Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P<0.0001) and the vacA s1/i1/m1 allele (P<0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P<0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression. Helicobacter pylori (H. pylori) is a successful pathogen, colonizing the gastric mucosa of over 50% of the world’s population , 42H. pycagA (EPIYA type), vacA (s/i/m type), hom loci A/B (hom type) and bab loci A/B/C (bab type). Each locus was assigned a discrete numerical character corresponding to its type. Consequently, each clinical isolate was distinguished by a seven-number code representing its genotype. For example, strain G27 has the following genotype: cagA EPIYA (ABCC), vacA (s1/i1/m1), hom locus A (homB), hom locus B (empty), bab locus A (babC), bab locus B (babB), and bab locus C (babA). This genotype corresponds to the number 2020321 in the input file. All resulting codes were utilized to generate the strain input files used in subsequent analyses (Phylogenetic trees were constructed using Phylip v3.695 (Phylogeny Inference Package) . First, analyses .We began constructing the trees via the SEQBOOT program to generate multiple data sets. With this program we used a combination of bootstrapping and character permutation to generate 100 data sets. These data sets were then used as the input file in the PARS program, a general parsimony program that carries out the Wagner parsimony method and allows for up to 8 discrete character states and the use of ‘?’ for states that are unknown. PARS was run using the default settings with the following exceptions: species order was randomized using a random number seed of 5, with 10 replicates and the trees were constructed from the 100 data sets generated in SEQBOOT. PARS searches for the best fit tree, saving the 100 best options. As a final step, we used the PARS output trees in the CONSENSE program to generate a majority-rules consensus tree. In doing this, the branch lengths of our final tree represent the number of times a given branching event occurred in the 100 trees generate by PARS. FigTree v1.4.2 is a tree editing program .bab genotype, hom genotype, cagA EPIYA polymorphisms, vacA s/i/m polymorphisms, and disease state were analyzed using the Fisher’s exact test. Log linear modeling was used to assess higher order associations that were significant at a 5% level. Using categorical values to represent our data sets, we fit a saturated model. We then applied a backward selection algorithm that eliminates the least significant associations at each step and then reforms the model to look for associations. Data analysis was conducted using SPSS version 22 software  or SAS version 9.3 software .Statistical analysis was conducted as previously described . Brieflybab genotypes in American and South Korean strains were deposited in the NCBI GenBank database with accession numbers KP339308 to KP339491.The sequences for Complete demographic data was available for all 80 AH  Tables. bab genes at locus A, B and C is shown in babA, babB and babC, three types of chimeric bab genes were also present: babAB, babBA and babBC. A total of 23 different bab genotypes, including a single strain where bab was absent from the three known loci (-/-/-), were identified within the 160 isolates chosen for this study. While 6.9% of strains (11/160) carried a single bab gene, the majority of the strains  carried two bab genes, one at locus A and one at locus B  and babC/babB/- (15.0%) showed a higher frequency in this population (babA in greater than 90% of isolates (74/80), most of the diversity within the bab genotypes for the AH was driven by locus A, which was significantly more variable (P<0.0001) than seen in KH. Indeed, babA occurred at locus A only 47.5% of the time (bab genes (28.8%), followed by babC (15.0%) and babB (7.5%).The distribution of identified  locus B . Of notebab gene . In the isolates . In comppulation . Whereasthe time . The nexbab genes, babAB, babBA and babBC that were the result of gene conversion. This gene conversion likely occurs in the 5′region (~60 to 240 bp from the initiation codon) and in the 3′region (~1200 to 2100 bp) but this later event is hard to detect due to the homology in this region. It can be speculated that the characteristic of the chimera would follow that of the second bab gene, which would constitute the majority of the chimeric gene. bab chimeric forms were relatively common in the AH group, occurring at one or more loci in 26/80 (32.5%) of isolates versus 6/80 (7.5%) in the KH group  and 83.3% in KH (5/6)  . Within  of babA , resulti present . In thesbab genes from the AH group and 72 bab genes from the KH group contained a run of CT repeats (5–14 CT repeats)  occurring at locus B (bab genes containing CT repeats were found at locus B (97.2%) . Although not significant, the trend was similar for all babB genes regardless of loci, where more babB genes were ‘OUT’-of-frame (54.9%) than ‘IN’-frame (45.1%) in the AH group (babB genes tended to be ‘IN’-frame (59.7%) as compared to ‘OUT’-of-frame (40.3%) (Eighty-two repeats) . In all  locus B . Similar (97.2%) . InteresAH group . The opp (40.3%) .babC, we took this opportunity to sequence 15 babC genes from our collection and compare the deduced BabCs. As shown in babC sequences, we identified only one chimeric babBC gene (AH-J68) and no babAC gene. Furthermore, we found that the N-terminus of BabC was most similar to that of BabA, and the C-terminus of BabC was homologous to that of both BabA and BabB  (P = 0.0028). When KH and AH were combined, there was also a significant association between locus A and locus B (P = 0.0131) . Furthermore, if BabB was being expressed from locus A, then babB at locus B was more likely to be ‘OUT’-of-frame . Yet interestingly, if babA occupied locus A, then babB at locus B was more likely to be ‘IN’-frame .To further understand the  0.0027) . Convers 0.0131) . Of notebab loci, we next assessed whether bab genotype was associated with other outer membrane proteins. We have previously looked at the association between homB and disease in the South Korean population [hom paralog (homA and homB) from the Hom protein family, which shares over 90% identity, are known to occupy two possible loci  [bab genes, and homA and homB. Once again, given the differences in the two populations, each was analyzed individually as well as combined. There were no significant associations when comparing strains for presence of homA versus homB. Given that homA and homB are homologous and share two conserved loci, locus A and locus B, we also considered hom gene number in which we looked at whether or not both hom locus A and locus B were occupied regardless of if it was homA or homB. We found a significant association with the bab paralog present at locus A and number of hom loci occupied. This association was seen in both the AH and KH groups   (babC was rare (n = 14), it occurred only in isolates with a single hom gene. Also of note in the AH group, in strains where locus C was occupied there was a significant difference between carrying a single hom gene versus two hom genes (P = 0.039). Although the finding wasn’t significant in the KH group, the same trend was seen; locus C was only occupied in strains with a single hom gene (Given the associations among pulation . hom parA and B) . Therefoctively) . In the e 61.5%,  Tables. m 97.3%,  Tables.  0.0029) . One faccagA and vacA polymorphisms are associated with more severe disease outcomes [bab genotypes were found in association with known cagA or vacA polymorphisms. There were no significant associations between cagA and bab when the groups were looked at separately; however, when considering the clinical isolates as a whole, there was a significant association between the bab gene at locus A and the cagA EPIYA polymorphism (P<0.0001) , whereas strains carrying a babB or babC at locus A were most likely to have a non EPIYA-ABD   . The i and m regions of vacA clustered together in that i1 compared to i2 and m1 compared to m2 resulted in the same distributions. babA at locus A was more frequently associated with i1 and m1 as compared to babC, which was much more commonly associated with i2 and m2. Once again, babB appeared to be found in relatively equal frequencies with i1m1 and i2m2; however, when we analyzed the vacA genotype of strains carrying babB at locus A, the strains with only an i2m2 vacA type were more likely to have a single hom gene  and strains with an i1m1 vacA classification were more likely to carry two hom genes  (data not shown).It has been well established that particular outcomes , 27, 46.<0.0001) . The cagctively)  Tables. or each) . Furtherhom loci . If we fcagA and vacA and the bab genes, particularly at locus A, we asked if there was an association between bab genotype and disease. The only significant association between bab and disease was seen in the KH group. In this group, there was a significant association between the bab genes present at locus B and disease outcome (P = 0.0101) (bab gene at locus B were more likely (66.7%) to come from individuals suffering from more severe disease outcome (gastric cancer and gastric ulcers) than from individuals suffering from gastritis and duodenal ulcers (33.3%). It was also interesting to note, although the finding was not significant (P = 0.176), that individuals with cancer (gastric carcinoma) or pre-malignant lesions (Barrett’s Esophagus) were more likely to have a babB gene that was ‘IN’-frame’  compared to ‘OUT’-of-frame’  (data not shown).Given the association between the virulence factors,  0.0101) . SpecificagA (EPIYA type), vacA (s/i/m type), hom locus A and B (hom type), and bab locus A, B, and C (bab type). As shown in cagA ABD EPIYA motif and vacA s1/i1/m1 type, their bab and hom genotypes more closely resembled the AH. Given this observation, we next constructed a separate consensus tree using only the hom and bab genes . Furthermore, these data suggest that OMPs may be a valuable tool to help develop a comprehensive classification system to identify H. pylori populations and sub-populations. In all, this study has expanded the profile of bab possible genotypes and characterized a new population. Furthermore, it highlights several associations that should be investigated next at a functional level.Taken phically ; however strains , the priS1 Fig(TIF)Click here for additional data file.S2 FigbabB gene reading frame in each population. (B) Number of ‘IN’ and ‘OUT’ babB gene reading frame at each locus.