Sula dactylatra. We further analysed whether ARS behaviour may be used as a proxy to identify important feeding areas. Depth-acceleration and GPS-loggers were simultaneously deployed on chick-rearing adults to obtain (1) location data every 4 minutes and (2) detailed foraging activity such as diving rates, time spent sitting on the water surface and in flight. In 82% of 50 foraging trips, birds adopted ARS behaviour. In 19.3% of 57 detected ARS zones, birds spent more than 70% of total ARS duration resting on the water, suggesting that these ARS zones were falsely detected. Based on generalized linear mixed models, the probability of detecting false ARS zones was 80%. False ARS zones mostly occurred during short trips in close proximity to the colony, with low or no diving activity. This demonstrates the need to account for resting on the water surface positions in marine animals when determining ARS behaviour based on foraging locations. Dive rates were positively correlated with trip duration and the probability of ARS behaviour increased with increasing number of dives, suggesting that the adoption of ARS behaviour in Masked Boobies is linked to enhanced foraging activity. We conclude that ARS behaviour may be used as a proxy to identify important feeding areas in this species.Identification of Area-restricted search (ARS) behaviour is used to better understand foraging movements and strategies of marine predators. Track-based descriptive analyses are commonly used to detect ARS behaviour, but they may be biased by factors such as foraging trip duration or non-foraging behaviours (i.e. resting on the water). Using first-passage time analysis we tested if (I) daylight resting at the sea surface positions falsely increase the detection of ARS behaviour and (II) short foraging trips are less likely to include ARS behaviour in Masked Boobies  Marine predators usually forage in an environment where food resources are patchily distributed in space and time Mirounga leonina  we investigated time budgets of individuals within detected ARS zones, including diving activity and amount of time spent resting at the sea surface;(II) we determined which factors influence the detection of ARS behaviour;(III) we examined whether ARS behaviour can be used as a proxy to identify important feeding areas;(IV) and finally, we developed a decision tree based on our findings to determine incorrectly-detected ARS zones resulting from high proportions of resting at the sea surface positions.This research study was carried out under permission of Norfolk Islands National Park (Project Permit No 2009/0003/01). The animal use protocol for this study was reviewed and approved by the Animal Ethics Committee of the University of Tasmania (Project Permit No A10756), meeting the requirements of the Australian Code of Practice for the Care and Use of Animals in Scientific Purposes (1997).The study was carried out on Phillip Island  in the south-west Pacific Ocean. Phillip Island (190 ha) is part of the Norfolk Island Group , located approximately 1670 km north-east of Sydney, Australia and 1070 km north-west of Auckland, New Zealand. The Norfolk Island Group lies within the “Norfolk Island Seamounts area”, a unique marine ecosystem surrounded by a high number of seamounts Approximately 300 Masked Booby pairs breed on Phillip Island Foraging movements of Masked Boobies were studied using GPS data loggers  recording time, latitude and longitude every 4 minutes. GPS loggers were attached on the top of three central tail feathers using waterproof adhesive TESA® tape , minimising the damage to the feathers upon retrieval Chick-rearing adults  were captured with a noose-pole and weighed using a spring balance . Adults were sexed by voice and by measurements: males have a distinctive higher pitched voice than females and are smaller than females within a pair lavielle of R package adehabitatLTchooseseg (adehabitatLT) to estimate the number of segments of each foraging track and the function findpath (adehabitatLT) to find the limits of the FPT segments (i.e. start and end of the segments). All segments of lower FPT values were then identified as ‘‘movement’’ and all higher segments were identified as ARS behaviour . Accordingly, the nocturnal portions of these trips were excluded because these resting positions may falsely increase the FPT variance Acceleration and dive data were analysed using IGOR Pro 6.21 . The component of the gravity acceleration along the surging axis was firstly separated from the high-frequency component resulting from wing beat activity, using a low-pass filter Each foraging trip was divided into individually numbered sections based on presence or absence of ARS behaviour. For example, if a foraging trip had one ARS zone present along the track, the trip was divided into three sections of known duration: (1) ARS absent, (2) ARS present and (3) ARS absent. If a foraging trip had two ARS zones, the foraging trip was divided into five sections of known duration: (1) ARS absent, (2) ARS present, (3) ARS absent, (4) ARS present and (5) ARS absent. For each section of a foraging trip, the proportions of time spent sitting on the water surface and in flight, as well as the total number of dives, were calculated.Due to battery exhaustion of the depth-acceleration loggers, nine foraging trips were excluded from the data analysis and only complete foraging trips recorded by both GPS and depth-acceleration loggers are presented in the results section.treefalse ARS zones in foraging trips of Masked Boobies for which only GPS information was available. ARS duration, scale of ARS zones (i.e. radius), mean distance of ARS zones from colony, as well as total foraging trip duration and total distance travelled  were used to classify false ARS. Jack-knifingJack-knifing is a re-sampling method systematically re-computing  the sampling set by leaving out one observation at a time, and using the resultant model to predict the nature of the excluded record.We used a classification tree to construct a decision tree in R , the proportion of time adults spent in flight was excluded from the model. Prior to checking the fit of the model, model simplification and checking of model assumptions following anova (chi-squared under one degree of freedom), including the nested random effect to a second model with same fixed effects, but with only trip ID as a random effect. As p>0.05, we concluded that the nested random effect “trip ID within season” did not improve the fit of the model and used only individual trip ID as a random effect for further statistical testing. Bayesian inference from our simplified mixed model was obtained by applying the function sim of R package armAll statistical analyses were performed using R 2.15.0 τ = 0.54, p<0.001, n = 50) and maximum distance from colony .A total of 50 foraging trips from 26 individuals  were recorded. Of the 26 individuals, 21 performed single-day foraging trips, while five adults  stayed overnight at sea, returning to the colony the next day. Masked Boobies had an averaged foraging trip duration of 6.52±3.79 h  and travelled on average a total distance of 160.74±111.43 km , at a mean maximum distance from colony of 74.41±68.06 km  . Trip duWelch’s t-test t = –0.726, df = 11.48, p = 0.482).First-passage time analysis of the tracks revealed the presence of ARS behaviour in 41 out of the 50 foraging trips (82%), while in the remaining nine foraging trips, no ARS behaviour was detected. Foraging trips with and without ARS zones lasted on average 6.71±3.78 h  and 5.67±3.93 h , respectively. The duration of foraging trips with and without the presence of ARS behaviour were not significantly different . Mean distance of ARS zones from the colony was further positively correlated with trip duration  and duration of ARS behaviour .A total of 57 ARS events with an average of 1.1±0.8 ARS zones per foraging trip  were detected in Masked Boobies . AverageA total of 537 dives were recorded, averaging 13.1±13.0 dives per foraging trip . Of all dives, 65.2% (n = 350) occurred inside ARS zones, with a mean of 8.3±9.4 dives being observed per ARS zone . Outside ARS zones, birds dived an average of 3.5±6.1 times . In 10 out of 57 ARS zones (17.5%) no diving activity was recorded. All ARS events without dives were located less than 52 km from the colony, presented a smaller scale of ARS and were of shorter duration than ARS zones containing dives . In addiThe time birds spent sitting on the water surface within ARS zones represented on average 44.11±26.14% of the total ARS duration , while outside ARS zones, birds spent a mean of 13.91±21.46% of their time sitting at the water surface . Inside ARS zones, flight time accounted for a mean of 52.68±25.63% of the total ARS duration , which was similar to the mean flight time outside ARS zones: 58.65±38.10% .b = 0.478, z = 0.969, p = 0.332), subsequently the term sex was dropped from the model. The probability of detecting ARS behaviour increased significantly with increasing time that adults spent sitting at the water surface   . In contrast, a shorter time spent sitting on the water surface combined with a higher diving activity decreased the probability of detecting ARS behaviour. Surprisingly, the probability of an ARS event occurring decreased with increasing foraging trip duration    and with= 0.013) . A signi= 0.001) .Based on our model, the predicted probability of detecting ARS behaviour in birds spending 50% of total ARS duration on the water surface was 0.65  and 0.8 for birds spending at least 70% on the water surface  . That isfalsely positive ARS events. Therefore, 