Based on stoichiometry and carbon stable isotope ratios, the source of organic carbon in these wetland sediments is derived from a combination of wetland macrophyte, algal, and phytoplankton sources. The reconstruction of natural wetland dynamics in Baja California provides a counterpoint to the history of wetland destruction elsewhere in North America, and measurements provide new insights on the control of carbon sequestration in arid wetlands.Although saline tidal wetlands cover less than a fraction of one percent of the earth’s surface (~0.01%), they efficiently sequester organic carbon due to high rates of primary production coupled with surfaces that aggrade in response to sea level rise. Here, we report on multi-decadal changes (1972–2008) in the extent of tidal marshes and mangroves, and characterize soil carbon density and source, for five regions of tidal wetlands located on Baja California’s Pacific coast. Land-cover change analysis indicates the stability of tidal wetlands relative to anthropogenic and climate change impacts over the past four decades, with most changes resulting from natural coastal processes that are unique to arid environments. The disturbance of wetland soils in this region (to a depth of 50 cm) would liberate 2.55 Tg of organic carbon (C) or 9.36 Tg CO While tThe global blue C sink is being diminished by anthropogenic activities and climatic change. A decline in the areal extent of vegetated coastal habitats is being driven by coastal development; the dredging, diking, and filling of coastal wetlands; and eutrophication, which is harmful for seagrass beds. Estimates of current declines in the global blue C sink range from 0.7% to 7% annually ,6. AlthoPrevious studies have focused on establishing wetland change rates over time and on determining country or habitat-level coastal wetland C stocks. Long-term wetland change has been calculated by comparing contemporary and historic wetland distribution patterns based either on historic maps ,17 or poThe objectives of this study were to produce a preliminary assessment of C sequestration in the upper horizons of coastal wetland soils for the Baja California peninsula, and to determine whether changes in C sequestration are resulting from modifications to wetland extent caused by coastal development or climate change ,28. The −1, and occurs during winter mid-latitude cyclonic activity and during summer convection storms and infrequent tropical storms and cyclones. Precipitation seasonality grades shift from Mediterranean (dry summer) in the north to North American Monsoonal (wet late summer) in the south. Lagoons are hypersaline inverse estuaries, with a typical salinity range of 34–39‰ [The climate of the Baja California peninsula is classified as arid, with a mean annual temperature on the Pacific Coast of 22 °C, a January mean temperature of 14–16 °C, and an August mean temperature of 22–29 °C. Precipitation is ~150 mm yearf 34–39‰ ,31. The Chelonia mydas) and loggerhead turtle (Caretta caretta). All five estuaries support extensive eelgrass populations, and are regionally significant overwintering grounds for waterfowl and shorebirds [The five largest areas of tidal wetlands found on the Baja California peninsula were identified based on spatial inventories developed by Pro Esteros, a Mexican conservation NGO based in Baja California. Focus sites include: Estero Punta Banda (EPB), Bahía San Quintín (BSQ), Laguna Ojo de Liebre (LOL), Laguna San Ignacio (LSI), and Bahía Magdalena (BM); all are tectonic estuaries protected by coastal barriers . Four oforebirds ,33.To establish a contemporary estimate of tidal wetland areal extent, and to identify changes in wetlands over time, wetland vegetation boundaries at study sites were photo-interpreted using aerial photographs from 1972 and high-resolution satellite imagery circa 2008. The 2008 imagery was downloaded as tiles from Google Earth Pro as color digital orthoimagery . Spatial referencing was preserved by saving associated world files , which record the location, scale, and rotation of the imagery. The 1972 imagery was obtained from INEGI  as color and B/W prints with scales ranging from 1:25,000 to 1:70,000, which were scanned at 500 dpi. The 1972 imagery was georeferenced in ArcGIS, version 9.1 using the 2008 orthoimagery as a base map. To ensure that changes detected were not a function of differing resolution, imagery was resampled to produce identical pixel dimensions (2.5 m), and the color imagery was rendered in grayscale using a red–green–blue to hue-saturation-intensity conversion, with the intensity component interpreted. Wetlands were digitized as a raster overlay using the remote sensing software ENVI  by employing a semi-automated procedure that used scene-specific operator-defined intensity thresholds to classify imagery . Site vin = 75), sediments were analyzed in duplicate for organic C, nitrogen composition and C stable isotope ratios using a Vario Micro Cube elemental analyzer coupled to an Isoprime stable isotope ratio mass spectrometer (IRMS). Samples were pretreated using hydrochloric acid fumigation prior to analysis to remove inorganic C. As soil organic C was a strong linear function of LOI values , organic C estimates were produced using LOI values for samples not analyzed for organic C. Soil C composition (stable isotope and OC:N stoichiometric ratios) was compared with potential C source end-members, including phytoplankton; macropyhte plant tissue from Ulva ; Zostera marina; C3 and C4 marsh plants; and mangroves from Bahia San Quintin [Sediment cores with a length of 50–200 cm were collected from each lagoon using a Russian peat borer, which is a side filling coring device that collects sediment samples free of compaction . Cores were X-rayed at the University of California Davis, School of Veterinary Sciences, and sub-sampled at 2-cm intervals for bulk density and organic content determinations. Density and organic content measures were made using loss on ignition (LOI) by sub-sampling a known volume of sediment, drying it to a constant weight, re-weighing, ashing the sub-sample at 550 °C for four hours, and measuring a final weight . For sam Quintin , the Tij Quintin , San Fra Quintin , and the Quintin .Sediment sub-samples were collected from 10-cm intervals and analyzed for particle size distribution and geochemistry. Multiple aliquots of heated hydrogen peroxide were used to oxidize organic material, and sodium hexametaphosphate was used as a dispersant , prior tTo aid in the identification of lithic sediment sources, the geochemistry of wetland sediment deposits was analyzed for similarity to potential source sediments . Geochemical concentrations were corrected for the influence of grain size, which varies independently of mineral composition, with the latter calculated as the inclusive graphic mean  for the For each lagoon, C storage estimates were made by taking the product of wetland area, soil bulk density, and soil fraction organic C, limited to the first 50-cm of cores only. Values were summed for the five focus sites studied. Estimates of soil organic C storage fluxes were also made by taking the product of wetland area and C storage rate approximates using actual soil organic C density and a minimum accumulation estimate derived from a C storage study conducted in California . To provAnalysis of aerial photography showed that the extent of tidal wetlands in the five largest estuary/lagoon complexes along the Pacific Coast of Baja California in 2008 approached 40,000 ha, and further suggested that the overall extent of Baja California’s tidal wetlands increased between 1972–2008 3.9%; . Net losExamination of aerial imagery revealed the processes responsible for the stability to slight expansion that had been found by image classification. Losses have resulted from coastal development, fluvial sediment burial by prograding river deltas, sand burial by active dunes, barrier transgression, and inlet widening . Gains iThe comparison of automated to heads-up imagery classification found high levels of agreement between the two methods with respect to areal wetland extent . The higThe 18 geochemical variables were effectively reduced to two principal components, which explained 61%, 67%, 68%, 69%, and 85% of the variability in the elemental data for lagoons Estero Punta Banda, Bahía San Quintín, Laguna Ojo de Liebre, Laguna San Ignacio, and Bahía Magdalena, respectively. Eigenvalues for the two principal components were 14.0, 14.3, 15.6, 13.8, and 17.0. For the three southerly lagoons, most of the sediment was large-grained , and the results of the PCA analysis  suggeste12 g C). Assuming that the Baja California wetlands are accumulating sediment at rates at the low end of those found in other California (United States) wetlands (2.5 mm year−1) [10 g C year−1.A north to south gradient in C density was identified, such that sediments deposited at Bahía Magdalena possessed about one-quarter the C density of the more northerly lagoons . Summing year−1) , yearly r2 = 0.698; p < 0.001). There was a strong relationship between precipitation and soil texture, such that low precipitation sites had soil with coarser particle sizes.Soil organic C density was found to vary significantly as a function of soil texture and climate . Higher 13C) of the organic material found in sediment cores (Plotting sediment organic carbon/nitrogen (OC/N) stoichiometric ratios and the stable C isotope ratio (δnt cores  shows th−1). Disturbance of these sediments through coastal development, or loss due to erosion linked with sea level rise, could result in the mineralization of buried C equal to 9.36 Tg CO2eq, or the yearly CO2 emissions of 1.97 × 106 passenger vehicles. Using a conservative accumulation rate of 2.5 mm year−1, C storage can then be estimated for these five lagoon complexes at a rate of 12 to 73 g C m−2 year−1 or 4.68 × 1010 g CO2eq year−1. Given that the areal extent of seagrass found in these lagoons is also significant, and that these seagrass beds likewise capture and sequester significant volumes of C [The amount of organic C stored in the top 50-cm of Baja California lagoon wetlands (2.55 Tg) is significant when compared with the yearly estimates of the global coastal marsh C sink  [Studies of C sequestration in saline coastal soils report highly variable rates of sequestration globally, spanning two orders of magnitude (ca. 10–1000 g m year−1) . ResultsThis study reports on the soil C density in coastal wetlands that are unusually pristine and have been impacted very little by coastal development and alterations to hydrological processes. We posit that developing a deep understanding of how wetlands are changing over time—in response to coastal development and climate change—is key to determining changes in C sequestration capacity. In addition, tidal wetlands in arid landscapes are not well studied, nor are pristine wetlands. This study thus provides new insights on natural coastal processes that occur in coastal wetlands in arid landscapes, as well as how these pristine wetlands will be affected by sea level rise, therefore identifying potential changes in C storage.This study reports on wetland change patterns that share both commonalities with and differences from those described for temperate regions of North America. Similar to what has been reported for both the mid-Atlantic and Pacific coast of the United States (U.S.), edge loss has occurred over the past few decades ,53,54. EThe upslope migration of wetland vegetation due to positive sea level anomalies, which has been noted for Bahía Magdalena by this and a previous study , has alsAnalogous to transgression, this study also identified a significant expansion of wetland areas in central and southern Baja California resulting from the formation of new lagoons . These lOur analyses of stratigraphic profiles also suggest that Baja California Pacific coast tidal wetlands will be relatively resilient to climatic change based on sediment composition and source. Wetlands with mineral soils and little anthropogenic alteration to sediment transport pathways are thought to