Gammaproteobacteria in this location, while GB locations with lower nutrient concentration favored Alphaproteobacteria and Flavobacteria. According to the subsystems (SEED) functional analysis, GB has a distinctive metabolic signature, comprising a higher number of sequences in the metabolism of phosphorus and aromatic compounds and a lower number of sequences in the photosynthesis subsystem. The apparent phosphorus limitation appears to influence the GB metagenomic signature of the three locations. Phosphorus is also one of the main factors determining changes in the abundance of planktonic vibrios, suggesting that nutrient limitation can be observed at community (metagenomic) and population levels .Structuring of bacterioplanktonic populations and factors that determine the structuring of specific niche partitions have been demonstrated only for a limited number of colder water environments. In order to better understand the physical chemical and biological parameters that may influence bacterioplankton diversity and abundance, we examined their productivity, abundance and diversity in the second largest Brazilian tropical bay , as well as seawater physical chemical and biological parameters of GB. The inner bay location with higher nutrient input favored higher microbial (including vibrio) growth. Metagenomic analysis revealed a predominance of  Vibrio splendidus and V. anguillarum species groups were among the most abundant taxa in cold water environments Microbes are recognized as major drivers of nutrient cycling in the Oceans and in coastal waters V. cholerae, V. vulnificus, and V. parahaemolyticus) and marine animal (e.g. V. harveyi species group) pathogenic species The environmental parameters and ecological factors involved in habitat and/or niche partitioning may comprise biotic and abiotic factors. Three levels of control over microbial abundance and diversity are recognized Gammaproteobacteria are known to respond swiftly to nutrient inputs We studied the bacterioplankton abundance and diversity of Guanabara Bay (GB) as a model to better understand the effects of water quality parameters on free-living bacterioplankton, in three representative locations of the GB, according to previous studies The study area is located in the Guanabara Bay (centered on lat. 22°50′S and long. 43°10′W), Rio de Janeiro State, a eutrophic estuarine system near one of the largest metropolitan areas in Brazil. No specific permits were required for the described field studies. Subsurface waters (1–2 m depth) were sampled from three different sites, representing different trophic levels , accordia analyses were performed after vacuum filtration (<25 cm of Hg) of 2L of water. The filters (Glass fiber Millipore AP15) were extracted overnight in 90% acetone at 4°C, and analyzed by spectrophotometry or fluorimetry. Inorganic nutrients were also analyzed: 1) ammonia by indophenol, 2) nitrite by diazotization, 3) nitrate by reduction in Cd-Cu column followed by diazotation, 4) total nitrogen by digestion with potassium persulfate following nitrate determination, 5) orthophosphate by reaction with ascorbic acid, 6) total phosphorous by acid digestion to phosphate, and 7) silicate by reaction with molibdate. Dissolved (DOC) and particulate (POC) organic carbon were analyzed as described previously All environmental parameters were analyzed by standard oceanographic methods Prokaryotic production and total prokaryotic counts were performed only in the first year of study. Abundance was determined from two replicates of seawater by flow cytometry with Syto-13 , with minor modifications pyrH) gene sequences were obtained in order to determine vibrio diversity. PCR amplification of the pyrH gene was performed as described previously pyrH sequences were analyzed using the software Kodon package 2.03, and compared to reference sequences (type strains) available from GenBank (NCBI). Phylogenetic tree was constructed based on pyrH gene sequences applying the Neighbor-joining method using the software MEGA Monthly seawater samples were serially diluted and plated in triplicate (100 µl) in Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar. Colony counts were performed 48 h after incubation at 28°C. All vibrio colonies obtained in February 2010 from the three locations were stored in Tryptone Soy Broth with 20% glycerol at −80°C. These colonies were purified and isolates were subjected to DNA extraction following 2) were calculated in each case, in Microsoft Excel (Microsoft®). Variable transformation was used to ensure normality wherever needed . Two additional multivariate analyses were performed using the STATISTICA (Statsoft®) software: a) an ordination analysis, the principal component analysis (PCA), performed on a correlation matrix between total prokaryotic abundance, HNA, LNA, Vibrios and abiotic variables, and b) a multiple regression analysis with total prokaryotic abundance, HNA, LNA and vibrio counts as dependent variables and abiotic parameters as independent variables.Simple correlations were initially used as exploratory analysis, through fit of data points in linear models. Total prokaryotic abundance and Vibrio counts were plotted against all other variables and fit to the linear model .For the multiple regression analysis, assumptions were respected through residual analysis. To avoid distortions in the statistical tests, outlier data (>2*sigma) were excluded and independence between explicative variables was assumed for a −5A deeper understanding of structuring and metabolic potential of the sampled sites involved metagenomic analysis. Two replicate (ca. 2L each) seawater samples collected in February 2010, from each of the three study locations. In total, six samples were pre-filtered in a 20 µm nylon filter, followed by a Sterivex 0.2 µm filter. Microbial cells retained in the Sterivex filter received SET buffer and were stored at −80°C until DNA extraction, which was performed as described previously a levels and lower pheophytin (chlorophyll degradation product) levels in this location, indicating active photosynthesis. Based on the physical chemical and biological parameters of the seawater, the three locations can be split in two groups: Group 1, corresponding to locations 1 and 7, represented an intermediate area of the GB with higher influence from the coastal waters; Group 2, corresponding to location 34, a heavily polluted area of the GB, and with considerable input of nutrients.Seawater parameters were measured monthly in three locations of the GB . The hig5 and 3,68×107, with the highest values occurring in April 2009  according to pyrH gene sequence analysis  was higher in locations 1 and 7 than in location 34 (results not shown).Total prokaryotic counts obtained by flow cytometry and vibrios CFU counts obtained by plating on TCBS were higher in location 34 than in the other two locations . Prokaryril 2009 . Vibriosril 2009 . A presuril 2009  and 3. Pril 2009  [1]. Ouranalysis . The pyr2 = 0.829), and between both these counts and microbial production (0.70<r2<0.76) (Supplementary 2 = 0.620) and ammonia (r2 = 0.443) showing direct correlation, as opposed to an inverse correlation when plotted against salinity (r2 = 0.511)  .The PCA supported the division between Group 1 (locations 1 and 7) and Group 2 (location 34) . All bioa decreases (p = 0.043). HNA cells responded to increases in orthophosphate and ammonia (0.036<p<0.038). LNA, on the other hand, was highly statistically dependent only to temperature (p = 0.0006), even though total phosphorus, chlorophyll-a and ammonia were also selected for the model with no statistical support (p>0.05). Vibrio abundance was directly dependent on orthophosphate (p = 0.012) and inversely dependent on salinity (p = 0.032), despite nitrite being also selected for the model, with no statistically significant contribution (p = 0.161).The more robust multiple regression analysis also highlighted the importance of ammonia and mainly phosphorus to all biotic variables . Total pTo better analyze the structure of the bacterioplankton and to further assess the metabolic potential of the microbes, a total of six microbial plankton samples were subjected to metagenomic analysis. Location 1 produced 72,907 and 96,780 sequences, from which 85,723 (50.52%) were classified in the MG-RAST subsystems hierarchy and 112,245 (66.15%) were classified in the SEED database . LocatioBacteria. Archaeal sequences corresponded to less than 1% of the total, mainly represented by methane metabolizing groups  were responsible for the higher relative abundance in the more polluted location 34  in location 34. An increased contribution of Betaproteobacteria sequences to several subsystems was also noted in location 34, while the opposite was observed in Bacteroidetes/Chlorobi group, also reflecting changes in abundance in these groups. Cyanobacteria had a higher proportional contribution to the photosynthesis subsystem in the location 34, despite the similar relative abundances observed. Actinobacteria was also proportionally more abundant in location 34 and contributed predominantly to sulfur metabolism in this site , suggesting that other controlling factors, such as predation by protists and viruses, may account for prokaryotic abundance variation in GB, particularly in the vibrio model (r2 = 0.350). Future studies are needed in order to evaluate their ecological role in (vibrio) abundance and diversity in GB. Nevertheless, linear model fits revealed phosphorus  as one of the main limiting factors for vibrio growth in the GB, and the more robust multiple regression models supported this view for total prokaryotic abundance, HNA and vibrios, but not for LNA cells  and the higher prevalence of phosphorus metabolism subsystem in metagenomes from all locations supports the idea of phosphorus limitation. Tropical estuarine systems are usually phosphorus limited, even though in general, coastal systems are limited by nitrogen pyrH allelic diversity was also lower in location 34 (results not shown). The metagenomic analysis allowed some inferences regarding the structuring of the microbial populations. Gammaproteobacteria, Betaproteobacteria and Actinobacteria were proportionally the most abundant groups in location 34. The contribution of the former two to the metabolism in this location were generally higher in most subsystems, while Actinobacteria seemed to explore a particular niche metabolizing sulfur compounds plankton, not analyzed in this study. Because the GB had a distinct metagenomic profile, we suggest that metagenomic signatures may reflect environmental effects on microbial populations of GB, supporting the view that metagenomic signatures are useful to describe environmental features Because most subsystems  were found to be equally abundant regardless of the sample location  and the taxonomic group, it appears that a selection of microbes adapted to the GB environmental conditions occurred. Overall, GB had a distinct metabolic profile compared to an average bay metagenome (pooled data from 19 public bay metagenomes obtained from MG-RAST). Phosphorus metabolism represented one of the distinctive features in the metagenomes and was more (relative) abundant in GB than in other bays. Indeed, phosphorus seemed to be a limiting factor in GB due to excessive C and N input. Metagenomic sequences belonging to the aromatic compounds subsystems were also proportionally more abundant in the GB than in other bays. Aromatic compounds are persistent pollutants that accumulate in the water because they are hard to digest Vibrio parahaemolyticus was among the most frequently found species in these waters. Additionally, Vibrio plating is a relatively inexpensive technique for monitoring bacterial numbers in the environment, unlike the more expensive and technically demanding flow citometry count.Vibrios are indigenous marine bacteria, which grow easily on plates and play important ecologic roles in the marine environment This is the first study on the Guanabara Bay aiming at a time series comprehensive analysis on the planktonic microbial diversity and abundance. Our approach comprised nutrient concentration measurements, microbial counts, and metagenomics, to unravel microbial population composition and metabolic potential. The integration of data allowed us to shed light on the main bottom up factors controlling microbial abundance in GB. Our data also shows that the GB has typical features (e.g. phosphorus metabolism) that differentiate it from other bay metagenomic signatures. These features may have been acquired in the course of the occupation of the GB area, leading to in the observed structuring of the microbial composition. We also show that nutrient (phosphorus) limitation may be inferred from community (metagenome) and population  levels.Figure S1Phylogenetic tree based on pyrH gene sequences of vibrio isolates from Guanabara Bay using the Neighbor-joining method. Distance estimation was obtained by the model of Kimura 2-Parameter. Bootstrap percentages after 2,000 replications are shown. Scale bar, 1% estimated sequence divergence. Evolutionary analyses were conducted in MEGA5.(TIF)Click here for additional data file.Figure S2Most meaningful fits to linear models between vibrio/microbial counts and physical chemical parameters. Data points from each location are marked differently according to the legend. An exponential fit (data log transformed) was deemed best in figures B, C, D, E, F, G and H to ensure normality.(TIF)Click here for additional data file.Figure S3Relative percentage of contribution of archaeal sequences to GB metagenomes, separated by locations. Different letters indicate significant difference  between samples, while repeated letters indicate no statistical difference. In all cases, a>b>c, regarding relative percentage values.(TIF)Click here for additional data file.Figure S4Relative percentage of contribution of viral sequences to metagenomes, separated by locations. Different letters indicate significant difference  between samples, while repeated letters indicate no statistical difference. In all cases, a>b>c, regarding relative percentage values.(TIF)Click here for additional data file.Figure S5Relative percentage of contribution of alphaproteobacterial sequences to metagenomes, separated by locations. Different letters indicate significant difference  between samples, while repeated letters indicate no statistical difference. In all cases, a>b>c, regarding relative percentage values.(TIF)Click here for additional data file.Table S1List of public Bay metagenomes and general data obtained from MG-RAST.(XLSX)Click here for additional data file.Table S2General data regarding GB metagenomes.(XLSX)Click here for additional data file.


