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POLL: Command and Conquer Games
Atal posted on Jan 05, 2011 4:50:10 AM - Report post
Zero Hour. I was a bit disappointed with Tiberian Wars and Kane's wrath.
[Edited by Atal, 1/5/2011 4:51:58 AM]
LordVenator posted on Jan 05, 2011 4:50:19 AM - Report post
C&C3: Tiberium* Wars
MSI Z87-G43 Gaming
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Stinger11 posted on Jan 05, 2011 1:23:29 PM - Report post
originally posted by Rise_Against
You missed one: Red Alert 2: Yuri's Revenge (Expansion)
But I believe Tiberium Wars was the best. Then they had to go and mess up CNC 4.
Edit: You also missed Tiberian Sun
[Edited by Rise_Against, 1/5/2011 2:32:55 AM]
Yeah, i forgot them
Stinger11 posted on Jan 05, 2011 1:24:18 PM - Report post
CNC 4 the special effects are cool but the units looked disappointing and cartonnish.
Stinger11 posted on Jan 05, 2011 1:24:58 PM - Report post
Generals Zero Hour was great a good game to pass time. Also that Contra mod is great you should see it: contra.cncguild.net/oldsite/Eng/index.php
[Edited by Stinger11, 1/5/2011 1:28:25 PM]
Stinger11 posted on Jan 05, 2011 1:29:15 PM - Report post
I liked Renegade it was a great First Person Shooter.
RageaholicRick posted on Jan 05, 2011 1:36:09 PM - Report post
I like Command And Conquer: Generals-Zero Hour the best. I still play that alot.
Varacka posted on Jan 05, 2011 1:48:38 PM - Report post
im bound to get flamed for this but i loved the idea of teamwork actually being needed in cnc4 instead of 1 man spamming 50k units and winning in under 5 mins
Anything less then peace will destroy us all.
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Does anybody have anything to say on this board - or are most SH's from Wisco, therefore engrossed in Favre news???
Well, I do not own this stock, but it has been a very strong performer. It's steadness in THIS market speaks volumes. It is the very lack of problems that makes posts on this message board in frequent. (In addition to it's snaller size.) So as a mere visitor and fellow utility investor, congratulations!ycapa
Think the other poster summed it up...this stock is exceptionally boring, hasn't done anything and only folks in southern wisconsin know about the company.I'm going to invest soon for the long term DRIP growth. Love their sustained and growing 4% dividend...
Do it - I think the initial investment in that program is only like $500. I havent been to Madison in 10 years - I look forward to getting up there before the Summer is out. Based on the mailings that I get from MGEE it appears much has changed!Brian
Yep.Many are from Wisconsin and many are from Madison.I have a coffee mug that says "Madison.... an alternative to reality"Farve seems to be looking for an alternative to reality also.MGEE seems to be chugging right along. I just wish that the price would move a bit more. I have been swing trading this stock for the past 4 years. sb
at what levels are you trading this?


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Jimmy Kimmel really does love Ben Affleck.
The talk show host – who memorably declared "I'm F---king Ben Affleck" in a music video Sunday night – cannot stop singing the actor's praises.
"Ben could not have been more sporting," he told PEOPLE of his video costar. (The song was designed as a retaliation against Kimmel's girlfriend Sarah Silverman for her clip, "I'm F---king Matt Damon.) "He told us, 'Listen, if we're going to do this, we've got to do it right. I don't want you guys to worry about my image.' "
In the video (available online), Affleck and Kimmel declare their love while backed by an A-list gospel choir including Cameron Diaz, Robin Williams, Ashlee Simpson and Don Cheadle.
There are also non-singing cameos by Brad Pitt and Harrison Ford, who blows the new couple a kiss before driving off in his convertible – revealing a "Honk if you're f---ing Ben Affleck" bumper sticker.
"That was his real car," Kimmel says of the legendary Indiana Jones star. "He's still got that [sticker] on there!"
So what did Silverman think of the new song? "I suspected Jimmy was on the down low for a long time now," she deadpanned to the New York Daily News. "I wish I could say I was surprised by this."
If she's plotting another salvo, Kimmel isn't quaking in his cut-off denim shorts. "I suppose it's possible that Sarah and Matt Damon are huddled in a cave somewhere planning their revenge," he said. "But one thing I know is, if they are, they won't tell me about it!"
Jimmy Kimmel: Ben Affleck 'Could Not Have Been More Sporting'
Richard Cartwright/ABC


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Students may wish to enhance their primary degree program by minoring in history. Those minoring in history take six history courses, four of which must be upper-division courses (200 level or above).
Education: B.A. and M.A., SUNY Buffalo; M.A. and Ph.D., University of Oklahoma
Areas of Academic Interest: American politics, political history, public history, history of science and medicine, African-American history, and women's history.
Read more about his interest in politics and American culture in Dr. Kneeland's Life of the Mind article.
Media expert: TV interview on Pope Francis' historical address to the U.S. Congress.


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Junebug 2011 is officially over...
Yes, we have celebrated the whole of the 2011 ArtFest in fine form and reached its conclusion. Both of the Guest Artists, who appearances you sponsored, were amazing and added... Read more
International Artist Sandy Skoglund to Speak at the Junebug
Yes, the time has moved along and this Thursday...thanks to YOU, our Kickstarter backers... we are able to bring Sandy Skoglund to Metuchen to give a presentation, regarding creativity and... Read more
Statewide Advertising!
Just had to share the news with all of you: The June issue of NJ MONTHLY magazine
did a FULL PAGE article with photos and story to the JUNEBUG... Read more
We are genuinely 'on the cusp' of the event now! All of the exhibiting artists, musicians and performers have received their assigned times and locations...a massive task by a dauntless... Read more
Just a note to let you know that things are "humming" at "Junebug Central"! Chairpersons and Coordinators are burning the midnight oil to wrap things up. All kinds of final... Read more
Thanks to many, many people with six days still to go, Metuchen's Junebug ArtFest has been successfully funded through its Kickstarter project! What a joy!
In addition to all... Read more
We Are Amazed...
With six days left of our Junebug fundraising campaign, we are 96% funded, needing just $100 more to reach our goal!!!
The generosity of Metuchenites, family members and friends has... Read more
METUCHEN appears with 'the big boys'...!!!!
Kickstarter has done us the honor of listing our little Borough on the main list of cities with Kickstarter projects...NYC, LA, Seattle, Chicago, Nashville, Boston...and yes, imagine in that list... Read more
This IS very exciting! We have 31 backers at this point and two full weeks to go. We can do this!
