text:
Skip to comments.Ana Navarro: Some Conservatives Oppose Amnesty Because They're 'Racist'
Posted on 01/30/2014 8:39:38 PM PST by 2ndDivisionVet
So-called Republican Ana Navarro told a left-of-center outlet this week that she believes some people on the right oppose amnesty because they are racist.
Navarro, the former "strategist" to liberal Republicans like John McCain and Jon Huntsman, has been elevating her profile among the mainstream press in the so-called "Acela corridor" by bashing conservatives, including former Alaska Gov. Sarah Palin and Sen. Ted Cruz (R-TX).
Though there is often hardly a difference between the mainstream media and left-of-center outlets like Talking Points Memo, the Huffington Post, or BuzzFeed, Navarro now wants to be adored by them as well. There is no easier way to do that than to imply that those who are opposed to amnesty are racists, even though the primary argument against amnesty has been that it would lower the wages of American workers of all races, ethnicities, and backgrounds--particularly those in black and Hispanic communities who have suffered in President Barack Obama's economy.
Navarro said that there are "certainly" some "racist people" on the right who oppose amnesty primarily because of race. She said that she wants to "think its a minority and thats not whats going to decide the immigration debate.(continued)
(Excerpt) Read more at breitbart.com ...
These people have a simple goal. Destroy the middle class, which is the uniqueness of America, and turn America into a third world nation that they can rule.
That's all it really boils down to.
Are you and Vidkun dating? Isn't he a bit old (and dead) for you?
Oh YES, Mr. Obama, since the country isn't going down the crapper fast enough, let us help you add 11 to 20 MILLION new DemonRAT voters to the rolls. Thank you, sir, may we have another?
I always enjoy watching the enemy reveal themselves. It adds more enjoyment to the hunt - - - .
#:o<> aaaaaaaaaaaaAAAAAAARHLLLOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOooooooooooooooooooooooooooooooooooooooooooooooooooooo - - - - - - - - - - - - - - - -
I don't like criminals of any race.
This silly argument is getting really old.
and some liberals are for amnesty because they just want more democrat votes.
and some liberals are for amnesty because they want another demographic to balkanize and exploit.
and some liberals are for amnesty because they are in favor of anything they believe will undermine and destroy the usa as it has historically been.
McCain? Huntsman?? CNN??? Republican???? Hardly.
She is cute though.
It's racist to save what jobs there are for the millions of unemployed people in this country by not letting millions more unemployed people in. Got it. Amazing, I didn't see that. I must be a racist.
What part of ILLEGAL do these anti-white Hispandering racists not understand?
is it three card monte or just a she'll game
Every single time I've seen this squatty little gal on CNN, she's trash-talking conservatives/tea-party/Palin/Cruz, and simultaneously laughing and yukking it up with her Dem counterparts.
People keep saying it, and it's true. The conservative right truly is at war with the quisling RINO party.
First, we were just "wacko birds" that need a good "punch in the nose". Now, they're openly calling us racists.
I'm just wondering when the RINO party is going to drop the charade and link arms with Obama.
Of course people who are racist because they object to illegal aliens coming here...
like Xavier Brown...
Xavier was a 3 year old black boy killed with his parents by a drunk illegal alien who smashed into their car at a stop light in downtown Dallas...at noon time...
Some Hispanic "Republicans" are pimping amnesty because they are La Raza racists who want to take control of the country. The way to do that is to load the electorate with Hispanics. Two can play that game Ana! You want to talk about "global warming" next?
Count me as one and I am not ashamed to say it. Illegals come here and DO NOT assimilate. They bring their culture (i.e., race, La Raza anyone?) with them and don't give two squats about my country. As well, inner-city blacks act like tribes instead of Americans. White trash do the same damn thing. Should we talk about "Chinatowns' across the USA? What about Dearbornistan in Michigan? You'll have to forgive me that I consider my "race" as being a doer. Someone who does not want the government providing for me from cradle to grave. Someone who does not want his compensation for work provided raped for redistribution by federal and state taxes. i am someone who is not the new race but the original; an INDIVIDUAL with inherit rights not "given" to me, but what I am now forced to defend not withstanding a feral government that wants nothing more than to remove my ability to do so. All other's are parasites and leeches and THAT is what we should start calling them by until they stop voting for politicians that squander their communities abilities to earn by way of weakening their will to stand on their own damn two feet and get off their lazy asses.
I'm quite sure plenty of democrats are in favor of amnesty because they're a bunch of racist effers. let's start with "la raza" for example which literally translates to "the race".
I applied to the IOP because I goofed-off and partied too much during high school. I couldn't have gotten into Harvard at the point, - - - "
Yup, just what the District of Corruption needs, another Liberal Party, party-girl.
- See more at: http://www.iop.harvard.edu/blog/meet-fall-fellows-ana-navarro#sthash.nTi1ir25.dpuf
There is one simple answer to race baiting. Read this ten times so you can recite it whenever someone accuses a certain category of people of being racists:
"As horrible as being a racist is, there is one type of person who is even more despicable, and that is one who believes he or she has the moral authority to look into the hearts of entire groups of people and delare them racist in an attempt to excise paltry political gain. Those who prejudge others in this way truly represent the crudest, lowest form of racism."
Ana Navarro
1/2 my twitter feed flooded by ppl pissed at amnesty & 1/2 by ppl pissed at creation of 2nd class citizens. Sounds like perfect compromise!
4:17pm - 30 Jan 14
Undocumented worker = Illegal alien.
Is she related to Rubin Navarro?
I'm pretty sure I'm become racist against republicans at this point ;/
To condem LEGAL immigrants to SLAVE wages is RACIST.
That is what happens when you flood the market with cheap labor.
That will be the result of granting amnesty to 20 MILLION ILLEGAL IMMIGRANTS
Twenty million? Yeah, maybe just in California!
Gee...that's original, Ana.
These people are projecting their own racial prejudices and are only pushing amnesty because of their own "white guilt."
If our country was being overrun by blond, blue eyed Russian illegal aliens who were gaming the welfare system, then I'd be just as pissed about them as I am about the ones coming from south of the border.
If being a loyal American means that I get called a racist, then I am a proud racist.
Piss on these race baiting America haters.
when they stop being racist to me I'll stop thinking racist about them . Until then , what goes around comes around .
McCain says they are friends,
she started on CNN as political analysis.
She hates conservatives and does a very weak act as playing Republican opposition to Dems, which is why she gets on. .
McCain says they are friends,
she started on CNN as political analysis.
She hates conservatives and does a very weak act as playing Republican opposition to Dems, which is why she gets on. .
Some La Raza activists refuse to get real jobs.
Obviously there is a race angle when an overweight hispanic tells non Hispanics they are lazy and won't do real work.
The MSM loves fake Republicans who come on and attack conservatives
For these Commie Alinsky practicioners, there is no argument against their agenda. If 2 + 2 = 4, they would say that was a racist conclusion. They don't operate on logic, they operate on demonization of the opponent. Remember, the ends justifies the means.
excuse my spelling.
practicioners = practitioners (the correct spelling)
A case where something incorrect equals something correct.
I'm gonna start calling myself a "Democratic strategist".
good idea
What Republican does this woman work for? How do we primary them?
I should call into one of these mainstream media programs, call myself a "lifelong moderate Democrat", then rag on the air about how deranged the RATs are.
I don't know who or what (besides MSNBC) is signing her checks at this time but she's worked for McCain, W Bush, Jeb Bush, Jon Hunstman. As well as the government of Nicaragua.
Very close to McCain AND the Bushes, what a gal.
She tweets way too damn much.
Any candidate that she actively helps should get the stink eye.
Just plain wrong.
(a) People on the right "KNOW" that people like Ana Navarro want amnesty b/c Navarro and her ilk despise America and her remarks show she loathes Americans.
(b) People on the right "KNOW" that amnesty is a diabolical plan to undermine US national security.
REFERENCE Quoting Miguel Perez of Cal State-Northridges MEChA chapter: The ultimate MECha ideology is the liberation of Aztlan (taking back the SW). Communism would be closest [to it]. Once Aztlan is established, ethnic cleansing (of Americans) would commence: Non-Chicanos (Americans) would have to be expelled opposition groups (that's us) would be quashed because latinos have to keep power (over the gringos they loathe).
Taxpayers demand answers WRT Ana Navarro's activities:
<><> Is she now, or has she ever been, a member of violent reconquista groups?
<><> how many govt checks is she cashing?
<><> did she falsify govt documents to get a Senate job?
<><> does she pay taxes on all of her income?
<><> does she have a valid SS number?
<><> does she have a valid driver's license?
<><> does she receive payment(s) from foreign entities?
<><> does she receive payments from tax-exempt latino groups?
<><> is she listed as a lobbyist for other latino organizations?
<><> how many identities is she using?
<><> was she claimed on multiple EITC refund forms?
<><> was she claimed on EITC forms by any other illegals?
<><> who pays her rent?
<><> does she have a rap sheet here? In a foreign land?
<><> did she falsify official govt forms to get a Senate job?
IRS toll free 1-800--829-1040
FBI TIPS PAGE https://tips.fbi.gov/ ========================================================
CONTACT CONG STEVE KING (R-Iowa) for assistance:
Washington DC Office
2210 Rayburn Office Building
Washington, DC 20515
Phone: 202.225.4426
Fax: 202.225.3193 ]
.....elevated the parasites to the USSC, cabinet positions, admin jobs, absolved them of all culpability for falsifying official documents, welfare fraud, EITC fraud, murders, border violations, arms trafficking, drug trafficking. He stopped deportations, gave em jobs taken away from Americans, free healthcare, education, financed every other whim w/ tax dollars.
LOOK AT EM--- OBAMA AND US LAWMAKERS IN LOCKSTEP---COLLUDING TO EFFECT A THIRD WORLD COUPE D'ETAT. The not so well-hidden amnesty agenda seems to have specific goals:
<><> (1) undermining US ntl security,
<><> (2) aiding and abetting crimes against Americans,
<><> (3) financing Third World political goals w/ US tax dollars,
<><> (4) suppressing dissent.
Heavens to Berchtesgarden.....IS A "PUTSCH" IN THE OFFING?
A chubby couch cow from Hell in the making... Udderly Disgusting!
Anyone desirous of living in a country full of Mexicans could simply move to Mexico !
Ditto re <insert transnational shitehole here>.
I liked your post, but how do you realy feel?
Tell me again how the Republicans are going to sweep to victory in the House and Senate later this year.
