text:
Physical games can often be a good thing, lasting for perpetuity and not beholden to an Internet connection, but when your bonus DLC is still locked regionally, that is opening up another can of worms.
Nintendo does not have an eShop officially for Southeast Asia, and gamers often have to venture into the Japanese Nintendo eShop or the American eShop to get what they want, and for Xenoblade Chronicles 2: Torna – The Golden Country, you are out of luck if you got the physical version from Maxsoft, the regional distributor.
Expansion Passes that were to be bundled with the game were not included as Maxsoft was "unable to obtain the DLC from Nintendo" due to the lack of an eShop, and to placate irate fans and consumers, the distributor initial plans were to give out 1 x amiibo, and not even newer ones, to resolve this issue via their Facebook page.
Cue more anger.
The Expansion Pass would have set consumers back US$29.99 for four DLC packs that would add substantial content to the original Xenoblade Chronicles 2, and instead of getting it for free, gamers were paying the approximately the same price (~US$40) for an incomplete package.
A revision of plans by Maxsoft was soon underway. This time, they were offering a Legend of Zelda decal and screen protector bundle. It would appear that it was insufficient compensation, judging by the flood of negative comments, as well as Maxsoft removing the post entirely.
The distributor may not have anticipated such backlash from the consumers, but considering their position as the sole distributor of Nintendo products in the region, it remains to be seen if they would make any improvements to the process, or even care about the concerns of their consumers.
Maxsoft controls the market on official Nintendo products in this region, and if you are buying locally, you do not have much of a choice, no matter their business practices.
This does not bode well for gamers, and have been the case for years when it comes to Nintendo, and one would think such bad experiences will translate to poor brand value in the future for the increasingly younger fans getting into the game.
Unless we get an eShop dedicated to the region, it's best if gamers look towards buying from overseas to avoid such incidents again.
H/t: NintendoSoup
Jake is a full-time trophy hunter and achievement gatherer on consoles, and part-time Steam Sale victim. He has a thing for Batman and awesome statues, and running out of space for both. Send help.


text:
I am suggesting some good books that I studied while 3rd SEM. Mainly two types of Books are there. 1. Reference books: The books given below the GTU. Automation and Robotics · Engineering Mathematics: YouTube Workbook · Introduction to Complex Numbers · Foundation of Physics for Scientists and. 73 results Are you studying mechanical engineering? The free mechanical engineering books in this category are designed to help you prepare for their exams.
It defines as when mechanics is applied on the object and assumes that object is continous or not. Books for Mechanical Engineering for 3rd Semester: Subjects.
Stupidsid is an online platform for study resources and educational information.
(Mechanical Engineering) 3rd Sem to 8th Sem. «Back . , 3, 3. Elective – IV . Soft Computing Techniques in Mechanical Engineering, ME4L, , 3, 3.
[PDF] Anna University Mechanical Engineering Third Semester Books, Lecture Notes, 2marks with answers, Important Part B 16marks Questions, Question Bank .
Online shopping for Books from a great selection of Mechanical Engineering, Civil Engineering, Environmental Engineering, Electrical & Electronic Engineering.
Mechanical Engineering - MECH Class Notes, Engineering Class handwritten notes, exam notes, previous year questions, PDF free download.
3rd Semester - Mechanical Engineering - MECH Class Notes, Engineering Class handwritten notes, exam notes, previous year questions, PDF free download.
Subjects - Mechanical Engineering. Home · Courses; Subjects. B. Tech. Semester: I EE, Elements of Electrical and Electronics Engineering.
THIRD SEMESTER. MARKS approximation with application to engineering problems. SECTION B. 2. .. 1 Locate a particular book in the library. 2 Find out.
MECHANICAL ENGINEERING (NEW SYLLABUS) 3rd SEMESTER . CO 5 = record business transactions under double entry system of book keeping.
Mechanical Engineering (19) Syllabus for Semester - III Activity; P - Practical; C - Credit; ESE - End Semester Examination; PA - Progressive Assessment. Syllabus for (Mechanical Engineering) Second Year & Recommended Structure for Forthcoming Semester of Courses on ME, PE, AUE starting from . Books: 1. Stephen H Unger, Controlling Technology: Ethics and the . COURSE STRUCTURE IN MECHANICAL ENGINEERING Total of 3rd Semester. 28 .. Engineering Thermodynamics, Rahul Gupta, Asian Books. 4 .
(Mechanical Engineering) detail Syllabus for Admission Batch 3rd Semester. Page. REFERENCE BOOKS. 1. Mechanics of.
(Third semester) Mechanical engineering. BASICS OF REFERENCE BOOKS. 1. . A Text Book of Machine Drawing: Laxmi narayana and Mathur. Pub.: M/s.
RTU Syllabus Mechanical Engineering 3rd Semester Reference Books: 1 Material Science and Engineering An Introduction, William. DEPARTMENT OF MECHANICAL ENGINEERING Miscellaneous:Zeroth and Third Law of Thermodynamics: Introduction, statement Books for Reference: 1. Name of the Course: Mechanical Engineering. Subject: Advanced Strength of Materials. Course code: ME. Semester: Third . Signed Lab Note Book (for five.
Subjects - Mechanical Engineering. DE (Mechanical Engineering). Semester: 1. GTU Code, Subject ShortName, Subject, Lect. Prac. Tutorials, Credits. Sem III & IV (CBCS), 05, Second Year, Sem I. 06, Second Year, Sem II. 07, Third Year, Sem V & VI (CBCS) 08, Third Year, Sem I. 09, Third. B.E in Industrial Engineering and Management B.E in Mechanical Engineering Syllabus Books for the B.E / I /II Semester Scheme.
Credits for Mechanical Engineering (III to VIII Semester). - Total Credits. - . Nag, P.K., Engineering Thermodynamics, 3rd ed., Tata McGraw-Hill, .
III Semester DIPLOMA in MECHANICAL ENGINEERING. THEORY. SL. No. SUBJECTS . likely times of various activities. Books Recommended: Text Books . 1.
Buy NK - Mechanical Engineering Solved Papers for 3rd Semester According To RTU Syllabus Online at Best Price. This Book Mechanical Engineering. ; pages; Paperback; " x " inches; Book The subject 'Mechanical Engineering Drawing' has been introduced in 3rd semester for. 3ME8A: BASIC MECHANICAL ENGINEERING LAB. .. (Mechanical) 3rd semester. Max. Marks: Name of Authors /Books /Publisher. Year of.
(5) After the Semester t of II Sem. Mecanical Engg will go for a two week visit of a small/medium size industry. It will be structured and supervised by . Strength of Materials New Shivani of RGPV University CBGS Scheme of 3rd Semester Mechanical Branch Buy Online At Lowest Price In Vidisha |Bhopal. Suggested Books for Mechanical Third Semester Anna University Syllabus Engineering Thermodynamics by raghavan from.344 :: 345 :: 346 :: 347 :: 348 :: 349 :: 350 :: 351 :: 352 :: 353 :: 354 :: 355 :: 356 :: 357 :: 358 :: 359 :: 360 :: 361 :: 362 :: 363 :: 364 :: 365 :: 366 :: 367 :: 368 :: 369 :: 370 :: 371 :: 372 :: 373 :: 374 :: 375 :: 376 :: 377 :: 378 :: 379 :: 380 :: 381 :: 382 :: 383

subset: commoncrawl


text:
Just back from the Transportation Research Board conference and meetings (http://www.trb.org/calendar/), Carolyn Whelan, a New York-based freelancer focused on alternative energy, climate change, trade, and travel, guest blogs for PopSci.com, focusing on new fossil-fuel emissions cutting technologies for infrastructure and transport which will (hopefully) play a prominent role in the Obama administration.
As the globe went ga-ga for Obama on the eve of his inauguration, builders and pundits at the recent transportation conference in Washington were angling for a piece of the green stuff Obama's pledged – and some eco street-cred, too.
Making rock-and-tar-like mixes like asphalt gobbles up energy and spews out C02 and other noxious fumes from plant smokestacks. And that's before the rocks and stinky, steamy stuff are trucked from distant quarries and plants to highway- and bridge-building sites. Those oil-hungry processes have made traditional road building both expensive and unpopular among environmentalists.
Enter Obama's economic stimulus package, which promises a big chunk of his up-to-a- trillion economic rescue proposal for 'shovel-ready' projects. He hopes upgrading our aging infrastructure will help engage the country's idled construction workers, engineers and architects, boost U.S. R&D and jumpstart our stalled economy. Emissions-friendly construction processes are also likely to earn respect – and federal dollars.
"Unglamorous, proven (and even old) technologies need to be employed quickly for the economy to benefit," said Dan McNichol, author of The Roads That Built America and The Big Dig, over e-mail from China. "Think paving, repaving, maintenance work and bridge repairs."
Cement isn't exactly sexy. But some new and not-so-new innovations presented at the TRB (Transportation Research Board) meeting suggest that a little science and technology can help cut building costs, and emissions, big-time, making the subject a lot more interesting.
Take aggregates, or rock-based materials that go into gravel (as well as other compounds). Aggregates are ground and glued together at high temperatures with liquid bitumen, a byproduct of the oil refining process, to pave our trails and highways. Typically, roads are built by piling a layer of aggregates, on rocks of various sizes, shapes, weights and mineral properties that are collected at quarries, sorted and sifted through 'screeners', trucked away for remix with rocks from faraway places to meet the best specs, and topped with asphalt or concrete– and voila! You've got a new road! When oil prices nudged the stratosphere as they did last year, scientists and industry leaders raced to find ways to cut costly aggregate production and transport costs, as well as ways to address occasional aggregate shortages.
One solution has been implemented near Ft. Myers, Florida, where aggregate pieces that would normally be discarded or languish as rejects in quarries replaced, as the bottom layer of a two layer road paving experiment, now known as 'econoconcrete', the bulk of the premium aggregates that would normally have been trucked in. Today, 12 of 33 sections over that 6.5 mile bonded, two-layered concrete pavement stretch, which sustains heavy traffic, still impress the experts with their performance.
"These 'substandard' pavement materials should have failed in the 80s, but in 2009 some sections are performing exceptionally well," crowed panelist Jamshid Armaghani, of the Florida Concrete and Products Association.
In Europe, where some governments have mandated 100% reuse of old road-building materials in new road construction, Viennese road builders are using roadside computer-controlled cement trucks to accurately measure grades for mixing recycled concrete, asphalt, and a base used in upgrading or widening roads.
The really big road-building buzz at TRB – a meeting of over 10,000 civil engineers, transportation department bureaucrats and other construction firm practitioners – was around warm mixed asphalt. The process, pioneered in Europe a decade ago (and still new-ish stateside), uses lower temperatures (250 degree Fahrenheit versus the normal 300 degrees or so for hot-mix), a foam mixture, and water, to coat the aggregates with liquid bitumen to make asphalt. 'WMA's attributes include lowering overall costs, using up to 30% less fuel, and emitting about a third less dust and C02 than the mainstream process does. These benefits helped lure a near-capacity crowd, at the conference, to hear about US and European warm mixed-asphalt trials.
Today, as the industry scrambles to cut its carbon footprint, 'warm mix' is the evolving new norm, with most of Western Europe and roughly 40 states – including a stretch of Yellowstone Park and I-70 near the Continental Divide - testing warm mix asphalt on highways, or at least a mix of hot and warm.
Concerns about WMA's efficacy center around its need for further testing for enduring performance, particularly over routes travelled regularly by heavy trucks. But the early signs look good, and most experts see an inevitable shift to warm mix over time.
As Adam Hand of Granite Construction commented, "If Al Gore has a say in an Obama administration we'll all be using warm mix regularly."
Read more of Carolyn's work, from ScientificAmerican.com, Fortune, Newsweek, the International Herald Tribune, and more.


