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The hotel has a wonderful location very close to Baker Street and Regent's Park, in one of London's most attractive areas containing Georgian architecture and quaint gardens. Regent's Park is one of London's most beautiful and expansive public parks, offering a perfect spot for guests to escape the hustle and bustle of the city and have some relaxing time to themselves. However, for those seeking excitement there are a huge amount of nearby attractions to enjoy, including an endless deluge of shops, bars and restaurants, as well as London Zoo and Madame Tussauds, all within walking distance of the hotel. For greater distances, such as travel into the West End, Baker Street tube station is a quick stroll away from the hotel, providing access to the Circle, Hammersmith and City, Metropolitan, Bakerloo and Jubilee lines, linking you to the rest of the capital.
For guests who will be working throughout their stay, free wireless is available throughout the hotel and there is also a free PC to use. Printing, photocopying and fax services are provided. There is also complimentary coffee provided all day in the hotel's comfortable lounge area.
There is a car park 200 metres away from the hotel at standard London rates.
The hotel has various types of room available, including single, double, and twin occupancy. All rooms are ensuite and have simple yet welcoming and attractive modern decor. There are also various helpful amenities provided, including digital television, complimentary wireless internet access, free toiletries, a safe for hire, and tea and coffee making facilities. Daytime room service is available.
Every day from 7.30am to 9.30am the hotel provides a tasty complimentary breakfast. For other meals, there are various fine dining establishments nearby to enjoy.
Young children staying in their parents' room can reside for free. Older children who require extra bedding will be charged at an extra £10 per night. The hotel does not provide cribs or highchairs, and there is no babysitting service on offer.
The hotel has an accessible entrance and one accessible room. There is a lift to all floors, and guide dogs are welcome to stay.


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This Laser soldering machine allows soldering of bared chips on bare metal lead frames. This precision machine performs the stacks to form a "sub-layer" and the automatic laying of the flow with micrometric accuracy.The chip is layed of by a dynamically controlled electromagnetic head in stress and displacement.This head (ISP System patent) controls the melting and solidification. Thus brazing is performed with optimum quality (no void).
The heat input is performed by the laserYAG laser controlled by the machine. This machine is designed for continuous production, high speed and clean room integrates traceability of manufacturing.

subset: commoncrawl


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About Patricia BootsmaIn Pursuit of the Heart of God
Patricia Bootsma, alongside her husband John, has been in full time pastoral ministry with Catch the Fire for over twenty-four years. As spiritual children of John and Carol Arnott, Patricia and John are presently the Sr. Leaders of Catch the Fire Canada. Patricia has been leading houses of prayer for over fifteen years and is an Internationally recognized prophetic voice, having led the Canadian Prophetic Counsel and is on the World Prophetic Team for Catch the Fire.
Patricia seeks to inspire individuals, churches and nations to passionately love Jesus, walk in their God-giving calling, living victorious in identity and destiny. Patricia is the author of three books: Convergence, Raising Burning Hearts and A Lifestyle of Divine Encounters. She and John are the parents of six amazing children and grandparents of five.
Book Patricia Bootsma
If you would like to invite Patricia to speak or minister at your Church, organisation or event, please email firstname.lastname@example.com. We will personally contact you after prayerfully considering your request.
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See your children walk in the fullness of their destiny


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Track Listing
Bad Flower
LolitA☆Strawberry in summer
Bad Flower -Royal Mirrorball Mix- (CD only)
Bad Flower (Instrumental)
LolitA☆Strawberry in summer (Instrumental)
Discord (Instrumental)
1. Bad Flower
As the leading track (and A-side), Bad Flower isn't too much different from "Limited addiction" (or any of its leading songs) since it has a long intro and the overall feel is similar. However, the song really stood out to me since it's darker and has an eerie feel to it. I've also noticed that these girls had some training because they sound great! Especially some of the back girls who really sound powerful in the song. It's just a sweet and badass track from them ^_^!
2. LolitA☆Strawberry in summer
So we learned of this track because of the "Senkou Ongen" from the last single. Though learning that it's another cover of a different SweetS track (their first being "Love like candy floss") is interesting…I wonder if avex is grooming them to be the next SweetS but more badass LOL. I didn't expect the track to open with acoustic guitar (almost made me think it was a country song). Though it speeds up and the song begins. I don't think the track is as strong or as powerful as "Bad Flower" but for being a cover song, it's a different take and I commend them for that.
3. Discord
The other original track on the single starts off on a really cool synth melody and follows through with a pretty nifty 80's new wave feel and guitarwork as well. I actually thought this was pretty nostalgic but also quite strong from them…it's dark (but not like "Bad Flower" dark….just dark tinted). I was surprised they filtered the verses out but everything is clear…different but I like it. Not bad for a B-side, one of their better ones in recent times.
4. Bad Flower -Royal Mirrorball Mix-
Of course the single ends with the signature remix and this time Bad Flower gets it. Unlike the original's dark rock feel, it's given more of an 80's feel (just like "Discord" but I kind of dig this one as well). It gives the vocal melody a different feel for something and the identity of the song shifted but I don't mind it because it's smooth and catchy! I'm starting to see Royal Mirroball really getting better at this now!
After taking a short break to train a little more, TG'S releases Bad Flower and I'm just surprised that this single was really good! Bad Flower might have the same direction as "Limited addiction" but you can't deny that the girls here sound AMAZING. It even follows through with Discord which sounds really nice as well bearing a synth-focused melody. Even the remix is just awesome to listen to and gives Bad Flower a different take. The only track that disappointed me was their SweetS cover…it just wasn't as good nor did it make much of an impact on the single…Yeah this single is a *RECOMMEND*


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Janine Slitheen
|Played By
|2 credits in
1 entry
(this image appears for illustrative purposes only and no attempt is made to supersede any copyright attributed to it)
Janine Slitheen
Florm Rox Fey Fenerill-Slam Slitheen was a member of the criminal family Slitheen.
Coldfire Construction had been taken over by the Slitheen around March 2007. Over the next eighteen months, they planned to build a loop of transducers to absorb all the energy out of the Sun, destroying the Earth, while the Slitheen lay safe inside a hidden room in the technology blocks across Earth. They would use the profits from Earth's remains to buy battlecruisers to destroy Raxacoricofallapatorius' Grand Council. Florm posed as Janine, a Coldfire secretary in London.
In September 2008, after warning Sarah Jane Smith not to interfere, Florm tried to kill her, but Sarah Jane escaped by spraying her perfume at Florm. Later, she teleported to Park Vale School and helped restrain Sarah Jane and her gang from stopping their plan.
The final transducer's capacitor at Park Vale started overloading and the process was reset. When the Slitheen tried to make Luke Smith repair it, Sarah Jane passed him the sonic lipstick, which he used to damage the machinery. Florm was killed trying to teleport away.
Biography from the TARDIS Data Core article, licensed under CC-BY-SA


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The Centre is contemplating to hike retail prices of urea by 10 per cent. The proposal is likely to be put up before the Cabinet Committee on Economic Affairs soon for its consideration.
A panel, led by Finance Minister Pranab Mukherjee, had last year approved a new draft urea policy, which proposed partial freeing of retail prices, allowing companies to hike urea prices by 10 per cent in the first year of policy implementation.
Urea is the only fertilizer that remains under the full price control.
Its current retail price stands at Rs.5,310 a tonne and the Fertilizer Ministry wants to increase the price by 10 per cent to Rs.5,841 a tonne to bring down the subsidy burden.
Mr. Mukherjee, on Wednesday, indicated that the government could take some 'unpopular' decisions in view of the difficult economic situation around the globe and its impact back home. In 2011-12, urea is estimated to have contributed Rs.24,500 crore to the fertilizer subsidy bill.
In fact, a Committee of Secretaries, headed by Planning Commission member Soumitra Choudhary, had proposed complete deregulation of the urea sector. However, the proposal was opposed by the finance, agriculture and fertilizer ministries.
Due to the subsidy burden, urea production has been stagnant at around 21-22 million tonnes and about 7-8 million tonnes are being imported to meet the domestic demand.
The Department of Expenditure, which has opposed freeing the urea prices totally, has supported the proposed 10 per cent hike every year for three years, provided it is rolled out with the planned changes in the New Investment Policy on urea projects.


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The rising demand for water is pinching those living in the flats as tanker operators have renewed their charges, which will push up the cost of water.
Water is now supplied through tankers at a cost that ranges between Rs. 500 and Rs. 1,000, depending on the distance travelled, according to a representative of the Water Tanker Lorries Association. A full tanker load carries 12,000 litres of water while a mini load has 6,000 litres.
The drinking water supply from the main station of the Kerala Water Authority at Aluva with a capacity of 225 million litres a day (mld) is now stretching to 250 mld. But, this meets only half the demand.
The groundwater level was down by at least one to three metres in July this year compared to July last year. And this has put a strain on the borewell and the well water resources. However, the groundwater department in the district, which is yet to find the latest data concerning the level of water, believes the rains in August and September would have pushed up the water level.
