fish did not show gross tissue damage at the time of death [F. columnare of high virulence was usually very limited. Apparently, death occurred before gross external manifestations of the disease appeared. However, some of the last fish to die did show macroscopically visible signs [occurred . Strainsof death . The samof death  and Foscof death . The grole signs . Besidesle signs ,35.In chronic cases, it takes longer before gill damage appears and skin lesions may develop as well ,32,35. OIn rainbow trout, the area around the adipose fin may become dark and show erosions. These lesions expand to the peduncle, hence the name “peduncle disease” . The lesUlceration of the oral mucosa also occurs, resulting in mouthrot. These mouth lesions are more lethal than are the skin lesions, since the painful oral lesions render the fish anorectic and lead to death due to starvation. Moreover, the disease spreads easily to the mandible and the maxilla. Secondary infections with fungi or other bacteria may deteriorate the situation and can be seen together with the filamentous bacteria . In tropF. columnare from internal organs without any external lesions appearing [The skin or gills need to be abraded, for bacteria to enter the bloodstream and cause systemic infections . Howeverppearing . Foscarippearing . The firppearing .F. columnare can be found on the cell surface and/or in between necrotic sites  or Giemsa stained sections from affected tissue where they appear bluish-purple and blue, respectively . When alScanning electron microscopic (SEM) pictures of affected gill arches reveal the presence of rod-shaped bacterial cells, approximately 0.3-0.5 μm wide and 3-10 μm long Figure . These lF. columnare in channel catfish and zebrafish (Danio rerio) using SEM [Bullard and McElwain were the first to describe the ultrastructural features of saddleback lesions associated with experimental infections of sing SEM . ChannelF. columnare also revealed anemia, but levels of mean corpuscular volume and mean corpuscular haemoglobin were higher [In a cutaneous columnaris challenge model in koi carp, significant changes in blood parameters were observed in the infected fish . For thee higher . Total pe higher .Timely detection of this pathogenic agent is important to prevent its spreading and to reduce the economic loss to fish farmers.F. columnare is possible from external lesions, provided that the samples are taken from the edge of recent lesions [F. columnare requires low nutrient media. No growth of F. columnare is reported on trypticase soy agar (TSA), nutrient agar or Marine 2216 agar [F. columnare was first cultured on Cytophaga agar, a nutrient poor medium [F. columnare is easily overgrown by contaminating bacteria, selective media have been developed based on inherent resistance of F. columnare to different antimicrobial agents, including polymyxin and neomycin [F. columnare[The isolation of  lesions . F. colur medium . Since tr medium  and TYESr medium  have beer medium ,12, withneomycin ,25. Decocolumnare.F. columnare largely displays two colony types on solid media; smooth and rhizoid [F. columnare are notorious for their sometimes strong adherence to the agar. This trait is also exhibited in broth cultures where yellow, filamentous clumps of bacterial cells can form a thick ring at the surface of a glass recipient [F. columnare is whole-cell protein analysis [F. columnare into three genomovars I-III [F. columnare[F. columnare based on the selective amplification of the 16S ribosomal RNA gene using species-specific primers [ rhizoid . Kunttu  rhizoid . The rhi rhizoid . Colonieecipient ,50. Adheecipient . A methoanalysis . Genomovrs I-III . Comparecolumnare. Polymer primers ,53. This primers .Flavobacterium columnare from infected fish organs  was established in channel catfish [F. columnare[F. columnare[F. columnare in tissues  of infected fish. Moreover, real-time PCR-based methods are distinctively more advantageous than conventional PCR as they eliminate the need for detection of amplified products by gel electrophoresis, thus reducing costs, time and labour [F. columnare in fish tissue [Besides isolation, other methods may be used for detecting this pathogen. These include serological methods such as the enzyme-linked immunosorbent assay  and the  catfish . PCR maycolumnare,53. In acolumnare. This PCd labour . Length d labour . LH-PCR h tissue .F. columnare strains [F. columnare, especially a plasmid-based inframe knockout system [F. columnare to improve electroporation efficiency and suggested that it would be of significant interest to examine the composition and diversity of R-M systems in strains of F. columnare in order to set up a suitable genetic manipulation system for the bacterium [F. columnare ATCC 49512 was sequenced [Various reports have noted the difference in virulence among  strains ,42,61-65t system , despitet system ,68. Li eacterium . Only veequenced .The colonization of the fish tissue is to be regarded as a complex multistep process which can be subdivided into the stages of attraction, adhesion and aggregation, requiring a step-by-step analysis. The exact factors mediating colonization have however not yet received the full attention they merit and to date remain largely unidentified.F. columnare[. The gliding motility of this bacterium is well known [F. columnare. Pate and Ordal described that the location of fibrillar structures spanning the gap between the outer membrane and the mucopeptide layer might play a role in the gliding motility of the F. columnare bacterial cells [F. columnare, the chemotactic response of the more virulent genomovar ll isolates suggested that chemotaxis could be associated with virulence [F. columnare to skin mucus using blind-well chemotaxis chambers [N-acetyl-D-glucosamine) associated with the capsule of F. columnare might be involved in chemotactic responses [gldH was found to be significantly (P < 0.001) upregulated in F. columnare as soon as five minutes post-exposure to catfish mucus. When pretreated with D-mannose, there was no upregulation of gliding motility genes [The research group of Klesius et al. demonstrated by means of the traditional capillary tube method that the mucus from the skin and gills of catfish promotes chemotaxis of columnare. The glill known ,12,41. Ial cells . Althougirulence . LaFrentchambers  generatichambers . At leasesponses . The glity genes .Poecilia reticulata) than did the low virulence strain [F. columnare to the gill tissue constitutes an important step in the pathogenesis of columnaris disease. Bader et al. adopted an adhesion defective mutant of F. columnare in immersion challenge trials and found that the mortality was reduced with 75% and occurred 24 h later compared to the strains that still possessed the adhesion capacities, confirming the findings of the former research group [F. columnare demonstrated a difference in the cumulative percent mortalities (CPM), with the genomovar II isolates inducing significantly higher CPM [F. columnare genomovar I and II strains to the skin and gill of channel catfish and the gill of zebrafish (Danio rerio) [F. columnare exhibit different levels of adhesion to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease. The same statement was previously made by Klesius et al. [The ability to adhere is a prerequisite for the successful colonization of the host tissue. Decostere et al. performed a series of both in vivo and in vitro experiments and found that a highly virulent strain adhered more readily to the gill tissue of black mollies  . At 0.5 s et al. .F. columnare to polystyrene and put forward the hypothesis that there is a link between virulence and rhizoid colony morphology [F. columnare changes colony morphology during experimental long-term storage in lake water, indicating the importance of different colony morphologies to bacterial survival [Kunttu et al. have shown that colony morphology affects the adhesion capacity of rphology ,74. The survival ,74,75. AF. columnare produces two types of mucus. The first one is an acidic polysaccharide and is made visible by ruthenium red staining. Another type of mucus is a basic, partially acetylated polygalactosamine, which cannot be stained with ruthenium red. Pate and Ordal described a capsular material which coated the surface of the bacterial cell that could be stained with ruthenium red [F. columnare strain to the gill were significantly reduced following treatment of the bacteria with sodium metaperiodate or incubating them with D-glucose, N-acetyl-D-glucosamine, D-galactose and D-sucrose [nium red . They st-sucrose . Treatme-sucrose . The samF. columnare bacterial cell aggregates around goblet cells in common carp [F. columnare binding of host RBL. Moreover, RBL expression was upregulated in fish fasted for 7 d (as compared to fish fed to satiation daily), yet expression levels returned to those of satiated fish within 4 h after re-feeding. These findings highlight putative roles for RBL in the context of columnaris disease and reveal new aspects linking RBL regulation to feed availability [Sun et al. conducted the first transcriptomic profiling of host responses to columnaris disease following an experimental immersion challenge by using illumina-based RNA-sequencing expression profiling . A rhamnmon carp  and catfmon carp  followinmon carp . Beck etmon carp . In the lability . FurtherF. columnare remains to be elucidated.The adhesion to the fish tissue was shown to be impacted by various environmental parameters. Using a gill perfusion model ,80, DecoF. columnare strain onto gill tissue is a distinct feature in both in vivo and organ culture experiments [F. columnare bacterial cells, most likely impeding oxygen uptake and causing death of the fish. These microcolonies are not observed when a low virulence strain is used. Biofilm formation capacity was demonstrated in vitro for one F. columnare strain when exposed to mucus [F. columnare, warranting its further investigation.An aggregative adhesion pattern of a highly virulent eriments ,42,53,61to mucus . These fN-acylhomoserine lactone (AHL) [Flavobacterium sp., but no AHL-presence could be confirmed using gas chromatography-mass spectrometry (GS-MS) [Tenacibaculum maritimum strains, biofilm-forming members of the phylum Bacteroidetes, formerly referred to as “Cytophaga-Flavobacterium-Bacteroides” group [F. columnare.The developmental switch to the biofilm state is commonly regulated by quorum sensing. Quorum sensing (QS) is a mechanism of gene regulation in which bacteria coordinate the expression of certain genes in response to the presence or absence of small signal molecules. In many Gram-negative bacteria, the signal molecule is an ne (AHL) . Additione (AHL) . Wagner- (GS-MS) . Using ls” group . To our F. columnare produces an enzyme that degrades chondroitin sulfates A and C and hyaluronic acid, the complex polysaccharides of connective tissue. This so-called chondroitin AC lyase acts specifically on a group of acidic mucopolysaccharides found primarily in animal connective tissue [F. columnare[F. columnare strains, both high AC lyase activity and gliding motility of the bacteria would be needed for F. columnare to be virulent [Flavobacterium columnare derived primarily from channel catfish raised in the southeastern United States [It is known that polysaccharide degradation in combination with the secretion of various extracellular enzymes participate in the destruction of skin, muscle and gill tissue , enhancie tissue . No corre tissue . AC lyascolumnare,65. Thouvirulent . Proteasvirulent . Newton d States . The bacd States .F. columnare release specific, non-transmissible, bactericidal substances equivalent to colicins of Escherichia coli into the environment to reduce competition from other bacterial strains [F. columnare also possess multiple specific receptors for the bacteriocins, and, consequently, the cells are sensitive only to those bacteriocins for which the cell possesses receptors [Pseudomonas sp. MT5 against F. columnare bacteria was found to be very strong in agar assays [Pseudomonas bacterial strain could not yet prevent nor treat a F. columnare infection following experimental challenge [Bacteria not only need to enter the host tissue, they also need to eliminate competitive bacteria. Different strains of  strains . It was eceptors . Antagonr assays . Howeverhallenge .F. columnare retrieved from channel catfish have been detected [F. columnare isolate lacked the high molecular weight components of LPS present in virulent isolates. Based on these differences of LPS and total protein profiles, the research group was able to discriminate the attenuated mutant from other F. columnare strains [F. columnare in rainbow trout [Differences in LPS composition between highly and low virulent strains of detected . Analysi strains . Kunttu ow trout . Colony ow trout .F. columnare have been demonstrated. In an experimental challenge trial of cutaneous columnaris disease in koi carp [F. columnare inoculated agar plates, a lower number of colonies was counted on plates to which mucus was added. Fluorescent microscopy of a stain-based bacterial viability assay also revealed a higher number of dead bacteria in F. columnare cultured with mucus [F. columnare strain cultured in media supplemented with juvenile Atlantic salmon  skin mucus with the same media without mucus [F. columnare, induced the bacteria to grow as a biofilm and increased extracellular protease activity [F. columnare strain which is critical in determining the antibacterial capacities of the mucus.When encountering an infection, the first system activated is the innate immune system. It is assumed that the surface mucus layer, as a first physical-immunological barrier, plays an important adhesive role and that it is part of the innate host resistance of fish to disease . Antibackoi carp , lesionsth mucus . Staroscut mucus . Interesactivity . This miAnguilla japonica) in a F. columnare suspension to monitor dermal nonspecific stress responses [The fish skin in itself also forms a barrier against pathogens. Aranishi et al. immersed Japanese eel  was inhibited by large amounts of sialic acid contained by Gram-negative bacterial pathogens, including F. columnare[F. columnare, as very little or no bactericidal activity was produced against this bacterium by the catfish ACP. Furthermore, the latter greatly increased after removal of sialic acid with neuraminidase [F. columnare infections, is the lack of an inflammatory response as observed upon inspecting affected tissues microscopically. This resulted in the hypothesis that F. columnare triggers the endogenous programmed cell death machinery of immune cells to evade the immune system. Do Vale et al. have already proven that apoptosis can be a very powerful pathogenic strategy by inducing lysis of phagocytic cells [F. columnare via secreted toxins [F. columnare infection. Rasputin plays an inhibitory role in negative regulation of apoptosis and was highly downregulated at all examined time-points [F. columnare as was described by Morrison et al. [Some studies reveal that columnare. Sialic minidase . Anotheric cells . Sun et d toxins . Furthere-points . This con et al. .F. columnare is resistant to the bactericidal action of serum of non-immunized catfish via the ACP [F. columnare[F. columnare can also be influenced by transferrin [F. columnare by intraperitoneal injection with iron-free human transferrin (Sigma) was reduced when iron was injected prior to exposure. The effect of iron was only evident in one of the two strains examined when the challenge was delivered via immersion. These results indicate that iron depletion may limit the virulence of F. columnare more in systemic infections than in external infections. This hypothesis is supported by data indicating that administration of transferrin prior to challenge increased survival after challenge by injection but had little or no effect on bath-challenged fish [Ourth and Wilson demonstrated that  the ACP . Ourth a the ACP , but alscolumnare. The virnsferrin . The surged fish . HoweverF. columnare infections by activating the adaptive immune system [F. columnare cells [6 live F. columnare cells [Oreochromis niloticus (L.)) could mount a significant humoral response in plasma and cutaneous mucus to F. columnare after intraperitoneal immunization with formalin-killed sonicated cells in Freund’s complete adjuvant [F. columnare in the fish feed of three-month-old coho salmon (Oncorhynchus kisutch) [F. columnare were not detected in sera from immunized fish [F. columnare, Aeromonas salmonicida and Yersinia ruckeri antigens, interference from A. salmonicida antigen was shown to suppress responses to two other antigens [Micropterus salmoides) fry and salmon, respectively [Several immunization experiments adopting different administration routes, have proven that fish can be protected from subsequent e system ,22,95-98re cells . Becker re cells . Tilapiaadjuvant . Protectkisutch) . Howeverzed fish . Song’s zed fish . Song wazed fish . Polyvalantigens . Commercectively . VaccinaF. columnare could be one of the many taking advantage of this phenomenon. Indeed, transmission of columnaris disease is more efficient in higher temperatures [Salmo gairdneri) or coho salmon (0. kisutch) experimentally infected with F. columnare were held in water at 12 to 20°C, mortality increased with temperature [F. columnare strain is enhanced at increased temperature [Karvonen et al. described the effect of global warming on the prevalence of different fish parasites and bacteria . F. colueratures ,105. Holperature . As statperature  and chonperature . The infF. columnare[F. columnare significantly increased as unionized ammonia concentrations increased [F. columnare increased the susceptibility of rainbow trout to the bacterial pathogen [Argulus coregoni and F. columnare[Columnaris disease is furthermore influenced by water quality. Decostere et al. observed significantly higher bacterial titers on the gills when organic matter or nitrite were added to an organ bath when performing ex vivo trials with columnare. They dicolumnare. Morris ncreased . These rncreased . Bandillpathogen . Comparecolumnare.F. columnare, the fine and delicate balance and complex interplay between the bacterial cells, fish and environment in the successful reproduction of columnaris disease.A reliable and reproducible experimental infection model is crucial for studying bacterium-host interactions and evaluating the efficacy of both curative and preventive measures. Most researchers recognize, in experimental challenges with Poecilia sphenops) [Notemigonus crysoleucas) [Hitherto, columnaris disease has been reproduced experimentally in black mollies (phenops) , channelphenops) ,110,111,phenops) , golden oleucas) , koi caroleucas) , rainbowoleucas) ,105,113,oleucas)  and zebroleucas) ,44.F. columnare growth medium broth [In the various experimental infection trials, several ways to both cultivate and harvest the bacterial cells for inoculation were adopted. Different media were used to cultivate the