I feel like we have reached that 'magic middle'... Read more
Greetings to all! Thanks to so many people, we are now "a hair shy" of the halfway mark! Things are now getting interesting. Our 'TEN DOLLARS IN TEN DAYS' project... Read more
Thanks to our most recent supporters, we are one third of the way to meeting our goal! Every person who has backed the Junebug has brought us this far. So... Read more
Update from the Junebug Sculptor!
Here's the latest word from Joy about the signature sculpture she is creating to preside over the event!
She even gives us a peek at the evolving 'Bug! ... Read more
This Week w/ 'The Bug'
Central Jersey weathered a snow-storm on April 1; I think that helped to keep our little Junebug in hibernation...while Team Junebug has done some amazing things to continue the project... Read more
How Exciting!
We have just reached $500! This seems like a real milestone in our process! Again, thanks to all who have helped to make that happen.
Tonight the Junebug Team meets... Read more
Our First Full Week of Kickstarter!
So now we begin our first full week of Kickstarter...exciting! The Junebug team is really hustling at this point to spread the word about the event and line up the... Read more
Project Grows!
First, this is such an exciting moment for us to have "launched"! So many people share talents so generously to make this event happen.
Last Friday evening, the sculptor for... Read more


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Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA
Logar, Katarina
Kopinč, Rok
Bandelj, Petra
Starič, Jože
Lapanje, Aleš
Ocepek, Matjaž
BMC Veterinary Research
Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle.
The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher's exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.
This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.
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Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples
Stabel, J. R.
Bannantine, J. P.
Journal of Clinical Microbiology
This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 102 CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 102 CFU/g for the IS900 element and 103 CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.
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High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle
Plain, Karren M.
Marsh, Ian B.
Waldron, Anna M.
Galea, Francesca
Whittington, Ann-Michele
Saunders, Vanessa F.
Begg, Douglas J.
de Silva, Kumudika
Purdie, Auriol C.
Whittington, Richard J.
Journal of Clinical Microbiology
Johne's disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemar's test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611–622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.
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Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces
Sevilla, Iker A.
Garrido, Joseba M.
Molina, Elena
Geijo, María V.
Elguezabal, Natalia
Vázquez, Patricia
Juste, Ramón A.
Applied and Environmental Microbiology
The enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused by Mycobacterium avium subsp. paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detect M. avium subsp. paratuberculosis in feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02 (an insertion sequence of M. avium subsp. paratuberculosis present in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10 M. avium subsp. paratuberculosis cells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900 and ISMap02 PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection of M. avium subsp. paratuberculosis in fecal samples from cattle and a very valuable tool to be used in PTB control programs.
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Contrasting Results of Culture-Dependent and Molecular Analyses of Mycobacterium avium subsp. paratuberculosis from Wood Bison
Forde, Taya
De Buck, Jeroen
Elkin, Brett
Kutz, Susan
van der Meer, Frank
Orsel, Karin
Applied and Environmental Microbiology
Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.
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Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis
Keller, Selina M
Stephan, Roger
Kuenzler, Rahel
Meylan, Mireille
Wittenbrink, Max M
Acta Veterinaria Scandinavica
Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.
Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.
By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).
There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.
Mycobacterium avium subspecies paratuberculosis; Cattle; Feces; Culture; PCR
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Composite testing for ante-mortem diagnosis of Johne's disease in farmed New Zealand deer: correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR
O'Brien, Rory
Hughes, Alan
Liggett, Simon
Griffin, Frank
BMC Veterinary Research
In the absence of overt clinical signs of Johne's Disease (JD), laboratory based tests have largely been limited to organism detection via faecal culture or PCR and serological tests for antibody reactivity. In this study we describe the application of quantitative faecal PCR for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in New Zealand farmed deer to quantify the bacterial load in cervine faecal samples as an adjunct to an existing serodiagnostic test (Paralisa™) tailored for JD diagnosis in deer. As ELISA has potential as a cheap, high throughput screening test for JD, an attempt was made to assess the sensitivity, specificity and positive/negative predictive (PPV/NPV) values of Paralisa™ for estimating levels of faecal shedding of MAP as a basis for JD management in deer.
Correlations were made between diagnostic tests (ELISA, qPCR, culture and histopathology) to establish the precision and predictive values of individual tests. The findings from this study suggest there is strong correlation between bacterial shedding, as determined by faecal qPCR, with both culture (r = 0.9325) and histopathological lesion severity scoring (r = 0.7345). Correlation between faecal shedding and ELISA reactivity in deer was weaker with values of r = 0.4325 and r = 0.4006 for Johnin and Protoplasmic antigens, respectively. At an ELISA Unit (EU) cutoff of >50 (Johnin antigen) the PPV of Paralisa™ for significant faecal shedding in deer (>104 organisms/g) was moderate (0.55) while the NPV was higher (0.89). At an EU cutoff of ≥150, the PPV for shedding >105 organisms/g rose to 0.88, with a corresponding NPV of 0.85.
The evidence available from this study suggests that Paralisa™ used at a cutoff of 50EU could be used to screen deer herds for MAP infection with sequential qPCR testing used to cull all Paralisa™ positive animals that exhibit significant MAP faecal shedding.
Johne's disease; Paratuberculosis; MAP; Quantitative PCR; ELISA; Deer; Cervus elaphus
Related Articles
Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis
Gao, Anli
Odumeru, Joseph
Raymond, Melinda
Hendrick, Steven
Duffield, Todd
Mutharia, Lucy
Canadian Journal of Veterinary Research
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn's disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne's-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.
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Diagnosis and Molecular Characterization of Mycobacterium avium subsp. paratuberculosis from Dairy Cows in Colombia
Fernández-Silva, J. A.
Abdulmawjood, A.
Bülte, M.
Veterinary Medicine International
The objective of this study was the serological, bacteriological and molecular diagnosis, as well as the molecular characterization of Mycobacterium avium subsp. paratuberculosis (Map) in adult cows of five Colombian dairy herds. Serum samples were tested by an indirect absorbed enzyme–linked immunosorbent assay (ELISA-C). All fecal samples were tested by pooled culture. After that, fecal samples of Map positive pools were tested individually by culture and polymerase chain reaction (PCR). In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. Map isolates were analyzed by the Multilocus Short Sequence Repeat (MLSSR) and the Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were Map positive by culture and PCR. MLSSR and MIRU-VNTR revealed two different strain profiles among eight Map isolates recovered. This study reports the first molecular characterization of Map in one dairy herd in Colombia, the limitations for individual diagnosis of subclinical Map infections in cattle, and the usefulness of pooled fecal samples and environmental sampling for Map diagnosis.