However, a 74-flat complex in Tripunithura, where the association had not so far been compelled to buy water from outside for the last one year , has now been buying water for the last 20 days for use in toilets and washing. There is very little seeping in of groundwater that used to provide the flat complex with all their needs. The association has spent about Rs. 20,000 in the last 20 days, paying Rs. 1000 a load. A separate storage for water supplied by Kerala Water Authority takes care of the drinking water needs.
According to the Ground Water Department official, the groundwater in a particular area is shared by a number of people and the share comes down with a rise in population.
At Silver Lawn apartments in Edappally, the association buys 90 to 100 loads every month besides the water authority supply. The Association manages to cut their costs of water during the rainy season by going for rainwater harvesting. The load of water bought from tankers is cut by half during the time. The association was billed at Rs. 800 a load of water till recently, but the tanker suppliers have now raised it to between Rs. 900 and Rs. 1000 a load, said an executive member of the association.


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(Want to get this briefing by email? Here's the sign-up.)
Good morning. Trade retaliation, murmurs of dissent in China and overlooked obituaries. Here's what you need to know:
• President Trump signed off on tough tariffs on steel and aluminum, promising to be "very flexible" after facing a global outcry.
The tariffs have raised fears of retaliation. "Choosing a trade war is a mistaken prescription," China's foreign minister said. (Our senior economics correspondent calls it more of a "skirmish.")
Mr. Trump suggested some allies, including Canada, Mexico and Australia, might be spared. But some nations aren't taking any chances. A group of 11 — including Australia, New Zealand and Japan — signed a broad free trade deal in a direct challenge to Mr. Trump's protectionist agenda.
And our news podcast, "The Daily," examines Mr. Trump's claims about tariffs and trade deficits.
• China is all but certain this weekend to throw out a 1982 rule that has restricted the country's presidents to two five-year terms. The beneficiary, President Xi Jinping, could be in power for life.
To position himself for this moment, our correspondent writes, Mr. Xi "deployed speed, secrecy and intimidation to smother potential opposition inside and outside the party." Here's a look at Mr. Xi's rise to power.
Despite the display of support, murmurings of opposition suggest that Mr. Xi and his subordinates have not fully sold the idea to a broader public. Such stirrings of discontent, our Beijing correspondents write, could build over time.
• The U.S.-China rivalry is increasingly focused on technology, and at the center of that dispute is 5G, shorthand for fifth-generation wireless technology. Here's a look at why companies and countries are fighting for 5G dominance.
Huawei was a bit player in shaping today's cellular networks. Now, as 5G approaches, the Chinese telecom giant, is determined to lead the pack, worrying Washington.
"It's a commercial advantage which parlays itself into a security advantage," one expert said. "Whoever controls the technology knows, intimately, how it was built and where all the doors and buttons are."
• The U.S. secretary of state, Rex Tillerson, is on a five-nation tour of Africa, warning against Chinese investment and wearing a tight smile when asked about President Trump's insulting description of the continent.
Separately, in our latest visual investigation, we show how villages in the Democratic Republic of Congo have been destroyed in recent attacks, forcing 140,000 people to flee.
This Times Insider piece explains how we used active-fire data from NASA and Openstreetmap to bring the atrocities to light.
• "There's a lot to fight for."
That's how one woman summed up the mood on International Women's Day, which was marked with a sense of urgency this year, in the era of #MeToo and Time's Up.
At The Times, we unveiled a project, "Overlooked," that starts with the stories of 15 remarkable women who did not receive a Times obituary.
The names are surprising: Sylvia Plath, Ida B. Wells, the Bollywood legend Madhubala and Qui Jin, China's Joan of Arc.
Our editor discussed how the idea came to her, and a veteran of the Obituaries desk pondered the historical imbalance. Do you have a suggestion for the series?
• Samsung's new flagship smartphone, the Galaxy S9, arrives with a new feature, AR Emoji, that uses a selfie to create an animated avatar. (Our reviewer called it a bit creepy. His avatar is above.) Base price: $720.
• China dominated a list of self-made female billionaires, taking the top four spots and five of the top 10. Zhou Qunfei, founder of Lens Technology, is the world's richest self-made woman, with $9.8 billion.
• How foreign is Broadcom? About 54 percent of its revenue comes from China, but 55 percent of its employees are in North America. Our DealBook column explores that and other issues of the day.
• They call it "alcopop." Coca-Cola plans to introduce a drink in Japan that combines flavored sparkling water with alcohol.
• U.S. stocks were directionless. Here's a snapshot of global markets.
In the News
• A flag dispute will keep the two Koreas from marching together at today's opening ceremony of the Winter Paralympics in Pyeongchang. [BBC]
• Two senior South Korean officials arrived in Washington to brief U.S. officials on their meeting with the North Korean leader Kim Jong-un and the prospect of nuclear talks. [Reuters]
• A former Russian spy, Sergei Skripal, and his daughter are in stable condition after what Britain's home secretary called a "brazen and reckless" attack involving a nerve agent. Officials suspect Russian involvement. [The New York Times]
• Iran prevented the widow of an environmental activist, who the authorities say hanged himself in jail, from leaving. [The New York Times]
• A Danish inventor goes on trial in the murder of the Swedish journalist Kim Wall. The inventor, Peter Madsen, is said to have killed and dismembered Ms. Wall on his submarine. [The New York Times]
• The Trump administration's plan to move the American embassy to Jerusalem has a hitch: The proposed site lies partly in disputed territory. [The New York Times]
• In a study of mice, scientists learned that a type of immune cell is key to making tattoos permanent, knowledge that might help improve methods for removing them. [The New York Times]
Smarter Living
Tips, both new and old, for a more fulfilling life.
• Plan a movie-themed vacation with these tips and see the places your favorite films were shot.
• The Global Entry system is not only for U.S. citizens.
• Recipe of the day: Want an elegant and easy breakfast? Try a "soufflazy."
• Women may be the answer to a severe labor shortage in Japan's construction industry. But workplace problems aren't being helped by a tone-deaf government recruiting campaign. (To attract female welders, for example, a cartoon depicts a woman wearing a pink uniform with a heart-shaped welding mask.)
• "Pachinko," Min Jin Lee's epic novel set in Korea and Japan returns to our paperback trade fiction best-seller list, and Peter Frankopan's "The Silk Roads" is on the paperback nonfiction best-seller list. You can find all of the lists here.
• And from K Road to Indigenous Drive. Our 36 Hours travel series explored Auckland, New Zealand, a city that is laid-back, but also sophisticated. If you go, here's what to pack.
Back Story
President Trump has recently conducted a series of televised conversations with lawmakers as well as with regular citizens.
Forty-one years ago, President Jimmy Carter held a similar unscripted discussion, answering questions from callers during a live radio broadcast from the Oval Office.
"Ask President Carter," moderated by the CBS anchor Walter Cronkite, was broadcast on the afternoon of March 5, 1977. (You can read a transcript here.)
Mr. Cronkite invited listeners to call the White House at 900‐242‐1611. According to CBS, more than 9 million Americans tried.
Some of the dozens of questions that Mr. Carter answered were distinctively of the late 1970s: about the gas tax, Mr. Carter's pardoning of draft dodgers in Vietnam and the early days of the U.S. space shuttle program.
Others — about tax overhaul, drug abuse and relations with Cuba — still resonate today.
One caller asked why members of Congress were getting a $12,000 raise while Mr. Carter was proposing that taxpayers receive a rebate of only $50.
"Gerald, that is a hard question for me to answer," Mr. Carter replied.
"I'm sure it is," the caller said. "That's why I thought I would throw it at you."
The program caught the public's attention ("Saturday Night Live" ran a spoof the next week), but it was never repeated.
Chris Stanford contributed reporting.
Your Morning Briefing is published weekday mornings and updated online. Sign up here to get it by email in the Australian, Asian, European or American morning. You can also receive an Evening Briefing on U.S. weeknights.
And our Australia bureau chief offers a weekly letter adding analysis and conversations with readers.
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The Sports Xchange
By Suzanne Halliburton, The Sports Xchange
Buie's 207 rushing yards lead West Virginia past Texas
AUSTIN, Texas -- Even though West Virginia can boast the best quarterback in the country, it was a Mountaineer running back who devastated Texas on Saturday night.
Andrew Buie ripped the Longhorn defense, rushing 31 times for 207 yards and two touchdowns to help No. 8 West Virginia to a 48-45 win over 11th-ranked Texas in front of 101,851 fans, the largest crowd in Royal-Memorial Stadium history.
West Virginia quarterback Geno Smith, who is considered the leading candidate for the Heisman Trophy, threw for 265 yards and four touchdowns. It wasn't anywhere close to his performance a week ago against Baylor, when he threw for 656 yards and eight touchdowns. But on a cool night in Austin, all Smith needed late in the game was Buie.
On the Mountaineers' final drive, with the WVU offense trying to stretch out a three-point lead, Buie rushed for 63 yards, capping it off with a 5-yard touchdown run. That gave West Virginia a 48-38 lead with 1:18 to play.
The Longhorns scored with 15 seconds remaining on a David Ash throw to Marquise Goodwin. But the onside kick was recovered by West Virginia.