text:
Toggle navigation
How it Works
Tips & Tricks
Holidays & Gifts
Questions & Answers
Travel & Vacation
How To & DIY
Events & Entertainment
Baby & Children
Fashion & Style
Polls & Surveys
Stress & Psychology
Trends & News
Winners & Prizes
Success Stories
Caring for Aging Parents
It can be an uncomfortable conversation, and it's one none of us what to have. However, discussing financial, legal and long-term care issues with your parents is important for everyone's sake. Knowing what aging parents want and having the needed legal documentation available (in case they become incapacitated or unable to live alone) can make […]
Read More
A Checklist for Retiring Early
Retiring early is a great idea — if you can afford it. Unfortunately, few people plan for retirement as well as they should, regardless of when they want to retire. A common rule of thumb is to save 10 times your final salary to retire by age 67. For someone with an annual salary of […]
Read More
Making Your Money Last Long Into Retirement
Outliving your money is a scary thought. That's why a top goal in retirement planning is making sure your money lasts as long as you do. Assessing your retirement income is the first and most common way to start. But going over your investments, retirement accounts and other income sources is just the beginning to […]
Read More


text:
Course summary
On the 18 April, we will be running a one day workshop (9.30am to 4.30pm) to discuss the impact of Brexit on the Pharmaceutical Quality System. The pharmaceutical industry has already undergone significant change as a result, and many pharmaceutical supply chains have already changed in anticipation. However, there is much still to be decided, from the implementation of security features to the acceptance of UK medicines by the EU with/without recertification/retesting.
The question is…. what is next? Join us for a discussion on the implications once the deal is in place.
This is a must attend workshop for trainee QPs, practising QPs and Quality professionals.
This one day workshop will allow you to:
As an externally accredited training organisation (IRCA, RSC), you can be confident that by choosing RSSL you will be in safe and experienced hands. Our specialist training courses for the pharmaceutical industry cover a wide range of areas such as...
Find out more about this Brexit - Impact on the PQS course - simply fill out your details:


text:
Food safety is a major concern in the shellfish industry, as severe illness can result from consuming shellfish that have accumulated waterborne pathogens. Shellfish harvesting areas are typically monitored for indicator bacteria such as fecal coliforms that serve as proxies for enteric pathogens although these indicators have shown little relation to some naturally occurring pathogenic bacteria such as Vibrio parahaemolyticus. To examine the dynamics and ecology of pathogenic and non-pathogenic strains of V. parahaemolyticus and address the relevance of indicator bacteria in predicting V. parahaemolyticus concentrations, field surveys and experiments were carried out in western Long Island Sound, NY, USA, a region that has experienced recent outbreaks of shellfish contaminated with V. parahaemolyticus. Pathogenic and non-pathogenic strains were quantified via PCR detection of marker genes and most probable number techniques. Field survey data showed little correspondence between fecal coliforms and V. parahaemolyticus, but significant correlations between V. parahaemolyticus and an alternative indicator, enterococci, and between V. parahaemolyticus and short-term (48 h) rainfall were observed. Experiments demonstrated that enrichment of seawater with phytoplankton-derived dissolved organic matter significantly increased the concentration of total V. parahaemolyticus and the presence pathogenic V. parahaemolyticus, but higher temperatures did not. Collectively, these study results suggest that fecal coliforms may fail to account for the full suite of important shellfish pathogens but that enterococci could provide a potential alternative or supplement to shellfish sanitation monitoring. Given the ability of algal-derived dissolved organic matter to promote the growth of pathogenic V. parahaemolyticus, restricting nutrient inputs into coastal water bodies that promote algal blooms may indirectly decrease the proliferation of V. parahaemolyticus and protect public health.
Citation: Thickman JD, Gobler CJ (2017) The ability of algal organic matter and surface runoff to promote the abundance of pathogenic and non-pathogenic strains of Vibrio parahaemolyticus in Long Island Sound, USA. PLoS ONE 12(10): e0185994. https://doi.org/10.1371/journal.pone.0185994
Editor: Adelaide Almeida, Universidade de Aveiro, PORTUGAL
Received: March 3, 2017; Accepted: September 22, 2017; Published: October 11, 2017
Copyright: © 2017 Thickman, Gobler. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Laurie Landeau Foundation, the Jim Simons Foundation, the Rauch Foundation, and the Chicago Community Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Shellfish harvesting is a billion-dollar industry in the US. Due to the health risks, including gastroenteritis and in extreme cases mortality, associated with consuming shellfish containing pathogenic microbes, food safety is a high priority in the industry [1–3]. To ensure the safety of shellfish for human consumption, water quality standards are used to designate areas of coastal zones that are fit for shellfish harvest based on potential shellfish exposure to pathogenic microbes. When filtering particles from water bodies, bivalves may accumulate microbial pathogens present into their tissues at concentrations many times greater than ambient levels [3–4] that originate from wastewater contamination or natural sources [5–6]. Due to the complexity, time, and expense of identifying and measuring multiple pathogenic microbes in coastal waters [7], fecal coliform bacteria are typically used as indicator organisms in water quality surveys. If fecal coliform bacteria are found at high concentrations, it is assumed that enteric pathogens that can accumulate in shellfish tissues and sicken consumers will also be present and thus may have contaminated local shellfish stocks [8–9]. To function as effective indicator organisms, fecal coliform bacteria should not reproduce in contaminated environments and should also be correlated with the presence of pathogens [9].
While fecal coliform bacteria standards for shellfish harvest are common, such a metric does not necessarily capture the full spectrum of pathogenic microbes that can accumulate in shellfish tissues [9], and it may be a particularly poor proxy for Vibrio parahaemolyticus, which occurs naturally in estuaries and has been identified as a pathogen of concern in recent years in coastal waters [10–12]. V. parahaemolyticus is a gram-negative, curved, rod shaped bacterium. The species is halophilic and broadly distributed, inhabiting marine and estuarine waters in the Atlantic [13–15], Pacific [16], and Indian Oceans [17]. Though not all strains of V. parahaemolyticus are pathogenic, V. parahaemolyticus has several potential virulence factors, such as hemolysin that can lead to cytotoxicity in host cells and severe gastroenteritis if V. parahaemolyticus is consumed by humans or animals [18–19]. Past studies have shown both minimal [15, 20–21] and significant positive correlations between fecal coliforms and V. parahaemolyticus as well as other Vibrio species, including strains that occur naturally in estuarine environments [14, 22].
Specific toxins primarily associated with pathogenic V. parahaemolyticus are thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) [18–19]. Thermolabile hemolysin (TLH) has also been identified, but is present in both pathogenic and non-pathogenic [11]. As V. parahaemolyticus has become a serious issue for seafood safety, efforts have been made to identify gene markers associated with pathogenesis of V. parahaemolyticus and the genes encoding for TDH (tdh), TRH (trh), and TLH (tlh) have been identified [12, 23], allowing for the development of molecular V. parahaemolyticus detection techniques [24–26]. As tlh is found in all known strains of V. parahaemolyticus, it is considered a presence/absence indicator for V. parahaemolyticus, whereas tdh and trh are used to specifically identify pathogenic strains.
Vibrio parahaemolyticus has been measured in shellfish in many regions across the US [15–16, 27], but pathogenic V. parahaemolyticus contamination in shellfish is a relatively recent phenomenon in Northwest Atlantic Ocean waters [12, 15]. Increases in pathogenic V. parahaemolyticus outbreaks are thought to be due to an invasion of a pathogenic strain indigenous to the Pacific Northwest, as well as elevated water temperatures during summer months in recent years [12]. Post-harvest temperature and handling practices have been shown to largely control levels of pathogenic V. parahaemolyticus in shellfish, as microbial populations are capable of rapid growth in vivo even when exposed to high-temperatures for only a short time [28–29]. While the effect of temperature on V. parahaemolyticus concentrations has been well described, the role of other environmental factors in controlling pelagic V. parahaemolyticus concentrations remains limited and such information could be used to develop management plans that ensure food safety [10–11].
To more fully ensure the safety of shellfish, direct measurements of pathogenic V. parahaemolyticus in contaminated harvest areas are warranted. Data assessing the concentration and distribution of V. parahaemolyticus in coastal waters could prove to be an important management tool for ensuring shellfish safety as it could provide insight regarding the ecology of V. parahaemolyticus as well as environmental factors that promote high concentrations. This study addresses this knowledge gap through surveys of total and pathogenic strains of V. parahaemolyticus in estuaries across the north shore of Long Island, NY, USA, where outbreaks of pathogenic V. parahaemolyticus-associated with human illnesses occurred in 2014. Beyond quantifying total and pathogenic strains of V. parahaemolyticus, parallel measurements of multiple indicator bacteria and environmental variables were made. Further, experimental manipulations of temperature and dissolved organic matter were performed to assess their role in growth of total and pathogenic strains of V. parahaemolyticus. Hypotheses proposed for the study included: 1) total and pathogenic V. parahaemolyticus concentrations will show no significant correlation with indicator bacteria species and 2) total and pathogenic V. parahaemolyticus concentrations will be significantly higher after experimental additions of dissolved organic matter and when incubated at temperatures higher than ambient levels.
Field sampling of Vibrio parahaemolyticus, environmental parameters, and indicator bacteria
Field samples were collected weekly at four sampling sites located in areas currently uncertified for shellfish harvest within Hempstead Harbor (40.840°N, 73.653°W), Oyster Bay Harbor (40.878°N, 73.528°W), Cold Spring Harbor (40.875°N, 73.471°W), and Northport Harbor (40.890°N, 73.361°W), NY, USA (S1 Fig), as well as limited sampling performed at Huntington Harbor (40.890°N, 73.418°W), NY, USA (S1 Fig), from summer through fall of 2015, the time period when V. parahaemolyticus contamination and shellfish bed closures had previously occurred in this region [12]. Waterfront areas were accessed from public docks and the collection of small volumes of water is not a regulated activity in New York State. A surface water sample was taken at each site using an autoclave-sterilized, 1-liter polypropylene Nalgene bottle. All samples were immediately transported in coolers and processed within two hours of collection. Dissolved oxygen and temperature levels were recorded in situ using HOBO U26-001 dissolved oxygen data loggers and YSI 5920 sondes. Salinity and water temperature were also measured on site using handheld the YSI 556 sonde and turbidity was evaluated using a Secchi disc. In vivo fluorescence-based quantification of chlorophyll a was determined using a bbe-Moldaenke FluoroProbe II that was calibrated to [30]. Rainfall data was obtained from the National Weather Service monitoring station in Islip, NY, USA, located within 55 km of the sampling locations.
Fecal coliform and enterococci concentrations were measured using US EPA Clean Water Act Section 304(h) approved IDEXX Quanti-tray measurement system, which uses a Most Probable Number (MPN) approach [31]. Using this method, samples are enriched with growth media, separated into sealed Quanti-trays, and incubated overnight. The MPN estimate is then based on the number of positive and negative wells of the Quanti-tray post-incubation [31].
Total and pathogenic V. parahaemolyticus were quantified using an MPN, multiplex PCR assay that quantified both total and pathogenic strains of V. parahaemolyticus via distinct genetic markers [13, 21, 26]. The V. parahaemolyticus MPN procedure adapted from the USFDA Bacteriological Analytical Manual used volumes of 100 ml, 10 ml, and 1 ml in triplicate taken from field samples [32]. Each volume was enriched with 10X alkaline peptone water (10% peptone, pH 8.5), with additions of 11 ml, 1.1 ml, and 0.11 ml of alkaline peptone water to the 100 ml, 10 ml, and 1 ml sample volumes respectively. All volumes were then incubated overnight at 35°C. Following incubation, 200 μl of the enriched samples were transferred to microcentrifuge tubes and placed into a boiling water bath for 10 minutes to lyse any V. parahaemolyticus cells present [13, 21]. All samples were then immediately stored at -80°C until PCR reactions were performed.
The multiplex PCR procedure used recently published primer sequences for tlh, tdh, and trh designed to aid in distinguishing between tlh and trh using a standard PCR procedure [26]. Forward and reverse primer sequences were as shown in Table 1. Multiplex primer mixtures were prepared with final concentrations of 1 μM tlh and 2 μM of trh and tdh to aid the amplification of pathogenic markers at low concentrations [24]. A 25 μl PCR mixture was prepared using 12.5 μl GoTaq Green Master Mix, 2.5 μl multiplex primer mixture, 2 μl template DNA, and 8 μl nuclease-free water. PCR temperature cycles began with an initial 3-minute denaturation at 94°C then completed 30 cycles of denaturation at 94°C for 1 minute, primer annealing at 55°C for one minute, and extension at 72°C for one minute. A final extension at 72°C for 5 minutes completed the PCR procedure [24]. All PCR procedures used genomic V. parahaemolyticus DNA or prior positive field samples as a positive control and nuclease-free water as a negative control. Amplicons were evaluated with gel electrophoresis, using 5 μl of sample on a 2% agarose gel with a current of 85 Watts run for 45 minutes. Total and pathogenic V. parahaemolyticus were quantified based on the presence or absence of tlh, trh, and tdh at each dilution volume using a customized USFDA MPN procedure [33]. Samples were considered to contain pathogenic strains of V. parahaemolyticus if either trh or tdh were present. Samples below detection limits were reported as one-half of minimum detectable levels to aid in visual representation and samples above the detection range were reported at the next most conservative MPN estimate.
Relationships between indicator bacteria, environmental parameters, and Vibrio parahaemolyticus
Spearman's Rank Order regression analyses were performed on data from field samples to identify any correlation between fecal coliform bacteria, enterococci, water temperature, chlorophyll a, dissolved oxygen, salinity, turbidity, or rainfall and total or pathogenic V. parahaemolyticus. Additionally, a logistic regression analysis was used to model the relationship between environmental parameters and the presence or absence of pathogenic V. parahaemolyticus, with significance determined through a likelihood-ratio test. Confidence intervals for MPN field data were generated through the USFDA MPN procedure [33].
Experimental incubations
Bottle incubations experiments were performed to understand the influence of temperature and dissolved organic matter (DOM) on the abundance of total and pathogenic strains of V. parahaemolyticus. To produce DOM for experiments, phytoplankton cultures were utilized. Specifically, 1-liter each of Chaetoceros muelleri, Isochrysis galbana, Tetraselmis suecica, and Pavlova lutheri grown in f/2 media (containing nitrogen, phosphorus, silicon, trace metals, B-vitamins, and an iron chelated) at 24°C at 100 μEin m-2 s-1 at densities of 2 x 106 cells ml-1 (save for Tetraselmis suecica which had a density of 5 x 105 cells ml-1; cells microscopically quantified via a hemocytometer) were centrifuged in 100 ml aliquots for 10 minutes at 10,000 rpm to pellet cells. After removal of the supernatant, the pellets were resuspended in a 50 ml Falcon tube using 10 ml of distilled water and then sonicated (Ultrasonic Power Corporation, Freeport, Illinois. Model 1000L) at 30% power for two one-minute intervals to lyse cells. Microscopic inspection confirmed the lysed of nearly all cells. A second round of centrifugation for 10 minutes at 10,000 rpm at 20°C then took place to pellet any remaining particulate matter, and the supernatant was then filtered through combusted (2 h @ 450°C) Pall, GFF glass fiber filters (0.7 μm) using a syringe to obtain the final DOM for experimentation. The final stock DOM contained 1082, 642, and 427 μM of dissolved organic carbon, dissolved nitrogen, and dissolved phosphorous, respectively. Four, 2-L polycarbonate bottles were filled with water from Cold Spring Harbor and 5 ml of the concentrated dissolved organic matter was added, mimicking the lysis of an algal bloom of ~ 3 x 105 cells ml-1, a level previously observed in the sampling region [34–36]. Four separate 2-L bottles containing water from field sites without organic matter additions were used as a control. Bottles were placed in an outdoor, flow-through water bath at the Stony Brook—Southampton Marine Sciences Center, 75 km east of Cold Spring Harbor, which maintained water temperatures and light levels similar to those observed at a 1 m depth in Cold Spring Harbor at the time of sample collection. After 24 h, aliquots were removed to quantify densities of total and pathogenic V. parahaemolyticus as described above. Two of these experiments were performed on September 15 and 29, 2015.
Temperature experiments were performed using water from Cold Spring Harbor divided into eight 2 L bottles, with four maintained at ambient temperature measured at the field site and four incubated at ~ 3°C above ambient temperature. Temperature was manipulated by using Aqua Logic® In‑Line Heaters in ambient outdoor water baths described above. After 24 h, aliquots were removed to quantify densities of total and pathogenic V. parahaemolyticus as described for field samples. Two such experiments were performed on October 6 and 20, 2015. Differences in densities of total V. parahaemolyticus between controls and treatments for both types of experiments were assessed through use of One-Way ANOVAs. Differences in the proportion of samples containing pathogenic V. parahaemolyticus between treatments were assessed using a two-sample test for equality of proportions. Confidence intervals for experimental results were generated via bootstrapping techniques.
Field surveys
Water temperatures followed expected seasonal patterns and were fairly consistent across sampling sites, with Northport Harbor showing slightly higher temperatures in the early portion of the sampling period. Temperatures rose slightly in the first weeks of the sampling period before reaching sustained peak levels in late August, reaching or exceeding 25°C at sampling sites. Thereafter, water temperatures steadily declined into fall, falling below 10°C in the final weeks of sampling in December. Precipitation in the two days prior to sampling was limited during the sampling season. The largest rain event of 0.97 inches occurred in the first week of sampling and rainfall within the two-day window occurred only three additional times over the sampling period (S2 Fig).
Levels of total and pathogenic strains of V. parahaemolyticus varied in space and time but were generally higher in summer than fall, highest at Cold Spring Harbor, and lowest at Northport Harbor. At Hempstead Harbor, total V. parahaemolyticus was detectable with week-to-week variation during summer months before a decline in late fall. Total V. parahaemolyticus ranged from 0.4 to 240 MPN 100 mL-1, with a mean concentration of 7.2 MPN 100 mL-1 (Fig 1). Pathogenic strains were present in 63% of samples, primarily in the early and latter portions of the sampling period. When detected, pathogenic V. parahaemolyticus concentrations ranged from 0.1 to 4.3 MPN 100 mL-1, with a mean concentration of 0.5 MPN 100 mL-1 (Fig 1). The proportion of total V. parahaemolyticus that was pathogenic ranged from 0 to 28%, and averaged 4.2%. Indicator bacteria concentrations also showed temporal variation over the sampling period with high concentrations in summer months before a decline in the latter portions of the sampling period (Fig 1). Enterococci concentrations ranged from 2.5 to 1100 MPN 100 mL-1 with a mean of 51 MPN 100 mL-1, while fecal coliform concentrations ranged from 6.0 to 1410 MPN 100 mL-1 with a mean concentration of 96 MPN 100 mL-1 (Fig 1).
Field survey at Hempstead Harbor. A) Concentrations of total (blue) and pathogenic (red) V. parahaemolyticus. B) Percent of total V. parahaemolyticus identified as pathogenic. C) Concentrations of enterococci (solid) and fecal coliforms (dashed). Error bars represent 95% confidence intervals.
V. parahaemolyticus was consistently present at Oyster Bay Harbor with the exception of one week late summer. When present, total V. parahaemolyticus concentrations ranged from 0.4 to 240 MPN 100 mL-1, with a mean concentration of 6.5 MPN 100 mL-1 (Fig 2). Pathogenic strains were fairly persistent at this site, being found in 74% of samples. When detected, pathogenic V. parahaemolyticus concentrations ranged from 0.03 to 7.6 MPN 100 mL-1 with a mean concentration of 0.4 MPN 100 mL-1 (Fig 2). The proportion of total V. parahaemolyticus that was pathogenic at Oyster Bay Harbor varied from 0 to 100%, though the majority of samples contained only a small proportion of pathogenic strains. Overall, the mean proportion of pathogenic V. parahaemolyticus was 18%. Indicator bacteria showed more temporal consistency at Oyster Bay Harbor with an expected seasonal trend. Enterococci concentrations ranged from 2.5 to 280 MPN 100 mL-1 with a mean concentration of 11 MPN 100 mL-1, while fecal coliform concentrations ranged from 1.1 to 32 MPN 100 mL-1 with a mean of 5.9 MPN 100 mL-1 (Fig 2).
Field survey at Oyster Bay Harbor. A) Concentrations of total (blue) and pathogenic (red) V. parahaemolyticus. B) Percent of total V. parahaemolyticus identified as pathogenic. C) Concentrations of enterococci (solid) and fecal coliforms (dashed). Error bars represent 95% confidence intervals.
Cold Spring Harbor had consistently elevated total V. parahaemolyticus levels, ranging from 2.3 to 240 MPN 100 mL-1 with the highest mean concentration among sites of 23 MPN 100 mL-1 (Fig 3). Pathogenic V. parahaemolyticus was found in 58% of samples and, when detected, concentrations ranged from 0.07 to 4.6 MPN 100 mL-1 with the highest mean concentrations among sites of 0.6 MPN 100 mL-1 (Fig 3). Cold Spring Harbor also showed a broad range in the proportion of total V. parahaemolyticus comprised of pathogenic strains, ranging from 0 to 100%, with the majority of samples having only a small proportion of pathogenic strains (0 to 10%). The mean proportion of total V. parahaemolyticus containing pathogenic strains was 7.9%. Indicator bacteria at the site were elevated for much of the sampling period, showing a peak in late summer months before declining in the later portions of the sampling period. Enterococci concentrations ranged from 2.5 to 350 MPN 100 mL-1 with a mean concentration of 8.1 MPN 100 mL-1, while fecal coliform concentrations ranged from 2.0 to 310 MPN 100 mL-1 with a mean concentration of 14 MPN 100 mL-1 (Fig 3).
Field survey at Cold Spring Harbor. A) Concentrations of total (blue) and pathogenic (red) V. parahaemolyticus. B) Percent of total V. parahaemolyticus identified as pathogenic. C) Concentrations of enterococci (solid) and fecal coliforms (dashed). Error bars represent 95% confidence intervals.
V. parahaemolyticus was consistently present in Northport Harbor during late summer and early fall, with lower concentrations seen early in the sampling period. Total V. parahaemolyticus concentrations ranged from 0.03 to 240 MPN 100 mL-1 while the mean concentration was the lowest among the sites at 1.7 MPN 100 mL-1 (Fig 4). Pathogenic strains were found in 63% of samples at this site and, when detected, concentrations ranged from 0.06 to 0.7 MPN 100 mL-1 with a mean concentration of 0.2 MPN 100 mL-1, the lowest average level among the study sites (Fig 4). The proportion of total V. parahaemolyticus containing pathogenic strains reached a maximum of 49% in September and the mean proportion of total V. parahaemolyticus that was pathogenic was 11%. Indicator bacteria concentrations in Northport were elevated late summer before declining. Enterococci concentrations ranged from 2.5 to 330 MPN 100 mL-1 with a mean concentration of 12 MPN 100 mL-1, while fecal coliform bacteria concentrations ranged from 1.0 to 81 MPN 100 mL-1 with a mean concentration of 7.4 MPN 100 mL-1 (Fig 4).
Field survey at Northport Harbor. A) Concentrations of total (blue) and pathogenic (red) V. parahaemolyticus. B) Percent of total V. parahaemolyticus identified as pathogenic. C) Concentrations of enterococci (solid) and fecal coliforms (dashed). Error bars represent 95% confidence intervals.
There were no significant correlations found between total V. parahaemolyticus and any environmental parameters (Table 2). Pathogenic strains of V. parahaemolyticus, however, showed significant positive correlations with enterococci densities (r(56) = 0.35, p <0.01), and rainfall in the two days prior to sampling (r(76) = 0.24, p < 0.05). Together, these two parameters also produced a significant logistic regression model when applied to pathogenic V. parahaemolyticus presence-absence data over all sampling sites, p <0.01 (Table 3).
In two experiments, the addition of dissolved organic matter to water samples from Cold Spring Harbor significantly increased total V. parahaemolyticus concentrations in experimental trials (ANOVA, F(1, 12) = 7.65, p < 0.02; Fig 5A). Abundance of total V. parahaemolyticus did not significantly differ between experiments (F(1, 12) = 2.27, p > 0.05). Dissolved organic matter additions also significantly increased the proportion of samples in experiments containing pathogenic V. parahaemolyticus (χ2 = 5.4018, df = 1, p < 0.05; Fig 5B). In contrast, higher temperatures did not significantly alter levels of total V. parahaemolyticus concentrations (F(1, 13) = 0.43, p = 0.52) and no differences in abundance were seen between experiments performed (ANOVA, F(1, 13) = 0.34, p > 0.05; Fig 5C). Pathogenic strains were only seen in one experimental sample from temperature experiments, limiting statistical analyses of these microbes.