11 out of 57 ARS zones (19.3%) were regarded as false , 15 out of 21 false ARS (71.4%) and 76 out of 79 true ARS (96.2%) were estimated correctly.Based on the decision tree, .0 hours . If fora90 hours . ThroughThis study aimed to establish which foraging parameters influence the detection of ARS behaviour in foraging locations of Masked Boobies and whether ARS zones were indicative of foraging. Our results provide strong evidence that resting at the sea surface positions leads to an incorrect detection and interpretation of approximately 19% of total ARS zones. This finding underlines the importance to account for resting at the sea surface positions when ARS behaviour is determined by means of foraging locations in marine predators and suggests that inferences should be drawn cautiously. In addition, we provide evidence that ARS behaviour is linked with enhanced diving activity, suggesting that ARS behaviour may be used as a proxy to identify important feeding areas in this species.Thalassarche carteriThalassoica antarcticaThe adoption of ARS behaviour is expected to occur in patchy environments with high prey densities in situ changes in relative body lipid content as derived from measured changes in buoyancy Cetorhinus maximusThe model containing the number of dives, the amount of time that individuals spent sitting on the water surface, as well as foraging trip duration, best explained the detection of ARS behaviour in Masked Boobies. Higher dive rates within ARS zones suggest that the adoption of ARS behaviour in Masked Boobies is linked with enhanced foraging activity. Consequently, ARS behaviour may be used as a proxy to identify important feeding areas in this species, in particular during foraging trips of longer durations (see below). Although prey capture rates were not estimated in Masked Boobies, it is reasonable to assume that at least some dives resulted in prey capture, as it was observed in Northern Gannets. In the latter, 43% of short and shallow V-shaped dives, in which birds used only the momentum of their fall to gain depth, and 95% of deep U-shaped dives, in which birds actively used their wings to reach greater depths and pursue prey, were associated with successful prey capture Foraging trip duration influenced the accuracy of FPT analysis in other species false ARS events, though such surface-feeding has been recorded in Cape and Northern Gannets false ARS events occurred mainly within close range of the colony because birds may have rested at the water surface after opportunistic or single diving events, which typically occurred without the adoption of ARS behaviour false ARS events in 7 out of the 10 false ARS zones.First-passage time analysis cannot differentiate between areas in which individuals adopt slow travel speeds due to searching for prey or due to a resting pause at the sea surface. It is unlikely that Masked Boobies carried out surface-feeding events during these Several analytical methods based on marine predators foraging movements have been developed e.g.  to proviWe provide empirical evidence that the detection of ARS behaviour is highly influenced by the amount of time Masked Boobies spend sitting on the water surface during daylight hours, leading to an incorrect detection and interpretation of approximately 19% of total determined ARS zones. Without the information on individuals’ time budgets, we would have misleadingly assumed that the probability of ARS behaviour was higher during short foraging trips in the vicinity of the colony, than during long foraging trips at greater distances. This clearly demonstrates the need to account for time budgets during daily foraging movements in order to correctly identify ARS behaviour in marine predators. Nevertheless, although approximately 35% of the dives occurred outside ARS zones, the probability for ARS behaviour to occur was strongly linked with increased dive rates, which in turn were negatively correlated with the time spent resting on the water surface by adults. This suggests that ARS behaviour may be used as a proxy to identify important feeding areas in this aerial marine predator. This study was developed on foraging data of Masked Boobies, but the methods employed and our findings have implications for the detection and interpretation of ARS behaviour in other marine predators.


Epipactis (Orchidaceae) species. Culturable bacteria and yeasts were isolated and identified by partially sequencing the small subunit (SSU) ribosomal RNA (rRNA) gene and the D1/D2 domains of the large subunit (LSU) rRNA gene, respectively. Using three different culture media, we found that bacteria were common inhabitants of the floral nectar of Epipactis. The most widely distributed bacterial operational taxonomic units (OTUs) in nectar of Epipactis were representatives of the family of Enterobacteriaceae, with an unspecified Enterobacteriaceae bacterium as the most common. In contrast to previous studies investigating microbial communities in floral nectar, very few yeast species (mainly of the genus Cryptococcus) were observed, and most of them occurred in very low densities. Total OTU richness  varied between 4 and 20. Cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species. This study extends previous efforts to identify microbial communities in floral nectar and indicates that the floral nectar of the orchids investigated mainly contained bacterial communities with moderate phylogenetic diversity.Floral nectar of animal-pollinated plants is commonly infested with microorganisms, yet little is known about the microorganisms inhabiting the floral nectar of orchids. In this study, we investigated microbial communities occurring in the floral nectar of seven  The orchid family (Orchidaceae) is renowned for its remarkable diversity in floral structures and breeding systems, which, since the early work of Darwin , has attAnacamptis morio would result in a 40% increase in geitonogamous pollination. Similarly, Jersáková and Johnson , microbes have been shown to alter nectar sugar composition, and as a result pollinator behavior , facultatively autogamous species , and one autogamous species (Epipactis muelleri). For each species, the presence of yeasts and bacteria was assessed in five individuals using culture-dependent detection methods and sequencing of the D1/D2 domains of the large subunit (LSU) ribosomal RNA (rRNA) gene and the small subunit (SSU) rRNA gene, respectively.In this study, we investigated microbial diversity in the floral nectar of seven Epipactis (Orchidaceae) is a widespread orchid genus occurring in the temperate and subtropical regions of Europe, America, and Asia , three species were facultatively autogamous , and one species was completely autogamous (E. muelleri) (van der Cingel E. helleborine) are extremely rare in Belgium, with in the case of E. microphylla only one population occurring. To limit damage to populations, for each species five flowers  were randomly collected and transported to the laboratory for further processing.In this study, we investigated microbial diversity in the floral nectar of seven 2O, yielding a total of 35 nectar samples. As floral nectar usually contains high concentrations of sucrose and other sugars and can also contain high levels of inorganic ions, nectar dilutions (even in distilled H2O) are not hypotonic and both bacteria and yeasts have been shown to remain viable in nectar dilutions in distilled H2O for several months  and diluted in 500 μL of sterile distilled HFor each culture, genomic DNA was extracted from 5-day old cultures, grown on the original isolation medium, by the phenol–chloroform extraction method described by Lievens et al. . Subsequhttp://rdp.cme.msu.edu/) , followed by trimming to consensus start and end motifs. Subsequently, phylogenetic trees were computed using MrBayes 3.2  were restricted to one representative sequence. In all cases, presumptive identifications based on top BLAST hits were confirmed by the nearest neighbor in the phylogenetic trees containing type strain sequences.Obtained sequences were compared with reference sequences from GenBank using the Basic Local Alignment Search Tool (BLAST) . Bacteria were recovered from all seven species  individuals in total  to over 300 colonies per plate (representing an “uncountable plate”) (Appendix 1). Highest microbial densities were observed in nectar samples from the species E. atrorubens and E. helleborine (on average >300 colony-forming units (CFUs) per plate for the different individuals and media tested). On the contrary, for E. muelleri and E. neglecta only a maximum of 6 and 7 CFUs per plate, respectively, was obtained. Intermediate counts (30–300 CFUs/plate) were obtained for the three other species, including E. microphylla, E. palustris, and E. purpurata (Appendix 1).Bacterial and yeast isolates were obtained from all three media used . Following isolation and purification, a total of 25 yeast and 163 bacterial isolates was obtained across the different isolation media from 28 individuals of the seven Epipactis species , and was recovered from three species , representing seven investigated plants , with an average of 2.1 OTUs per sample. The total number of bacterial OTUs that could be associated with the plant species ranged from 2 (E. muelleri) to 12 (E. microphylla), whereas the average number of bacteria per plant species varied between 0.4 (E. muelleri) and 3.8 (E. purpurata) , with an average of 0.5. On the species level, the total number of yeast OTUs associated with the investigated Epipactis species varied between 0  and 8 (E. microphylla), with an average of 0–2 (E. microphylla) OTUs per species  varied between 4 (E. atrorubens) and 20 (E. microphylla). Finally, cluster analysis revealed that microbial communities of allogamous species differed from those of autogamous and facultatively autogamous species   were observed in our study, and these occurred in very low frequencies.Using three different media , we found a wide variety of culturable microorganisms inhabiting the floral nectar of several E. helleborine on Öland (Sweden) and isolated three different bacterial strains, some of which had a high incidence based on phenotypic features (>50%). However, they did not further identify the bacteria, making it impossible to compare our findings to theirs. Álvarez-Pérez et al.  