The Mountaineers (5-0, 2-0 Big 12), the newest members of the Big 12 Conference, took a huge early step to claim the league title. They'll come back to the state next week for a date against Texas Tech. The Longhorns (4-1, 1-1) will have to immediately regroup to play archrival Oklahoma in Dallas.
Longhorn defensive coordinator Manny Diaz said he wanted the Mountaineers to run more, thinking such plays would keep the ball out of Smith's hands and negate receivers Tavon Austin and Stedman Bailey. However, Diaz just didn't anticipate the success of the running plays. Before Saturday, Buie's best game came had come last Saturday, when he rushed for 82 yards.
"The last drive was poor, of course," Diaz said.
Buie easily outclassed UT's much more publicized running attack.
"He averaged 6.7 yards per run and that's phenomenal," WVU coach Dana Holgorsen said. "We did commit to the run. That was something we talked about earlier in the week. And there weren't any tricks, either. We just lined up and ran it right at them."
Longhorn tailback Joe Bergeron scored four touchdowns, but he gained just 49 yards.
Ash, who was second in the country behind Smith in passing efficiency, was 22 of 29 for 269 yards and a touchdown. After leading Texas to a comeback victory over Oklahoma State a week ago, Ash faltered late Saturday.
The Longhorns had a chance to take the lead midway through the fourth quarter. Defensive end Alex Okafor had an incredible play, using a bull rush to push into the backfield. He then reached to swat the ball away from Smith. Texas, down 41-38, took over at the West Virginia 12.
But on a third-and-6, Ash momentarily diverted his eyes and wasn't ready for the snap. He was sacked for a 16-yard loss. Longhorn kicker Anthony Fera, a transfer from Penn State playing in his first game at UT, missed a 41-yard field goal.
"It was my fault," Ash said. "That's on me."
West Virginia then put the game away with Buie's runs. Stopping the run has become a problem for UT. Although the Longhorns beat Oklahoma State, Cowboys tailback Joseph Randle rushed for 199 yards.
The first half was offensive, as expected, but Texas was able to lean a bit on its defense to take a 28-27 lead at intermission.
The Longhorns sacked Smith three times and held him to a somewhat pedestrian stat line: 15 of 24 for 168 yards and two touchdowns. He also was sacked three times. Smith was able to regroup on West Virginia's final drive of the second quarter as he directed the Mountaineers on an eight-play drive that culminated in a 41-yard field goal by Tyler Bitancourt.
West Virginia had owned a 21-7 lead early in the second quarter, thanks to Smith's 8-yard touchdown pass to Stedman Bailey, his 40-yard scoring pass to Tavon Austin and Buie's 4-yard run. But the Longhorns quickly erased the deficit with a combination of offense, defense and special teams.
Carrington Byndom partially blocked a punt to give Texas the ball at midfield. The Longhorns, with three quick-hitting running plays, slashed the score to 21-14 on Joe Bergeron's second 2-yard TD run of the half.
On West Virginia's next possession, Texas defensive end Alex Okafor sacked Smith and stripped him of the ball inside the 10-yard line. It was recovered by fellow end Jackson Jeffcoat for the touchdown.
Texas took its first lead with 1:14 remaining in the half when Bergeron scored his third touchdown, from a yard out.
NOTES: Coming into the game, West Virginia had failed to convert on four fourth-down attempts. The Mountaineers were 5 for 5 against Texas. ... Bailey had three touchdowns, giving him 13 for the season and 29 for his career, a school record. ... The Longhorns had two blocked kicks -- one punt and a field goal -- giving them five for the season. ... UT sacked Smith three times. In West Virginia's previous four games, Smith had been sacked four times. ... Okafor forced two Smith fumbles and also blocked a field goal.


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The high journey months are on the area now. Individuals from all parts of society around the world are active searching for the best delivers of atmosphere and terrain traveling, lodgings, getaway bundles to accommodate their ambitions, needs and budgets. A few of them have even began to make their reservations. On this page I would want to focus on one particular truly essential issue, which is a must to consider when scheduling a visit, but ignored by a great number of travelers.
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Let's say, you might have been organizing the journey of your respective goals for a long time; you might have eventually found the ability to help make your long time hopes for an exclusive getaway becoming reality. You might be so fired up! You may have considered of all of the specifics. You have been regularly getting ready for this journey; contemplating, development, shopping . . . spending a lot of time, money, and energy! You happen to be proud of oneself which you have eventually been able to materialize your ideal!
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Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.
Cryptosporidium parvum, a coccidian parasite of the mammalian intestinal epithelium, has been identified as the agent responsible for numerous outbreaks of diarrheal disease (5, 7, 8, 11, 13). In immunocompetent hosts, the disease is self-limiting, but in immunocompromised individuals, the disease can become chronic and debilitating (4, 6, 23). Infection of both humans and animals with C. parvum elicits the development of characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against low-molecular-mass parasite antigens in the 27- and 17-kDa ranges (12, 15-17, 20, 21, 25, 26). Preliminary work has suggested that the presence of antibody against these two immunodominant antigens is associated with protection from symptoms during infection (17). These antigens may be important for invasion of the host cell, since oral administration of monoclonal antibodies against the 27-kDa antigen is capable of reducing the infection load in neonatal mice (2).
In the past, serum antibody responses to C. parvum infection have been tracked by using a crude extract of disrupted oocysts as antigen with either an enzyme-linked immunosorbent assay (ELISA) (28) or a Western blot assay (15, 16, 27). Recent work has shown that the crude oocyst antigen ELISA is not a sensitive means of detecting antibodies against the immunodominant low-molecular-mass C. parvum antigens (16, 17). Although the Western blot assay is quite sensitive (it serves as the reference, or "gold standard," in our work), it suffers from a limited linear range and the antibody levels are difficult to quantitate by densitometry. The assay is also technically challenging and labor intensive, in that a gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel is required for optimal antigen separation. A new assay capable of high sample throughput and easy quantitation is required for planned population-based studies of the risk factors forC. parvum infection in both immunocompetent and immunocompromised persons. We report here the development of ELISA-based techniques for the detection and quantitation of serum IgG antibodies against the 27- and 17-kDa C. parvum antigens.
Preparation and purification of native antigen. C. parvum isolates from Maine and Iowa were maintained by serial passage in Holstein calves (12, 13). Oocysts were isolated from collected feces by discontinuous sucrose gradient centrifugation as described by Arrowood and Sterling (3). A crude antigen supernatant fraction was prepared by sonication and freezing-thawing of purified oocysts followed by centrifugation for 30 min at 24,000 × g, as described elsewhere (14).
A partially purified fraction of the 17- and 27-kDa antigens was prepared from crude antigen by a modification of the Triton X-114 phase partition extraction protocol described by Ko and Thompson (9). Briefly, crude antigen at 1 to 3 mg/ml was mixed with an equal volume of extraction buffer to achieve final concentrations of 20 mM HEPES at pH 7.4, 150 mM NaCl, 2% Triton X-114, 1 mM phenylmethylsulfonyl fluoride, 1 mM p -chloromercuribenzenesulfonic acid, and 5 mM EDTA. After a 30-min incubation at 4°C, insoluble material was removed by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant was frozen for 24 h at −20°C, thawed at 4°C, mixed well, and subjected to two rounds of phase partitioning at 37°C for 10 min. The detergent-rich phase from the final partition was dissolved in 20 mM HEPES and 150 mM NaCl and centrifuged at 12,000 × g for 15 min at 4°C. Partially purified antigens in the collected supernatant were precipitated with 4 volumes of acetone at −20°C overnight. The precipitated proteins were collected by centrifugation at 12,000 × g for 15 min at 4°C and dried at room temperature. The pellet was dissolved either in a nonreducing buffer, for SDS-polyacrylamide gel electrophoresis (10), or in a minimum volume of buffer containing 0.5% SDS and 20 mM HEPES at pH 7.4, for use in ELISA. Both solutions were heated at 95°C for 5 min to insure solubilization.
Crude antigen from the Iowa isolate was used for all of the Western blot analyses of serum samples. Crude antigen from both the Iowa and Maine isolates was used for the development of the Triton X-114 extraction procedures. Triton X-114-purified antigen from the Maine isolate was used for all of the ELISAs.
Recombinant protein expression.The following two deoxyoligonucleotides were designed for the directional cloning of theC. parvum 27-kDa antigen (22) (GenBank accession no. U34390) into the BamHI and EcoRI restriction enzyme sites of the pGEX 4T-2 expression vector (Pharmacia Biotech, Uppsala, Sweden): Cp23-5′ primer (5′-CGC GGA TCC ATG GGT TGT TCA TCA TCA AAG-3′) and Cp23-3′ primer (5′-GCG GAA TTC ATT AGG CAT CAG CTG GCT TG-3′). The 27-kDa-antigen coding sequence was amplified from 260 ng of genomic DNA by using 100 μM concentrations of Cp23-5′ and Cp23-3′ and AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) as directed by the manufacturer. The following amplification protocol was used: 30 cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, followed by 1 cycle of 72°C for 15 min. Plasmids containing inserts were transformed into Escherichia coli HB101 cells (Life Technologies, Frederick, Md.). The sequence of the resulting clone was confirmed by automated DNA sequencing. A recombinant C. parvum 27-kDa antigen–Schistosoma mansoniglutathione- S -transferase (GST) fusion protein was purified from isopropyl-β-d-thiogalactopyranoside (IPTG)-induced cell cultures by using glutathione Sepharose 4B as directed by the manufacturer (GST bulk purification module; Pharmacia Biotech). TheC. parvum protein with an additional GlySer dipeptide at the amino terminus was released by overnight cleavage with thrombin at room temperature and then separated from uncleaved fusion protein and the GST cleavage product by passage over glutathione Sepharose 4B resin. Protein purity was monitored by both SDS-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody against the native 27-kDa antigen (C6B6 [12]) and with serum samples from infected humans.