bacteria, including Hsu-Shotts or modified Hsu-Shotts medium ,114, Ordum broth  and Shieum broth -45,105. um broth ,109-111.F. columnare strain, to the aquarium water in which fish were immersed [F. columnare[To reproduce columnaris disease experimentally, largely two inoculation routes were adopted, viz. bath (immersion) and injection. With regard to immersion challenges, mostly full-grown broth cultures were added to the water with water temperatures of the immersion water varying from 18°C to 30°C -44,111. immersed . In someimmersed ,94,105 wcolumnare.The infection route, which determines the way the bacterial cells are to be grown/collected, also defines the disease producing capacity of the adopted strains and the disease picture they elicit. Indeed, especially for low virulent strains, higher morbidity and mortality rates were noted after injection of fish as compared to inoculation through immersion . Pacha aOncorhynchus mykiss), Chinook salmon (O. tshawytscha) and coho salmon with F. columnare, via the water-borne route. At a water temperature of 9.4°C, there were no mortalities attributable to F. columnare. By increasing the temperature to 12.2°C, mortality became as high as 4-20%; at 20.5°C, all the steelhead trout and coho salmon and 70% of the Chinook salmon died [Environmental conditions can also highly influence morbidity and mortality rates during a challenge trial. To illustrate the dramatic effects of water temperature on the level of mortalities, the investigation of Holt et al. is particularly relevant . This temon died . High  revealed decreasing mortality as salinity goes up, with significantly lower and no mortalities when salinity reaches values of 1.0‰ and between 3 and 9‰, respectively [F. columnare, since deprivation reduced innate resistance of catfish to columnaris disease [Management plays a key role in the prevention of the disease. Cunningham et al. showed that some commonly recorded production variables  were associated with columnaris disease outbreaks and, if monitored, could help identify “at risk” ponds prior to disease outbreaks . Suomalacolumnare, and theevention . Salt ancolumnare. An in vectively . If the  disease .4) bath at 37 mg/L (1:30 000) or by adding copper sulfate to pond water at 0.5 mg/L [4) to the water at 2 mg/L [4 at doses around 2 mg/L [F. columnare infection from 84–100% to 6–14% [F. columnare[Besides optimizing and adjusting management practices, chemical agents can also be adopted as a preventive approach. Davis concluded that the development or intensification of columnaris disease could be prevented by treating the fish for 20 min in a copper sulfate CuSO bath at t 2 mg/L . Darwishd 2 mg/L . Prophylto 6–14% . Thomas-columnare.F. columnare incorporated into fish feed [F. columnare challenge. This vaccine was also proven to be efficient in largemouth bass fry resulting in RPS values between 74 and 94%, depending on the vaccine dose [F. columnare during the field trial [F. columnare was also brought to the market in the USA as an aid in the prevention of columnaris disease in healthy salmonids of over three grams [Another method to prevent columnaris disease is through vaccination. Although vaccination trials have not always been successful, success rates have increased as knowledge on fish immunity and its role in the defense against bacterial diseases continues to expand. Bath immunization with a bacterin was shown to protect carp against experimental challenge, but no agglutinin could be found in sera from immunized fish . Immersiish feed . Ransom ish feed . Ourth aish feed . As statish feed . At 10 wish feed . An atteish feed . Fry betine dose . Bebak eine dose . Vaccinald trial . An immeee grams .Salvelinus fontinalis) which in vitro revealed antagonistic effects against F. columnare[F. columnare at any concentration of the stimulants used [F. columnare[Probiotics appear to be a promising way in the prevention of different bacterial diseases in aquaculture . Boutin columnare. The strnts used . The rescolumnare.F. columnare[I. furcatus)) (C×B) [F. columnare belonging to genomovar II compared to channel catfish and blue catfish. Beck et al. also found one of the two investigated catfish families to be completely resistant towards F. columnare resulting in no mortality after inoculation with the bacteria [F. columnare resistant families could be of great financial importance in the catfish industry. Interestingly, LaFrentz et al. found that the two families that exhibited the highest CPM after F. columnare challenges, had the lowest CPM following E. ictaluri challenge, the latter being the most prevalent bacterium to cause disease and mortality in the catfish industry [E. ictaluri and resistance to F. columnare, the two leading bacterial diseases in the catfish industry [Recent studies have demonstrated genetic variation in resistance towards columnare,78,124. industry . Furtherindustry .Salmo salar) [A. hydrophila and F. columnare in Sunshine bass  [F. columnare has its negative attributes though [F. columnare strains originating from ornamental fish toward several clinically important antibiotics, such as quinolones and tetracyclines [Treatment of columnaris disease using antimicrobial agents has known different success rates. External treatments are possible only in early stages of the disease, when the infection is still superficial . Drugs wo salar) ,133. Laco salar) . Sulfonao salar) ,135. Nito salar) ,96,130. o salar) . Darwishis male) . The excs though . These is though . Potentis though . Declerccyclines . The res®  was shown to significantly reduce channel catfish mortalities to zero percent after challenge with F. columnare[F. columnare infections in channel catfish when treated in an ultralow flow-through system during 4 h [F. columnare infection [Flavobacterium sp. phage lysates to inhibit growth or lyse the bacterial cultures [F. columnare phage FCP1 to combat columnaris disease in walking catfish (Claries batrachus) [C. batrachus. The result of this study opens new perspectives for the treatment of columnaris disease elicited by antimicrobial resistant F. columnare strains [Besides resorting to antimicrobial agents, chemicals have also been adopted in the curative treatment of columnaris disease. In a study by Thomas-Jinu and Goodwin, the herbicide Diquatcolumnare. The hercolumnare,139. Copcolumnare and potacolumnare are amoncolumnare. Darwishring 4 h . Thomas-ring 4 h . An in vnfection . Laanto cultures . The auttrachus) . Phage t strains .F. columnare strains remains to be unraveled. Full genome sequencing of the reference strain has been an important step towards gaining insight into the pathogenesis of columnaris disease. Efficient molecular manipulation systems could be of great importance to further explore this domain. Genetic knockouts could in effect illuminate virulence factors involved in the pathogenesis of this disease. Although various models to experimentally induce columnaris disease are available, there is a lack of a standardized experimental model hampering the comparison of retrieved data in between research groups and the drawing of consistent conclusions. Consistency in the cultivation protocol of the bacteria in terms of incubation temperature, infection dose, exposure time and infection route is desirable. Extrapolation of a standardized model from one to another fish species is not possible rendering it necessary to set-up an experimental model for each fish sensitive species. Besides further elucidating the pathogenesis of columnaris disease, the development of efficient curative and effective preventive measures also needs to continue. With the upcoming antimicrobial resistance, focus should be laid on the search for preventive measures. Management plans trying to optimize or adjust fish densities and control water quality parameters are a first critical step in controlling columnaris disease. Vaccines, chemotherapeutics and probiotics could possibly have a promising future in the prevention and control of the disease as some studies have already demonstrated. Another interesting envisaged method to mitigate columnaris disease is the creation of a fish breeding program introducing F. columnare resistant fish. Data generated through pathogenesis studies will allow for the further development and optimization of cost-effective and environmentally friendly prevention methods of columnaris disease in the various susceptible fish species.Despite the worldwide importance of columnaris disease and the multitude of studies focusing on its causative agent, only snippets of the bacterium-host interactions have hitherto been exposed. This results in major knowledge gaps still prevailing on amongst others how the pathogen is able to establish and maintain a grip on the skin and gill tissue and elicit disease and mortality. Methods for rapid identification of the bacterium in the environment, host tissue or in culture have indeed evolved rapidly and some techniques permit to trace even a few bacterial cells. However, the rationale for the difference in virulence among The authors declare that they have no competing interests.AM Declercq: Gathering information and papers, writing the outlines, writing the paper; A Decostere: Proposing the subject, reviewing and editing the paper; F Haesebrouck: Critically reviewing the manuscript; W Van den Broeck: Interpretation of histological, SEM- and TEM- pictures, critically reviewing the manuscript, P Bossier: Critically reviewing the manuscript. All authors agreed on outlines and the final version of the paper.