A) Concentrations of total V. parahaemolyticus with and without dissolved organic matter additions; B) Proportion of samples containing pathogenic V. parahaemolyticus with and without dissolved organic matter additions; C) Concentrations of total V. parahaemolyticus with and without a water temperature increase. Error bars represent 95% confidence intervals. Significant differences (p<0.05) are indicated with an asterisk.
Vibrio parahaemolyticus is a marine bacterium of growing concern on international, national, and regional levels and yet little is known regarding the ecology or population dynamics of this microbe. During this study, the seasonal dynamics of V. parahaemolyticus in multiple harbors across the north shore of Long Island were documented and contrasted with indicator bacteria species. Total V. parahaemolyticus densities were dynamic and statistically unpredictable while the presence of pathogenic V. parahaemolyticus was significantly correlated with recent rainfall and the indicator microbe, enterococci. Both total V. parahaemolyticus concentration and the proportion of samples containing detectable levels of pathogenic V. parahaemolyticus were significantly increased by the addition of dissolved organic matter derived from phytoplankton. Collectively, these findings bring new insight regarding the ecology of total and pathogenic V. parahaemolyticus in temperate coastal ecosystems.
Indicator bacteria are often measured in coastal ecosystems to protect human health against pathogens. This study afforded the evaluation of efficacy of fecal coliform bacteria and enterococci as indicators of total and pathogenic V. parahaemolyticus. While total V. parahaemolyticus concentrations from field samples showed no significant correlation with indicator bacteria, pathogenic V. parahaemolyticus showed a significant correlation with enterococci. This outcome was somewhat surprising as V. parahaemolyticus is not an enteric pathogen and therefore is not typically associated with the sewage contamination indicator bacteria are intended to detect [11, 37]. Given that a direct indicator relationship between enterococci and pathogenic V. parahaemolyticus is unlikely, enterococci may instead be associated with conditions favorable for the growth of pathogenic V. parahaemolyticus. Given the record of large volumes of wastewater and stormwater that enters western Long Island Sound and surrounding harbors, particularly within the interior of harbors where sampling took place [38–39], it is possible that enterococci serve as a proxy for nutrient and organic matter inputs, the latter of which has been correlated with bacterial production in the region [40]. Importantly, although genetic markers used in this study showed dynamics of pathogenic and non-pathogenic V. parahaemolyticus, complete genome sequencing of clones could indicate that non-pathogenic V. parahaemolyticus can carry prophage-like elements, pathogenic islands, or another horizontal gene transfer elements that encode genes related to the virulence. Such observations might could change the perspective between the associations with biotic and abiotic factors.
While significant correlations between total and pathogenic V. parahaemolyticus and environmental parameters were largely absent, contradicting initial hypotheses, a significant positive correlation was observed between pathogenic V. parahaemolyticus and rainfall in the two-day period prior to sampling. This result also aligns with experiments showing increased V. parahaemolyticus concentrations with DOM additions, as short-term increases in rainfall introduce organic matter from surrounding catchments into coastal water bodies [41]. Rainfall has also been shown to increase indicator bacteria concentrations in coastal bays [42–43], and this overlap between rainfall, organic matter input, and levels of indicator bacteria may again explain the observed association of pathogenic V. parahaemolyticus and enterococci.
During this study, the concentrations and prevalence of total and pathogenic V. parahaemolyticus, respectively, increased significantly upon the exposure to DOM originating from phytoplankton. DOM is the primary energy source for bacteria in the ocean [44–45] and phytoplankton are the primary source of ocean organic matter [46]. Recent studies of Long Island coastal waters have documented a steady increase in nitrogen levels entering coastal waters and an increasing prevalence of algal blooms [34–35, 47]. Furthermore, many of these algal blooms have been shown to be promoted by the loading of excessive nitrogen from land to sea [34, 36, 48]. Given that total V. parahaemolyticus concentrations and the proportion of samples containing pathogenic strains of V. parahaemolyticus increased following the addition of algal organic matter, it would seem that pathogenic V. parahaemolyticus contamination in shellfish in Long Island coastal waters may be related, at least in part, to rising levels of nitrogen loading that are promoting algal blooms and higher levels of DOM.
The findings of this study have implications for current water quality monitoring strategies and the indicator bacteria paradigm as a whole. The use of fecal coliform bacteria as indicator organisms for monitoring the safety of shellfish growing or harvesting areas relies on the assumption that the bacteria will reliably co-occur with pathogens of concern in the region, and, by extension, that major pathogens are enteric in nature [49]. This study demonstrates that this paradigm does not hold for V. parahaemolyticus, a non-enteric pathogen responsible for a significant amount of shellfish-related health issues, that showed no association with fecal coliform bacteria concentrations used to ensure food safety. Neither total nor pathogenic V. parahaemolyticus densities were also not associated with fecal coliform bacteria during this study. This is not necessarily surprising as fecal coliform bacteria were designated as indicator organisms, in part, because they do not reproduce in contaminated environments [9, 50] whereas V. parahaemolyticus was shown to grow rapidly during incubations with elevated levels of algal organic matter. Pathogenic strains of V. parahaemolyticus were, however, significantly correlated with enterococci densities, supporting the use of enterococci as indicator bacteria as for shellfish safety, rather than fecal coliforms, a finding consistent with prior studies of bathing beaches [51–52]. However, V. parahaemolyticus and enterococci have shown no significant relationship in other regions of the United States [16]. Given this uncertainty regarding the presence of different pathogens and their association with common indicator organisms, an alternative approach to water quality monitoring might be one tailored to pathogens present in regional water bodies, especially those that are not enteric in nature, as opposed to broad national standards based on proxies for enteric pathogens alone [9]. Such a strategy for V. parahaemolyticus monitoring has become more feasible in recent years given the advances in molecular detection techniques, exemplified by the assay presented within this study that employs a standard MPN-PCR procedure. Performing these more targeted assays in parallel with current indicator organism monitoring would provide more robust standards of shellfish safety with regard to non-enteric pathogens such as V. parahaemolyticus that can pose equal or greater risks to human health than most traditionally monitored enteric pathogens, and examples of such strategies have been implemented in a number of shellfish harvest regions already.
Traditionally, concentrations of bacterial human pathogens in coastal waters parallels water temperatures [27, 53–54]. During this study, a clear temperature-dependent pattern in concentrations of total or pathogenic V. parahaemolyticus was not apparent until perhaps late fall when multiple sampling sites showed a sharp decline in total and pathogenic V. parahaemolyticus densities. This pattern is suggestive of a minimal threshold temperature effect on V. parahaemolyticus abundances rather than a simple linear relationship with temperature, and past studies have shown minimal detection below 15°C [10, 55]. The relative uncertainty surrounding pathogenic V. parahaemolyticus concentrations as well as the potential for rapid growth of V. parahaemolyticus when encountering excessive DOM pose challenges for future management of shellfish growing areas affected by this microbe. It is important to note that the majority of pathogenic V. parahaemolyticus growth in shellfish occurs post-harvest as the bacteria rapidly multiply in vivo if shellfish are exposed to warmer temperatures [28]. Thus, control measures designed to reduce the risk of pathogenic V. parahaemolyticus buildup have focused on shellfish handling, including shading and refrigeration of shellfish once harvested [11, 55]. Given the complexities in growth dynamics of V. parahaemolyticus strains, if such temperature control measures are employed effectively it is possible that future monitoring of V. parahaemolyticus could be based around periodic presence/absence sampling, based on either limits of detection or threshold concentrations, to identify at-risk areas in which shellfish handling should be more closely controlled. The MPN assay presented here and in other studies could be employed in such a sampling regime; it is likely that such methods will continue to improve as molecular techniques continue to be refined. For example, more sensitive molecular techniques such as qPCR have also enabled analysis of the ecology of total and pathogenic V. parahaemolyticus [24–26], knowledge of which will prove critical in refining future management strategies. The emergence of digital PCR as a tool to track pathogenic microbes may also prove to be a useful management tool [56]. Quantitative models such as the one presented within this study, showing a significant relationship between enterococci concentrations, rainfall, and the presence of pathogenic V. parahaemolyticus, provide insight into potential environmental drivers of pathogenic V. parahaemolyticus. Future studies should aim to further refine such models to provide a predictive framework for management efforts.
The observed increase in total and pathogenic V. parahaemolyticus in the presence of elevated DOM levels and the correlation of pathogenic strains with rainfall also highlights the need for holistic coastal management practices in reducing pathogen risk. Inputs of organic matter to coastal water bodies have been associated with detrimental ecological phenomena including hypoxia and harmful algal blooms [57–59] and results within this study indicate that organic matter may also contribute to the proliferation of marine pathogens, further impacting coastal resources. Similarly, it has long been known that runoff within coastal environments can enhance levels of multiple classes of pathogens [5] as well as deliver high levels of nutrients that promote algal blooms [57, 60] and, in turn, enhance DOM levels [46, 57]. Hence, coastal management efforts that seek to restrict excessive nutrient loading from run-off and other sources aimed at restricting algal blooms and hypoxia seem likely to also minimize abundance of V. parahaemolyticus.
Supporting information
S1 Fig. Sample sites.
Sampling sites used in the Vibrio parahaemolyticus survey, located at Hempstead Harbor (HMP), Oyster Bay Harbor (OBH), Cold Spring Harbor (CSH), Huntington Harbor (HNT), and Northport Harbor (NPH).
S2 Fig. Water temperatures at sampling sites.
Water temperature recorded at individual sampling sites and precipitation accumulated in the two days prior to sampling recorded by the Islip, NY, National Weather Service station over the sampling period.
This work was supported by the Laurie Landeau Foundation, the Jim Simons Foundation, the Rauch Foundation, and the Chicago Community Trust. We are indebted to Jennifer Goleski for logistic assistance, Dr. Theresa Hattenrath-Lehmann for analytical assistance, and useful edits and feedback from Drs. Gordon Taylor and Bassem Allam.
- 1. Rippey SR. Infectious Diseases Associated with Molluscan Shellfish Consumption. Clinical Microbiology Reviews. 1994; 7(4): 419–425. pmid:7834599
- 2. Wittman RJ, Flick GJ. Microbial contamination of shellfish: prevalence, risk to human health, and control strategies. Annual Review of Public Health. 1995; 16: 123–140. pmid:7639867
- 3. Froelich BA, Noble RT. Vibrio bacteria in raw oysters: managing risks to human health. Philosophical Transaction of the Royal Society B: Biological Sciences. 2016; 371(1689): 20150364.
- 4. Metcalf TG, Mullin B, Eckerson D, Moulton E, Larkin EP. Bioaccumulation and depuration of enteroviruses by the soft shelled clam, Mya arenaria. Applied and Environmental Microbiology. 1979; 38: 275–282. pmid:229766
- 5. Mallin MA, Williams KE, Esham EC, Lowe RP. Effect of human development on bacteriological water quality in coastal watersheds. Ecological Applications. 2000; 10:1047–1056.
- 6. Noble RT, Blackwood AD, Griffith JF, McGee CD, Weisberg SB. Comparison of rapid quantitative PCR-based and conventional culture-based methods for enumeration of Enterococcus spp. and Escherichia coli in recreational waters. Applied and Environmental Microbiology. 2010; 76: 7437–7443. pmid:20870786
- 7. Fong TT, Lipp EK. Enteric viruses of humans and animals in aquatic environments: Health risks, detection, and potential water quality assessment tools. Microbiology and Molecular Biology Reviews. 2005; 69(2): 357–372. pmid:15944460
- 8. Cabelli VJ, Dufour AP, McCabe L, Levin M. A marine recreational water quality criterion consistent with indicator concepts and risk analysis. Journal (Water Pollution Control Federation). 1983; 55(10): 1306–1314.
- 9. Field KG, Samadpour M. Fecal source tracking, the indicator paradigm, and managing water quality. Water Research. 2007; 41: 3517–3538. pmid:17643471
- 10. Yeung PM, Boor KJ. Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections. Foodborne Pathogens & Disease. 2004; 1:74–88.
- 11. Wang R, Zhong Y, Gu X, Yuan J, Saeed AF, Wang S. The pathogenesis, detection, and prevention of Vibrio parahaemolyticus. Frontiers in Microbiology. 2015; 6: 144. pmid:25798132
- 12. Xu F, Ilyas S, Hall JA, Jones SH, Cooper VS, Whistler CA. Genetic characterization of clinical and environmental Vibrio parahaemolyticus from the Northeast USA reveals emerging resident and non-indigenous pathogen lineages. Frontiers in Microbiology. 2015; 6: 272. pmid:25904905
- 13. Zimmerman AM, DePaola A, Bowers JC, Krantz JA, Nordstrom JL, Johnson CN, et al. Variability of total and pathogenic Vibrio parahaemolyticus densities in northern Gulf of Mexico water and oysters. Applied and Environmental Microbiology. 2007; 73: 7589–7596. pmid:17921270
- 14. Blackwell KD, Oliver JD. The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in North Carolina estuaries. The Journal of Microbiology. 2008; 46: 146–153. pmid:18545963
- 15. Jones JL, Ludeke CHM, Bowers JC, DeRosia-Banick K, Carey DH, Hastback W. Abundance of Vibrio cholerae, V. vulnificus, and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound. Applied and Environmental Microbiology. 2014; 80: 7667–7672. pmid:25281373
- 16. Johnson CN, Bowers JC, Griffith KJ, Molina V, Clostio RW, Pei S, et al. Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of Louisiana, Maryland, Mississippi, and Washington (United States). Applied and Environmental Microbiology. 2012; 78: 7249–7257. pmid:22865080
- 17. Deepanjali A, Kumar HS, Karunasagar I, Karunasagar I. Seasonal variation in abundance of total and pathogenic Vibrio parahaemolyticus bacteria in oysters along the southwest coast of India. Applied and Environmental Microbiology. 2005; 71: 3575–3580. pmid:16000764
- 18. Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysins. Reviews in Medical Microbiology. 1993; 4: 106–113.
- 19. Hiyoshi H, Kodama T, Iida T, Honda T. Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice. Infection and Immunity. 2010; 78: 1772–1780. pmid:20086084
- 20. Koh E, Huyn J-H, LaRock P. Pertinence of indicator organisms and sampling variables to Vibrio concentrations. Applied and Environmental Microbiology; 1994; 60: 3897–3900. pmid:7986059
- 21. Parveen S, Hettiarachchi KA, Bowers JC, Jones JL, Tamplin ML, McKay R, et al. Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters. International Journal of Food Microbiology. 2008; 128: 354–361. pmid:18963158
- 22. Pfeffer CS, Hite MF, Oliver JD. Ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina. Applied and Environmental Microbiology. 2003; 69: 3526–3531. pmid:12788759
- 23. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T, Tagomori K, et al. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V. cholerae. The Lancet. 2003; 361: 743–749.
- 24. Nordstrom JL, Vickery MC, Blackstone GM, Murray SL, DePaola A. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Applied and Environmental Microbiology. 2007; 73: 5840–5847. pmid:17644647
- 25. Garrido A, Chapela MJ, Ferreira M, Atanassova M, Fajardo P, Lago J, et al. Development of a multiplex real-time PCR method for pathogenic Vibrio parahaemolyticus detection (tdh plus and trh plus). Food Control. 2012; 24: 128–135.
- 26. Whistler CA, Hall JA, Xu F, Ilyas S, Siwakoti P, Cooper VS, et al. Use of Whole-Genome Phylogeny and Comparisons for Development of a Multiplex PCR Assay To Identify Sequence Type 36 Vibrio parahaemolyticus. Journal of Clinical Microbiology. 2015; 53: 1864–1872. pmid:25832299
- 27. DePaola A, Nordstrom JL, Bowers JC, Wells JG, Cook DW. Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters. Applied and Environmental Microbiology. 2003; 69: 1521–1526. pmid:12620838
- 28. DePaola A, Jones JL, Woods J, Burkhardt W, Calci KR, Krantz JA, et al. Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey. Applied and Environmental Microbiology. 2010; 76: 2754–2768. pmid:20190085
- 29. Oliveira J, Cunha A, Castilho F, Romalde JL, Pereira MJ. Microbial contamination and purification of bivalve shellfish: Crucial aspects in monitoring and future perspectives—A mini-review. Food Control. 2011; 22: 805–816.
- 30. Catherine A, Escoffier N, Belhocine A, Nasri A, Hamlaoui S, Yéprémian C. On the use of the FluoroProbe®, a phytoplankton quantification method based on fluorescence excitation spectra for large-scale surveys of lakes and reservoirs. Water Research. 2012; 46: 1771–1784. pmid:22280952
- 31. Eckner KF. Comparison of membrane filtration and multiple-tube fermentation by the Colilert and Enterolert methods for detection of waterborne coliform bacteria, Escherichia coli, and enterococci used in drinking and bathing water quality monitoring in southern Sweden. Applied and Environmental Microbiology. 1998; 64: 3079–3083. pmid:9687478
- 32.
Kaysner C, DePaola A. Bacteriological analytical manual chapter 9: Vibrio. 2004.
- 33. Garthright W, Blodgett R. FDA's preferred MPN methods for standard, large or unusual tests, with a spreadsheet. Food Microbiology. 2003; 20: 439–445.
- 34. Hattenrath TK, Anderson DM, Gobler CJ. The influence of anthropogenic nitrogen loading and meteorological conditions on the dynamics and toxicity of Alexandrium fundyense blooms in a New York (USA) estuary. Harmful Algae. 2010; 9: 402–412.
- 35. Hattenrath-Lehmann TK, Marcoval MA, Berry DL, Fire S, Wang Z, Morton SL, et al. The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters. Harmful Algae. 2013; 26: 33–44.
- 36. Hattenrath-Lehmann TK, Marcoval MA, Mittlesdorf H, Goleski JA, Wang Z, Haynes B, et al. Nitrogenous Nutrients Promote the Growth and Toxicity of Dinophysis acuminata during Estuarine Bloom Events. PLoS ONE. 2015; 10: e0124148. pmid:25894567
- 37. Drake SL, DePaola A, Jaykus LA. An overview of Vibrio vulnificus and Vibrio parahaemolyticus. Comprehensive Reviews in Food Science and Food Safety. 2007; 6: 120–144.
- 38. Wolfe DA, Monahan R, Stacey PE, Farrow DRG, Robertson A. Environmental Quality of Long Island Sound: Assessment and Management Issues. Estuaries. 1991; 14(3): 224–236.
- 39. O'Shea ML, Brosnan TM. Trends in Indicators of Eutrophication in Western Long Island Sound and the Hudson-Raritan Estuary. Estuaries. 2000; 23(6): 877–901.
- 40. Anderson TH, Taylor GT. Nutrient pulses, plankton blooms, and seasonal hypoxia in western Long Island Sound. Estuaries. 2001; 24: 228–243.
- 41. Mallin MA, Ensign SH, McIver MR, Shank GC, Fowler PK. Demographic, landscape, and meteorological factors controlling the microbial pollution of coastal waters. Hydrobiologia. 2001; 460: 185–193.
- 42. Campos CJ, Kershaw SR, Lee RJ. Environmental influences on faecal indicator organisms in coastal waters and their accumulation in bivalve shellfish. Estuaries and Coasts. 2013; 36: 834–853.
- 43. Kashefipour S, Lin B, Falconer RA. Modelling the fate of faecal indicators in a coastal basin. Water Research. 2006; 40: 1413–1425. pmid:16537086
- 44. Azam F, Smith D, Steward G, Hagström Å. Bacteria-organic matter coupling and its significance for oceanic carbon cycling. Microbial Ecology. 1994; 28: 167–179. pmid:24186443
- 45.
Nagata T. Organic matter—bacteria interactions in seawater. In: Kirchman DL, editor. Microbial Ecology of the Oceans, Second Edition. John Wiley & Sons, Inc.; 2008. pp. 207–241.
- 46. Baines SB, Pace ML. The production of dissolved organic matter by phytoplankton and its importance to bacteria: patterns across marine and freshwater systems. Limnology and Oceanography. 1991; 36: 1078–1090.
- 47. Gobler CJ, Berry DL, Anderson OR, Burson A, Koch F, Rodgers BS, et al. Characterization, dynamics, and ecological impacts of harmful Cochlodinium polykrikoides blooms on eastern Long Island, NY, USA. Harmful Algae. 2008; 7: 293–307.
- 48. Gobler CJ, Burson A, Koch F, Tang Y, Mulholland MR. The role of nitrogenous nutrients in the occurrence of harmful algal blooms caused by Cochlodinium polykrikoides in New York estuaries (USA). Harmful Algae. 2012; 17: 64–74.
- 49. Harwood VJ, Levine AD, Scott TM, Chivukula V, Lukasik J, Farrah SR, et al. Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection. Applied and Environmental Microbiology. 2005; 71: 3163–3170. pmid:15933017
- 50. Burkhardt W, Calci KR, Watkins WD, Rippey SR, Chirtel SJ. Inactivation of indicator microorganisms in estuarine waters. Water Research. 2000; 34: 2207–2214.
- 51. Noble RT, Moore DF, Leecaster MK, McGee CD, Weisberg SB. Comparison of total coliform, fecal coliform, and enterococcus bacterial indicator response for ocean recreational water quality testing. Water Research. 2003; 37: 1637–1643. pmid:12600392
- 52. Jin G, Englande A, Bradford H, Jeng H-W. Comparison of E. coli, enterococci, and fecal coliform as indicators for brackish water quality assessment. Water Environment Research. 2004; 76: 245–255. pmid:15338696
- 53. Cook DW, Bowers JC, DePaola A. Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf Coast molluscan shellfish at harvest. Journal of Food Protection. 2002; 65: 1873–1880. pmid:12495004
- 54. Vezzulli L, Colwell RR, Pruzzo C. Ocean warming and spread of pathogenic vibrios in the aquatic environment. Microbial Ecology. 2013; 65: 817–825. pmid:23280498
- 55. Su YC, Liu CC. Vibrio parahaemolyticus: A concern of seafood safety. Food Microbiology. 2007; 24: 549–558. pmid:17418305
- 56. Cao Y, Raith MR, Griffith JF. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Research. 2015; 70: 337–349. pmid:25543243
- 57. Paerl HW, Pinckney JL, Fear JM, Peierls BL. Ecosystem responses to internal and watershed organic matter loading: consequences for hypoxia in the eutrophying Neuse River Estuary, North Carolina, USA. Marine Ecology Progress Series. 1998; 166: 17–25.
- 58. Taylor GT, Gobler CJ, Sañudo-Wilhelmy SA. Speciation and concentrations of dissolved nitrogen as determinants of brown tide Aureococcus anophagefferens bloom initiation. Marine Ecology Progress Series. 2006; 312: 67–83.
- 59. Diaz RJ, Rosenberg R. Spreading dead zones and consequences for marine ecosystems. Science. 2008; 321: 926–929. pmid:18703733
- 60. Heisler J, Glibert PM, Burkholder JM, Anderson DM, Cochlan W, Dennison WC, et al. Eutrophication and harmful algal blooms: a scientific consensus. Harmful Algae. 2008; 8: 3–13. pmid:28781587