Western blot assay.Crude oocyst proteins from the Iowa isolate of C. parvum were resolved on gradient SDS-polyacrylamide gels (10 to 22.5% acrylamide) with the buffer system of Laemmli (10). The proteins were electrotransferred onto polyvinylidene difluoride membrane (Immobilon P; Millipore Corp., Bedford, Mass.) and cut into 2-mm-wide strips. Each strip was incubated overnight at 4°C with a 1:100 dilution of serum in phosphate-buffered saline (0.85% NaCl and 10 mM Na2HPO4 at pH 7.2) (PBS) containing 0.3% Tween 20 detergent. Bound antibodies were detected with a biotin-labeled mouse monoclonal antibody against human IgG (clone HP6017; Zymed Laboratories, South San Francisco, Calif.) and alkaline phosphatase-labeled streptavidin (Life Technologies). Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolylphosphate were used to visualize the bound antibodies. The presence or absence of antibodies against the 17- and 27-kDa antigens was determined visually by three independent readers. Eight ambiguous samples (3.3% of the total) were reassayed, and four of these were assayed a third time until a consensus was reached by at least two of the readers.
Western blots that used a mouse monoclonal antibody for visualization of bound antibodies to the 27-kDa antigen (C6B6 [12]) or to the 17-kDa antigen (C6C1 [1]) were developed with a horseradish peroxidase-labeled goat anti-mouse antibody in 0.3% Tween 20-PBS. After incubation for 1 h at room temperature, the antibodies were visualized with diaminobenzidine substrate and H2O2.
ELISA.Antigens diluted in 0.1 M NaHCO3 buffer (pH 9.6) were used to sensitize 96-well plates (Immulon 2 flat-bottom microtiter immunoassay plates; Dynatech Industries, Inc., McLean, Va.) overnight at 4°C. Each well contained 50 μl of either the recombinant 27-kDa antigen (0.2 μg/ml) or the Triton X-114-extracted antigens (0.14 to 0.28 μg/ml) (BCA protein microassay; Pierce Biotechnology Company, Rockford, Ill.). The plates were blocked with 0.3% Tween 20-PBS for 1 h at 4°C. After a series of three washes (subsequent washes were all with 0.05% Tween 20-PBS), 50-μl aliquots of serum diluted 1:50 with wash buffer were added to all wells. All serum samples were tested in duplicate. A twofold serial dilution (1:50 to 1:6,400) of a strong positive control was used to generate a standard curve on each individual plate. One buffer blank and a battery of seven serum samples known by Western blot assay to be negative for C. parvum antibodies were also included on each plate. The plates were incubated for 2 h at room temperature and then washed four times with wash buffer. A biotinylated mouse monoclonal antibody against human IgG (clone HP6017; Zymed Laboratories) (50 μl of a 1:1,000 dilution in wash buffer) was added to each well and incubated for 1 h at room temperature. Following four washes, the wells were filled with alkaline phosphatase-labeled streptavidin (Life Technologies) (50 μl of a 1:500 dilution in wash buffer) and incubated for an additional hour at room temperature. After four washes (the final wash was for 10 min at room temperature), p -nitrophenylphosphate substrate was added in 3 mM MgCl2 and 10% diethanolamine at pH 10, and the color was allowed to develop until the 1:50 positive control wells had reached an absorbance of about 1.5 at 405 nm. Absorbances were measured with a Molecular Devices UVmax kinetic microplate reader. Antibody levels of the unknown samples were assigned a unit value based on the eight-point positive control standard curve with a four-parameter curve fit. The 1:50 dilution of the positive control was arbitrarily assigned a value of 6,400 U. Unknown samples with absorbance values above the standard curve were diluted further and reassayed. Arbitrary unit values were expressed per microliter of serum.
For the crude oocyst antigen ELISA protocol, a modification of the protocol of Ungar et al. (28) was used. Each well contained 50 μl of crude oocyst antigen in bicarbonate buffer at a protein concentration of 2.0 μg/ml. Test serum samples were diluted 1:50 in 0.05% Tween 20-PBS, and the plates were developed as described above with a biotinylated monoclonal anti-IgG antibody and alkaline phosphatase-labeled streptavidin. Quantitation of the test serum samples was done as described above and was based on the same positive control serum dilution.
Serum samples.Banked serum specimens were available for analysis, collected in 1988 from 74 employees at the Centers for Disease Control and Prevention who had no history of foreign travel and no documented exposure to C. parvum (referred to as the nonoutbreak serum set). Banked specimens were also available from individuals known to have been exposed to Cryptosporidiumduring outbreaks of waterborne infection: 129 from the 1987 outbreak in Carrollton, Ga. (7), and 35 from the 1994 outbreak in Walla Walla County, Wash. (5). The 129 serum samples from the Georgia outbreak were divided into two sets. The Georgia "early-outbreak" serum set consisted of 8 samples collected from individuals 2 to 26 days before symptom onset and 25 samples collected from symptomatic individuals at or less than 10 days after the onset of their diarrheal illnesses. Of these 33 samples, 22 were collected from individuals with laboratory-confirmed cases of cryptosporidiosis. The Georgia "late-outbreak" set consisted of 76 samples from patients who met the clinical case definition for cryptosporidiosis (collected between 28 and 66 days after the onset of their diarrheal illness) and 20 samples from asymptomatic individuals who were exposed to the contaminated water supply (collected approximately 4 weeks after the outbreak) (7). Paired samples were available from four symptomatic individuals.
Of the 35 samples in the Washington outbreak serum set, 25 were collected from individuals who met the clinical case definition for infection with C. parvum and 10 were collected from exposed individuals who were asymptomatic or who had mild symptoms that did not meet the case definition (5). Four of the serum donors who met the clinical case definition had Cryptosporidium oocysts detected in their stools. All of the samples in the Washington outbreak set were collected approximately 6 weeks after the peak of the epidemic.
Statistical analysis.ELISA absorbance values for the various sample sets were converted into unit values as described above, and geometric means were calculated. Geometric means were then compared by using a multiple-comparison t test. Blot positives were compared between groups with the Freeman-Tukey multiple-comparison test.
Partial purification of native antigens.During the course of infection with C. parvum, the host's IgG antibody response is directed against two low-molecular-mass sporozoite antigens. Using an optimized gradient SDS-polyacrylamide gel system, we have determined that the 27-kDa-antigen family contains approximately 5 proteins in the 23- to 27-kDa range and the 17-kDa-antigen family contains approximately 10 proteins in the 15- to 17-kDa range (Fig.1) (see below). As shown by the representative Western blots in Fig. 1A, IgG antibodies against the 27- and 17-kDa-antigen families were consistently detected in late-outbreak serum samples collected from the Georgia patients 28 to 66 days after the onset of diarrhea. No other antigens were as consistently recognized by this battery of serum samples. Furthermore, antibodies against these antigens were detected more frequently in the late-outbreak sera than in early-outbreak sera (Fig. 1B) collected from Georgia patients. These observations suggested that antibodies against the two antigens might serve as useful markers for past infection with the parasite.
Both the 17- and the 27-kDa antigens have been detected on the sporozoite surface by immunolocalization studies (12). As a first step in the purification of the antigens and to determine whether they are membrane associated, Triton X-114 phase partition extraction was performed on a crude sonicate of oocysts. As shown in Fig.2, the 17- and 27-kDa antigens recognized by serum samples from an infected patient were partitioned unequally between the two phases following detergent extraction. Of the family of 10 17-kDa antigens recognized by the C6C1 monoclonal antibody, the five largest proteins were completely extracted into the detergent phase, leaving five proteins in the 15-kDa range in the aqueous phase. The proteins were recognized by the monoclonal antibody even after total chemical deglycosylation with anhydrous trifluoromethanesulfonic acid (24). Similarly, of the family of five 27-kDa antigens recognized by the C6B6 monoclonal antibody, the three proteins with the highest apparent molecular masses were extracted into the detergent phase, leaving two lower-molecular-mass proteins in the aqueous phase. These proteins were also recognized by the monoclonal antibody following total chemical deglycosylation (24).
Surprisingly, the majority of the oocyst antigens that were recognized by the antibodies in human postinfection serum remained in the aqueous phase following extraction. Many of these antigens are clearly glycosylated, since cleavage of the carbohydrate epitopes with periodate significantly reduced the Western blot reactivity in the high-molecular-mass range without affecting serum antibody binding to the 17- and 27-kDa antigens (24). An AuroDye-stained blot of the Triton X-114 extraction (Fig. 2) suggested that a total of about 10 to 20 proteins were partitioned into the detergent phase. This represents about 3% of the total protein present in the initial oocyst sonicate (Micro BCA protein assay; Pierce).