Related Articles
Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland
Good, M
Clegg, T
Sheridan, H
Yearsely, D
O'Brien, T
Egan, J
Mullowney, P
Irish Veterinary Journal
A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johne's disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.
herd ELISA-prevalence; Ireland; Johne's disease; Mycobacterium avium subspecies paratuberculosis; Paratuberculosis
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Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis in a Longitudinal Study of Three Dairy Herds▿
Pradhan, Abani K.
Mitchell, Rebecca M.
Kramer, Aagje J.
Zurakowski, Michael J.
Fyock, Terry L.
Whitlock, Robert H.
Smith, Julia M.
Hovingh, Ernest
Van Kessel, Jo Ann S.
Karns, Jeffrey S.
Schukken, Ynte H.
Journal of Clinical Microbiology
The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis were passively shedding or truly infected with M. avium subsp. paratuberculosis. We also investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by contemporary high-shedding animals (supershedders). The M. avium subsp. paratuberculosis isolates were obtained from a longitudinal study of three dairy herds in the northeastern United States. Isolates were selected from fecal samples and tissues at slaughter from all animals that were culture positive at the same time that supershedders were present in the herds. Shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for the animals at each culture-positive occasion were determined. Using a multilocus short-sequence-repeat technique, we found 15 different strains of M. avium subsp. paratuberculosis from a total of 142 isolates analyzed. Results indicated herd-specific infection patterns; there was a clonal infection in herd C, with 89% of isolates from animals sharing the same strain, whereas herds A and B showed several different strains infecting the animals at the same time. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection. The results of M. avium subsp. paratuberculosis strain typing and observed shedding levels showed that at least 50% of low shedders have the same strain as that of a contemporary supershedder. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis. The sharing of strains between low shedders and the contemporary supershedders suggests that low shedders may have been infected by environmental exposure of M. avium subsp. paratuberculosis.
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Evaluation of a Rapid Fecal PCR Test for Detection of Mycobacterium avium subsp. paratuberculosis in Dairy Cattle▿
Wells, Scott J.
Collins, Michael T.
Faaberg, Kay S.
Wees, Carrie
Tavornpanich, Saraya
Petrini, Kristine R.
Collins, James E.
Cernicchiaro, Natalia
Whitlock, Robert H.
Clinical and Vaccine Immunology
A high-throughput TaqMan PCR assay for detection of bovine paratuberculosis was evaluated by using fecal samples from 1,808 dairy cattle in seven naturally infected herds and 347 dairy cattle in seven herds considered free of paratuberculosis. Fecal, blood, and milk samples were submitted to laboratories where the PCR-based assay, three different fecal culture procedures for Mycobacterium avium subsp. paratuberculosis (centrifugation, sedimentation, and the BACTEC filter concentration method), two serologic enzyme-linked immunosorbent assays (ELISAs), and one milk ELISA were performed. Results from testing of dairy cattle in herds free of M. avium subsp. paratuberculosis showed that the PCR assay's specificity was 99.7%. Twenty-three percent of the dairy cows that were fecal culture positive by at least one of the three methods were positive by the PCR assay. By Bayesian non-"gold standard" analysis methods, the TaqMan PCR assay had a higher specificity than the serum ELISAs (99.3%; 95% confidence interval [CI] = 98.6 to 99.7%) and a test sensitivity similar to that of the serum ELISAs (29%; 95% CI = 24 to 35%). By classical methods, the estimated relative sensitivity of the fecal PCR assay was 4% for light and moderate fecal shedders (compared to 12 to 13% for the ELISAs) and 76% for heavy fecal shedders (compared to 67% for the milk ELISA). The PCR assay has higher sensitivity for detection of heavy fecal shedders than the evaluated milk ELISA but lower sensitivity than a serum or milk ELISA for detection of light and moderate fecal shedders. This assay can be used as a quick test for detection of cattle with heavy fecal shedding, those cattle with the highest risk of transmitting infection to susceptible cattle.
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Risk factors for the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection on 59 Irish dairy herds
Cashman, W
Buckley, J
Quigley, T
Fanning, S
More, S
Egan, J
Berry, D
Grant, I
O'Farrell, K
Irish Veterinary Journal
Since 1994, Irish cattle have been exposed to greater risks of acquiring Mycobacterium avium subspecies paratuberculosis (MAP) infection as a consequence of the importation of over 70,000 animals from continental Europe. In recent years, there has been an increase in the number of reported clinical cases of paratuberculosis in Ireland. This study examines the prevalence of factors that promote the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) on selected Irish dairy farms in the Cork region, and the association between these factors and the results of MAP screening tests on milk sock filter residue (MFR). A total of 59 dairy farms, selected using non-random methods but apparently free of endemic paratuberculosis, were enrolled into the study. A questionnaire was used to collect data about risk factors for MAP introduction and transmission. The MFR was assessed on six occasions over 24 months for the presence of MAP, using culture and immunomagnetic separation prior to polymerase chain reaction (IMS-PCR). Furthermore, blood samples from all entire male and female animals over one year of age in 20 herds were tested by ELISA. Eighteen (31%) farms had operated as closed herds since 1994, 28 (47%) had purchased from multiple sources and 14 (24%) had either direct or indirect (progeny) contact with imported animals. Milk and colostrum were mixed on 51% of farms, while 88% of farms fed pooled milk. Thirty (51%) herds tested negative to MFR culture and IMS-PCR, 12 (20%) were MFR culture positive, 26 (44%) were IMS-PCR positive and seven (12%) were both culture and IMS-PCR positive. The probability of a positive MFR culture was significantly associated with reduced attendance at calving, and with increased use of individual calf pens and increased (but not significantly) if mulitiple suckling was practised. There was poor agreement between MFR culture and MFR IMS-PCR results, but moderate agreement between MFR culture and ELISA test results. This study highlights a lack of awareness among Irish dairy farmers about the effect of inadequate biosecurity on MAP introduction. Furthermore, within-herd transmission will be facilitated by traditional calf rearing and waste management practices. The findings of viable MAP in the presence of known transmission factors in non-clinically affected herds could be a prelude to long-term problems for the Irish cattle and agri-business generally.
culture; dairy farms; IMS-PCR; Johne's disease; milk filter; paratuberculosis; risk factor
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Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk
Tasara, T.