Sw. On the other hand, we found very little support for yeasts being common inhabitants of the floral nectar of Epipactis species occurring in Belgium, as only a few yeasts were observed, mainly Cryptococcus species. Additionally, these yeasts occurred in very low densities, with only a few colonies per plate. Previous research  amplification using both bacterial universal primers (577F [5′-AYTGGGYDTAAAGNG-3′] and 926R [5′-CCGTCAATTCMTTTRAGT-3′]) . Although the reasons for the low microbial diversity are not totally clear, recent studies have indicated that several factors may contribute to the low species diversity in floral nectar, including dispersal limitation  was almost devoid of microorganisms  and that cell densities as measured by the number of colonies on plates were very low (<30 cells per plate), whereas allogamous and partially autogamous species showed remarkably higher OTU richness and higher cell densities.Despite the relatively large number of OTUs detected , the total number of OTUs per orchid species and the average number of OTUs per individual were low, confirming previous findings that microbial species richness in floral nectar is low . However, if nectar viscosity or the presence of antimicrobial compounds were the main factors driving microbial communities in Epipactis, no differences between species with different breeding systems should be obtained. Although sampling size was quite small, possibly impeding generalization of our results, they suggest that dispersal limitation (insect visits) to some extent has contributed to microbial community organization in Epipactis. Clearly, more research is needed to elucidate the precise factors determining microbial community structure in orchids.On the other hand, the nectar of a et al.  have shoEpipactis species was commonly infested with microorganisms, mainly bacteria, some of which reached high abundances. Bacteria and yeasts have the potential to modify nectar chemical properties, and therefore pollinator behavior and ultimately plant reproductive success and fitness (Herrera et al. E. helleborine was beneficial for the plant, as the production of alcohol reduced the efficiency of grooming by wasps and therefore increased reproductive success. As most Epipactis species are pollinated by wasps, which are efficient groomers, Ehlers and Olesen (We have shown that the floral nectar of several d Olesen  suggested Olesen . We ther


I), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(−39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world’s sheep breeds.DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular “parentage SNP” varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families , each parent of every lamb was identified without using the other parent’s genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (P Significant gains in efficiency are realized in production systems that use teams of rams for breeding ewes. These advantages include fewer enclosures and equipment, reduced labor, and increased mating efficiency. However, the success of genetic evaluations systems is directly affected by the accuracy of pedigrees. Misidentification of parents reduces the genetic gain and is economically disadvantageous E) is the probability that an alleged parent would be excluded from parentage There are numerous theoretical approaches for DNA-based parentage assignment. These range from simple exclusion, to categorical and fractional allocation, to genotype reconstruction E value in as many breeds as possible. SNPs with high PE values also tend to have a low probability of identity (PI) values, i.e., the probability that two animals selected at random from the same population would have identical genotypes An important and challenging use of SNP parentage testing is the assignment of one parent without knowledge of the other parent’s genotype. For this application, a candidate parent with a homozygous genotype is excluded when the offspring has the opposing homozygous genotype. Achieving accurate parentage assignment without the other parent’s genotype, while also keeping the number of SNPs  to a minimum, requires that each “parentage SNP” has a high PFour sets of parentage SNPs have been used with Australian and New Zealand sheep since the Ovine SNP50k BeadArray was reported by the International Sheep Genomics Consortium (ISGC) The present article describes the identification and characterization of 163 parentage SNPs with the exceptional overall qualities for use in diverse breeds of sheep, and a subset of 109 parentage SNPs developed for use in North American sheep. Of these 109 parentage SNPs, 34, 44, 55, and 56 were also contained within the four international parentage sets, respectively, and thus provide reference markers for standardization between sets. The set of 109 parentage SNPs also contained 22 that had not previously been identified or used in any parentage SNP set. The combined power of the 109 SNPs for determining parentage and tracing animals appeared to be suitable for use in many breeds throughout the world.Starting with 47,693 autosomal SNPs on the Ovine SNP50 Bead Array, markers were evaluated in a step-wise fashion by multiple criteria to identify those with desired properties . A SNP wThe 163 parentage SNPs were, on average, highly informative in 48 of the ISGC breed groups (±5.1). The average MAF for 163 SNPs across all 74 breed groups was 0.33±0.04. The names, MAFs, GenBank accession numbers, and other features of the 163 parentage SNPs are provided in Multiplex assays of 57 and 52 SNPs were developed starting from the 163 parentage SNPs, as described in the Materials and Methods. In the first round of development, 119 parentage SNPs were selected for testing and 117 markers produced quality genotypes in a U.S. panel of 96 rams (assay conversion rate 98.3%). Comparison of these genotypes with those derived from Sanger Sequencing and the OvineSNP50 Bead Array indicated that eight of the 117 SNPs did not meet the cutoffs for 97% scoring rate  and 99% accuracy (data not shown). These eight MALDI-TOF MS assays were omitted from subsequent rounds of MALDI-TOF MS multiplex assay development. The remaining MALDI-TOF MS assays for 109 parentage SNPs (multiplexes of 57 and 52 SNPs) were used to genotype 95 tetrad families . ThirteeIn the U.S. panel of 96 rams, there were 10,464 SNP genotypes possible from the 109 SNPs and 10,452 of them were reported . When the 10,452 MALDI-TOF MS genotypes were compared to those from Sanger sequencing and the Ovine SNP50k Bead Array, 10,431 were in agreement and 21 were discordant owing to MALDI-TOF MS genotyping errors (99.80% SNP accuracy). In the set of 380 sheep from the 95 tetrad families, there were 41,420 genotypes possible from the 109 SNPs and 41,037 of them were reported . The SNP call rate for complete tetrad families was also relevant for parentage analysis . In the 95 tetrad families, genotypes from all four family members were reported in 10,061 of the 10,355 possible cases . In these 10,061 sets of tetrad genotypes, there were 10,047 inheritance patterns that were consistent with Mendelian expectations (99.86%).The 95 tetrad families were used to evaluate the use of the 109 parentage SNP MALDI-TOF MS assays for assigning exactly two parents to each offspring without using genotypes from the other parent. These families had germplasm derived from 11 breeds of sheep . A notewI and PE for each SNP was estimated from its genotype and allele frequencies as described in the Materials and Methods  and 9.3×10−45 (Gulf Coast Native), respectively. Thus, for the purposes of traceability, it would be unlikely that two samples with matching genotypes would be from different animals.For each of the 74 ISGC breed groups, the P Methods . AlthougAfshari, . The witE was estimated under stringent conditions where genotype information from only one parent was used . Among unrelated candidate parents, the median within-breed PE for the combined 109 parentage SNPs was estimated to be 0.999987  to the Namaqua Africaner (0.992), there was no apparent geographic bias. Assuming unrelated parents, breed groups within 0.000005 of the median PE were from Australia, Bangladesh, Brazil, China, Germany, Indonesia, Iran, Jamaica, Spain, Sumatra, Switzerland, and the United Kingdom. As expected, close relationships between candidate parents reduced the PE by two to six orders of magnitude, depending on the breed and the relationship  to the predicted frequency of opposing homozygotes derived from the average genotype frequencies . In this analysis, no correction was made for any family relationships among the parents. The average measured PE was 0.113±0.037 for comparisons of 190 adults with the 190 offspring in the 95 tetrad families. The average predicted PE calculated for 109 SNPs in the same 380 members of the 95 tetrad family panel (i.e. a random adult and a random offspring) was 0.129±0.021. The