Expression of recombinant 27-kDa antigen.The 11.2-kDa coding sequence identified by Perryman et al. (22) as the 27-kDa antigen was cloned into the pGEX expression vector in frame with GST. Induction with IPTG resulted in the appearance of a fusion protein band at an apparent molecular mass of 43 kDa (Fig.3, lanes 2 and 3). In contrast to the native antigen, the fusion protein was not membrane associated in the bacterial lysate nor could it be extracted into Triton X-114 (data not shown). The fusion protein bound to the glutathione Sepharose 4B column and could be eluted with only a few minor contaminants in the 30- to 32-kDa range (Fig. 3, lane 4). Cleavage of the glutathione Sepharose 4B-bound fusion protein with thrombin (Fig. 3, lane 5) resulted in a preparation that contained a single, weakly staining protein band at an apparent molecular mass of 27 kDa (Fig. 3, lane 6). The approximate yield of this purified protein was 0.2 mg (Micro BCA assay) per liter of E. coli culture.
The 27-kDa apparent molecular mass of the purified, thrombin-cleaved recombinant antigen was unexpected, given the length of the coding sequence that was cloned into the expression vector. A monoclonal antibody (C6B6) raised against the native 27-kDa antigen was used in a Western blot of the proteins resolved as shown in Fig. 3 to confirm that the protein band at 27 kDa was indeed the recombinant 27-kDa antigen (data not shown). The fusion protein, like the purified antigen, reacted with the monoclonal antibody, but no reactivity was noted in the lane loaded with GST alone (data not shown). The weak staining with Coomassie blue and the aberrant migration may reflect the high alanine, proline, and acidic-residue content of the cloned protein sequence (25% Ala, 19% Pro, and 20% Asp plus Glu). As with the fusion protein, the purified recombinant antigen could not be extracted into Triton X-114 detergent (data not shown).
Western blot analysis of serum sets.Serum sets that were available for use in the ELISA sensitivity and specificity determinations were assayed for the presence of anti-C. parvum antibodies by using a modification of the Western blot assay of Moss et al. (16). The Western blots shown in Fig. 1are representative of the early- and late-outbreak serum sets available from Carrollton, Ga. Of the early-outbreak serum samples, 33% were positive by immunoblotting for IgG antibodies to the 17-kDa antigen and 52% were positive for antibodies to the 27-kDa antigen (Table1). In contrast, 95 and 99% of the late-outbreak serum samples were positive for antibodies against the 17- and 27-kDa antigens, respectively (Table 1). No significant differences were observed between the Western blots of the serum samples from symptomatic individuals and those from asymptomatic individuals in the Georgia late-outbreak set. The Washington outbreak serum set had frequencies of positive blots similar to those observed for the Georgia late-outbreak set: 80 and 97% were positive for antibodies against the 17- and 27-kDa antigens, respectively (Table 1). As with the Georgia late-outbreak set, no significant differences were observed between the Western blots of the serum from symptomatic and asymptomatic individuals in the Washington set. The nonoutbreak serum set had an intermediate frequency of positives for antibodies against the 17-kDa antigen (62%) and a high frequency of positives for antibodies against the 27-kDa antigen (92%).
ELISA analysis of serum sets.The serum sets were assayed by ELISA with both the Triton X-114-extracted antigens and the recombinant 27-kDa antigen. The mean values, medians, and ranges (in arbitrary units) for the two assays are given in Table2. The Georgia early-outbreak set had the lowest mean reactivity for both the Triton antigen (11.5 U) and the recombinant 27-kDa antigen (102.5 U). The mean ELISA values were significantly lower than those determined for the Georgia nonoutbreak set (P = 0.0001 and 0.0012, respectively) and the Georgia late-outbreak and Washington outbreak sets (P = 0.0001 for all comparisons). The mean Triton antigen and recombinant-27-kDa-antigen ELISA values for the nonoutbreak set were seven- and fourfold higher, respectively, than those for the Georgia early-outbreak set but were still significantly lower than those determined for the Georgia late-outbreak (P = 0.0001 for both assays) and Washington outbreak (P = 0.0104 and 0.0226, respectively) sets.
In contrast to the qualitative assessment of the 27-kDa group in the Western blot assay, the recombinant-27-kDa-antigen ELISA was able to demonstrate a significant difference (P = 0.0243) between symptomatic and asymptomatic members of the Georgia late-outbreak set. Symptomatic individuals had a mean value of 2,015.7 arbitrary units (median = 2,060; range ( R) = 87 to 34,456) compared to 492.6 U for the asymptomatic individuals (median = 940; R = 0 to 4,599). Similar trends were noted for the Triton antigen ELISA (mean values of 454.0 and 203.2 for symptomatic and asymptomatic subsets, respectively) and for the symptomatic and asymptomatic subsets from the Washington outbreak (recombinant-27-kDa-antigen ELISA mean values of 1,854.7 and 565.1; Triton antigen ELISA values of 439.2 and 147.0, respectively), but the differences did not approach statistical significance. Neither ELISA assay detected a significant difference between the Georgia late-outbreak and Washington outbreak sets.
Sensitivity and specificity of ELISAs.Based on an overall comparison of the ELISA responses and the Western blotting results (which served as the reference, or gold standard), positive threshold unit values were chosen for both ELISAs so as to maximize the sensitivity and specificity of the ELISAs relative to the Western blot. These values were generally greater than the cutoff values that would have been assigned based on a mean plus 3 standard deviations of the ELISA responses of the seven Western blot-negative controls that were included on each ELISA plate. Using a positive cutoff of 206 U, the recombinant-27-kDa-antigen ELISA was able to predict 90% of the samples that were positive by blotting for antibodies against the 27-kDa antigen and 92% of the samples that were negative by blotting. Using a positive cutoff of 76 U for the Triton-purified antigen ELISA, the assay was able to correctly identify 90% of those samples that were positive by blotting for antibodies against the 17-kDa antigen and 94% of the samples that were negative by blotting. We do not yet know if these values will be applicable to other serum sets and to other lots of purified and recombinant proteins.
The Triton antigen ELISA response did not correlate well with the 27-kDa-antigen blot response even though the 27-kDa antigen was present in the partially purified preparation used to sensitize the plates. Of 39 samples that were positive by blotting for antibodies against the 27-kDa antigen but negative by blotting for antibodies against the 17-kDa antigen, only 3 (8%) were defined as positive by the Triton-purified-antigen ELISA. Antibodies against the 27-kDa antigen were certainly present in most of these samples, since 30 (77%) were correctly identified as positive when the recombinant-27-kDa-antigen ELISA was used. Thus, the 17-kDa antigen appears to be responsible for most of the response seen in the ELISA with the partially purified Triton fraction.
Others (16, 17) have reported that the blot responses to the 27- and 17-kDa antigens are not well correlated with the ELISA response when a crude oocyst antigen preparation is used to sensitize the plates. In our hands a modified version of the crude antigen ELISA (at a 117-U positive cutoff value chosen to optimize sensitivity and specificity relative to the Western blot) had a 91% sensitivity and a 60% specificity for detection of antibodies against the 17-kDa antigen. At the same unit value cutoff, the crude antigen assay accurately predicted 83% of the samples that were positive by blotting for antibodies against the 27-kDa antigen and 69% of those that were negative. However, of 23 samples that were negative by blotting for antibodies against both the 17- and 27-kDa antigens, 5 (22%) appeared to be positive by the crude antigen ELISA. Only one of these same samples was misclassified by the Triton antigen ELISA, and only two were misclassified by the recombinant-27-kDa-antigen ELISA. Of 42 samples that were positive by blotting for only one antigen, the crude antigen ELISA detected 21 (50%) while the recombinant-27-kDa-antigen and Triton-purified-antigen ELISAs together correctly identified 30 (71%).
ELISAs on paired sera.Both ELISAs were used to monitor changes in antibody levels in paired serum samples from four symptomatic individuals (two of whom had laboratory-confirmed cryptosporidiosis) from the Carrollton, Ga., outbreak. The initial serum samples were collected in the early-outbreak period (0 to 4 days after symptom onset), and the follow-up samples were collected in the late-outbreak period (41 to 68 days after symptom onset). A third sample was available from one individual (74 days after onset). As shown in Fig. 4A, two individuals who were initially negative for IgG antibodies by both ELISAs developed positive responses by the second time point. The seroconversion detected by ELISA for these two individuals was also evident by immunoblotting. IgG antibodies against the 27- and 17-kDa antigens as well as IgA antibodies against the 17-kDa antigen and IgM antibodies against the 27-kDa antigen were present in the late-outbreak serum specimens but absent from the early-outbreak samples (data not shown). One individual was ELISA positive by both assays at all time points and had peak levels of antibody approximately 40 days after onset. The remaining individual, who was initially negative for antibody by Triton antigen ELISA but positive by the recombinant-27-kDa-antigen ELISA, was positive by both assays at the later time point. All of the individuals experienced increases in antibody levels by both assays after the initial sample was collected. The Triton antigen ELISA and the recombinant-27-kDa-antigen ELISA were able to track changes in antibody levels in single individuals and to correctly identify those individuals who had anti-17-kDa and anti-27-kDa antibodies by Western blotting in each instance (blots not shown).