Given this garbage, I certainly won't be voting for any of them.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


text:
" To Wozniak, it showed a fundamental difference in their characters.Ugg Kids Bailey Bow.Ugg Classic Short Chestnut "My college fund came from my dad paying $50 for a Ford Falcon or some other beat-up car that didn't run, working on it for a few weeks, and selling it for $250—and not telling the IRS. It emphasized rationality and functionality by employing clean lines and forms. The modernist International Style championed by the Bauhaus taught that design should be simple, yet have an expressive spirit. I was fifteen, and then began using pot regularly. Deckers is headquartered in Goleta, California1 with an e-commerce division located in Flagstaff, Arizona. ugg canada sale He lived at home and worked at Atari; she had a small apartment and spent a lot of time at Kobun Chino's Zen center.Ugg Factory Outlet Online I was fifteen, and then began using pot regularly. The nurse likes to getaway from me and then Martha comes. "Most of our generation scorned computers as the embodiment of centralized control," Brand later noted. When Jobs was looking for someone to write a manual for the Apple II in 1976, he called Raskin, who had his own little consulting firm. He did, however, spend years studying and trying to practice the tenets of Zen Buddhism.ugg boot outlet onlineUgg Boots With Fur Even in small everyday rebellions, such as not putting a license plate on his car and parking it in handicapped spaces, he acted as if he were not subject to the strictures around him.
He took a bunch of us snowshoeing in Yosemite. Ugg Kids Bailey Bow [40] In 1999, Deckers registered the trademarks for "UGG" in the US.Grey Ugg When Jobs was looking for someone to write a manual for the Apple II in 1976, he called Raskin, who had his own little consulting firm. Polyphemus is son to Neptune by the nymph Thoosa, daughter to the sea-king Phorcys; therefore though he will not kill Ulysses outright, he torments him by preventing him from getting home. These are even easier to get on because of the slit down the side and more versatile also because you can fold the top down. Uggs makes their own spray and it works well. [Ugg Kids Bailey Bow] Fortunately there was a place nearby for entrepreneurs who had outgrown their garages.
our trucks are rear wheel drive! moving on.ugg boot outlet online Then the bottom fell out of the market. The son of an Auschwitz survivor who became a prosperous Chicago architect, he had originally gone to Bowdoin, a liberal arts college in Maine. The partnership paved the way for what would be a bigger adventure together. There was little that could shield you from the force, Hertzfeld discovered. [Ugg Kids Bailey Bow] " The disagreements were more than just philosophical; they became clashes of personality.
" Concisely but fully, Kindell told of Myra's request, and of the parcel which she had asked him to take to England. ugg canada sale "They said it would force us to use a phosphor that was a lot less persistent and would flicker more. "Jef was pretty firm and stubborn, and Steve was right to take it over. I can't believe how happy I am with this product. [ugg canada sale] The epitome of relaxed luxury, the Slipper Collection is defined by a timeless fusion of fashion and function.
«il vino è un miracolo della natura coadiuvata dall'intelligenza e dalla fatica dell'uomo. e' cultura, è storia, in una parola è civiltà…il vino!»
|benvenuti nel sito dell'azienda monsupello!
|welcome to the english website of monsupello!
|anteprima di alcuni riconoscimenti delle guide 2015
cl. nature
si � aggiudicato i
tre bicchieri
della guida vini d'italia 2015
leggi la recensione
| l'azienda monsupello
si � aggiudicata la
e il brut cl. nature
il riconoscimento vino slow
della guida slow wine 2015
si � aggiudicato la
golden star e la corona
della guida vini buoni d'italia 2015
| l'azienda monsupello
si � aggiudicata le
tre stelle
della guida vini essenziali d'italia 2015
recensioni di franco ziliani dei nostri vini:
leggi l'articolo
franco ziliani
|sito in aggiornamento
ultima modifica 23 dicembre 2014
monsupello eredi di boatti carlo società agricola
via san lazzaro 5 - 27050 torricella verzate - pavia - italia
tel. 0383.896043 - fax 0383.896391

subset: commoncrawl


text:
Easy Solo Ad Advertising That Gets Results...
Grow your business with our easy to use advertising system.
- Professional Ad Writing Included: Our ad writers are happy to write ads for you at no extra charge.
- Responsive & Effective Advertising: We only send your ad to the highest qualified prospects.
- Solo Ad Exposure: We only send your ad out by itself (solo ad) to the highest quality verified prospects.
- Targeting Niches & Geotargeting: Choose the specific states and countries we send your campaign to.
Our Customers Love Our System Because
It is So Simple to Use...
"Every week I get new business from my ad campaign. I am consistently building my business with your service."
Shelly B, Ameriplan Distributor
"I know that I can rely on your ads for consistent and reliable results. Thanks for another great campaign."
Chris Allen, Blogger & Entrepreneur
"From start to finish I was thouroughly pleased. Thanks for helping me with my ad set up and for a great experience."
Nancy, Empower Network
No Previous Experience Is Required;
Easy Use To Use & Expert Help When You Need It...
As a business owner or entrepreneur; you already know that finding new clients and customers can be a difficult task. Targeted email advertising is one of the most effective methods of growing your business, winning customers, and earning more money.
At EasySoloAds.com, we are proud to provide an effective advertising solution for you or your business. Our affordable solo ad advertising services cover every aspect of reaching new potential customers. We send your ad campaign out alone (solo ads) with no others attached. Each solo ad that we send goes to new prospects that have been verified and qualified through our network of targeted ads.
Easily Grow Your Audience, Increase Conversions
& Boost Sales...
We match your ad to our targeted lists of potential clients. Targeting your email advertising will boost your conversions and results because you are contacting people who are interested in products & services in your industry. Every time we send an ad out for you it will send out to new, fresh, verified prospects.
Trusted By Leading Distributors...
Fresh Verified Prospects...
We pre-qualify all prospects before sending them your ad. Every prospect is verfied by email and indicate their areas of interest (targeting categories.)
Each ad we send for you goes to new people. We never show your ad twice to the same person.
Real Time Campaign Stats...
You will receive a third party tracking link with each campaign that we send for you. The tracking link will show you real time stats from your campaign.
Here Are Just A Few Of The Powerful Features...
Easy To Use Service
Send us your ad (or website link) and we will write your ad & suggest the best possible campaign settings.
Campaign Targeting
Laser target your campaign by choosing from our extensive list of verticals (targeting categories.)
Real Time Campaign Stats
View link statistics from your campaign through the third party real time tracking link we provide.
Fast Results
In most cases we can start your campaign within 24 hours of ordering. Our goal is to provide a fast turn around.
Customize Every Detail
Tweak your campaign settings or use the suggestions we provide. Full customization is available.
Powerful Results
Our goal is to provide you with the best results possible. From ad writing, to campaign targeting, we get you results.
Award Winning Customer Service
Our goal is to provide the fastest and friendliest support in this industry. We are here to help you with every aspect of using our service. We are here to answer any questions or address any concerns you might have.
YES! I'm Ready To Get Started Today
You can start sending ads the same day you order
Reach Thousands Every Week!
Easy To Use Service
Full Support & Set Up Help
Mark Harris, empowernetwork.com
"I recommend your service to all of my affiliates and JV partners. You always provide me great advertising."
Here's What You Get When You Order Today:
- Your Ad Sent To Thousands Of New People Per Week
- Our Lists Contain Only Qualified & Verified Prospects
- Choose From Our Extensive List Of Industries & Targeting Categories
- Full Support & Help With Your Campaign
Plus These Amazing Bonuses
- Real Time - Third Party Verified Click Tracking
- Geotarget Your Campaign To Specific States & Countries
All orders 100% SAFE using our SECURE server
Have A Question...? Ask One Of Our Experts
Below are answers to some of the most common questions we receive. Please read the questions below, if you don't see an answer to your question please contact us!
Q.What are your available targeting categories?
A.We offer an extensive list of categories. You can view the list here. If you don't see a category that matches please send us an email and we will locate a source for you.
Q.Do offer targeting to specific cities or states?
A.We currently offer geotargeting with all campaigns. We can target your campaign by state or country at this time. With the geotargeting feature you can choose the specific states or countries we send your campaign to.
Q.How soon can you start my campaign?
A.We can have you up and running the same day you order. If you have our ad writers create your ad the turnaround time is usually 24 hours.
Q.I don't know how to write an ad campaign...
A.We offer an optional ad writing service. Our professional ad writers will be happy to write an ad for you. The turnaround time to have an ad written is usually about 24 hours.
Q.Will I have problems with spam?
A.We use only opt-in lists. The lists are closely maintained and monitored. We handle all email compliance and we only send whitehat opt-in campaigns. All mailing is handled on our whitelisted mailing servers.
Q.How do I track my results?
A.When your campaign begins sending we will email you a tracking link. The tracking link will allow you to monitor the results of your campaign in real-time.
Q.How often do you send ads?
A.Our extensive lists allow us to send campaigns on a daily basis. Prospects on our lists receive no more than 2 campaigns per week. Your ads will never be sent to the same person twice.
Q.I have never done this before...
A.We are happy to help you every step of the way. For most people our system is very intuitive and easy to use. If you need any assistance we are happy to set up your campaign for you or suggest targeting categories that would be a good match for your website.