Little is known about exactly which elements of alcohol brief motivational intervention (BMI) make it work. A causal chain between therapist motivational interviewing behaviors, subsequent client change talk (CT), and actual behavior change has been postulated. Other researchers have proposed the notion of a working alliance between counselor and client to explain better outcomes. However, these links have never been tested within a single mediation framework. We investigated the articulation of counselor behaviors, CT, working alliance, and six-month alcohol use outcomes (drinks per week at follow-up adjusted for drinks per week at baseline) during BMI with young men using regression analyses. There were no direct links from counselor skills to alcohol outcomes. Some counselors skills predicted more CT (MI-consistent behaviors frequency [p = 0.02] and MI spirit rating [p = 0.04]) as did working alliance (p < 0.001). However, CT did not predict the alcohol outcome. One subdimension of CT, ability/desire/need to change (ADN), did predict outcome (p = 0.04). None of the tested counselor skills predicted this dimension, but working alliance did (p = 0.03), giving a first significant model . Adding a step in this model, we showed that the percent of MI-consistent behaviors and the MI spirit, empathy, and acceptance ratings predicted better working alliance scores . As the final step, we will calculate the mediated effects in these models. We found that MI skills and behaviors predicted a better working alliance, which, in turn, predicted more ADN change talk, which, in turn, predicted better alcohol use outcome. This gives support to previous findings but also highlights the importance of working alliance. Such findings might give precious clues for clinicians by giving an indication of the most determinant elements in BMI.


Fusobacterium necrophorum subspecies funduliforme, a normal flora found mainly in the oral cavity, appears to be the etiologic organism.Post-partum, post-sterilization tubo-ovarian abscess is a rare event. Fusobacterium necrophorum subspecies funduliforme. Progressively severe symptoms started 3 weeks after her third vaginal delivery with a tubal sterilization on the following day. On admission, she presented with peritonitis and impending shock. An exploratory laparotomy showed a ruptured left tubo-ovarian abscess. A segment of her ileum had to be resected because of severe inflammation.In this case report, a 25-year-old Thai woman had a post-partum, post-sterilization tubo-ovarian abscess caused by the strictly anaerobic bacterium, Fusobacterium necrophorum subspecies funduliforme can be an etiologic organism of a ruptured tubo-ovarian abscess following tubal sterilization in a healthy host. Tubo-ovarian abscess (TOA), characterized by an inflammatory complex mass in the pelvis, accounts for 15% of all pelvic inflammatory diseases (PIDs) . The posParticularly in women who have had tubal sterilization (TS), the incidence of TOA is minimal because the procedure blocks communication between the genital tract and the pelvic cavity . TheoretFusobacterium necrophorum subspecies funduliforme.Three possible explanations of TOA after previous occluded tubes (TOAPOT) have been proposed . The mosgravida 3, para 3) had her third uneventful vaginal delivery followed by TS by modified Pomeroy technique on the following day. She experienced a persistent low-grade fever and abdominal pain that began in the third post-partum week and deteriorated over time. Her medical history was unremarkable. She denied any history of intravenous drug abuse, smoking, alcohol intake, or abdominal trauma. She reported being monogamous. Her partner was healthy and denied any history of sexually transmitted infections. Her previous obstetric history was unremarkable at 8 years and 5 years prior to this delivery. The vaginal delivery of her third baby took place 2 hours after a spontaneous rupture of membranes and progressed uneventfully. Post-partum TS was performed by request without any remarkable findings. She recovered well and was discharged with her baby from the hospital 3 days later.A 25-year-old Thai woman , tachycardia (pulse rate of 112 beats per minute), tachypnea (respiratory rate of 26 per minute), and a blood pressure of 110/60mmHg. An abdominal examination showed a small transverse subumbilical incision scar (from the TS) without any signs of wound infection. A 16-week, pregnancy-sized, non-mobile pelvic mass was found on the left side of her pelvis. The mass and other areas of her pelvis and abdomen were tender, suggesting peritonitis. A pelvic examination revealed a large amount of purulent vaginal discharge, a marked degree of pain elicited upon cervical excitation, a bulging cul-de-sac, and the tender pelvic mass as described in the abdominal examination findings. The results of the rest of the physical examination were unremarkable.3 (90.2% were polymorphonuclear neutrophils), and a platelet count of 422,000/mm3. Levels of blood urea nitrogen and creatinine were 6.2 and 0.5mg/dL, respectively. Blood electrolytes were as follows: sodium 138mmol/L, potassium 3.4mmol/L, chloride 106mmol/L, and bicarbonate 22mmol/L. A slightly prolonged coagulogram was found: a prothrombin time of 15.3 (10 to 13) seconds and a partial thromboplastin time of 34.7 (21 to 30) seconds. The results of a liver function test were normal. The results of a cervical swab and blood culture were negative.Ultrasonograms showed a uterus of 10.1×6.2×5.1cm in size and a smooth thin endometrial lining. A multiloculated mass of 6.2×8.2×10.9cm in size was seen over the left adnexal region. The right adnexal region was unremarkable. Approximately 50mL of free fluid was noted in the cul-de-sac. Laboratory testing revealed a hematocrit level of 32.2%, a white blood cell count of 33,170/mmThe clinical diagnosis was a ruptured TOA. After counseling for a diagnosis and a plan for an urgent exploratory laparotomy, intravenous clindamycin 2700mg per day and gentamicin 240mg per day were initiated. Fluid resuscitation and preparation of blood components were executed.The operative findings showed 50mL of bloody purulent intraperitoneal fluid. A left tubo-ovarian complex Figures  and 2 ofFusobacterium necrophorum. The organism was identified as F. necrophorum subsp. funduliforme by microscopic examination, biochemical characteristics, and 16S ribosomal deoxyribonucleic acid (rDNA) sequencing. The isolate was designated as SIRD333. The 658 base-pair fragment of SIRD333 16S rDNA was submitted to GenBank (accession number JX103157). The surgical pathology showed chronic endometritis, TOA, and ileitis.A total hysterectomy with left salpingo-oophorectomy, ileal resection with end-to-end anastomosis, abdominal toileting, and Penrose drain placement were performed. The operation took 4 hours, and the estimated blood loss was 2000mL. A Gram stain of pus from the abdominal cavity demonstrated numerous polymorphonuclear cells and numerous coccoid pleomorphic Gram-negative rods. An abdominal fluid culture revealed no growth, whereas the pus culture showed Our patient recovered well after the operation and had further treatment with intravenous antibiotics (clindamycin and gentamicin) until being clinically stable for 48 hours . She was discharged on the 10th post-operative day. She continued oral antibiotics (clindamycin) for six more weeks. She was clinically well during all of her follow-up visits.F. necrophorum as the etiologic organism of TOA developing during the post-partum period in a patient who had undergone TS. At our institution, where more than 5000 TS procedures are performed annually by using the modified Pomeroy technique, post-sterilization TOAs are rarely encountered.To the best of our knowledge, this is the first case report of Time intervals from tubal occlusion to TOAPOT had been reported to range from as early as 36 hours to up to 12 years  appearedFusobacterium is a strictly anaerobic, Gram-negative bacillus. Among this group, F. necrophorum is the most virulent species. This species has been divided into two subspecies: F. necrophorum subsp. funduliforme and F. necrophorum subsp. necrophorum. The former subspecies is usually associated with human infection, whereas the latter is usually associated with other mammals [ mammals .F. necrophorum subsp. funduliforme normally colonizes in the oral cavity [F. necrophorum has been found to be related to preterm labor, chorioamnionitis, dead fetus in utero, and TOA [l cavity  but can l cavity . It can l cavity . Transie and TOA . As our et al. [In general, TOA responds well to the empirical antibiotic regimen recommended by the Centers for Disease Control and Prevention . Howeveret al.  found thF. necrophorum subsp. funduliforme can be an etiologic organism of a ruptured TOA following TS in a healthy host. Early detection and early treatment by a multidisciplinary care team can significantly reduce the maternal mortality and morbidity..Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journalPID: pelvic inflammatory disease; rDNA: ribosomal deoxyribonucleic acid; TOA: tubo-ovarian abscess; TOAPOT: tubo-ovarian abscess after previous occluded tubes; TS: tubal sterilization.The authors declare that they have no competing interests.CC helped to review the case and complete the case summary and reviewed the literature. PR and MT helped to review the case and complete the case summary. AL performed the microbiological examination. All authors read and approved the final manuscript.