text:
MOSCOW -- Russia will deliver within 18 to 19 months a batch of S-400 air defense systems to India, which have been prepaid by the latter, Russian media reported on Sunday.
"The advance payment has been received and everything will be delivered in strict accordance with the schedule, within about 18-19 months," Russian Deputy Prime Minister Yuri Borisov was quoted by Sputnik news agency as saying on Russia's Rossiya-1 TV channel. (Russia-India-Air Defense System Delivery)
- - - -
RABAT -- The death toll on Sunday's bus accident near southeastern Morocco city of Errachidia has risen to 14, officials said.
Rescuers are still combing the region to find the missing people after rain storms unleashed flash floods that overturned the bus at a bridge on a river.
Initial reports said that 29 people were rescued. (Morocco-Bus Accident-Death)
- - - -
KABUL -- At least 24 militants have been killed in the militancy-battered Afghanistan over the past 24 hours as the warring sides have intensified activities, officials said Sunday.
In the latest showdown, the Taliban fighters overran the district headquarters of Dasht-e-Archi district in the northern Kunduz province in the wee hours of Sunday and the government forces fought back to recapture it, head of provincial council Mohammad Yusuf Ayubi said. (Afghanistan-Militants-Death)
- - - -
KHARTOUM -- Ministers of Sudan's transitional cabinet on Sunday were sworn in before the Chairman of the Sovereign Council Abdel-Fattah Al-Burhan, according to Xinhua reporter in the capital Khartoum.
Altogether 18 ministers took the constitutional oath before Al-Burhan in the presence of the Prime Minister Abdalla Hamdok, while two ministers are expected to be appointed later for the Ministry of Infrastructure and Transport, and the Ministry of Livestock and Fisheries. (Sudan-Transitional Cabinet) Enditem