have low vulnerability to sea level rise. Physical feedbacks promoting sediment accumulation occur when marsh flooding increases, allowing marshes to accumulate at rates similar to that of inundation increases ,63. In cIn summary, this study suggests that the lagoonal tidal wetlands of Baja California appear to have low vulnerability to loss with sea level rise, and as such are an important C storage resource. Wetland sediment accumulation is supported by mineral sediment, and unlike many regions of North America, sediment supply is abundant and not restricted by issues such as upstream dam retention and land-use change . The genRecent publications have highlighted the high rates of C emissions that are associated with accelerated losses of vegetated coastal ecosystems through climate and land-use change ,65,66. TAlthough this study found little loss of wetlands over the past four decades that could be attributable to anthropogenic impacts, past and future coastal development projects have and will threaten Baja California coastal wetlands and the valuable services they provide, including C sequestration. While the government of México has shown a strong commitment to wetland conservation, applicable protections are not consistently preventing habitat loss . FederalHowever, wetlands remain highly threatened in México despite their protected status. Estimates of potential loss that are based on maps of actual versus potential coastal wetlands have suggested that 62% of Mexican coastal wetlands have been lost due human impacts . In La P2eq. While México has developed strong protections for coastal wetlands, these policies have not been successful at preventing wetland destruction, and Baja California wetlands are very vulnerable to future impacts from coastal development. The continued persistence of these unique landscape features in such an undisturbed state, and the results presented here, argue for the preservation of the current tidal wetland acreage to help mitigate the impacts of greenhouse gas emissions as well as to maintain the other valuable functions these wetlands provide.Efforts to mitigate greenhouse gas emissions have increasingly focused on preserving and restoring forests and wetlands; as such, these activities provide strong benefits to wildlife and humanity in addition to those of C sequestration. In this study, we summarized the context of C storage functions for the five largest areas of coastal wetlands on the Pacific Coast of Baja California, including land-cover change trajectories and interactions with management and sea level rise. Our remote sensing analysis found a slight increase in wetland extent over past decades (+3.9%), and little evidence for direct anthropogenic disturbance, which forms a strong counterpoint to the history of wetland destruction that has occurred over the past centuries elsewhere in North America. Natural coastal processes unique to arid landscapes drove changes in wetland extent, including the burial of wetlands by active dunes, inlet breaches, and delta front progradation. No evidence for wetland fragmentation and drowning in response to sea level rise was observed, although some upslope migration of mangroves was found, suggesting that inundation increases are occurring. Soil C density was found to vary along the climatic gradient of the Baja California peninsula, with less C dense soils found in southern Baja, in association with warmer and drier conditions, and sandier sediments. We found that disturbances to wetland soils in this region (to a depth of 50 cm) would liberate 2.55 Tg of organic C or 9.36 Tg CO


It is clear that specific intestinal bacteria are involved in the development of different premalignant conditions along the gastrointestinal tract. An analysis of the microbial constituents in the context of pancreatic cystic lesions has, however, as yet not been performed. This consideration prompted us to explore whether endoscopically obtained pancreatic cyst fluids (PCF) contain bacterial DNA and to determine the genera of bacteria present in such material.Total DNA was isolated from 69 PCF samples. Bacterial 16S rRNA gene-specific PCR was performed followed by Sanger sequencing and de novo deep sequencing for the V3-V4 variable region of 16S rRNA gene.n = 33) were positive for bacterial microbiota as determined by next generation sequencing (NGS). Comprehensive NGS data analysis of PCF showed the presence of 408 genera of bacteria, of which 17 bacterial genera were uniquely abundant to PCF, when compared to the Human Microbiome Project (HMP) database and 15 bacterial microbiota were uniquely abundant in HMP only. Bacteroides spp., Escherichia/Shigella spp., and Acidaminococcus spp. which were predominant in PCF, while also a substantial Staphylococcus spp. and Fusobacterium spp. component was detected.We observed that 98.2% of the samples were positive in conventional PCR, and that 100% of selected PCF samples  contains supplementary material, which is available to authorized users. Pancreatic cysts are fluid-filled neoplasms that can be detected with a frequency of up to 2% in the general asymptomatic adult population and have a low, but not negligible risk for evolving into pancreatic ductal adenocarcinoma, while accounting for up to 5% of the total incidence of pancreatic cancerous lesions , 2. The Progress with respect to understanding the nature and natural history of pancreatic cysts is compounded by the presence of different types of this lesion. Grutzmann et al. and Farrell et al. have attempted to classify the different types of pancreatic cyst lesions and distinguish among others as intra-ductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCN), serous cystadenomas (SCA), and pseudocysts –5. In geHelicobacter pylori remains the archetypical example of a prokaryotic organism that can initiate a cascade of molecular events finally leading to full-blown cancer, whereas in the colon, various organisms and especially Fusobacteria have been linked to the appearance of dysplasia , the supernatant and pellet were separated by centrifugation at 13000 rpm for 1 min and both pellet and supernatant were used for total DNA isolation, using the Wizard DNA isolation kit as specified by the manufacturer’s protocol . Isolated DNA was equilibrated in the DNA rehydration solution from the kit and quantified on nanodrop-2000 spectrophotometer . Pancreatic cyst fluid DNA was diluted to 1 ng/μl for the PCR analyses and subsequently stored at − 20 °C.® Flexi DNA polymerase kit  and universal 16S rRNA primers were used . The 16S rRNA gene amplicons were run on 1% agarose gels , and the positive samples were selected by the presence of 16S rRNA gene 1394 bp band , MCN (n = 7), Pseudocysts (n = 8), and SCA (n = 9)] was selected for de novo 16S rRNA gene amplicon sequencing by Macrogen (South Korea). 16S rRNA gene amplicon sequencing was performed on the V3-V4 variable region using Illumina Miseq adapter PCR followed by clustering and sequencing. The raw images generated are used by MCS (MiSeq Control Software v2.4.1.3) for system control and base calling through an integrated primary analysis software called RTA . The BCL binary  is converted into FASTQ utilizing illumine package MSR (MiSeq Reporter).From this cohort of 69 PCF samples, DNA of 33 samples  were considered significant.All the statistical analysis were done using excel, Graphpad Prism 5.0., and STAMP statistical analysis software. When appropriate, results were Bonferroni corrected in graphpad analysis and Benjamini-Hochberg multiple test corrections in STAMP. For analysis of the potential microbial component of pancreatic cyst fluid (PCF), material collected from 69 patients was used. The characteristics of these patients are listed in Table P value > 0.99; χ2 test): in mucinous cystic neoplasms, 100% of samples contained significant amounts of bacterial DNA; in IPMNs, this number was 92.6%; in pseudocysts, 88.9% of PCF were positive for bacterial DNA; in serous cystadenomas, 88.9%; whereas in the group of others which included GIST, NET, and clinically undefined samples, 90.9% displayed significant amounts of bacterial DNA  out of the 69 samples . Another predominating bacterium in PCF was Bacillus spp. which was present in 16 out of 69 (23.19%) samples. The presence of other bacteria was also noted which included Ruminococcus spp., Staphylococcus spp., Caldimonas spp., Arthrobacter spp., Acinetobacter spp., Bacteroides spp., Orpinomyces spp., and Anaerococcus spp.  were considered, it appeared that such findings were limited to very low abundant organisms  that were relative low in one of the groups compared, more indicative of technical detection problems rather than a reflection of true biological differences between the groups  .To confirm the presence of these bacteria and gain more insight into the bacterial composition of the PCF, DNA obtained from the 23 fluids containing the highest apparent concentration of 16S rRNA copies were sent out for 16S rRNA de novo sequencing of V3-V4 region, using universal 16S rRNA primers which should allow identification of the bacteria present in such fluid at least on genus level. The results  bacterial populations are shown in Fig. t test was performed to identify the specific bacterial populations having high and low abundance in the PCF when compared to HMP selected body sites. A total of 136 bacteria . A role for many of these bacteria in initiation and progression of colon, lung, and liver cancer has been suggested [Streptococcus spp., Propionibacterium spp., Lactobacillus spp., Fusobacterium spp., Corynebacterium spp., Veillonella spp., Neisseria spp., Staphylococcus spp., Porphyromonas spp., Prevotella spp., Leptotrichia spp., Actinomyces spp., Capnocytophaga spp., Gemella spp., and Selenomonas spp. (P < 0.0001). These were also the least abundant bacteria present in the PCF. This comparison suggests that the PCF fluid bacterial colonization is unique and characterized by high abundant genera which have a tendency to propagate in the cancerous microenvironment, feeding the tumor  with the PCF bacterial population showed 26 unique bacterial genotypes in PCF when compared with stool, 27 genotypes compared with throat, 28 genotypes compared with tongue dorsum, and 36 unique bacterial genotypes in the PCF which are not present within any of the other body sites selected Fig. . ANOVA aStreptococcus constitute by far the major component of the bacterial ecosystem in the esophagus [H. pylori. The genus Helicobacter, however, was rare in our analysis, this genus the 67th genus in our ranking of our samples. Other bacteria found regularly in the stomach include Streptococcus, Neisseria, and Lactobacillus, and these species are not dominant in our analysis  [Propionibacterium, which, although fairly often detected, was relatively rare in our analysis of pancreatic cyst fluids, with its abundance not even approaching 1% of the entire flora in any of the samples tested. Other genera dominating the duodenal microbiota include Porphyromonas (ranked 76th in cyst fluid), Streptococcus, Neisseria, and Heamophillus . Hence, the bacteria we observed in cyst fluid widely diverges from that expected if duodenal contamination was a major factor. Indeed in an analysis of microbiome of the healthy proximal tractus in our own institution (manuscript in preparation), we observed that Streptococcus, Veillonella, Prevotella, and Pseudomonas have the highest relative abundance from distal esophagus to the jejunum and proximal ileum. This situation is markedly different from that observed in pancreatic cyst fluids . In apparent agreement, in a preliminary series of experiments employing fluorescent in situ hybridization on surgically obtained pancreatic cyst material, bacteria were apparent (not shown). Thus, the most straightforward interpretation of results is that the pancreatic cyst is home to a previously unsuspected and also unique bacterial ecosystem.In the current study, we have demonstrated that 92.8% of a large and clinically representative collection of pancreatic cyst fluids (PCF) is host to a complicated ecosystem of bacteria. The