In the original report of the Carrollton, Ga., C. parvum outbreak (7), the IgG antibody seroprevalence was reported to be 76% in symptomatic individuals (n = 68) and 56% in asymptomatic individuals (n = 18) when a crude antigen ELISA was used. Using the Western blot assay, we reexamined this same late-outbreak serum set along with an additional eight samples from symptomatic individuals and two samples from asymptomatic individuals. The seroprevalences for IgG antibodies against the immunodominant 17- and 27-kDa antigens were much higher than anticipated, based upon the earlier ELISA results: 96% for the 17-kDa antigen and 100% for the 27-kDa antigen in the symptomatic individuals and 90 and 95% for these antigens, respectively, in the asymptomatic individuals. Discrepancies between the crude antigen ELISA and the Western blot assay have been described earlier by our laboratory and by others. Moss et al. (16) reported that, of 20 patients with confirmed or probable cryptosporidiosis who were classified as positive for antibodies against the low-molecular-mass antigens by immunoblotting, only 11 showed a significant change in crude antigen ELISA response between initial and follow-up serum samples. Of 14 noninfected individuals who did not meet the case definition for cryptosporidiosis and who were negative by immunoblot, 5 showed a significant change in crude antigen ELISA response between the two time points. Similarly, using a different version of the crude antigen ELISA, Ungar et al. (28) were able to identify only 70% of those individuals who had a 27-kDa-antigen response by Western blotting and 71% of those who were blot negative. Given that the antibody responses to the 27- and 17-kDa C. parvum antigens appear to be characteristic of infection and may be relevant to the development of symptomatic disease (17), a new ELISA with a sensitivity and specificity equivalent to those of Western blotting is required for future work.
A careful analysis of the 17- and 27-kDa antigens demonstrated that they are actually complex families of related proteins, some of which are membrane associated and some of which are soluble. We believe that the 10 antigens in the 17-kDa-antigen family and the 5 in the 27-kDa-antigen family share the same protein backbone but are modified to various extents, perhaps by endopeptidase cleavages and by the addition of carbohydrates and fatty acids. The five soluble antigens in the 15-kDa range and the two soluble antigens in the 23-kDa range probably represent cleaved forms of the 17- and 27-kDa antigens, respectively, that lack carbohydrates and a membrane anchor. The observation that the recombinant form of the 27-kDa antigen runs on SDS-polyacrylamide gels at an apparent molecular mass of 27 kDa but cannot be extracted into Triton X-114 detergent supports this hypothesis. We are currently studying the possibility that both antigens are linked to the membrane via glycosyl phosphatidylinositol anchors.
Since the 17-kDa antigen has not yet been cloned, we exploited the membrane association of the antigens and their extractability into Triton X-114 detergent in order to partially purify a native protein fraction for use in one of the new ELISAs. The ELISA response with the Triton-extracted antigens correlated well (at least 90% sensitivity and specificity) with the presence or absence of antibodies against the 17-kDa antigen, as determined by Western blotting. The 27-kDa antigen present in the Triton fraction apparently does not contribute significantly to the ELISA response (at least at low antibody titers). This observation may reflect the low concentration of this antigen relative to the 17-kDa antigen. Based on the AuroDye-stained blot of the partially purified proteins, the 27-kDa antigen is probably present at a 10- to 20-fold lower concentration than the 17-kDa antigen. At 14 ng of antigen per well in the Triton ELISA, each well would contain less than 1 ng of the 27-kDa antigen compared with 10 ng per well in the recombinant-27-kDa-antigen ELISA.
Our inability to detect the 27-kDa-antigen response with the Triton-extracted-antigen ELISA led to the development of a second ELISA that uses a recombinant protein based on the 27-kDa-antigen sequence of Perryman et al. (22). Despite the absence of carbohydrates on the recombinant protein, the sensitivity and specificity of the recombinant-27-kDa-antigen ELISA (at least 90% compared to the Western blot assay) were similar to those observed for the native 17-kDa antigen in the Triton-extracted-antigen ELISA. This suggests that a significant proportion of the human antibody response is directed towards the protein component of the 27-kDa antigen. This result is consistent with our observation that antibody recognition of the 27- and 17-kDa antigens by Western blotting is retained following carbohydrate cleavage with periodate or anhydrous trifluoromethanesulfonic acid.
Using the new assays, we were able to demonstrate statistically significant differences in ELISA responses between a nonoutbreak set of serum samples and sets of acute- and convalescent-phase sera from the Carrollton, Ga., outbreak. Late-outbreak serum samples from an outbreak in Washington State had ELISA responses similar to those of the Georgia outbreak. This suggests that there were no gross differences in the antigenic makeup of the isolates that were responsible for the two waterborne infection outbreaks, despite the widely separate geographic locations. We have recently examined serum samples from a food-borne infection outbreak with similar results (24). Surprisingly, we were also able to demonstrate a significant difference between the responses of the nonoutbreak serum set and the early-outbreak set. The early-outbreak set from Carrollton had a mean response by both ELISAs unexpectedly lower than that of the set collected contemporaneously from Centers for Disease Control and Prevention employees in nearby Atlanta. Although these serum sets are not representative of populations, it is interesting to speculate that the residents of Carrollton who became ill may have been at increased risk for symptomatic Cryptosporidium infection because of lower overall antibody titers. Human volunteer studies have suggested that the presence of an IgG antibody response to the 17- and 27-kDa antigens is able to prevent illness but not infection upon oocyst challenge (17). If this hypothesis is correct, the exposed but asymptomatic late-outbreak subsets should contain serum both from individuals who were not infected and from individuals who were infected but who never developed overt symptoms. Indeed, the mean ELISA values for these subsets were between those determined for the nonoutbreak set and those determined for the symptomatic late-outbreak subsets, but the observed differences were only statistically significant for one comparison (symptomatic versus asymptomatic Georgia late-outbreak sera by recombinant-27-kDa-antigen ELISA). Unfortunately, we do not know the levels of anti-17- and anti-27-kDa antibodies in any of the exposed, asymptomatic individuals prior to the two outbreaks, since paired serum samples were not available.
With paired serum specimens we have demonstrated that two symptomatic individuals who lacked an IgG response by both of the new ELISAs and the crude antigen ELISA and lacked IgG, IgA, and IgM responses by Western blotting were able to seroconvert to IgG positive by blotting and by ELISA upon C. parvum infection. IgA and IgM responses were also detected by immunoblot assay. In one of these individuals, infection was confirmed by microscopy of stool after acid-fast staining. These results are consistent with our previous experience with immunoblotting (17, 18). In contrast, Okhuysen et al. (19) were able to detect an IgM and an IgA response but not an IgG response upon primary challenge of volunteers with C. parvum. This apparent discrepancy may reflect the fact that Okhuysen et al. used a crude antigen ELISA for their work and based their assignment of seroconversion on an ELISA change of 0.1 optical density unit between a baseline serum and a postchallenge serum. When the crude antigen ELISA was used on the two individuals who seroconverted in our paired serum study, one demonstrated a 0.15-optical density unit change and the other had only a 0.062-unit change (data not shown). Our results suggest that most infected persons develop IgG antibody responses that can be detected with appropriately sensitive and specific assays.
In future studies, we hope to use these new assays to examine sample sets that are representative of the general population and of outbreak populations in order to determine basal IgG antibody levels and to relate changes in antibody levels to rates ofCryptosporidium exposure and infection.
We are indebted to William MacKenzie for his critical comments and helpful suggestions on the manuscript. We would also like to recognize the assistance provided by Jan Mead and Maria-Teresa Bonafonte during the early stages of this work.
- Received 11 November 1998.
- Returned for modification 2 February 1999.
- Accepted 10 February 1999.
- Copyright © 1999 American Society for Microbiology


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As a Financial Analyst for StreetLights Residential, Terence Huffines is responsible for pro forma underwriting and assisting in acquisitions, dispositions, capital markets, and asset management. His responsibilities also include coordinating RFPs, due diligence, and investor reporting.
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Cricketer Chris Gayle is at it yet again, igniting new controversy with disparaging comments directed towards teammates and cricket legends alike in his autobiography, Six Machine. Nothing like a little innuendo in a book's title to tip the nature of the literature betwixt the covers.
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Andrew Flintoff was also targeted by some entertaining Gayle vitriol for Flintoff's admission of having been run off while batting for England after having taken the ultimate performance-enhancing drug, Viagra, the night before the match.


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ONMA has everything about Internet Advertising
If you are in search of a top-quality web marketing expert in Switzerland, Austria and Germany, you are fortunate to have roughly 10,000 possibilities. And if you look into this industry on a more expansive reach, you will notice over a hundred thousand recognized online marketing consultants earthwide. Our clients in Zirndorf are cheerful to get wonderful quantity of leads that actually purchase their merchandise. That is why our experts is undefeated regardless of the hard competition between web advertising agencies. Because ONMA use this strategy not just only to online marketing campaigns but also to a wide selection of other advertising tasks, we were able to make a name for ourselves. In order for our customers to achieve their specific marketing targets, we meticulously designed fantastic service packages as choices to take.