average difference between the measured PE and the predicted PE of a SNP was 0.016±0.033. Thus, the predicted PE indicated that 1.6% more parents would be excluded by each SNP than were actually excluded. This likely reflects the impact of family relationships between some of the ewes.The accuracy of the PThis report describes the identification of 163 SNPs with exceptional qualities for use in parentage testing and traceability in globally diverse breeds of sheep. The application of stringent selection criteria identified SNPs that have a high degree of informativity and are amenable to accurate scoring by a variety of genotyping technologies. These SNPs are relatively unencumbered with negative attributes such as indels, repetitive structures, and unknown flanking SNPs, and thus more likely to perform well when interrogated by present and future genotyping technologies. A subset of 109 SNPs was also developed for a MALDI-TOF MS platform and used with success in “one-parent” parentage testing in U.S. sheep breeds. All 163 SNPs and the multiplex MALDI-TOF MS assays for the subset of 109 SNPs are available for world-wide use without restriction. Alternatively, other subsets from the 163 SNPs could be tailored to specific breeds and still have substantial overlap with existing SNP sets. If needed, more SNPs with similar properties could be developed and added. However, genetic linkage between SNP alleles increases as their distance decreases. The current average distance between the 163 SNP markers (15.3 Mb) is already small enough that a significant degree of haplotype sharing is expected between breeds Until recently, commercial and research laboratory parentage testing in sheep was accomplished with sets of eight to 24 multi-allelic simple tandem repeat markers  E estimates, poor quality assay designs, inefficient genotyping platforms, or degraded DNA samples from the field could result in parentage tests without sufficient discriminatory power. Ultimately, the usefulness of any set of parentage SNPs in a given population is determined locally by those who use it. The present report describes a commercial test for U.S. sheep that shares significant overlap with other contemporaneous international tests and provides a starting point and a rationale for designing other sets customized for local breeds.Several factors may reduce the chances of success when applying the present results to other breeds and real world production settings. Inaccurate PPrior to their implementation, all animal procedures were reviewed and approved by the care and use committees at the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) Meat Animal Research Center (USMARC) in Clay Center, Nebraska.The ISGC collected and genotyped samples from 2,819 sheep from 74 breeds as part of a large study into genetic diversity and the impact of selection after domestication Ovis aries. Additional animals were recruited that either had previously been used in the construction of genomic resources for the sheep genome Ovis Canadensis) and Thinhorn  populations of North America. Each genome was sequenced to a depth of approximately 10-fold mapped read coverage with Illumina GAII (unpublished). Prepublication access to the .bam files was provided under the Toronto guidelines for data users The ISGC had selected 75 animals for WGS to extend its investigation of genetic diversity and selection in the world’s sheep breeds The USMARC Sheep Diversity Panel version 2.4 (MSDPv2.4) consists of 96 rams from Dorper, White Dorper, Dorset, Finnsheep, Katahdin, Rambouillet, Romanov, Suffolk, Texel, USMARC composite , has been reclassified as a non-family member because the genotypes from multiple disperse loci indicate it is not the sire of the twin offspring. The corresponding composite ewe (200023372) and her twin lambs (200440264 and 200440265) have also been removed. The remaining 95 families . The frequency of minor homozygotes in a population is critical for parentage exclusion based on opposing homozygous genotypes. For SNPs with a MAF≥0.3, the minor allele nucleotide is often different among breeds. A simple average of the MAF among all animals leads to an inflated estimate. Thus, to correctly calculate the average MAF among breeds, the MAF must first be estimated within breed, regardless of which nucleotide is the minor allele. The minor allele for each of the 163 parentage SNPs is identified in For each of the 356 candidate SNPs , set D, A multi-phase iterative strategy was used to validate assay development and check concordance of diplotypes derived from MALDI-TOF MS with those derived from the Ovine SNP50k Bead Array and Sanger sequencing. Prior to the development, the cutoffs for call rate and accuracy were set at 97% and 99%, respectively. Although, these cutoffs are relatively high, they are well within the capability of today’s DNA testing technology and lend substantial efficiency to testing when met. In each phase of development, the samples were blinded, scored, and decoded. Adjustments in assay conditions were made between phases of development. Genotyping was performed at GeneSeek  with the Sequenom MassARRAY platform and iPLEX GOLD chemistry according to the manufacturer’s instructions . In the first phase, two multiplex assays were attempted with approximately 60 of the 163 parentage SNPs in each multiplex. The expectation was that some SNPs would not advance to subsequent rounds. Within each multiplex design, software settings were adjusted and markers grouped to maximize the number of autosomes represented, spacing between markers, and overlap with parentage SNPs from the other sources. Multiplex information and primer sequences are provided in I for locus A with SNP alleles A1 and A2, was the sum of the squares of the three genotype frequencies: PI =  (χ11)2+ (χ12)2+ (χ22)2, where χ11, χ12, and χ22 were the relative genotype frequencies of A1A1, A1A2, and A2A2, respectively I for multiple SNP markers was the product of the PI for each individual marker. The underlying assumption was that the marker spacing was sufficient for meiotic recombination to cause alleles to be randomly associated with one another. However, as parentage SNP density increases, the validity of this assumption is decreased. Thus, it is recognized that the combined PI for 163 parentage SNPs is an underestimate of the probability of a coincidental match between random animals from the population owing to linkage disequilibrium between SNPs on the same chromosomes.The PE were estimated without the use of the other parent’s genotype information and, thus, exclusion was based only on the frequency of the opposing homozygous SNP genotypes in the offspring and the purported parent. Briefly, the probability of opposing SNP homozygotes (POH) between a random offspring and a random eligible adult at SNP locus A with alleles A1 and A2, was calculated as follows: POH = (χ11offspring)(χ22adult)+(χ22offspring)(χ11adult), where χ11 and χ22 were the relative genotype frequencies of A1A1 and A2A2, respectively for the adults or offspring groups. The frequencies of homozygous SNP genotypes were assumed to be the same within a breed group regardless of age. Thus, for a single biallelic SNP, PE = POH = 2(χ11)(χ22) when one of the parent’s genotypes are unavailable. This represents the fraction of eligible adults that would be excluded from parentage at one locus, averaged over all comparisons between offspring and adults. Without using the other parent’s genotype information, the combined PE for multiple SNPs was as follows: PE(SNPn) = PE(SNP1)+R1PE(SNP2)+R2PE(SNP3) …+Rn-1PE(SNPn), where PE(SNP1) represents the fraction of eligible adults excluded by the first SNP and R1 is the remaining fraction of unexcluded adults. R2 to Rn-1 are remaining fractions of unexcluded adults after each round of subsequent testing with n parentage SNPs. Thus, for 163 parentage SNPs, the combined PE for unrelated parents is given by: PE(163) = PE(1) + R1PE(2) + R2PE(3) …+ R162PE(163). As was the case with combined PI, the combined PE for 163 parentage SNPs is an underestimate of the probability that a random alleged parent would be excluded from parentage owing to linkage disequilibrium between SNPs on the same chromosomes. For related parents, the PE for each SNP was multiplied by a coefficient of relatedness (r), where r = 0.125, 0.250, or 0.500 E(163) for related parents = (rPE(1)+rR1PE(2)+rR2PE(3) …+rR162PE(163)).In this report, all PFigure S1Distribution of SNP informativity in ISGC breed groups. The MAF was calculated for 47,963 autosomal SNPs in the Ovine SNP50k Bead Array for each of the 74 ISGC breed groups. SNPs with a MAF greater than or equal to 0.3 in an ISGC breed group were defined as highly informative in that group.(TIF)Click here for additional data file.Table S1Features of 163 sheep parentage SNPs.(XLSX)Click here for additional data file.Table S2I, and PE for each 163 sheep parentage SNPs in breeds from around the world.Statistics for allele frequency, P(XLSX)Click here for additional data file.Table S3OvineSNP50k Bead Array genotypes for 2,819 ISGC sheep and 163 parentage SNPs.(XLSX)Click here for additional data file.Table S4List of 587 SNPs from four parentage sets.(XLSX)Click here for additional data file.Table S5Consensus reference genotypes for 96 U.S. sheep (MSDPv2.4) and 163 parentage SNPs.(XLSX)Click here for additional data file.Table S6Oligonucleotide sequences for multiplex MALDI-TOF MS assays of 109 parentage SNPs.(XLSX)Click here for additional data file.Table S7MALDI-TOF MS genotypes for 380 U.S. sheep  and 109 parentage SNPs.(XLSX)Click here for additional data file.