AbstractMorphogenia struhligen. et sp. n. represents the possible sister taxon of the abundant and speciose genus Barrojuba Park, sharing with it the putatively derived condition of anterolaterally shifted vertexal foveae, producing a smoothly convex vertex devoid of fovea or sulci. However, unlike Barrojuba, Morphogenia retains a plesiomorphic antebasal sulcus on the pronotum in both sexes, and additionally lacks elaborate abdominal fovea-like pockets and teeth on the lateral margins of the pronotum that are typical of Barrojuba. The genus is also unusual among jubine genera in lacking the characteristic V- or Y-shaped gular carina. In contrast to the commonly-collected Barrojuba, specimens of Morphogenia are absent in extensive jubine collections housed in museums in the United States, indicating that the new taxon may be relatively scarce or localised.A new genus and species of the large Neotropical pselaphine tribe Jubini is described from Manaus, Brazil, based on material preserved in the Natural History Museum, London.  Jubini is an entirely Neotropical radiation and a major component of the South American rove beetle fauna  to the vicinity of the postantennal notches.Thirteen genera of escribed , with seBarrojuba, but differing in crucial aspects that warrant their placement in a new genus. Here, the new genus is described and illustrated, its sexual dimorphism documented, and putative phylogenetic relationships discussed.Investigation of the pselaphine collection at the Natural History Museum, London (NHM), revealed undescribed jubine material collected during the 1990s from the Smithsonian Tropical Research Institute/INPA Biological Dynamics of Forest Fragments Project in Manaus, Brazil. Among this material were specimens with apparent affinities to For morphological description, specimens were removed from mounts and relaxed in a Sodium Dodecyl Sulphate-based buffer with Proteinase K, and then transferred into ethanol for observation with stereo microscopy, or into 1:1 ethanol : glycerol for imaging with a Zeiss compound microscope. Genitalia and terminal segments were dissected and mounted in temporary glycerol slide mounts, and are stored in glycerol microvials pinned with their respective specimens. Photographs of specimens were taken by using a Visionary Digital photomicrographic apparatus with Infinity optics and a Canon 60D camera, installed at the American Museum of Natural History, New York. Montage images were constructed from stacks using Helicon Focus. DNA from two paratype females was extracted according to a non-destructive protocol outlined previously . The symPageBreakhttp://zoobank.org/4368E5BC-73B0-443D-AE2C-020F58EFD36Chttp://species-id.net/wiki/MorphogeniaMorphogenia struhli, here designated.Morphogenia, and its only species, Morphogenia struhli, can be distinguished from all other known jubine genera by the following combination of characters: (1) smoothly convex vertex lacking sulci, and afoveate due to anterolateral shift of vertexal foveae into vicinity of postantennal notches anterior to eyes; (2) absence of V- or Y-shaped gular carina; (3) pronotum with margins smooth, lacking lateral spines, and with a simple, well-defined transverse antebasal sulcus in both sexes; (4) abdomen lacking fovea-like cuticular pockets at bases of tergites V–VII and sternites V–VII .Body length ~3 mm . Form reHead: Approximately triangular . Five tergites (IV–VIII) evident. Tergites with broad, angularly-projecting paratergites on segments III–VI and smaller paratergites on VII  evident; penial plate apparently internalized, not externally visible, with genital aperture formed by contiguous apical margins of tergite and sternite VIII . Femora and tibiae simple, lacking modifications. Profemora somewhat thickened. Tarsi : “89 7 // Leaf litter, Winkler method. Terra firmé fst. // BRAZIL: Manaus, A.M. INPA/Smithsonian Res. 2°25'S, 59°50'W, R. Didham, i. 1994//BMNH{E} 2003-84 // (pink label) 0749”. “68 12 // Leaf litter, Winkler method. Terra firmé fst. // BRAZIL: Manaus, A.M. INPA/Smithsonian Res. 2°25'S, 59°50'W, R. Didham, i. 1994 // BMNH{E} 2003-84”. All material is deposited in the Coleoptera collection of the Department of Entomology, Natural History Museum, London.Holotype ♂: “88 1 // Leaf litter, Winkler method. Terra firmé fst. // Body length 2.9 mm . HolotypPageBreakHead: Length 0.71 mm from occipital constriction to clypeus; width across widest point (posterior to eyes) 0.55 mm. Margins broadly and smoothly rounded from eyes to base  0.57 mm. Tergite lengths: IV= 0.32 mm, V = 0.12 mm, VI = 0.10 mm. Apical margin of sternite IV with male-specific medially positioned small, round tubercle covered in small setae. Male tergite VIII . struhli  may servPageBreakMorphogenia, is supported by cladistic interpretation of the suit of adult character states presented by the NHM material. Among jubines, Morphogenia appears to be allied to Barrojuba based on its putatively derived dorsal head morphology: the lack of sulci, and the proposed synapomorphy of anterolaterally shifted vertexal foveae that sit in the post-antennal regions anterior to the eyes. A smoothly convex vertex is present in Arctophysis Reitter, another jubine genus, but here the vertexal foveae have been evolutionarily lost rather than repositioned, and vestiges of the apodemes of the tentorium remain connected to the medial vertex (visible in cleared specimens). The dorsal head morphology of Barrojuba and Morphogenia is perhaps most closely approached by Endytocera Sharp, where the vertexal foveae sit somewhat anterolaterally on the head vertex, but are recessed into deep S-shaped sulci that extend from the gular region dorsally between postantennal notch and eye, merging apically at the interantennal area of the frons.Creation of a new genus, Morphogenia and Barrojuba differ markedly in several important characters. Barrojuba lacks a typical (straight and simple) antebasal sulcus on the pronotum in the male sex. Instead, this part of the pronotum is variously modified and impressed . In contrast, Morphogenia retains the plesiomorphic condition of a conventional antebasal sulcus in both sexes. A further autapomorphy of Barrojuba is the presence of large, fovea-like cuticular invaginations at the bases of tergites V–VII and sternites V–VIII. Such invaginations are absent in Morphogenia. Hence, Morphogenia possesses only one of three putatively derived character states of Barrojuba, arguing for its distinctiveness and proposed sister taxon status.Despite their similar vertexal morphologies, Barrojuba and Morphogenia, in combination supporting the reciprocal monophyly of the two genera. However, for several such characters, whether these differences represent gains or losses in the respective genus is presently unclear. Barrojuba has tooth-like acuminations at the lateral edges of the pronotum that are absent in Morphogenia, but seemingly homologous structures are present in many jubine genera, implying a possible loss in Morphogenia. Most strikingly, Morphogenia lacks any trace of a V- or Y-shaped gular carina—a character found in all jubine genera except Balega Reitter, Pselaphomorphus Motschulsky, and Phamisus Aubé. Such an absence may again be consistent with a loss occurring in Morphogenia. The tarsal claws of Morphogenia are equal in size, while in Barrojuba they are unequal. Equally-sized claws are plesiomorphic in pselaphines, but instances of evolutionary reversal from inequality may have occurred .A large number of other characters of systematic utility separate occurred , and thihe tribe . A furthPageBreakMorphogenia yielded no further specimens, implying that the genus may be relatively scarce or geographically localised. Fortunately, despite the age of the three known specimens, which were collected almost twenty years ago, DNA extraction from the two female paratypes of Morphogenia struhli was successful. Partial gene sequences have been obtained that may help resolve the placement of the new genus in a future molecular phylogenetic analysis of Jubini.Interpreting transitions in these more broadly distributed or sexually dimorphic characters will require a thorough phylogenetic analysis of the entire tribe. Extensive collections of Neotropical pselaphines, accumulated from expeditions spanning decades, are housed in the Field Museum of Natural History, American Museum of Natural History, Louisiana State Arthropod Museum and University of Kansas Biodiversity Institute. Screening these collections for