Stephan, R.
Applied and Environmental Microbiology
A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 × 101 to 2 ×106 copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.
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Culture- and Quantitative IS900 Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment
Moravkova, M.
Babak, V.
Kralova, A.
Pavlik, I.
Slana, I.
Applied and Environmental Microbiology
The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 103 were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 102 after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.
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Mycobacterium avium subsp. paratuberculosis Antibody Response, Fecal Shedding, and Antibody Cross-Reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-Infected Cattle Herds Vaccinated against Johne's Disease
Tewari, Deepanker
Hovingh, Ernest
Linscott, Rick
Martel, Edmond
Lawrence, John
Wolfgang, David
Griswold, David
Clinical and Vaccine Immunology : CVI
Vaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serum M. avium subsp. paratuberculosis antibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity to M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination against M. avium subsp. paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P < 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity to Mycobacterium bovis, no cross-reactivity was observed.
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Highly Specific and Quick Detection of Mycobacterium avium subsp. paratuberculosis in Feces and Gut Tissue of Cattle and Humans by Multiple Real-Time PCR Assays▿
Imirzalioglu, Can
Dahmen, Heinrich
Hain, Torsten
Billion, Andre
Kuenne, Carsten
Chakraborty, Trinad
Domann, Eugen
Journal of Clinical Microbiology
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples.
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Suspicion of Mycobacterium avium subsp. paratuberculosis Transmission between Cattle and Wild-Living Red Deer (Cervus elaphus) by Multitarget Genotyping
Fritsch, Isabel
Luyven, Gabriele
Köhler, Heike
Lutz, Walburga
Möbius, Petra
Applied and Environmental Microbiology
Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit–variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.
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Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic
Ayele, Wuhib Y.
Svastova, Petra
Roubal, Petr
Bartos, Milan
Pavlik, Ivo
Applied and Environmental Microbiology
Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.
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Dam Mycobacterium avium subspecies paratuberculosis (MAP) infection status does not predetermine calves for future shedding when raised in a contaminated environment: a cohort study
Eisenberg, Susanne WF
Rutten, Victor PMG
Koets, Ad P
Veterinary Research
Uptake of Mycobacterium avium subsp. paratuberculosis (MAP) by calves in the first days of life from colostrum, milk and faeces is regarded an important moment of transmission. The objective of this study was to quantify the association between the MAP status of dams as determined by the presence of MAP DNA and antibody in colostrum and that of DNA in faeces and the environment with subsequent MAP shedding of their daughters. A cohort of 117 dam-daughter pairs giving birth/being born on eight commercial dairy farms with endemic paratuberculosis was followed where colostrum, faecal and environmental samples (dust) were analysed for the presence of MAP using an IS900 real-time PCR. Antibodies in colostrum were measured by ELISA. Analysis of dust samples showed that on all farms environmental MAP exposure occurred continuously. In significantly more colostrum samples (48%) MAP DNA was detected compared to faecal samples (37%). MAP specific antibodies were present in 34% of the colostrum samples. In total MAP DNA was present in faecal samples of 41% of the daughters at least once during the sampling period. The association between faecal shedding in the offspring and the dam MAP status defined by MAP PCR on colostrum, MAP PCR on faeces or ELISA on colostrum was determined by an exact cox regression analysis for discrete data. The model indicated that the hazard for faecal shedding in daughters born to MAP positive dams was not significantly different compared to daughters born to MAP negative dams. When born to a dam with DNA positive faeces the HR was 1.05 (CI 0.6; 1.8) and with DNA positive colostrum the HR was 1.17 (CI 0.6; 2.3). When dam status was defined by a combination of both PCR outcomes (faeces and colostrum) and the ELISA outcome the HR was 1.26 (CI 0.9; 1.9). Therefore, this study indicates that neither the presence of MAP DNA in colostrum, MAP DNA in faeces nor the presence of MAP antibodies in colostrum of the dam significantly influences the hazard of MAP shedding in their subsequent daughters up to the age of two years when raised in a contaminated environment.
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Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h☆
Swift, Benjamin M.C.
Denton, Emily J.
Mahendran, Sophie A.
Huxley, Jonathan N.
Rees, Catherine E.D.
Journal of Microbiological Methods
The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.
•Optimisation of efficient MAP cell capture in blood using magnetic separation•Found a limit of detection of 101 pfu ml− 1 in spiked blood•Optimised a PCR to detect signature MAP DNA sequences from just one plaque•We successfully detected viable MAP in naturally infected animals within 48 h
PMMS, Peptide mediated magnetic separation; FPTB, FASTplaqueTB assay; MP, Media Plus; Bacteriophage; Johne's disease; Magnetic separation; Paratuberculosis; Rapid detection
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Peptide aMptD-Mediated Capture PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Bulk Milk Samples
Stratmann, Janin
Dohmann, Karen
Heinzmann, Julia
Gerlach, Gerald-F.
Applied and Environmental Microbiology
A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 × 102 CFU ml−1 for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 × 102 CFU ml−1) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 × 103 and an association constant of 1.33 × 109. The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.
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Occurrence of Mycobacterium avium subsp. paratuberculosis in Untreated Water in Northern Ireland
Whan, Lynne
Ball, Hywel J.
Grant, Irene R.
Rowe, Michael T.
Applied and Environmental Microbiology
Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources.
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Novel Secreted Antigens of Mycobacterium paratuberculosis as Serodiagnostic Biomarkers for Johne's Disease in Cattle
Facciuolo, Antonio
Kelton, David F.
Mutharia, Lucy M.