distinct bacterial community had a rich species diversity which is very different from that observed elsewhere in the proximal human digestive tract. Although the EUS-FNA is not a sterile procedure, 7.8% of the PCF samples were negative for bacterial DNA arguing that the procedure did not have a major impact on our cultivation-independent bacteriological findings. Furthermore, the ecosystem observed by deep sequencing of pancreatic cyst material is inconsistent with that expected from contamination by the oral, nasal, pharyngeal, oesophageal, gastric, or duodenal flora. Members of the genus sophagus , but whit genus) . Thus, tt genus) . The mosH. pylori being the most important example. The genus Helicobacter was only marginally detected in pancreatic cyst fluid; other bacterial species linked to transformation in the intestine were, however, more prevalent. In particular, F. nucleatum, which is relatively predominant in a fraction of the samples and has been convincingly linked to neoplasm formation in the colon, excites interest in this respect. Emerging evidence suggests that Fusobacterium species detected in pancreatic cancer tissues is associated with a worse clinical outcome in pancreatic cancer patients [Bacteroides genus compared to control subjects [Bacteroides spp. were the most predominant genus in PCF. In addition, a recent pilot study using 16S rRNA gene sequencing on saliva specimens showed that while there was no difference in diversity of oral microbiota between patients with pancreatic ductal adenocarcinoma (PDAC), IMPN, or healthy controls, pancreatic cancer patients had higher levels of members of the phylum Firmicutes while healthy individuals were associated with higher relative abundances of Proteobacteria [The presence of such a bacterial ecosystem in pancreatic cysts raises important questions as to the role of the bacteria present in such cysts in the development of such structures. In general bacteria have been linked to transformation and trans-differentiation of endodermal epithelia, with patients . But alspatients , 30. Altbacteria . EvidencVeillonella and Bifidobacterium spp. which were identified in pancreatic abscesses [E. coli, Enterobacter spp., Klebsiella spp., and Staphylococcus spp. but also fungal isolates including Candida albicans (15 case studies) [Clostridium perfringens infections in five patients leading to pancreatitis and pancreatic cyst formation, which required surgical interventions [milieu interieur formed by cyst fluid and hence have a profound effect on the bacterial composition. As such, changes in the bacterial communities may serve to detect such transformation in cyst-forming structures and may become useful for guiding clinical management.Pancreatic infections mainly arise from translocation of bacteria from the small bowel, and rarely from the colon and oropharyngeal route as demonstrated by study on bscesses . A studybscesses . The mosstudies) . Importaventions . Such stIrrespective, however, of its potential as future diagnostic and prognostic marker, the present study shows an as yet unknown bacterial ecosystem in pancreatic cyst fluid. As it is evident that bacteria influence physiology and pathophysiology of their interacting epithelia everywhere in the gut, it is well possible that such interactions also exist in the pancreas and its cysts, and that the biology of the pancreatic cysts is in important ways shaped by this ecosystem. Studies addressing this possibility are currently in progress.The study reveals previously undescribed bacterial diversity present in human pancreas and its cyst fluids. As specific bacteria are associated with this body site, we propose that such bacteria may carry the potential to influence the development of pathophysiological processes in the pancreas. The study points out to the need to further explore the microbiome in this specific niche for diagnostic and therapeutic purposes.Additional file 1:Detailed patient characteristics, samples characteristic of samples taken for NGS, gene accession numbers of Sanger sequenced samples and SRA numbers for the NGS selected samples, NGS sequenced V3-V4 variable regions of 16S rRNA. (XLSX 26 kb)Additional file 2:Detailed quality of reads, taxonomic profiles, taxonomic distribution and taxonomic distribution rank. (XLSX 432 kb)Additional file 3:kb)SRA numbers of Human Microbiome Project used for comparisons. Additional file 5:ANOVA analysis statistics table for the PCF and selected body sites of bacterial distribution. (XLSX 89 kb)Additional file 6: Figure S1.p < 0.01 showing high (54) and low (82) abundance distribution between the PCF and 13 body site selected. This image constitutes the comparison between the PCF and 13 body site microbiomes , A. PCA of antecubital fosa and pancreatic cyst fluids microbiome, B. PCA of anterior_nares and pancreatic cyst fluids microbiome, C. PCA of buccal_mucosa and and pancreatic cyst fluids microbiome, D. PCA of gingiva and pancreatic cyst fluids microbiome, E. PCA of hard_palate and pancreatic cyst fluids microbiome, F. PCA of mid_vagina and pancreatic cyst fluids microbiome, G. PCA of posterior_fornix and pancreatic cyst fluids microbiome, H. PCA of palatine_tonsils and pancreatic cyst fluids microbiome, I. PCA of retroauricular_crease and pancreatic cyst fluids microbiome, J. PCA of stool and pancreatic cyst fluids microbiome, K. PCA of saliva and pancreatic cyst fluids microbiome, L. PCA of tongue_dorsum and pancreatic cyst fluids microbiome and M. PCA of throat and pancreatic cyst fluids microbiome. Figure S2. Difference of mean of selected high and low abundance bacterial microbiome in PCF and 13 body sites together, respectively. Difference of mean between the bacterial genus distribution of pancreatic cyst fluid and 13 different body site microbiome selected from Human Microbiome Project database. When compared 17 bacterial genus (with p < 0.01) showing high abundance in PCF and 15 bacterial genus (with p < 0.01) showing high abundance in 13 body sites selected. (PPTX 7223 kb)PCA of pancreatic cyst fluid (PCF) and 13 body site microbiome comparisons. PCA showing the difference between pancreatic cyst fluid and 13 different body site microbiome selected from Human Microbiome Project database. When compared 136 bacterial genus with Additional file 7:t test statistics run for the bacterial genotypes comparison calculation of the PCF and 13 different body sites microbiota. (XLSX 9711 kb)Welch’s


This dating of the materials indicates that the sediments in which the soils were formed were deposited during the Pleistocene, while the carbonates are from the Holocene. The basin-like format of the laminar structures suggests that part of the more clayey material was deposited in lacustrine environments. The more humid climate in the Holocene intensified argilluviation, which at an advanced stage, led to a more pronounced textural gradient, reducing drainage and leading to ferrolysis and thickening of the E horizon. Besides pedogenic processes, more erosive flooding during the Holocene began reducing and rounding the landscape’s more elevated structures . In the final stage, these structures were occupied by termites to shelter from flooding. Thereafter, the bio-cementation action of the termite nests has increased the resistance of the vegetated mounds to processes of erosion.Vegetated mounds are an important geomorphological feature of the Pantanal, where the influence of floods dictates not only hydropedological processes, but also the distribution and ecology of the flora and fauna. This work aimed to identify factors and processes that influence the formation and spatial distribution of the mounds, which are commonly associated with termite activity. In order to characterize pedological processes, macro and micro morphological descriptions, satellite image interpretation, dating of the sandy sedimentary material using OSL and carbon dating using  The majority of studies on vegetated mounds in tropical environments recall their origin through biological activity –6. Termiet al.  (RPPN) belonging to the Pantanal Social Service of Commerce [Serviço Social do Comércio] (SESC), in the municipality of Barão de Melgaço, North Pantanal. The area is located between the coordinates of 16°32’S to 16°49’S and 56°03’W to 56°26’W , Cerradão (Savanna forest formations), Cambarazal forest (Vochysia divergens), Seasonal Forest with Acuri  palm trees, Fields and ecotones.A wide variety of phytophysiognomies can be observed in the RPPN, from species characteristic of the semi-arid climate to vegetation types associated with flood environments, with a predominance of savanna vegetation identified by Hasenack  as folloPantanal of Mato Grosso (Brazil), which extends beyond the borders of Bolivia and Paraguay, is the largest continental sedimentary floodplain in the world. The annual flooding of Pantanal plains occurs from November to April and the remaining months are marked by severe drought , the sum of exchangeable bases (S = Ca2+ + Mg2+ + K+ + Na+), base saturation [V = (Ca2+ + Mg2+ + K+ + Na+) × 100 / CEC], aluminum saturation [m = Al3+ × 100 / (Ca2+ + Mg2+ + K+ + Na+ + Al3+)], and exchangeable sodium saturation (ESP = Na+ × 100 / CEC) [The hydrometer method was used for particle-size analysis. The pH was determined in water (potentiometer), using a soil:solution ratio of 1:2.5 after agitation and 1-hour rest. Exchangeable aluminum (Al Embrapa . The Ca20 / CEC) .et al. [14 dating using Accelerator Mass Spectrometry (AMS). It was not possible to collect samples for dating from the other sites, as these did not have a sandy layer at more than 50cm deep or precipitations of carbonate.In order to see if the studied soils had recently undergone climatic change, during the Pleistocene or Holocene (from 2.58 Ma until today), samples of sandy layers were collected from Sites 2, 3 and 5 in PVC tubes and these were dated using optically stimulated luminescence (OSL), following the single-aliquot–regenerative dose (SAR) protocol proposed by Walinga et al. , at the + and clay accumulation (illuviation) in the subsurface , such as pore and channel walls coated with Fe or clay material (illuviation), Fe impregnation of the matrix inside pore walls (hypocoatings) and Fe and Mn removal from the soil matrix (depletion) were commonly observed on thin sections . CarbonaThe most intensive Fe depletion and clay illuviation were observed at site 5. The related distribution between coarse and fine particles was identified as predominantly porphyric, with few voids or connected channels porphyric, while horizons with more porosity were identified as having a chitonic distribution. Sandy material was observed in the entire profile at the edge of site 5, coinciding with the lateral edges of the Bt horizon. No significant termite activity was observed at the five studied sites, neither through field observation nor micromorphological features such as excrement in voids or channels. Specific descriptions of the 5 sites are given below:Soil morphology presents clear smooth transition for all horizons . Subsupegeo–earth, phagy–feeding on), this therefore configures a geophagic site. The main reasons for wildlife soil consumption are mineral nutrition (mainly sodium) or dietary detoxification  [+ and clay content.Through visual examination in the field, we observed a depression in the central part of the mound, aligned to its extension. Near the depression wildlife was found to practice intentional ingestion of soil, known as geophagy  . Once arinerals) . GeophagFor the thin section, an undisturbed sample was collected from Btn1. In the micromorphological description , the coaGeophagy was also observed at this site. The described profile presents a 40cm thick E horizon, which is followed in depth by Btn1- and Btn2-horizons until 150cm . UnderlyOn a thin section from Btn1 horizon, micromorphological description indicates a single spaced porphyric c/f related distribution pattern , with abAt around 200cm in depth, a laminar clayey layer (C-horizon) with no evidence of pedogenic processes was observed. In order to understand parental material, an undisturbed