Aprasia parapulchella), a small legless lizard belonging to the endemic Australian family Pygopodidae.The infraorder Gekkota is intriguing because it contains multiple chromosomal and environmental sex determination systems that vary even among closely related taxa. Here, we compare male and females karyotypes of the pink-tailed worm-lizard n and (AC)n, have been amplified multiple times on the Y chromosome.Delma inornata and Lialis burtonis), suggesting that the common ancestor of Pygopodidae also had XY sex chromosomes. However, the morphology and size of the Y chromosomes are different among the three species, suggesting that the processes underlying the evolution of sex chromosomes in the Pygopodidae involved chromosome rearrangements and accumulation and amplification of repeats.XY karyotypes are found in other pygopodids ( Sex determination in reptiles shows astonishing diversity in comparison with other amniotes. It includes temperature-dependent sex determination (TSD) in many turtles, all crocodiles, the tuatara and many lizards, a wide variety of female heterogamety and male heterogamety sex chromosomal forms including species with multiple sex chromosomes, and species in which temperature and sex chromosomes combine influence to determine sex -5. NowheAprasia, Delma, Lialis, Ophidiocephalus, Paradelma, Pletholax and Pygopus. Lineages represented today by Delma and then Lialis are the first genera to diverge from a common ancestor  and AC (reverse complement of GT) – to chromosomes using FISH and inferred the process of sex chromosome differentiation in this species.In this study, we examined karyotype of A. parapulchella to be 2n = 42 8, and (AC)15, were mapped by FISH on to the chromosomes of both sexes. The (AATG)8 probe showed no specific signal in either male or female metaphases (data not shown) whereas (AGAT)8 showed intense hybridization signal at the centromeric region of one small size chromosome in the male metaphase e Figure A, but noe Figure B. This se Figure C, D while Figure C, D. We A. parapulchella is 2n = 42, the largest among all pygopodids thus far examined. The most common karyotype found among species of Diplodactylidae and Carphodactylidae is 38 acrocentric chromosomes [Delma, Ophidiocephalus and Pygopus, suggests that the chromosome number in the common ancestor of the Pygopodidae was also 38. It therefore appears likely that the chromosome number has increased in the lineage of A. parapulchella possibly through chromosome inversions and subsequent fissions.Our analysis has demonstrated that the chromosome number of omosomes , shared 15 and (AGAT)8, were mapped with intense fluorescent signals on to the Y chromosome also suggest accumulation and amplification of repetitive sequences. The lengths of the microsatellite repeats in the two microsatellite loci, APP6 and APP40, are 16 bp and 40 bp  and FISH analysis can not localize accurately such short length of DNA fragments. Therefore, our FISH mapping did not identify exact location of the two loci. However, the results clearly indicate that the two microsatellite repeats are in high copy number on the Y chromosome, and that the two microsatellite repeats, (AGAT)n and (AC)n, form the main components of the centromeric region and the long arm of the Y chromosome, respectively. It is commonly thought that suppression of recombination between the sex chromosomes favors the accumulation of repetitive DNA sequences such as retrotransposons, ribosomal DNAs and microsatellite repeats on the non-recombining regions increasing the level of differentiation between sex chromosomes [A. parapulchella.The intense CGH signal on a single chromosome with the male genomic DNA reveals a Y chromosome that is highly differentiated from the X chromosome not only in morphology but also in DNA content. The Y chromosome of this species has therefore probably degenerated and accumulated large quantities of male specific DNA, much of which is likely to be repetitive ,19. Two omosomes . Such acomosomes -30), andAprasia parapulchella, Delma inornata, and Lialis burtonis – exhibit male heterogamety. Given that these species come from three major clades within the Pygopodidae, including those representing basal lineages, it is likely that the common ancestor also had male heterogamety  were collected from sites in the ACT for cell culture. Animal collection, handling, sampling and all other relevant procedures were performed following the guidelines of the Australian Capital Territory Animal Welfare Act 1992 (Section 40), and conducted under Permit LT2012587 (ACT Government) and CEAE 11/12 . Two males and two females were used in the experiments.Tail tips cut from two male and two female Pink-tailed worm-lizards  was added to the culture flask at 75 ng/ml as the final concentration prior to harvesting. Following harvesting, cultured cells were fixed in 3:1 methanol:acetic acid and the cell suspension dropped onto glass slides, air-dried and stored at −80°C.Metaphase chromosome spreads were prepared from fibroblast cell lines of tail tissues following protocol described in Ezaz et al. . Briefly8, (AATG)8 and (AC)15, were purchased from GeneWorks .Total genomic DNA was extracted from cultured fibroblasts using the DNeasy kit  and following the manufacturer’s protocol. The oligonucleotides labeled with Cy3 of three microsatellite motifs, (AGAT)FISH and CGH were conducted according to our previous study  with sliGenomic DNA was labelled by nick translation incorporating SpectrumGreen-dUTP  for males and SpectrumOrange-dUTP (Abbott) for females. The labelled male and female DNA was coprecipitated with 20 μg glycogen as carrier, and dissolved in 15 μl hybridization buffer. The hybridization and washes were carried out as above with slight modification. Specifically, hybridization was carried out for 3 days and the slides were washed by 0.4xSSC, 0.3% IGEPAL at 55°C for 2 min and then 2xSSC, 0.1% IGEPAL at room temperature for 1 min.in situ hybridization; SSC: Saline sodium citrate; DAPI: 4′,6-diamidino-2-phenylindole; CCD: Charge-coupled device; dUTP: Deoxyuridine triphosphate; GSD: Genotypic sex determination.TSD: Temperature-dependent sex determination; CGH: Comparative genomic hybridization; FISH: Fluorescence The authors declare that they have no competing interests.The authors have made the following declarations about their contributions. TE and KM conceived, designed and directed the experiments. KM performed the experiments. KM wrote the first draft and all coauthors contributed in reviewing the paper. All authors read and approved the final manuscript.


PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children.In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of  Congenital and hereditary disorders of skin adhesion are collectively grouped under the umbrella of epidermolysis bullosa (EB), and these diseases are further separated depending upon the level of intracutaneous cleavage First described in 1997 by McGrath et al. Spontaneously arising hereditary skin diseases resembling suprabasal variants of human EBS have been reported only exceptionally in animals. A disease similar to human acantholytic EB has so far been described only in cattle: the so-called “familial acantholysis” was first reported in Angus calves nearly 30 years ago PKP1 was found to cause the absence of PKP1.In this paper, we characterize a novel genodermatosis of juvenile Chesapeake Bay retriever (CBR) dogs that clinically, histologically and ultrastructurally reproduces the phenotype of human ED-SFS. In these dogs, a mutation of the canine orthologue of Researchers were not at all involved in the breeding of these dogs. Outside of a standard physical examination, the authors did not perform any research-related work before death. Due to the pain and incurability of their disease, the puppies were euthanized within a couple of hours of their arrival to our hospital.Skin samples from neonatal CBR puppies, and skin and internal organ samples collected at post mortem examination from three month-old CBR puppies, were fixed in 10% neutral-buffered formalin, processed routinely in paraffin, cut to 5 micrometer histological sections and stained with hematoxylin and eosin for histopathology evaluation. Per NC State University's Animal Care and Use Policy, an application for vertebral animal use was not required as tissues were collected after the animals' death.For transmission electron microscopy (TEM), 1 mm thick skin samples from carpal pad, trunk and concave aspect of ear pinnae of three month-old CBR puppies  were placed in McDowell's and Trump's fixative  within less than 30 min of euthanasia. Samples were rinsed twice with 0.1 M sodium phosphate buffer (pH 7.2), prior to incubation in 1% osmium tetroxide  in 0.1 M phosphate buffer for one hour, rinsed twice with distilled water, and dehydrated in a concentration gradient series of ethanol baths, followed by 100% acetone double rinse. Samples were then placed in a mixture of Spurr resin  and acetone (1∶1) for 30 minutes, followed by two changes of 100% resin for two hours each. Subsequently, samples were polymerized in resin at 70°C for eight hours. Semi-thin sections (0.25 µm) were cut, stained with 1% toluidine blue-O in 1% sodium borate and used to confirm epidermal areas. Ultrathin sections (70–90 nm) were stained with methanolic uranyl acetate , followed by lead citrate, and were examined by transmission electron microscope . Reagents were obtained from Fisher Scientific, Pittsburgh, PA, unless otherwise indicated.The presence and expression pattern of selected keratin and desmosomal adhesion molecules was assessed on biopsies from clinically affected and unaffected footpad and ear skin. We performed indirect IF using a panel of antibodies specific for the human proteins. Details on the validation of these antibodies for use in dogs can be found in a recent article Tissue and blood samples were obtained from three CBRs with ED-SFS, two pairs of their parents, ten clinical normal littermates and related dogs, as well as one unrelated normal dog. Genomic DNA was extracted using the QIAamp DNA Extraction kit  following manufacturer's protocol.PKP1 spanning 4021 base pairs were sequenced using genomic DNA samples from an affected CBR, its sire, and an unrelated normal dog , 0.5 µl of each 20 µM primers (forward and reverse), 0.25 µl of platinum Taq polymerase (5 U/µl) and sterile de-ionized water (diH2O) up to a total volume of 24 µl  and synthesized by Integrated DNA Technologies, Inc., San Diego, CA). The general PCR conditions were as follows: initial denaturing at 94°C for 5 minutes, followed by 35 cycles of 94°C for 30 seconds for denaturing, 62°C for 30 seconds for annealing . Of this mixture, 10 µl was mixed with 10 µl of the PCR product. Digestion took place in a 37°C water bath overnight; the digested products were separated on a 1% agarose gel by electrophoresis and stained with ethidium bromide for visualization.Once an informative nucleotide polymorphism was discovered, endonuclease reactions were carried out using Alu1 restriction enzyme to distinguish the variants. The digestion reaction mixture included NEB Buffer 4 (10×), AluI  and dIHBetween 2008 and 2009, nine  out of 28 CBR puppies were diagnosed with ED-SFS; six of the affected puppies died within hours or days of birth. The affected puppies were born in four different litters to eight related, clinically normal parents. A seven-generation pedigree was evaluated in which all of the affected dogs could be traced back to one common ancestor. The degree of inbreeding ranged from 0.031 to 0.125 (mean = 0.036), which is fairly typical of a tightly bred purebred dog population. One affected dog (C18) was produced from a mother-son mating (inbreeding coefficient = 0.25). Pedigree analysis was most consistent with an autosomal recessive mode of inheritance.At birth, the skin of affected dogs was abnormally pale and translucent on the ears, feet, nose and mouth area, otherwise the puppies appeared normal. In all affected dogs, clinical signs occurred immediately after birth with spontaneous sloughing of the nose and footpad epithelium and bleeding of the ear tips if traumatized. Within 48 hours of birth, the lips and facial superficial skin layers also sloughed when rubbed dry or licked by the mother. Three dogs were kept alive by their breeder for three months; all exhibited waxing and waning superficial skin sloughing with erosions and fissures at areas of friction , concave ear pinnae and mucocutaneous junctions  . RubbingIn neonates that died within hours of birth, histopathology of the ear, trunk, distal limb and footpad areas revealed separation of keratinocytes and common, multifocal acantholysis of suprabasal keratinocytes, which coalesced in some areas . This dyExamination of lesional epidermis by TEM revealed widened intercellular spaces but retention of convoluted, interdigitating plasma membranes . In seveIndirect immunofluorescence (IF) performed with a panel of antibodies specific for selected epidermal keratins and desmosomal proteins revealed notable differences in staining patterns between samples of normal and affected CBR dogs . In the Overall, the immunomapping profile of affected dogs was consistent with lesion formation due to an absence of PKP1 leading to a detachment of desmoplakin from the desmosomal plaque and its retraction to the cell cytoplasm. As a likely consequence of these changes, keratin filaments condensated and aggregated around the nucleus.PKP1 including the exon-intron boundaries were sequenced and the sequence data compared to those in GenBank (NCBI). The product sizes are listed in All exons of Furthermore, the G-to-C mutation created a restriction enzyme cutting site within the PCR product used to sequence exon 1, which resulted in two fragments of 493 and 501 bp in length. These restriction fragment length polymorphisms were then used to genotype samples from 19 CBRs: three puppies from two litters affected with ED-SFS, each of their four clinically normal parents, and ten related clinically normal CBRs. One unrelated normal dog (American bulldog) was also tested as a normal control. A partial pedigree with the RFLP results is shown in PKP1, which results in a very short protein, abnormal and dysfunctional superficial epidermal desmosomes, profound keratinocyte acantholysis and superficial epidermal sloughing occurring at birth. These changes are characteristic of ED-SFS with PKP1 deficiency, a very rare genetic skin disease of humans. The availability of an inbred line of dogs harbouring such spontaneous mutation could be useful for the generation of PKP1-null puppies in whom, at least theoretically, gene therapy could be attempted.In this paper, we report a novel spontaneously-arising congenital and hereditary disease of dogs caused by a mutation in Puppies with ED-SFS exhibit most of the clinical signs seen in children with this syndrome . Acantholysis is associated with the aggregation of the keratin cytoskeleton (dyskeratosis). As dogs age, the epidermis becomes hyperplastic and hyperkeratotic while acantholysis decreases. Transmission electron microscopy is useful to demonstrate a reduced number of hypoplastic desmosomes with separation of keratin intermediate filaments from desmosomal plaques. These ultrastructural anomalies are shared with acantholytic suprabasal EB with desmoplakin deficiency In children and puppies with ED-SFS, immunostaining for desmosomal proteins usually reveals a marked reduction to a complete absence of PKP1 although some carboxyterminal mutations might lead to residual PKP1 immunostaining in some children with ED-SFS PKP1 have been described in humans, an unusually large proportion of them lie in splice donor and acceptor sites such as the IVS1. PKP1 (www.mutationtaster.org). However, because of a sequence difference between human and canine PKP1, a premature stop codon was encountered before an alternate splice donor site could be used in our dogs. In theory, the premature stop codon is predicted to lead to a truncated protein (67 amino acids plus 8 from the translation of intron 1 instead of the predicted 749 amino acid long canine PKP1). Importantly, this mutation is expected to result in a loss of most of the head and all nine armadillo-domains that are critical to the binding of PKP1 to its partners desmoglein-1, desmocollin-1 and desmoplakin, thereby rendering PKP1 completely dysfunctional While a variety of mutations in PKP1 mutation characteristics of ED-SFS of humans. With the selective mating of mutation-carrying CBRs and the generation of PKP1-null puppies, one could envision the possibility of evaluating the feasibility of gene therapy for this rare disease of children, as such intervention cannot be translated directly from the laboratory to human individuals. The main limitation of this spontaneous animal model, however, is the requirement of purpose-mating rare carrier dogs to obtain affected puppies that, in the absence of treatment effect, would have to be euthanized promptly for humane purposes. Alternatively, with a genetic test now available, ED-SFS could be rapidly eliminated from this breed with the testing of all at risk CBRs and the subsequent removal of mutation carriers.In summary, we reported herein a novel canine skin disease that mirrors clinical, microscopic, ultrastructural and Table S1PCR primers used for amplification and sequencing of the dog PKP1 gene.(DOC)Click here for additional data file.