Proficient Internet Store and Website Designing Services in Zirndorf
Because we value honesty, each one of us is devoted to offer you a high level of honesty when it comes to our charges and services rendered. Just contact us if you need a site for your office. We are so proud in our ability to offer our partners in Zirndorf an attention grabbing and responsive website as we are dedicated to high quality of outputs in the line of website development and online advertising. Being scarce on the budget doesnt necessarily imply that company owners will miss the possibility to stand-out among the competitors. We can build your business website for as low as 499 and your e-commerce shop for just 1000!
Skilled Web Programming Services for low Prices
Different from other companies that limit themselves by merely working with small jobs, ONMA accepts all tasks, big and small! Because of the sensitivity of particular tasks, some assignments require countless working hours. If thats how you identify your next web development projects, we can spend more hours on accomplishing your tasks. Our packages begin with a very cheap price of 30 per hour. Our price are one of the most affordable in Zirndorf. Your business rivals are driven these days to influence Google, Bing and Yahoo, thats why it is definitely never adequate to depend on your web site or online store exclusively. For you to have targeted traffic, website administrators have to focus on search engine optimization, online content creation and reputation management not only on creating and setting up the web site or e-commerce website.
Online Articles for A Discounted Price? We Will Create it for You!
Your web business attainments relies on the mode you popularize your capacity and products by using persuasive website articles. Because of a long time of practice, we gained great expertise regarding these diverse fields that we are able to offer in Zirndorf special service for extremely decently pricing which can barely offer by our business rivalry.
Decently Priced Proficient Articles – 1 Euro for DIN-A4-Page Text – Get Limitless Articles Right Now!
Looking to buy hundreds of texts but not sure on how it would would it imply? You may definitely benefit on our rock bottom prices for 'handwritten' article. You can have one page DIN-A4 page article for just 1! Due to this immense discounted price, your web site will never again be inadequate of excellent articles permanently! Our text writers are excited to help you with meta-tag posting, internal link creation, short codes, call-to-action-buttons, and others. We are also capable to enhance existing contents and give it a fresh appeal!
Highly Proficient Content in Zirndorf
Most of the contents available on the internet are unverifiable so we do not resort to reuse online texts. Instead, we conduct extensive study and affirm facts by means of professional conversations so we are able to ensure that we write out is strictly dependable. Highly skilled articles regardless of niche or subject are capable to be definitely created by means our researched unique strategies. Compared to some writing agencies in Zirndorf, our prices starts for as minimum as 60 per DIN-A4-page, a truly low pricing considering the processes implemented in this in depth kind of service.
Photography Specialist / Videographer in Zirndorf
Are you at Zirndorf and is currently planning to hold unforgettable events? Our agency is specializing in pictorials and is ready to serve you with your professional photography jobs. Memorable events are worth celebrating. ONMA is also competent of recording professional video clips, video editing and digital animations effect.
Digital Identity Managementconsultants for Your Business
Permanent success of any web company will respond on the manner your identity was accepted by your relevant group of people. If your company is not making any effort to build your online impression, then you must be left out by your business rivals. Our team are capable to build your company internet identity as an aspect of our brand administration services.
Zirndorf Reputation Services and Public Relation Service
What do we mean by reputation and how does it affect your branding? Reputation is the same as the manner target your officemates talk about you particularly when you're not listening. Your Reputation cause your potential buyers use or not use your product or services. Since your business competitors in Zirndorf do anything to be the best, your business reputation needs to be regularly progressing. Our team has the skills and ability to influence reputation in a good way, so that your web reputation in Zirndorf continually improves web product evaluations on your goods and offered services are able to alter your web brand in a constructive or distructive way. ONMA are able to render you limitless number of prominent product evaluations and comments. We are able to also develop and pass on great PRs as well as published articles to various articles sharing sites. We truly realize how social media operates and how could it impact your business impression. Facebook, Twitter, Instagram, YouTube and Google Plus marketing has long been a an integral part of our methods.
Your Web Advertising Scheme Must Consider SEO
Talking about internet marketing elements, search engine optimization is the most problematic. But because getting your website listed at searches will supply you infinite possible advantages, you may like to accept the challenge of providing Google, Bing and Yahoo what it need for you to on showing up the results. As we all know, there is no exact rule that deals with search engine optimization. Majority of the search engine advertising consultancy firms that are in your community are competent to rank sites in a very period of time. But because Google, Bing and Yahoo improves its parameters regularly, that sites got irreparably hit by Google, Bing and Yahoo. Our search engine marketing assistance is penalty proof! We are qualified to promote website utilizing 100% legal scheme. We assure that your rankings will supply you long lasting benefits.
Relationship between Backlink Creation and Search Engine Marketing
ONMA renders a limited discount rates on our charges regarding our all-inclusive packages on our web setup, article creation and search engine optimization bundles, specially for product managers that simply don't have time in managing their own projects. For as minimum as 99 EUR per month, we will supply you 150 German website website backlinks that are cautiously handpicked to make sure that you will get the best quality website backlinks possible! As we all know, backlinks are crucial for search engine optimization because search engines render more value to domain that have a high quantity of quality website backlinks. We promise to provide you that!
Ranking value as an important measure in defeating against your business rivals
Search engine optimization schemes such as online press releases, Google News, jobs board posting, social networking, social bookmarks and publishing contents to article sharing websites are recognized to be several of the criteria in maximizing your site ranking value. We are capable to effectively do online PR these strategies along with other marketing methodologies to ensure that your website is getting sufficient traffic to obtain awareness. By this manner, we are qualified to generate increasing traffic based on search engines and social networks to an increased fashion that no other search engine advertising professionals can provide you in Zirndorf with.
ONMA has the Capability to Assure Successful Campaigns
Some clients need the best possible outcome for their SEO projects while hiring a second-rate SEO agency in Zirndorf. If you need dependable search result position, contact us and lets get it done. When we profess that we areprofessionals in search engine optimization, we are proud that we are masters at it. Even Google.com has documented our team to be one of their partner consultants. Another type of SEO that has been very common At the moment is the local search engine marketing. Local SEO is the method of optimizing your product website so that you are capable to gain enhanced local search rankings. We are one of theSEO consultants that invented local search engine advertising campaigns. Since comapanies in Zirndorf wants to have service guarantee to make it absolutely sure that they will get the top-quality service out of their limited amount of money, we will give you this limited promotion: if in case half of your chosen keyphrases didn't make it to show up at Googles page one, you will only need to pay half of your monthly bill!
Google Adwords as a Prompt Source for Highly Targeted Traffic
Added fountain of targeted clicks hits is the Google Adwords. Are you in search of an Google Adwords consultancy service that will assist you manage your advertisement campaigns? We are dignified to say that Our team is proclaimed as one of the best Google Adwords experts in Germany. If we are capable to deliver fantastic service bundles to German clients, we are assured that we are capable to work for clients in each city! Our capability to manage customer assistance in the most professional manner and our ultra-low charges makes our company apart from other web advertising agencies.
More Data About Zirndorf
Talking about statistics, a considerable number of the 25.957 resides in Zirndorf, that is situated in Germany from Europe. Acquiring the average of the citizens established in Zirndorf to the world population per habitant, you will get plenty businesses for Zirndorf. Some may believe that getting a professional advertising consultant is a waste of funds. But that line of thinking was applicable a long time ago! Since the struggle is so heavy and anyone are anxious to dominate, employing a professional internet advertising specialists is definitely a requisite. Why are we so certain that we are confident of pushing your online impression and brand? We are a registered Google partner and search engine optimization is our strong point. Our SEO and web marketing packages are all created from Deutschland, a country that emphasizes quality more than everything We does not restrain this fantastic assistance to European companies. We are contented to serve business partners in Warstein also. To see is to believe! Let's work hand in hand on your future assignments and see how achievements is a reality for us!
ONMA: Your Zirndorf Reliable Yet Inexpensive Internet Advertising Specialists
Our over fifteen years of practical knowledge has made us one of the biggest name in the online advertising business. We can show off to you more than a thousand of flourishing tasks listed in our record book. What is more, our being an official Google partner adviser gave us numerous recognition on the line of internet marketing. We provide cheap solutions in anything with regards about internet advertising like web design, article generation and marketing, social accounts management and sophisticated SEO. We also make practiced utilization of AdWords in order improve your profit. While you are qualified to create great videos, we are competent to support you advertise your videos by the use of our video advertising services.
To wrap it up, we have integrate each and every online advertising services that business in Zirndorf will require all in all in one shop! We are proficiently giving our loyal clients professional consultancy assistance, information analysis, world class content creation, website development and search engine optimization. Additionally, we are competent to ensure highest profit for every penny that you expend on advertisements!
We realize your online marketing visions
Whether we solve your online marketing problems or realize your internet visions. Were burning with curiosity, which idea you have. Lets get together and lets develop a professional, creative concept so you can realize your idea. Step by step. ONMA – Were delivering sucess in online marketing.