text:
Today, I was told I needed to start carrying bandaids with me at work because practically every day I hurt myself. FML
by anonymous / 09/24/2016 at 9:10am / United States (Washington) / Work
Today, I've been recovering for a week from my medically needed circumcision. I'm 30, and they advise you wear essentially a jock strap for the first week to help. It wasn't too bad until I went to take the thing off and it caught a stitch on my manhood. That's the most unique pain ever. FML
by T3kM4n / 09/20/2016 at 2:08am / United States (Washington) / Health
Today, my girlfriend told me that she didn't know we were actually dating. FML
by anonymous / 09/05/2016 at 11:05pm / United States (Washington) / Love
Today, I overheard two classmates wondering who Joan of Arc was. They agreed among themselves that she had to be the wife of Noah. We're in college. FML
by Emmereen / 08/22/2016 at 10:21pm / United States (Washington) / Miscellaneous
Today, I woke up and heard the shower running. Would have been nice to know my father in law was here before I walked in naked. FML
by Pikababy19 / 08/18/2016 at 7:12pm / United States (Washington) / Miscellaneous
Today, I had an appointment. After 2 hours of waiting in the sun, my mom finally picked me up and lectured me on how irresponsible my dad was for not communicating with her. Not 5 minutes after I got home, my dad showed up and gave me the same exact lecture about my mom. FML
by Still Waiting / 08/16/2016 at 2:54pm / United States (Washington) / Transportation
Today, I logged on to my Gmail account and to find that my brother had sent my math teacher a picture of Bigfoot peeing into a urinal. Using my account. FML
by Hey, thats not math!? / 08/09/2016 at 4:20pm / United States (Washington) / Work
Today, I thought I'd spice things up by kissing my husband on the lips and then working my way down. But about halfway, I got some of his chest hairs lodged in my throat and started gagging. To avoid ruining the mood, I kept going, silently gagging, until we finished. I swallowed the hair. FML
by so unsexy / 08/04/2016 at 5:32pm / United States (Washington) / Intimacy
Today, I got so anxious while trying to fight the ticket I got earlier this month that I passed out in front of the judge. FML
by so embarrassing / 08/02/2016 at 2:27am / United States (Washington) / Transportation
Today, I sent a Snapchat the girl I've been flirting with all week. Her response was the back of her Coke Zero, which had the quote "You've Got a Friend in Me." I got rejected by a soda can. FML
by Anonymous / 07/25/2016 at 5:28pm / United States (Washington) / Geek
Today, I dropped my knitting project while I was sitting down, but I managed to catch it between my legs. So my knitting needles also caught me. FML
by Anonymous / 07/20/2016 at 2:40am / United States (Washington) / Health
Today, I told my parents that I was going on a diet to lose weight and I wanted their support. They brought home donuts and pizza for dinner. FML
by mattlikesfunions / 07/18/2016 at 2:56pm / United States (Washington) / Health
Today, an elderly patient complained because I used the words "vaginal" and "breast". I work at an OBGYN's office. FML
by Anonymous / 07/15/2016 at 10:08pm / United States (Washington) / Work


text: Revisit Your Old Flickr Photos with Photojojo's Time CapsuleTamar Weinberg3/08/08 10:00amFiled to: Digital PhotographyDigital PhotosFlickrPhotographyTime CapsuleWebapps3EditPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Experience nostalgia by receiving mailings of your old Flickr photos twice a month with Photojojo's Photo Time Capsule. Previously, we suggested that you mail your future self. Instead, receive emails from the past. The tool integrates with your Flickr account and emails you weekly (or twice a month) with photos that you've taken 1 month ago, 3 months ago, 6 months ago, 1 year ago, or two years ago (the time setting is totally customizable). With the Photo Time Capsule, you'll never forget your precious photographs that you took a while ago and you won't have to manually dig into your old pictures to view those precious memories either. Photo Time Capsule [Photojojo]Gear from Kinja DealsYour Pick For Best Cheap Bluetooth Earbuds: Anker SoundBudsRepel 100 Is Your Pick For Best Bug SpraySave A Whopping $55 On These Popular Mackie Reference SpeakersReply3 repliesLeave a reply


text:
It's there in black and white... English rugby is now a laughing stock
There are many sporting institutions
known by their colours, many reds and blues and even baggy greens; but
there is only one team in world sport that is entirely defined, tip to
toe, by the chromaticity of its kit.
The All Blacks.
Bitter rivals such as Liverpool and Manchester United both lay claim to redness and last season three teams in the Premier League alone could have been considered the Blues (Birmingham City, Chelsea and Everton). Yet mention the All Blacks and recognition is instant. No imagination is even needed to conjure a mental image. The All Blacks are New Zealand's rugby union team and nothing else.
So powerful is the name that no rugby follower would even speak of an encounter with New Zealand. Rugby nations play the All Blacks, as if the opponent is the shirt, with its tradition, heritage and inky totality. This is no mere length of material. It has a back story. The All Blacks were named by JA Buttery, rugby correspondent of this newspaper, in 1905. 'The only colour not black was the silver fern on the left breast and the white of their bootlaces,' he wrote. New Zealand's shirt had been changed from dark blue in 1893 and by 1901 the rest of the uniform followed.
The Originals: Billy Wallace kicks a conversion in the All Blacks's 55-4 win against Devon in 1905
The All Black team that Buttery saw was the stuff of legend. Touring the United Kingdom, France and North America it played 35 matches and won 34, the only defeat coming to Wales, 3-0, in highly controversial circumstances.
The Originals — as they were known before the rechristening — scored 976 points and conceded 59. Their first win, 55-4, over Devon at the County Ground, Exeter, on September 16, came as such a shock that many newspapers attributed the higher score to the home side in error.
By the time they returned home, New Zealand's rugby players were the most feared in the world, and had a new billing, with them ever since. The Daily Chronicle described a team that turned 'defence into attack with such bewildering rapidity... there is nothing in the game at which they do not excel.'
A lot of pride there, then; a lot of history. So what colour strip will England unveil in a match against Wales on August 6? What colour will England's second kit be at the World Cup in New Zealand later this year? All black. England will play Argentina in their first World Cup group game on September 10, having stolen the sacred attire of the host nation.
This after coach Martin Johnson stuffed his training squad with New Zealanders and other foreign nationals. Maybe England will walk out and do a haka, too. There are Maori markings on their shirts, believe it or not.
More from Martin Samuel - Sport for the Daily Mail...
- Phil Mickelson: We're prepared to succeed this time... as USA try to banish eight years of Ryder Cup hurt 28/09/16
- Fallen England boss Sam Allardyce is a piteous figure undone by greed 27/09/16
- Why is it always Wayne Rooney? Give the Manchester United man a break 27/09/16
- Sorry Pep Guardiola, Manchester City fans are right to boo UEFA hypocrites 18/09/16
- MARTIN SAMUEL'S 5 POINTS: The Champions League is being hijacked by greed but its future could be even more terrifying 15/09/16
- Europe's elite are shafting the poor so enjoy Leicester City while you can 13/09/16
- Tony Pulis' style of football may be basic but it's harsh to blame him for West Brom's woes 11/09/16
- Manchester City and their quest for world domination takes another boost as Yankees link becomes key step in the global project 09/09/16
- MARTIN SAMUEL'S 5 POINTS: Jurgen Klopp might not solve Liverpool's problems but he'll thrill us by giving it a go... plus why the Premier League doesn't need the Old Firm 08/09/16
Has there ever been a marketing exercise quite as crass as this? If it was intended to annoy the hosts, who are clearly the team to beat on home soil, then it is hopelessly ineffectual. A few locals are upset, but not the ones that matter, who appear to be looking on with wry bemusement.
If English rugby wishes to tick off the Kiwis, try beating them, as Sir Clive Woodward's team did in Wellington, the summer before the 2003 World Cup. They hated that. Nicking their colours is just seen as ill-mannered and boorish and further evidence of an ill-founded superiority complex, patronising the former colonies.
It also seems a desperate measure, like the time Sunderland chose to celebrate the magnificent history of Benfica by naming their ground the Stadium of Light (Estadio da Luz, as it is in Lisbon), rather than acknowledging their own noble past (what was wrong with New Roker, the way Barcelona play in Nou Camp, having left their old one, Camp de Les Corts, in 1957).
Are England, having swiped a few New Zealand nationals, now trying to acquire more All Blacks magic by stealing their look? Either way, it makes the Rugby Football Union appear weak and foolish, acting as if the colours contain the inspirational strength, rather than the men within. FC Basel of Switzerland play in the same red and blue as Barcelona, but there similarity ends. Dan Carter would still be the world's best fly-half, whatever his shirt; Toby Flood in black remains Toby Flood.
Most likely, this is simply a headline-grabbing ploy by the marketing department at Nike, manufacturers of England's shirts. There will be an ad campaign to accompany the launch, no doubt with a catchy slogan presumptuously playing up the rivalry with New Zealand, as if England do not first have to find a way to beat Ireland with their dander up before worrying about taking on the world's finest.
Tour of duty: The New Zealand side won plaudits and a fierce reputation that has stuck ever since
In the bubble of sports branding and sales, stealing New Zealand's threads is a cheeky little wind-up and cheap publicity. Dismal commercial considerations over-ride all. To anyone who cares for sport, however, it is disrespectful, wrong and makes English rugby a laughing stock. Imagine if the footballers turned up for the next World Cup in a strip of yellow, light blue and white? Yet, while Brazil's colours are evocative, they cannot compare to the powerful statement of New Zealand's black.
England could have changed into just about any colour at the World Cup, even ones long taken, or even purple, without seeming quite so ridiculous. France and Scotland wear blue, Wales red, South Africa and Ireland green, Australia yellow, and all are teams with great histories, but no proprietary right.
Had England adopted a changed strip of red — the colour of the cross of St George — it would not have been regarded as an affront to Welsh rugby. There are plenty of countries that nominate white as an away kit, including New Zealand, but nobody knows or cares. All black is the exception. In rugby, all colours are not equal.
This idea should not have got beyond the first brain-storming session at headquarters.
Nike's clever dicks should have been firmly directed back to the drawing board the moment they came up with a plan of such clumsiness. Instead, the RFU played along, and are now in damage limitation mode, claiming the black change strip was adopted only after consulting the New Zealand Rugby Union, who claimed to have no issues.
Yet there is the clue. If it is right for England to wear black, why were the RFU asking permission of another country? The stunned silence on the end of the line was not, in fact, approval, chaps. Once they had got over the shock, far from complaining, the Kiwis were probably too busy trying to stifle their laughter.
Posturing: Adebayor
Adebayor is not the Real deal
Tediously, Emmanuel Adebayor is cutting up rough at Manchester City after being left out of the squad for a tour of America. Adebayor is pouting and posturing and clearly desires a move to Real Madrid.
His problem is that if Jose Mourinho really wanted him, he would surely have moved by now.
Adebayor was on loan at Madrid last season and City want to sell; so there is nothing to stop the deal going through other than reticence on Madrid's part. Mourinho seems to prefer Neymar from Brazilian club Santos. He may think Adebayor is too much trouble. He may well be right.
Blatter's bad Korea move...
In his restless search for a Nobel Peace Prize, Sepp Blatter, president of FIFA, fought long and hard to get North Korea back into the fold. He succeeded, but the rewards have not been great.
Blatter is probably about as far away from being recognised for his contribution to world harmony as Rebekah Brooks is from a lifetime achievement citation at the next British Press Awards, and North Korea are a bunch of cheats.
At the 2010 World Cup in South Africa, they tried to slip an extra outfield player into the squad by naming a striker, Kim Myong-Won, as one of their three goalkeepers; and now five North Korea players have tested positive for steroids at the women's World Cup.
The official explanation — and you'll like this because even by the standards of drug-cheating liars it's a cracker — was that the disgraced players were part of a group that was struck by lightning at a training camp on June 8 and were treated with a Chinese remedy based on musk deer gland. It was this gland, hairy and 4cm long, that was somehow contaminated with steroids.
Strangely, the North Koreans did not mention the lightning until after they lost their first game against the United States. Maybe they were waiting to see if their players would develop special powers after the lightning/musk deer gland combo, the way Peter Parker did
when he was bitten by that radioactive spider. Maybe they decided not to wait, and gave them some special powers from a nearby medicine cabinet anyway.
Whatever occurred, North Korea are sport's equivalent of a rogue nation and FIFA should have nothing more to do with them. If Sepp reckons this will hurt his mission to be recognised as a forger of world peace, someone must take him aside for a quiet word.
Between you and me, Sepp, that was always going to be a bit of a non-starter.
Lewis is a standard bearer
Sounding very much like a stern father, Sir Clive Woodward, director of sport for the British Olympic Association, has drawn up a list of standards for British athletes competing at London 2012.