sample was collected from C horizon for thin section confection. Its related distribution is massive, with fissural pores in horizontal orientation . Several-1. The underlying horizon is reddish, shows powdered carbonate associated with fissures and old root channels, with a pH value of 8.8, an ESP of 35% and low EC, identifying this as a Btnk horizon. Beneath 130cm and underlying Btnk, the soil matrix is sandy with great variation in color. Despite decreases in pH and EC values in C horizons, ESP remains higher than 30%. This soil was identified as Abruptic Stagnic Solonetz [Soil morphology shows a 15cm thick E horizon. The underlying horizon has 3.5 times more clay content, pH values of 9.1 and an ESP of 34% , it is aThe Btnk thin section presents fractured carbonate associated with voids among peds (channels), with creamy color interference . Quartz -1 in the A and E horizons, reaching 450g kg-1 in the Cg horizon  and temporal saturation with surface water (stagnic properties) is evidenced by Fe and Mn nodules, at a frequency of <5% in volume, with strong effervescence in contact with H2O2 [The albic horizon (E) has a thickness of 80cm, with 800g kgP of 33% . The undith H2O2 . The soiith H2O2 .Remnants of the Btgn were observed in the midst of the E horizon . SeveralIn the micromorphological description we observed that quartz grains from E and Btgn horizons are of similar size and shape . For theIn profile 5B, broken and abrupt lateral transition can be observed from the Btgn/E horizon . Small cSome features observed in mound soils are not compatible with the current hydropedological conditions or biological activity, and we therefore associated them with past processes and climates. An example of these features is high ESP and pH values that commonly occur in semiarid regions and soils with quite low drainage. Despite not being currently observed, there is strong evidence that in the past there were lakes in the study area.Morphological features corroborate this hypothesis, for example the depression observed at site 1, is probably related to a former river channel enclosed by fluvial avulsion, which created a lake on the upper part of the landscape (oxbow lake). The laminar clayey structures at Site 2  also sug+ in the soil exchange complex, resulting in a major pathway for sodification (ESP ≥ 15%) and natric horizon formation in mound soils [+, other basic cations were concentrated, elevating the pH value. The Bt thin section from site 3 shows quartz fragments embedded in carbonate, with serrated border and optical continuity, suggesting that these nodules were formed a long time ago  or nodules distributed in a circular shape . This larainfall .Concomitant to carbonate dissolution and partial elimination, other soluble salts tend to be leached, so ESP becomes greater and greater. High ESP is a major factor in argilluviation, which can be observed through coating and infilling pedofeatures, as observed on the thin sections ,48. WhenArgilluviation was observed in soils from more preserved mounds to more eroded mounds, suggesting that even at incipient stages of pedogenesis, it is one of the principal processes in the hierarchy. This process is essential to E horizon formation, which has a sandy texture and is more susceptible to erosion processes. Chronologically, argilluviation is a current process, evidenced by impure reddish clay coatings in the middle of carbonate nodules . The lam3+ to Fe2+) and depletion at the extremities of aggregates Click here for additional data file.S1 FileRPPN borders and sampled points.(KML)Click here for additional data file.


The human pineal gland is a small neuroendocrine organ which produces melatonin. The main goal of this study was to provide a reference range for pineal volume in all age groups and to determine calcified and noncalcified tissue and their proportions, which may be a reflection of melatonin production in all age groups, by using very thin computerized tomography (CT) slices. A total of 167 outpatients had undergone cranial CT. Each of the subject’s total pineal volume (TPV), calcified pineal volume (CPV) and noncalcified pineal volume (NPV) according to age groups were calculated in cubic millimeters. Also, proportion of calcification (POC) was noted.3 (12.3 mm3–411mm3) for TPV, 74.3 mm3 (12.3 mm3–298 mm3) for NPV, and 3.9 mm3 (0 mm3–141 mm3) for CPV. POC showed a gradual increase from 0–49 years. In the ≥70 group, when compared with the 60–69 age group, CPV and POC values were significantly lower . The median values were 88.5 mm This study brings a radiological point of view to the distribution of pineal calcification according to age that has a link with melatonin secretion. The human pineal gland is a small crescent-shaped neuroendocrine organ that connects with the diencephalon by the pineal stalk. The pineal stalk has an inferior lip that links the pineal gland to the posterior commissure, and a superior lip to the habenular commissure. The base of the pineal stalk possesses a recess that is related with the third ventricle. It functions in the secretion of melatonin. About 80% of the pineal gland consists of pinealocytes with the main secretory product being melatonin, which has a major role in circadian rhythm 2. Pineal345789A total of 167 outpatients  were recruited for this cross-sectional designated study at our university hospital and it was approved by the local ethical committee.All patients were selected from outpatient clinics of neurology department with the complaints of predominantly severe headache, syncope or dizziness. Each of the patients had one set of non-contrast cranial CT scans. Depending on the retrospective design of the study we excluded some disorders according to the patients’ hospital records. Exclusion criteria for the patients were: having intracranial mass, cerebrovascular disease, cancer, psychiatric diseases such as Alzheimer’s disease, Parkinson’s disease, bipolar disease or schizophrenia, which can be linked with pineal gland pathology according to the literature [111213141516171819CT examinations were performed on an 80-row detector CT (160 slice) scanner  with the following parameters: collimation, 80 × 2 mm; pitch factor, 0.625; rotation time, 0.75 sec; 120 kVp; 250 mAs, FOV: 220.3 mm and with a slice thickness of 0.5 mm. Adaptive Iterative Dose Reduction 3D (AIDR 3D) was used as an iterative dose reduction software and Sure Exposure 3D was used as a mA modulation software for all the examinations.Axial CT images were transferred to a workstation . Axial CT, coronal and sagittal MPR images with a slice thickness of 0.5 mm and with section intervals of 0.3 mm were reconstructed with the AquariusNET program , dividing the computer screen into four equal quadrants in multiview mode. The quadrigeminal cisterna, the mamillary bodies, the ambient cisterna, and the posterior part of the third ventricle were taken into account for anatomical borders for the selection of the pineal region. The pineal gland was outlined using the free hand selection tool, by verifying on axial, sagittal and coronal planes. The appropriate plane was used to calculate the volume in which we could draw the borders of the tissue perfectly and in order to restrict the parenchyma by excluding the pineal recess mostly using the sagittal plane. Then the volume of the outlined region was calculated by computer software, given in cubic millimeters. This analysis was performed on all slices on which the pineal gland, or parts of the pineal gland, were displayed, with 10–35 slices, depending on the measured volume. The noncalcified volume was calculated by excluding the regions more than 70 Hounsfield units. The calcified volume was then manifested by subtracting the noncalcified volume from the total pineal gland volume Figures . Proportt test was used for continuous variables with normal distribution, and Mann–Whitney U test was used for continuous variables without normal distribution. Volumes among different age groups were analysed using the Kruskal–Wallis test. The correlation coefficients and their significance were calculated using the Pearson test while investigating the associations between normally distributed variables (TPV and NPV) and Spearman test was used for non-normally distributed variables (CPV). For all tests a significance level of 0.05 was used.For statistical analysis, SPSS statistical software  was used. Continuous variables are presented as mean and standard deviation, and categorical variables are expressed as percentage. Kolmogorov–Smirnov test was used to evaluate the distribution of variables. Student 3 (12.3 mm3–411 mm3) for total pineal volume (TPV), 74.3 mm3 (12.3 mm3–298 mm3) for noncalcified pineal volume (NPV), and 3.9 mm3 (0 mm3–141 mm3) for calcified pineal volume (CPV). In both females and males, TPV, NPV, and CPV had high inter-individual variations in all age groups . 87 patients (52%) were male , 80 patients (48%) were female . The median values were 88.5 mmfountain of eternal youth [9Melatonin was firstly isolated by Lerner A. et al. in 1958 and in the literature it was described as the al youth 21. Some l youth 9. Intracrl youth 9. Accordil youth 9.Cross-sectional imaging studies have not yet provided elaborate evaluation of calcification of the pineal gland with thin section CT series to demonstrate calcification and noncalcified volume in healthy people 8. A reviIn the study by Tapp and Huxley, a gradual increase in the weight of the pineal gland from puberty to old age was demonstrated . In a stThere was a large variation in size among all age groups and no difference in size was noted between males and females . AlthougComputed tomography (CT) scans can better show calcification than MR imaging (MRI). Recently, radiological studies of the pineal gland have been mainly conducted by CT on the existence of pineal calcification and the proportion of calcification on axial CT images over different populations 456, 27. 45Our study has some limitations that may influence the results. Depending on the retrospective design of the study, we excluded some disorders according to patients’ hospital records, that may have a link with pineal failure. We couldn’t exclude phospho-calcic metabolism diseases according to these records in some patients. The enormous inter-individual variation of the pineal gland complicates radiological evaluations 8. In addIn the study by Nolte et al., they evaluated 15 healthy male subjects’ (20–27 years) pineal parenchyma volume and circadian melatonin profiles, 24-hour melatonin per volume of pineal tissue and melatonin profiles of cystic and solid glands. They found that parenchyma volume correlates linearly to melatonin plasma concentration. So pineal cysts may have a negative effect on melatonin secretion by compressing the parenchyma and, therefore, the pinealocytes . AccordiA previous study by Liebrich LS et al., compared melatonin levels in saliva and pineal volume by means of three-dimensional T2 turbo spin echo (3D-T2-TSE)—solid parenchyma—and susceptibility-weighted imaging  in a population of 103 young and healthy participants. Results of this study demonstrated a negative correlation between pineal volume and sleep rhythm disturbances . Using hWe assume that optimized volumetry of active pineal tissue and therefore a higher correlation of melatonin and pineal parenchyma can potentially be improved by a combination of MR and CT imaging in addition to serum melatonin levels. Moreover, in order to improve MR quantification of pineal calcifications, the combined approach would possibly allow an optimization and calibration of MRI sequences by CT and then perhaps even make CT unnecessary . In addiCalcifications of the pineal gland and decreased production of melatonin have the same trend. This study brings provides a comprehensive assessement of the pineal gland morphology on imaging in different age groups. Future studies are necessary to verify our results in a large population of advanced age to reach stronger statistical association between the pineal gland morphology and function.The authors declare that they have no competing interests.