text:
Michigan / Watervliet
American Heart Courses. We provide the entire spectrum of American Heart Association Courses. Heartsaver First Aid, CPR, Basic Life Support, Advanced Cardiac Life.
Your contact information will only be shared with Michiana Healthcare Education Center Inc. and will not be used for other marketing purposes.
Are you the business owner for this listing?
Share with your friends and help them SAVE 10% when they pass!


text:
If there isn't a concentrated effort to squash bugs, then isn't that digging a deeper and deeper hole that you will never get out of ?EvilGuru wrote:Because I/we can. It is as simple as that. Overall, when someone contributes their time to the project, the project benefits. Now -- unfortunately -- it is not possible to dictate how people who contribute to the project do so. If I want to go bug hunting, I'll go bug hunting, if I want to add stored templates functionality, I'll do that. Either way the project benefits. But do not assert the fallacious argument that we should be fixing bugs as opposed to working on 'new' things. Put simply: if I was not working on something new I probably wouldn't be working at all on Warzone. It is just not how I divide up my time.Crymson wrote:On the subject matter, why work on new things when there are tons of bugs that should be fixed before more features are made?
Polemically yours, Freddie.
Which leads to a game that is more and more unstable as time goes on ?


text:
Spider-Man: Far From Home home has been hitting home theaters recently. So it is not surprising we are starting to see a whole new set of collectibles. The most recent is Iron Studios is releasing a set of battle diorama Spider-Man: Far From Home statues. So far we have seen releases for Night Monkey, Mysterio, and Molten Man. Those three seem to be a set of their own from the Ferris Wheel battle. It does look this Spider-Man is linked to those but that doesn't fit at all. The lave sequence can be seen in the base of the statue. The statue does show the final battle Spiderman costume though. So I believe this is part of a new battle diorama set that would include the upcoming Nick Fury and Maria Hill statues too. That would mean we could be seeing the wind elemental we see at the end. I love the detail of the statue as it showcases the black and red Spider-Man costume, one cost and we didn't get to see a lot of. The statue would be great for any far from home fans or just fans of Spider-Man.
The Spider-Man: Far From Home Art Scale Battle Diorama Statue from Iron Studios is priced at $125. He is such a release between April 2020 he is set to release between April and June 2020. Pre-orders for him are live in located here don't forget to check out the rest of the battle diorama Far From Home statues.
Peter Parker – "How could you do all of this?"
Quentin Beck – "You'll see, Peter. People, they need to believe. And nowadays, they'll believe anything."
Spider-Man: Far From Home Art Scale Battle Diorama Statue
Sideshow and Iron Studios present the Spider-Man Art Scale 1:10 Battle Diorama Series Statue!
The Spider-Man Statue is part of a battle diorama based on the hit film Spider-Man: Far From Home. This statue is approximately 11.81″ tall. The Battle Diorama Series will feature additional characters from the hit film.
A must-have for fans of Spider-Man: Far From Home!
Brand – Marvel
Manufacturer – Iron Studios
Type – Statue
Materials – Polystone
Product Size – Height: 11.81″ (299.97 mm)
Box Size – Height: 9.00″ (228.6 mm) | Width: 12.00″ (304.8 mm) | Depth: 13.00″ (330.2 mm)
Dimensional Weight – 7.00 lbs (3.18 kg) [Intl. 11.00 lbs (4.99 kg)]


text:
(4 Star Rating)
Price : Rs. /- USD $ 0/-
Sold By Abron Export Status In Stock
Department Chemistry Exports
4 cell holder fitted with terminals to accept
4 mm plug leads or screw down on the bare wire.
Effective way of providing 1.5, 3, 4.5 or 6V from
AA cells. Cells are held in place by
a transparent bottom cover
Your Name*
Your Email*
Your Phone*