Keep hands clean, be punctual, tidy your room, switch mobile telephones off at night and refrain from swearing in public are among the orders. And much fun is being had at Sir Clive's expense.
Woodward, however, is deadly serious in regarding these issues as 'one per centers', the seemingly trivial matters that make the difference in a high performing team.
He notes that the most successful British sports — cycling, for instance — insist on the most rigorous standards of behaviour. He has a point.
Walking around Royal St George's with Tom Lewis's group on Friday, I talked with John Petrie, the chief executive of the English Golf Union.
He has lots of fond memories of Lewis's contribution in amateur tournaments, events and conditions that will help him on turning professional later this year, but a seemingly insignificant detail stood out.
Lewis was the only member of the Walker Cup team who knew how to iron his shirts and was therefore perfectly turned out every morning.
He really cared about all of it, and he sweated the small stuff. Along with the most wonderful ability, this is one reason he is destined for the top.
Disciplined performance: Tom Lewis is destined for the big time
Murderous talk
Since when did killing become acceptable in boxing?
Not doing it, talking about it, throwing it in to the trash talk mix as if there is a paying audience for murder.
We know so much of the antagonism before a fight is there to sell tickets or pay-per-view packages, but people still bought both in times when boxers had too much respect for each other to talk of death and decapitation.
Heavyweight Tyson Fury yakked about killing Dereck Chisora more than once when publicising their fight on Saturday.
These outbursts revolt the public and offend true fight fans. David Haye had lost the respect of the British public with his mouth, long before his little toe sealed the deal.
Making a killing: David Haye (right) pocketed £10million from his farcical bout with Wladimir Klitschko
Fletcher's tough to replace
Continued problems with Darren Fletcher's fitness may cause Manchester United to return to the transfer market. Sir Alex Ferguson will find there are not too many out there like him — and none in this country.
Self-serving protectionism
The forces of vested interest — elite clubs mistakenly convinced they are entitled to more success than they are getting — are lining up against Manchester City over the Etihad deal.
Arsenal and Liverpool are the latest to call for a thorough investigation into the £400m sponsorship.
Strange, isn't it, that the small clubs financial fair play was supposed to encourage and protect have long ceased to care about its implementation, while the wealthiest by tradition are keenest on its enforcement.
It is almost as if there is something in it for them.
Wenger's talk is cheap
Arsene Wenger describes it as Arsenal's destiny to win the Barclays Premier League. On the same day it is revealed the club has made a bid of £14m for Anderlecht teenager Romelu Lukaku.
Chelsea are also in the mix, and the player is clearly very talented, but somehow these destinies can sound alarmingly like another of Wenger's five-year plans in which a squad is assembled that is going to be absolutely unbeatable.
Destination unknown: Romelu Lukaku is a long-term Chelsea target
Olympic debate is a non-starter
The debate over whether Rory McIlroy should represent Great Britain or Ireland when golf enters the Olympics in 2016 forgets a significant factor: only two players per nation qualify, based on world rankings.
If the competition began tomorrow, Britain's pair would be Lee Westwood and Luke Donald and McIlroy's only method of entry would be via a united Ireland team, partnering Graeme McDowell, with the south — in particular the popular Padraig Harrington — unrepresented.
That should go down well.
(Also missing the cut as the list stands: Tiger Woods, Ernie Els and Sergio Garcia. The sponsors will be delighted with this format, if it stays.)
Most watched Sport videos
- Hector Bellerin goes on dinner date with Bollywood star Esha...
- Arsenal vs Basle, UEFA Champions League LIVE: Plus coverage...
- 'Juan Mata was one player at Chelsea but at Manchester...
- Celtic 3-3 Manchester City: Fernandinho, Raheem Sterling and...
- Eight Premier League managers accused of receiving bungs in...
- Danny Willett 'bitterly disappointed' after brother brands...
- Arsenal's Alex Iwobi videos girlfriend Clarisse Juliette as...
- Jamie Vardy's amazing confession: I drink port out of a...
- Jose Mourinho feels sorry for Sam Allardyce after he was...
- Arsenal wear yellow away strip for home win against Basle as...
- Fallen England boss Sam Allardyce is a piteous figure undone...
- Son Heung-min is in unstoppable form for Tottenham... but he...


text:
We use cookies to help us deliver our services. By continuing to browse the site you are agreeing to our use of cookies. Find out more here.
Search Commercial Property in Swansea with PropertySales.com,
a leading portal for commercial property listings.
PropertySales.com is home to a huge range of
Commercial Property,
with prime locations available in every region.
Inform your search for the perfect property by visiting our commercial property News & Insight section,
where you will find news, opinion, features, industry reports and guides relating to Commercial Property,
Swansea and all other commercial property sectors and locations.
Residential Development Opportunity, Swansea. Outline Planning Permission for demolition of existing property and construction of 4 residential dwellings (outli ...
See more:
Bars in Waunarlwydd
KEY POINTS. Nearest Train Station: Llansamlet. Nearest Road Link: M4.
See more:
Serviced Offices in Llansamlet
A very spacious detached property that is presently operating as a convenience stores with an off license and full lottery service. The business has been run do ...
See more:
Offices in United Kingdom, High Street Retail Properties in United Kingdom, Other Residential Properties in United Kingdom and Mixed Use Properties in United Kingdom
KEY POINTS. Nearest Train Station: Swansea. Nearest Road Link: B4290.
See more:
Offices in Swansea
KEY POINTS. Cost Per Person: 150. Minimum Size Available: 1 Person(s).
See more:
Serviced Offices in Swansea
Type: Serviced/furnished Offices in Swansea for rent. Sizes: Private or shared offices for 5 - 60 people. Prices: £329 per person/per month (all inclusive).
Refurbished Georgian Residential Accomodation in Swansea to Let, Swansea. Recently renovated. Stunning Gower location in close proximity to Oxwich Bay.
See more:
Other Residential Properties in Reynoldston
**Guide Price �289,950 To �299,950**. Viewing is essential to appreciate this four bedroom modern, detached property which is located on the outskirts of Swanse ...
See more:
Other Residential Properties in Swansea
Ground floor retail premises avaiable for leasehold, Llanelli. Ground floor retail premises available for leasehold. Prominent ton centre location within the pe ...
See more:
Other Retail Properties in Llanelli
We use cookies to help us deliver our services. By continuing to browse the site you are agreeing to our use of cookies. Find out more here.


text:
Written on: 29/07/2010
Totally waste of time and money. Accommodation is old, smelly & dirty. Bed linen old, dirty and has holes. Entertainment is first come first served basis, so no seats! Toilet facilities all dirty and smell of urine. Chalets and Queen Vic infested with ants. Drinking glasses dirty but smell and taste of cleaning fluid. Swimming pool over crowded and dirty. Changing rooms are filthy. Food is over priced and disgusting. Nothing for kids under 5 to do.
Check in procedure is a joke, have to queue up in main hall and wait your turn. Very Hot and no air conditioning. 70 free activities?? You have to pay for anything worth doing! Pontins need to clean up their act. We shouldn't be paying them to stay there, they should be paying us!!
As rated by our community of reviewers
" Nice Park, But " Read More
" Longleat Center Parcs Pinewoods Apartments disgrace " Read More
" Worst park we've ever stayed at! " Read More
" Don't book this site if your wanting a quiet site. " Read More
Written by Tallygo
" Excellent is an understatment " Read More


text:
arcticscythe arcticscythe is offline
WRXtuners Member
Last Activity: Today 11:47 AM
About Me
- About arcticscythe
- City
- Santa Clarita
- State/Country
- California
- WRX Club Region
- SoCal
- WRX Model
- WRX Wagon
- WRX Year
- 13
- Non-WRX
- 2012 Impreza Limited
- Mods-speed
- Cobb Accessport V3
Torqued Performance Stage 2+ E-tune
Grimmspeed Master Cylinder Brace
Escort i9500ix
SF Intake
Cobb Intake Air Box. Mylar wrapped for temp handling
Cobb CATed Downpipe - Stock Catback
GFB Mach 2 Bypass Valve ( replaced when stock unit starting leaking )
Gorilla Flaps to stop speeding rocks
- Mods - engine
- Stock
- Mods - drivetrain
- STi Short throw Dealer installed
Cobb knob
- Mods -suspension
- Whiteline 22mm Adjustable front/rear - stiff on rear and medium on front
Whiteline Braces
Kartboys endlinks
Fade to black bushings
White lines Poly Bushing Fills
- Parts Wishlist
- Koyo Radiator
Engine Dress up Belt Covers
Strut tower brace - cause they look cool
Pro-tune ** E-tune handled by Eric at Torqued Performance **
- Racing Series
- Autocross, Open Track Events
- 0.000
- 0.000
- Turbo Type (Timeslip)
- Stock
- Pump or Race Gas? (Timeslip)
- Pump gas
- Nitrous? (Timeslip)
- No Nitrous
- Tires (Timeslip)
- Street Radials
- Tranny type (Timeslip)
- 5-speed
- Track Name (Timeslip)
- jason
- Sponsored Car? (Timeslip)
- No
- Nitrous? (on Dyno)
- No Nitrous
- Favorite WRX shop/Vendor
- Yimi Sport Santa Clarita
- Occupation
- Director - Information Technology
- Other Interests
- Mountain Biking, Airsoft, Yoga, Parkour
- - Jason
Slow is Smooth > Smooth is Fast
- - Jason
Total Posts
- Total Posts: 1,472
- Posts Per Day: 1.44
- Last Post: Muffler delete need tune ? Today 11:47 AM
- Find all posts by arcticscythe
- Find all threads started by arcticscythe
Visitor Messages
- Total Messages: 9
- Most Recent Message: 03-22-2016 09:46 PM
- Visitor Messages for arcticscythe
General Information
- Last Activity: Today 11:47 AM
- Join Date: 12-16-2013
|11:21 AM - arcticscythe mentioned ic3b3rg in post [post=579633]Radar Detectors?[/post]
...uracy as I can. ic3b3rg, The way I see it...
|01:31 PM - arcticscythe mentioned titter in post [post=579345]Radar Detectors?[/post]
titter, thats not entirely ...
|07:52 AM - wrxnh mentioned arcticscythe in post [post=578681]LED Light Bar Install on GR WRX[/post]
arcticscythe What's the width of ...
|10:47 AM - arcticscythe mentioned mosc in post [post=575625]Need new wheels[/post]
What mosc said, High offset an...
|10:13 AM - arcticscythe mentioned sleepr in post [post=573866]Swapped my 13 for a 17[/post]
sleepr, the 1st gear with t...
|09:49 AM - arcticscythe mentioned gbarbur in post [post=572377]2016 stage 1 with intake questions[/post]
...sional e - tune. gbarbur, The dealership, ser...