To the Editor: Bats are distributed worldwide and are among the most diverse and species-rich mammals on earth. They exist in a large variety of distinct ecologic niches. Many bat species roost near humans, which is of particular interest for research on bat-to-human transmission of potential zoonotic pathogens. Moreover, migratory bats could act as long-distance vectors for several infectious agents. In recent decades, scientific interest in chiropteran species has markedly increased because bats are known hosts to zoonotic agents, such as henipaviruses, Ebola virus, and severe acute respiratory syndrome (SARS)–like corona viruses , were subjected to necropsy and investigated by using routine histopathologic and bacteriologic methods. During necropsy, instruments were dipped in 70% ethanol and moved into a Bunsen burner flame after every incision to prevent any cross-contamination. For bacteriologic examination, tissue samples were treated accordingly to prevent environmental contamination. A freshly cut tissue surface was plated onto Columbia agar , Gassner agar (Oxoid), and MacConkey agar (Oxoid) and incubated at 37°C for 24–48 hours.Yersinia spp., i.e., Y. pseudotuberculosis and Y. enterocolitica. The first Yersinia strain (Y938) was cultured from lung, heart, kidney (pure cultures), liver, spleen, and intestine (mixed cultures) of a greater mouse-eared bat (Myotis myotis). This isolate was identified as Y. pseudotuberculosis by Api 20E , Micronaut-E , and 16S rRNA gene analysis  43 in all tissue samples investigated. During necropsy, severe enlargement of the liver and a marked hemoperitoneum were seen. Microscopic examination showed multifocal severe necrotizing hepatitis and splenitis associated with numerous intralesional gram-negative coccobacilli and a moderate to marked interstitial pneumonia. The remaining organs, including heart, kidney, and intestine, had no pathologic changes.Twenty-five bacterial genera were cultured from bats, including 2 known human-pathogenic analysis . The seqYersinia strain (Y935), Y. enterocolitica, was isolated in pure culture from spleen and intestine of a common pipistrelle (Pipistrellus pipistrellus) and identified by the methods described above (Y. enterocolitica serotype O:6, biovar 1A. Necropsy and histopathologic examination showed no inflammatory changes, suggesting a subclinical state of infection.The second ed above . The 16SY. pseudotuberculosis and Y. enterocolitica are frequently isolated from a variety of wild and domestic animals (Y. pseudotuberculosis infection in captive flying foxes have been published (Y. pseudotuberculosis strains belonging to serogroup 1 are most common and cause most Y. pseudotuberculosis infections in humans and animals (Y. enterocolitica biovar 1A has been found in a wide range of human, animal, and environmental sources. Although often considered nonpathogenic, this biovar is described as an opportunistic pathogen (Yersiniosis is a bacterial disease with a wide distribution and host range. Yersinia species generally occurs after ingestion of contaminated food or water. All bat species in Germany are insectivorous, and insects can be infected with various microbial agents. Investigations concerning bacteria–insect interactions showed that insects may carry pathogenic bacteria, including Yersinia (Transmission of both Y. pseudotuberculosis and Y. enterocolitica were isolated from 2 bat species in Germany, representing evidence of Yersinia spp. in free-ranging vespertilionids. Histopathologic findings of the greater mouse-eared bat were consistent with those of systemic Y. pseudotuberculosis infection, rendering this species pathogenic for bats. The common pipistrelle was subclinically infected with Y. enterocolitica. The role of wild animals as reservoir hosts for bacterial pathogens such as Yersinia spp. is well known, underlining the need for biologists and persons handling wildlife to be aware of these zoonotic infectious agents.In conclusion,


Gossypium barbadense is an important species of cotton because of its extra-long staple fibers with superior luster and silkiness. However, a systematic analysis and utilization of cDNA sequences from G. barbadense fiber development remains understudied.Cotton is the leading fiber crop worldwide. G. barbadense Hai7124 fibers and ovules. After assembly processing, a set of 8,653 unigenes were obtained. Of those, 7,786 were matched to known proteins and 7,316 were assigned to functional categories. The molecular functions of these unigenes were mostly related to binding and catalytic activity, and carbohydrate, amino acid, and energy metabolisms were major contributors among the subsets of metabolism. Sequences comparison between G. barbadense and G. hirsutum revealed that 8,245 unigenes from G. barbadense were detected the similarity with those released publicly in G. hirsutum, however, the remaining 408 sequences had no hits against G. hirsutum unigenes database. Furthermore, 13,275 putative ESTs InDels loci involved in the orthologous and/or homoeologous differences between/within G. barbadense and G. hirsutum were discovered by in silico analyses, and 2,160 InDel markers were developed by ESTs with more than five insertions or deletions. By gel electrophoresis combined with sequencing verification, 71.11% candidate InDel loci were reconfirmed orthologous and/or homoeologous loci polymorphisms using G. hirsutum acc TM-1 and G. barbadense cv Hai7124. Blastx result showed among 2,160 InDel loci, 81 with significant function similarity with known genes associated with secondary wall synthesis process, indicating the important roles in fiber quality in tetraploid cultivated cotton species.A total of 21,079 high quality sequences were generated from two non-normalized cDNA libraries prepared by using a mixture of G. hirsutum and G. barbadense.Sequence comparisons and InDel markers development will lay the groundwork for promoting the identification of genes related to superior agronomic traits, genetic differentiation and comparative genomic studies between  Gossypium spp.) is the leading fiber crop worldwide. There are four cultivated cotton species, two diploids from Africa-Asia, G. herbaceum L.  and Gossypium arboreum L. , and two tetraploids from Americas, G. hirsutum L.  and G. barbadense L. . At present, G. hirsutum is the most widely cultivated cotton species, accounting for more than 95% of the world cotton production , followed by G. barbadense (accounting for 2 ~ 3%) and G. arboreum (accounting for 1 ~ 2%), while G. herbaceum is rarely cultivated. G. hirsutum has the characteristics of high yield, broad adaptation, and medium fiber quality. G. barbadense typically has a longer growing period, matures later, and produces smaller bolls that give a yield significantly lower than that of G. hirsutum. In spite of these drawbacks, however, G. barbadense possesses superior fiber properties, which makes it an important raw material for high-grade or special cotton fiber textiles [Cotton . Among them, 297,214 ESTs were from G. hirsutum, 63,577 from G. raimondii, 41,781 from G. arboreum, while only 11,446 and 247 were from G. barbadense and G. herbaceum, respectively. Compared with great amount of EST resources from G. hirsutum, ESTs for G. barbadense are relatively scarce and have hindered the exploration of its economic importance.Recently, a number of genome resources have been developed from the genus age maps -7, constage maps ,9, and aage maps ,11. Of tLarge scale transcript sequences offer an efficient resource for targeted marker development. In cotton, SSR markers have been mined widely based on existing sequences data from different cotton species ,12-17, aG. barbadense cv. Hai7124. In total, 21,079 ESTs were generated by a large-scale 5′ end single-pass sequencing of randomly picked cDNA clones from the two libraries. Upon adding the released 11,446 G. barbadense EST sequences in the National Center for Biotechnology Information (NCBI), EST assembly for G. barbadense was performed and functional categories of unigenes were assigned. Further, by meta-analysis of fiber ESTs between G. barbadense and G. hirsutum, some putative G. barbadense-specific expressed fiber genes and InDel loci of orthologous or homoeologous variation between/within G. barbadense and G. hirsutum were developed, which put the foundation for promoting the identification of genes related to superior agronomic traits, genetic differentiation and comparative genomic studies between G. hirsutum and G. barbadense.In this study, we constructed two non-normalized cDNA libraries from fibers and ovules mixtures at −3 to 5 days post-anthesis (DPA) and fibers at 6 to 24 DPA, for efficient generation of unique ESTs from G. barbadense