Critical illness due to 2009 H1N1 influenza has been characterized by respiratory complications, including acute lung injury (ALI) or acute respiratory distress syndrome (ARDS), and associated with high mortality. We studied the severity, outcomes, and hospital charges of patients with ALI/ARDS secondary to pandemic influenza A infection compared with ALI and ARDS from other etiologies.t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient subgroups. Data that were not normally distributed were compared with the Wilcoxon rank-sum test.A retrospective review was conducted that included patients admitted to the Cleveland Clinic MICU with ALI/ARDS and confirmed influenza A infection, and all patients admitted with ALI/ARDS from any other etiology from September 2009 to March 2010. An itemized list of individual hospital charges was obtained for each patient from the hospital billing office and organized by billing code into a database. Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student’s p = 0.15). H1N1 patients had lower APACHE III scores  and had higher Pplat and PEEP on days 1, 3, and 14. Hospital and ICU length of stay and duration of mechanical ventilation were comparable. SOFA scores over the first 2 weeks in the ICU indicate more severe organ failure in the noninfluenza group (p = 0.017). Hospital mortality was significantly higher in the noninfluenza group . The noninfluenza group tended to have higher overall charges, including significantly higher cost of blood products in the ICU.Forty-five patients were studied: 23 in the H1N1 group and 22 in the noninfluenza group. Mean ± SD age was similar (44 ± 13 and 51 ± 17 years, respectively, ALI/ARDS secondary to pandemic influenza infection is associated with more severe respiratory compromise but has lower overall acuity and better survival rates than ALI/ARDS due to other causes. Higher absolute charges in the noninfluenza group are likely due to underlying comorbid medical conditions. Severe nce 1968 ,8,13-17.Limited data exist that compare the clinical differences between ALI in H1N1 patients and ALI arising from other etiologies. Furthermore, whereas a number of studies have assessed different aspects of the economic impact of the recent pandemic -21, few We report the severity, clinical outcomes, and hospital charges of ALI/ARDS secondary to pandemic influenza A infection compared with ALI/ARDS from other etiologies during a similar period of time. Based on clinical bedside observations and published reports ,5,8, we The study was approved by the Human Investigation Committee of the Cleveland Clinic Foundation (CCF)  as a retrospective, single-center study at the CCF Medical ICU.2/FiO2 ≤300; acute bilateral infiltrates; positive pressure ventilation via endotracheal tube; and no clinical evidence of left atrial hypertension or congestive heart failure) between the months of September 2009 to March 2010—the time that influenza infection was most prevalent. Diagnostic methods for influenza A virus detection consisted of rapid antigen testing, polymerase chain reaction (rtPCR), and viral culture from nasopharyngeal swabs, tracheal aspirates, and bronchioalveolar lavage specimens. The patients were grouped into two categories: those with laboratory-proven H1N1 infection; and those in whom H1N1was not clinically suspected. Only patients with confirmed infection were included in the influenza group to ensure that the clinical course of the disease was accurately captured. Patients were excluded from the study if they did not meet the above criteria for ARDS, or if clinical suspicion pointed to a likely pandemic viral infection with negative diagnostics.Patients were identified from a unit-based acute lung injury screening database (Cleveland Clinic is one of the centers participating in the ARDSnetwork) and the H1N1 patient log maintained during the fall-winter season of 2009–2010. Patients were included if they met criteria for ALI (PaOA Research Electronic Data Capture (REDCap) database was constructed with a complete listing of the patient’s demographic and clinical information, including age, gender, height, weight, body mass index (BMI), presenting symptoms, past medical history, primary reason for admission to the ICU, vital signs, presence of vasopressors, laboratory values, ventilator settings and respiratory parameters, Acute Physiology and Chronic Health Evaluation (APACHE) III and Sequential Organ Failure Assessment (SOFA) scores on admission to the MICU, number of intubated days, duration of ICU and hospital stay, mortality, and rescue therapies  . The dat2/FiO2: worst daily ratios were recorded; 3) plateau pressure (Pplat): for patients on volume control ventilation the airway pressure was measured after a 5-second inspiratory hold without concomitant active inspiratory efforts, and for patients on pressure control ventilation (PCV) the highest total system pressure (PEEP + inspiratory pressure) was recorded; 4) positive end expiratory pressure (PEEP): the value corresponding to the highest PEEP for the day was recorded; 5) tidal volume (Vt): the largest daily volume was recorded. Respiratory data were captured on the first day of intubation (day 1) and then on subsequent days 3, 7, and 14 of mechanical ventilation. There were no differences in ventilator protocols or management between the two groups.As part of the routine MICU respiratory therapy protocol, mechanical ventilation parameters are recorded every 4 hours. All patients are managed according to a mechanical ventilation protocol that incorporates the use of nonconventional modes when a lung protective strategy on conventional modes failed to provide adequate oxygenation. The following criteria were used to define the analyzed parameters: 1) mode of ventilation: the mode of ventilation that was used for the longest time for a given day; 2) PaOAn itemized bill of individual charges for each patient was obtained from the hospital billing office and was organized by billing code into the following categories: room/board, pharmacy, supplies, laboratory, radiology, surgical , blood products, respiratory services, dialysis, and miscellaneous . The values represent the hospital charges for the aforementioned services rather than the actual reimbursement, which may be subject to more variability. The single-center nature of the study removes interfacility differences in clinical and billing practices.t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient groups. In instances where the data were not normally distributed, the groups were compared with the Wilcoxon rank-sum test. Differences were considered statistically significant if the p value was <0.05.Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student’s Fifty-one patients were identified in the acute lung injury screening database between September 2009 and March 2010. Twenty-two met criteria for ALI and did not have confirmed or suspected H1N1 infection and were thus included in the noninfluenza group (ALI/ARDS secondary to noninfluenza etiologies). Thirty-six patients in the H1N1 patient log had confirmed influenza A testing. Of those, 23 had ALI requiring mechanical ventilation (MV) during their MICU stay and were included in our analysis.p = 0.135) and had increased requirement for mechanical ventilation on admission to the ICU . On the other hand, the noninfluenza group had a higher propensity to present with shock requiring vasopressors . The primary cause of ALI in the H1N1 group was pneumonia (n = 23), whereas in the noninfluenza group the etiologies were more varied, including pneumonia (n = 9), sepsis (n = 5), aspiration of gastric contents (n = 2), transfusion reaction (n = 1), and other (n = 5). Whereas seven patients (30%) in the H1N1 group were considered healthy, only one patient (5%) in the noninfluenza group had no comorbid medical conditions on admission to the ICU (Table p = 0.015), despite similar SOFA scores .Demographics, presenting symptoms, past medical history, and acuity on admission are shown in Table p = 0.017; Table There were no statistically significant differences between the two groups for initial laboratory data, including white blood cell count, platelets, serum creatinine, bilirubin, and creatinine kinase. The number of patients who developed acute renal failure that required dialysis throughout their ICU stay was the same (n = 8) in both groups. SOFA scores on days 1, 3, 7, and 14 of mechanical ventilation indicate that patients in the noninfluenza group had more severe organ failure during their ICU stay (p = 0.108); however, similar numbers of patients in both groups required rescue therapies over the duration of MV . Rescue therapies in the H1N1 group included inhaled NO (n = 4), ECMO (n = 2), prone ventilation (n = 3), and high-frequency ventilation (n = 1), and in the noninfluenza group only inhaled NO (n = 3) and prone ventilation (n = 2).Table p = 0.53) as were 28-day ventilator-free days . Four patients in the H1N1 group and seven in the noninfluenza group underwent a tracheostomy procedure. Hospital and ICU LOS were comparable . Mortality was significantly higher for patients in the noninfluenza group . Interestingly, a Kaplan-Meier curve of ICU mortality , supplies (p = 0.09), radiology (p = 0.08), and miscellaneous (p = 0.09) were large but not significant due to considerable variation. The proportion of charges in each of the major categories was similar between the groups  and the average ICU cost per patient per day  tended to be higher in the noninfluenza group.Even though all charges were higher in the noninfluenza group, only the difference in blood products utilized in the ICU was significant , and in relatively good health . There were no pregnant patients in either group. Compared with other studies of pandemic influenza patients who required mechanical ventilation, SOFA scores (mean 8.3) were similar, although APACHE II (25 ± 9) scores were higher [In accordance with other descriptive reports of pandemic influenza -12, patiACHE II 2 ± 9 scorres mean .3 were se higher ,16,17,23res mean .3 were se higher ,14,16,23res mean .3 were se higher ,14,16,232/FiO2 ratios in the two groups may be a reflection of higher PEEP values used in the H1N1 group for lung recruitment, rather than being indicative of comparable degrees of lung injury. Although assessing recruitability from this retrospective analysis is difficult and may be inaccurate, the higher PEEP used and the implication of lower compliance observed are predictors of potentially recruitable lung [Looking at the different patient characteristics between groups, it may be tempting to postulate that the higher rate of patients with pulmonary ARDS in the H1N1 group, in contrast to prevalent nonpulmonary ARDS in the noninfluenza group, would correlate with a higher PEEP response among the latter . Our finble lung . These oble lung .A number of important differences between the two cohorts emerged as well. As expected, the noninfluenza group was older, had more comorbid medical conditions, and less often presented to the ICU with respiratory failure. The degree of ventilator support was significantly higher in the H1N1 group on days 1, 3, and 14, and there was a trend to more severe hypoxemia during that time as well. Nevertheless, the use of use of APRV and rescue therapies was comparable in both groups. Despite more severe respiratory compromise, H1N1 patients did not have longer time on the ventilator, longer ICU or hospital stays, or higher mortality. Although SOFA scores were similar, the noninfluenza group had significantly higher APACHE III scores, likely secondary to points assigned to comorbid medical conditions. The high acuity of illness, as well as the presence of severe comorbidities, such as solid and hematologic oncologic conditions (7 patients), chronic renal insufficiency (6 patients), and cirrhosis of the liver (4 patients), likely contributed to the poor outcomes in the noninfluenza group. Conversely, despite more severe respiratory compromise, patients in the H1N1 group were more likely to recover due to their younger age and better overall health histories.The 77% mortality in the noninfluenza group was much higher than typically reported in clinical trials, with one notable exception . HoweverARDS is among the most expensive conditions encountered in the ICU . In 1984The overall similarity of charges in room and board and respiratory therapy between the two groups is likely indicative of the comparative durations of hospitalization and mechanical ventilation. Interestingly, despite higher ventilatory requirements and more severe hypoxemia in the H1N1 group, respiratory charges were similar between the two groups, suggesting that the high cost of maintaining a patient on mechanical ventilation is independent of the degree of ventilator support necessary. Thus, respiratory charges are more likely a reflection of duration of mechanical ventilation rather than the degree of ventilator support necessary. Absolute ICU charges for room and board, blood products, pharmacy, radiology, average daily charge, and overall charge per patient were larger in the noninfluenza group. ICU charges for blood products in the noninfluenza group were greater by a factor of four, and pharmacy charges double that of the H1N1 group. This finding is likely a reflection of the higher prevalence of underlying comorbid medical conditions in the noninfluenza group, such as malignancy and cirrhosis, which require expensive medications and predispose to anemia. Moreover, the high mortality in this cohort likely precluded even higher hospital charges. Nevertheless, the H1N1 cohort amassed charges of similar magnitude to the most ill and expensive patients in the ICU, indicating the abundant health care resources consumed by severe pandemic influenza infection.There are a number of limitations to our study. As a retrospective chart review rather than a prospective investigation, the information was culled from sources that were at times incomplete. Second, the study contained a relatively small number of patients, and measures taken to ensure internal validity of each group, such as limiting the influenza group to confirmed H1N1 infection and the noninfluenza group to the duration of the influenza season, further limited its size. Additionally, whereas our study provides descriptive information relevant to the patient population of our institution and tertiary referral centers with similar acuity, other ICUs may be exposed to a different cohort of patients. On the other hand, as a single-center study, potential differences in clinical and billing practices could be minimized. Although a comprehensive charge profile of each patient was generated, trends in the timing of charges could not be obtained. Finally, the hospital charge data were mined from an extensive database divided by charge coding, and therefore, some charges may have been mislabeled or inappropriately categorized.Our study provides interesting observations about the clinical course, outcomes, and cost of the H1N1 influenza pandemic. Although patients with severe pulmonary complications of pandemic influenza infection have poor oxygenation and require significant ventilatory support and rescue therapies, their younger age and tendency to have fewer comorbid medical conditions contribute to their improved prognosis compared with patients with ALI from other causes. Both groups of patients consume enormous amounts of hospital resources, and physicians and policy makers must be aware of this when future pandemics arise.The authors declare that they have no competing interests.JW and JK were responsible for the data input. JW and JG composed the manuscript. HW provided editorial assistance. EW provided the statistical analysis. All authors read and approved the final manuscript.