|09:49 AM - arcticscythe mentioned Mweger in post [post=572377]2016 stage 1 with intake questions[/post]
...parts installed. Mweger, unfortunately you c...
|09:49 AM - arcticscythe mentioned Modifiedamerican in post [post=572377]2016 stage 1 with intake questions[/post]
...ading the blind. Modifiedamerican, you can purchase an...
|11:10 AM - arcticscythe mentioned TravsWRX in post [post=561929]Fuel injectors and Pump[/post]
TravsWRX If your looking to m...
|08:40 AM - arcticscythe mentioned stemp661 in post [post=560970]2015 wrx... What should I do[/post]
... in the engine bay. stemp661 has a 2016 that cook...
|05:21 PM - arcticscythe mentioned Fidrat in post [post=560626]Fuel injectors and Pump[/post]
Fidrat, Ive got that bookma...
|03:39 PM - arcticscythe mentioned boo-key in post [post=559546]FEAL 441 Max Travel[/post]
...o local - Thanks to boo-key for the heads up on ...
|10:52 AM - stemp661 mentioned arcticscythe in post [post=559185]Tire rubbing.. Stock everything.[/post]
...r shocks rechecked. arcticscythe has this car im sure...
|01:46 PM - arcticscythe mentioned stemp661 in post [post=557969]Cobb stage 1 loosing boost[/post]
stemp661, thats exactly the i...
|08:39 AM - arcticscythe mentioned boo-key in post [post=557497]Adding hp to 2005 wrx[/post]
...for a stage 2 setup boo-key has the most cost ef...
|10:02 PM - arcticscythe mentioned clamer in post [post=557473]Looking for wheels[/post]
clamer, the Original Poster...
|11:51 AM - arcticscythe mentioned pjkatz04 in post [post=557313]Cobb stage 1 loosing boost[/post]
pjkatz04 didnt you have the s...
|08:24 AM - arcticscythe mentioned ton in post [post=556362]Tony W[/post]
tony, Welcome to our f...
|04:37 PM - titter mentioned arcticscythe in post [post=553386]Shift Knobs?[/post]
hey arcticscythe any progress on thos...
|04:23 PM - arcticscythe mentioned stemp661 in post [post=553186]Center console won't latch closed.[/post]
... cover from a 2016. stemp661 got the upgraded sli...
|11:19 AM - sleepertc quoted arcticscythe in post [post=580617]My Airflow project 2015wrx[/post]
That looks pretty good. If I may...
|05:46 AM - titter quoted arcticscythe in post [post=579729]Radar Detectors?[/post]
There is no way for a lidar or ra...
|03:33 PM - ic3b3rg quoted arcticscythe in post [post=579689]Radar Detectors?[/post]
The way I see it a speeding ticke...
|11:21 AM - arcticscythe quoted titter in post [post=579633]Radar Detectors?[/post]
ya and they have the techno...
|07:46 AM - wrxnh quoted arcticscythe in post [post=578841]LED Light Bar Install on GR WRX[/post]
Th bumper bar was a 22 inch mictu...
|11:54 PM - hempy quoted arcticscythe in post [post=578577]Accessport Auto-Shutoff Feature[/post]
I haven't had any issues with the...
|02:48 PM - Emilio246 quoted arcticscythe in post [post=578345]2016 wrx CVT[/post]
:(Welcome to our forums, To an...
|01:35 PM - brfatal quoted arcticscythe in post [post=576698]TBE and CAI with tune?[/post]
Thats been the common knowledge a...
|11:20 AM - arcticscythe quoted brfatal in post [post=576682]TBE and CAI with tune?[/post]
Ugh, this old game of teleph...
|12:50 PM - brfatal quoted arcticscythe in post [post=576578]TBE and CAI with tune?[/post]
Cold air intakes are not useless ...
|09:41 PM - rdb quoted arcticscythe in post [post=576394]04 sti for 12k$[/post]
Thats a gamble, and my dad always...
|09:36 AM - arcticscythe quoted GSMNZ in post [post=576145]Turbo blowing oil through intercooler?[/post]
I feel pretty strongly tha...
|06:49 PM - gtbigup quoted arcticscythe in post [post=576097]H16 Yellow[/post]
Im typically a fan of GE bulbs bu...
|09:02 PM - rdb quoted arcticscythe in post [post=575065]WRX at 400HP[/post]
Honestly, If you dont have the mo...
|04:24 PM - rdb quoted arcticscythe in post [post=575033]WRX at 400HP[/post]
you will need to replace almost e...
|10:27 AM - RogueMantis quoted arcticscythe in post [post=575009]WRX at 400HP[/post]
Even with a slightly higher stock...
|03:46 PM - KryptoNyte quoted arcticscythe in post [post=574857]2017 WRX - Strange Brake Pedal Vibration[/post]
Also i always try honey before vi...
|05:40 AM - wrxham quoted arcticscythe in post [post=574458]LED Light Bar Install on GR WRX[/post]
|07:53 AM - mlavoie83 quoted arcticscythe in post [post=573914]Loss of Boost[/post]
Boost levels that high in the fir...
|01:55 PM - arcticscythe quoted sleepr in post [post=573682]Re-key lock cylinder[/post]
I can't believe someone tri...
Feedback Score
|iTrader Profile
|Recent Ratings


text:
In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus.
Maria Alejandra Diaz, 1102 Bates Ave, Feigin Center, Ste. 830, Houston, TX 77030, USA. E-mail: firstname.lastname@example.com
In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus.
We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti.
Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals.
This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals.
Little is known about the composition and functions of the symbiotic gastrointestinal microbiota of marine mammals, or the exact contribution of various microbes to disease in these animals (Venn-Watson et al. 2008). Gastroenteritis occurs in bottlenose dolphins (Tursiops truncatus), and diagnosis can be based upon abnormal faecal or gastric content appearance, changes in gut motility and appetite, or overgrowth of Candida spp. or Clostridium perfringens in faeces. While the aetiology of gastroenteritis in dolphins is not commonly identified, Campylobacter spp., Cryptosporidium spp., Edwardsiella tarda, enteropathogenic Escherichia coli (EHEC), Giardia spp., Listeria spp, Salmonella spp. and Vibrio spp. are common terrestrial mammalian gastrointestinal pathogens that are found in marine mammals (Minette 1986; Higgins 2000; Venn-Watson et al. 2012).
Dolphins appear to be particularly susceptible to gastric ulcers due to a lack of glands in the forestomach to protect itself against digestive fluids and hydrochloric acid (Gaskin 1978). Further, Helicobacter infections may affect the health status of dolphins by contributing to the pathogenesis of gastric ulcers. In effect, novel helicobacters have been found in wild and captive marine mammals (Goldman et al. 2011). Helicobacter cetorum has been isolated from the gastric mucosa of dolphins (Harper et al. 2000, 2002), although its relation to gastric disease remains unclear. Given the variety of potential causes of gastroenteritis in dolphins, and the difficulty in acquiring a definitive diagnosis, there is a need for broad-spectrum protection against disease.
Probiotics, as defined by the Food and Agricultural Organization of the United Nations, are 'live micro-organisms which when administered in adequate amounts confer a health benefit on the host' (FAO/WHO 2001). Previous studies, extensively reviewed elsewhere (Rastall et al. 2005; Guarner et al. 2012), have enumerated the necessary traits and potential benefits of probiotic use for prevention and treatment of disease in animals and humans. Some of the beneficial features include inhibition of pathogen growth and prevention of colonization, suppression of virulence factor expression, modulation of host microbiota, modification of energy utilization and of pain perception, enhancement of epithelial cell function, protection from physiological stress and modulation of host immune responses, including alteration of cytokine and antibody production by host cells and regulation of T-lymphocyte function (Ryan et al. 2009; Thomas and Versalovic 2010). Beneficial microbes and probiotics have been delivered to various animals of agricultural importance such as cattle (Nader-Macias et al. 2008), swine (Mori et al. 2011), poultry (Pascual et al. 1999; Brisbin et al. 2011) and fish (Balcazar et al. 2007; Gatesoupe 2008), in applications such as animal feed, for growth promotion, modulation of the gut microbiota and prevention of infectious diseases (Bernardeau et al. 2006; Czarnecki-Maulden 2008; Gaggia et al. 2010).
Lactic acid bacteria, which include the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus and others, have received particular interest as probiotics, because many of them are classified as generally regarded as safe (GRAS) organisms (Bernardeau et al. 2006; Heczko et al. 2006). Lactobacillus spp. produce antimicrobial factors and bacteriocins which make them attractive candidates for prevention and treatment of a variety of infectious diseases (Aiba et al. 1998; Pascual et al. 1999; Corr et al. 2007). In effect, lactobacilli have been used to reduce Salmonella loads and eradicate various pathogens from chickens, pigs and other animals (Pascual et al. 1999; Walsh et al. 2008; Chen et al. 2012), making them a sensible choice to evaluate for marine mammal health promotion.
With the aim of exploring complementary health management strategies for marine mammals, we embarked on an effort to isolate and identify candidate probiotic lactobacilli from the indigenous microbiota of bottlenose dolphins (T. truncatus).
Samples were collected from 38 bottlenose dolphins at the Navy Marine Mammal Program (MMP), a programme that has been active for more than 50 years. Navy dolphins are housed in netted enclosures in San Diego Bay, California, and routinely work in the open ocean. They are fed restaurant-quality frozen–thawed fish (mackerel, herring, capelin and squid), receive routine antihelminthics, are observed daily, have routine physical examinations by highly experienced veterinarians and are part of a vigilant preventive medicine programme. Voluntary, trained behaviours are used to aid in routine sample collection, including blood, gastric fluid and faecal samples, and blood panel reference ranges amongst healthy animals have been published for this group of dolphins (Venn-Watson et al. 2007, 2011b). While the average age of dolphins in the wild is approximately 24 years, an increasing number of Navy dolphins are living 40–50 years (Venn-Watson et al. 2011a).
The Navy Marine Mammal Program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and adheres to the national standards of the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Animal Welfare Act. As required by the Department of Defense, the MMP's animal care and use programme is routinely reviewed by an Institutional Animal Care and Use Committee (IACUC) and the Department of Defense Bureau of Medicine.
Samples included in this study were oral swabs (from the gingival sulcus), rectal swabs and approximately 5 ml of gastric fluid from each dolphin, all taken from each animal on the same date. Oral and rectal swabs were collected using BD BBL™ CultureSwab™ Plus Amies Medium (Sparks, MD, USA) and subsequently stored at −80°C. Gastric fluid samples were collected and frozen in Brucella broth supplemented with 20% glycerol. Thirty-one dolphins were considered healthy at the time of sampling. Of the remaining seven dolphins in the study, gastric endoscopic evaluation confirmed that two dolphins had erosive or ulcerative esophagitis and five animals had erosive or ulcerated gastritis. Dolphins were sampled at multiple time points from November 2007 to December 2008, creating a total of 119 oral swabs, 119 rectal swabs and 119 gastric fluid specimens. In addition, milk samples were collected from three lactating female dolphins as lactobacilli have been isolated from human breast milk and that of other mammals (Diaz-Ropero et al. 2007; Lara-Villoslada et al. 2007), and oral swabs were collected from their nursing calves as part of routine health assessments. For milk collection, the skin surrounding the teat was wiped with 70% isopropyl alcohol and then rinsed and wiped with sterile water prior to application of the modified manual breast pump for sample acquisition. Milk samples were stored in Brucella broth with 20% glycerol. All samples were immediately stored at −80°C and shipped frozen to our (M.A.D., E.P.G., J.V.) laboratory on dry ice.