Minimally invasive total hip arthroplasty (MITHA) remains considerably controversial. Limited visibility and prosthesis malposition increase the risk of post-surgical complications compared to those of the traditional method.A meta-analysis was undertaken of all published databases up to May 2011. The studies were divided into four subgroups according to the surgical approach taken. The radiological outcomes and complications of minimally invasive surgery were compared to traditional total hip arthroplasty (TTHA) using risk ratio, mean difference, and standardized mean difference statistics.In five studies involving the posterolateral approach, no significant differences were found between the MITHA groups and the TTHA groups in the acetabular cup abduction angle (p = 0.41), acetabular anteversion (p = 0.96), and femoral prosthesis position (p = 0.83). However, the femoral offset was significantly increased . Additionally, there were no significant differences among the complications in both the groups  and revision rate (p>0.05). In three studies involving the posterior approach, there were no significant differences in radiological outcomes or all other complications between MITHA or TTHA groups (p>0.05). Three studies involved anterolateral approach, while 2 studies used the lateral approach. However, the information from imaging and complications was not adequate for statistical analysis.Posterior MITHA seems to be a safe surgical procedure, without the increased risk of post-operative complication rates and component malposition rates. The posterolateral approach THA may lead to increased femoral offset. The current data are not enough to reach a positive conclusion that lateral and anterolateral approaches will result in increased risks of adverse effects and complications at the prosthesis site. Total hip arthroplasty (THA) is considered one of the most successful operations in orthopedic surgery for over 40 years, during which improvements in the design of implants and biological materials have resulted in positive clinical outcomes. However, for the past decade, considerable interest has been devoted to the development of minimally invasive surgical techniques The advantages of minimally invasive surgeries include less soft-tissue trauma , reduced blood loss and fewer blood transfusion requirements. Postoperative benefits include less pain, shorter hospital stay, quicker return to function and a better cosmetic appearance Despite the increase in adopting MITHA, its risks and benefits still generate debates among orthopedic surgeons. Many of them believe that MITHA introduces additional risks due to the limited visibility of anatomical landmarks and vital structures There have been several related meta-analysis studies We conducted a meta-analysis using the guidelines of the Cochrane Collaboration Any randomized controlled trials comparing MITHA and TTHA for the treatment of hip disease.Participants of the 2 treatment groups were similar demographically, and there were no statistically significant differences with respect to the variables of age, gender, body mass index (<35.0).We focused on comparing MITHA and TTHA with a common operation approach that is, posterior, posterolateral:, anterolateral, lateral, so that there would be a consistency between the two groups.We excluded studies in which the surgical approach was inconsistent . In addition, we also excluded studies that examined the double incision surgical approach.The primary outcomes were:Imaging outcomes , and femoral offset.Postoperative complications .The following search terms were used: total hip arthroplasty (or THA), total hip replacement (or THR), prosthesis (or prostheses or implant), minimally invasive (or less invasive), mini-incision . The literature range was defined as between February 1990 and May 2011. The databases searched included PubMed, Cochrane Central Register of Controlled Trials, Embase, and CBMdisc. A manual search was performed for relevant publications from European Federation of National Associations of Orthopaedics and Traumatology and British Hip Society.Both review authors (BaoHui Yang and HaoPeng LI) assessed potentially eligible trials for inclusion, any disagreement were resolved through discussion. Titles of journals, names of authors or supporting institutions were not masked at any stage.Data were extracted independently by both authors using piloted forms. The data included the general characteristics of each study and the outcomes measured. General characteristics included study design, first author, year of publication, sample size, and interventions. Only the primary outcomes were measured.To avoid inherent problems with scale validity For continuous data such as acetabular cup abduction angle, the mean and standard deviation were used to calculate the weighted mean difference (WMD) and 95% confidence interval (CI); for count data such as post-operative complications, the odds ratio (OR) or relative risk (RR) and 95% CI were used. Data processing was performed using Review Manager 5.0 software (the Cochrane Collaboration).2 was used to estimate the size of the heterogeneity. I2>50% indicated considerable heterogeneity among the included studies.The heterogeneity test P values revealed by the forest plot were used to determine the heterogeneity of the included studies. IWe planned separate outcome analyses according to the different surgical approaches, and if there is no heterogeneity internal each groups, data were pooled using the fixed-effect model and 95% confidence intervals. Where there was clear or significant heterogeneity, we viewed the results of the random-effects model, but in such cases opted not to pool data where the outcome measures were clearly different.A total of 552 citations were identified from the search strategy. Twelve studies were deemed appropriate . Eleven The surgical interventions included 4 studies that used the lateral approach A summary of methodological domain assessment for each study is detailed in Although there were ‘lost to follow-up’ phenomenon in some studies, missing outcome data balanced in numbers across intervention groups, with similar reasons for missing data across groups, so attrition bias was considered as a low risk of bias.Abduction angle of the acetabular cup was again used for the funnel plot analysis of publication bias , which rNo significant differences were found between the MITHA and the TTHA groups in the imaging data of the acetabular cup abduction angle (p = 0.41), acetabular anteversion (p = 0.96), femoral prosthesis position (p = 0.83). The average femoral offset was significantly increased in the MITHA group than in the TTHA group  .No significant differences were noticed in the complications that occurred , and revision rate (p = 0.44)  and S4.No significant differences were found between the MITHA and the TTHA groups in the imaging data of the acetabular cup abduction angle (p = 0.46), acetabular anteversion (p = 0.67), femoral prosthesis position (p = 0.25), and femoral offset (p = 0.16)  and S4.No significant differences were found between the two groups in the complications that occurred , and revision rate (p = 0.41)  and S4.No significant differences were found between the two groups in the acetabular cup abduction angle (p = 1.00) and proximal femoral fracture (p = 0.16). However, data on acetabular anteversion, femoral prosthesis position, femoral offset, dislocations, nerve injury, infection, deep vein thrombosis, and revision rate were not adequate for analysis  and S4.No significant differences were found in the imaging data of the acetabular cup abduction angle (p = 0.88) and femoral prosthesis position (p = 0.30). Acetabular anteversion and femoral offset were not evaluated  and S4.No significant differences were found among complications such as dislocations (p = 1.00), infection (p = 1.00), and deep vein thrombosis (p = 0.33). Nerve injury, proximal femoral fracture, and revision rate were not evaluated  and S4.Minimally invasive total hip arthroplasty has often been the subject of debate in recent years. No clear definition exists for what constitutes MITHA, but there is a relative consensus that hip arthroplasties performed with any incision less than 10 centimeters can be included. Proponents of MITHA believe that this approach leads to a faster functional recovery, quicker hospital discharge, and increased patient satisfaction. Opponents believe that compared with TTHA, MITHA leads to increased iatrogenic nerve injury, prosthesis malposition, and revision rate, because of the limited field of vision during the surgery During THAs, the position of prosthesis is directly related to the efficacy of the procedure, and malposition of the prosthesis can cause dislocation, impaction, and pain post-operatively. The bony marks can be clearly visualized in TTHA. In contrast, any approach that is taken in MITHA, is plagued by limited surgical vision and inadequate exposure, thus resulting in difficult, and often, a malpositioning of the prosthesis. Hence, in our study, X-ray assessment of the acetabular abduction angle, anteversion, femoral prosthesis positions were generally considered reliable indicators of successful MITHA.Total hip arthroplasty -related complications can severely compromise the efficacy of the surgery, such as infection, deep vein thrombosis, fracture adjacent to the prosthesis, and dislocation. The commonly seen complications after THA include dislocation (3.9%), pulmonary embolism (0.9%), and deep infection (0.2%) Minimally invasive surgeries for THAs can be divided into five categories: posterior, anterolateral (OCM: a modified Watson-Jones approach) This approach can spare the gluteus medius and the hip flexors. In addition, this approach is a modification of the conventional posterolateral one and is easy for surgeons to master.In this meta-analysis, no significant differences were found in the acetabular cup abduction angle (p = 0.41), acetabular anteversion (p = 0.96), and femoral prosthesis position (p = 0.83). In addition, there were no significant differences observed with respect to complications. However, one study found that the average femoral offset was significantly increased in the MITHA group (1.6±0.76 cm) than in the TTHA group (1.3±0.81 cm) . Femoral offset is the distance from the center of rotation of the femoral head to a line bisecting the long axis of the femur. A decrease in the femoral offset moves the femur closer to the pelvis, which can result in impingemen at the extremes of movement. Moving the femur medially results in soft tissue laxity. Both of these problems can cause instability and possible dislocation In our meta-analysis, the results showed the radiological outcomes  and complications  were not statistically significant between the two treatments by the posterior approach. The possible reason might be that the posterior approach is well-established and the anatomical structures are well understood. The minimally invasive incision can easily be determined in THA through the posterior approach. Additionally, the correct use of retractors can increase the operation field. Particularly, this approach can clearly expose the medullary cavity of the femur at the proximal end of the incision, and expose the acetabulum side at the distal end of the incision. The long-term effects, however, still need further observation.The minimally invasive anterolateral and lateral approach was thought to be difficult to perform as the acetabular cup installation was prone to excessive anteversion and abduction, causing post-operative anterior dislocation In our meta-analysis, only the acetabular cup abduction angle (p = 1.00) and proximal femoral fracture (p = 0.16) were analyzed and no statistical significance was found (p>0.05). However, no adequate data was available to effectively evaluate the acetabular anteversion, femoral prosthesis position, femoral offset, dislocations, nerve injury, infection, deep vein thrombosis, and revisions. Therefore, no positive conclusion can be drawn from the current study until more high-quality randomized controlled studies are available.The lateral approach also has a relatively lower risk of postoperative dislocation. he acetabulum can be adequately exposed and the prosthesis can be easily implanted. Nevertheless, the major shortcoming of this procedure is that part of the gluteus medius and gluteus minimus is spliced, causing weakness of the abductor, damage to the superior gluteal nerve, ipsilateral post-operative lameness, and increased risk of heterotopic ossification around the hip Our findings are mainly limited by the quality and the low number of included studies. This limited our assessment of potential publication bias and unpublished research having negative results that cannot be identified. Therefore, publication bias may exist, which could result in the overestimation of the effectiveness of interventions. Third, the methodological quality of the all trials was found to be medium risk of bias. Due to these limitations, the combined results of this meta-analysis should be cautiously accepted, and more independent high-quality RCTs with effectiveness analyses are needed.Our meta-analysis indicates that the posterior approach in MITHA is a safe surgical procedure, without increased operative complication rates and component malposition rates. The posterolateral approach may lead to increased femoral offset. No thorough conclusion can be drawn from the lateral or anterolateral approach as the risks of adverse effects and complications are increased at the site of prosthesis. It must be pointed out, that this result is only a research report from140 patients and the results need to be treated with caution. More high-quality studies are needed to assess the best surgical approach in minimally invasive hip arthroplasties.Table S1The Cochrane Collaboration's tool for assessing risk of bias.(DOC)Click here for additional data file.Table S2Characteristics of included randomized controlled trials (RCTs).(DOC)Click here for additional data file.Table S3Minimally invasive versus traditional total hip arthroplasty .(DOC)Click here for additional data file.Table S4Minimally invasive versus traditional total hip arthroplasty (complications outcomes).(DOC)Click here for additional data file.


Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ∼30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1–10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ∼76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used. Respiratory infections are an important cause of morbidity and mortality, with a worldwide disease burden estimated at almost 98,000,000 disability adjusted life years (DALYs) and more than 4,000,000 deaths per year Virus detection and typing is a tedious task because the presence of multiple viral species and strains blurs the image, and infection with a wide range of pathogens may result in a similar clinical outcome Molecular methods may be roughly divided into sensitive and specific assays such as real-time PCR or loop-mediated isothermal amplification (LAMP) methods, and techniques with broad specificity that are capable of detecting a wide variety of pathogens. The latter group includes sequence-independent methods (e.g. virus discovery based on cDNA-AFLP (VIDISCA), Sequence-independent, single-primer amplification (SISPA), differential display) In the present study, human ciliated airway epithelial cell cultures were used To design the sensitive and specific assays, all available sequences from public databases were analyzed. Primer design was based on sequence analysis facilitating the identification of conserved regions within viral genomes. Primer blasting showed no significant similarity to human genomic sequences and primer pairs with the best thermodynamic parameters were chosen for subsequent studies. The selected primer pairs are listed in in silico were evaluated in an experimental set-up. Briefly, primer sets were tested on positive control material and the resulting products were analyzed on agarose gels. Several optimization steps allowed the selection of PCR conditions common for all reactions, including primer concentration, amount of template, and PCR profile.All primer sets developed To test assay performance, a set of plasmids containing genetic elements recognized by certain primers was prepared. All plasmids were used as an input for nested PCR, and the resulting products were analyzed on agarose gels. All reactions resulted in the appearance of specific bands Figure 150 per mL of 1. For pathogens that were not cultured, samples were diluted 10 times and used as an input for amplification. Selected clinical samples from patients not presenting with obvious clinical symptoms of respiratory disease or tested negative for respiratory viruses were used as dilution media. The resulting PCR products were visualized on agarose gels and are shown in To show whether it is possible to detect viral pathogens in clinical material, a set of virus culture supernatants, or clinical isolates, was prepared, which contained HCoV-NL63, HCoV-229E, HCoV-HKU1, RSV, influenza A and B viruses, parainfluenza 1, 2, 3 viruses, human bocavirus, human adenovirus, human metapneumovirus or human respiratory enterovirus. Virus titers were determined for all cultivable pathogens, and samples were diluted to a TCID5 copies/µl and serially diluted to 100 copies/µl. One microliter of such a template was further used as an input for the PCR reaction. The results presented in Serial dilutions of control plasmids containing genetic elements recognized by the PCR primers were used to determine the sensitivity of each assay. Briefly, after assessment of plasmid concentration using two independent methods , the plasmid copy number was set to 10To determine whether the newly designed assays were specific for the selected pathogen, all primer pairs were tested for cross-reactivity against all other respiratory pathogens included in the study. Briefly, RNA or DNA was isolated from concentrated cell culture samples or clinical specimens and subjected to reverse transcription, where appropriate. The resulting cDNA was used as an input for nested PCR using all the primer sets shown in , A total of 34 samples were subjected to analysis. Briefly, RNA or DNA was isolated and samples processed as described in “,As mentioned previously, an etiological agent cannot be found in ∼30% of clinical specimens from patients suffering from respiratory tract illnesses in silico analysis reveals that the majority of these methods are based on the sequence of only a single viral isolate, and that their performance in laboratory settings is limited (data not shown). In the current study, a broad and sensitive panel of assays based on a two-step nested PCR was developed for the detection of a majority of human respiratory viruses. Selected primer sets, designed based on complete GenBank information, are complementary to 95–100% of the sequences of archival and contemporary viral strains. Such an approach limits the influence of intra–species sequence variability and facilitates the detection of contemporary and (most likely) newly emerging strains. The developed assays may thus be considered not only as detection techniques but also as a basic tool for virus discovery. The main disadvantage of developed tests is that detection is limited to viruses that belong to specific viral families/subfamilies, and they cannot be used for all viruses. Furthermore, the diversity of viral species in combination with their low load in clinical samples may result in false-negatives At present, the most sensitive and specific methods for detection, typing and identification of viruses are based on molecular biology techniques. Numerous molecular assays have been described, though The detection of unknown viral pathogens in respiratory clinical material is difficult using sequence-independent virus discovery methods, as the low viral load and high background signal from cellular nucleic acids frequently hinders detection. Until now, sequence independent virus discovery techniques were mostly used for virus culture supernatants  ex vivo cell culture techniques based on fully differentiated human airway epithelium.In the current study, detection of viral pathogens was possible in ∼76% of clinical samples, leaving ∼24% of cases undiagnosed. Such a detection rate is consistent with that in previously published reports HAE cultures constitute a semi-natural environment for the replication of viral pathogens, and pre-amplification of viruses contained in otherwise negative clinical samples resulted in the detection of viral agent(s) in 100% of cases. This observation is compliant with the fact that the HAE model is superior to any other culture system in terms of amplifying respiratory viruses. HAE cultures allow replication of majority of the respiratory viral species, including those that cannot be cultured in cell lines.Again, one may question whether using HAE cultures will bias the results, as it is known that human metapneumovirus, for example, does not replicate in such a model (unpublished observation). This assumption is undoubtedly correct, although as mentioned above, this culture model is superior for the majority of respiratory viruses and is unrivalled in terms of virus discovery.ex vivo HAE cultures. Such amplification may result in enhanced detection of viruses in clinical samples during the second round testing with universal primers and, eventually, in the identification of obstacles that hinder the detection of pathogens . Furthermore, these cultures may serve as a source of input material for sequence-independent amplification, which require high virus yield and good signal/background ratios.The system developed in the present study for the detection of respiratory pathogens represents a two-stage screening system, in which clinical samples can be tested for known viruses, and samples that remain undiagnosed may be amplified in In conclusion, the present study describes a set of assays with sensitivity as low as 1–10 copies per reaction that can be used in a routine diagnostic laboratory. The obtained results clearly show that the evaluated tests are highly specific and sensitive. Thorough testing showed that the developed primer sets do not cross react with other respiratory pathogens and are able to detect viruses in a wide range of clinical samples. Further, HAE cultures can be used for pre-amplification of the signal contained in the sample and to facilitate detection of pathogens by standard PCR or sequence-independent methods. Based on these observations, one may hypothesize that the reported lack of an etiological agent in a proportion of patients suffering from respiratory disease may result not only from the presence of unknown viral species, but also from a lack of