The rising global temperature is predicted to expand the distribution of vector-borne diseases both in latitude and altitude. Many host communities could be affected by increased prevalence of disease, heightening the risk of extinction for many already threatened species. To understand how host communities could be affected by changing parasite distributions, we need information on the distribution of parasites in relation to variables like temperature and rainfall that are predicted to be affected by climate change.Plasmodium, Haemoproteus, Leucocytozoon and Trypanosoma) in 403 birds. The overall prevalence of the four genera of blood parasites was 32.3%, with Haemoproteus the predominant genus. A total of 48 unique lineages were detected. Independent of elevation, parasite prevalence was positively and strongly associated with annual temperature. Parasite prevalence was elevated during the dry season.We determined relations between prevalence of blood parasites, temperature, and seasonal rainfall in a bird community of the Australian Wet Tropics along an elevation gradient. We used PCR screening to investigate the prevalence and lineage diversity of four genera of blood parasites (Low temperatures of the higher elevations can help to reduce both the development of avian haematozoa and the abundance of parasite vectors, and hence parasite prevalence. In contrast, high temperatures of the lowland areas provide an excellent environment for the development and transmission of haematozoa. We showed that rising temperatures are likely to lead to increased prevalence of parasites in birds, and may force shifts of bird distribution to higher elevations. We found that upland tropical areas are currently a low-disease habitat and their conservation should be given high priority in management plans under climate change. Plasmodium, for example, can occur between 16–30°C, with optimal temperatures around 28–30°C, whereas temperatures lower than 16°C greatly inhibit parasite development Many studies have described trends in the structure of assemblages along elevational gradients, and have found temperature to be one of the main variables controlling elevational distribution across a diverse taxonomic and ecological range of species In contrast to predictions for vector borne parasites, many studies have reported reductions in geographical range size and abundance, and shifts to lower latitudes or high altitudes, in a wide range of organisms that are potential hosts for these parasites The main aim of this study was to determine how temperature and rainfall influence prevalence of blood parasites in tropical birds. To do this we studied bird communities along elevation gradients in the Australian Wet Tropics. This bioregion is one of the best-studied tropical rainforests in the world. It consists of a strip of coastal plains and a series of adjacent mountain systems, with an altitude range from sea level to 1600 meters above sea level Plasmodium, Haemoproteus, Leucocytozoon and Trypanosoma) in birds of the Australian Wet Tropics in relation to elevation. We test for relations between parasite prevalence, elevation, temperature, and seasonal rainfall. These studies are not only important to implement future models on how increase of temperature will affect parasite loads but also how host communities could be affected by parasites.We present data on the prevalence and lineage diversity of four genera of blood parasites . Birds were caught and banded under the license number 2664 from the Department of the Environment and Heritage, Australia. All birds were released after blood samples were taken.2 and is primarily distributed along the mountain ranges The Australian Wet Tropics bioregion (AWT) is located in far North Queensland between 19°30’S and 15°30’S. The region is dominated by tropical rainforest, which covers an area of 10,000 kmhttp://www.bom.gov.au/jsp/awap/. Temperature decreased at an approximate rate of 1°C per 200 m altitude and there was approximately 1°C difference between the two areas sampled at the same elevation  and the Carbine Range . These localities are around 125 km apart. Nevertheless, they are within the same bioregion and have similar vegetation structure and almost identical bird faunas levation . The monlevation .We collected blood samples from 403 individual birds belonging to 40 species in sixteen different families: Acanthizidae, Alcedinidae, Climacteridae, Columbidae, Dicaeidae, Dicruridae, Estrildidae, Eupetidae, Meliphagidae, Muscicapidae, Nectariniidae, Pachycephalidae, Paradisaeidae, Petroicidae, Ptilonorhynchidae and Zosteropidae  Plasmodium, Haemoproteus and Leucocytozoon the first PCR step was carried out in a 10 µl reaction, using approximately 50 ng of DNA, 1x GoTaq Green Master Mix (Promega) and 0.5 of each primer .DNA was extracted from all samples using silica fines h primer . Cycling primers  and 2 µlh primer  and apprh primer  and 1 µlHaemoproteus with 80 infected birds (19.9%). Trypanosoma and Leucocytozoon showed very similar prevalence with 28 (6.9%) and 25 (6.2%) infected birds respectively, whereas Plasmodium was present in only 7 (1.7%) birds. An additional 15 (3.7%) individuals were infected with Haemoproteus and/or Plasmodium but the parasite could not be identified to genus due to low PCR amplification, poor-quality sequence or unresolved multiple infections. Among well-sampled host families  to 60.3% (Petroicidae). The family with the highest prevalence of Plasmodium and Trypanosoma was Pachycephalidae with 3.1% and 15.6% respectively, whereas Dicruridae had the highest prevalence of Leucocytozoon with 16.3%. Prevalence of the four genera of parasites was similar across different host families.Of the 403 individual birds screened, 130 (32.3%) tested positive for one or more parasite genera. The predominant parasite was Haemoprotueus was the genus exhibiting the highest number of lineages (30). Trypanosoma and Leucocytozoon presented 7 and 6 haplotypes respectively. Finally, for Plasmodium only 5 unique lineages were detected  was detected. Analysis of parasite lineages along the elevation gradient showed that most of the lineages were present only in certain elevation sites. This was probably due to the observed high lineage diversity and the specific trends of host distribution along the gradient. Only a few lineages of two well-represented families  were distributed along the entire gradient. Nevertheless, due to the great diversity of lineages found, sample sizes of each of these well-distributed lineages are not large enough to determine significant trends of distribution in relation to elevation, temperature or rainfall.1,4 = 52.45, P<0.002, R2 = 0.93) and positively to mean annual temperature  but only temperature contributed significantly to the model . We checked for relationships of parasite prevalence to host characteristics, including each species’ geographic range size, body mass and body size, but found no significant relationships  was negatively related to elevation  and Dicruridae , whereas Meliphagidae  and Pachycephalidae  displayed positive relationships that were not significant. Finally, Petroicidae was divided into the two species that make up this family and both showed a positive but statistically non-significant relationship of parasite prevalence to temperature: Tregellasia capito ; and Heteromyias albispecularis .Relationships of overall parasite prevalence to temperature in well sampled families  were positive in Acanthizidae . Relationships for Leucocytozoon , Trypanosoma  and Plasmodium  were also positive but were not statistically significant.Testing relationships of temperature to prevalence for each genus of parasite showed that prevalence of the predominant parasite 1,10 = 14.44, P<0.003, R2 = 0.59; 1,10 = 1.43, P<0.02, R2 = 0.04). The multiple regression model including both independent variables  to predict parasite prevalence was significant  but again only temperature contributed significantly to the model .The positive relationship between parasite prevalence and temperature held even when the data were divided into monthly averages  and the upland (600–1200 m) distinction of climatic zones, based on forest structure Plasmodium, and elevation Plasmodium occurs between 16–30°C, temperatures lower than 16°C inhibit parasite development, whereas optimal temperatures fluctuate between 28–30°C Haemoproteus) and sucking lice  Philornis on Argentinean forest birds found that temperature and rainfall were positively correlated with intensity of infection Protocalliphora (blow flies) on swallows showed that the number of blow flies varied in a curvilinear fashion with temperature, with parasite loads highest in nest around 25°C and decreasing at both higher and lower temperatures One of the mechanisms that could explain these results is that abundance of vectors is directly related to temperature. Bird haematozoa are transmitted by arthropod vectors The AWT are characterized by two marked seasons, the wet and dry. The dry season begins in May and is extended and acute until October or November, when the wet season begins. There was an interesting trend for parasite prevalence during the dry season to be higher, and lower during the wet season. However, we found no significant relationship between monthly parasite prevalence and rainfall. Further research is needed to show the influence of seasonal shifts that include both changes in rainfall and temperature.Haemoproteus and Leucocytozoon in passerine birds Haemoproteus can also cause severe disease and high mortality in avian hosts Average global temperatures increased 0.6°C in the period 1901–2000 The predicted increase of parasite prevalence due to increased temperature could interact with, and further exacerbate, the projected impacts of decreased range size, increased fragmentation, and decreased population size of birds due to climate change; all these can lead to an increased risk of extinction, specially for species inhabiting the uplands Table S1The full list of frequency of detection of blood parasites.Haemoproteus (Hae), Plasmodium (Pla), Leucocytozoon (Leu) and Trypanosoma (Try) in all the avian species presented alphabetically by family. Number of infected individuals/number of individuals sampled are shown.(DOC)Click here for additional data file.Table S2Primer sequences used for the two PCR step reactions to detect bloodparasites. Primers used to amplify Cytochrome b (Cyt-b) in Plasmodium (Pla), Haemoproteus (Hae) and Leucocytozoon (Leu), and 18 S rRNA (18 S) in Trypanosoma (Try).(DOC)Click here for additional data file.Table S3The full list of parasite lineages and host species. MalAvi lineage names , GenBank accession numbers, Parasite genus, Host Family and Host species.(DOC)Click here for additional data file.Table S4Regressions between Parasite prevalence and host ecological variables. Relationships between Parasite prevalence and host: a) Geographic range size, b) Body mass and c) Body size. All regressions are low and none significant.(DOC)Click here for additional data file.