Samples were either plated directly on de Man, Rogosa and Sharpe (MRS), Brucella, sheep blood or chocolate agar, or enriched for 24–48 h in MRS or Brucella broth and then plated on these agar media. Blood and chocolate agar were obtained from Remel (Lenexa, KS, USA); MRS and Brucella media were obtained from BD (Franklin Lakes, NJ, USA). Blood, Brucella and chocolate media were used to obtain greater isolate diversity, while MRS media was chosen for enrichment and selection of lactic acid bacteria. A greater emphasis was given to screening rectal swabs and gastric fluid, as these specimens were likely to contain lactobacilli, according to studies carried out in other animals (Neville and O'Toole 2010) and to preliminary data obtained by 16S rDNA clone library sequencing from these dolphin specimens (E.M. Bik et al., personal communication). All samples were incubated under anaerobic or microaerobic culture conditions at 37°C to simulate the host environment, for as long as 7 days, until colony growth was apparent. Isolates displaying different colony morphologies (about 2–3 per plate) were subcultured for further study. Pure cultures made from isolated colonies were stored at −80°C in Brucella broth supplemented with 20% glycerol.
Cultured bacterial isolates were identified by analysis of the 16S ribosomal RNA gene (rDNA). Partial 16S rDNA sequencing was performed to screen for lactobacilli; only isolates identified as Lactobacillus spp. were fully sequenced. PCR was performed using the FastStart PCR Master Mix (Roche, Indianapolis, IN, USA) and universal bacterial primers 27f (5′-GAG TTT GAT CCT GGC TCA G- 3′) and 1525r (5′-AGA AAG GAG GTG ATC CAG CC-3′) (Rainey et al. 1996). Other primers used include universal bacterial primers B-V3 (5′-ACG ACA GCC ATG CAG CAC CT-3′) and BR5-V1 (5′-GAA GAG TTT GAT CAT GGC TCA G- 3′) (Luna et al. 2007), and 18S rRNA gene primers ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) to detect yeasts (Zachow et al. 2009). Template DNA was obtained by colony lysis, or purified from pure bacterial cultures using MO BIO UltraClean DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) or MagNA Pure Nucleic Acid Isolation kit (Roche). A variety of DNA extraction and PCR sequencing conditions were employed as these can vary considerably for different organisms. Purified amplicons (QIAquick kits; Qiagen, Valencia, CA, USA) were sequenced at SeqWright. Sequence analysis was performed using VectorNTI (Invitrogen, Carlsbad, CA, USA), SeqMatch, available through the Ribosomal Database Project II (RDPII database, http://rdp.cme.msu.edu/index.jsp) (Maidak et al. 2001), and BLAST (Altschul et al. 1997) (National Center for Biotechnology Information, available through http://www.ncbi.nlm.nih.gov/BLAST/). Isolates identified as Lactobacillus spp. by 16S rDNA sequencing were further characterized biochemically using API 50 CHL (bioMérieux, Marcy l'Etoile, France) and by DiversiLab repPCR (bioMérieux) genomic fingerprinting. The repPCR fingerprinting was performed as instructed by the manufacturer (Woods et al. 1993; Healy et al. 2005), using genomic DNA extracted from pure bacterial cultures with the MO BIO UltraClean DNA isolation kit.
Ribosomal RNA sequences of isolates identified as Lactobacillus spp. were aligned using the Greengenes NAST aligner (DeSantis et al. 2006) and phylogenetically analysed using the Greengenes version of the ARB software package (Ludwig et al. 2004). A neighbour-joining tree was generated using a 1323-bp column filter, a Jukes-Cantor correction and a bootstrapped version building 1000 trees.
Pathogen growth inhibition assays were performed to assess the probiotic properties of candidate probiotic strains, as previously described (Schillinger and Lucke 1989; Jacobsen et al. 1999; Tzortzis et al. 2004), with modifications. These assays involved growth of spot inocula of probiotic candidate strains ('effectors') on agar to allow secretion and diffusion of growth inhibitory factors. This step was followed by overlaying the plate with a pathogen culture ('indicator') and ultimately measuring the pathogen growth inhibition zone. Pathogens used as 'indicator' organisms included a marine mammal-derived isolate of Salmonella enterica serotype Enteritidis (strain MMP-3466467), a human enterohaemorrhagic E. coli (EHEC; strain EHEC-JV.112) and a human-derived enterotoxigenic E. coli (ETEC; strain ETEC-JV.3A5). Strains EHEC-JV.112 and ETEC-JV.3A5 were clinical isolates obtained from the Microbiology Laboratories of the Department of Pathology and Microbiology at Texas Children's Hospital, Houston, TX. The Salmonella MMP-3466467 strain had been isolated from a sea lion faecal sample collected in June 2006 at the MMP location in San Diego. The animal was diagnosed with gastroenteritis and mixed viral (PCR positive for Calicivirus) and bacterial infections (heavy growth of E. coli and Salmonella). Clinical signs included anorexia, lethargy and an erosive oral lesion.
Candidate probiotic 'effector' strains were grown 18–24 h anaerobically at 37°C in MRS broth. Two microlitre (2 μl)-droplets of standardized culture broth containing approximately 109 cells ml−1 (as estimated by OD600 absorbance readings) were spotted onto MRS agar and incubated anaerobically for 24 h at 37°C. The three pathogen 'indicator' strains were grown aerobically in brain–heart infusion (BHI) broth for 18–24 h at 37°C and used to inoculate 7 ml soft BHI agar (0·7% agar; molten and tempered to 45°C) to obtain standardized bacterial suspensions of 107 cells per plate. Plates containing effector strain growth spots (3 mm radius each) were carefully overlaid with indicator organism cell suspensions and incubated aerobically for 18–24 h at 37°C to obtain bacterial lawns. The radius of each pathogen growth inhibition zone was measured in millimetres. Assays were performed twice, both times in triplicate, and results are presented as the means and standard deviations. Statistical analyses (anova, P < 0·05) were performed using Stata (StataCorp, College Station, TX, USA). An inhibition radius <4 mm was considered noninhibitory; a radius between 4 and 7 mm was designed as intermediary inhibitor, and a radius exceeding 7 mm was considered strongly inhibitory. Lactobacillus salivarius ATCC 11741, originally isolated from the human oral cavity (Rogosa et al. 1953), and recently sequenced as a reference genome by the NIH Human Microbiome Consortium (http://genome.jgi.doe.gov/HumanMicr/HumanMicr.info.html), was used as a reference strain and positive control in these assays.
A high-throughput quantitative immunoassay was developed and used to screen for candidate bacteria with the ability to affect TNF secretion by host cells, based on previously described methods (Lin et al. 2008; Jones and Versalovic 2009), with the following modifications. Wells from 96-well plates containing MRS broth were inoculated using a fresh colony of the probiotic candidate and incubated anaerobically at 37°C for 24 h. Cultures were subcultured using fresh 96-well plates with MRS broth, incubating anaerobically at 37°C for 24 h. OD600 readings were taken to ensure bacterial growth to late log phase and to make adjustments when necessary. Conditioned media containing secreted bacterial factors were prepared by filtering the culture material (0·22 μm pore size) to remove bacterial cells, then vacuum drying the filtrates and resuspending them in equal volumes of RPMI 1640 media (Sigma-Aldrich, St Louis, MO, USA). Plates with conditioned media were kept at 4°C for 24 h and then stored for up to 1 week at 4°C or at −20°C until use.
THP-1 human monocytoid cells (ATCC TIB-202) were cultured in RPMI 1640 media supplemented with 10% foetal bovine serum. To challenge THP-1 cells, conditioned media (1% v/v; 200 μl final volume) were added to cell cultures containing approximately 50 000 THP-1 cells and incubated at 37°C with 5% CO2 for 3 h 30 min. MRS broth-matched controls (no bacteria; also vacuum-dried and resuspended in RPMI) were used to detect the effects of culture media on THP-1 cells. Supernatants of the THP-1 cells were tested for TNF production, which was measured by quantitative ELISA (R&D Systems, Minneapolis, MN, USA) as previously described (Jones and Versalovic 2009). These assays were performed in duplicates, and results are presented as the means and standard deviations of TNF quantities secreted by THP-1 cells exposed to bacterial factors. Statistical analyses (anova, P < 0·05) were performed using Stata. As references, we tested a collection of type strains of Lactobacillus, which included Lact. acidophilus ATCC 4796, Lact. brevis subsp gravesensis ATCC 27305, Lact. buchneri ATCC 11577, Lact. casei ATCC 334, Lact. delbrueckii subsp bulgaricus ATCC 11842, Lact. fermentum ATCC 14931, Lact. gasseri ATCC 33323, Lact. johnsonii ATCC 33200, Lact. paracasei ATCC 25302, Lact. plantarum ATCC 14917, Lact. reuteri ATCC 53609, Lact. reuteri ATCC 55148, Lact. reuteri ATCC 55730, Lact. reuteri ATCC PTA 6475, Lact. rhamnosus ATCC LSM2-1, Lact. ruminus ATCC 25644 and Lact. salivarius ATCC 11741.
A total of 307 isolates were cultured from 21 rectal swabs, 25 gastric fluid specimens, 2 oral swabs and 3 milk specimens obtained from 38 dolphins, using MRS and Brucella media. Each isolate corresponded to a distinct morphotype obtained per plate with the sample and culture conditions employed.
The majority of isolates were obtained using MRS (150 isolates, 48·9%) and Brucella (118 isolates, 38·4%) media; some isolates were cultured using sheep blood (27 isolates, 8·8%) and chocolate (12 isolates, 3·9%) agar. One hundred and fifty-three (49·8%) isolates were recovered from rectal swabs, 97 isolates (31·6%) were obtained from gastric fluid, 53 isolates (17·3%) were derived from milk, and four isolates (1·3%) were cultured from nursing calf oral swabs.