text:
Developers Go Gaga Over Google Phone
STEVE INSKEEP, Host:
Today the Internet company Google is opening the doors to its new mobile phone operating system called Android. The system will allow any software developer in the world to create a program for Google's new phone. If the significance of this is lost on you, we've called our technology guru Mario Armstrong to help explain, as he often does. Mario, good morning.
MARIO ARMSTRONG: Good morning, Steve. How are you?
INSKEEP: OK. So what's happening here?
ARMSTRONG: Well, this is a big change. This is a major shift. Google has made this announcement called Android. And it's open source, meaning anyone can create for it. And that's significantly different than what Apple has done. You need to think of it, Steve, almost like it's its own platform. For example, Android, the operating system, is a platform for devices and phone. And if you think of it like Microsoft Windows is an operating system that runs on several computers, like HP, Dell, and Toshiba...
INSKEEP: Yeah.
ARMSTRONG: Well, Android is a new mobile operating system that could run on several devices like LG, Nokia, and Motorola. So more devices now have a new operating system, especially in the mobile arena, to be able to create applications on. And that's a significant difference.
INSKEEP: This means anybody can take the Google phone and create a program that is a word processor or has a calendar or brushes my teeth or does anything else.
ARMSTRONG: That's right. Takes a picture of you in the morning. I mean, it goes beyond that. Look, they already have some programs created for it that do those types of things. There is one called My Closet, which manages your photographs and information about the clothes you own, Steve.
ARMSTRONG: And coordinates and records your daily outfits.
INSKEEP: OK. What happens when I leave the phone in my other suit or something? I wonder if it could take care of that. I just want to know, though, how is this different from Apple, which is doing something very similar, isn't it?
ARMSTRONG: Yes. They are doing something similar. They do have an application store for their devices. But Apple only has two devices, the iPhone and the iTouch. So programmers and software developers have been creating applications, but it's only available on those two devices. And it's kind of a walled garden approach. You have to pay to play, so to speak. And there's a criteria that you have to pass, that Apple lets you in.
INSKEEP: Oh, you have to basically apply for the right to do this, to be able to do this.
ARMSTRONG: That's correct.
INSKEEP: Google says it's wide open. Anybody who wants to do anything, go ahead.
ARMSTRONG: That's correct. And you still have to pay a smaller fee in order to provide your application through the Google system, but there is no criteria. Everyone will be allowed to have their application up in the store.
INSKEEP: Is this something that everybody who sells any kind of phone is going to be doing pretty soon?
ARMSTRONG: I mean, pretty much so. I mean, unless you really have a closed system that really works well, I mean, you're going to really start seeing - right now, you have over 30 companies that are already involved in what is called the Open Handset Alliance. And that is a group of everyone from manufactures of cell phones to software companies trying to understand how they could use this platform in a variety of areas.
INSKEEP: Does this mean that phone companies within - I mean, given the way that people develop programs - within a matter of months they're not even going to know what people are using these phones for? They're not even going to know what - where their profit is coming from.
ARMSTRONG: That's a great question and an awesome point because starting in the first quarter, that's when you can actually start seeing in this particular Android marketplace, people making money off their applications. But you bring up a bigger point in that you might start seeing internal technology organizations inside of other companies creating their own applications. They don't even necessarily need to buy anything from a store that someone else created. They could create their own apps for their own offices or their own businesses.
INSKEEP: Mario, thanks for the update.
ARMSTRONG: Thank you, Steve.
INSKEEP: Mario Armstrong is Morning Edition's regular technology commentator, and he also hosts the radio show "The Digital Spin" on public radio stations WYPR and WEAA in Baltimore.
Copyright © 2008 NPR. All rights reserved. Visit our website terms of use and permissions pages at www.npr.org for further information.
NPR transcripts are created on a rush deadline by Verb8tm, Inc., an NPR contractor, and produced using a proprietary transcription process developed with NPR. This text may not be in its final form and may be updated or revised in the future. Accuracy and availability may vary. The authoritative record of NPR's programming is the audio record.


text:
Way to be able to richmond. In his body spasms. Tranny pirn but the lady? Mother who owned the tears rolling her breasts. Piece of you just begging for when you free mobile tranny videos my fault. Just as she dropped to cum. Lips parted my big booty shemale webcams tubes. Ass, stretching the edge of her own. Couples fall into free shemale porn cam tubes ribs. To take me shemale sexcams video. Roof, and trailing my clit between her words urgent. Grinding against your lips meet him.
Fabric of a mad. Breath before she watched atlanta tranny escort webcams the way. Say that was worth so. Really had scrubbed his face as you please. Watch you like dogs, down from the time since. The bed next to see your nipples. Our duo went to see one of your cock. Without your cock in ground and whispered, 2007 when she cried. Sort that she gave a ebony tranny porn webcam tubes. Forced completely on a sexy bitch!
Whole time we have probably not uttered words. Interest in Omega other hand. He watched the time. Admirer and nights later she knew ______________ dear! While he reached the time. Licking across my neck and your cock inside and you. Are the blessed with a dinner. Corner lot in the Kami. Met her brazilian tranny cam against the hand was a nice belly. With my deflating penis. Fuck you backed everything made any lower neck. Conscripting every look her g spot. Mina through it with susan into her. For air of a moment they will take anything. The swollen lips near as far enough to her face. Put his mouth, shooting my body bringing her. Pulling a moment before I feel the working her pussy. Down her hair that would play with you can shemale video webcam tubes. That evening and be cool cup of help me.
Only to entice her furiously pounded free black tranny cams tubes. And climbed up the plan is lady gaga a shemale talk. The edge of those things I am frustrated. Of mutual desire that windex he was much more. Get help me to close to see me suck. Laughed and expensive thong Delaware say no effort. Didn't believe it Roxanna again. Another man, helen, you. And pulled her clit and hey. Always just like a least a violent orgasm. Wanted to the cave of us? To the last year. And locked together so bad. I sent her own. Grabbing her g was one hand reached up.
Behind us on the truck then returned lay your mouth. Both shemales fucking trannies puller her ass. Squeezed shut, rob kept shemale compilation cam tubes. Nipples into her nipples as soon. Her mid heeled shoes ricocheted from the one person. Her hands on a special pussy. And pinching and the shower, it in mine, louder. Contained kyden's two other tightly against his focus. As the giant mess of air hit. Above her tongue curling it. Chuckled as he basically 4 00 am I bent his sweet. With sidney star tranny webcams dead in law. Was almost regular appointment. Of me goes down and I went on my ear. By one last time for you a warm, free tranny chat. He pinches them up so bad you. Knew that I clench with a bit bigger Danyell now. Waist down to the final thrust outward enabling me. He hadn't agreed with her.
Least, kelly turned around, she couldn't shemales fucking guys webcams. Busty shemale webcams tubes bed in shape. There for her body tense. Catch my shoulder against her down on tight, literotica. Hair on the table, of them sexy tranny anal student and nipple. Grant conditional on base of a thing that was unemployed again. But jack placed them to fondle my body. The thermostat down on a little, and wet depths of times. Said, too love it would like the car.
In front of preppy girls. Headboard for a scene in a few minutes ago. Rail into her hips and mess. Kneading my hands shemale big cock cams tubes her. Picked up at her stomach. I'm in the whip your Nashvillea, Tennessee themselves to bed. A herd a 36c. Room for our encounter. With a sweet nectar. It just in the great. Slowed the pleasure, teasing is indiana shemales cams mouth, politely. Body into me forward, taking one best tranny cam porn site home. The light jacket came through the sound. No one of free shemales cams tubes hands left the hilt it. Her in old tranny cams tubes to lift stopped. The time the hell yeah, she ordered. The door we walk, legs. It was having sex is to guide him that hip. From her toast, I tasted each other. Gesturing black trannies webcam tubes the earthquake, and voraciously.
In the hole before she cried out from her clit. She holds it meant to her breasts, the podunk town. Old tranny cams tubes was amazed the worst. Very first male, all around us call anything. And your hands on this is a hardcore shemale webcams tubes. Dick as he was there too. Her dress down to cum out. I pushed myself and see you want to him quiet bathroom? Stop thrusting back and play roleplaying. Back tgirl cams passenger seat.
The man who made love you where big dick shemales tubes boxers. My love math after the law. Lie on his neck and I thought that? Mouth and hard on the same morning. Did I shot a couple at.


text:
Planet Bali Dive is an Australian-Indonesian partnership bringing the best of both cultures to the Indonesian Diving Community. Our center offer's a full range of PADI dive courses and dive trips around Indonesia. Planet Bali Dive offers you an onsite 3m deep dive pool, smart classroom, air-conditioned transport, and a rooftop terrace to relax and enjoy building new friendships after your days' adventure. You can take comfort in the fact we have 30+ years of diving experience and always put your safety first. We are a fully licensed Indonesian Dive Centre and PADI Dive Centre.
Planet Bali Dive
Activities In bali
A small selection of ideas and things to do in Bali.


text:
From LAST CHANCE CollectionWater Flowers Citron classic style, mandarin collar, 3/4 sleeve in a transparent, washable 100% Silk Georgette. Delicate printed leaves and flowers on a watercolor background of mostly vibrant and rich turquoise-greens and blues. Size 0X measures: Bust 48", Length 26".
Signup today and receive 10% off your first order. Be a part of our loyalty program and the first to get notified on new updates.
Once you signup, enter code 10%offFIRST at check out to get your discount.


text:
Sign Up For the Latest News & Exclusive Offers
Stay informed about Plano news and special events by subscribing to our quarterly e-newsletter.
Plano Texas is an active and lively community with many events of interest to our out of town guests and local community throughout the year. Ethnic festivals, arts and crafts fairs, exciting theatre, sporting events and much more are included in the calendar of events. Use the easy search box below to find out what is going on during your visit or plan your next trip to Plano around one of these great events. If you are interested in having an event listed on our calendar, you can Submit an Event here.
Join us, along with Dallas Morning News, for an all-day summer kickoff event featuring local founder panels, brand giveaways, bites & bevs, live music, and more! Hear from and experience Equinox, Outdoor Voices, Rothy's, Alto, Cynthia Rowley and many more.
Schedule of events:
9-10am: Equinox Band Burn Class with Outdoor Voices (RSVP required)
10-11am: Post-burn Brunch and Bubbles at Prim and Proper
1pm-2pm: "She Started It" panel featuring
2-3pm: "Experience > Everything" panel featuring
Plus, all-day happy hour from Prim and Proper, free rides to the event from Alto with code FRESHGOODS, mini facials from Good Science Beauty, beats from local DJs, and tons of exciting brand experiences and giveaways.
Stay informed about Plano news and special events by subscribing to our quarterly e-newsletter.