Clinical and Vaccine Immunology : CVI
Johne's disease is a chronic gastroenteritis of cattle caused by Mycobacterium avium subsp. paratuberculosis that afflicts 40% of dairy herds worldwide. M. avium subsp. paratuberculosis-infected cattle can remain asymptomatic for years while transmitting the pathogen via fecal contamination and milk. Current serodiagnosis with enzyme-linked immunosorbent assays (ELISAs) fails to detect asymptomatic M. avium subsp. paratuberculosis-infected cattle due to the use of poorly defined antigens and knowledge gaps in our understanding of M. avium subsp. paratuberculosis components eliciting pathogen-specific immune responses. We set out to (i) define a subset of proteins that contain putative antigenic targets and (ii) screen these antigen pools for immunogens relevant in detecting infection. To accomplish our first objective, we captured and resolved M. avium subsp. paratuberculosis-secreted proteins using a 2-step fractionation method and reverse-phase liquid chromatography to identify 162 unique proteins, of which 66 had not been previously observed in M. avium subsp. paratuberculosis culture filtrates. Subsequent screening of M. avium subsp. paratuberculosis-secreted proteins showed four antigens, of which one or more reacted on immunoblotting with individual serum samples from 35 M. avium subsp. paratuberculosis-infected cows. Moreover, these novel antigens reacted with sera from 6 low M. avium subsp. paratuberculosis shedders and 3 fecal-culture-positive cows labeled as ELISA seronegative. The specificity of these antigens was demonstrated using negative-control sera from uninfected calves (n = 5) and uninfected cows (n = 5), which did not react to any of these antigens in immunoblotting. As three of the four antigens are novel, their characterization and incorporation into an ELISA-based format will aid in detecting asymptomatic cattle in early or subclinical stages of disease.
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Diagnostic accuracy of a Mycobacterium phlei-absorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows.
Bech-Nielsen, S
Jorgensen, J B
Ahrens, P
Feld, N C
Journal of Clinical Microbiology
The purpose of this study was to describe the responses of sera from five groups of cattle to an enzyme-linked immunosorbent assay (ELISA) for paratuberculosis by using serum absorbed with Mycobacterium phlei at a single working dilution. The infection status of the cattle was determined by fecal culture. Cattle with different levels of exposure (high versus low prevalence and test negative) and disease manifestation (clinically suspect infection versus subclinical infection) were examined, as follows: (i) two paratuberculosis-negative herds; (ii) a fecal culture-confirmed, clinically suspect cases of paratuberculosis; (iii) cows from a paratuberculosis-infected herd with a high infection rate, as determined by fecal culture, but with no clinical cases at the time of sampling; (iv) cows from three paratuberculosis-infected herds known to have paratuberculosis diagnosed on the farm (low infection rate determined by fecal culture); and (v) one fecal culture-negative herd with known serologically positive cattle. Results generally showed a decreased ELISA response when absorbed rather than nonabsorbed serum from each animal was used. The results of the fecal culture confirmed clinically suspect cases, which were analyzed in relation to the amount of colonies isolated from the animals on fecal culture (0, +, ++,+++ , ++++, and above). There was a significant increase in the ELISA response for animals with heavy Mycobacterium paratuberculosis shedding ( ++++ or above), when both unabsorbed and absorbed sera were used, compared with the response in animals that were fecal culture negative or that shed M. paratuberculosis at lower levels (less than +) (P less than 0.05). The effects on sensitivity and specificity by using different cutoff points for the five groups of cattle with different levels of exposure is described, since sera were not discretely segregated into distinct groups of positive and negative samples. The specificity of the ELISA in the two fecal culture-negative herds was 100% at an ELISA cutoff of an optical density (OD) of 0.1 and above for absorbed serum. For unabsorbed serum the specificity was 62.9% at a similar cutoff value. Similarly, the specificity of the fecal culture-negative, serologically positive herd increased from 37.5 to 72.2 at an ELISA cutoff value of 0.1 to 0.2 (OD) by using absorbed versus unabsorbed serum from 75.0 to 94.4 at an ELISA cutoff value of 0.2 to 0.3 (OD).
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Chat with Austin Ward
Welcome to BuckeyeNation! On Thursday, reporter Austin Ward stops by to chat about the state of Ohio State football.
Ward joined ESPN.com in 2012 after five years as a college football writer at the Casper (Wyo.) Star-Tribune and the Knoxville (Tenn.) News Sentinel. The award-winning journalist lives with his wife in Columbus, Ohio.
Send your questions now and join Ward Thursday at 2 p.m. ET!
More Austin Ward: @BuckeyesBeat | Search | email@example.com | Blog
Austin Ward (1:59 PM)
Hey everyone, welcome back. Fire when ready.
Adam (Ohio)
With O'Connor and Crowell gone, are we expecting 2 or 3 guys left in this 2013 class?
Austin Ward (2:02 PM)
As it stands now -- and it's always subject to change with a bit more attrition still possible -- it looks like the Buckeyes should have room to pick up at least two more. My colleague Brad Bournival is the expert on the names and how the class might shake out, so I will leave it to him to provide that kind of analysis. But as the roster stands now and with 22 players committed, the Buckeyes should be able to add at least two more.
Adam (Ohio)
What are your thoughts on the talk of ticket prices being raised?
Austin Ward (2:15 PM)
I think there's a risk Ohio State could be pricing a large number of fans out of the stadium as the tickets get more expensive, and there's already a nationwide trend of decreased attendance thanks to the convenience of watching games at home on big-screen, high-definition televisions. So, while people are going to continue to fill the Shoe at a higher rate than almost any other stadium in the country and that's something the athletic department needs to cash in on to run other programs, it obviously comes with some element of risk. Certainly I'm fortunate enough to be in the press box on Saturdays, so the bigger question is what do you all think of the ticket prices being raised?
Luke (Connecticut)
Any chance we land Elliot and Wilson? Or is it an either or situation?
Austin Ward (2:17 PM)
Wish I could give you an answer that would really provide some enlightenment, Luke -- but the way we structure our coverage of the Buckeyes, my focus is on the guys already on campus and my partner Brad Bournival is the expert on the recruiting aspect. So, while I'm not completely in the dark or anything on that stuff, it's definitely best if I leave that analysis up to him. Sorry about that.
Adam (Ohio)
As a Clevelander, I attend about a game per year and so I'm more likely to spend more as a result of buying through secondary ticket site (Ebay, Stubhub, etc.) but I can definitely see a different crowd of fans attending games, which could certainly change the fan environment.
Austin Ward (2:17 PM)
I can definitely see that being an outcome of this potential move, Adam.
Shane (Statesboro)
Do you think that if Urban and the Buckeyes win the MNC soon, the SEC will try to take credit for that one, too?
Austin Ward (2:23 PM)
Surely they would in some way, right? That it took a coach with SEC experience to end the run would certainly be pointed out by a few diehards down South. In so many ways, though, it would be quite a story if the man who played such a big role in getting this run of dominance started at Florida is able to bring it to an end in a different league.
Luke (Connecticut)
I have heard rumors about a possible out of conference game being worked on for next year by ESPN. Any truth to this? Our schedule is so bad that I have worries we could get left out of the tittle game even if we went undefeated.