Two hundred and fifty isolates (81·4%) were identified by partial 16S rDNA sequencing; 58 (18·9%) isolates remained unidentified. The most prevalent genera cultured from dolphin samples under the conditions employed were Staphylococcus (77 isolates, 25·1%) and Escherichia/Shigella (61 isolates, 19·9%), not surprisingly as both are able to grow in MRS media. Staphylococci were isolated from all specimen types and were predominant in gastric fluid and milk, whereas Escherichia/Shigella were mainly isolated from rectal swabs. Other genera recovered included Achromobacter, Actinobacillus, Alcanivorax, Bacillus, Citrobacter, Clostridium, Corynebacterium, Desulfovibrio, Edwardsiella, Enterococcus, Eubacterium, Lactobacillus, Macrococcus, Ochromobactrum, Peptostreptococcus, Photobacterium, Pigmentiphaga, Pseudomonas, Rhodococcus, Stenotrophomonas and Streptococcus. Although 52 isolates were recovered from dolphin milk samples, no lactobacilli were recovered from these samples; the isolates were predominantly staphylococci. A detailed list of isolates recovered and their specimen of origin is provided in Table 1.
|Rectal swab
|Gastric fluid
|Oral swab
|Total (%)
|Achromobacter denitrificans
|1 (0·3%)
|Actinobacillus scotiae
|6 (2·0%)
|Alcanivorax dieselolei
|2 (0·7%)
|Bacillus thuringiensis
|1 (0·3%)
|Citrobacter braakii
|1 (0·3%)
|Citrobacter freundii
|4 (1·3%)
|Clostridium ghonii
|1 (0·3%)
|Clostridium perfringens
|3 (1·0%)
|Clostridium sordellii
|2 (0·7%)
|Corynebacterium tuberculostearicum
|1 (0·3%)
|Desulfovibrio spp.
|1 (0·3%)
|Edwardsiella ictaluri
|1 (0·3%)
|Edwardsiella tarda
|13 (4·2%)
|Enterococcus casseliflavus
|3 (1·0%)
|Enterococcus silesiacus
|2 (0·7%)
|Enterococcus termitis
|13 (4·2%)
|Eubacterium tenue
|1 (0·3%)
|61 (19·9%)
|Lactobacillus salivarius
|4 (1·3%)
|Lactobacillus spp.
|3 (1·0%)
|Macrococcus caseolyticus
|1 (0·3%)
|Ochrobactrum tritici
|1 (0·3%)
|Peptostreptococcus stomatis
|2 (0·7%)
|Photobacterium damselae
|7 (2·3%)
|Pigmentiphaga kullae
|1 (0·3%)
|Propionibacterium avidum
|2 (0·7%)
|Pseudomonas otitidis
|1 (0·3%)
|Rhodococcus corynebacteroides
|1 (0·3%)
|Staphylococcus caprae
|12 (3·9%)
|Staphylococcus cohnii
|1 (0·3%)
|Staphylococcus delphini
|9 (2·9%)
|Staphylococcus epidermidis
|22 (7·2%)
|Staphylococcus hominis
|10 (3·3%)
|Staphylococcus pasteuri
|10 (3·3%)
|Staphylococcus warneri
|13 (4·2%)
|Stenotrophomonas maltophilia
|1 (0·3%)
|Streptococcus australis
|2 (0·7%)
|Streptococcus mitis
|1 (0·3%)
|Streptococcus parasanguinis
|2 (0·7%)
|Possible yeast (18S rDNA positive)
|26 (8·5%)
|Not sequenced or identified
|58 (18·9%)
|154 (50·2%)
|97 (31·5%)
|4 (1·3%)
|52 (16·9%)
Of the 307 bacterial strains isolated from dolphin samples, seven were Lactobacillus spp. Four Lact. salivarius isolates were recovered from rectal swab samples, all from dolphin 'C'. The isolates had 16S rDNA sequences that were more than 99·8% identical to each other. White, circular, smooth colonies formed on MRS and Brucella agar after 24 h of anaerobic incubation at 37°C, and an alpha-haemolytic phenotype was evident when they were cultured on sheep blood agar. These isolates were nonmotile and did not form spores. They were Gram-positive, catalase-negative, facultative anaerobic bacilli that appeared as single cells and as pairs of cells by microscopy.
In addition, we obtained three novel Lactobacillus spp. isolates from the gastric fluid of dolphin 'Z', with 16S rDNA sequences 99·54% identical to each other and with 96·3% 16S rDNA sequence identity to Lactobacillus ceti, a species previously isolated from beaked whales (Vela et al. 2008). The isolates formed small, grey, nonhaemolytic round colonies after 3–7 days of incubation on sheep blood agar at 37°C, under anaerobic conditions. They were Gram-positive, anaerobic, catalase-negative bacilli present as single cells and as pairs of cells. The 16S rDNA sequences obtained from these isolates were 99·8% identical to sequences detected directly in gastric fluid and rectal swab samples from several dolphins studied by whole-community, broad-range 16S rDNA PCR and clone library sequencing (E.M. Bik et al., personal communication). The novel Lactobacillus sp. and the Lact. salivarius isolated in this study had 92·71% 16S rDNA sequence identity to each other. The results of a 16S rDNA-based phylogenetic analysis of lactobacilli found in both studies are provided in Fig. 1. These sequence data have been submitted to the GenBank database under accession numbers JX142127 through JX142133.
RepPCR genomic fingerprinting of lactobacilli isolated in this study revealed that, as with ribosomal DNA sequence analysis, the four Lact. salivarius isolates, MMP005, MMP006, MMP007 and MMP077, obtained from dolphin 'C', grouped together to form one clade, as did the novel Lactobacillus isolates MMP239, MMP241 and MMP242, obtained from dolphin 'Z' (data not shown). Further comparative genomic analyses using microarray or whole-genome sequencing technologies will allow us to refine the phylogenetic relation and genome variability amongst the Lact. salivarius and the novel Lactobacillus sp. isolates, respectively, and determine whether these isolates represent distinct strains of each species (Raftis et al. 2010).
Lactobacillus salivarius isolates were characterized using API 50 CHL strips. These biochemical test kits consist of 50 carbohydrate utilization/fermentation assays used to characterize lactobacilli and related micro-organisms. Substrate utilization results, summarized in Table 2, showed that dolphin-derived Lact. salivarius isolates MMP005, MMP006, MMP007 and MMP077 yielded identical biochemical and carbohydrate fermentation profiles and were positive for d-ribose, d-adonitol, d-galactose, d-glucose, d-fructose, d-mannose, d-mannitol, d-sorbitol, N-acetylglucosamine, d-maltose, d-lactose, d-melibiose, d-saccharose (sucrose), d-trehalose and d-raffinose. These profiles differed slightly from reference strain Lact. salivarius ATCC 11741, which displayed a typical carbohydrate fermentation profile of Lact. salivarius (Jacobsen et al. 1999; Neville and O'Toole 2010), and did not utilize d-ribose or d-adonitol, but was positive for l-rhamnose. The three novel Lactobacillus isolates (MMP239, MMP241 and MMP242) were fastidious and failed to yield sufficient growth for API 50 CHL assays.
|Enrichment media
|Isolation media
|Pathogen growth inhibition
|TNF modulation
|API-CH profileb
|Lactobacillus salivarius
|Rectal swab
|Lact. salivarius
|Rectal swab
|Lact. salivarius
|Rectal swab
|Lact. salivarius
|Rectal swab
|Novel Lactobacillus sp.
|Gastric fluid
|Novel Lactobacillus sp.
|Gastric fluid
|Novel Lactobacillus sp.
|Gastric fluid
|ATCC 11741c
|Lact. salivarius
Lactobacillus salivarius candidate probiotic isolates MMP005, MMP006, MMP007 and MMP077, obtained from dolphin 'C' rectal swabs, strongly inhibited growth of the marine mammal-derived Salm. enterica serotype Enteritidis (strain MMP-3466467) and human pathogens EHEC-JV.112 and ETEC-JV.3A5 (Fig. 2). Similarly, Lact. reuteri ATCC 55730 and Lact. salivarius ATCC 11741, references established as probiotic strains (Rogosa et al. 1953; Valeur et al. 2004), also inhibited these enteric pathogens. Other marine mammal-derived isolates tested included Staphylococcus sp. MMP123 and strains of Bacillus thuringiensis, Edwardsiella ictaluri, Enterococcus casseliflavus, Escherichia/Shigella, Photobacterium damselae and Staphylococcus spp., all of which yielded inhibition radii smaller than 4 mm and were noninhibitory (not shown). As mentioned earlier, the novel Lactobacillus sp. isolates were not studied further as they were fastidious and yielded insufficient growth. The growth medium alone had no growth inhibitory effect on any of the bacterial pathogens tested (not shown).
Secreted factors from Lact. salivarius isolates MMP005, MMP006, MMP007 and MMP077 were assayed for their ability to modulate TNF production by THP-1 human monocytoid cells. Bottlenose dolphin cell lines (Beineke et al. 2010) were not available for this study, so use of a human cell line was a valuable model for screening purposes. We found that conditioned media from Lact. salivarius isolates stimulated TNF production significantly more than the MRS medium control (Fig. 3). Secreted factors from the bottlenose dolphin-derived Lact. salivarius strains exhibited TNF-stimulatory features similar to the effects of human-derived reference probiotic strains Lact. salivarius ATCC 11741 and Lact. reuteri ATCC 55730. The latter is a known TNF-stimulatory strain previously studied in our laboratory and elsewhere (Valeur et al. 2004; Lin et al. 2008; Jones and Versalovic 2009). Other reference lactobacilli tested yielded various degrees of TNF inhibition.
The main objectives of this study were to isolate gastrointestinal bacteria from bottlenose dolphins and to evaluate selected isolates for beneficial properties. Following the laboratory screening of 307 bacterial isolates from oral, gastric and rectal specimens collected from 38 dolphins in the U.S. Navy Marine Mammal Program, gastrointestinal lactobacilli and other Gram-positive bacteria were evaluated for defined beneficial features. Novel Lactobacillus spp. phylogenetically related to Lact. ceti were identified by direct sequencing and bacteriological culture. Gastrointestinal Lact. salivarius isolates obtained by bacteriological culture from T. truncatus demonstrated the ability to suppress the proliferation of enteric pathogens and stimulated TNF production in mammalian myeloid cells. These new isolates represent an initial foray into the characterization of lactobacilli with potentially beneficial health-promoting features in marine mammals.
Microbiome studies suggest that microbial communities have co-evolved with their hosts (Ley et al. 2008; Spor et al. 2011) and highlight the roles that micro-organisms may play in their co-habitation with them (Muegge et al. 2011; Clemente et al. 2012). The mammalian gastrointestinal microbiota includes a diverse group of micro-organisms that exert functions with potential probiotic activities (Ley et al. 2008; Gerritsen et al. 2011). Probiotics may confer beneficial effects as modulators of the host microbiota, enhancing resilience and resistance to pathogens and other perturbations by promoting a healthier microbial ecology of the gut (Gerritsen et al. 2011; Wallace et al. 2011). These studies bring to light the value of culture-based methods to complement the predominantly molecular-based approaches used to study the microbiome at bacterial membership and functional levels (16S rDNA surveys, metagenomics, metabolomics, etc.). Efforts to culture symbiotic microbes enable a better understanding of the biology of the host microbiota, particularly given the historical priority given to studying organisms of traditional medical and veterinary relevance, namely pathogens (Spor et al. 2011).