(A) Percentage of ‘IN’ and ‘OUT’ (TIF)Click here for additional data file.S3 FigVariable regions 1 and 2 are indicated, VR1 and VR2, respectively, above the BabC amino acid sequence.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Filoviridae viruses, is a NIAID category A, lethal human pathogen. It is responsible for causing Ebola virus disease (EVD) that is a severe hemorrhagic fever and has a cumulative death rate of 41% in the ongoing epidemic in West Africa. There is an ever-increasing need to consolidate and make available all the knowledge that we possess on EBOV, even if it is conflicting or incomplete. This would enable biomedical researchers to understand the molecular mechanisms underlying this disease and help develop tools for efficient diagnosis and effective treatment. In this article, we present our approach for the development of an Ebola virus-centered Knowledge Base (Ebola-KB) using Linked Data and Semantic Web Technologies. We retrieve and aggregate knowledge from several open data sources, web services and biomedical ontologies. This knowledge is transformed to RDF, linked to the Bio2RDF datasets and made available through a SPARQL 1.1 Endpoint. Ebola-KB can also be explored using an interactive Dashboard visualizing the different perspectives of this integrated knowledge. We showcase how different competency questions, asked by domain users researching the druggability of EBOV, can be formulated as SPARQL Queries or answered using the Ebola-KB Dashboard.Ebola virus (EBOV), of the family Database URL:http://ebola.semanticscience.org. Zaire ebolavirus and is one of the five known viruses within the genus Ebolavirus. The other members of this genus are Bundibugyo virus (BDBV), Reston virus (RESTV), Sudan virus (SUDV) and Taï Forest virus (TAFV) (Marburgvirus [Marburg Virus (MARV) and Ravn Virus (RAVV)]. Ebolavirus  and Marburgvirus (MARV and RAVV) constitute a part of the family Filoviridae (Ebola virus (EBOV) is a member of the species s (TAFV) . Four ofs (TAFV) . BDBV was (TAFV) . TAFV was (TAFV) . EBOV, ts (TAFV) . EBOV haoviridae . These voviridae  were conoviridae . The 41%oviridae , 8.The Viral Hemorrhagic Fever Consortium sequenced a set of 99 EBOV sequences from 78 confirmed patients in Sierra Leone to 2000× coverage  RNA-dependent RNA Polymerase’, and the adjoining widgets are automatically updated to facet on this selection, and show only the associated publications and annotations from an initial listing. The user can then click on any particular publication to read its abstract and other information in the adjoining tab.Moreover, the Ebola-KB Dashboard makes this information just a click away from the biomedical researcher. The user of the Dashboard can click on the desired gene ‘Polymerase’, the researcher would now like to proceed on the next step of his analysis. He would like retrieve knowledge available in KEGG and DrugBank on all the putative small molecule ligands which may bind to protein product of ‘Polymerase’ or other similar proteins, as determined by different in silico experiments . He could formulate the SPARQL CONSTRUCT Query as shown in Supplementary Appendix I (Listing II and III), which uses a Sub-Select SPARQL query against the respective SPARQL endpoint of Bio2RDF KEGG and DrugBank. Information like chemical name, molecular formula, molecular weight, SMILES notation of the ligands contained within PDB structures associated with ‘Polymerase’ can also be obtained. Using the Ebola-KB Dashboard, clicking on the relevant gene constrains the list of ligands automatically. The properties of each ligand entry in the list can then be viewed in the separate tab, or the three-dimensional PDB structure can be visualized , however on individual inspection of each corresponding PDB entry, we found that they were related to our initial EBOV search term ‘Polymerase’.Using the subselect queries, we obtain the pharmacology, action mechanism and packagers of these set of ligands from DrugBank, and activity, drug targets and implicated pathways from KEGG using the kegg:x-drugbank predicate. The aggregated RDF graphs can be combined to create an exemplary data table as shown in ‘Polymerase’, the researcher would like to know which scientific publications provide evidence to the binding of these ligands to other similar proteins or domains. Before starting in vivo experiments for the druggability of the EBOV domain using the binding ligand, this step is relatively important for the domain researcher. The researcher may be interested to determine if the ligand under question is also effective against wild type or rapidly mutating strains, and does not bind to other unrelated human proteins causing adverse side effects. For instance, after checking the exemplary table retrieved in the previous step, the researcher may be interested to investigate further into the drugs ‘Rifampicin (RFP)’, ‘Rifabutin (RBT)’ and ‘Rifapentine (RPT)’, which fall under the KEGG category of ‘Rifamycins’, are known to target bacterial DNA-Dependent RNA Polymerase  would allow us to deal with the heterogeneity of the data sources and provide integrated access across several biomedical data sources, as shown in the KEGG/DrugBank use case, but it also necessitates the availability and better uptime of SPARQL Endpoints, which may not always be the case  could be considered  (Supplementary data are available at Database Online.Stanford University start-up fund (M.D.) Funding for open access charge: XXX.Conflict of interest. None declared.


BVD and IBR are contagious viral diseases highly prevalent in Irish cattle. Despite their significant reproductive and economic impact very little is known about the BVD and IBR status of stock bulls (a bull used for breeding purposes). There are still a high proportion of dairy farms in Ireland that rely on the use of a bull for breeding cattle and ensuring the fertility of the bulls is of paramount importance for the efficiency of the farms. The prevalence of BoHV-1 and BVD in stock bulls in Irish dairy herds has never been investigated. The objectives of this study therefore were: (i) to provide descriptive, observational data on the use of stock bulls on Irish dairy farms; (ii) to investigate the BVD and BoHV1 status of a sub-set of stock bulls; (iii) to investigate factors associated with BVD and BoHV1 status of stock bulls and (iv) to investigate factors associated with dairy herd status for BVD and BoHV1, including any associations with the use of stock bull.A total of 529 blood samples from bulls involved in the dairy breeding process were analysed for BVD virus using RT-PCR, and BoHV-1 antibodies by ELISA test. A total of 305 different dairy herds took part in the study and the overall BVD and BoHV-1 herd status was determined by ELISA using four bulk tank milk samples over the 2009 lactation. Logistic regression was used to investigate the associations between the stock bulls and BVD and BoHV-1 herd and individual status.Of the 305 total participating farms, 235 farms (77 %) had at least one bull and 167 farms had purchased bulls. Two bulls (0.4 %) out of 529 tested were found positive for BVD virus and 87 (16.7 %) tested seropositive for BoHV-1. Some significant associations were identified between the purchase of bulls and both viral diseases. Purchased bulls were three times more likely to be seropositive for BoHV-1 than homebred bulls. In the same way, herds with purchased bulls were three times more likely to be classified as seropositive for BVD and four times more likely to have evidence of recent BoHV-1 circulation than farms where all the bulls were homebred.The prevalence of BoHV-1 and BVD in stock bulls in Irish dairy herds has never been investigated. This study highlights the widespread use of stock bulls in Irish dairy herds, as well as the high rate of exchange of bulls between farms. Significant associations were found between the origin of the bull and their serological BoHV-1 status. In keeping with these results, bulls with higher number movements between farms were more likely to be seropositive for BoHV-1. Artificial insemination (AI) has become the breeding norm on the majority of dairy farms in the Republic of Ireland . HoweverA key breeding goal in a spring-calving system is to achieve high 3 and 6-week pregnancy rates from breeding start date to achieve a concentrated calving pattern the next season . Use of Cattle movement has been explored by many authors as a root cause of disease spread , 10. ManThe levels of BVD and BoHV-1 infection amongst dairy stock bulls in Ireland have never been examined nor has the degree of bull movement between farms. Therefore, the objectives of this study were (i) to provide descriptive, observational data on the use of stock bulls on Irish dairy farms; (ii) to investigate the BVD and BoHV1 status of a sub-set of stock bulls; (iii) to investigate factors associated with BVD and BoHV1 status of stock bulls and (iv) to investigate factors associated with dairy herd status for BVD and BoHV1, including any associations with the use of stock bulls.The study was carried out in 2009 and licensed by the Irish Department of Health and Children meeting all legislative requirements for research involving animals in the Republic of Ireland at the time of the study.A detailed description of the sample population used in this study has previously been outlined , 16. BriStudy farms were visited to sample all bulls involved in the dairy breeding process. Bulls being raised for beef production were not included. Samples were taken by coccygeal venepuncture into plain glass vacutainers. Each sample was centrifuged at 5000 g for 5 min, serum aspirated and samples stored at −20 °C until analysed. Subsequently, a 100 μl aliquot of each serum sample was pooled in batches of ten for BVD virus testing. In addition, four bulk milk samples, and blood samples from 20 % of the replacement heifer group with a minimum of five heifers less than 9 months were sampled from each herd over the 2009 lactation These were used to determine herd BVD and BoHV-1 serostatus, both historical (bulk milk) and recent seroconversion (weanlings) .Pooled serum samples were tested for the presence of BVD virus by commercial laboratory  using a Real Time–Polymerase Chain Reaction (RT-PCR). The detection limit of this assay was 50 to 100 viral particles in 1 ml whole blood . Where avs. non-spring-calving), number of bulls per farm (no bull vs. 1 bull vs. >1 bull), region (high density dairy vs. low density dairy), herd size (31–65 cows vs. 66–99 cows vs. >99 cows), type of farming enterprise (dairy livestock only vs. mixed livestock (farms with other cattle enterprises)), herd vaccination status (vaccinated vs. unvaccinated). Farms were also classified with regard to the origin of the bulls on each farm i.e. all bulls homebred vs. at least 1 bull purchased.Herds were classified according to a previous study  in diffeWith regard to disease status, two herd classification methods were employed. Firstly herds were classified as seropositive or seronegative for each of the diseases BVD or BoHV-1 according bulk milk ELISA results . For theSecondly, herds were classified on the basis of having ‘evidence of recent viral circulation’ i.e. those herds having at least one weanling serologically positive for either BVDV or BoHV-1. Herds that did not record a positive weanling were classified as ‘not having evidence of recent viral circulation’ .Finally, herds were classified according to ‘overall bull BoHV-1 status’, positive herds having at least one serologically positive bull in the farm.A questionnaire survey was used to determine the IBR vaccination status of each study farm, the vaccine product used, and the date the vaccine was administered. In the case of purchased bulls, the IBR vaccination history prior to purchase was not available. Any purchased bull testing positive for IBRgB, therefore, was re-tested using the IBRgE assay, as only marked vaccines (gE deleted) are available in republic of Ireland, in order to rule out prior vaccination as the source of seropositivity.vs. purchased). As a number of homebred bulls had moved off and back to the herd of origin, bulls were also classified according to their number of off-farm movements prior the time of sampling. Other categories included vasectomized status (vasectomized [Vas] vs. not vasectomized [NVas]), BoHV-1 individual vaccination status (vaccinated [Vacc] vs. unvaccinated [Unvacc]), and region in which the study herd was located (Region 2: high density dairy vs. Region 1: low density dairy). With regard to disease status, all individual animals were classified as positive or negative for BVD virus or BoHV-1 antibody.Bulls were categorised on the basis of breed and herd of origin . Pearson’s chi-squared, Fisher’s exact, univariable and multivariable logistic regression, were carried out using Stata (Version 11).Number of bulls per farm, number of vasectomized bulls, number of bulls per farm across regions, and number of non-homebred bulls was investigated. The age of each bull on the date of sampling was calculated, as well as the number of total movements recorded per bull and the time spent in the study herd prior to sampling.vs. homebred) and reproductive use of the bulls (vasectomized vs. no vasectomized). Only BoHV-1 seropositivity of individual bulls was investigated using logistic regression due to the small number of BVD virus-positive bulls.Examination of relationships between BoHV-1 seropositivity of individual bulls and a number of independent variables was completed. Independent variables investigated comprised the age of the bulls at the sampling time, movements between farms pre-sampling, herd of origin , as the dependent variables by a number of independent variables. Independent variables included having a bull, number of bulls, herd of origin of bulls and having vasectomized bulls in the farm. To investigate the relationships between the purchase of bulls and the herd status only herds with purchased bulls that had spent at least 3 months in the farm prior the sampling were considered.A total of 305 study herds took part in the study and 529 individual breeding bulls were sampled. Of the 305 participating farms, 235 (77 %) had at least one bull involved in the breeding process, and 70 (23 %) had no bulls in the farm. Of the 235, 68 (29 %) farms had bulls which were homebred only. The remaining 167 (71 %) herds contained at least one purchased bull. The proportion of herds in each bull classification is outlined in Table In relation to the individual bull data 243 out of the 529 bulls analysed for this study were non-homebred. The number of bulls of each breed and vasectomized bulls in each breed is shown in Table Movement data relating to 486 bulls, of the 529 total participants, was available for the period prior to sampling. Demographic information on the 43 remaining animals was not available and was, therefore, not included in the statistical analysis. Examination of movement data highlighted that 217 bulls had recorded at least 1 between farm movement, and 40 bulls recorded more than three movements prior the sampling point. The average number of movements was 1.8 (range 1 to 7). The number of animals recording between-farm movement prior the sampling is included in Fig. n = 87). Regarding the use of vaccines, only 75 bulls were vaccinated against BoHV-1 on the farm on which they residing at the time of the study. A total of 16 (21.3 %) tested seropositive for BoHV-1, using the gE assay. Also, 12 purchased bulls positive for BoHV-1 (test gB) were classified as negative after being re-tested using gE.In all, the BoHV-1 status of 8 bulls couldn’t be confirmed and these animals were excluded from statistical analysis. Two bulls out of 529 tested positive for BVD virus yielding an apparent prevalence of 0.4 %. One bull, aged 9 months at the time of sampling, was homebred; the other aged 21 months was purchased. The apparent prevalence of BoHV-1 amongst bulls tested was 16.7 % (P = 0.49).With regard to herd demography, the prevalence of bull exposure to BoHV-1 in region 1 (low dairy- density) was 18 %, meanwhile in region 2 (high dairy-density) 20 % of the bulls were detected positive. No significant differences were found between regions . Also, the bulls with higher numbers of movements prior to sampling were more likely to be seropositive (Table P = 0.07).The number of movements pre-trial was significantly associated with an exposure to BoHV-1, with animals with one or more recorded movements being 1.3 times more likely to be seropositive (ve Table . FurtherP = 0.064).Results of the final multivariable logistic regression model of herd status are shown in Table P = 0.027). Similarly to BVD herd status, farms with purchased bulls were approximately four times more likely to be categorised as having evidence of recent BoHV-1 circulation (P = 0.039). Although significant associations weren’t found between the presence of purchased bulls and BoHV-1 herd status (bulk milk and heifer serology), farms with purchased bulls were almost three times more likely to have at least one positive bull on the farm over farms where all the bulls were home born (P = 0.009).Regarding BoHV-1 herd status, a significant association with the number of bulls was recorded. Farms with more than one bull were twice as likely to be categorised as BoHV-1 positive over those who had a single bull . Although there are a number of studies carried out in beef and dairy cattle, it would appear that no studies have been carried out to investigate the degree of infection in the stock bull population. Despite the economic advantages of artificial insemination (AI) over natural service (NS), a high number of dairy farmers in Ireland that still rely on NS, compared to other European countries . Among tA number of countries have reported BVD PI prevalence of between 0.4 and 1.1 %. The prevalence of PIs amongst Irish calves ear-notched as part of the national BVD eradication scheme in 2013 was 0.68 %It is also of note that one of the BVDv positive bulls identified was vasectomized. This bull was homebred, underweight and ill-thrifty as a calf, and could therefore not be sold at an early age with herd cohorts. The calf was therefore maintained on the farm and subsequently vasectomized, highlighting the ease with which a PI can be maintained on farm and may ultimately contribute to the breeding process on a dairy farm. Based on these results, the use of vasectomized bulls and choice of animal to be vasectomized deserves careful consideration on dairy farms. Vasectomized bulls act as a cost-saving measure for heat detection purposes , but if Based on the results of the current study, the likelihood of introducing of BoHV-1 seropositive bull would appear greater than BVDv positive bulls. While a previous Irish study reported 6 % of beef bulls selected for performance testing by the national cattle breeding centre as being seropositive for BoHV-1 , just ovPurchased bulls were three times more likely to be BoHV-1 seropositive in the current study. Cattle trade and movement, in general, have been reported by a number of authors as major risk factors for the spread of BoHV-1 , 26, 27.An additional noteworthy finding of this study was the significant association between purchase of bulls with both BVD herd status and BoHV-1 viral circulation. The high probability of having recent BoHV-1 viral circulation in herds that purchased bulls has also been identified in previous studies , where pFarms with non-homebred bulls were three times more likely to be bulk milk positive for BVD than those farms where all the bulls were homebred, in contrast to BoHV-1, where no relationship was found. This most likely relates to the differences in the dynamic of spread of both viruses across a herd population. In the case of BoHV-1, a large proportion of latent carriers exist within an adult dairy herd allowing for the possibility of continuing re-infection from within the herd itself. In contrast to this, transmission of BVDv in the adult herd is dependent on contact with a PI. Many PIs die, however, before reaching breeding age , leadingTwo factors were significantly associated with an increased BoHV-1 herd serostatus. This included farm location and the number of bulls present on each farm. Farms in Region 1 (lower cattle density) had a higher probability of being positive for BoHV-1, which has already been investigated by other authors , who proMinimal attention has been dedicated internationally to the role of stock bulls in transmission of infectious diseases in dairy farms. This study recorded widespread use of stock bulls on Irish dairy farms, a high degree of ‘between-farm’ movement of these animals, and the potential for bulls to act as a vector of disease between farms. Further studies into the role of stock bulls in transmission of viral diseases, in countries where NS is common practice, is required, as is the role of these breeding animals in transmission of other infectious pathogens.