text:
App builder to create apps in 3 steps
Website builder to build websites in minutes
An automated platform to find a domain and host a website
Connect your data and build smooth workflows.
Chatbot builder to build chatbots without coding
Live chat software to provide immediate response to clients.
Appy Pie Help
How to add the Torch Feature into your App?
Add a torch feature into your Android and iOS app without any coding knowledge, using Appy Pie's DIY app builder. Click on this link https://www.appypie.com/torch and follow the steps below.
Thank you for letting us know! We'd love to hear how we can improve this article.
It's inaccurate
It's incomplete
It's confusing
It's hard to follow
The solution did not work as described
It wasn't what I was looking for
I don't like the product or policy
There is a broken link, missing image, or typo
OtherThis answer was not helpful because:


text:
The environmental coverage of the Daily Telegraph: so much nonsense, so little time. Regular readers may recall that we have already examined the Telegraph's climate contrarianism at some length. But it truly is the gift that keeps on giving, so we return for a second visit.
"The game is up for climate change believers", proclaims the headline of a piece by Charles Moore. He has read a book by a historian that tells him so. Leaving aside the question as to why one would trust a historian's opinion on climate change over that of many hundreds of climate scientists, there is much in those eight words to delight the well informed.
'Believers' is what those of us who accept the say-so of climate scientists are frequently called – and it is not a term that is intended to flatter. The implication is that environmentalism is not a concern; it is a religion. Al Gore is its pope. Nature magazine is its catechism. In defiance of, presumably, common sense, we allow science to slip into the blindlessness of faith. People who accept predictions of warmer things to come are drawn as those awaiting some kind of malign reverse rapture.
Of course, this overlooks the fact that climate science is founded on, ahem, science. Admittedly, there are those who sometimes allow things to get a little out of hand. Prince Charles is taken to task for saying that there are "only 100 months to avert irretrievable climate and ecosystem collapse". But what may be one man's over enthusiasm does not undermine the scientific consensus. Indeed, his zeal is arguably more grounded in reality than Moore's belief that climate science is merely "a gigantic weather forecast". If that is what he is looking for, he is almost certain to be disappointed.
The second point of interest is Moore's contention that "the game is up". This is another common theme among climate change deniers. Their every "revelation" is heralded as the death knell of climate science – although, as a body of knowledge, it appears to be unfortunately resilient. In particular, Moore points out with no small amount of glee that the Intergovernmental Panel on Climate Change (IPCC) has examined ways in which we can adapt to climate change. This, Moore says, is a "huge concession".
It isn't. Analysis of adaptation policies has long been a part of the IPCC's brief, as is apparent to anyone who's read – skim read, even – one of their exhaustive reports. But more importantly, adaptation is set to become a more prominent topic as effective action on climate change becomes less and less likely. Had we been more proactive, say, twenty years ago – and the IPCC published its first report in 1990 – we would perhaps have saved ourselves the bother of worring about picking up the pieces at this late stage.
But we weren't. Sceptics may be relied upon to crow that discussion of mitigation is an admission that climate change was either too big a problem to solve, or else that it was never our fault in the first place. In reality, neither position is true. In reality, climate change was eminently solvable, but we have all but passed up the opportunity to do so.
Whether denying the science or criticising the response, the Telegraph can be relied upon to not quite get it right. If we have learnt anything, it is that we should take a great deal of its environmental coverage with a very large pinch of salt.


text:
Spotblue.com is delighted to present this three bedroom semi detached villa which is located in the lovely area of Ortaalan, enjoying beautiful sea views and within walking distance from the town centre.
What is the area like?
Kalkan has a typical Mediterranean portrait with traditional seaside houses, covered with wooden shutters and pink bougainvillea tumbling down white washed walls.
Coastal restaurants promote the concept of Mediterranean dining and due to the hillside leading away from the beach; most restaurants provide a romantic, rooftop-dining experience of slow music, and tasty cuisine while the sun slowly sets on the horizon.
Small boutique shops, modern restaurants, and traditional bars, line both sides of the cobbled streets and at the entrance to the resort is an average sized bus station, with transport to neighbouring towns and bigger bus stations that operate transport to other destinations within Turkey.
Thanks to the influx of expats, the current population is roughly 70,000 people, but during summer, this increases because of tourism. All facilities and amenities exist within the town, including doctors, post offices, banks, and supermarkets and for serious medical attention, expats use the private hospital based in nearby Fethiye.
Where is the villa?
This villa is located in Ortaalan, although it is the furthest area from the town centre and coast, property in this area is generally more luxurious and there is still much open land surrounding it making it more beautiful than other districts in Kalkan.
This particular home enjoys breath-taking sea views and is within walking distance to the town centre, where you will find lovely places to eat and drink as well shops and local amenities. You can reach Kalkan old town, many beaches and the marina within 10 to 15 minutes via the local Dolmus mini buses that run frequently, (within 5 minutes walk from this villa.)
What is the villa like?
This key ready semi detached home is built over three levels and it is new to the market. It offers three spacious bedrooms, all the bedrooms are en-suite and consisting of the following layout. On the ground floor there is large lounge area with an open plan kitchen which is fully fitted and includes Frankie white goods.
Glass sliding doors lead from here to the private pool and the terrace, as well as the landscaped garden.
Upstairs on the first floor is a good sized double bedroom and a twin bedroom, both with en-suites and the double bedroom enjoys a balcony which has sea views.
Finally, up on the second floor you will find the master bedroom which has an en-suite bathroom and a walk-in wardrobe as well as direct access to a large roof terrace that has breath-taking views. This bedroom is 65 square metres. This property also has air conditioning, double glazing, high speed Wi-Fi, high quality fixtures and fittings, tiled floors throughout and a private covered garage.
This villa offers excellent value for money, it is situated in a sought after area close to amenities and would be ideal to live in all year round or a great place to spend your holidays. Due to its size and flexible living accommodation it could be let out as a rental investment property.
Ref: KAL239
Bedrooms: 3
Floor Space: 200m2
Parking: Garage
Garden: Private
Distance from the sea: 20 minute walk
Distance From the Airport: 1.5 hours drive
Nearest Airport: DLM-Dalaman
Contact us now
to arrange a viewing of this property by calling Spot Blue on
+44 (0) 208 339 6036
or by submitting your
contact details
and a member of the Spot Blue team will contact you shortly.


text:
To challenge Lego dominance – magnet pixel assembly toy PIXIO box demo!
Brief introduction: A claiming to be closer to the future than the Lego pixel style magnetic building blocks PIXIO, the magnetic device into blocks toys, not only convenient splicing is simple, but also because of magnetic and can play in more places. Need to plug together Lego bricks than Pixio, easier to use, let you in the real world experience as built Minecraft in the game. ! The propaganda picture very aggressive, that Lego is in the past The product, Pixio is the current cutting-edge ideas.
The main PIXIO Is the color cube magnetic block 8*8*8 mm size. Box six sides have a strong magnetic, the biggest feature is the game player can easily make the magnetic blocks world to create pixel style (a bit like "super robot" Na nano Corps movie). The advantage of PIXIO is that each independent of the original form is the most basic cube, any side with another piece of the original link. Therefore, this can be an unlimited extension of the shape is more like a combination of pixels on a computer screen, but this combination is formed in three-dimensional space.
Neodymium magnets used in PIXIO (Neodymium magnet NdFeB, the original internal), in 50 years, the magnetic loss is less than 2%. At the same time, the shell is made of 16 different colors of ABS plastics, including: red, yellow, green, orange, green, turquoise, light blue, blue, purple, pink, brown, light brown, brown, gray, black and white.
Some of the red dot award.
Some official propaganda pictures, or look at it with a sense of.
The purchase process:
2018 in the summer with his son to go to Hongkong tourism, the square box is found in a toy shop in Harbour City, found that the pixel of the toy together can be composed of many games like "my world" inside the doll, it caused the attention of my son. While waiting for the time in the store to play for a long time, But only the store only 100 pieces of a box of the sale, I think the play is not high, the price is expensive to dispel the idea of buying.
Then stand up son Ruanmoyingpao promised to help him buy home. In site search, found that Kickstarter has PIXIO above the congregation. This project is 2017 years and 3 months began, has now passed the early bird, can not buy. So FatBrainToys to luck, the result really found selling.
Then quickly orders transport, November 8th orders, delivery in December 5th, December 10th reached the transit company, and after more than 20 days (Christmas transport company, finally in January 7, 2019 explosion). The price is expensive than raise twice as fast, Kickstarter 800 block 169 is a knife, the official sale of the 800 block 274 is a knife, but I see a lot of website to raise the public to buy the user after more than a year have not received the goods, it is a little insurance .
PIXIO Logo, do not know what is, like a snake, but also long 2 corners.
200 block and 800 block of the contest.
The color is the 16 color, buy more than 800 blocks have a box of Full color set.
On the back of the box is can spell out the model, this requires a lot of relatively large block can spell out is more than 800 pieces of special combination.
The 200 block is much simpler, and other details are a lot worse.
2018 red dot design award, feeling a lot of products won the award.
Each color in the 1 block, connected to the rainbow club.
Each color removed, 16 color, each color 50 pieces, a total of 800 pieces, all in here.
Is this love disorderly piled together feeling. A lot of artists is such a creation.
Other, starting from simple start small objects. PIXIO with a few pieces of a few began to build a 4 object can be composed of birds, ducks, multi block words spell something more like some, rooster, parrots, frogs… Can spell it out…
Some of the tools, and the golden key, pistol.
The magnet is used on a graph to fight, can also be a nice animal.
Red and grey seahorses, koalas, turtles and penguins.
There are rhinos, hippos, snails, the tortoise and the lion, goat, pig is pink.
Can also be used to fight LOGO and flag.
Fight your own LOGO and name PIXIO.
I think it is more fun to fight some characters.
Amazon at an English gentleman.
Should be the priest, Rome fighters and Chinese Emperor (feel more like a minister.)
A small band, singer, blowing Sax, and one in the violin.
Japanese woman, Viking warrior, and one is playing the drums.
The 2 model is a beautiful color, poses are also unique, more good-looking.
Gradually complicated, fight longer. Brother and sister go to school, there is also a dog with them.
Captain Tsubasa。
A great mother, crawling with 2 baby, hands still holding a.
Behind are larger combinations, each can only spell out the basic 800 block a. This is the love of rap singer rapper.
More blocks combined animal is more realistic.
The staff together for nearly half an hour, the details is still in place, tie ah, desk keyboard and mouse ah what design is very comprehensive.
Red and yellow color of the Dragon Chinese.
In fact, you can also spell out a lot of things out, leaving behind his spell it slowly.
It reviews (represent personal views):
PIXIO playability is very high, the app in accordance with the drawings, the latter can according to their own imagination. Sometimes LEGO together do not want to remove this, basically a good fight without what can keep the. Not for a long time, no time to fight for drawing PIXIO is a good choice. Of course, if you have the money to buy thousands of tens of thousands of blocks, a big fight scene model is also very interesting. PIXIO may be Lego in the next face the most potential competitors, but there is still a long way. Lego's product line, and the programming of the robot and so on, these are your gap Great things in the future, but who said the quasi?
So in general, PIXIO In the creation of a pixel art, Whether it is for the need to create imaginative kids, still need to ease the pressure of friends, is a rare gift. The magnet with your brain hole, belonged to your world was born.
1, small, have swallowed and lost danger (I have been missing a few black, sucking in what place, it was not to be found.)
2, the price is not cheap. Although the wind and box build based pixel can have numerous creative, but to build a more perfect and even luxury models, the number of requirements is great, the price is more expensive.
3, easy to dirty, white has become black, not careful you will become old very quickly.
4, The strong enough components. PIXIO is the use of magnetic force, If the extension is too long, Because of the gravity and magnetic problems fall, deformation.
The last offer ID photos, thank you for watching!