Lactobacillus salivarius is part of the indigenous microbiota of the mammalian gastrointestinal tract and was first isolated from the human oral cavity (Rogosa et al. 1953). The species has also been isolated from human breast milk (Martin et al. 2006), a plausible route for gastrointestinal colonization (Martin et al. 2006; Jara et al. 2011), and from food and environmental sources (Neville and O'Toole 2010). Lactobacillus salivarius displays a high level of genomic diversity (Raftis et al. 2010). The 2·13-Mb genome of strain UCC118 has been annotated (Claesson et al. 2006), revealing a circular genomic megaplasmid, which is a novel feature amongst lactobacilli, and may be implicated in its adaptation to a wide range of environments and probiosis (Raftis et al. 2010). Comparative genomics studies will provide further understanding of strain diversity within the species as genomic characterization of various Lact. salivarius strains isolated from different habitats is completed by scientists at the NIH Human Microbiome Consortium and other laboratories (Raftis et al. 2010; Kergourlay et al. 2012; Langa et al. 2012).
To our knowledge, Lactobacillus spp. have not yet been reported in dolphins. Given the growing scientific evidence supporting successful probiotic applications of lactobacilli in humans and other animals, these organisms were the focus of this study. We successfully recovered Lactobacillus spp. from bottlenose dolphin specimens. Lactobacillus salivarius isolates were recovered from dolphin rectal swab samples, and a novel Lactobacillus species was recovered from dolphin gastric fluid. Lactobacillus-like 16S rDNA sequences were also detected in dolphin gastric fluid and rectal swab samples by broad-range rDNA PCR and clone sequencing (E.M. Bik et al., personal communication), with some of the amplified sequences being 99·8% identical to the 16S rDNA sequences of the novel Lactobacillus strains that were cultured in this study. The 16S rDNA sequences of the novel Lactobacillus are 96·3% identical to the sequence of a cultivated, marine mammal-derived Lact. ceti (Vela et al. 2008). Isolates of the novel Lactobacillus species were fastidious and grew too slowly to perform standard biochemical assays.
Our focused search for lactic acid bacteria yielded isolates from genera other than Lactobacillus and may include organisms with beneficial features. Given the limited information available on marine mammal indigenous gastrointestinal microbiota (Venn-Watson et al. 2008), with existing studies primarily addressing their role in the digestion of food (Olsen et al. 1994), the isolation and identification of a diverse set of members of the bottlenose dolphin indigenous microbiota, including lactobacilli, should significantly contribute to the understanding of the dolphin microbiome. Future studies characterizing the dolphin gastrointestinal microbiota will help elucidate the contribution of these organisms to their host's health.
Pathogens that affect gastrointestinal dolphin health have been previously reviewed elsewhere (Baskin 2006; Venn-Watson et al. 2008) and include astroviruses (Rivera et al. 2009), Campylobacter spp. (Foster et al. 2004; Stoddard 2005; Goldman et al. 2011), Clostridium spp. (Buck et al. 1987), Edw. tarda (Coles et al. 1978), Giardia and Cryptosporidium species (Hughes-Hanks et al. 2005), Helicobacter (Harper et al. 2000, 2002) and Salmonella spp. (Foster et al. 1999; Smith et al. 2002).
The action and effectiveness of probiotics can be evaluated by a variety of screening techniques, which include assessment of antimicrobial activities, modulation of cytokine production, host colonization and resistance to acid or bile stress (Rastall et al. 2005; Guarner et al. 2012). Antagonistic properties of probiotic organisms have been demonstrated in vitro and in vivo in bifidobacteria, lactobacilli and combinations of probiotics (Neville and O'Toole 2010; Jara et al. 2011; Langa et al. 2012). Adaptation of Lact. salivarius to the gastrointestinal niche has entailed the capacity to inhibit pathogens and to tolerate the host's antimicrobial defences, granting it considerable interest as a promising probiotic species (Neville and O'Toole 2010). Lactobacillus salivarius have been shown to exhibit antagonistic properties against Listeria (Barrett et al. 2007; O'Shea et al. 2011), Salmonella (Pascual et al. 1999; Casey et al. 2007), Campylobacter (Robyn et al. 2012) and other pathogenic bacteria (Corr et al. 2007; Neville and O'Toole 2010) and have been utilized to eradicate Helicobacter infection in humans and other mammals (Aiba et al. 1998; Canducci et al. 2002). The capacity of Lact. salivarius to prevent infection has been attributed to a number of features including the production of salivaricin (Barrett et al. 2007) and other bacteriocins (Corr et al. 2007; O'Shea et al. 2011), prevention of colonization (Kabir et al. 1997), interference with cytokine induction and virulence gene expression (Ryan et al. 2009), and modulation of human intestinal cells (O'Hara et al. 2006). In addition, standard Lactobacillus metabolism produces antimicrobials such as hydrogen peroxide, lactic acid and other organic acids that can inhibit pathogen growth by chelating essential nutrients or sensitizing bacteria to antimicrobial assault (Neville and O'Toole 2010) and can decrease pathogen toxin production, such as Shiga toxin by EHEC (Carey et al. 2008). In this study, we evaluated the probiotic potential of dolphin-associated Lact. salivarius isolates by testing their ability to inhibit the growth of several pathogens in vitro. The strong inhibition of selected marine mammal and human pathogens by dolphin-derived Lact. salivarius isolates MMP005, MMP006, MMP007 and MMP077 confirms studies carried out in humans and other animal models and supports the selection of these organisms for further consideration as candidate probiotics for marine mammals.
Lactobacillus salivarius have been used for the capacity to modulate both humoral and cell-mediated immunity in humans and various animal models (Neville and O'Toole 2010). In effect, Lact. salivarius have been shown to increase antibody production and reduce cell-mediated immune responses in intestinal cells (Brisbin et al. 2011), to evoke intestinal immunomodulatory responses (Walsh et al. 2008) and to modulate cytokine induction by pathogens (Ryan et al. 2009). The production of exopolysaccharides contributes to its immunomodulatory, antitumorigenic and prebiotic properties and is valued in the food industry (Neville and O'Toole 2010; Raftis et al. 2010). The functional immunoassays we performed to study TNF modulation in vitro have been validated in various in vivo studies (Pena et al. 2005; Lin et al. 2008; Oksaharju et al. 2013) and are useful to screen for bacteria with immunomodulatory capacities (Thomas et al. 2012). In this study, we determined that TNF production by human monocytoid cells is stimulated by secreted factors from Lact. salivarius strains isolated from dolphins. This is in contrast to other well-characterized Lact. salivarius strains, reported to induce an anti-inflammatory environment in the host (Neville and O'Toole 2010). Reduction in TNF expression has been shown to be of benefit to the host in cases involving chronic inflammatory conditions such as IBD, IBS and autoimmune diseases such as rheumatoid arthritis, Crohn's disease and psoriasis (Jones and Versalovic 2009; Thomas et al. 2012), as TNF contributes to the pathogenesis of inflammatory diseases. However, the TNF signalling pathway plays a crucial role in the activation of the innate immune response to pathogens (Secher et al. 2009) and has been proven necessary for protection against intracellular microbes such as Salmonella and Listeria, Mycobacterium tuberculosis and other Gram-positive and Gram-negative organisms (Dinarello 2003). TNF induction has been shown to be beneficial to the host in specific contexts, such as protection against pathogens (Secher et al. 2009) and neoplasia (Calzascia et al. 2007; Iyer et al. 2008). The benefits of induction of pro-inflammatory cytokine secretion by probiotic lactobacilli have been well documented for established immunostimulatory probiotic Lact. reuteri 55730 (Valeur et al. 2004). Desired immunoprobiotic traits should be chosen in accordance with specific treatment needs. Hence, Lact. salivarius-enhanced stimulation of host immunity by TNF induction can be beneficial to dolphins by contributing to the dolphin-derived Lact. salivarius strains' anti-infective properties, a salient beneficial feature contingent upon clinical trial validation. Immunomodulatory compounds secreted by dolphin-derived Lact. salivarius strains can be characterized as previously described for other probiotic lactobacilli (Thomas et al. 2012), ideally using dolphin cell lines, and thus facilitate understanding of cytokines and pathways that are activated in the cells of the natural host.
The Human Genome Sequencing Center (HGSC) at Baylor College of Medicine has engaged in a study to sequence the genomes of 24 mammals. The genome of the bottlenose dolphin has been sequenced from DNA obtained from a healthy female dolphin from the Navy Marine Mammal Program (http://www.hgsc.bcm.tmc.edu/). Comprehensive studies of the marine mammal microbiome and genome should allow us to better understand the co-evolution of these unique hosts with their microbiomes and environment (Muegge et al. 2011; Spor et al. 2011). With a more complete molecular survey of the dolphin microbiota, and the use of functional metagenomics, a more targeted, customized approach for identifying and culturing probiotic bacteria in dolphins will be greatly facilitated. We hope to further evaluate the isolates obtained in this study by monitoring colonization and effects on the dolphin microbiota, characterizing the active immunoprobiotic compounds secreted by dolphin lactobacilli, assessing immune pathway activation and production of other indicators of immune function such as IL-6, IL-10 and IL-12, using dolphin host cell lines, and examining the contributions to health outcomes. Additional characterization of these lactobacilli will aid in the understanding of their biology and function within their cetacean hosts.
In conclusion, we isolated and characterized Lactobacillus spp. indigenous to the dolphin microbiota. To the best of our knowledge, this is the first study to isolate Lactobacillus spp. from the bottlenose dolphin. Dolphin-derived Lact. salivarius strains appear to be capable of stimulating TNF production by mammalian cells and inhibiting the growth of select marine mammal and human pathogens. As has been the case for other Lact. salivarius strains in other host species, these dolphin-associated isolates deserve further consideration as candidate probiotics in future clinical studies. Further studies are needed to characterize these isolates fully, as well as to optimize culturing of the novel Lactobacillus sp. and establish whether it might also have beneficial properties and applications. The introduction of candidate probiotic strains indigenous to marine mammals will offer new possibilities for the prevention and treatment of infectious diseases and gastrointestinal disorders in these unique animals.
This work was supported by Office of Naval Research Grant N000140710287 (J.V., D.A.R.). E. P. Gaston was supported by the SMART program. We would like to thank Laura A. Millecker and Ruth Ann Luna for assistance with DiversiLab strain typing, and Angela Major for microscopy work. D.A.R. is supported by the Thomas C. and Joan M. Merigan Endowment at Stanford University.