The aim of our work was to evaluate the potential impact of the European policy of testing for HIV all individuals presenting with an indicator disease, to prevent late diagnosis of HIV. We report on a retrospective analysis among individuals diagnosed with HIV to assess whether a history of certain diseases prior to HIV diagnosis was associated with the chance of presenting late for care, and to estimate the proportion of individuals presenting late who could have been diagnosed earlier if tested when the indicator disease was diagnosed.We studied a large cohort of individuals newly diagnosed with HIV infection in 13 counselling and testing sites in the Lazio Region, Italy (01/01/2004-30/04/2009). Considered indicator diseases were: viral hepatitis infection (HBV/HCV), sexually transmitted infections, seborrhoeic dermatitis and tuberculosis. Logistic regression analysis was performed to estimate association of occurrence of at least one indicator disease with late HIV diagnosis.In our analysis, the prevalence of late HIV diagnosis was 51.3% (890/1735). Individuals reporting at least one indicator disease before HIV diagnosis (29% of the study population) had a lower risk of late diagnosis  compared to those who did not report a previous indicator disease. 52/890 (5.8%) late presenters were probably already infected at the time the indicator disease was diagnosed, a median of 22.6 months before HIV diagnosis.Our data suggest that testing for HIV following diagnosis of an indicator disease significantly decreases the probability of late HIV diagnosis. Moreover, for 5.5% of late HIV presenters, diagnosis could have been anticipated if they had been tested when an HIV indicator disease was diagnosed.However, this strategy for enhancing early HIV diagnosis needs to be complemented by client-centred interventions that aim to increase awareness in people who do not perceive themselves as being at risk for HIV. Combination antiretroviral therapy (cART) has dramatically changed the natural history of HIV infection by substantially reducing associated morbidity and mortality . HoweverDifferent strategies have been proposed to address this problem. In 2006, the US Centres for Disease Control and Prevention (CDC) recommended routine HIV testing for all individuals aged 13 to 64 years who come into contact with the health system . HoweverIn Europe another approach has been proposed, which is based on routine testing of individuals presenting with an “HIV indicator disease”, independent of any risk assessment. These diseases include certain infections that share a common mode of transmission with HIV, diseases whose onset is favoured by HIV-induced immunodeficiency, and any other medical condition associated with an undiagnosed HIV prevalence greater than 0.1%, the prevalence value for which delivering routine HIV testing resulted to be cost-effective ,12.The main objective of this study was to assess retrospectively the potential impact of this policy on preventing late HIV diagnosis in a cohort of newly HIV-diagnosed individuals. In particular, we estimated the association of HIV indicator disease occurrence with late HIV diagnosis, identifying variables associated with HIV testing after the diagnosis of an indicator disease and estimating the proportion of late presenting individuals who could have been diagnosed earlier if tested when the indicator disease was diagnosed.Since January 2004, a prospective multi-centre observational study on newly diagnosed adults with HIV infection (SENDIH Study) has been conducted in 13 regional counselling and testing sites of the Lazio region. Characteristics and methods of the study have been previously described . BrieflyIn particular, based on the frequencies observed in the pilot study, the questionnaire collected information about the following HIV indicator diseases: hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, syphilis, gonorrhoea, genital herpes, genital warts, infectious vaginitis, seborrhoeic dermatitis and tuberculosis.Regarding tuberculosis, even if it is already a generally accepted practice to screen TB patients for HIV, often, this recommendation is not followed. In addition, a number of studies demonstrated that TB represents a missed opportunity for HIV infection diagnosis ,16.The ethics committee of the coordinating centre, the “L. Spallanzani” National Institute for Infectious Diseases has approved the study .In the analysis, we included all individuals enrolled up to April 30, 2009 with a CD4 count determination available within 3 months of HIV diagnosis.HIV exposure category was classified according to the following modes of acquisition: intravenous drug use (IDU); men who have sex with men (MSM); heterosexuals. Individuals infected through blood products and those without a defined HIV exposure risk factor were classified as “other/unreported”.In a subset of individuals who also completed a self-administered behavioural and clinical questionnaire, we measured the concordance between the information on each single indicator disease reported in the questionnaire and the data collected at enrolment by Cohen's kappa statistic (K coefficient).For patients who reported a previous HBV or HCV infection at enrolment, we also checked serological status in clinical and laboratory records and measured the agreement between reported information and serological data.A descriptive analysis was performed to describe the occurrence of an indicator disease before HIV diagnosis.Univariable and multivariable logistic regression analysis were performed to estimate the association of HIV indicator diseases with late diagnosis, categorized as “late presentation” or “presentation with advanced HIV disease”. As a measure of association, we calculated the odds ratio (OR), multivariable logistic regression odds ratio (MLR-OR) and their 95% confidence intervals (95% CI).Exposure variables: having had at least one HIV indicator disease before HIV diagnosis (model 1); HIV testing after an HIV indicator disease (model 2) and type of indicator disease ; Seborrhoeic dermatitis/Tuberculosis) (model 3).Outcome variables: “Late presentation” was defined when the individual had a CD4 count <350 cells/mmc or an AIDS-defining event within 3 months of HIV diagnosis .“Presentation with advanced HIV disease” was defined when the individual had a CD4 count <200 cells/mmc or an AIDS-defining event within 3 months of HIV diagnosis .The covariates introduced in the multivariable models were: gender, age (as a continuous variable), area of birth , HIV exposure category  and a previous HIV- negative test.In order to estimate the chance that a person with at least one HIV indicator disease will be diagnosed late, we calculated the positive predictive value (PPV) of the indicator diseases for late presentation.Moreover, for 120 persons reporting more than one indicator disease, we performed the chi-square test to study the association between different combinations of multiple indicator diseases and late presentation.We performed a multivariable regression analysis in order to compare the characteristics of individuals who were tested for HIV with those who were not, after the diagnosis of an indicator disease, adjusted for age, gender, CD4 cell count, area of birth, risk factors, previous negative test and type of indicator disease. A patient was classified as tested for HIV after an indicator disease if the patient first tested HIV-positive or tested negative within 6 months after the indicator disease. For individuals with more than one indicator disease, we considered the most recent one.Since tuberculosis is considered an AIDS-defining illness, all statistical analysis were also performed excluding all individuals who reported tuberculosis in their clinical history.According to data derived from the CASCADE collaboration on HIV seroconverters, we estimated the presumed time since HIV infection for each individual. For the purpose of the analysis we assumed that HIV infection occurred 1.19, 4.19 and 7.94 years before the first HIV positive test, respectively, for people with a CD4 cell count <500 cells/mmc, <350 cells/mmc and <200 cells/mmc at the time of HIV diagnosis . Taking Data were entered into an Access database, verified by double entry and analyzed using SPSS package .Out of the 1,864 individuals enrolled up to April 30, 2009, 1,735 had a CD4 cell count performed within 3 months of the first HIV- positive test and were included in the analysis. Late presenters were 51.3% (890/1735) and presenters with advanced HIV disease were 34.2% (594/1735). Table Twenty-nine percent (504/1735) of the individuals reported at least one HIV indicator disease before HIV diagnosis and 120 reported more than one, for a total of 641 indicator diseases. Median time from indicator disease to HIV diagnosis was 15.4 months (IQR: 0.9-63.1 months).Distribution of reported indicator diseases was: 210 viral hepatitis cases ; 382 STI cases , 38 cases of seborrhoeic dermatitis and 11 cases of tuberculosis.Concerning the 120 persons reporting more than one indicator diseases, 37 reported two or more STI, 49 reported STI and hepatitis, 20 reported both HBV and HCV infection and 14 reported seborrhoeic dermatitis/tuberculosis with a STI or one type of hepatitis.In a subset of 683 individuals who completed a self-administered questionnaire, for each single indicator disease considered we observed a good concordance  between the questionnaire and the data collected at enrolment.Among the 126 individuals who reported HBV infection, serological data were available for 113 and 95% of them (107/113) were HBsAg and/or HBcAb -positive. Among the 84 individuals who reported HCV infection, serological data were available for 79, and 90% of them (71/79) were anti-HCV- positive or HCV-RNA- positive.Figure Univariable regression analysis showed that individuals who reported at least one indicator disease before HIV diagnosis had a lower risk of late presentation . The risk of late presentation significantly increased for older individuals  and female gender , while it decreased for MSM , IDUs  and individuals with a previous HIV negative test . The above estimated associations were unchanged after all individuals who reported tuberculosis were excluded from the analysis.With regard to the type of indicator disease, compared to individuals who did not report an indicator disease before HIV diagnosis, the risk of late presentation was lower for those reporting a STI  while it increased for those reporting seborrhoeic dermatitis/tuberculosis . Excluding all individuals with previous tuberculosis, the risk of late presentation for individuals reporting a STI was not modified, while it increased for those reporting seborrhoeic dermatitis .Multivariable logistic regression models, shown in Table If we consider the type of indicator disease, the multivariable analysis (model 3) showed that the risk of late presentation was significantly lower for individuals who reported diagnosis of a STI  compared to individuals who did not report a previous indicator disease, while individuals with seborrhoeic dermatitis or tuberculosis had a higher risk of late presentation . Excluding all individuals with tuberculosis from the analysis, the measures of association (MLOR) were unchanged in all 3 models, except for seborrhoeic dermatitis in model 3 .The