cotton fiber cDNA libraries were constructed using Hai7124 developing fiber tissues, one for −3 to 5 DPA (named as GB1 library) and the other for 6 to 24 DPA (named as GB2 library) respectively. Once each library was constructed, the lengths of inserts were identified by amplifying randomly 100 clones with universal T7 primers. The results showed that most of the inserts were among 0.5-1.2 kb, with an average length of 0.7 kb for the GB1 library and 0.8 kb for the GB2 library. Based on this, 22,329 cDNA clones  were randomly isolated and sequenced to generate ESTs from the 5′ end. After the removal of low quality sequences below the Q20 threshold, short sequences less than 100 bp in length, and those with vector sequences and poly-A tails, 21,079 high-quality sequences with scores ranging from 40 to 60 were obtained for further analysis. Most of the sequences ranged from 500 to 900 bp, with an average sequence length of 652 bp with the longest EST at 877 bp. All high quality sequences have been deposited in the dbEST division of GenBank under the accession numbers JK790134 to JK811212.Two high-quality G. barbadense EST sequences available in Genbank and herein were combined for EST processing and assembly analysis. Of 32,525 ESTs, 4,938 were singletons and the remaining were assembled into 3,715 contigs, yielding 8,653 unigenes for further analysis. The average sequence length of unigenes was 712 bp, and the longest unigene was 2,331 bp. We subjected 8,653 unigenes to Blastx against the non-redundant (nr) database to identify their similarities with known proteins  of genes with too short of a coding sequence, or novel genes [To reduce the redundancy and facilitate the process of gene annotation and mapping, all el genes .Of the 8,653 unigenes, 7,316 were mapped to the GO hierarchy with characterized biochemical and physiological functions using Gene ontology annotation , the metabolic-related enzymes encoded by 4,436 unigenes were also located in metabolic maps based on the KEGG pathway classification Table d.The level of representation in a cDNA library generally correlates with transcript abundance in non-normalized conditions. We assessed the distribution of contigs based on the number of assembled ESTs. Compared with singletons, most of the contigs consisted of 2 to 5 ESTs and a few were comprised of more than 100 ESTs  threshold of 10-10. As a result, 8,245 unigenes from G. barbadense were detected the similarity with G. hirsutum. The remaining 408 sequences had no hits against the G. hirsutum unigenes database.To find putative specific expression genes in G. barbadense were assigned functions by Blastx against the nr protein database. Of these, 213 sequences had one or more similarities with proteins in the non-redundant protein database, however, 195 sequences had no significant similarity. According to ESTs abundance, we also confirmed the expression specificity/predominance of these genes by selecting seven putative genes at random to analyze their expression patterns at 10 and 20 DPA in the fiber developmental process between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124 using RT-PCR method  database (http://www.arabidopsis.org/index.jsp) to identify genes involved in secondary wall synthesis. As a result, 81 InDel loci with significant function similarity with known genes associated with secondary cell wall synthesis process of Arabidopsis thaliana trichomes  thickening is one of the most important events for fiber quality. To further confirm whether insertion-deletion EST variation in tetraploid cultivated cotton species might be related to fiber quality difference between GhCesA  [GhFLA [GhAGP  [Rac13[GhCTL (chitinase-like) [Sus (sucrose synthase) [In these predicted genes, some family genes have been identified as a major role in cotton fiber secondary wall synthesis, such as subunit) -38, GhFLgalactan),40, GhAGprotein) , Rac13[4n) [Rac13, GhCTL (se-like) , and Susynthase) . The insG. barbadense has been used more commonly as a gene donor for the high-quality cotton fiber trait. The hybrids produced with G. hirsutum are expected to have the desirable characteristics including a high yield, exceptional fiber length, fineness and strength. However, the lack of G. barbadense genomic resources has seriously hampered the exploration of modern functional genomic approaches for selective breeding purposes.G. barbadense dataset was publicly released, which included 11,446 ESTs from G. barbadense cv. 3–79 fiber tissues [G. barbadense ESTs available in public databases.Expressed Sequence Tag (EST) sequencing strategies are efficient in identifying a large number of genes expressed in a given tissue, which are particularly relevant when no genomic data are available . Prior tth and 24th days after flowering [In addition, a high level of representation in a cDNA library generally correlates with transcript abundance in non-normalized conditions. Abundance analysis revealed that large clusters existed (high expression), such as lipid-transfer protein precursors, copper-zinc superoxide dismutase, E6, and arabinogalactan protein, which was in agreement with many previously reported cotton fiber key genes. Orford and Timmis (2000) suggested lipid transfer protein gene family might play an important role in fiber development . John etlowering . Superoxlowering . Other hlowering , pectatelowering , arabinolowering  and fibeG. hirsutum and G. barbadense will facilitate the identification of genes related to the important agronomic traits such as fiber growth and development. TblastX comparisons between G. barbadense fiber sequences and those from G. hirsutum released publicly in GenBank revealed that 8,245 unigenes (95.3%) from G. barbadense were detected as similarities with those from G. hirsutum. The remaining 408 sequences had no hits against the G. hirsutum unigenes database. Though the E-Value setting, UTRs regions' interference, and insufficient coverage of G. hirsutum ESTs database might inevitably cause certain false positives in the process of similarity analyses, the expression predominance of selected seven unigenes in G. barbadense was confirmed experimentally by comparing 10 and 20 DPA fiber tissues between G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. Based on the fact that G. barbadense possesses superior fiber quality, key genes related to fiber quality in G. barbadense may be exploited in cotton molecular breeding programs for improving fiber quality. The predominantly expressed sequences found here will provide an insight into exploring interspecific fiber quality divergence between G. barbadense and G. hirsutum. Further function confirmation of these sequences will be verified by experimental analyses.Comparative analysis of ESTs from fiber development between Currently, there is an increasing focus on polymorphisms of short insertions and deletions (InDels) types in genomic research of model species such as humans , mice 4 and riceG. barbadense and G. hirsutum, and 2,160 potential Gh-Gb InDel markers were further developed for genotyping and evolutionary research. Further, the InDel-derived ESTs/genes related to fiber growth and development was detected. In the paper, we found some genes involved in secondary cell wall synthesis process with potential EST InDel loci, such as those encoding cellulose synthase, sucrose synthase, beta-1,3-glucanase, glycosyl transferase, fasciclin-like arabinogalactan protein, arabinogalactan protein, chitinase-like protein, and other transcriptional factor genes involved in secondary cell wall synthesis. In Arabidopsis, InDel mutations of cellulose synthase genes in secondary cell wall synthesis had been known to cause structural weaknesses in vascular bundles presumably due to cellulose deficiency [In allopolyploids, mutational variation may arise between homologous sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies . When inficiency ,53. So, G. barbadense ESTs were collected from different stages of fiber development, Gh-Gb InDels enriched many genes related to cotton fiber development, which could greatly promote their deep research for structural and expressional differences related to the fiber development processes in G. barbadense and G. hirsutum. By randomly selecting 90 InDels primer pairs to conduct PCR analysis of genomic DNA, with the sequencing confirmation from 9 InDel loci in four cotton species, more than 70% InDel polymorphic loci (64/90 = 71.11%) were detected effectively. From the result, not only homologous and homoeologous loci difference were found, but also some evolutionary events could be inferred. The