Endostar is a new endogenous angiogenic inhibitor with implicated anti-tumor activity. This study was to investigate whether thoracic perfusion of Endostar could be used to control malignant pleural effusions (MPE).We searched the databases of MEDLINE, Web of Science, EMBASE, Goggle, Cochrance Library and CNKI to select the studies regarding the efficacy of Endostar to treat MPE. A total of 13 randomised controlled trials (RCTs) with 1066 patients were included.P < 0.001; odds ratio = 3.58) and disease control rate (DCR)  of Endostar combined with chemotherapeutic agents were significantly higher than those of chemotherapeutic agents alone. In addition, Endostar combined treatment remarkably promoted quality of life (QOL) of patients  compared with that of chemotherapeutic agents alone. Moreover, Endostar combined treatment did not have an impact on the incidence of adverse reactions (AEs) (P < 0.05).The overall response rate (ORR) (The efficacy of Endostar combined chemotherapeutic agents was superior to chemotherapeutic agents alone through thoracic perfusion in treating MPE, which indicated that Endostar could be an effective agent for controlling MPE. In China, lung cancer has been becoming a major cause of death in malignant tumors due to increasingly air pollution and deterioration of the natural environment. In 2015 in China, There is dreadful fact that 733,000 lung cancer cases were diagnosed and 610,000 patients will die from this disease . Today, Endostatin is a naturally-occurring, 20-kDa C-terminal fragment derived from type XVIII collagen, which was first reported by Folkman. Endostatin has been reported to inhibit angiogenesis in a wide range of tumors, and may interfere with the pro-angiogenic effects of growth factors. Capillary endothelial cells are the targets of endostatin, endostatin blocks endothelial cell proliferation and formation of new blood vessels, and affects the progress and metastasis of malignant tumors . A new rRecent years, some studies have specially investigated the clinical effect and safety of Endostar combined with chemotherapeutic agents versus chemotherapeutic agents alone in treating MPE via thoracic perfusion. Here, we performed a systematic literature review to assess the clinical benefit of Endostar combined therapy in treating MPE.We searched and identified relevant randomized controlled trials (RCTs) from the databases of MEDLINE/PubMed, EMBASE, Cochrance Library, SCI, and CNKI database (from January 2005 to April 2016). We adopted various MeSH terms and key words that related to MPE and Endostar as follows: “malignant pleural effusion,” “MPE,” “rh-endostatin,” “endostatin,” “chemotherapy,” “Endostar,” and “recombinant human endostatin injection.” In addition, if we found useful information that was intimately associated with Endostar in the reference lists of those studies, we should further look for additional studies and identified them.The data that we extracted included: (1) the number of patients of each RCT, (2) publication date of literature, (3) the clinical characteristics of data, (4) the ways of clinical intervention, (5) overall response (ORR) and disease control rate (DCR) and (6) quality of life (QOL) and adverse effects (AEs).Inclusion criteria: (1) studies must be designed to compare Endostar plus chemotherapeutic agents to chemotherapeutic agents alone; (2) patients must be given drugs through thoracic perfusion; (3) patients must be diagnosed with MPE; (4) outcome measures must be reported; and (6) the total cases of patients must be greater than or equal to (but not less than) 50. Exclusion criteria: (1) studying on animals not human; (2) patients were given excessive other adjutant drugs; (3) studies were sponsored by pharmaceutical manufacturers; and (4) study was short of efficient control group.Trial design: RCTs of Endostar combined with chemotherapeutic agents versus chemotherapeutic agents alone through thoracic perfusion for treating MPE. The ways of interventions: the dosage was defined according to the statement of manufacturers and frequency of administration at least 2 times; Evaluation indicators of therapeutic efficacy: ORR, DCR, QOL, and AEs.We utilized the evaluation criteria shaped by Cochrane Handbook (Version 5.0.1) to assess the included trials, which included: (1) methods to random group of patients; (2) how to perform an adequate setting blinding; (3) how to perform an adequate allocation and conceal the sequence; and (4) a description of intention to treat. Eventually, the quality of trials was divided into three levels: low risk of bias, unclear risk of bias, and high risk of bias , 14.P-values <0.05 were considered to be statistically significant. The identification of homogeneity was studies was calculated by the χ2 statistic and was quantified with the I2 statistic. In our study, we adopted fixed effects model preferential to perform meta-analysis. We also used meta-regression to evaluate whether the results were different between two groups. In order to assess the bias of literature, we omitted each study from the estimated pool to analyze the influence of each study to overall effect. In addition, funnel plots, Egger’s test, and Begg’s test were performed to assess publication bias. We used SPSS  and Stata version 15.0  to implement the statistical analysis and used a significance level of P <0.05.After sufficient data were collected and identified, the process of meta-analysis was implemented. The odds ratio (OR) with 95 % confidence intervals (CI) were major statistical data that were applied to explore the difference of efficacy. The overall effect was calculated by Z-scores and Originally, 122 potentially relevant studies were identified. Of them, 66 studies were removed because they were not original literature such as review, abstract and meeting records. Remaining 56 studies were identified as requiring RCTs, but 31 studies were excluded subsequently because of the following reasons: did not describe a clear control; did not have usable end points; duplicate of another study; non-human studies; low quality of statistics; and too small sample size. Of the remaining 25 trials, 12 were excluded further because complicated combination therapy and low design quality. Finally, 13 –27 studiThe design of 8 studies were that Endostar combined with cisplatin versus cisplatin alone through thoracic perfusion for treating MPE , 24, 27,Two investigators independently reviewed and determined the quality of each study. The discrepancies were resolved by consensus with the third expert. The evaluation was performed according to the evaluation criteria established by Cochrane Handbook Version 5.0.1) . As show.0.1 . Asp < 0.001; Fig. p = 0.767). Moreover, sensitivity analysis revealed the odds ratio and 95 % CI did not change when we omitted anyone study, with an odds ratio pool changing between 2.08 to 6.82.We identified 13 RCTS –27 pertap = 0.803). The pooled odds ratio was 2.97 , which indicated that Endostar combined with chemotherapeutic agents promoted the DCR, compared with chemotherapeutic agents alone   Fig. .Fig. 4Cop =0.423)    Fig. . The inc14) Fig. . Five st05) Fig. .Table 4CP = 0.562) , and moment-based estimate of between studies variance was zero , which indicated that no obvious variation between groups was observed in this meta-analysis.The shape of Begg’s funnel plot seems to be symmetrical , suggesting that publication bias did not have an impact on the results Fig. . The Egg62) Fig. . Get togIn clinical work, malignant pleural effusions (MPE) is a common problem that physicians, oncologists and thoracic surgeons often face. Although many malignant tumors directly lead to accumulation of pleural effusions, the mainly causes for MPE are lung cancer (37.5 %), breast cancer (16.8 %), and lymphoma (11.5 %). It is reported that 8 to 15 % of lung cancer patients presented symptom of MPE . The locMPE is a common manifestation of disease progression to patient with advanced lung cancer and other cancers. In order to control symptoms and improve the quality of life, careful evaluation of pathology and patient treatment individualization is very crucial . In addiWe found that myelotoxicity and digestive reactions are most common adverse reactions, but most of which were grade 1 or 2 and were well tolerated. Through the further analysis, we noticed that the incidence of myelotoxicity, digestive reactions, liver and renal injury, arrhythmia and fever in treatment of Endostar combination was as high as that in chemotherapeutic agents alone, suggesting that the Endostar did not have an extra impact on the incidence of the AEs. The detection of heterogeneity is very important to meta-analysis, because it will affect the pooled statistical efficacy. We carefully assessed the included studies and found that those studies had a good clinical homogeneity. Moreover, the Egger’s test and the Begg’s test did not imply the possibility of publication bias.However, there are some deficiencies in included trials. First, most of studies lack adequate analysis of subgroup data such as age, sex, smoking, histology, and treatment status and so on. Second, design quality of some is relatively low. Third, sample size of some is too small. The last, and mostly importantly, most of patients were from China (because Endostar was approved by the China State Food and Drug Administration and applied in treatment of lung cancer), which may lead to geographical and ethnic differences. In spite of this, these studies still propose a credible suggestion pointing toward that the Endostar is effective and safe for treating MPE, and it is a new choice for treating MPE. Nevertheless, Endostar, as a new molecular targeted drug, still needs to be investigated in the future. Especially, rigorously randomized control trials with large sampler size and multi-centered cooperation should be done before it could be recommended in clinic extensively.Thoracic perfusion of Endostar combined with chemotherapeutic agents has a better benefit of ORR and DCR for treating MPE and improves the QOL of MPE patients, compared with chemotherapeutic agents alone. Moreover, the participation of Endostar does not have an extra influence on the incidence of AEs. However, rigorously randomized control trials should be required before it is used widely.