Austin Ward (2:30 PM)
I haven't heard anything of substance about a nonconference game being worked on for next season, and at this stage in the year, I would be very surprised if any further adjustments were made. The Vanderbilt cancellation and addition of the San Diego State matchup already forced a change that is considered pretty late in the game, and leaving opponents in the cold in January would be a harsh deal. The schedule certainly looks pretty easy, Luke, there's not much doubt about that. But the Buckeyes still have games on the road that will be difficult in the league (Michigan, obviously, and Northwestern), and if the Big Ten is even just slightly better, a conference title win and an undefeated record won't keep them out of the BCS if it reaches that point.
Austin Ward (2:31 PM)
I should say -- won't keep them out of the BCS title game, that is.
Luke (Connecticut)
In your mind who steps up at the big time receiving threat next year?
Austin Ward (2:38 PM)
Philly Brown is going to continue to be the primary target given his ability as a short-to-intermediate threat, which puts the ball in his hands pretty often and allows him to pile up catches. In the latter half of the season, he was turning those into bigger gains and starting to show Meyer he could be the weapon the coach thought he could be -- and now has another offseason to build on that. But the guy who I expect to really take a step forward is Devin Smith. He's got all the tools to stretch the field, and if he becomes a more deep-to-intermediate weapon that complements Brown, that alone will give the passing attack a big boost. As for some guys who didn't do as much last season, I anticipate Michael Thomas taking on a bigger role after he got more comfortable on the field in November, and Evan Spencer is another one to watch.
Adam (Colummbus OH)
With the declaration of Big Hank to the NFL who do you think is going to step up to fill his spot?
Austin Ward (2:43 PM)
That's probably not a one-person job, but I think Tommy Schutt pairing with Michael Bennett on the interior is a pretty good start to filling those shoes up front. Schutt didn't see all that much playing time last fall, but just getting his feet wet and holding his own as a true freshman bodes well. Combine that with the encouraging reports from the coaching staff about his skills and approach to the game and a body that should really benefit from this upcoming full year in the strength program (already at 6-2, 295), and Schutt could set the tone at tackle. Bennett, if healthy, really is capable of doing positive work and would have been in line for a substantial role last season if not for the nagging groin. I think Washington is best suited at end, and certainly Spence will fill that other role.
Luke (Connecticut)
Assuming Ohio State continues to roll, who is the first coach in the Meyer staff to leave for another job?
Austin Ward (2:45 PM)
To me, there's not much question that Tom Herman is going to have plenty of suitors for a top job. He's an offensive innovator, relates well to his kids, knows how to work with the media and deal with all the outside pressures that come with the job -- it's hard for me to think that he's not at the front of the line, particularly if the spread attack is as explosive next fall as it figures to be with all that talent returning.
Adam (Columbus OH)
who is the leader in the clubhouse to get Vonn Bell?
Austin Ward (2:49 PM)
Hard for me to say since I have never had the chance to speak with the kid or covered him in any way. Have to leave that up to my man Brad Bournival to handicap, but I think in his last update on Bell, the kid didn't have a clear-cut favorite among the finalists at this point.
Austin Ward (2:51 PM)
All right, folks. Gotta run -- thanks as always for the questions and comments. See you next time!
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Upstate News
Doretta Royer 315 464-4833
Puppet workshop promotes acceptance of kids with autism and Asperger's Syndrome
SYRACUSE, N.Y. — An interactive puppet workshop called "Kidspeak" created by SUNY Upstate Medical University to foster understanding and acceptance of children with autism and Asperger's syndrome will be available for use in classrooms throughout the county in the coming year.
The workshop, which made its debut Nov. 13 in Syracuse, is supported by the Kohl's Foundation "Kohl's Cares for Kids Program." SUNY Upstate's Margaret L. Williams Developmental Evaluation Center created the Kidspeak puppet show.
Since 2005, the Kohl's Cares for Kids Program has donated more than $400,000 to SUNY Upstate Medical University, with more than $325,000 in support of autism. Included in this amount is a recent donation of $141,741 that was raised through the 2007 Kohl's Cares for Kids campaign.
Kidspeak will be primarily available to teachers and students in grades one through three. The workshop includes a puppet show to help students understand the unique learning styles of children with autism spectrum disorders. In addition, a companion worksheet will offer teachers suggestions on ways that they can promote a successful academic experience for children with ASD.
Kohl's Cares for Kids program raises funds for children's health and educational opportunities across the country through the sale of special gift items. All net profits from the sale of these items supports children's health and educational opportunities in the market areas served by Kohl's.
Search Upstate News
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Hotel Calamidoro - Calcinaia
This hotel offers free self parking, a nightclub and gift shops/newsstands. Take a splash in one of the 4 outdoor pools during your stay. Fancy sipping a cool drink poolside? Don't miss out as there is a poolside bar as well as a bar/lounge if you prefer to sip your beverage indoors. The leisure facilities include a Turkish bath/hammam.
Multilingual staff are available at the front desk 24 hours-a-day and can provide express check-in and express check-out. Free WiFi is provided so you can check your in-box whenever you like.
Hotel Calamidoro - Calcinaia
Hotel Info: 1-877-214-4288from $86
- Suite
- Classic Double Room
- Classic Double Room Single Use
- Superior Single Room
- Superior Triple Room
- Royal Suite
- Superior Room
- Suite (Duplex)
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 70 rooms
- Check-in time starts at 2 PM
- Check-out time is noon
- Express check-in/out
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets not allowed
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
- Free RV/bus/truck parking
In the hotel
- Buffet breakfast (surcharge)
- Bar/lounge
- Poolside bar
- Room service (during limited hours)
- Number of outdoor pools - 4
- Outdoor seasonal pool
- Golf lessons on site
- Golf driving range on site
- Nightclub
- Children's pool
- Number of meeting rooms - 3
- Conference space
- Meeting rooms
- 24-hour front desk
- Concierge services
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Wedding services
- Multilingual staff
- Porter/bellhop
- Number of buildings/towers - 6
- Elevator/lift
- ATM/banking
- Safe-deposit box at front desk
- Garden
- Accessible bathroom
- In-room accessibility
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Minibar
- Slippers
- Hypo-allergenic bedding available
- Blackout drapes/curtains
- Private bathroom
- Bidet
- Designer toiletries
- Hair dryer
- Satellite TV channels
- Free WiFi
- Daily housekeeping
- In-room safe
- Connecting/adjoining rooms available
Hotel Calamidoro - Calcinaia's small print
Also known as
- Calamidoro
- Calamidoro Calcinaia
- Calamidoro Hotel
- Hotel Calamidoro
- Hotel Calamidoro Calcinaia
- Calamidoro Hotel Calcinaia, Province Of Pisa, Italy
- Calamidoro Hotel Pisa
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport.