sensitivity of the available assays. Further studies, including VIDISCA analysis, will provide greater insights into the epidemiology of different strains and species, and may possibly allow the detection and identification of previously unknown viruses.Virus stocks were generated by infecting HeLa cells (ATCC: CCL-2) with adenovirus; RD cells (ATCC: CCL-136) with enterovirus; LLC-MK2 cells  with parainfluenza type 1, 2 and 3, HCoV-NL63, hMPV and hRSV; and MDCK.2 cells (ATCC: CCL-2936) with influenza A and B. In all the cases, cells were lysed after appearance of the cytopathic effect by two freeze-thawing cycles. The virus-containing fluid was aliquoted and stored at −80°C. A control cell lysate from mock-infected cells was prepared in the same manner as the virus stock.Virus yield for all pathogens was assessed by titration on fully confluent susceptible cells, according to Reed and Muench formula Human coronavirus 229E, parainfluenza 1 and 2 viruses, and bocavirus originated directly from clinical samples processed in our laboratory. Stock samples containing influenza A virus, human echovirus 9, RSV, human parainfluenza 3 virus and human adenovirus were kindly provided by Marcel Muller; human coronavirus NL63 was a kind gift from Lia van der Hoek. Human metapneumovirus and human coronavirus HKU1 were kindly provided by Oliver Schildgen and Astrid Vabret, respectively. Influenza B virus was successfully isolated in our laboratory in the past.5 cells per well on permeable Transwell inserts  supports in BEGM. Cells were cultured at 37°C in presence of 5% CO2 until confluence. Human airway epithelium (HAE) cultures were generated by changing the media to Air Liquid Interface media (ALI) and provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures that resemble in vivo pseudostratified mucociliary epithelium. All procedures were performed as previously described Human tracheobronchial epithelial cells were obtained from airway specimens resected from patients undergoing surgery under Silesian Center for Heart Diseases – approved protocols. This study was approved by the bioethical committee of the Medical University of Silesia in Katowice, Poland . A written informed consent was obtained from all patients. Primary cells detached from human bronchi and trachea with pronase E were expanded on collagen-coated  plastic in bronchial epithelial growth media (BEGM) to generate passage 1 cells and plated at density of 3×10For visualization of HAE structures, cultures on Transwell inserts were washed one time with 1× PBS and fixed with 400 µl of 4% paraformaldehyde in 1× PBS. Samples were incubated for 15 min at room temperature and membranes were detached from the plastic frame. Subsequently, membranes were washed with 1× PBS, dehydrated in a gradient of ethanol and xylene, paraffin embedded, sectioned (5 µm) and mounted on silanised glass slides. Prior to staining, slides were deparaffinized with xylene and rehydrated in ethanol.Morphology of HAE cultures was visualized by means of standard haematoxylin and eosin method. For fluorescence microscopy, slides were incubated consecutively in 0.2% Tween 20  and 2% bovine serum albumin . Subsequently, for immunolocalisation of β5-Tubulin and mucin 5AC samples were incubated with mouse monoclonal anti-recombinant β5-Tubulin  and mouse monoclonal anti-recombinant Mucin 5AC, Santa Cruz; dilution 1∶50), respectively. Goat anti-mouse IgG (H+L) Alexa Fluor 488  was used as a secondary antibody. Nuclear staining was performed using Hoechst 33258 . Sections were examined with fluorescent microscope (Nicon Eclipse Ti).Clinical samples from adult patients with asthma exacerbations were submitted to the Jagiellonian University Medical College, Krakow, Poland. Samples  were collected as a part of standard procedure for diagnosis of respiratory pathogens. This study was approved by the Bioethical Committee of the Jagiellonian University in Krakow, Poland . A written informed consent was obtained from all patients. Following the collection samples were stored at −80°C.http://www.ebi.ac.uk/). For influenza A sequence alignments, sets containing 500 sequences were generated, due to limitations of the alignment software. Conserved sites in sequence sets were identified with BioEdit and inspected by eye. For larger data sets, a visual basic script was developed to identify the presence of sites conserved in more than 95% of analyzed genomes. All positions were further verified in terms of primer quality and specificity with Blast server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and with OligoAnalyzer 3.1 . In order to correct for strain variability, 3′ part of the primer was designed at the conserved site, while 5′ part was degenerated, if required.In order to design primers that would recognize all strains of a given virus, irrespectively of natural variability, all genomic sequences available via GenBank were obtained. Sequences were organized with BioEdit software and further aligned with ClustalW script , according to manufacturer's protocol. DNA was isolated using Viral DNA/RNA mini kit , according to manufacturer's instructions. Isolated nucleic acids were stored at −80°C.E. Coli and plasmid isolation with Plasmid mini kit , identity of cloned cDNA fragments was confirmed with DNA sequencing. Subsequently, plasmid DNA was linearized with EcoRI restriction enzyme (Fermentas), separated with gel electrophoresis and purified from the gel, as described above. DNA concentration was assessed spectrophotometrically and by gel analysis and number of copies per mL was calculated. Generated plasmids were further used as positive controls for PCR reaction.In order to develop positive controls that can be easily quantified, a set of plasmids containing DNA fragment targeted by dedicated primers was prepared. Briefly, isolated RNA was reverse transcribed with High Capacity cDNA Reverse Transcription (Applied Biosystems), according to manufacturer instructions, using 15 µl of the previously extracted RNA. Isolated DNA from samples containing DNA viruses was used as input for PCR without any processing. First PCR reaction was conducted as described below and resulting mixtures were layered on 1.5% agarose gel. Following gel separation of DNA fragments, bands corresponding in size to the expected products were extracted from the gel and total DNA was purified with Gel-out kit . DNA concentration was further assessed spectrophotometrically and cloned with InsTAclone PCR Cloning kit (Fermentas) into pTZ57R/T vector. After amplification in DH5α st PCR: 200 nM each; 2nd PCR: 600 nM each) and template DNA . The first PCR (touch-down PCR protocol) cycling conditions included initial denaturation for 3 minutes at 95°C followed by 13 cycles of 20 sec at 95°C, 30 sec at 68°C (which was decreased by 1°C per cycle), 40 sec at 72°C. A further 27 cycles of 20 sec at 95°C, 30 sec at 56°C, 40 sec at 72°C then followed and 5 min at 72°C for the final elongation.Isolated nucleic acids (reverse transcribed for RNA viruses) were used as an input material for two-round PCR reaction with selected primers. Amplification was carried out in a total volume of 10 µl with DreamTaq PCR Master Mix (Fermentas), in presence of forward and reverse primers  was analyzed on a 2.5% (influenza A assay) or 1.5% (other assays) agarose gel and visualized with ethidium bromide.For the nested PCR, reaction was prepared in the same manner, with 2.0 µl of the 10–105 copies per reaction. PCR and nested PCR were conducted as described above. Analysis was performed with standard gel electrophoresis. Further, assays were evaluated with cell culture material or clinical samples positive for HCoV-NL63, HCoV-229E, HCoV-HKU1, RSV, influenza A and B viruses, parainfluenza 1, 2, 3 viruses, human bocavirus, adenovirus type 4, human metapneumovirus and human echovirus 9. Virus stocks were diluted in culture media or clinical samples tested negatively for all known pathogens, including sputum, bronchioalveolar lavage, and nasal lavages.In order to determine the sensitivity of each assay, serial dilutions of linearized positive control plasmids were used in concentrations ranging from 10Viral nucleic acids were isolated as described above and reverse transcribed where appropriate. Concentrated viral samples were tested with all primer sets by two-round PCR and validity of assays was evaluated. Viruses used in this study include HCoV-229E, HCoV-NL63, HCoV-HKU1, influenza A and B viruses, parainfluenza 1, 2, 3 viruses, bocavirus, RSV, human adenovirus, human metapneumovirus and human echovirus 9.