Low-grade inflammation appears to play an etiological role in cognitive decline. However the association between an inflammatory dietary pattern and cognitive decline has not been investigated. We aimed to investigate dietary patterns associated with inflammation and whether such diet is associated with cognitive decline.We analyzed 5083 participants (28.7% women) from the Whitehall II cohort study. Diet and serum interleukin-6 (IL-6) were assessed in 1991–1993 and 1997–1999. We used reduced rank regression methods to determine a dietary pattern associated with elevated IL-6. Cognitive tests were performed in 1997–1999 and repeated in 2002–2004 and 2007–2009. The association between dietary pattern and cognitive decline between ages 45 and 79 was assessed using linear mixed models.We identified an inflammatory dietary pattern characterized by higher intake of red meat, processed meat, peas and legumes, and fried food, and lower intake of whole grains which correlated with elevated IL-6 both in 1991–1993 and 1997–1999. A greater decline in reasoning was seen in participants in the highest tertile of adherence to the inflammatory dietary pattern  compared to those in the lowest tertile  after adjustment for age, sex, ethnicity, occupational status, education, and total energy intake (p for interaction across tertiles = 0.01). This association remained significant after multivariable adjustment. Similarly for global cognition, the inflammatory dietary pattern was associated with faster cognitive decline after multivariable adjustment (p for interaction across tertiles = 0.04). Associations were stronger in younger participants (<56 years), reducing the possibility of reverse causation.Our study found that a dietary pattern characterized as higher intake of red and processed meat, peas, legumes and fried food, and lower intake of whole grains was associated with higher inflammatory markers and accelerated cognitive decline at older ages. This supports the case for further research. •The derived inflammatory dietary pattern was associated with repeated signs of inflammation.•The derived dietary pattern is characterized by higher intake of red meat, processed meat, peas and legumes, and fried food, and lower intake of whole grains.•This derived dietary pattern associated with systemic inflammation.•This inflammatory dietary pattern predicted faster decline in reasoning over ten years of follow-up. It is predicted to reach 65.7 million in 2030, and 115.4 million in 2050 Inflammation has been recognized as a risk factor for age-related neurodegenerative diseases including cognitive impairment, Alzheimer's disease, and vascular dementia A recent randomized controlled trial showed improved or maintained cognitive function in the intervention group , compared to the control group In this study, our aim was to examine the associations of diet pattern with inflammatory markers and changes in cognitive function within a single analytic setting. We hypothesized that a dietary pattern related to increased IL-6 concentration in midlife predicts accelerated cognitive decline in a large sample of middle-aged population.22.1The Whitehall II cohort is an ongoing study established in 1985 among 10,308 (67% men) British civil servants. The detail of the cohort and follow-up has been described previously 2.23 clinical examinations over 10 years were administered for the cognitive test battery , and consisted of 4 standard tasks: Alice Heim 4-I, short-term verbal memory, phonemic fluency, and semantic fluency 2.3Dietary intake was assessed by using a 127-item food frequency questionnaire. The validation of this questionnaire with 7-day diary has been reported previously 2.4In 1991–1993 and 1997–1999, fasting serum was collected between 8 AM and 1 PM and stored at −80 °C. IL-6 was measured with a high-sensitivity ELISA . Values lower than the detection limit (0.08 pg/mL) were set to half the detection limit.2.5Demographic variables included age, sex, ethnicity, education, and total energy intake. Health related variables included body mass index (kg/m2), diabetes mellitus, hypertension, smoking, and leisure time physical activity. Ethnicity , occupational position (6 levels), education , smoking history , and leisure time physical activity  were based on self-report. Body weight and height were measured, dressed in a cloth gown and underclothes, in standardized fashion and body mass index (kg/m2) was calculated. Diabetes mellitus was defined by fasting glucose ≥7.0 mmol/L or a 2 h post load glucose ≥11.1 mmol/L, self-reported physician-diagnosed diabetes, or use of diabetes medication. Hypertension was defined as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, or use of antihypertensive drugs. Since alcohol consumption was included as one of the food components in our dietary pattern, we did not include alcohol in covariates.2.6The dietary pattern associated with cognitive decline was assessed using reduced rank regression A linear mixed model was used to estimate the association between dietary pattern and cognitive decline over 10 years Logistic regression analysis was used to identify the association between derived dietary pattern and decline in MMSE, defined as reduction of 3 points or more in MMSE from 2002-2004 to 2007–2009. Decline of 3 or more MMSE points has been reported as a reliable change We conducted several sensitivity analyses. We excluded the participants diagnosed with diabetes mellitus and hypertension to exclude misclassification since participants with diabetes and hypertension might have changed their dietary habits at the baseline making diabetes mellitus and hypertension potential confounders. We also excluded participants with the lowest 10% score on global cognition to exclude potential cases of dementia, and to avoid reverse causation on dietary habits at baseline due to low cognitive function. In addition, we further conducted a stratified analysis with median age and the BMI to evaluate whether the association would be different across the strata. Two-sided p < 0.05 was considered statistically significant in all analyses. The software package SAS  was used for all statistical analyses.2.7Written informed consent was given by all participants, and the University College London ethics committee approved this study.3A total of 7870 participants completed the medical examination in 1997–1999, the baseline of this study. Of these, 7495 individuals participated in at least one of the three cognitive function assessments over the following 10 years. Of these, 4678 (62.4%) contributed to all 3 phases, 1670 (22.3%) to 2 phases, and 1147 (15.3%) to a single phase. Participants with missing diet assessment data in 1991–1993 and 1997–1999 (n = 2303), and participants with implausible total energy intake  (n = 109) were excluded, such that 5083 participants remained in the analysis .The mean age of the participants at the baseline was 56 years, and the proportion of women was 28.7%. The prevalence of diabetes and hypertension at baseline was 4.4% and 28.2%, respectively. Among the participants, 8.7% were currently smoking, and 53.0% reported moderate and/or vigorous physical activity during leisure time.Two dietary patterns were identified using reduced rank regression. Scores for these two patterns explained 3.54% of the total variation of the response variables (IL-6 in 1991–1993 and 1997–1999). The first dietary pattern accounted for 95% of explained total variation. Thus, we named the first dietary pattern the inflammatory dietary pattern (IDP) and used this for further analyses. IDP score positively correlated with IL-6 both in 1991–1993  and IL6 in 1997–1999  adjusting for age and sex.The factor loadings of the 37 food groups represent the magnitude and direction of each food group's contribution to IDP . A positIn cross-sectional analyses , IDP scoIn sensitivity analyses, we excluded the participants diagnosed with diabetes mellitus and hypertension and this did not change the results (data not shown). Excluding participants with the bottom 10% score of global cognition also did not change the results (data not shown). Furthermore, in stratified analyses with median age (56 years), higher IDP score showed significantly faster cognitive decline in reasoning in the age<56 group, but the association was not significant in the ≥56 age group . In addi4In this large cohort of middle-aged men and women, a dietary pattern associated with systemic inflammation at two measurement occasions five years apart predicted faster decline in reasoning over ten years of follow-up. The inflammatory dietary pattern was associated with repeated signs of inflammation and is characterized by higher intake of red meat, processed meat, peas and legumes, and fried food, and lower intake of whole grains. This is, to the best of our knowledge, the first longitudinal study to link diet, inflammation and risk of cognitive decline over 10 years within a single analytic setting. This may support the mechanisms that an inflammatory diet pattern increases the risk of faster cognitive decline through low-grade systemic inflammation, and this gives caution and provides helpful information to the general public in choosing their daily food to eat.So far, dietary pattern analyses have generally been conducted using either a principal components analysis that determines, post hoc, dietary patterns specific to the targeted population or a hypothesis-oriented approach based on an ideal dietary pattern e.g. the Mediterranean diet. Principal components analysis reflects the dietary background but is not able to include any evidence about predictors of targeted disease risk. On the other hand, hypothesis-oriented analyses of an a priori dietary pattern are based on previous information but ignore the dietary background of the targeted population. So it is beneficial to use reduced rank regression since the derived dietary patterns combine the advantage of both approaches. The approach incorporates evidence about predictors and reflects the dietary background of the targeted population.Reduced rank regression has been employed with nutrients as hypothesized intermediate or response variables to identify a dietary pattern which can be tested as a predictor of dementia risk A previous study based on an a priori index of diet-related inflammation did not find an association with Alzheimer's disease or with serum C-reactive protein (CRP) We chose IL-6 as the response variable, since our previous study suggested an association between IL-6 and cognitive function that was not evident with CRP The mechanisms underlying the proposed association between an inflammatory dietary pattern and cognitive function are likely to involve several processes. Although the IDP diet is not an immediate cause of cognitive decline, to the extent that dietary pattern may produce systematic inflammation, several lines of research suggest pathological pathways involving inflammatory processes may operate. Animal studies indicate that deficits in cognitive function with increased amyloid-β deposition are due to elevated oxidative stress and inflammation Depression, which is a risk factor for dementia The derived IDP is consistent with previous studies showing associations between overall dietary pattern, food group intake and inflammatory markers Paradoxically, we found that intake of peas and legumes loaded strongly on the IDP factor. This finding conflicts with expectations for a dementia-protective diet In our present study, the association between IDP and cognitive decline was observed for reasoning, and there was no dietary effect on verbal fluency. These findings parallel the inverse association of serum IL-6 with reasoning and lack of association with verbal fluency in our earlier study We should consider reverse causation as an alternative explanation for the observed link between dietary pattern and cognitive decline. Such an effect might arise if the presence of sub-syndromal mild cognitive impairment led both to a low-grade inflammatory state and to the dietary pattern we have identified here. Our study participants are relatively healthy and young, and therefore it appears unlikely that preclinical disease might generate the links of the small degrees of cognitive decline we observed with IL-6 and dietary habit. In addition, the association was the clearest in younger participants. Reverse causation, whereby cognitive decline may affect dietary habits, is unlikely to be important in this group, and the dietary effect on cognitive decline probably drives the association. Furthermore, after excluding the participants with the bottom 10% of global cognition score at baseline, results did not change.The strength of this study includes the large sample size and repeat measurements of dietary intake, IL-6 and cognitive function. Participants were prospectively followed over 10 years with three measurements of cognitive function. IL-6 was measured twice, at a five-year interval. To avoid misclassification and improve validity of dietary intake derived from food frequency questionnaire, we used the average intake from 1991 - 1993 and 1997–1999. Our study also has limitations. First, estimated intake of nutrients derived remains subject to errors of recall of average diet in previous year. The validation of food frequency questionnaire using biomarkers and a 7-day estimated food diary showed a relatively good performance in our cohort In conclusion, an IDP characterized by high intake of red meat, processed meat, and peas and legumes, and low intake of whole grain, was associated with high circulating IL-6 levels and accelerated cognitive decline over a 10 year follow-up. Further research in other cohorts is needed to replicate our finding, followed ideally by intervention studies to determine whether the avoidance of IDP would reduce inflammation and, in the long term, slow future cognitive decline.UNESCO- L'Oreal Foundation, Astellas Foundation for Research on Metabolic Disorders, the British Medical Research Council (K013351), the Economic and Social Research Council and the National Institute on Aging (R01 AG034454). Archana Singh-Manoux receives research support from the National Institutes of Health , R01AG034454 (PI)). Eric Brunner is supported by the British Heart Foundation (RG/13/2/30098).This study was supported in part by Mio Ozawa contributed to the study concept, design, interpretation of data, statistical analysis, and writing the manuscript. Martin Shipley contributed to the study concept, interpretation of data, statistical analysis, and writing the manuscript. Mika Kivimaki, and Archana Singh-Manoux are study coordinators and contributed to the study concept, interpretation of data, and writing the manuscript. Eric Brunner is a study coordinator and contributed to the study concept, interpretation of data, statistical analysis, and writing the manuscript.The authors report that there are no disclosures relevant to this publication.