multivariable regression analysis for presentation with advanced HIV disease confirmed the results of three multivariable regression models for late presenters (data not shown).The overall PPV of these indicator diseases for late presentation was 0.4, varying from 0.9 for seborrhoeic dermatitis to 0.5 for hepatitis and 0.4 for STI. In particular, both for hepatitis and STI, women had higher chance to be HIV late presenter  than heterosexual men  and MSM .Concerning the 120 persons reporting more than one indicator diseases, the proportion of late presenters who reported two or more STI (27.0%) was significantly lower than the proportion of those who reported STI and hepatitis (38.8%) or both HBV and HCV infection (65.0%), or seborrhoeic dermatitis/tuberculosis with a STI or one type of hepatitis (64.3%) (p = 0.013).Among the 504 individuals who reported at least one indicator disease, with respect to this diagnosis, 202 (40.1%) first tested HIV-positive within 6 months, 156 (30.9%) tested HIV- negative at least once in the following period and 123 (24.4%) were not tested for HIV following the indicator disease. For 23 (4.6%) individuals, information on previous HIV- negative tests was not available.The multivariable regression analysis Table  showed tBased on our assumption of the presumed time since HIV infection, 59 individuals (3.4% of the study population) who were diagnosed with an indicator disease and were not tested for HIV thereafter could already have been HIV- infected at the time of the indicator disease and their diagnosis could have been anticipated a median of 18.6 months.Among the 890 late presenters (CD4 < 350 cells/mmc), 52 individuals (5.8%) could already have been infected with HIV at the time of the indicator disease, which was diagnosed a median of 22.6 months before HIV. The majority of these 52 individuals were male (84.6%) with a median age of 39 years (range 22–71 years) and Italian (88.5%) With regard to HIV exposure category, individuals infected through homosexual contacts accounted for 57.7%, heterosexual contacts accounted for 28.8%, and those through injecting drug use for 9.6% of the total. More than one half (31/52) reported a STI as an indicator disease and 44.2% were never tested before HIV diagnosis. An additional 27 (3%) late presenters reported an indicator disease before HIV diagnosis that, according to our assumption, occurred before the estimated time of HIV infection.Among the 594 presenters with advanced HIV disease (CD4 < 200 cells/mmc), 33 (5.5%) could already have been infected at the time of the indicator disease which was diagnosed a median of 41.6 months before HIV.Routine HIV testing in individuals presenting with diseases/conditions which may indicate the presence of HIV infection, the so-called indicator diseases, has been recommended to reduce undiagnosed HIV infection ,12. In pIn our multi-centre study, we found that almost thirty percent of newly diagnosed adults with HIV infection reported at least one indicator disease before HIV diagnosis, and that being tested for HIV within six months of being diagnosed with an indicator disease reduced the risk of late HIV presentation by 50%.More than half of our study population had a late HIV diagnosis, i.e. at a stage of their disease when, according to current guidelines, they should have already started cART, and 39% of the individuals were first diagnosed with HIV at an advanced stage. These figures are consistent with previous reports from industrialized countries. In Europe among 10,222 newly diagnosed HIV infection cases with CD4 cell counts reported in 2009, 51% had a first CD4 cell count below 350/mmc . SimilarFactors associated with late diagnosis and presentation with advanced HIV disease in our study included older age, being foreign born and having acquired HIV infection through heterosexual contact. These results agree with those from previous studies -22 and sOn the other hand, we found that a previous diagnosis of an HIV indicator disease followed by an HIV test within six months significantly reduced the risk of late presentation.Almost thirty percent of our study population reported at least one indicator disease before HIV diagnosis and more than ten percent were diagnosed with HIV within six months of being diagnosed with an indicator disease. Taken together, these data support the hypothesis that testing for HIV following a diagnosis of an indicator disease may significantly prevent late diagnosis.To our knowledge, few reports have analyzed the association between diagnosis of an indicator disease and probability of receiving an earlier HIV diagnosis -28. KleiIn contrast with these results, in the study conducted between 2001 and 2005 in a South Carolina health care facility, Duffus et al. found that approximately 80% of the health care visits before HIV diagnosis for both late and early testers were for conditions not likely to prompt HIV testing in a non-routine testing environment . The autIn our study, the association with late diagnosis differed according to the type of indicator disease considered. Individuals reporting a STI had the lowest risk of late diagnosis, while those reporting tuberculosis and seborrhoeic dermatitis had an increased chance of being diagnosed at an advanced stage of immunosuppression. This finding is not surprising since the risk of persons with HIV developing tuberculosis significantly increases, parallel to their increasing level of immunodeficiency . NeverthIn our study population, although diagnosis of an indicator disease reduced the overall risk of late diagnosis, almost one fourth of the individuals were not tested for HIV within six months of being diagnosed with the indicator disease.The probability of being tested for HIV following an indicator disease didn’t differ for age, gender, CD4 cell count or place of birth. Individuals who reported to have been previously tested for HIV had a higher probability of being tested after an indicator disease; on the contrary, those with an unreported mode of HIV acquisition had a lower probability of being tested after an indicator disease.It is difficult to interpret this result since individuals with unreported modes of HIV acquisition may actually include people with different risk behaviours . HoweverRegarding the potential further impact of this strategy, our data suggest that HIV diagnosis could have been anticipated by a median of 22.6 months in almost 6% of late presenters if they had been tested at the time the indicator disease was diagnosed. We may have underestimated this proportion. In fact, an additional 3% of late presenters reported an indicator disease which occurred before our estimated time of HIV infection, based on CD4 cell counts at HIV diagnosis. Since there is variability in progression of HIV infection, not taken into account in our analysis, it is possible that some of these individuals could already have been infected at the time the indicator disease was diagnosed.The indicators diseases considered in our study, are conditions that a population-based case control study conducted in Denmark  and the Other potential limitations of our study need to be considered. First, HIV-testing history and clinical history were collected by patient interviews and this may imply a recall bias. However, we found a good concordance between the information on indicator diseases collected at enrolment and those reported in a self-administered questionnaire. Additionally, serological data provided evidence of past HBV or HCV infection, respectively in 95% and 90% of individuals who reported these infections at enrolment. Second, for individuals who were not tested for HIV following an indicator disease, we do not know if HIV testing was recommended by the health care provider but was refused by the patient. Third, our study population accounts for approximately 60% of the newly diagnosed HIV infections reported every year to the Regional HIV Surveillance System in Lazio region  and therA recent survey conducted in European countries has shown that an HIV prevalence greater than 0.1% can be recorded among patients presenting with some indicator medical conditions, including those considered in the present study . Based oOur data suggest that testing for HIV following diagnosis of an indicator condition significantly decreases the probability of late HIV diagnosis, and thus reinforces the indication to implement this strategy as an important component of a control policy for the HIV epidemic.However, this policy needs to be complemented by client-centred interventions that aim to increase awareness in people who do not perceive themselves as being at risk for HIV.cART: Combination antiretroviral therapy; US: United States; CDC: Centres for Disease Control and Prevention; IDU: Intravenous drug use; MSM: Men who have sex with men; HBV: Hepatitis B virus; HCV: Hepatitis C virus; STI: Sexually Transmitted Infections; MLR-OR: Multivariable logistic regression odds ratio; PPV: Positive predictive value.EG has received honoraria for presentations at workshops and/or travel grants from Abbott, Bristol-Myers Squibb, Boheringer Ingelheim. PS, GC, GDC, MG, CMM, SAB, ARB, SG, AS, AC, NO, VP and GI declare that they have no competing interest.EG designed the study and drafted the manuscript. PS participated in the design of the study and drafted the manuscript. GC conducted the statistical analysis and contributed to writing the manuscript. GDC; MG; CMM; SAB; ARB; SG; AS; AC; NO contributed with data collection and critically revised the manuscript. VP; GI participated in the design of the study and critically revised the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/473/prepubSENDIH Study questionnaire.Click here for file