ONMA Online Marketing International
Mail: email@example.com
Tel.: 0044 2038 8500 27


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Privacy Policy
This privacy policy sets out how we use and protect any information that you give us when you use this website.
We are committed to ensuring that your privacy is protected. Should we ask you to provide certain information by which you can be identified when using this website, then you can be assured that it will only be used in accordance with this privacy statement. Bedhead respects your privacy and the confidentiality of information provided by you, and reiterate that Bedhead, its subsidiaries and director-related parties adhere to the national privacy principles.
This Privacy Policy has been developed to inform you of how we take steps to both respect your privacy and to protect the personal information that we collect, use, disclose and keep secure. It also covers how we make the personal information that we hold available for access to and correction by you and has been drafted in accordance with our obligations under the Privacy Act 1988 (Cth) and the Privacy Amendment (Enhancing Privacy Protection) Act 2012 which includes the Australian Privacy Principles (collectively, the Privacy Act).
Personal Information when used in this Privacy Policy has the meaning given to it in the Privacy Act but it generally means any information that can be used to personally identify you. For example, your name, gender, contact details (including phone numbers and email addresses), age and possibly financial information, including your credit card information. The personal information may be collected through our website, over the phone, or otherwise.
Please take a few minutes to review this Privacy Policy. By using our website and our online services, you are consenting to the collection, use and disclosure of your personal information as set forth in this Privacy Policy.
We may change this policy from time to time by updating this page. You should check this page from time to time to ensure that you are happy with any changes.
What personal information about customers do we collect?
The information we learn from customers helps us personalise and continually improve your shopping experience at Bedhead. We will, from time to time, receive and store personal information you enter on our website or give to us in other forms. You may provide basic information such as your name, phone number, address, and email address to enable us to send information or process your product order. We may also collect additional information at other times, including but not limited to, when you provide feedback, change your content or email preferences, respond to a survey, or communicate with Bedhead's customer support or enquiries department. You may change your details at any time by advising us in writing via email.
We may collect the following information:
• name and job title
• contact information including email address
• demographic information such as postcode, preferences and interests
• other information relevant to customer surveys and/or offers
Automatic Information:
We also receive and store certain types of information whenever else you may interact with us. For example, like many web sites, we use "cookies," and we obtain certain types of information when your web browser accesses Bedhead. Scroll to the bottom of this page to see examples of the information we receive. A number of companies offer utilities designed to help you visit web sites anonymously. Although we will not be able to provide you with a personalised experience at Bedhead if we cannot recognise you, we want you to be aware that these tools exist.
E-mail Communications:
To help us make e-mails more useful and interesting, we often receive a confirmation when you open e-mail from Bedhead if your computer supports such capabilities. Bedhead may use personal information collected from you to provide you with information and updates regarding our website and changes to the law from time to time. Bedhead may also make you aware of their new and additional products and services from time to time. This email newsletter service is free. You may change your email details at any time by advising us in writing via email. If you do not want to receive e-mail or other mail from us, please adjust your account to reflect this by selecting "Unsubscribe" in the Newsletter menu.
What we do with the information we gather?
We require this information to understand your needs and provide you with a better service, and in particular for the following reasons:
• Internal record keeping.
• We may use the information to improve our products and services.
• We may periodically send promotional emails about new products, special offers or other information which we think you may find interesting using the email address which you have provided.
• From time to time, we may also use your information to contact you for market research purposes. We may contact you by email, phone, fax or mail. We may use the information to customise the website according to your interests.
We are committed to ensuring that your information is secure. In order to prevent unauthorised access or disclosure, we have put in place suitable physical, electronic and managerial procedures to safeguard and secure the information we collect online.
Bedhead strives to ensure the security, integrity and privacy of personal information submitted to our site and updates its security measures to keep up with current technologies. All information that Bedhead receives from its customers is protected from unauthorised use or access by our secure servers and all customer information is encrypted before it is sent to us. Credit/Debit card information is not stored by us on our servers. We take reasonable precautions to ensure that the personal information we collect, use and disclose is complete, relevant and up-to-date.
However, the accuracy of that information depends to a large extent on the information you provide. That's why we recommend that you:
• let us know if there are any errors in your personal information; and
• keep us up-to-date with changes to your personal information such as your name or address.
We will take reasonable steps to protect the personal information from misuse, interference, loss and unauthorised access or disclosure. This may include taking reasonable steps to destroy or permanently de-identify personal information once it is no longer needed for any purpose for which the information may be used or disclosed.
We will not attempt to match de-identified or anonymous data collected through surveys or such online devices as "cookies", with information identifying an individual, without the consent of the relevant individual.
We require our employees and contractors to perform their duties in a manner that is consistent with our legal responsibilities in relation to privacy, including those in this Privacy Policy. We note that we take reasonable steps to ensure that personal information is only accessible by people who have a genuine "need to know" as well as "right to know" particularly in the carrying out of our services to you.
How we use cookies
A cookie is a small file which asks permission to be placed on your computer's hard drive. Once you agree, the file is added and the cookie helps analyse web traffic or lets you know when you visit a particular site. Cookies allow web applications to respond to you as an individual. The web application can tailor its operations to your needs, likes and dislikes by gathering and remembering information about your preferences.
We use traffic log cookies to identify which pages are being used. This helps us analyse data about webpage traffic and improve our website in order to tailor it to customer needs. We only use this information for statistical analysis purposes and then the data is removed from the system.
Overall, cookies help us provide you with a better website by enabling us to monitor which pages you find useful and which you do not. A cookie in no way gives us access to your computer or any information about you, other than the data you choose to share with us.
You can choose to accept or decline cookies. Most web browsers automatically accept cookies, but you can usually modify your browser setting to decline cookies if you prefer. This may prevent you from taking full advantage of the website.
Links to other websites
Our website may contain links to other websites of interest. However, once you have used these links to leave our site, you should note that we do not have any control over that other website. Therefore, we cannot be responsible for the protection and privacy of any information which you provide whilst visiting such sites and such sites are not governed by this privacy statement. You should exercise caution and look at the privacy statement applicable to the website in question.
Controlling your personal information
You may choose to restrict the collection or use of your personal information in the following ways:
• whenever you are asked to fill in a form on the website, look for the box that you can click to indicate that you do not want the information to be used by anybody for direct marketing purposes
• if you have previously agreed to us using your personal information for direct marketing purposes, you may change your mind at any time by writing to or emailing us.
We will not sell, distribute or lease your personal information to third parties unless we have your permission or are required by law to do so. We may use your personal information to send you promotional information about third parties which we think you may find interesting if you tell us that you wish this to happen.
Personal information is only supplied to a third party supplier when it is required for delivery of goods or services which you have purchased, or for the purposes of audit, liaison with and reporting to regulators. We may from time to time need to disclose certain information, which may include your personal information, to comply with a legal requirement, such as a law, regulation, court order, subpoena, or warrant in the course of a legal proceeding or in response to a law enforcement agency request.
Bedhead may also use your personal information to protect the copyright, trademarks, legal rights, property or safety of Bedhead, its customers or third parties. If there is a change of control in one of our businesses (whether by merger, sale, or otherwise), or a sale or transfer of its assets, customer information, which may include your personal details, could be disclosed to a potential purchaser under an agreement to maintain confidentiality, or could be sold or transferred as part of that transaction. We would only disclose your information in good faith and where required by any of the above circumstances.
If you believe that any information, we are holding on you is incorrect or incomplete, please write to or email us as soon as possible at the above address. We will promptly correct any information found to be incorrect.
Changes to Privacy Policy
To ensure our privacy policy remains current, this policy is subject to change. We may modify this policy at any time, in our sole discretion and all modifications will be effective immediately upon our posting of the modifications on this site. Please return periodically to review our privacy policy.
The APPs place more stringent obligations on APP entities when they handle 'sensitive information'. Sensitive information is a type of personal information and includes information about an individual's:
• health (including predictive genetic information)
• racial or ethnic origin
• political opinions
• membership of a political association, professional or trade association or trade union
• religious beliefs or affiliations
• philosophical beliefs
• sexual orientation or practices
• criminal record
• biometric information that is to be used for certain purposes
• biometric templates.


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Electronic elements on new album by Shawn McDonald
OHIO-based singer/songwriter Shawn McDonald has the followup to 2013's unique live-to-tape re-recorded collection 'The Analog Sessions' released on 15th April. Speaking about the 'Brave' album on Sparrow McDonald said, "I've been a singer/songwriter for close to 16 years now and have seen so many elements progress within my music. I may have started with just an acoustic guitar and some simple stories, but I've since sought to create art with meaning, messages, thought and depth. This record feels like the deepest I've ever dived in that direction, all while adding considerably to my sound, which is why I called the record 'Brave'. I feel like I'm making art with the same intentions, but there is definitely an increasingly noticeable electronic element on top of the acoustic foundation." The project was produced by Chris Stevens (TobyMac, Carrie Underwood), Jamie Kenney (Marc Broussard, Erin McCarley) and David Garcia (Group 1 Crew, Britt Nicole).The opinions expressed in this article are not necessarily those held by Cross Rhythms. Any expressed views were accurate at the time of publishing but may or may not reflect the views of the individuals concerned at a later date.