The rapid spread of Zika virus in the Americas and current outbreak of microcephaly in Brazil has raised attention to the possible deleterious effects that the virus may have on fetuses.th gestational week. Ultrasound examinations in the 2nd and 3rd trimesters demonstrated severe microcephaly, hydranencephaly, intracranial calcifications and destructive lesions of posterior fossa, in addition to hydrothorax, ascites and subcutaneous edema. An induced labor was performed at the 32nd gestational week due to fetal demise and delivered a female fetus. ZIKV-specific real-time polymerase chain reaction amplification products were obtained from extracts of cerebral cortex, medulla oblongata and cerebrospinal and amniotic fluid, while extracts of heart, lung, liver, vitreous body of the eye and placenta did not yield detectable products.We report a case of a 20-year-old pregnant woman who was referred to our service after a large Zika virus outbreak in the city of Salvador, Brazil with an ultrasound examination that showed intrauterine growth retardation of the fetus at the 18This case report provides evidence that in addition to microcephaly, there may be a link between Zika virus infection and hydrops fetalis and fetal demise. Given the recent spread of the virus, systematic investigation of spontaneous abortions and stillbirths may be warranted to evaluate the risk that ZIKV infection imparts on these outcomes. The rapid spread of Zika virus in the Americas and outbreak of microcephaly in Brazil has raised attention to the possible deleterious effects that the virus may have on fetuses. We report a case of a 20-year-old pregnant woman from Salvador, Brazil whose fetus had developed hydrops fetalis, a condition where there is abnormal accumulation of fluid in the fetus, as well as severe central nervous system defects such as microcephaly and hydranencephaly. After fetal demise, ZIKV RNA was detected in central nervous system tissues and amniotic fluid. The case report provides evidence that in addition to microcephaly, there may be a link between Zika virus infection and hydrops fetalis and fetal demise. Given the recent spread of the virus, systematic investigation of spontaneous abortions and stillbirths may be warranted to evaluate the risk that ZIKV infection imparts on these outcomes. The current outbreak of microcephaly has raised speculations that Zika virus (ZIKV) causes a congenital syndrome. ZIKV, a mosquito-borne flavivirus, was detected in Brazil in early 2015 . A largeWhile conducting an outbreak investigation in Salvador, Brazil, we identified a patient who was referred to Hospital Geral Roberto Santos with an abnormal fetal ultrasound examination and followed during outpatient evaluations. After fetal demise and induced labor, tissues aspirates and fragments were collected by needle aspiration and thoraco-abdominal viscerotomy, respectively, since an autopsy could not be performed. RNA was extracted and tested by a ZIKV-specific reverse transcriptase-polymerase transcriptase assay (RT-PCR) .th week of gestation whose ultrasound examination showed low fetal weight. The patient procured prenatal care during the 4th gestational week in July, 2015 at which time she was found to have a negative serology for HIV, HTLV and hepatitis C viruses and positive IgG and negative IgM ELISA results for toxoplasmosis, rubella virus and cytomegalovirus. She had an uneventful course of pregnancy with a normal ultrasound evaluation at the 14th gestational week. In the 18th week, ultrasound examination found that the fetus had a weight three standard deviations below the mean value for gestational age.We assumed the care for a 20-year-old woman  in the 18th and 30th gestational weeks showed microcephaly, hydranencephaly with minimal residual cortical parenchyma  of ZIKV infections are asymptomatic , it is lThe finding of an association between ZIKV infection and hydrops fetalis suggests that the virus may cause damage to tissues in addition to the fetal central nervous system. Recent autopsy studies found that histopathologic findings and detection of ZIKV in newborns and fetuses with microcephaly were limited to the brain and in some cases, placenta ,7, indicWe cannot extrapolate from this single case the overall risk for developing hydrops fetalis and fetal demise among pregnant women exposed to the virus. The strain detected in this case of fetal demise appears to be the same as the epidemic strain that has spread across the Americas and Caribbean ,11. Give


Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers  of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus  The importance of hybridization in plant speciation and evolution has been debated for decades, with opposing views of hybridization as either a creative evolutionary force or evolutionary noise Ligularia Cass. Ligularia distributed in the Hengduan Mountains region are endemic Ligularia is considered to have arisen from rapid and continuous allopatric speciation in small and isolated populations, combined with interspecific diploid hybridization in the QTP and adjacent areas of eastern Asia LigulariaLigularia × maoniushanensis X. Gong & Y. Z. Pan is the first natural hybrid reported as a result of comprehensive study L. lamarum (Diels) C. C. Chang and L. subspicata (Bureau & Franchet) Handel-Mazzetti, Saito et al. Ligularia species in certain populations. A study on natural hybridization between L. subspicata and L. vellerea (Franchet) Handel-Mazzetti confirmed this conjecture Ligularia species. Based on morphological and molecular data , bidirectional but asymmetric hybridization and introgression were detected between L. subspicata and L. nelumbifolia (Bureau & Franchet) Handel-Mazzetti. Hybridization is very common in Ligularia, with various forms and progenies. However, these instances were all found in a single plot, and there has been no natural hybridization reported from multiple sites to date.The genus Ligularia cymbulifera (W. W. Smith) Handel-Mazzetti and L. tongolensis (Franchet) Handel-Mazzetti belong to the series Lapathifolia S. W. Liu L. cymbulifera and L. tongolensis based on their intermediate morphological traits and the partial overlap of the flowering period.trnK-rps16, trnL-rpl32 and trnQ-5'rps16) for all collections to determine the relationship between these progenies and the two putative parents. In particular, we paid attention to the extent, direction and dynamics of introgression between the two species. In addition, we also took advantage of the existence of four independent hybrid zones to compare the outcome of hybridization in each zone and test the hypothesis that hybrid zones can evolve differently and / or result from independent hybridization processes.In the current study, the ITS region of the nuclear ribosomal DNA of all the sampled individuals was sequenced to determine the hybrid status of the morphological taxon. If hybridization was confirmed, we sequenced three chloroplast intergenic spacers  were also chosen as references 2SO4, 1.3 µL MgCl2 (25 mM), 0.5 µL dNTP (2.5 mM each), 0.3 mM each primer and 0.75 unit of Taq polymerase (Takara). The amplifications were performed as follows: 1 cycle, 94°C, 5 min; 30 cycles, 94°C, 45 sec; 55°C, 1 min; 72°C, 40 sec, followed by a final extension of 1 cycle, 72°C, 5 min. The PCR products were purified by electrophoresis through a 1.2% agarose gel, and then an E.Z.N.A. Gel Extraction Kit  was used. All accessions were subjected to sequencing with amplification primers in an ABI 3700 DNA automated sequencer with the BigDye Terminator Cycle Sequencing Kit . The sequences were aligned and compared in SeqMan  L. cymbulifera and L. tongolensis, it produced chimeric or unreadable peaks in the chromatograms for the putative hybrids. Hence, cloning sequencing was performed subsequently for the putative hybrid. Purified PCR products were cloned into plasmids using the pUM-T vector system . Four to 20 positive clones were selected for each amplification product and cultured to isolate plasmids. Positive clones with the correct size inserts were confirmed using colony PCR. The plasmids with correct inserts were sequenced using ITS4 and ITS5 primers.The ITS region of the nuclear ribosomal DNA of all the sampled individuals was amplified using primers ITS4 and ITS5 trnL-rpL32, trnQ-5′rps16, and trnK-rps16 4)2SO4, 1.0 µL MgCl2 (25 mM), 1.0 µL dNTP (2.5 mM each), 0.3 mM each primer and 1.5 unit of Taq polymerase (Takara). The amplification conditions were as follows: 1 cycle, 94°C, 4 min; 30 cycles, 94°C, 45 sec; 53°C, 45 sec; 72°C, 1 min or 50 sec, followed by 1 cycle, 72°C, 7 min. PCR products were purified and then directly sequenced using the methods mentioned above. New DNA sequences were deposited in Genbank under accession numbers KM036217-KM036281.Four cpDNA fragments were amplified using the following universal primers: Ligularia species, L. lingiana, L. subspicata, and L. nelumbifolia, were downloaded from GenBank  as outgroups in the parsimony analyses. The nrITS networks and chloroplast networks were constructed using the program TCS version 1.21 Minor variation  between some clones may have resulted from PCR errors caused by Taq DNA polymerase. Putative PCR-mediated recombinants were excluded before further phylogenetic analysis. All sequences were aligned in Clustal X Ligularia vellerea, which appeared in the hybrid population, showing 18 specific variable sites , could be clearly distinguished from the putative parental species and hybrids (Hap 15)  in the mixed-growing populations were pure and shared haplotype H1, which was also present in the reference population , L. tongolensis shared haplotype H8 in Desha, H9 in Xiaoxueshan, H10 in Pachahai, and H12 in the Jiajinshan reference population and the Jiawa mixed population  were homologous and independent, as no double peaks were present in the direct sequences. For one of the putative parental species, pulation . These tpulation . All theL. vellerea formed a cluster with one of the outgroups, L. lingiana. In cluster A, H1 and H3 represent homologous individuals of L. cymbulifera that were detected in direct sequences in most locations (except for in Desha). Haplotypes H1–H7 were all detected in cloned sequences. In cluster B, H8–H10 and H12 represented homologous individuals of L. tongolensis that were detected in direct sequences. Haplotypes H8–H14 were all detected in cloned sequences. Among the cloned sequences (L. cymbulifera and L. tongolensisL. vellerea is apparently not involved in the hybridization between L. cymbulifera and L. tongolensis.A strict consensus tree from the maximum parsimony (MP) analysis was generated to show the phylogenetic relationships . The MP equences , each clL. cymbulifera, L. tongolensis, their putative hybrids and sympatric L. vellerea were generated to show their relationships , the nrITS network resolved two major parts   of L. tongolensis were introgressed by L. cymbulifera. In part B of the network, the four mixed populations had a special haplotype. There were also three individuals  of L. cymbulifera having L. tongolensis clones, which indicates that L. cymbulifera were introgressed by L. tongolensis in Desha. In the Desha