diversity among the allotetraploid cottons such as orthologous and/or homoeologous polymorphisms could be traced back to ancient diploid ancestors by banding patterns analysis, which indicated the independent evolution or different degrees of colonization by comparing G. barbadense and G. hirsutum, with two diploids, G. herbaceum and G. raimondii, as controls [Due to the fact that all InDel loci were discovered based on transcriptional level and all G. barbadense or G. hirsutum germplasm in cotton breeding. It also offers a novel model for the study of the origin, evolution, and genetic differentiation of G. barbadense and G. hirsutum and their adaptation to various environmental changes.Taken together, InDel molecular detection provides a new tool for effectively mining genes related to superior agronomic traits, and selecting appropriate G. barbadense fiber cDNA libraries and obtained 21,079 high-quality sequences. A systematic analysis and utilization of ESTs was further performed including assembling, annotation, GO classification and comparative analysis with G. hirsutum. The resulting dataset yielded nearly 8,653 putative unigenes, from which over 80% had similarities with publicly available proteins. Furthermore, putative ESTs InDels loci involved in the orthologous and/or homoeologous difference between/within G. barbadense and G. hirsutum were discovered by in silico analysis and confirmed by experimental analysis. The large-scale G. barbadense ESTs in the study were a significant contribution for public G. barbadense ESTs databases, and either expression or candidate EST InDel difference will provide a new tool for effectively mining genes related to superior agronomic traits. These data will provide a solid foundation for molecular breeding, functional genomics studies, and comparative genomics analysis in Gossypium. With the continuous growth of sequence information from non-model organisms such as cotton, we suggest that InDels will be a crucial source for next-generation mapping of key genes in these accessions.Transcriptome analysis from different tissues and organs provides the basis for functional genomics research. In the present work, we constructed two G. barbadense cv. Hai7124, a commercial Sea-island Verticillium-resistant cultivar, was planted under standard field conditions at Jiangpu Breeding Station, Nanjing Agricultural University, Jiangsu Province, China, using normal farming practices in 2008. All necessary permits were obtained for the described field studies from Nanjing Agricultural University. Developing ovules of Hai7124 were excised from each boll at −3, 0, 3, 5, 6, 9, 12, 15, 18, 21, and 24 day post-anthesis (DPA) and used for RNA extraction. Of these, the samples at −3 to 5 DPA were collected from mixture of fibers and ovules, and the samples from 6 to 24 DPA were derived from fiber cells dissected from the ovules. All harvested plant materials were immediately frozen in liquid nitrogen and stored at −70°C.http://www.clontech.com). Automated DNA sequencing of >10,000 random cDNAs for each library from the 5′ -termini using universal T7 primers was performed with a Big Dye Terminator sequencing kit using Applied Biosystems (ABI) 3730 automated sequencers .Total RNA from different fiber developmental stages was extracted using the CTAB-sour phenol extraction method . Poly  with the following parameters: minmatch 20, minscore 20. PolyA tails and the “X” character were also removed by the EST_trimmer.pl script (http://pgrc.ipk-gatersleben.de/misa/download/est_trimmer.pl). After processing, sequences less than 100 bp were excluded from the analysis. ESTs larger than 100 bp length and Q20 quality after removing vectors, adapters, and ploy-A tails were performed to generate a unigene set using Cap3 [All by Phred  for baseby Phred  from theing Cap3  with theThe unigenes (contigs and singletons) were subjected to a similarity analysis using Blastx  against The set of unigenes were submitted for GO (Gene ontology)  annotatihttp://www.genome.jp/kegg/kaas/) provided a functional annotation of putative genes by Blast comparisons against the KEGG GENES database. The output included KO (KEGG Orthology) assignments and automatically generated KEGG pathways.Unigenes were assigned into metabolic pathways with the tools supplied by the Kyoto Encyclopedia of Genes and Genomes (KEGG) . The uniG. barbadense and G. hirsutum, 297,214 ESTs from G. hirsutum released in NCBI were downloaded in FASTA format and saved on a local computer. TBlastx program was used to screen for differential genes between G. barbadense and G. hirsutum with an expectation value of 1e-10. Differential genes then were assigned a putative function using Blastx against the nr protein database.For comparative analysis of fiber cDNAs between http://www.ncbi.nlm.nih.gov/dbEST/). Of them, 329,749 ESTs from the two tetraploid cultivated cotton species, with 297,214 from G. hirsutum and 32,525 from G. barbadense , were assigned respectively as two distinct data sets for mining all possible InDels loci. In silico  PCR strategy was used to predict the possible InDel difference among orthologous and/or homoelogous loci between/within G. barbadense and G. hirsutum. In detail, each G. barbadense EST was first randomly cut into several segments about 300 bp for designing nested PCR primer pairs, which ensured the amplified regions to cover the whole EST sequence. Then, the set of primer pairs from G. barbadense EST sequences were run against the G. hirsutum EST sequences dataset based on in silico PCR analysis with a threshold less than 3 mismatch bases. At last, PCR products at given region with different size were used for mining potential InDel difference among orthologous and/or homoelogous loci between/within G. barbadense and G. hirsutum. As a result, InDel size larger than 5 bp was preferentially selected to confirm the reality by the gel electrophoresis combined with sequencing analysis, and homology analysis against previous reported genes associated with secondary wall synthesis of Arabidopsis thaliana trichomes [As of Jan. 20, 2012, approximately 414,271 cotton EST sequences were available in Genbank ESTs database (richomes -65 were G. barbadense ESTs were designed using Primer3 [Forward and reverse flanking primer pairs based on  Primer3  by runniG. barbadense) and TM-1 (genetic standard line in G. hirsutum) were used as parents for detecting orthologous and/or homoelogous loci polymorphisms between/within G. barbadense and G. hirsutum, with the two diploid progenitors, G. herbaceum and G. raimondii, as controls. The genomic DNA extraction followed that of Paterson et al. [in silico PCR analysis and gel electrophoresis confirmation, the accuracy of InDel loci was further evaluated by selecting at random the amplification products to sequence. For sequencing analysis, standard PCR reactions were performed using High-fidelity ExTaq DNA polymerase . The PCR products were cloned into the pMD18-T Vector (TaKaRa) according to the manufacturer’s instructions and sequenced from plasmid DNA templates. At least three positive clones were chosen to sequence. Sequencing was performed by GenScript Inc. .Hai7124  protein database and assigned with GO (Gene Ontology) and KO (KEGG) terms.Click here for fileStatistic analysis of KEGG classification with corresponding metabolism maps. Note: KO (KEGG) annotation and metabolism maps were performed with BLAST2GO. Classification showed in second level and third level.Click here for fileExperimental verification of candidate unigenes expressed specifically in G. barbadense using RT-PCR.Click here for fileG. hirsutum EST databases were identified and finished function annotation against nr (non-redundant) protein database.Function annotation of 408 putative G. barbadense -specific unigenes. Note: 408 unigenes without significant match to any sequence in the current Click here for fileG. barbadense (Gb) and G. hirsutum (Gh), and size range was supplied in the table.Summary for putative Gb-Gh Indel loci by in silic analysis. Notes: The information on insert size both in Click here for file2,160 Indel markers developed by using InDel > =5 bp EST sequences between G. barbadense and G. hirsutum.Click here for filePCR amplification and banding patterns of randomly selected 90 Indel markers. Note: 1: No difference in tested accessions; 3: showed orthologous and/or homoeologous loci polymorphism in tested accessions.Click here for fileFunction identification related to secondary wall synthesis using sequences detected InDels by tblastx analysis with databases from Arabidopsis.Click here for file