This study aims to develop and to standardize a polymerase chain reaction (PCR) assay that will diagnose clinical as well as carrier state of the disease and to compare the results with conventional microscopy technique.cytochrome b gene sequence of Theileria annulata were used in the PCR assay that was standardized to use in the laboratory.A herd of crossbred cattle with the previous history of theileriosis in village Lahli, district Rohtak, Haryana, was selected for this study. A total of 29 blood samples were collected randomly from cows including five clinically ill cattle. Blood smears from all animals and lymph node biopsy smears from animal with swollen lymph nodes were examined microscopically after conventional Giemsa staining. Phenol chloroform isoamyl alcohol method was used for extracting DNA from blood. Previously published primers targeting Out of 29 samples tested,18 (62.06%) were found positive for theileriosis by PCR assay, whereas only 10 (34.48%) samples were detected positive by conventional microscopic technique using Giemsa staining method.T. annulata or low parasitemia in cows. Therefore, PCR assay can serve as a more sensitive tool to detect Theileria for detection of theileriosis even in asymptomatic carrier cattle which is important for the implementation of successful control programs.On the basis results of comparative studies, it can be concluded that PCR assay is a more sensitive than microscopic examination for detection of theileriosis. This can be attributed to the ability of PCR assay to detect small amounts of genomic DNA of  Theileria annulata. The disease is characterized by a marked rise in body temperature, reaching 40-41.5°C, depression, lacrimation, nasal discharge, swelling of the superficial lymph nodes, and anemia. It is transmitted by Hyalomma anatolicum, a three host tick which act as a biological vector for T. annulata. Recovered cows from acute or primary theileriosis, remain infected for a long period and even for the rest of their lives, acting as reservoirs of infection for ticks which cause natural transmission of the disease [In India, bovine tropical theileriosis is an important endemic hemoprotozoan disease caused by  disease .Theileria in the animals suspected to be suffering from clinically acute theileriosis, but it is not applicable for the detection of presymptomatic or carrier animals, where parasitemia is very low [The conventional method for identification of organism includes an examination of blood smears using Giemsa staining. Indeed, Giemsa-stained blood smears can be used as a suitable method to detect very low . IdentifTheileria species in persistently infected cattle [T. annulata – infected asymptomatic carrier animals and to compare PCR assay with microscopic examination.Molecular methods, with a high degree of sensitivity and specificity, have been developed to identify various d cattle . TherefoSamples were collected by qualified veterinarians as per standard sample collection method without any stress/harm to animals and with due permission of the Institutional Animal Ethics Committee.Crossbred cattle herd with a recent history of three clinical cases of theileriosis detected microscopically was selected for sampling. A total of 29 blood samples (10% approximately) from a herd of 250 cattle comprising 67 calves and 183 adult animals were collected by random sampling including five clinically ill cattle in vials containing ethylene diamine tetra acetic acid. The clinically affected animal was having a fever, pale mucous membrane, and swollen lymph nodes. Thin blood smears were prepared from each sample at the site of sample collection and fixed using methanol and remaining part of the sample was brought to the laboratory for DNA extraction. Lymph node biopsy was also taken from four animals having swollen pre-scapular lymph nodes, and a smear was made and fixed in methanol at the site of sample collection.Age, sex, lymph node examination, mucous membrane examination, and rectal temperature of all 29 animals included in the study were recorded.T. annulata piroplasm under 100×objective magnifications. About 20 microscopic fields, per slide, were observed to view the parasite. The presence of single piroplasms was recorded as positive for T. annulata. Fixed lymph node biopsy smears were examined under 100×magnifications in the search for characteristic Koch blue bodies, the presence of which is confirmatory diagnosis of theileriosis.Fixed thin blood smear and lymph node biopsy smears were stained with Giemsa stain for 30 min . Blood sth volume of 3M sodium acetate and three volumes of chilled absolute ethanol was added and incubated overnight at 4°C. Precipitate was obtained by centrifugation at 12,000 rpm for 20 min. The precipitate was washed with 1 ml of 70% ethanol and centrifuged at 12,000 rpm for 10 min. The pellet was air dried and dissolved in 50 μl of nuclease free water and stored at −20°C for further use.DNA was extracted using phenol-chloroform isoamyl alcohol method. To 500 μl of whole blood three volume of red blood cell lysis buffer was added and kept for 15 min at 37°C. To this 600 μl of DNA extraction buffer was added followed by 10% sodium dodecyl sulfate (260 μl). Proteinase K (20 μl) was added and the sample was incubated at 50°C for 1h. After 1 h of incubation, an equal volume of saturated phenol was added and kept for 15 min at 37°C. Centrifugation at 12,000 rpm was done and upper aqueous phase was separated. To aqueous phase, the equal volume of phenol-chloroform isoamyl alcohol was added and centrifuged at 12000 rpm for 15 min. Upper aqueous phase was separated in a fresh Eppendorf and to it, chloroform isoamyl alcohol was added in equal volume, mixed and centrifuged at 12,000 rpm for 15 min. To aqueous phase 1/10Cytochrome b gene of T. annulata was targeted for its detection and identification. A primer set (Forward: 5’-ACT TTG GCC GTA ATG TTA AAC-3’/Reverse: 5’-CTC TGG ACC AAC TGT TTG G-3’) was used for amplification which was earlier reported by Bilgic et al. [c et al. . PCR assBabesia bigemina and Trypanosoma evansi.Out of 29 samples, 10 (34.48%) samples were found positive in Giemsa’s stained blood smears showing characteristic shaped piroplasm , whereasT. annulata is one of the most prevalent intra erythrocytic hemoprotozoan affecting crossbred cattle in India. Identification of carrier cattle is important as they are main source for the maintenance and perpetuation of Theileria in the environment and plays an important role in control program for theileriosis. Cattle become carriers when they survive the acute phase of the disease. After recovery, a low number of T. annulata piroplasms still remains in the body. In the most laboratories, detection of theileriosis is done by examining 50 microscopic fields for detection of parasites in blood smears which is a specific diagnosis but lacks sensitivity. In spite of the fact that method being cheaper, quicker, and easier to perform still requires high parasitemia, good smear preparation, proper staining and a well-trained microscopist. Although microscopic examination remains the convenient technique for day-to-day diagnosis of clinical cases, yet it is not able to detect carrier cattle due to the very low numbers (10−3× 5x 10−1) of infected erythrocytes [hrocytes .Theileria infections, particularly to determine carrier animals [T. annulata species-specific primer based on cytochrome b genesequences for the detection of this parasite in the carrier animals [PCR is now routinely used around the world for investigations of  animals ,8. The f animals .Babesia sp. and Trypanosoma sp. and can be used specifically to diagnose theileriosis in herd and differentiate it from other hemoprotozoan diseases.In this study, the prevalence of theileriosis was found to be 62.06% using PCR assay, whereas only 34.48% using microscopy which clearly indicates that PCR assay is the more sensitive tool in diagnosing theileriosis. It has been revealed that microscopic examination is a less sensitive method which has also been reported by other workers -15. HoweBy relating clinical findings with results from conventional staining and PCR assay, it has been revealed that a large section of screened herd is asymptomatic carrier.From this study, it can be concluded that detection of asymptomatic carrier state is difficult to diagnose by conventional staining, whereas PCR assay can precisely determine the carrier state. Results received from PCR assay will certainly help in planning control programs.GC and NKR designed and planned the study. GC and NKR collected the samples and prepared the onsite smears slides. SM, AK, and GC performed biotechnological part of the study. NKR, TK, and RJ examined the stained smear. GC and AK drafted the manuscript. SM critically reviewed the manuscript. All authors read and approved the final manuscript. NKR finalized the manuscript and sent for publication.