population, the number of L. tongolensis was much greater than that of L. cymbulifera, with nearly no typical L. cymbulifera to be found; in other populations, however, L. cymbulifera was very easy to recognize. Unlike the Desha population, in which individuals of L. tongolensis shared only one ITS haplotype (H8), L. tongolensis in other populations were heterogeneous. In the three mixed populations, individuals of L. tongolensis had different haplotypes for pure individuals; additionally, a portion of the actual introgressed individuals shared haplotypes with both L. tongolensis and L. cymbulifera. For putative hybrids, both the L. tongolensis and L. cymbulifera ITS types were found in cloned sequences.The networks based on haplotypes identified from the nuclear ribosomal internal transcribed spacer (nrITS) of ionships . Sympatrolensis) . BecausetrnK-rps16, trnL-rpl32 and trnQ-5'rps16 spacers aligned sequences were 923, 928 and 978 bp, respectively. The combined length varied from 3523 to 3571 bp, containing a total of 20 variable sites  and already excluded from the possibility of participation in nuclear donors based on ITS, shared their special haplotypes H23-H27. Because chloroplast DNA is maternally inherited in LigulariaL. vellerea being a plastid donor was rejected. In contrast to the ITS4-5, each taxon in each population had more than two chloroplast haplotypes . The putative hybrids between the putative parents were commonly found near Zhongdian, where the two species share a sympatric existence. The wintry highland climate requires species to evolve certain common adaptive mechanisms. Ligularia species often share the same pollinators in the highlands L. vellerea, L. tongolensis and L. cymbulifera flower from July to August Ligularia and shuttled frequently among flowers. This behavior was highly likely to transfer one species' pollen to another species' flowers. Because the three species have nearly the same peak flowering times, it is difficult to reject any of them as possible participants in interspecific hybridization.L. vellerea was absolutely uninvolved in natural hybridization in these mixed populations. The ITS network definitely supports the hypothesis that the morphologically intermediate individuals are the products of hybridization between L. tongolensis and L. cymbulifera.Based on data of ITS4-5 and three cpDNA, LigulariaL. tongolensis and L. cymbulifera. Yu et al. Ligularia subspicata and L. nelumbifolia in a mixed location where the directions support the prediction that the minority species usually acts as the maternal parent. Zhou et al. Because chloroplast DNA is maternally inherited in Ligularia cymbulifera and L. tongolensis were almost the same . Morphological classifications were mostly in accordance with molecular results except for three individuals (Pachahai) and two individuals (Jiawa) of L. tongolensis, which proved to be introgressed individuals. In Pachahai, four hybrids shared a cpDNA haplotype (H12) with pure L. cymbulifera, and six other hybrids shared a common haplotype (H7) with both pure parents. In Jiawa, half of the ten hybrids shared a cpDNA haplotype (H4) with both pure parents, and another four hybrids showed three particular haplotypes that were not detected elsewhere. It is certain only that L. cymbulifera acted as a chloroplast donor (Pachahai). The complementary direction may exist but was not detected because of hybridization and introgression.In Pachahai and Jiawa, the numbers of L. cymbulifera, and almost all the collections were L. tongolensis and putative hybrids. Only the three L. cymbulifera-like collections actually proved to be introgressed products. One hybrid and two L. cymbulifera-like collections shared a cpDNA haplotype (H2) with most of the reference L. cymbulifera in Suochonghe, which was not too far from Desha; nine hybrids shared a haplotype (H4) with one pure L. tongolensis. We can infer that hybridization was initially bidirectional but occurred predominantly in the form of L. tongolensis ♀ × L. cymbulifera ♂. The hybridization and introgression may continue for a long time, so the actual process cannot be determined with existing methods.In Desha, the exceptional case in our field research, it was extremely difficult to identify typical L. cymbulifera and L. tongolensis were almost equally represented , the site was previously dominated by fir and was destroyed by a fire during the 1980s. In the network, this near equality was unique to Xiaoxueshan. Except for three individuals of L. cymbulifera that shared the most common haplotype (H7), other collections at this site revealed population-specific haplotypes. Most L. tongolensis shared haplotype H21, and most hybrids shared haplotype H18, which was the closest to H21 in the network. Most likely, the chloroplast donor at the initial instance of hybridization was L. tongolensis.In Xiaoxueshan, although L. cymbulifera and L. tongolensis occurred in multiple places and diverse forms. However, the direction of hybridization is not simply correlated with the number of parent individuals. Many factors, such as differences in flowering times, floral organs, pollen tube germination on different stigmas, and the ratio of mature individuals, may codetermine the direction of hybridization.In summary, natural hybridization and also introgression between sympatric L. cymbulifera, morphological identifications were generally in accordance with the molecular results, except for Desha, which had introgressed individuals. For L. tongolensis, except in Desha, another three mixed locations had a portion of introgressed individuals. Based on ITS data, all the morphological and pure L. cymbulifera had low ITS sequence biodiversity, and L. tongolensis had relatively high ITS sequence variability. The results were consistent with the results of a study of chemical and atpB-rbcL data for 19 individuals of L. tongolensis and 13 individuals of L. cymbulifera from different plots Continuous hybridization may affect the independence of the parental taxa, even resulting in reticulate evolution. For the parental species Ligularia indicated that L. cymbulifera and L. tongolensis had identical sequences for three optional sequences  . Therefore, three other fragments were selected in the present study. The trnK-rps16, trnL-rpl32 and trnQ-5'rps16 spacers were known to be more variable than the other chloroplast regions L. cymbulifera and L. tongolensis. However, the three spacers still generated a problematic network. There are several reasons for the difficulty in clarifying the phylogeny: (A) convergent mutations may have caused the same haplotype to arise independently in different species; (B) shared haplotypes may have been inherited from a polymorphic common ancestor due to incomplete lineage sorting; and (C) one species may share haplotypes with another species via introgressive hybridization within a framework of stable species ranges, perhaps assisted by selection. The genus Ligularia has been estimated to have occurred primarily within the last 20 million years, and interspecific diploid hybridization has played a role in the species diversity of this genus L. cymbulifera and L. tongolensis. However, based on a study by Hanai et al. L. cymbulifera and L. tongolensis in different populations. Our ITS data in allopatric reference populations also confirmed this pattern. Accordingly, the explanations of convergent mutations (A) and incomplete lineage sorting (B) can be excluded in the present study. In addition, our ITS data do show that natural hybridization and introgression occurred between sympatric L. cymbulifera and L. tongolensis.With respect to the three cpDNA sequences, the network was disordered and unintelligible. Part of our previous study on the barcode L. tongolensis in Pachahai and Jiawa. The diverse hybrid individuals may adapt to more niches and form new taxa, as is the case for hybridization in sunflowers Ligularia, but more credible measures, such as SSR, spot investigation, and artificial hybrid experiments, would help provide a better understanding of the way in which hybridization occurred and affected species evolution.Species distinctiveness is maintained if hybrids are selected by extrinsic and intrinsic selection despite continuous gene flow Ligularia, appearing between different species, series and subgenera. In this case, the direct and cloned ITS sequences and the three chloroplast regions of the individuals in four populations confirmed that hybridization occurs between L. cymbulifera and L. tongolensis, and this phenomenon was demonstrated simultaneously in several locations separated by long distances. Although gene flow is common in Ligularia, most species maintain their independence. Because of the lack of study of reproductive isolation in Ligularia and selection on hybrid descendants, the impact of natural hybridization on the evolution and speciation of Ligularia is still not clear. Future work should focus on comparing the fitness of hybrids and their parental species and on considering the effect of natural hybridization.Hybridization is very frequent in S1 FileTable S1, Sample locations for 4 sympatric locations and 2 reference Ligularia populations. Table S2, Variable sites from the aligned sequences (both direct sequencing and cloned sequencing) of ITS4-5 in the 15 haplotypes (H1-H15) of all the collections. Table S3, Variable sites from the aligned sequences of the three chloroplast DNA spaces in the 22 haplotypes (H1-H22) of all the collections.(DOCX)Click here for additional data file.S2 FileFigure of sample locations for the four mixed populations (dotted area) and two reference populations (triangular area) of Ligularia from south-western China.(DOCX)Click here for additional data file.


Trichoplusia ni (TnSNPV) and another that has a broad host range (AcMNPV). Virus-challenged larvae performed better on more protein-biased diets, primarily due to higher survival, whereas unchallenged larvae performed best on a balanced diet. The environmental conditions that maximized host performance differed with virus identity because TnSNPV-challenge inflicted fitness costs  whereas AcMNPV-challenge did not. The performance of TnSNPV-challenged larvae rose with increasing P:C across all temperatures, whereas temperature modulated the optimal P:C in AcMNPV-challenged larvae (slightly protein-biased at 16 °C to increasingly higher P:C as temperature increased). Increasing temperature reduced pupal size, but only at more balanced P:C ratios, indicating that nutrition moderates the temperature-size rule. Our findings highlight the complex environmental interactions that can alter host performance after exposure to pathogens, which could impact the role of entomopathogens as regulators of insect populations in a changing climate.Specialist and generalist pathogens may exert different costs on their hosts; thereby altering the way hosts cope with environmental variation. We examined how pathogen-challenge alters the environmental conditions that maximize host performance by simultaneously varying temperature and nutrition  after exposure to two baculoviruses; one that is specific to the cabbage looper,  The harm a microorganism inflicts on its host is dependent on numerous factors, such that under some conditions it will act as a pathogen and in others not. Nowhere is this more evident than in the effects of malnutrition and climate warming on the changing prevalence or severity of some infectious diseases1Plodia interpuctella, the magnitude, and even the direction, of the individual effects of temperature, food quality and larval density on immunity were altered by complex interactions among the three factorsAnopheles stephensi, dietary supplementation to boost midgut immunity reduced infection by the rodent malaria parasite, Plasmodium yoelii, at intermediate temperatures but not at low and high temperature extremesIn ectotherms, temperature and nutrition individually play powerful roles in host-pathogen interactions. They can have direct effects through thermal sensitivities and nutrient requirements of hosts and pathogens35To further complicate these interactions, hosts are often susceptible to a large number of diverse parasites10Trichoplusia ni (Hübner) after exposure to one of two ecologically relevant species of baculovirus, Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) and Autographa californica multiple NPV (AcMNPV). Baculoviruses are highly pathogenic obligate killers that only infect the larval stage of susceptible insects. Transmission only occurs after the death of the infected larva. Both viruses occur in wild T. ni populations14T. niWe examined the effects of temperature and dietary nutrients on the survival and development of the cabbage looper T. ni can potentially feed on a vast array of plant species that can vary substantially in macronutrient content20215624612626Generalist insects such as X2 = 2.79, P = 0.10; Dietary P:C ratio affected survival after challenge by an equal dose of either TnSNPV or AcMNPV . TemperaTnSNPV-challenged larvae took significantly longer to reach pupation compared to unchallenged-control larvae . In contPupal weights of larvae challenged with TnSNPV were significantly lighter , while the pupal weight of AcMNPV-challenged insects did not differ significantly from unchallenged controls . P:C ratThe composite measure of performance, which was estimated at the individual level by multiplying an individual’s probability of survival, pupal weight and the inverse of development time, was influenced by a complex three-way interaction between P:C ratio, virus treatment  and temperature . PerformT. ni across temperatures occurred at a relatively balanced P:C ratio, similar to that selected when T. ni choose their own dietary nutrient ratios , increasing survival with P:C ratio combined with a more moderate effect of P:C ratio on pupal weight compared to control larvae, caused performance to increase with dietary protein content across all temperatures. In contrast, challenge by the ‘generalist’ virus (AcMNPV) caused maximal performance to shift to a slightly more protein-biased P:C ratio at 16 °C in comparison to unchallenged control larvae, and as temperature increased, maximal performance was achieved on increasingly more protein-biased P:C ratios. The response of the AcMNPV-challenged larvae differed from those challenged by TnSNPV because the influence of diet on survival was less and pupal weight was more strongly influenced by the marked interaction between diet and temperature.Our results clearly demonstrate that the response to pathogen exposure can be influenced by environmental factors. Host survival can change depending on dietary constraints, and overall host performance can be modulated by the interaction between diet and temperature. In addition, we show that a host’s response to environmental variation differs with the identity of the infecting pathogen . Maximals 1.3p:1c. For T. As we predicted, P:C ratio more strongly affected the survival of TnSNPV-challenged larvae, suggesting that the dietary boost in immune functioning or other resistance mechanisms was more effective against the specialist virus. In addition we found significant fitness costs  associated with surviving TnSNPV-challenge, but not AcMNPV. As baculoviruses are obligate-killers requiring host death for transmission and observable infection always results in death before the adult stage, we have taken any differences in host fitness traits as costs of surviving virus challenge, rather than a direct cost of virus infection (virulence). A key first line of defence against NPVs that may affect survival costs is the sloughing of infected midgut cells. Survival costs may also have resulted from an increased protein demand of immune responses to NPVT. ni in response to TnSNPV-challenge compared to AcMNPV-challenge, suggests that it might be more costly for T. ni to fight TnSNPV challenge. Spodoptera exigua cells up-regulated more immune-related genes in response to infection by the specialist SeMNPV than by AcMNPVT. ni, an increased response to TnSNPV challenge may be adaptive given that TnSNPV infection is more common in field-collected T. ni cadavers than AcMNPV14T. ni does not result in cross-resistance to AcMNPVThe greater survival costs incurred by Drosophila melanogaster743444546Gryllus texensis is more resistant to the Gram-negative bacterium Serratia marcescens at an elevated temperature, but is more susceptible to the Gram-positive bacterium Bacillus cereus at temperatures below or above the average field temperatureT. ni after AcMNPV or TnSNPV exposure, consistent with the thermal ecology of the western tent caterpillar and its NPV7434445464951Some insects can alter their temperature through thermoregulatory behavior as a means to increase their ability to fight off pathogens. This research has focused primarily on behavioural fever, where infected locusts, grasshoppers and houseflies survive longer after fungal infections at warmer temperatures. When given the choice, infected insects will move to warmer areas of their environment37383940Manduca sexta on a poor quality host plantT. ni followed the temperature-size rule at moderate P:C ratios but not at the extremes, indicating that dietary macronutrient ratios can moderate the temperature-size rule. Increased energy used at higher temperatures to fuel the increased basal metabolic rate associated with rapid growth can reduce lipid storage for energy52Increasing rearing temperature reduces the adult body size of most ectotherms (temperature-size rule), potentially through thermal sensitivity of growth rates and smaller cell size492 associated with climate warming can increase plant defensive traits, such as leaf toughness and plant secondary compounds5960T. ni survival and performance after virus-challenge are greater at higher dietary P:C ratios, the negative effects of pathogen-challenge will be exacerbated as the availability of dietary nitrogen decreases with climate warming. Moreover, larvae of another Noctuid moth, Spodoptura litura, have been shown to select a lower dietary P:C ratio at higher temperatures when given an opportunity to compose its own diet to meet higher energy demandsT. ni performance varies more with temperature after exposure to the generalist virus (AcMNPV) than the specialist (TnSNPV), it is clear that nutrition will be a key factor influencing host-pathogen interactions in a warming climate. This has implications for both the use of entomopathogens as biological control agents and more generally in terms of their regulation of natural insect populations. This study highlights a complex interplay of nutritional and temperature related effects on host performance and hence population density that may not be predictable from one pathogen to another.Rising temperature from climate change is predicted to increase pathogen transmission and disease outbreak intensity in insect herbivores, as increased feeding rate would elevate the herbivore’s risk of encountering pathogensTrichoplusia ni, (Lepidoptera: Noctuidae) is a widespread, generalist pest in many parts of North America, Europe, Asia and Africa, where it feeds primarily on crucifers but can also consume many other plant species. T. ni larvae were obtained from a laboratory colony that was originally collected from a commercial tomato greenhouse in British Columbia, CanadaT. ni were reared individually until the late fourth instar. Samples of TnSNPV isolate FV#34T. ni. Viral occlusion bodies (OBs) were counted using an improved Neubauer brightline haemocytometer  at 400x magnification on a phase contrast microscope, and serial dilutions were made to produce the experimental doses.The cabbage looper, T. ni, contained 12–37% dry weight in protein (% nitrogen multiplied by conversion factor of 6.25) and 11–60% dry weight in digestible carbohydrateFive isocaloric, chemically defined artificial diets were producedth instar larvae were allowed to complete their moult in individual wells of a 24-well cell culture plate  without food. Freshly moulted, unfed final (5th) instar larvae were weighed, and then assigned to “virus treatment” groups consisting of a no virus control or virus challenge with AcMNPV or TnSNPV (300 larvae/virus treatment group). Larvae were fed a dose of 360 OBs  of either AcMNPV or TnSNPV in 2 μl of distilled water applied to a 3 × 2 mm (length x diameter) diet plug or just water for controls. Each larva was left in individual wells of a 24-well cell culture plate with a single diet plug for 24 h at 25 °C to consume the entire diet plug. All larvae consumed the entire viral dose.Moulting late 4Larvae were then reared individually in 30 ml plastic cups  at one of three constant temperatures  with a block of one of five treatment diets (~0.7 g). Thus, it was a 3 × 3 × 5 design with three virus treatments , three temperatures and five P:C ratio diets for a total of 45 treatment groups, and 20 larvae reared in individual cups per treatment group. Death or pupation was recorded every 12 h and diets were replaced every 24 h. Surviving pupae were collected immediately after the pupal case hardened and were placed in a drying oven at 50 °C for 48 h until constant mass  and then weighed. Three larvae did not feed on the treatment diets and were excluded from analyses.The impact of virus treatment , and environmental variation (dietary P:C ratio and temperature) on larval survival was analyzed using generalized linear models . Pupal weight, development time and insect performance (see below) were analysed using analysis of covariance (ANCOVA) with temperature and virus treatment as categorical terms and diet (P:C) as both linear (diet) and quadratic diet continuomin]/[xmax − xmin] + 1) before calculating performance. Non-parametric thin plate splines were used to fit a three-dimensional response surface of the bivariate effects of temperature and diet on insect performance using the fields package in R (version 3.0.1)54Performance was estimated at the individual level by multiplying an individual’s probability of survival , pupal weight and the inverse of development time6How to cite this article: Shikano, I. and Cory, J. S. Impact Of Environmental Variation On Host Performance Differs With Pathogen Identity: Implications For Host-Pathogen Interactions In A Changing Climate. Sci. Rep.5, 15351; doi: 10.1038/srep15351 (2015).


Cluster analysis (CA) is a frequently used applied statistical technique that helps to reveal hidden structures and “clusters” found in large data sets. However, this method has not been widely used in large healthcare claims databases where the distribution of expenditure data is commonly severely skewed. The purpose of this study was to identify cost change patterns of patients with end-stage renal disease (ESRD) who initiated hemodialysis (HD) by applying different clustering methods.A retrospective, cross-sectional, observational study was conducted using the Truven Health MarketScan® Research Databases. Patients aged ≥18 years with ≥2 ESRD diagnoses who initiated HD between 2008 and 2010 were included. The K-means CA method and hierarchical CA with various linkage methods were applied to all-cause costs within baseline (12-months pre-HD) and follow-up periods (12-months post-HD) to identify clusters. Demographic, clinical, and cost information was extracted from both periods, and then examined by cluster.n = 113); Cluster 2: Very High to High (n = 89); Cluster 3: Average to Average ; or Cluster 4: Increasing Costs, High at Both Points (n = 1554). Median cost changes in the 12-month pre-HD and post-HD periods increased from $185,070 to $884,605 for Cluster 1 (Average to High), decreased from $910,930 to $157,997 for Cluster 2 (Very High to High), were relatively stable and remained low from $15,168 to $13,026 for Cluster 3 (Average to Average), and increased from $57,909 to $193,140 for Cluster 4 . Relatively stable costs after starting HD were associated with more stable scores on comorbidity index scores from the pre-and post-HD periods, while increasing costs were associated with more sharply increasing comorbidity scores.A total of 18,380 patients were identified. Meaningful all-cause cost clusters were generated using K-means CA and hierarchical CA with either flexible beta or Ward’s methods. Based on cluster sample sizes and change of cost patterns, the K-means CA method and 4 clusters were selected: Cluster 1: Average to High (The K-means CA method appeared to be the most appropriate in healthcare claims data with highly skewed cost information when taking into account both change of cost patterns and sample size in the smallest cluster. Cluster analysis (CA) is a statistical technique that helps reveal hidden structures by grouping entities or objects  with similar characteristics into homogenous groups while maximizing heterogeneity across groups , 2. EntiCA has been widely used in varied applications including finding a true typology, prediction based on groups, hypothesis generation, data exploration, and data reduction-or grouping similar entities into homogeneous classes, consequently organizing large quantities of information and enabling labels that facilitate communication , 4, 5. NLess well investigated is the utility of CA in identifying macro-structures associated with changes in treatment outcomes documented in large healthcare claims databases. A particular challenge for the use of CA in healthcare claims datasets is that the distribution of healthcare expenditure data are commonly severely skewed, which complicates analyses , 9. In s. Two types of dialysis are widely used: hemodialysis (HD) and peritoneal dialysis (PD). The most common and costly of the two, HD, uses a dialysis machine and a special filter called a dialyzer to clean blood outside of the body  = 14.1); 46 % were aged ≥65 years, and 29 % were aged 55 to 64 years. Of the total individuals, 58 % were males, 84 % had FFS insurance plans, and 14 % had HMO or POS capitation plans. At baseline, average ECI scores were 5.8 (SD = 2.6) in the full sample and CCI scores were 4.6 (SD = 2.3); at follow-up, ECI scores had increased to 7.1 (SD = 3.0) while CCI scores had increased to 5.3 (SD = 2.4).After applying the entry criteria for this study and from 140,720 individuals, a total of 18,380 individuals were identified in the MarketScan Database . Empirically, the 4-cluster solution was judged to be more appropriate and more easily interpretable than either the 3- or 5-cluster solution. Thus, a 4-cluster K-means solution was chosen for further investigation , which can be difficult to interpret. Among the models suggested by K-means CA, a 4-cluster solution appeared to be the most appropriate for these data: associated criteria suggested a 4-cluster solution offers maximum separation of clusters compared with either a 3- or 5-cluster solution. In addition, a 4-cluster solution was more interpretable, and thus more appropriate to apply than other methods.Mean all-cause medical costs in this sample of privately insured patients ranged from approximately $45,000 (USD) prior to the initiation of HD to $49,000 (USD) after; median costs ranged from $17,000 in the 12 months before HD initiation to $16,000 in the 12 months following HD initiation. Interestingly, these reported costs are generally lower than those found in other analyses in other populations. In 2004, the average annual Medicare expenditure for an ESRD patient started on HD was reported to be $72,000 (USD) , increasper se is not an important driver of cost for the majority of patients. A minority of patients drive an increase in overall costs after HD initiation.More specifically, CA demonstrated that the data could be reasonably represented by 4 clusters of patients: those with average costs before and after initiating HD (90 % of the full sample); those with high costs before and high/increased costs after (8 %); those with average costs who incur high costs after initiating HD (0.6 %); and a cluster with very high costs prior to initiating HD who see their annual costs reduced to a high level (0.5 %). Thus, overall costs stay stable for most ESRD patients initiating HD, suggesting transition to HD Because of the different cost patterns in each group, it is worthwhile to better understand patients in each cluster to help predict and contain the costs of HD. Comorbidities seem to be particularly relevant to costs, with increasing comorbidity scores from baseline to follow-up periods in those clusters associated with an increase in costs during follow-up, and more stable comorbidity scores associated with more stable costs (or even declining costs). This is consistent with other research: one study demonstrated that an increased level of comorbidity was associated with higher cost in the 2 years prior to starting HD , while aIn aggregate, costs are high at an absolute level, both before and after the initiation of HD, suggesting that the healthcare costs of the majority of ESRD patients not treated with HD are not substantially lower than the costs of care for these patients immediately after starting HD. Thus, HD does not add substantial costs for most patients and seems like an economically feasible option in most patients with CKD, given the overall high cost of care for these patients prior to initiating HD. True cost containment for patients with ESRD likely requires more aggressive or widespread intervention before patients reach this advanced stage of disease, where costs are high before and after HD. One overall strategy that may reduce costs includes early referral to a nephrologist in the period before starting HD . HD is nInterpretation of these results must be informed by limitations of these analyses. First, these analyses were conducted only in those employed individuals with commercial insurance coverage and some individuals with Medicare coverage; thus, these results from a relatively healthy population may not be fully generalized to individuals with Medicare, Medicaid, other insurance, and no insurance. Second, administrative claims data cannot capture deaths and changes of employment; therefore, the cost not captured due to loss to follow-up may lead to selection bias. In addition, administrative claims data are not collected for research purposes and measurement error may have been introduced by coding that was in error or driven by reimbursement needs more so than research needs. Further, administrative claims data does not collect clinical information that would have been valuable additions to these analyses, such as laboratory test results or vital signs. Access to patients’ claims prior to their enrollment in MarketScan databases is not available. Retrospective analysis limits the study to those who are clinically diagnosed and incur health care resource utilization through claims; resource utilization not identified by claims would not be included in these analyses. Finally, treatment costs in future studies should examine what cost drivers may have influenced increases or decreases in costs for each cluster.CA was a useful statistical technique for evaluating a claims data set that included skewed healthcare cost data. One implication of these analyses is that costs for most patients with ESRD stay relatively stable after starting HD; a minority of patients drive overall increasing annual costs after initiation of dialysis. These increasing costs may be driven, in part, by a greater comorbidity burden among these patients.


Our living sphere is constantly exposed to a wide range of pathogenic viruses, which can be either known, or of novel origin. Currently, there is no methodology for continuously monitoring the environment for viruses in general, much less a methodology that allows the rapid and sensitive identification of a wide variety of viruses responsible for communicable diseases. Traditional approaches, based on PCR and immunodetection systems, only detect known or specifically targeted viruses. We here describe a simple device that can potentially detect any virus between nanogap electrodes using nonlinear impedance spectroscopy. Three test viruses, differing in shape and size, were used to demonstrate the general applicability of this approach: baculovirus, tobacco mosaic virus (TMV), and influenza virus. We show that each of the virus types responded differently in the nanogap to changes in the electric field strength, and the impedance of the virus solutions differed depending both on virus type and virus concentration. These preliminary results show that the three virus types can be distinguished and their approximate concentrations determined. Although further studies are required, the proposed nonlinear impedance spectroscopy method may achieve a sensitivity comparable to that of more traditional, but less versatile, virus detection systems. Our environment is posed a constant threat of exposure by pathogenic viruses, whether to humans or pets, domestic animals, or agricultural and marine products . No established methodology exists for inactivating most viruses that could cause widespread communicable viral infection. It is therefore imperative to realize a technology that can monitor the environment for viruses and prevent their spread and disease transmission. The key is to develop a practical sensor that can continuously monitor the environment for viruses. Such virus sensors could be installed at airports, seaports, and other points of entry, and where necessary at farms and ranches, at restaurants, in air conditioning units, in sewage systems, and in other public facilities or utilities. These sensors would continuously monitor for viruses, and if they were detected, measures could be implemented to prevent infection, in contrast to their existing diagnostic application. In cases where detection and countermeasures are too late to prevent an outbreak of a communicable disease, a network of virus sensors could facilitate rapid identification of affected areas, allowing medical resources to be focused in those areas. This would lead to early-stage detection and treatment, and maximize the efficient use of medical resources.Currently, there is no technology that can sense viruses in our daily environment. Existing virus detection methods are focused on diagnosing infected individuals, and can be classified into three main categories: (1) those that utilize genomic information and are based on the polymerase chain reaction (PCR) and metagenomic analysis; (2) those that utilize molecular recognition or receptor linking functions of compounds such as antibodies, sugar chains, and peptide aptamers; and (3) those that are based on the electrical or optical properties of viruses  or plaque forming units (PFU) leaving the number of virions is unclear, making it difficult to quantify the sensitivity of the sensing and to compare the results with those of other methods. To prevent this problem, we first visualized and counted the number of virions by fluorescence labeling and observation, then expressed the concentration in terms of virions per unit volume. 1 mM KCl buffer was used, and a fluorescent dye  was added to solution for labeling the viruses. The labeled virions were counted under a fluorescence microscope. Figure 1 shows a typical result, with each bright spot representing a single baculovirion. The virion concentrations were estimated from the bright spot counts using image analysis software (Image J)  with a dielectric interface , which were controlled with SMART software , and the data were analyzed using Zview software .5 V/m or more would not be significantly damaging to the virus. It is, however, not confirmed whether the effect of high electric field is how much hazardous to the viruses in their biological activities.The electrical impedance of liquid samples suffer from several problems, including unavoidable electrode decomposition and substantial changes in the conductivity due to the release of ions, and damages to the virus with these ions in high concentrations. These problems were apparently appeared in low frequency region less than 100 kHz in our preliminary measurements, so that the measurements were conducted in the range 100 kHz–6.3 MHz. Prior to each measurement, open/short calibration was performed, and the effects of parasitic capacitance and other parasitic components, wiring induction coupling, and leakage were eliminated. The applied frequency for all of the measurements was 100 kHz or higher, to prevent background effects from the solid-liquid double layer capacitance that tends to form at the interface of the measuring electrode  in the subsequent experiments.11 virions/mL, this volume would contain approximately 0.3 virions. Under the assumption that the number of virions measured with a sample concentration of 1011 virions/mL would actually be one to several, we set 1011 virions/mL concentration as the lower limit for subsequent experiments.The electric field was designed by the finite element method using the commercially available solver COMSOL 4.4 (COMSOL Inc.). Based on simulation, the region between the electrodes for an electric field of 100 kV/m or higher was obtained with a gap length of 510 nm, an electrode width of 5 μm, and a depth of groove approximately 530 nm, corresponding to a volume of about 1.4 fL. This is a rough estimate due to the errors inherent in the measurements; regardless, for a sample concentration of 1011–1014 virions/mL for baculovirus, TMV, and influenza virus, as shown in Figure Our investigation of the impedance dependence on virus concentration showed that the impedance varies with virus concentration in the range 10As shown in Figure It would be difficult to distinguish quantitatively between the three virus types using only the impedance spectra shown in Figure 11–1014 virions/mL. The horizontal and vertical axes represent the phases of the imaginary impedance component somewhat arbitrarily at 100 kHz and the peak frequency, fp, respectively. Although some overlap is apparent between the three clusters of data points, the graph clearly shows that it is possible to distinguish between virus types on this basis, independent of their concentrations.We therefore compared the three virus types, and particularly the baculovirus and influenza virus, for differences in phase effects. Figure It should be noted, however, that this investigation was performed for only these three viruses, and it would be premature to extrapolate the results to other virus types.Here we proposed a virus detection method by nonlinear impedance spectroscopy under a strong electric field between two nanogap electrodes.In sweeping frequencies from 100 Hz to 6.3 MHz while varying the electric field strength, we found an apparently marked nonlinear effect on impedance at electric field strengths of 100 kV/m or higher. Further measurements were performed for those electric field strengths and frequency while varying the concentrations of baculovirus, TMV, and influenza A (H1N1) virus solutions. The data clearly showed that the virus concentration can be quantified by the impedance value. Furthermore, the three virus types could be distinguished by plotting the phase at 100 kHz against the phase of the peak value of the imaginary component of the impedance for each virus type.11 virions/mL used in this study, one to several virions occupied the effective measurement space. This is a rough estimate, and a more detailed study is required for verification. The results nevertheless show that the proposed nonlinear impedance spectroscopy method may achieve a sensitivity comparable to that of PCR and immunodetection systems. The response time to sweep the frequency for each measurement was about a few minutes, which will be enough fast for the continuous monitoring of environment. One of the large remaining issues is to evaluate the detection and identification ability of some specific viruses from the heterogeneous mixture of crude sample. That is the universal challenge for any types of biosensing methods, and also the future work to put this method into practical use.At the minimum sample concentration of 10The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We studied how the separation of dispensing and prescribing of medicines between pharmacies and clinics (the “separation system”) can reduce internal medicine costs. To do so, we obtained publicly available data by searching electronic databases and official web pages of the Japanese government and non-profit public service corporations on the Internet. For Japanese medical institutions, participation in the separation system is optional. Consequently, the expansion rate of the separation system for each of the administrative districts is highly variable. The data were subjected to multiple regression analysis; daily internal medicines were the objective variable and expansion rate of the separation system was the explanatory variable. A multiple regression analysis revealed that the expansion rate of the separation system and the rate of replacing brand name medicine with generic medicine showed a significant negative partial correlation with daily internal medicine costs. Thus, the separation system was as effective in reducing medicine costs as the use of generic medicines. Because of its medical economic efficiency, the separation system should be expanded, especially in Asian countries in which the system is underdeveloped. Previous studies have examined the factors that influence medical expenditures. In Japan, the separation system became optional in the 1960s. This system involves separating the prescriber from the dispenser of medicines, both duties formerly carried out by the Japanese Ministry of Health, Labour, and Welfare (JMHLW). Proponents of the system have argued that it reduces medicine costs, but since its introduction, there have been conflicting reports about whether this system does, in fact, do so . FurtherThe economic effects of the separation system have rarely been studied with quantitative data because it is difficult to precisely compare the objects under study. Therefore, it is unclear whether the system promotes the proper prescription of medicines or reduces medicine costs . A few pMore recently, Japan has a universal national health insurance system. Consequently, all Japanese people belong to a specific public health insurance system. Therefore, by analyzing public insurance data, we can estimate the health and medical costs of the whole nation. Importantly, because of the frequent changes in the Japanese insurance system, it is difficult to evaluate the exact effect of each factor on medicine costs. Here, we examined the correlation between medicine costs per prescription and the factors believed to influence medical expenses by analyzing objective nationwide prefecture-level data derived from three sources: the Japanese Statistics Bureau of the Ministry of Internal Affairs and Communications (JSB), the JMHLW, and the Japan Pharmaceutical Association (JPA), a non-profit public service corporation.Japanese prefectures are broad-based common municipal corporations. They divide Japan into 47 administration districts as shown in In the present study, we examined the financial efficiency of separating pharmacies from clinics by evaluating data from each of Japan’s prefectures.The result would be a clear understanding of the cost efficiency of the separation system. To do so, we performed a multiple linear regression analysis with daily medicine costs for internal use or other medicine costs per prescription as the dependent variables and the expansion rate of the separation system, the base ratio of generic medicine replaced with brand name medicine, and the proportion of people 75 years or older age in the population, as independent variables. We hypothesized that these variables would have a considerable influence on daily medicine costs, as explained in the following paragraphs.Currently, while the separation system is optional for hospitals and clinics, its use is gradually increasing throughout the country. The separation of prescription and medicine sales enables doctors to refrain from prescribing specific name brand medicines.Generic medicines can be sold only when the patent on the name brand of the pharmaceutical formula expires. The market price for generic medicines is much lower than that of brand-name medicines. Consequently, daily medicine prices should be lower in the areas that dispense more generic medicines than name brand medicines compared to the areas that dispense more name brand medicines than generic medicines.Finally, medical expenses tend to increase as people age. In 2011, 33% of the total amount of money spent on medicines was for people 75 years or older . Thus, we expected prefectures with a greater proportion of elderly people to have higher medical costs.We obtained 2011 fiscal data on average medicine cost and dosage days per prescription for each prefecture from the JMHLW website . We then divided the average medicine charge per prescription by the dosage days per prescription to obtain the daily medicine cost.Nationwide data on prescriptions are available from insurance applications for medical expenses received from pharmacies, 99.9% of which are currently covered by medical insurance. We could access these data freely because insurance applications for medical expenses from pharmacies have been made available on the JMHLW website since 2009.This rate refers to the number of separation system upgrades among the administrative divisions in Japan; we obtained these data for the period of April 2011 to March 2012 by the JPS (2011).These data were taken from the JMHLW website—specifically, they were based on trends in the composition of medical expenses (for computation processing) in 2011 . From these data, we calculated the rate of generic medicines replaced by brand name medicines  for each prefecture. These data are exact because they are based on universal health care and medical insurance  data.The elderly were defined as people 75 years or older. We obtained data on the 75 years or older age group in the population  for the fiscal year 2011 from the JSB website . The proA multiple regression analysis was conducted using the daily internal medicine cost per prescription  as the dependent variable and the expansion rate, generic medicine replacement ratio, and proportion of the elderly as independent variables. All statistical analyses were performed by Excel Statistics 2010 .The number of observations = the number of Japanese prefectures.1) and the generic medicine replacement ratio (X2) for the items included in the analysis. A correlation analysis between daily medicine charge and the independent variables yielded the following regression functions:1 = -0.0070X1*−0.0290X2*+0.0116X3+3.59, R = 0.712, P < 0.0001Y (1)where the variables are defined as follows:1 = the daily internal medicine cost, X1 = the expansion rate, X2 = generic medicine replacement ratio, and X3 = the proportion of the elderly. The asterisk (*) indicates that the multiple regression analysis showed a significant partial correlation for these items. The results were rounded to the fourth decimal place.YWe found that daily internal medicine costs had a significant multiple correlations with two of the three factors hypothesized to influence medicine costs—expansion rate and generic medicine replacement ratio, which were examined for the first time in this study.We observed a significant negative correlation for the relationship between expansion of the separation system and medicine costs. This finding indicates that the separation system in Japan might reduce daily medicine costs.We observed a strong, positive multiple regression coefficient for daily internal medicine cost. Thus, we found that the separation system had a constant effect in reducing medicine costs; this is especially important, considering that internal medicine costs are 84% of the total outpatient medicine cost in Japan . AlthougThe separation of prescriptions and medicine sales enables doctors to refrain from prescribing specific medicines, as they are no longer personally interested in drug-related profit margins for their institutions or themselves. Moreover, many doctors believe that prescriptions will be reviewed by pharmacists, who are experts in dispensing medicines, unlike the doctors themselves. In addition, because doctors are not controlled by the stock available in their own institutions, they are free to prescribe any medication and can, therefore, choose less expensive medicines.Very little data exist regarding the constant effect of the separation system on medicine cost reduction. However, we were able to identify a fixed effect here because the separation system is not mandatory in Japan; instead, it is the choice of the prescribing doctors in the medical institutions. Consequently, we were able to calculate the daily medicine costs data under various expansion rates and the effect of the separation system on medicine costs.We observed a statistically significant negative partial correlation between the generic medicine replacement ratio and daily internal medicine costs. Using generic medicines to reduce medicine costs is a well-known practice. The magnitude of the partial correlation coefficient for the generic medicine replacement ratio was similar to that of the expansion rate. Thus, with regard to reducing medicine costs in Japan, the separation system is as important as the use of generic medicines.We observed a positive, non-significant partial correlation for the ratio of the elderly and internal medicine cost. Though this result seems consistent with the finding that medicine costs for the 75 years or older population accounts for 35% of national medicine costs, the proportion of the elderly did not have a strong effect on outpatient internal medicine costs. This is possibly because seriously ill patients are rapidly hospitalized in Japan; consequently, the medicine costs of outpatients would be lower than those of inpatients.The results suggest that the separation system is as effective in reducing medicine costs as generic medicine usage. In light of the effects of the separation system on medical economic efficiency, the separation system should be expanded, especially in Asian countries in which the system is underdeveloped.