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Reservations are required for massage services and spa treatments and can be made by contacting the property before arrival at the number on the booking confirmation.
Optional extras
Rollaway beds are available for EUR 30 per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.


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Welcome to the website of the Australian Embassy in Cairo. This website is designed to provide you with information about Australia and Egypt, and the services the Embassy is able to offer.
The Australian Embassy in Cairo is responsible for covering Australia's relations with not only Egypt, but also Sudan, Syria and Eritrea, where we do not maintain Embassies.
There is an extensive array of information online about Australia and the Australian Government and this site may be used as a tool to direct you to these sites.
For those seeking information about Egypt and travel to Egypt, a good place to start is with the website of the Egyptian Embassy in Canberra.
Please feel free to contact us if you require more information or assistance. The Embassy's e-mail address is: email@example.com or follow us on Facebook at https://www.facebook.com/AustralianEmbassyEgypt
Neil Hawkins
If you are an Australian citizen requiring urgent consular assistance
- Call the chancery switchboard on +202 2770 6600. If we cannot take your call you will be provided the option to be transferred to the Department of Foreign Affairs and Trade's 24 hour Consular Operations Centre in Canberra.
- Call the Department of Foreign Affairs and Trade's 24 hour Consular Operations Centre direct on +61 2 6261 3305, SMS +61 421 269 080 or call 1300 555 135 (from within Australia).
- Email us at firstname.lastname@example.com
- Visit us at: Level 11 World Trade Centre, 1191 Corniche El Nil, Boulac, Cairo.
Personal information provided to the Department of Foreign Affairs and Trade (DFAT) is protected by law, including the Privacy Act 1988. We may collect, use and disclose your personal information, including to overseas recipients where we reasonably believe it is necessary, to provide you with consular assistance. Important information about the privacy of your personal information is contained in our Consular Privacy Collection Statement, which you should read and understand. Copies of the Statement are available at http://www.dfat.gov.au/dept/consular/privacy.html or by requesting a copy from the Department.
On November 2, Foreign Minister Julie Bishop launched a new Smartraveller campaign to help better inform and prepare you for your next overseas trip.
The campaign features a revamped Smarttraveller website, an updated registration system and the release of a new smartphone application for android devices to make it even easier to get the information you need to stay safe overseas.
General travel advice is also now available in six languages, including Arabic.
Check out some of the new Smartraveller videos on the DFAT YouTube account and visit the website at www.smartraveller.gov.au
- Register your travel plans;
- Subscribe to Travel Advice updates;
- Insure yourself and your belongings.
Visit the smartraveller website for more information.
Direct Aid Program
The Direct Aid Program (DAP) is a flexible, small grants program which focuses on relieving humanitarian hardship and advancing developmental objectives in Egypt, Sudan and Eritrea.
Expression of Interest for DAP 2016-2017 funding is now open. We invite organisations interested in receiving DAP funding to submit an Expression of Interest (EOI) form. This requires a brief description of the organisation and the project that requires funding. The deadline for submitting an EOI is Sunday 7 August 2016.
For tips on how to put together your EOI form, please refer to this link: http://egypt.embassy.gov.au/caro/cooperation.html


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Your Views
Voter ID safeguards voting
Allow me to dissect the editorial in opposition to the voter ID law ("Don't change voter ID law; get rid of it completely," Our View, Nov. 8).
The Journal Sentinel makes two main points of objection to the voter ID law. First, it might intimidate voters. (It is a highly remote possibility and not demonstrated to be fact, but don't let speculation stand in the way of demagoguery.) Second, it is unneeded, creating a solution to a problem that doesn't exist.
What happened to the argument "it is the law of the land, upheld by the Supreme Court, quit whining" used in defense of the Obamacare law? Oh, yes, that defense can only be used when rationalizing Democratic Party-endorsed laws.
The Journal Sentinel may not be aware of it, but Wisconsin has experienced a rash of voters voting twice in an election. Wisconsin also has had issues with the voter registration rolls. The voting process cannot be made into a trivial matter. The sanctity of the vote process is vital to democracy. Right now, because of the continuous corruption and lax oversight of the voting process, my vote really doesn't count. My vote is being suppressed by the current voting process. I am being disenfranchised.
The Editorial Board cited a ridiculous hypothetical situation posed by Democrats in which someone's religion might prohibit a person from being photographed and thereby failing to produce a photo ID. The comment made me wonder if the cult of the Democratic Party religion may now impose a photographic sanction. Or does this ban of photographs only pertain to elections and election-related materials?
The Editorial Board's claim that the veracity of the current voter registration system isn't a problem is a stridently deceptive claim. "The answer is to get rid of the law altogether so that everyone has equal protection under the law to exercise the right to vote." No, that is not the answer. No one is being prohibited from the right to vote; safeguards are being added to ensure that the process is not being abused.
Adding safeguards to our freedoms and rights are a common occurrence. For example, U.S. citizens have the right to bear arms. I am sure that the Editorial Board would agree that this right needs to be properly administered to ensure that this right is not abused. The same concept holds true for the right to vote.
The current law benefits only the Democratic Party. The answer is that any law that makes the voting process more accurately reflect one legal lawfully registered citizen equating to one vote is a step in the right direction.
Bernie Zeibart
Voter ID suppresses voting
I applaud the Nov. 8 editorial on getting rid of voter ID and not trying to circumvent the court cases in the process by passing an Indiana-type voter ID law ("Don't change voter ID law; get rid of it completely").
The whole idea behind voter ID is to suppress Democratic votes. Fear is always used as a way to bring democracy down. The fear of fraud is bogus with only a handful of cases in our state, and most of those have been related to felons voting not knowing they must wait until they are eligible to vote.
Most well-off Americans have no idea there are over 200,000 people in our state without acceptable photo ID passed in our state. Does one even know that you need to pay for birth certificates and you often need a photo ID to order one? Division of Motor Vehicles offices are distant in most counties and don't have evening hours.
The targeted are students, minorities, the disabled and the elderly. I may have trouble voting because my new driver's license has my maiden name on it instead of my initial for the middle name. That's how picky photo ID can be to deny voters their constitutional right to the franchise.
Dorothy Sherman
Obamacare will be better
The writer of the letter headlined "Nixon, Clinton and now Obama" clearly has never had to buy individual health insurance on the open market (Opinions, Nov. 9).
The individual insurance marketplace has always been an underregulated minefield with routine arbitrary cancellations, huge premium increases and poor coverage.
The difference now is that the Affordable Care Act dictates minimum standards that individual policies previously could not meet.
The writer's comment that it is unfair that maternity care coverage is required for post-menopausal women shows a misunderstanding as to how insurance works at a very basic level. The purpose of the ACA's mandate is to make insurance more affordable for everyone by spreading the risk.
Yes, there are glitches, but ultimately a national health insurance exchange will be better — better coverage and lower costs.
Terry Schwartz
Whitefish Bay
Southridge and bus stops
Apparently the writer of the letter "Why complain about bus stops?" has never been to Southridge Mall (Your Views, Nov. 7).
Bus stops have never been at the mall entrance, even before the remodeling started in 2010. Then the bus stop was moved about two blocks from the Sears entrance.
Remarks about the distance to walk around the mall are irrelevant. There are no benches or places to rest outside of the mall and across the parking lot for seniors or people with walkers or canes (the majority of people who use the bus to reach the mall). Also, there is no rain, snow, wind or 90-degree weather inside the mall to contend with.
I guess a person from a small town who is dependent upon his or her car would not think of that.
Mary Strobbe
Across the Board
The Journal Sentinel Editorial Board's blog on the day's happenings as they happen.


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Unsweetened gives you the goods without the extras. It's a direct "Hey, let's do this!" kind of way to start your day with unadulterated opportunity. Grab life by the bottle - be bold, unsweetened, authenthic, fearless, delicious.
Nutrition Facts
Serving Size 8 fl oz (237 ml)
Servings Per Container 6
Amount Per Serving
Calories 35 Calories From Fat 30
% Daily Value*
Total Fat 3g 5%
Saturated Fat 0g0%
Trans Fat 0g
Cholesterol 0mg0%
Sodium 160mg 7%
Total Carbohydrate 1g0%
Dietary Fiber 1g 4%
Sugars 0g
Protein 1g
Vitamin A 10% Vitamin C 0%
Calcium 45% Iron 2%
Vitamin D 25% Vitamin E 50%
Riboflavin 25% Vitamin B12 50%
Zinc 10%
*Percent Daily Values are based on a 2,000 calorie diet
Almondmilk (Water, Almonds). Contains Less Than 2% Of The Following: Vitamin/ Mineral Blend (Calcium Carbonate, Vitamin A, Vitamin E, Vitamin D2, Vitamin B12, Vitamin B2, Zinc), Sunflower Lecithin, Sea Salt, Potassium Citrate, Natural Flavors, Locust Bean Gum, Gellan Gum.
Allergen Statement: Contains Almonds.
Made from non-genetically engineered almonds.


text:
Exit Hwy 680 at Danville's Diablo Road (West). Travel west and turn left on Hartz Ave.
We offer a wide range of insurance products and will get you the best price available. We would love to meet you and say hello - please call us for a complementary consultation. Our office is open from 9:00 a.m. to 6:00 p.m., Monday through Friday. We serve clients in the San Francisco Bay Area as well as throughout California. We look forward to hearing from you.
"And...it is personal."


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Monday was a day off for the USC Aiken baseball team. But the pacers still made news, dropping in the latest rankings.
USCA is 10th in the newest Collegiate Baseball Newspaper NCAA Division II Poll. They were No. 6 last week, but likely fell because of they lost two of three games in a weekend series against Peach Belt Conference rival Montevallo, surrendering 24 runs in the three-game set.
The Pacers (27-7, 13-5) have now been ranked in 26 consecutive national polls. Although USCA dropped slightly because of the results of the weekend series, Montevallo checked into the poll at No. 16. They're joined by two other PBC teams in the poll, with Lander at third and UNC Pembroke at No. 11.
Delta State is the No. 1 team in the latest national tally. Mount Olive is second.
The Pacers will have another day off today and return to action on Wednesday, when they will travel to Gaffney for a 3 p.m. non-conference game against Limestone. It will be the second meeting between the teams this season with USCA taking a 10-6 victory over Limestone at Roberto Hernandez Stadium on March 13.
The Pacers will close out the week with a three-game home series against PBC rival Augusta State. The series will begin with a 7 p.m. contest on Friday and will conclude with a doubleheader on Saturday. The first game of the twin bill is set to get underway at 1 p.m.
USCA and Augusta State have already met once this season with the Pacers winning 4-1 in 10 innings during one of the Outback Cup contests played in Augusta. USCA owns a 65-20 all-time series lead over Augusta State.


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Top Definition
Used to define something that excites you sexually as well as providing entertainment. Usually used to describe an amazing stunt or martial talent.
"Kurt, did you just flying jump kick that chick in the uterus? That was awesrotic."
por James Gil 20 de Maio de 2008
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|Doll/Doll Clothing
I am a retired art educator of 30 years. Now my creative time is my own! I am a weaver, knitter, felter and all around jack- of all-trades fiber artist!! I've been doing this for about 100 years!!!View all patterns by designer (3) »
As my grand daughter needed some clothes for her Bitty Baby doll, I started trying my hand at designing clothes for her! It is a very easy, very quick pattern that uses only a bit of yarn! Scraps that we all save can be used for these cute little boots!!
5 stitches = 1" in garter stitch
200 yards of Worsted weight


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Alna Store - Alna, ME - Phone: (207) 586-5515
A variety store or a price-point retailer in Alna is a retail store that sells inexpensive items, often with a single price for all items in the store. Typical merchandise includes cleaning supplies, toys, household goods, and gardening equipment. Other variety store products include cooking supplies, small tools, personal hygiene supplies, kitchen supplies, organizational supplies, office supplies, holiday decorations, electronics supplies, home d�cor, novelties, pet supplies, out of print books, DVDs, food products and automotive supplies. Some items sold at a certain price point would cost that much anyway, whereas other items offer a substantially lower price than usual.
NYS Comptroller Thomas DiNapoli Addresses Clean Energy Leadership Task Force
Audits Confirm Savings from Energy Efficiency Improvements at Long Island School Districts (Long Island, NY) On Friday, February 19th, New York State Comptroller Thomas DiNapoli spoke before the Clean Energy Leadership Task Force which is a project directed by the Sustainability Institute at Molloy College located at Republic Airport in Farmingdale NY. Mr. DiNapoli discussed the results of audits..