Finfish, particularly shark, had high Hg content whereas bivalves generally had high Se content. Both taxonomic differences and variability were larger for Hg than Se, and Hg content explained most of the variation in Hg:Se molar ratios among taxa. Finally, Hg was more strongly associated with length and trophic level across taxa than Se, consistent with a greater degree of Hg bioaccumulation in the body over time, and biomagnification through the food web, respectively. Overall, our findings indicate distinct taxonomic and ecological Hg and Se patterns in commercially important marine biota, and these patterns have nutritional and toxicological implications for seafood-consuming wildlife and humans.Marine fish and shellfish are primary sources of human exposure to mercury, a potentially toxic metal, and selenium, an essential element that may protect against mercury bioaccumulation and toxicity. Yet we lack a thorough understanding of Hg and Se patterns in common marine taxa, particularly those that are commercially important, and how food web and body size factors differ in their influence on Hg and Se patterns. We compared Hg and Se content among marine fish and invertebrate taxa collected from Long Island, NY, and examined associations between Hg, Se, body length, trophic level (measured by δ Seafood is an important source of lean protein for humans and piscivorous wildlife. However, the overall nutritional value of individual marine fish and shellfish species is complex and depends on numerous other nutritional factors and contaminants such as mercury. Seafood is a primary source of both mercury (Hg) and selenium (Se) exposure. Mercury is a nonessential, toxic metal that often triggers fish consumption advisories While many studies have examined Hg  approved field collection.Alopias vulpinus), Oak Bluffs, MA (thresher shark), and Montauk, NY  in August 2010. Sharks from each of these areas are considered to be a part of the same regional population, thus were combined in our data set. Striped bass (Morone saxatilis) were collected by seine haul in 2009 from Jamaica Bay on the south shore, and Little Neck and Manhasset Bays on the north shore as part of a separate study conducted by the New York State Department of Conservation Fish and shellfish were collected from bays on the north and south shore of Long Island, NY, in accordance to an approved animal protocol at Stony Brook University (IACUC project number 20081587). Fish species, blue crab and squid were collected from three study sites between April and September in 2007 and 2008. Details regarding study sites and sample collection methods are published elsewhere Frozen fish and invertebrates were thawed before dissection for trace element and stable isotope samples. Prior to dissection, individual, whole-bodied organisms were measured for wet weight and length. Specifically, we measured length as total length for finfish, carapace length for blue crabs, from the end of longest arm to the posterior end of mantle for squid, and shell length for bivalves. Bivalve wet weights included soft tissue only. Weight and length measurements were not available for individual shark specimens. Instead, we used average total length measurements estimated for each shark species collected for this study  for all data analyses.Anchoa mitchilli), smaller scup (Stenotomus chrysops), and killifish (Fundulus sp.). We removed muscle tissue from blue crabs , external mantle tissue and arms for squid , and muscle tissue at the base of the dorsal fin from sharks. For bivalves, we included all soft tissue from the shell. Trace element sample tissues were added to 15 mL polypropylene metal-free centrifuge tubes and measured for wet weight. We collected sample blanks by rinsing sample preparation tools and surfaces between samples and adding rinse water to blank sample tubes to account for background contamination due to sample preparation.We removed commonly consumed, edible tissues from organisms for trace element  analysis. Individual, whole-bodied organisms were rinsed with 0.2 µm filtered Milli-Q water. All tools and surfaces were acid-cleaned using trace metal clean techniques 13C, 13C/12C and δ15N, 15N/14N) using a continuous-flow Europa Hydra 20/20 IRMS fit with a Europa ANCA sample combustion unit  at the Stable Isotope Facility at UC Davis. Trace element samples were closed vessel microwave digested  with HCl and HNO3 .We collected a parallel set of samples from the same tissues to be measured for C and N stable isotope analyses. Animal tissue samples for stable isotope analyses were placed in Whirlpack bags. All samples were placed in a −80°C freezer overnight and then lyophilized, after which dry weights were recorded. Trace element samples were acid-digested and analyzed for Hg and Se with an inductively coupled plasma mass spectrometer  at the Trace Element Analysis Core Laboratory at Dartmouth College. We measured total mercury content rather than methylmercury primarily because total mercury is more commonly reported in the literature, thus is easier to compare with other studies. Finally, almost all of the mercury in finfish is in the form of methylmercury We digested and analyzed standard reference materials for external quality control of trace metal samples, including NIST SRM mussel tissue 2976, DOLT4 and DORM-3, NRC-CNRC Canada. Additional trace metal quality control procedures included the use of sample duplicates, spike analysis and laboratory blanks. Recoveries between 80 and 120% of the spike amount were accepted to validate the calibration. Gold (200 ppb) was added to in the internal standard mix and the rinse solution to reduce carryover of Hg. Duplicate samples were analyzed after every 10–20 samples. Percent differences between digestion duplicates ranged from approximately <1 to 10%, indicating that Hg carryover did not affect our results. Percent recovery of SRM for Hg were 107±3% (n = 6) for Dolt 4, 116±9% for Dorm 3 and 116±10% for Tort 2. For Se percent recovery of SRM were 100±5% (n = 6) for Dolt 4, 107±6% for Dorm 3 and 101±1% for Tort 2. Average Se and Hg concentrations from sample blanks were negligible , thus blank corrections were not necessary. Dry weight sample detection limits for Hg and Se were 0.03 and 0.25 ppm, respectively, based on 3 standard deviations of the blank digests and an average sample digestion dilution of 500. Trace element concentrations were converted to a wet weight basis using dry weight to wet weight ratios measured for each sample.Data were log-transformed to normalize and improve homogeneity of variance of individual variables. We tested for taxonomic patterns of Hg content, Se content and Hg:Se molar ratios with MANOVA, followed by Welch's ANOVA for individual variables in order to account for unequal variance among taxa. We examined Hg:Se ratios because Hg:Se molar ratios >1 are thought to indicate the presence of Hg in the body that is unbound to Se, potentially resulting in more toxic conditions than when Hg:Se values are <1 Mya arenaria) were higher from Stony Brook Harbor than those from Smithtown Bay.We found no significant differences in Hg concentrations for taxa collected across multiple seasons, sites or years, when such comparisons were possible. Therefore, we pooled data for the same taxa across sites and collection dates for all analyses. One exception was that Hg concentrations in softshell clams  Se concentration and δ15N, and 3) δ13C and δ15N across taxa.We conducted a Principal Components Analysis (PCA) to examine general relationships between Hg, Se food web and body size factors across taxa. We used mean values for each taxon sensu, allowin63, 534 = 7.67, P<0.0001). In general, Hg was highest in shark, moderate in other finfish and lowest in bivalves. In contrast, Se was generally highest in bivalves and moderate to low in finfish and shark, depending on the species. Only mako shark had a Hg:Se ratio >1. Interspecific variability was higher in Hg than Se , and Se content explained 77% of the variation in molar Hg:Se. Similarly, when shark were excluded from the analysis, the Se-Hg:Se relationship for fish became marginally significant  and Se content explained 41% of the variation in molar Hg:Se. Although the slope of the Hg-Hg:Se relationship estimated for fish was greater than the slope for invertebrates, these differences in slope were not statistically significant .We found clear taxonomic patterns in Hg, Se and Hg:Se molar ratios . There w than Se , with th than Se . Finally than Se . When blFinally, we found that mean mercury concentrations were lower in most invertebrate taxa (8 of 10) and finfish taxa (7 of 9) from our study compared with Hg concentrations in these taxa across other studies summarized in the Seafood Hg Database of US commercial seafood taxa 15N), consistent with bioaccumulation and biomagnification, respectively  were excluded  and trophic level  to a broader taxonomic group consisting of multiple squid species from the database. Overall, sedimentary production is the primary source of methylmercury to the Long Island Sound Menidia menidia) were another species of forage fish that were relatively high in Hg, consistent with another study that found similar, high Hg in a congener species (Mississippi silverside) in San Francisco Bay While the taxonomic Hg and Se patterns we found were similar to other studies, taxon-specific Hg concentrations from this study were, on average, slightly lower but within the range of values from the Seafood Hg Database of US commercial seafood samples from multiple geographic regions The higher variability of Hg concentrations than of Se, as observed previously Regardless of the underlying causes of higher Hg variability, the comparatively narrow range of Se concentrations in teleosts that we found may result in toxic conditions if methylmercury concentrations are high, either because the latter is not bound by free selenium or because the mercury reduces available selenium concentrations to sub-optimal levels 13C values) was moderately associated with higher content of both Hg and Se. At least two other studies found positive associations between pelagic feeding and Hg content In general, Hg concentrations were more strongly linked with food web and body size factors than Se concentrations, reflecting clear differences in bioaccumulation and trophic transfer processes between these two elements. First, our finding that increasing length across taxa is strongly associated with higher Hg content, in contrast with slight decreases in Se content, reflects taxonomic differences in Hg and Se content . These intraspecific body size relationships are analogous to interspecific body size relationships in which Hg content strongly increases, while Se content nonlinearly decreases with body size While relationships between Hg concentration, trophic level and body size are established for fish from the Long Island Sound and elsewhere Figure S115N).Relationship between Hg content and trophic level .(PDF)Click here for additional data file.Figure S215N).No significant relationship between Se content and trophic level .(PDF)Click here for additional data file.Figure S313C) and trophic level (δ15N).No significant relationship between habitat-specific feeding .(PDF)Click here for additional data file.