Renal disease is considered the main cause of natural mortality in dogs with canine leishmaniosis. The pathological mechanisms associated with kidney injury in canine leishmaniosis include immune complex glomerulonephritis, tubulointerstitial nephritis and occasionally renal amyloidosis. Proteinuria is a frequent finding in canine leishmaniosis and its quantification by the urine protein-creatinine ratio (UPC) is an important parameter in the staging of canine lesihmaniosis as presented by the LeishVet group.Leishmania infantum was positive with high antibody levels. The dog was hospitalized and treated with intermittent hemodialysis, feeding through an esophageal feeding tube, medical treatment for protein losing nephropathy and antileishmanial treatment with allopurinol. Kidney function gradually improved and the dog’s creatinine levels and proteinuria decreased until complete normalization two years after the acute insult. However, rhinitis and sneezing persisted and although the anti-leishmanial antibodies decreased over time, the dog remains constantly seropositive.A 4.5 year-old spayed female Belgian Malinois dog was presented to the Hebrew University Veterinary Teaching hospital with epistaxis and rhinitis and diagnosed also with proteinuria and acute kidney injury (AKI IRIS grade V) associated with canine leishmaniosis that developed to LeishVet stage III with chronic kidney disease (CKD) after stabilization. Clinicopathologic abnormalities included azotemia with a peak creatinine concentration of 7.76 mg/dl , hypoalbuminemia , hyperglobulinemia  and proteinuria . Serology by the enzyme-linked immunosorbent assay (ELISA) for To our knowledge, this is the first report of hemodialysis management of AKI associated with canine leishmaniosis. Hemodialysis was imperative in stabilizing the dog’s renal disease and controlling its azotemia. It demostrates that hemodialysis can be beneficial in the management of acute deterioration of kidney disease in canine leishmaniosis.The online version of this article (10.1186/s13071-018-2719-6) contains supplementary material, which is available to authorized users. Renal injury is common in canine leishmaniosis and ranges from sub-clinical to severe and fatal end-stage kidney disease. Although dermal manifestations and lymphadenomegally are the most common manifestations of canine leishmaniosis, renal disease is considered the main cause of mortality in dogs with this disease and it occasionally presents without typical skin abnormalities , 2. The Acute kidney injury (AKI) leading to severe uremia, regardless of its underlying cause, is associated with high morbidity and mortality –9. Most The aim of this report is to describe the management and follow up of a dog with canine leishmaniosis and associated kidney disease by medical treatment and hemodialysis. To the best of our knowledge, this is the first report of the use of hemodialysis in the treatment of canine leishmaniosis.3/mm3 (reference range 5.2–13.9 × 103/mm3), azotemia with elevated creatinine (3.1 mg/dl) and urea , hypoalbuminemia , hyperglobulinemia  and an albumin/globulin ratio of 0.39 . Urinalysis indicated a high magnitude of proteinuria (+4 by stick) and urine protein/creatinine ratio (UPC) of 15.2 . The dog was diagnosed with AKI and hopitalized for fluid and medical treatment and monitoring. Serology for leptospirosis, was negative using the microscopic agglutination test (MAT). At this stage, due to the hyperglobulinemia, proteinuria and kidney injury it was suspected for canine leishmaniosis and tested by an ELISA quantitative serology using Leishmania infantum antigen as previously described [Leishmania serology was highly positive with an optical density (OD) of 1.6 (cut off level 0.3) and treatment against leishmaniosis was started with allopurinol  at 10 mg/kg q 12 h PO. Despite fluid and medical therapy, the dog’s azotemia worsened within the next 3 days , reaching a state of AKI International Renal Interst Society (IRIS) grade V [A 4.5 year old spayed female Belgian Malinois dog was presented to a veterinary clinic in Central Israel with complaints of episodic right nostril epistaxis initially seen by the owners 10 weeks prior to presentation. Blood tests which included a complete blood count (CBC), serum biochemistry panel and a blood coagulation panel were within normal limits. Rhinoscopy performed at the referring veterinerian’s clinic showed that the right nasal mucosal surfaces were thickened and edematous and an ulcer was noted on the dorsal meatus, while the left side of the nose appeared normal. The dog was referred to the Hebrew University Veterinary Teaching hospital (HUVTH) for further evaluation. Physical examination was unremarkable and results of the CBC, serum biochemisty and coagulation tests consisting of prothrombin time (PT), partial thromboplastin time (PTT) and buccal mucosal bleeding time (BMBT) were within normal limits  except for the BMBT which was prolonged (5 min). Computed tomography (CT) of the nasal cavity showed bilateral thickening of the caudal nasal cavity conchal walls without obvious bone involvement, and accumulation of fluid in the cranial nasal cavity. A second rhinoscopy was performed and biopsies were taken for histopathology. Lethargy, inappetance and severe nasal bleeding were noted by the owners 3 days following rhinoscopy and biopsies. Epistaxis was unresponsive to local treatment with adrenalin, sedation with acepromazine  at 0.05 mg/kg IM and gelfoam  placement, finally necessitating surgery with bilateral carotid ligation. The dog was discharged without further bleeding and with a normal serum creatinine  2 days following surgery, with borad spectrum antibiotic treatment against potential bacterial infection of the surgical sites . Biopsy results indicated lymphocytic eosinophilc rhinitis with no evidence of bacterial or fungal infection. Three weeks after surgery, the dog was admitted after 2 days of decreased appetite and vomiting. It had increased episodes of sneezing and stretor, and nasal secretions had reappeared. Blood tests revealed mild normocytic-normochromic anemia  with leukocytosis of 19 × 10 grade V . HemodiaHemodialysis was performed using routine technique as previously described . BrieflyThe dog was discharged after 20 days of hospitalization at the HUVTH with a creatinine of 2.4 mg/dl. Treatment at home included allopurinol (10 mg/kg q 12 h PO) for leishmaniosis, famotidine  at 1 mg/kg q 24 h PO against gastric ulceration, the antibiotic amoxillin-caluvalonic acid (25 mg/kg q 12 h PO) against bacterial infection, and the antiemetics maroptinat citrate  at 1mg/kg q 24 h PO and metoclopramide  at 0.5 mg/kg q 8 h PO. Blood tests at a recheck 11 days after discharge revealed further improvemnt in kidney function (creatinine 1.9 mg/dl) and treatment with enalapril  at 0.25 mg/kg q 12 h PO was started for decreasing proteinuria.L. infantum antigen by ELISA, although with a lower antibody level compared to its initial testing , despite continuous treatment with allopurinol and a course of miltefosine  at 2 mg/kg q 24 h PO for 28 days, administered five months after discharge from hospital when the ELISA serology result was 1.32 OD. The dog is currently classified as having leishmaniosis LeishVet stage IIa [A detailed clinical history followup of the dog during 15 weeks after its discharge from the hospitalization with hemodialysis is included in Additional file tage IIa , compareL. infantum antigen over two years of treatment, it was brought to a state of partial remission from severe disease.This study describes the treatment and management of a dog with canine leishmaniosis associated with kidney disease. Hemodialysis was imperative in stabilizing the dog’s renal injury and controlling its azotemia. Although the dog did not completely recover from canine leishmaniosis despite continuous antileishmanial treatment, as indicated by its persistent positive serology to This first report of hemodialysis management of kidney disease associated with canine leishmaniosis demostrates that hemodialysis can be beneficial in the management of renal disease associated with canine leishmaniosis and should be considered as an optional treatment in leishmaniosis with AKI.Ehrlichia canis [Leishmania amastigotes [Epistaxis has been described in studies of leishmaniosis in 6–10% of dogs reported with this clinical disease in Italy and Greece , 14. Theia canis , 15, 16.stigotes . The prostigotes , 19. DoxSome degree of renal pathology can be found by histology in almost every dog with canine leishmaniosis; however, this does not always progress to decreased kidney function. A study of 55 seropositive dogs from Brazil found glomerulonephritis in all dogs, of which 13 were infected sub-clinically. Interstitial nephritis was found in 78% of the dogs, and glomerular deposition of parasite antigen in 91% . HoweverThe dog in this report is an example of chronic rhinitis and AKI which developed into CKD associated with canine leishmaniosis. Possible causes for the acute decrease in kidney function in this dog include untreated leishmaniosis and possibly decreased kidney perfusion due to dehydration (i.e. decreased water consumption) associated with rhinitis and anesthesia and blood loss during the dog’s first visit. Other common causes of canine AKI include leptospirosis which was ruled out by serology, hypoadrenocorticism which is frequently associated with electrolyte disorders, which were not found in this case, toxicities including ethylene glycol toxicity, which were not likely due to the dog’s history, ultrasonographic finding and recovery without specific antidote treatment, and nephrotoxic medications which were not administered prior to the event. It was initially classified as having LeishVet’s leishmaniosis stage IV and AKI IRIS grade V, and has improved with treatment to reach LeishVet stage IIa with CKD stage I , 6, 11. Hemodialysis management of kidney disease associated with canine leishmaniosis is described here for the first time in dogs. Management of AKI associated with canine leishmaniosis by hemodialysis and long term treatment of CKD were able to stabilize and prolong the life of the treated dog for more than two years. Therefore, hemodialysis is an important treatment modality that can be used, when available, for the management of kidney injury associated with leishmaniosis.Additional file 1:Table S1. Follow up of clinical findings and treatment of the dog during 15 weeks after its discharge from the Hebrew University Veterinary Teaching Hospital. Earlier findings are described in the text. (DOCX 14 kb)