The superficial vein of the resting limb constricts sympathetically during exercise. Central command is the one of the neural mechanisms that controls the cardiovascular response to exercise. However, it is not clear whether central command contributes to venous vessel response during exercise. Tendon vibration during static elbow flexion causes primary muscle spindle afferents, such that a lower central command is required to achieve a given force without altering muscle force. The purpose of this study was therefore to investigate whether a reduction in central command during static exercise with tendon vibration influences the superficial venous vessel response in the resting limb.vein) and blood velocity of the basilic vein in the resting upper arm were assessed by ultrasound, and blood flow (BFvein) was calculated using both variables.Eleven subjects performed static elbow flexion at 35% of maximal voluntary contraction with (EX + VIB) and without (EX) vibration of the biceps brachii tendon. The heart rate, mean arterial pressure, and rating of perceived exertion (RPE) in overall and exercising muscle were measured. The cross-sectional area (CSAP <0.05). Increases in heart rate and mean arterial pressure during exercise at EX + VIB were also lower than those at EX (P <0.05). CSAvein in the resting limb at EX decreased during exercise from baseline (P <0.05), but CSAvein at EX + VIB did not change during exercise. CSAvein during exercise at EX was smaller than that at EX + VIB (P <0.05). However, BFvein did not change during the protocol under either condition. The decreases in circulatory response and RPEs during EX + VIB, despite identical muscle tension, showed that activation of central command was less during EX + VIB than during EX. Abolishment of the decrease in CSAvein during exercise at EX + VIB may thus have been caused by a lower level of central command at EX + VIB rather than EX.Muscle tension during exercise was similar between EX and EX + VIB. However, RPEs at EX + VIB were lower than those at EX (Diminished central command induced by tendon vibration may attenuate the superficial venous vessel response of the resting limb during sustained static arm exercise. Venomotor response is considered to play an important role in the transfer of blood from veins to the heart. Many studies have suggested that sympathetic activation has an impact on the venomotor responses in the resting limb during static exercise -3 and dySustained static exercise produces significant activation of the sympathetic nervous system, and the sympathetic activation during exercise is governed by both central  and peripheral  mechanisms -16. MetaIn addition to muscle metaboreflex, an essential central command role has emerged from a preliminary study . The convein). Tendon vibration during active muscle contraction excites the primary afferents of muscle spindles of the contracting muscle, thereby inducing reflex tension via the monosynaptic tendon reflex, which in turn aids voluntary tension development and consequently reduces the amount of central command required to generate a given force [Based on these considerations, we investigated whether central command affects venomotor tone in the contralateral limb during sustained static exercise. In the present study, using vibration of the biceps brachii tendon reported previously ,25, we een force ,25,26. Ien force .Eleven healthy subjects  volunteered to participate in the study. Their mean ± standard deviation age, height, and weight were 21.3 ± 0.9 years, 165.0 ± 6.6 cm, and 55.2 ± 5.7 kg, respectively. All subjects were nonsmokers. The participants were asked not to drink beverages containing caffeine or alcohol for 24 hours and not to eat for at least 2 hours before the start of the experiment. The purpose, procedures, and risks of the study were explained to the subjects, and their informed consent was obtained. The study was approved by the Human Ethics Committee of the Japan Women’s College of Physical Education and was conducted in accordance with the Declaration of Helsinki.Before the main protocol, the subjects were examined to establish the force produced by tendon vibration at rest. A custom vibrator  was used to induce left biceps brachii muscle contraction by reflex stimulation of the biceps brachii distal tendon on the cubital fossa . The oscIn a room maintained at 25.1 ± 0.2°C, each subject stayed in a semi-reclined position in a chair in which body position could be maintained, while the left elbow was kept at a 90° angle on a padded armrest with the wrist attached to an arm lever by a Velcro strap. The subjects rested for at least 20 minutes before data collection began. After baseline data were collected for 5 minutes, subjects performed: static elbow flexion at 35% MVC without vibration of the biceps tendon for 2 minutes (EX); and static elbow flexion at 35% MVC with vibration of the biceps tendon for 2 minutes (EX + VIB). Each exercise period was followed by a recovery period of 1 minute. Static elbow flexion was produced using the same dynamometer that was used to measure the MVC (VINE), with visual feedback of the achieved force provided via an oscilloscope display. For EX + VIB, tendon vibration was initiated 1 minute before starting exercise and continued during the exercise. Immediately after exercise, subjects read instructions for the 6 to 20 rating of perceived exertion  category scale developed by Borg  and instBeat-to-beat changes in arterial pressure were assessed by finger photoplethysmography . The monitoring cuff was placed around the middle finger. The heart rate (HR) and mean arterial pressure (MAP) were determined from the blood pressure waveform using the Modelflow software program, taking into account sex, age, height, and weight .Muscle oxygenation (oxyhemoglobin (oxy-Hb) and deoxyhemoglobin (deoxy-Hb) concentration) in the left exercising upper arm and right resting forearm was monitored using a near-infrared spectroscopy system  at dual wavelengths (760 nm and 850 nm). The near-infrared spectroscopy probe consisted of an optically dense holder containing an emission and detection probe and was secured to the skin with tape to minimize extraneous light.vein) and cross-sectional area (CSA), non-invasive ultrasound imaging of the basilic vein  of the resting upper arm was performed 5 to 6 cm proximal to the cubitus using an 8.7-MHz linear array transducer . A large quantity of ultrasound transmission gel was used to prevent direct contact with the skin and to avoid compression of the vein. Vvein and CSA were simultaneously measured on a transverse scan of the vein with the transducer tilted at 60°. Positioning of the transducer was determined at the beginning of each experiment, and it remained unchanged to limit potential errors in Doppler angle. Vvein was the result of the mean velocity of spectral Doppler recording every 12 seconds. CSA was calculated by manually tracing the edge of the offline transverse venous image at an arbitrary three points every 12 seconds, and then the three CSA values were averaged. Because CSA was obtained from the image measured at 60°, an accurate CSA (CSAvein) was determined as follows:To measure blood velocity , using time and condition as fixed factors. If a main effect of condition and/or interaction was detected, post hoc analysis with a paired t test was performed; and if a main effect of time was detected, post hoc analysis with a Bonferroni test was performed. To compare the baseline data of the circulatory response, CSAvein, Vvein, and BFvein between EX and EX + VIB, a paired t test was performed. In addition, differences in Overall RPE and Arm RPE between conditions were evaluated by paired t test. P <0.05 was considered significant.To compare the time-course changes, two-way analysis of variance with repeated measures was applied to the circulatory responses, CSAvein (from 0.20 ± 0.03 cm2 to 0.20 ± 0.03 cm2), Vvein (from 4.1 ± 0.5 cm/second to 3.9 ± 0.5 cm/second), and BFvein of the basilic vein (from 51.6 ± 10.3 ml/minute to 52.7 ± 11.9 ml/minute) did not change.Vibration of the biceps tendon for 2 minutes elicited a reflex force equivalent to 5.3 ± 2.3% of MVC. However, the HR (from 63 ± 3 beats/minute to 64 ± 2 beats/minute), MAP (from 78 ± 3 mmHg to 78 ± 3 mmHg), CSAvein, Vvein, and BFvein between EX and EX + VIB .Muscle tension during EX was similar to that during EX + VIB Figure . Howevervein, Vvein, and BFvein of the resting upper arm during EX and EX + VIB. The increase in HR during exercise from 96 to 120 seconds at EX + VIB was less than that at EX   , but CSAvein during EX + VIB did not change from baseline throughout the protocol. In addition, CSAvein at 120 seconds of exercise and during recovery at EX was lower than at EX + VIB (P <0.05) . Δoxy-Hb of the exercising upper arm during recovery at EX was lower than at EX + VIB  and returned to baseline during recovery in both EX and EX + VIB .Figure B Figure B. In theB Figure C,D. Howevein decreased from baseline during static elbow flexion alone, although CSAvein during static elbow flexion with tendon vibration did not change, and that BFvein did not change significantly during static exercise with or without tendon vibration. These results suggest that a reduction in central command during static exercise with tendon vibration may attenuate the superficial venous vessel response of the resting limb during sustained static arm exercise.The primary findings in this study were that CSAvein did not change throughout the protocol during EX and EX + VIB  of exercise. In addition to feed-forward control, there is evidence that the effects of central command on cardiovascular responses are closely related to the intensity or perceived effort of the exercise ,35. CentVibration is a powerful stimulus for primary muscle spindle afferents when applied to the biceps tendon during static exercise. When the biceps brachii was contracting, activation of its muscle spindle primary afferents provided reflex activation, which in turn aided voluntary tension development compared with contraction only of the biceps brachii. The afferent input of decreased voluntary tension during exercise with tendon vibration might thus cause interactions between perception of effort and central command, such that the activation of central command might alter .vein during static exercise between EX and EX + VIB might not be due to the differences in activation of the reflex neural mechanism under different conditions.The increase in sympathetic nervous system activity during exercise is caused not only by central command but also by the reflex neural mechanism that is activated by exercise (muscle mechanoreflex and muscle metaboreflex) -11,13,16Although the specific regions of the brain involved in exercise-related responses remain speculative, the following theory can be considered. Animal studies suggest that subthalamic regions are capable of generating both motor and cardiovascular responses . In humaStewart and colleagues reported that venoconstriction during static exercise, which occurs not only in the splanchnic area but also in the resting extremities, may contribute to an increase in venous return to the heart to increase cardiac output . Taking vein) is dependent on the venous pressure level – but we did not measure venous pressure. As mentioned above, however, BFvein and Δdeoxy-Hb (an index of venous blood volume) of the resting forearm did not change from baseline during both EX and EX + VIB (Figures Several limitations should be considered when interpreting our results. First, due to the large compliance of veins, volume (that is, CSA Figures E and 3D.vein in the resting upper arm when compared with static exercise alone, although BFvein was similar during exercise both with and without tendon vibration. These findings suggest that central command may contribute to the superficial venous vessel response of the resting limb during sustained static elbow flexion.Static elbow flexion with vibration of the biceps brachii tendon, which caused a decrease in central command during exercise, inhibited the increase in circulatory response and the decrease in CSAvein: Blood flow of the basilic vein; CSA: Cross-sectional area of basilic vein before correction; CSAvein: Accurate cross-sectional area of the basilic vein after correction; Δ: Change; EX: Elbow flexion without vibration; EX + VIB: Elbow flexion with vibration; oxy-Hb: Oxyhemoglobin; deoxy-Hb: Deoxyhemoglobin; HR: Heart rate; MAP: Mean arterial pressure; MVC: Maximal voluntary contraction; RPE: Rating of perceived exertion; Vvein: Blood velocity of the basilic vein.BFThe authors declare that they have no competing interests.AO designed and coordinated the study, carried out the experiment, and drafted the manuscript. KS participated in the design of the study and helped draft the manuscript. AH helped carry out the experiment. TS participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript.