text:
Court of Appeal orders new trial for Felix brothers …
This morning the Court of Appeal ordered that two brothers convicted of murder should get a new trial. In October 2006 Maurice and Emory Felix were found guilty of the murder of Belize City mechanic Mitchum Heredia. Justice Adolph Lucas subsequently ordered that both Felixes should be jailed for life. In connection to injuries sustained by another man during the same shooting, Maurice Felix was additionally sentenced to fifteen years for Attempted Murder, which was to be served concurrently. But this morning the judges of the Court of Appeal quashed those convictions, set their sentences aside and ordered a retrial of the case. According to court reports, the appeals were allowed because no identification parade was held. Apparently, the trial judge, Adolph Lucas had told the jury that holding an identification parade would have been a waste of time because the prosecution's chief witness named Maurice and Emory Felix as the gunmen. Following the ruling, Defence attorney Simeon Sampson admitted to us that he was surprised by the court's decision.
Simeon Sampson, Attorney, Felix Brothers
"I was somewhat disappointed, I thought they were going to be scotch free and never to return again but the interest of justice might be best served if they are retried."
Janelle Chanona
"Why were you hoping for otherwise?"
Simeon Sampson
"Because in my arguments, the fact was there was no other supporting evidence beside this incomplete identification evidence which was in my view, ought not to be sustained or could not sustain a country at all but there have been no other evidence. That was the reason why I was hoping that this would have been the end of the matter."
The brothers will remain on remand until the conclusion of new case. During the four-day trial almost two years ago, both men took the stand in their defence to maintain their innocence. In April of 2006, Mitchum Heredia had just pulled into his driveway on T Street when two men jumped from behind a tree in his yard and shot him multiple times.


text:
One of the things I've noticed about older regional galleries is that many of them seem to have been built from the same design. The vestibule of the Queen Victoria Museum and Art Gallery in Launceston looks a lot like the entry to the Ballarat Regional Gallery in Victoria. It makes sense – an art gallery needs a big, sweeping staircase, triple height ceilings and polished floorboards, and both these venerable galleries are overflowing with old world charm.
Many feature a commissioned drawing of the town as if seen from the air, or from an elevated tower or nearby hill. On opening day the crowds would have thronged to view the collection of English and select European paintings, and delight at the novelty of their very own town being pictured from afar. Other novelties in these galleries include portraits of local grandees, and my favourite, commissioned pictures of oversized cows and sheep, animals so large that the owner, invariably standing proudly next to the beast, barely manages to reach its shoulder.
Not every country town or city thought to build a permanent local gallery, or if they had, many didn't survive to the present day.
The Western Plains Cultural Centre, aka Dubbo Regional Gallery, out where the ground starts to turn red in western NSW, has a gallery space that was built from scratch: a large, high-ceilinged rectangular box with moveable interior walls. It's a perfect space in that it can be reconfigured into seemingly countless layouts, and the architectural design yokes the new gallery to buildings that were once a high school. With a permanent collection of local history in the older building, there's a harmony of experience as you wander from one space to the next, from old into new.
The Maitland Regional Gallery (MRG), in the Upper Hunter, is an older building that has been extended several times. The ground floor galleries are well-lit spaces that afford a museum quality hang while the upstairs and side rooms are a sprawling reminder of the timeworn building, an eccentric maze of short corridors, ramps and stairwells. Finding art there is a fun game of hide and seek. MRG also has what every decent regional gallery needs – a killer café/restaurant, one so good that when I'm travelling that way, I'll make a detour off the Hunter Expressway to eat there.
About 45 minutes east is Newcastle and its formidable 1970s brutalist concrete gallery. It looks and feels like an extension of the Art Gallery of NSW, from the stairwell and its galleries, to the Brett Whiteley sculpture at the door, to the same Eurostyle Bold font on all the interior signage.
These galleries are the opposite ends of the regional gallery spectrum – the contemporary makeover of a heritage building, or a near-contemporary building that almost got a makeover. (Perhaps one day the Newcastle Council will come to its senses and, instead of spending the millions it had been given to upgrade the art gallery on local roads, it realises the commercial value of a major regional gallery.) Somewhere in the middle of that spectrum are regional galleries that are basically make-do spaces in 1980s era postmodern architecture, galleries that look identical to the council's library, chambers and mothers' health clinic. I won't name names but they know who they are…
Of course, the thing that makes these galleries so valuable is their exhibitions.
While some wealthier museums and galleries can attempt to replicate the 'Bendigo Effect' (leapfrogging city art museums and bringing shows from overseas straight to the regions) most provide instead a venue for contemporary Australian artists to show their work.
For me, this is the most valuable aspect of regional galleries. Outside the commercial gallery system, and a handful of artist run initiatives in cities, there are remarkably few opportunities for artists to show their work, doubly so if they live in the regions. Regional galleries are usually run on a tight budget but they have proven to be remarkably adventurous, staging curated exhibitions, solo shows and local artists side-by-side. That regional galleries in close geographical proximity to each other haven't yet got together to jointly promote themselves and stage a multi-venue exhibition is a great idea still waiting to happen.

subset: commoncrawl


text:
Love yourself before someone else loves you
Thursday March 26 2020
"Love your neighbour as you love yourself." The Bible commands. Well, most people do not take time to love themselves in fear of being labeled 'selfish' but the command is; you love yourself first.
Most people go through life waiting and praying for someone to love them. It is great to be loved and everyone should experience it but before you fall in love with someone else, fall in love with yourself.
Maybe no one ever told you but you deserve to be treated with dignity and love. You should not allow people to step on you, let alone call you inferior. The more time you spend trying to appreciate who you are, the stronger you build your esteem.
Esther never felt beautiful regardless of the expensive make-up she wore. She stayed in an abusive relationship because she thought that she could not find someone else to love her and six months ago, she moved in with her boyfriend James. Her family never approved of the relationship but in most cases, it is hard to stand between lovers regardless of what you may say.
Two weeks ago, Esther was in hospital because she had had a miscarriage. She spent most of the time either buried in thought or crying and refused to eat or drink anything. We all thought that it was because of the loss she had encountered until James entered the room on day three.
She turned to the wall and sobbed bittery. When James came close to hold her, she burst out in anger, threw the flowers he had brought and ordered him to leave the room. "Calm down Essie," her mom intervened.
The nurse came in and asked us to leave the room but we could still hear James apologising. "I am sorry, I did not mean to do it," he said. "You have been saying that every time you hit me.
It ends now. Get out and never return." She shouted as she pushed him outside.
Finally, Esther had remembered that she deserved more than just a bouquet of flowers. A few of us knew about the mistreatment but she always defended his actions.
This is what happens to most people who think they do not deserve true love. They stick with the wrong person because they fear that maybe no one will love them.
However, if you do not love yourself, you might not be able to differentiate between true love and fake love because a person who loves him/herself knows what true love is and knows how to love others.


text:
Sep 6th, 2019
Bodie, California
Photography in any type of light offers up many challenges. Night photography in a city full of lights is even trickier for getting that right exposure. What happens when you leave the light of civilization and wander into Bodie Ghost Town, one of the best preserved Ghost Towns anywhere on the planet?!
Welcome to the Dark Side of Photography: Bodie Edition!
The pun is intended. Night photography is tricky, and left to figure it out on your own can be a frustrating endeavor for sure. Join the instructors of the Aperture Academy for an in-depth workshop that will teach you how to capture star trails, the Milky-Way, light painting and more! Already have some experience? No problem — with our small group everyone can learn at their own pace and skill level.
This workshop will be based out of Lee Vining California. After orientation, we will start with a classroom presentation to talk about the various techniques that will be taught so that once in the field, everyone will understand the camera settings and techniques. Then, after dinner the journey into the dark of night begins. We will photograph the many buildings and feautures that give Bodie its charm, as well as capture a variety of skies, all designed so you come away with stunning images and a solid understanding of how to get the most out of your night photography experiences.
On the second day of the workshop we will once again visit Bodie and capture the now familiar Ghost Town in daylight. After a mid-day break we will photograph sunset over the surreal tufa formations of Mono Lake, one of the most recognizable and iconic locations for landscape photography in the Western United States.
Our third day will be spent importing, organizing, and post-processing the photos using Adobe. Your instructors will help you learn how to enhance and edit your images to make them stand above!
Results may vary based on weather and atmospheric conditions, however, you WILL leave this workshop with a solid understanding of the proper techniques for capturing the night sky.
Day 1:
3pm: Orientation
Classroom presentation to talk about gear and techniques
5pm-5:30pm: Depart for in-field shooting (transportation provided)
In-field shooting (approx 5-7 hours)
Return to Lee Vining (transportation provided)
Day 2:
Morning shoot at Bodie
11:30am Break
Sunset shoot at Mono Lake (transportation provided)
Day 3 (Classroom post-processing):
9am: Downloading images, sorting & organizing
10am – 12pm: In-depth processing session
12pm: Conclusion
Ability to walk 1-2 miles each day
Headlamp for safely walking during on-location visits
A Laptop with Adobe Photoshop® CS4 or later (or Adobe Creative Cloud @ $10 per month)
What is/isn't included?
The workshop/tour price includes:
All workshop instruction
All on-location transportation
The workshop/tour price DOES NOT include:
Transportation to/from Lee Vining California
Instructor gratuities
Travel insurance


text:
Marirosa Molina. Ecosystems Research Division National Exposure Research Laboratory Athens, GA 30605
- Hillary Louise Black
- 1 years ago
- Views:
1 EPA/600/R-05/107 September 2005 Temporal and Spatial Variability of Fecal Indicator Bacteria: Implications for the Application of MST Methodologies to Differentiate Sources of Fecal Contamination by Marirosa Molina Ecosystems Research Division National Exposure Research Laboratory Athens, GA U.S. Environmental Protection Agency Office of Research and Development Washington, DC 20460
2 Notice The information in this document has been funded by the United States Environmental Protection Agency. It has been subject to the Agency's peer and administrative review, and it has been approved for publication as an EPA document. Mention of trade names of commercial products does not constitute endorsement or recommendation for use. 2
3 Abstract Temporal variability in the gastrointestinal flora of animals impacting water resources with fecal material can be one of the factors producing low source identification rates when applying microbial source tracking (MST) methods. Understanding how bacterial species and genotypes vary over time is highly relevant when the fecal material used to create a source library is collected under very different seasonal conditions than the environmental sample. Our objective was to identify and compare the temporal and spatial variability of fecal indicator bacteria from a specific host in manure and water samples and evaluate the implications of such variability on microbial source tracking approaches and applications. We selected Enterococcus as the model fecal indicator, given the supposedly high specificity of some of the species of this genus to the host organism. Cattle was chosen as the model host organism because of the documented high impact that cattle has on impairment of surface waters. The sites studied were located at a farm where cattle have unrestricted access to the stream. Enterococci were isolated monthly from water and manure samples using membrane-enterococcus Indoxyl-β-D-Glucoside agar (mei) as described in EPA method The isolates were identified using a multiplex PCR procedure that targets the genus and the species-specific gene superoxide dismutase. Eight species were identified in cattle manure, of which E. casseliflavus (37%), faecium (22%) and hirae (18%) were the most abundant. Nine species were identified in stream samples with E. faecalis (43%), casseliflavus/flavescens (34%), and hirae (11%) being the most abundant. September exhibited the highest species abundance in manure samples while March had the highest species abundance in stream water samples. E. assini and E. malodoratus were only detected in manure samples, but were not detected in water samples. In contrast, E. durans, gallinarum and sulfureous were only isolated from the stream samples. In general, the enterococci distribution pattern and species richness found in manure samples did not correlate with those found in the stream samples at the individual species level. However, cluster analysis revealed strong seasonal and spatial variability of groups of enterococci, and indicated that some clusters that seem specific to manure can be found in the water only during certain seasons. In addition to the enterococci library development, 16S rdna host-specific Bacteroides markers were also applied to the water samples. The results indicate that data obtained with the Bacteroides markers (BM) generally agreed with the enterococci data showing higher occurrence of the cattle BM in areas under obvious cattle impact. However, no seasonality was identified in conjunction with any of the BMs used. In addition, the cow marker was also detected at an upstream-of-the-farm location that was not under obvious cattle influence. This study suggests that in order to increase the validity of MST methods, it is necessary to consider temporal variability when designing the sampling scheme of the source material and constructing source libraries, and increase the specificity and field testing of DNA-based markers. 3
4 Table of Contents TEMPORAL AND GEOGRAPHIC STABILITY OF FECAL INDICATOR BACTERIA...6 TEMPORAL STABILITY... 6 GEOGRAPHIC STABILITY... 8 STUDY SITE AND SAMPLE DESCRIPTION...10 METHODOLOGY...13 PROCEDURE FOR VERIFICATION OF ENTEROCOCCI SPECIES MULTIPLEX PCR PROCEDURE DNA EXTRACTION AND AMPLIFICATION WITH BACTEROIDES PRIMERS DNA Extraction from Fecal and Water Samples Amplification using Bacteroides Primers STATISTICAL ANALYSIS RESULTS AND DISCUSSION...19 TOTAL ENTEROCOCCI COUNTS IN STREAM WATER SAMPLES AND COMPARISON OF FLUORESCENT ASSAY AND MEMBRANE FILTRATION PROCEDURE COMPOSITION AND TEMPORAL VARIABILITY OF ENTEROCOCCUS SPECIES IN MANURE AND WATER 23 SEASONAL AND SPATIAL VARIABILITY OF ENTEROCOCCI COMMUNITIES IN MANURE AND WATER COMPARISON OF BACTEROIDES MARKERS AND ENTEROCOCCI CLUSTERS CONCLUSIONS AND FINAL CONSIDERATIONS...36 REFERENCES
5 List of Tables Table 1: Description of stream water sampling locations at Chandler Farm, Georgia...13 Table 2: Enterococcus species isolated from cattle manure and stream water at a farm in Georgia Table 3: Seasonal % composition (mean ± sd) of the most common Enterococcus species isolated from cattle manure samples collected at a cattle farm in Georgia Table 4: Seasonal % composition of the most common Enterococcus species isolated from water samples collected in a stream located at a beef cattle farm in Georgia Table 5: Composition and abundance (%) of Enterococcus species in clusters that appeared 3 or more times in water and manure samples collected at a Georgia cattle farm from September 2003 through January Table 6: Composition of Bacteroides clusters identified in stream water collected at a cattle farm in Georgia...35 List of Figures Figure 1: Study site location...11 Figure 2: Sampling locations at Chandler Farm, Georgia Figure 3: Procedure diagram for counting, isolating, verifying and speciating enterococci in environmental samples...14 Figure 4: Seasonal enterococci counts in water samples collected at a Georgia cattle farm using the mei membrane filtration procedure Figure 5: Percent of isolates with a blue halo isolated from mei that tested positive for the genus Enterococcus with a multiplex PCR procedure Figure 6: High % occurrence of Enterococcus clusters (EC) in samples collected from different sources at a cattle farm in Georgia...29 Figure 7: Low % occurrence of Enterococcus clusters in samples collected from different sources in a cattle farm...30 Figure 8: Low % occurrence of enterococci clusters during different seasons in samples collected at a cattle farm in Georgia Figure 9: High % occurrence of enterococci clusters during different seasons in samples collected at a cattle farm in Georgia Figure 10: Cluster distribution and occurrence (%) per Chandler Farm sampling site and source Figure 11: Relationship of Bacteroides and enterococci clusters in stream water samples collected at a cattle farm in Georgia
6 Temporal and geographic stability of fecal indicator bacteria An ideal source tracking organism must be stable in the environment. Sampling performed over time should not reveal significant genotypic or phenotypic variability within host individuals or within host populations. In addition, variable environmental conditions such as temperature and ph, and host factors such as quality and type of feed or antibiotic treatments, etc., should not affect an ideal indicator. All these conditions affect the host organism and, in turn, the inside environment that the source indicator bacteria inhabit. To date, very few microbial source tracking (MST) studies have addressed the temporal stability of fecal indicators, making it difficult to reliably identify sources over time. In this study, we sampled manure and impacted manure water monthly at a Georgia farm site over a year to determine: the temporal variability of various species of Enterococcus; the spatial distribution and stability of enterococci species in stream water; and which species might be the most relevant and promising specific indicators of the host organism, i.e., beef cattle. Temporal stability When addressing temporal variability, it seems important to establish the difference between transient and resident populations of source indicators. This is of particular relevance if, for example, the range of clones estimated in natural populations of Escherichia coli ( per host species) (Selander et al. 1987) are found to be comparable for other fecal indicator bacteria. Caugant et al. (1981) defined a transient population as one observed at only one sampling time, while a resident population is observed at more than one sampling time. In order for MST methods to be effective, the source indicator bacteria selected 6
7 should be part of the resident population of the source species. In addition, it should be part of a clonal population that is stable through time as suggested by Gordon (2001) for E.coli. In a study performed over an 11-month period on a single human host, Caugant et al. (1981) found a significant difference between the resident and the transient populations of E. coli. The resident population accounted for only 5.6% of the 53 electrophoretic types identified using multilocus enzyme electrophoresis. Jenkins et al (2003) also found a rather low percentage of E. coli ribotypes to be part of the resident population in yearling steers sampled four times over a 129-day period. Specifically, only 8.3% of 240 ribotypes were determined to be resident in the host species. In addition, no ribotype was found at all four sampling times or in all of the steers sampled from a total of 20 resident ribotypes. Also using E. coli, Ochman et al. (1983) observed that the resident population from multiple hosts accounted for only 8% of all the electrophoretic types identified, and only 5 types were found in more than 7 hosts. These results suggest that there is a high probability that the majority of ribotypes obtained from a single host species at any given time belong to transient populations. This observation has major repercussions relative to the establishment of host origin libraries, that could require continuous updating in order for a particular MST methodology to be able to track the host species (Jenkins et al. 2003) over an extended period of time. It should be noted that although a general lack of temporal stability seems to be a big limitation in the identification of suitable source indicators, there are certain source genotypes that have been recovered from environmental samples after extended periods of time. These periods range from a few weeks to a year 7
8 (Faith et al. 1996; Jenkins et al.2003; Wiggins et al. 2003). Restriction endonuclease digestion profiles (REDP) performed in dairy cows from 70 farms in Wisconsin revealed that two isolates exhibited the same REDP even though they were sampled 7 months apart. Results from the same study also indicated that a herd or animal can contain isolates of E. coli O157:H7 that have multiple, but similar profiles; however, most of these profiles were found to change over time (Faith et al. 1996). Long-term temporal stability has also been observed for some indicator organisms using phenotypic tests such as antibiotic resistance patterns (ARA) (Wiggins et al. 2003). Geographic stability. Three main assumptions can be made when investigating the geographical stability of an ideal source indicator. These are that: a) a bacterial source indicator exhibits geographical structure, that is, the similarity of the bacterial indicator in various populations of a given host animal species is directly proportional to the geographical distance of the members of such population; b) a bacterial source indicator sampled from one population of a given host animal species will be similar to a bacterial source indicator sampled from any other population of the same host animal species, and a predictive relationship can be established between the two; and c) a bacterial host indicator sampled from various populations of a given animal host species separated by great geographic distances exhibits a high similarity index and accurately tracks the host species. Studies indicate that the first assumption regarding geographical structure for populations of the same host animal species is hard to verify for 8
9 human hosts. This is attributed to the mobility of humans among geographic areas. On the other hand, isolates from non-domesticated animals seem to exhibit more geographic structure due to their restricted movement patterns (Gordon 2001). Caugant et al. (1984) reported that geographic structure was hard to demonstrate for E. coli in families living within the same city, where only 6% of the diversity was explained by the geographical separation, and 1% of the diversity was explained by the distance separating families living in different cities. Another possible factor affecting geographic stability or structure is that the host animal digestive system can select for particular resident bacterial strains, generating a very specific gut flora in each host (Souza et al. 2002), making it difficult to identify genotypes and/or phenotypes over broad geographic areas. An important consideration when trying to assess spatial stability is the analysis methodology used. In a study performed across a broad geographical area in Florida, researchers used a one-enzyme ribotyping procedure to determine the accuracy of this MST methodology to identify beef and dairy cattle, poultry, swine and human host species using E. coli isolates (Scott et al. 2003). Although the methodology was accurate differentiating human vs. non-human hosts, it failed to distinguish among the different non-human host species across the broad geographical region. In contrast, Hartel et al. (2002) were able to successfully apply a two-enzyme ribotyping methodology to discriminate among E. coli ribotypes isolated from cattle and horses from two locations (Georgia and Idaho). The results from this study support the first assumption for geographic stability, but do not support the second and third assumptions. The latter 9
10 researchers indicated that up to a distance of 260 km, there is ribotype sharing among isolates obtained from horses. Cattle exhibited some ribotype sharing up to a distance of 350 km, but not at a distance of 2900 km (Georgia and Idaho isolates). However, for swine and poultry, the percent sharing was rather low and not significantly different from locations closer together (locations within Georgia) than far apart (Georgia and Idaho). Using a similar ribotyping method, human vs. non-human hosts were also accurately identified from E. coli isolates across an extended area in the Apalachicola region of Florida (Parveen et al. 1999). For library-based methods, the size of the library seems to be a determinative factor supporting the second and third assumptions presented in this section. In a study using ARA, results indicated that merging 6 watershed libraries to encompass a total of 6,500 isolates produced a library large enough to be representative and capable of being used to accurately identify enterococci host species across a broad geographic area (Wiggins et al 2003). These latter study results also suggested that the minimum size of a library should be about 2,300 isolates in order for it to be representative, in this way, it is possible to create multiwatershed databases representative enough for the reliable identification of fecal bacterial sources. Study Site and Sample Description Water and manure samples were collected at Chandler Farm (CF), a beef cattle farm located in Madison County, Northeast Georgia (Figure 1). 10
11 Figure 1: Study site location Water samples were collected from a first order stream that crosses the farm from west to east and is a tributary of the South Fork Broad River (Figure 2). Seven sampling locations were located along the stream within the Chandler farm site (Table 1) covering a distance of 2.3 km. One liter water samples were collected at each location once per month from September 2003 through January During each sampling campaign, five fresh cattle manure samples were also collected from different individuals after collecting the water samples. 11
12 Figure 2: Sampling locations at Chandler Farm, Georgia. 12
13 Table 1: Description of stream water sampling locations at Chandler Farm, Georgia. Site ID CFS-1 Description of Site Creek headwaters, upstream from cattle impact Direct Cattle Impact on Site Distance from Origin of Creek (km) None 0.21 CFS-2 Stream at cattle crossing area High 0.89 CFS-3 Unrestricted access of cattle to creek High 0.93 CFS-4 Intermittent unrestricted access of cattle to creek Medium 1.05 CFS-5 Stream by side of pond, cattle was never observed in this location Low 1.32 CFS-6 Stream at outlet of pond, cattle was never observed at this location Low 1.45 CFS-7 Stream outside of property fence, no direct access by cattle Low 1.48 Methodology After preparing slurries of the manure samples, both the manure and stream water samples were processed by membrane filtration to obtain the total number of enterococci in the water and to isolate enterococci species for library development. The specific procedure is depicted in Figure 3. 13
14 Sampling and Membrane Filtration If positive in salt broth, proceed to speciate Incubate mei plates for C Inoculate BHI Slant with individual colony & incubate for C Colonies w/blue halo = enterococci Filter water samples through 0.2 um polycarbonate filters Extract DNA and amplify using Bacteroides primers Isolate colonies in BHI plates, incubate for C Inoculate BHI broth, incubate for C ID species using Multiplex PCR Prepare template w/ single colony in 50µL H2O Isolate colonies in BHI plates, incubate for C Inoculate BHI broth + 6.5% salt, incubate for C positive result = enterococci proceed w/ verification step from BHI broth or slant neg result = discard isolate 10-20% Verification Procedure Freeze 2-3 colonies in BHI broth + 30% glycerol Inoculate BHI broth, incubate for C Inoculate BEA agar, incubate for C Perform Gram stain Figure 3: Procedure diagram for counting, isolating, verifying and speciating enterococci in environmental samples. Procedure for Verification of Enterococci Species A modification of EPA Method 1600 was used to count, isolate and verify enterococci from the environmental samples. Briefly, 1, 5, 10, and 50 ml of stream water and 10 and 25 ml of a 1 x10-6 dilution of manure slurry were filtered through 0.45um cellulose membranes and incubated on membrane- Enterococcus Indoxyl β-d-glucoside (mei) agar plates at 41 ± 0.5 C for 24 hours. After incubation, all colonies having a blue halo were considered to be presumptive enterococci. Five colonies from each location water sample and ten colonies from each manure sample were isolated on brain-heart infusion agar 14
15 (BHIA) slants and in a tube of brain-heart infusion broth (BHIB), using a 1ul loop. The BHIA and BHIB samples were then incubated at 35 ± 0.5 C for 48 and 24 hrs, respectively. After incubation, a loop-full (1ul) from each BHIB tube exhibiting growth was transferred to a tube of BHIB plus 6.5% NaCl, and incubated at 35 ± 0.5 C for 48 hrs. Any isolate not exhibiting growth on BHIA, BHIB or BHIB + NaCl was considered to be non-enterococci, and was not used any further in the procedure. About 20% of the isolates exhibiting growth on the media mentioned aboved were further verified as Enterococcus using the following procedure: a 1ul loop of sample was taken from a BHIA slant and transferred to a Bile- Esculine Agar (BEA) slant, and a tube of BHIB. The BEA slant was incubated at 35 ± 0.5 C for 48 hrs and the BHIB tube was incubated at 45 ± 0.5 C for 48 hrs. Finally, a Gram stain was performed on the final isolates. Growth in each medium combined with and identification of the final isolate as Gram positive cocci verified the isolate as an Enterococcus. Multiplex PCR procedure All polymerase chain reactions (PCR) were conducted within currently established EPA Quality Assurance/ Quality Control guidelines. The workflow was conducted such that the opportunity for sample contamination was reduced as much as possible. It was imperative that reagent preparation, sample preparation, DNA extraction and PCRs followed a one-directional flow in separate areas with separate pipettes and equipment to prevent crosscontamination. All reagents were prepared in working volumes in a positive pressure room on a clean bench after exposing the bleach-disinfected area to UV 15
16 light for 10 minutes. DNA extractions were conducted in a separate laboratory. PCRs were run in a third location, physically separated from the reagent and DNA prep rooms. Pipettes and lab coats were dedicated to the different steps of the procedure. Environmental samples were processed following standard microbiological aseptic techniques. Positive controls for the PCR were used in each run to insure that the PCR was not inhibited by contaminants. Negative controls (reagent blanks) were used to insure that amplified DNAs were only coming from the environmental samples, and not introduced to the samples at the laboratory. PCR optimization for the Bacteroides work was performed at the beginning of the study. Speciation of enterococci isolated from manure and stream water samples was performed as depicted in the lower left side of Figure 3. After verifying the isolates as Enterococcus, whole cell templates were prepared in molecular grade sterilized water. These templates were used for up to three weeks. Seven master mixes were used to identify up to 23 species of Enterococcus using a multiplex PCR procedure based on the superoxide dismutase gene (Jackson et al., 2004). The procedure was performed testing the isolates with the master mixes in the following order: 1, 2, 6, 4, 3, 5, and 7. The majority of the isolates could be speciated by applying only the first three master mixes in the sequence, thereby achieving the best use of resources and the most time and cost effective approach. PCR products were separated and identified using a 2% 1X TAE agarose gel containing 2 µg/ ml ethidium bromide. Gel analysis was performed using a EpiChemi Darkroom BioImaging System (UVP, Inc.) equipped with a transilluminator, and fitted with Labworks 4.5 software. Band sizes were 16
17 identified by comparing the sample DNA to the positive controls included with each run, and by comparing the band size to a 100 bp DNA ladder. Once the isolates were identified, the templates were plated again on BHIA and 3 to 4 single colonies were inoculated into BHIB containing 30% glycerol. The inoculated medium was stored at -80 C. DNA Extraction and Amplification with Bacteroides Primers DNA Extraction from Fecal and Water Samples. Manure fecal samples were stored at -20 o C immediately upon arrival at the lab until DNA could be extracted. DNA was extracted with a MoBio UltraClean fecal DNA mini kit using 0.25 gram of fecal material according to the manufacturer s instructions. Water samples (100 ml aliquots) were filtered through 0.4 µm cellulose filters and DNA was extracted from the membrane filters using a Qiagen DNeasy tissue kit, following the Qiagen protocol for DNA extraction with a micro-centrifuge and an additional wash of Buffer AW2 (included in the kit). Amplification using Bacteroides Primers. One general, two cowspecific and two-human specific-bacteroides primers (Bernhard and Field, 2000) were used to test water samples. After applying PCR optimization procedures, the following program was used: initial denaturation at 94 C for 2 minutes, product amplification by 30 cycles of denaturation at 94 C for 1 minute, annealing at 53/54 C for 1 minute, and elongation at 72 C for 1.5 minute. Amplification was followed by a final extension at 72 C for 3 minutes. Bacteroides- PCR products were identified (presumptive positive result) in a 1% agarose gel containing ethidium bromide by comparing the band intensities under UV light to the intensities of a commercially available 100 bp DNA mass ladder. 17
18 Statistical Analysis A hierarchical cluster analysis was performed (using Minitab v.12 statistical software) on the165 samples taken from cow manure patties, upstream-of-the-farm stream water, and on-the-farm stream water. The objective of this analysis was to group together samples that showed similar relative abundances of the most common species of Enterococcus. Enterococci species that were found in only a few of the 165 samples were not included in the analysis. The following five species were seen frequently enough to be included: E. casseliflavus, E. faecalis, E. faecium, E. flavescence, E. hirae. In addition, we included one category that was the sum of all unidentified enterococci species. In the first step of the clustering algorithm, the two samples with the most similar Enterococcus species relative abundances are grouped together. These two observations are now designated as a cluster, and this cluster is represented by a centroid, or mean value, of the two samples that compose it. In step two, all remaining samples are examined and the next two that have the most similar relative species abundance are grouped or clustered. In each subsequent step, the two samples (or possibly clusters) that exhibit the greatest similarity are grouped together. Hierarchical clustering requires that a subjective stoppingpoint be chosen as the algorithm progresses. If this is not done, the algorithm will eventually form one large group of all observations. We stopped the procedure after step 121, prior to the formation of two large clusters. At this point, 15 clusters had been formed with member species (could be the same or different species) appearing 3 or more times in each one of the 165 samples; 134 of the 165 total samples were found within these 15 clusters. The other 31 18
19 samples (19% of the total sample pool) were identified as outliers, meaning that their enterococci communities did not match well with communities seen in the other samples. After stopping the algorithm, we recorded the centroid of each cluster (i.e., the mean relative abundances for the five enterococci species used in the analysis) plus the general enterococci classification. The centroids for the 15 clusters with member species that appear 3 or more times are given in Table 5. In our final step, we used the cluster designations for each of the samples to perform ANOVA and MANOVA analyses to test for differences in the clusters found for manure, upstream-of-the-farm stream water, and on-the-farm stream water, as well as changes in the seasonal occurrence of the clusters. Results and Discussion Total Enterococci Counts in Stream Water Samples and Comparison of Fluorescent Assay and Membrane Filtration Procedures. As indicated previously, total enterococci counts were performed using EPA method Accordingly, sample volumes of 1, 5, 10, and 50 ml were used to target total counts into the suggested range of 20 to 66 colonies/100 ml. A defined-substrate assay method from IDEXX laboratories that applies a methyl-umbelliferyl-βglucuronide (MUG)-based medium (Enterolert ) for detection of enterococci was also used on the water samples. The objective of the test was to perform a comparison of the results obtained from both methodologies and to evaluate their accuracy. Figure 4 shows the total enterococci counts obtained by the membrane filtration procedure (mei) for the samples collected at the seven water 19
20 sampling locations. The results indicate that the highest counts were always obtained at locations CFS-2 thru -4, locations directly impacted by cattle, including a cattle crossing point (CFS-2). Locations CFS-5 thru -7 exhibited oneto two-fold less total counts than the upstream locations (with summer values being slightly higher than those in other seasons), indicating a decrease (due to dilution, settling, dye-off, etc.), of the fecal bacteria in the water column. Although the highest counts were observed mostly from April through September that tend to be months of low precipitation, monthly variability of the counts didn t allow us to establish significant differences Enterococcus colonies/100 ml Spring Summer Fall Winter 0 CFS-1 CFS-2 CFS-3 CFS-4 CFS-5 CFS-6 CFS-7 Chandler Farm Stream Location Figure 4: Seasonal enterococci counts in water samples collected at a Georgia cattle farm using the mei membrane filtration procedure. The accuracy of the mei procedure was determined by comparing the number of isolates originally obtained from the mei plates per site with the number of isolates identified as Enterococcus with the multiplex PCR. In mei, 20
21 colonies of any color that produce a blue halo are presumptive enterococci. Our results indicate that in water, an average of 99% of all isolates that produced a blue halo in the mei, also tested positive in the salt broth (which is one of the biochemical tests run to verify the presence of enterococci). However, this agreement was down to 21% for the manure samples. Those isolates that tested negative in the salt broth were discarded, as a test of them with the multiplex PCR demonstrated that they were not of the genus Enterococcus. Of the isolates that tested positive in the salt broth, 99 and 97.5% were identified as enterococci in the water and manure samples, respectively, indicating that salt tolerance was indeed a good indicator for the presence of enterococci in this type of stream water and manure samples. In contrast, the presence of a blue halo in the mei was not a good indicator of the presence of enterococci in manure samples, since only 43.8% of those isolates with a blue halo were identified as enterococci by the multiplex PCR. For water, however, 97.6% of all those isolates exhibiting a blue halo were identified as enterococci by the PCR procedure (Figure 5). 21
22 mei % Accuracy Manure Water 0.0 Mar- 04 Apr May Jun Jul Aug Sep Oct Nov Dec Jan- 05 Month Figure 5: Percent of isolates with a blue halo isolated from mei that tested positive for the genus Enterococcus with a multiplex PCR procedure. The higher than 100% accuracy indicated for some of the samples in Figure 5 is due to the presence of additional enterococci colonies mixed with isolates believed to be only one type of colony when they were originally isolated from the mei. The mixed colonies were separated into pure cultures and identified as Enterococcus by the multiplex PCR. We compared the precision of the Enterolert method relative to the mei for determination of the total number of enterococci in water samples by calculating the relative percent difference (RPD) of the total counts per 100 ml obtained with each method. The RPD was calculated using the following equation: RPD= (mei counts-enterolert counts/(mei counts + Enterolert counts)/2) *
23 The calculated results indicated that there is an average of 32 ±13.7% underestimation in the total Enterococcus counts obtained with the Enterolert procedure relative to the mei method for the type of water samples used in this study (data not shown). In addition, no correlation was observed between the two methodologies. Kinzelman et al. (2003) also reported a lack of correlation between these two methods; however, they found that the Enterolert procedure generated false positive results that produced an overestimation of the actual number of enterococci, contrary to the underestimation found in our study. Therefore, it is possible that the performance of the Enterolert procedure is highly dependent on the physical/chemical conditions of the environment tested, and probably more studies are necessary to determine its general efficacy in freshwater systems. Composition and Temporal Variability of Enterococcus Species in Manure and Water. A total of 11 species of enterococci were identified in water and manure samples collected during our study using a multiplex PCR procedure (Jackson et al., 2004). E. malodoratus and asini were only found in manure while E. sulfureus, gallinarum and durans were only found in the water samples (Table 2). Because E. malodoratus, asini and gallinarum were only found once during the whole sampling period, they were categorized as transient species in the system (Caugant et al., 1981). E. durans and sulfureus were found in several occasions during different seasons (data not shown) and were 23
24 not believed to be transient species, but were not observed frequently enough as to be considered important members of the enterococci community. 24
25 Table 2: Enterococcus species isolated from cattle manure and stream water at a farm in Georgia. Enterococcus species Manure Water E. casseliflavus E. casseliflavus E. faecalis E. faecalis E. faecium E. faecium E. flavescence E. flavescence E. hirae E. hirae E. mundii E. mundtii E. malodoratus E. sulfureous E. assini E. gallinarum E. durans The % abundances of the most common enterococci species found in manure and water samples are presented in Tables 3 and 4, respectively. We found greater variability in the seasonal abundances of individual species in manure than in water. In manure, E. faecalis was the most, while E. casseliflavus and E. flavescens were the least abundant abundant during spring (Table 3). During summer, E. casseliflavus, E. faecium and E. flavescens were all in high abundance (Table 3). It is clear that the relative % abundance of the individual species in manure varies as a function of season; indeed, the results indicate that E. hirae and E. faecium were completely absent during summer and winter, respectively. In contrast, it was not possible to identify any clear seasonal trend in the % composition of the different species in water due to the 25
26 high degree of seasonal variability observed. For instance, E. faecalis was found in high abundance during the fall in both farm and upstream-of the-farm locations. In contrast, during spring, it was found at relatively high abundance only in manure. High seasonal variability was noted in the Enterococcus populations isolated from water at the upstream-of-the-farm location, indicating that the background Enterococcus populations (in wildlife and possibly poultry due to the proximity of chicken houses to this site) are as variable as the Enterococcus populations isolated at the farm sites. In general, these results suggest that the five most abundant enterococci species are ubiquitous in the environment, given the fact that they were found in water samples that are not supposed to be impacted by cattle (CFS-1). In addition, the general use of individual species to establish seasonal and/ or source trends is likely to be a difficult task due to the high degree of variability observed. Table 3: Seasonal % composition (mean ± sd) of the most common Enterococcus species isolated from cattle manure samples collected at a cattle farm in Georgia. Season E. casseliflavus E. faecalis E. faecium E. flavescens E. hirae Spring 4 ± 7* 34 ± 32* 6 ± 14 3 ±7* 22 ± 32 Summer 40 ± 43 4 ± ± 32* 28 ± 39* 0 Fall 43 ± ± ± ± ± 27 Winter 49 ± 29 4 ± ± ± 27 *Significantly different than winter. 26
27 Table 4: Seasonal % composition of the most common Enterococcus species isolated from water samples collected in a stream located at a beef cattle farm in Georgia. Season Sample location E. casseliflavus E. faecalis E. faecium E. flavescens E. hirae Spring CFS-1 27 ± ± ± 12 Summer CFS-1 17 ± ± 44 6 ± 10 6 ± 10 0 Fall CFS ± 38* 7 ± ± ± 20 Winter CFS-1 11 ± ± ± 25 8 ± 14 Spring CFS-2 thru 7 27 ± ± 24 7 ± 5 3 ± ± 19 Summer CFS-2 thru 7 27 ± ± 21 1 ± 6 29 ± 26 1 ± 5 Fall CFS-2 thru 7 10 ± ± 32* 5 ± 8 14 ± 18 4 ± 11 Winter CFS-2 thru 7 14 ± 15 9 ± ± ± ± 24 *Significantly different from Winter; CFS-1: upstream from sites impacted by cattle; CFS-2 thru 7: farm sites potentially impacted by cattle. Seasonal and Spatial Variability of Enterococci Communities in Manure and Water. Cluster analysis was performed on the relative % abundances of the five most common enterococci groups and the general enterococci category found in the stream water and manure samples. The purpose of the analysis was to determine if a community approach could produce useful information related to developing more reliable MST data analysis. The analysis produced 15 clusters of species that were identified as being present in the system three or more times through out the whole sampling period (Table 5). 27
28 Table 5: Composition and abundance (%) of Enterococcus species in clusters that appeared 3 or more times in water and manure samples collected at a Georgia cattle farm from September 2003 through January % Composition of each Enterococcus species Cluster Composition Cluster 1 Cluster 2 Cluster 3 Cluster 4 Cluster 5 E. casseliflavus E. faecalis E. faecium E. flavescens E. hirae All other Enterococcus Cluster 6 Cluster 7 Cluster 8 Cluster 9 Cluster 11 E. casseliflavus E. faecalis E. faecium E. flavescens E. hirae All other Enterococcus Cluster 12 Cluster 13 Cluster 16 Cluster 17 Cluster 18 E. casseliflavus E. faecalis E. faecium E. flavescens E. hirae All other Enterococcus Six enterococci clusters were found in high relative % occurrence for the three different sample sources, i.e., upstream-of-the-farm, at-the-farm sites and manure (Figure 6). Cluster 4 was only found at the upstream and farm locations where E. casseliflavus was usually more abundant. Clusters 8, 9, and 13 were not only present in manure, but were also frequently found in the upstream and farm samples; E. faecalis was overall the most abundant species in these three clusters. Clusters 6 and 7 were only found at-the-farm sites and manure 28
29 samples, and had a higher occurrence in the manure samples. E. faecium and E. casseliflavus were the most abundant species in these two clusters (Figure 6) Upstream site Farm sites Manure % Cluster Occurrence EC-4 EC-6 EC-7 EC-8 EC-9 EC-13 Cluster # Figure 6: High % occurrence of Enterococcus clusters (EC) in samples collected from different sources at a cattle farm in Georgia. Eight clusters were found only in manure and at-the-farm samples (Figure 7). However, these clusters were generally composed of species found at lower abundances (8% or less) in the sampled material. E. casseliflavus and E. hirae were predominant in clusters 1, 2, 3, 5, 11, and 16; in addition, clusters 1 and 3 also contained significant contributions of E. faecalis that also dominated cluster
30 10 8 Upstream site Farm sites Manure % Occurrence EC-1 EC-2 EC-3 EC-5 EC-11 EC-16 EC-17 EC-18 Cluster # Figure 7: Low % occurrence of Enterococcus clusters in samples collected from different sources in a cattle farm. These data suggest that the % distribution of specific species of enterococci in a community (cluster) might be an indication of the source environment. The results also indicate that same communities (clusters) found in manure are also found in stream water samples impacted by cattle (Figure 7) despit their low % abundance. However, it seems that such indicators could be highly seasonal. For instance, those clusters only found in manure and farm water samples were present during the cooler months (spring, fall, and winter), but were never observed during the summer (Figure 8). The most frequently observed clusters were found through out the four seasons, but seasonal differences can still be seen. For instance, EC-4 is clearly more frequently 30
31 observed during spring, while for EC-9 is clearly more abundant during fall (Figure 9) Spring Summer Fall Winter % Occurrence EC-1 EC-2 EC-3 EC-5 EC-11 EC-16 EC-17 EC-18 Cluster # Figure 8: Low % occurrence of enterococci clusters during different seasons in samples collected at a cattle farm in Georgia. The higher observed frequency for EC-9 during fall is basically due to the significantly higher concentration of E. faecalis in the water samples (Table 4). In the summer, E. casseliflavus and E. flavescens were highly abundant in clusters 7 and 8, respectively. (Note: E. casseliflavus and flavescens have been reclassified as the same species under E. casseliflavus (Gilmore, 2002)). 31
32 40 30 Spring Summer Fall Winter % Occurrence EC-4 EC-6 EC-7 EC-8 EC-9 EC-13 Cluster # Figure 9: High % occurrence of enterococci clusters during different seasons in samples collected at a cattle farm in Georgia. Figure 10 shows the distribution of clusters and % occurrence of each cluster per sampling site. The figure shows that only one cluster, although widely observed in the water samples, could not be found in the manure samples (EC-4). Likewise, EC-12 and EC-17 were only found in manure but not in the water. These clusters are composed of E. flavescence and enterococci that could not be speciated. Although a variety of clusters could be found at any given time at each water sampling station, the most clusters identified per site was 8, while 14 clusters were identified in the manure samples, which indicates a much larger diversity of enterococci communities in the cattle GI system. The upstream site (CFS-1) with only 4 clusters, had the least diversity found in the system. The 32
33 clusters present at CSF-1 are part of the background composition of the system because they are present at all the water sampling sites. In almost all the farm stream sites, the cluster diversity was higher, probably reflecting the effect that the manure added to the system. 100% EC-18 EC-17 Cluster occurrence (%) 80% 60% 40% 20% EC-16 EC-13 EC-12 EC-11 EC-9 EC-8 EC-7 EC-6 EC-5 0% CFS-1 CFS-2 CFS-3 CFS-4 CFS-5 CFS-6 CFS-7 Manure EC-4 EC-3 EC-2 Chandler Farm Sources EC-1 Figure 10: Cluster distribution and occurrence (%) per Chandler Farm sampling site and source. Comparison of Bacteroides Markers and Enterococci Clusters. The Bacteroides markers (BM) were organized in five different clusters (Table 6) and this information compared to the presence of the enterococci clusters (EC) in the water samples (Figure 11). The human-bm was found twice concurrent with EC- 8 and once with EC-9. These two clusters had high abundance of E. faecalis and E. flavescens. The cow-bm was found concurrent with 6 different ECs, but 33
34 again most frequently with EC-8 and EC-9. In addition, both BMs were found with EC-1 at least once. No spatial trend for either BM could be established, which means that the markers were found at various locations in the stream through out the year. Two possible conclusions can be drawn from these results. The fact that the human-bm was found in various locations in the farm stream water suggested that some of the E. faecalis and E. flavescence in the water may not be coming only from cattle or wildlife, but also from human contamination. The sources for this contamination could be leaky septic systems given the rural aspect of the location where the samples were obtained. Alternatively, these results could suggest that the human-bm was amplifying Bacteroides DNA from sources other than human. This latter hypothesis also applies to one of the cow-bms that was amplified at CFS-1, the site upstream from obvious areas of cattle contamination. This site could be affected by run-off coming from various chicken houses located in fairly close proximity to the stream headwaters (see Figure 2). These chicken houses could also be responsible for the higher-than-expected enterococci diversity in the stream water. 34
35 Table 6: Composition of Bacteroides clusters identified in stream water collected at a cattle farm in Georgia Presence of marker 1 in cluster Bacteroides Cluster # General Marker Cow Marker 2 Human Marker 2 1 Yes No No 2 Yes No Yes 3 Yes Yes No 4 Yes Yes Yes 5 No No No 1 Reference for all Bacteroides markers: Bernhard and Field, The two human markers and two cow markers were combined to develop clusters 2 and 3, respectively 35
36 5 Bacteroides Cluster # EC-1 EC-2 EC-4 EC-6 EC-7 EC-8 EC-9 EC Frequency of Bacteroides Clusters Figure 11: Relationship of Bacteroides and enterococci clusters in stream water samples collected at a cattle farm in Georgia. Conclusions and Final Considerations The general conclusions for this study follow: From a total of 11 Enterococcus species that were identified at Chandler farm, 2 were only found in cattle manure, but were not recovered in stream water. This makes such species unreliable markers of cattle fecal contamination in surface waters since they do not survive in this environment. The relative abundance of individual Enterococcus species isolated from cattle manure that were also observed in the stream samples exhibited a high degree of seasonal variability. This finding suggests that when tracing back cattle contamination, season should be an important consideration to include in the criteria to select the species that can be used as tracer. However, the high degree of seasonal variability in some of the most common species makes it very difficult to establish significant differences between seasons and /or the sampled sources. 36
37 The 5 most common enterococci species identified were found in the water samples at-the-farm and upstream-the- farm locations, suggesting that these species are widely spread in the environment. Wildlife, an adjacent-to-the-farm chicken house, and a few scattered single-family houses could be contributing these same species in high numbers, therefore creating high background concentrations. Cluster analysis seems to be a good approach to identify species groups or enterococci communities that are specific to a location or source, and suggests that a community fingerprint rather than an individual species could be an alternative approach to trace back stream fecal contamination to its source. Results with the Bacteroides markers generally agreed with the enterococci data in that water sampled from stream locations CFS-2 thru 4 was highly impacted by cattle contamination, while locations CFS-5 thru 7 had occasional hits apparently affected by the season of the sampling event. However, the cow marker was also detected at location CFS-1 that was not under obvious cattle influence. The human Bacteroides marker was also detected occasionally throughout all stream locations, except for CFS-1, indicating either human fecal contamination in parts of the stream or non-specific amplification of the human- and cow-bacteroides markers due to other sources, such as poultry manure which is frequently used to fertilize cattle pasture sites. The two methodologies applied in this study differ greatly in terms of cost effectiveness and turn-around time of results. Building an enterococci library is a time-consuming, expensive approach that has the potential to provide a great deal of information when the proper statistical analytical approach (in this case it was cluster analysis) is used to interpret the results. Time availability (when are results expected or needed) and funding support (large quantities of consumable laboratory supplies are needed) are two important considerations to keep in mind when a librarydependent method for microbial source tracking is planned. Application of a library-independent approach, such as the Bacteroides markers allows for a much faster and possibly less expensive results. However, the need still exists for highly specific, reliable markers that will allow one to separate specific sources and not only human vs. non human contamination. In the case of Bacteroides, there remains a lack of thorough temporal, spatial and specificity analyses of the few genetic markers available so far. 37
38 Acknowledgements I like to thank Julie Maimes and Jared Fisher for the technical support offered on the construction, maintenance and speciation of the enterococci library. I like to acknowledge Dr. Charlene Jackson and Benny Barrett for the multiplex PCR technology transfer and Paul Smith, Dr. Caragwen Bracken and Lourdes Prieto for all the field and sample processing support. Thanks are also extended to Jorge Santodomingo and his laboratory staff for support with the 16S rdna analysis. Finally, I like to thank Dr. Mike Cyterski and the UGA Statistical Consulting Office for providing statistical support for data analysis. 38
39 References: Bernhard, A. E., and K. G. Field A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides- Prevotella genes encoding 16S rrna. Appl. Environ. Microbiol. 66: Caugant, D. A., B. R. Levin, and R. K. Selander Genetic diversity and temporal variation in the E. coli population of a human host. Genetics 98: Caugant, D. A., B. R. Levin, and R. K. Selander Distribution of multilocus genotypes of Escherichia coli within and between host families. J. Hyg 92: Faith, N.G., J. A. Shere, R. Brosch, K. W. Arnold, S.E. Ansay, M. S. Lee, J. B. Luchansky, and C.W. Kaspar Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. Appl. Environ. Microbiol. 62: Gilmore, M.S The enterococci : pathogenesis, molecular biology, and antibiotic resistance. ASM Press, Washington, D.C. 439p. Gordon, D. M Geographical structure and host specificity in bacteria and the implications for tracing the source of coliform contamination. Microbiology 147: Gordon, D.M The genetic structure of Escherichia coli populations in feral house mice. Microbiology 143: Hartel, P.G., J. D. Summer, J. L. Hill, J. V. Collins, J. A. Entry, and W. I. Segars Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia. J Environ Quality 31: Jackson, C. R., P. J. Fedorka-Cray, and J. B. Barrett Identification of enterococci using a genus and species specific multiplex PCR. J Clin Microbiol. 42: Jenkins, M. B., P. G. Hartel, T. J. Olexa, and J. A. Stuedemann Putative temporal variability of Escherichia coli ribotypes from yearling steers. J. Environ. Quality 32: Kinzelman, J. C. Ng, E. Jackson, S. Gradus, R. Bagley Enterococci as indicators of Lake Michigan recreational water quality: Comparison of two methodologies and their impacts on public health regulatory events. Appl. Environ. Microbiol 69: Ochman, H., T. S. Whittam, D. A. Caugant, and R. K. Selander Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella. J Gen Microbiol 129 (Pt 9): Parveen, S., K. M. Portier, K. Robinson, L. Edmiston, and M. L. Tamplin Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution. Appl Environ Microbiol 65: Scott, T. M., S. Parveen, K. M. Portier, J. B. Rose, M. L. Tamplin, S. R. Farrah, A. Koo, and J. Lukasik Geographical variation in ribotype 39


text:
Sell my car Central Coast
(Data Source: Car Intelligence, 2018)
- 1
- 2
I can inspect you car today
Limited spots available!
Just a few more details for your
Instant Estimate!
Thank you! Please find below your
Instant Estimate
Sold in: Sydney
2012 Toyota Hilux – sell my car
2012 Toyota Hilux – sell my car
Condition report: The car has no sunroof, leather or sat nav. Tailgate dented slightly. A few minor scratches around the body. It's due for a service and needs a full detail inside and out.
Odometer shows – 250484 kms, Engine size 4, manual transmission, seats 5, doors 4, 4×4, diesel engine.
It was bought in Sydney 2251
Paid cash for the car.
The spread of used 2012 Toyota Hilux cars for private sale ranges from roughly as low as $9,500 for a two-door, Ute tray Workmate, 2.7 Litre ULP engine with a 5-speed manual transmission, up to $45,000 for the SR5 4×4 with a 3.0L Diesel engine and a 4-speed automatic transmission.
The 2012 Hilux range had a slight revamp, setting it apart from previous models. These Toyotas have new fenders, bumpers, grilles, bonnets, and even headlamps. Some variations gained newly styled wheels and the whole range now has anti-skid brakes. All variants now have either a standard inclusion of cruise control, or a limited slip-differential if its a 4×4 Workmate. Stability control and side and curtain airbags were also added to several versions, as well as a new and improved audio system and climate control feature.
With so many options for body kits, engines, and transmissions, there are nearly 40 different standard variations of the 2012 Toyota Hilux that you may be selling. The body kits include:
-The 4×2 Single Cab (in Manual or Auto, either workmate or SR variations with Cab Chassis or pick-up option, and Turbo Diesel or V6 engine). The Workmate CC TD Manual is a new variant for the 2012 range.
-The 4×2 Xtra Cab (in Auto or Manual with option for TD or V6 engines). The SR TD Manual is a new variant in the 2012 range.
-The 4×2 Double Cab (Options for Workmate/SR/SR5 with manual or auto with L4, TD or V6 engine). The SR5 V6 manual was discontinued in the 2012 range.
-The 4×4 Single Cab (In Auto or Manual as either Workmate or SR, both with Cab Chassis, option for V6 or TD engine). The Workmate CC TD Auto and Manual are both new variants in the 2012 range. The SR CC V6 auto and manual were both discontinued in this year.
-The 4×4 Xtra Cab (All manual with either SR, SR CC, or SR5 with option for TD or V6 engines). The SR5 V6 manual was discontinued, and the SR TD manual is a new variant added in the 2012 range.
– The 4×4 Double Cab (In manual or auto as Workmate/SR/SR5, with TD or V6 engines). The Workmate TD manual and auto are both new additions to the 2012 Toyota Hilux Range.
Much of the range saw either new additions, or price decreases from as little as a $400 cut in the 4×4 double cab SR V6s, to a $5,650 cut in the 4×4 Xtra Cab SR5 TD manual. The greatest price increase was $900 in the 4×2 Single Cab SR CC V6 manual and auto, and $800 in the 4×2 double cab SR V6 manual and auto. Keep in mind that these price changes reflect new vehicles and that sales of used Toyota Hiluxs will vary greatly depending on the odometer, service history, and condition of the car at the time of sale.
Don't have a Hilux? Check out the buying and selling guides for the Toyota Tarago, Toyota Hiace, Toyota Corolla, and Toyota Aurion.
Sold in: Sydney
2010 Holden Commodore – are you selling
2010 Holden Commodore – are you selling
Condition report – This car has no spare key or Sat Nav. The front panel is all scratched and there's a few small dents. Car due for service. It needs a full detail inside and out.
Odometer reads – 188117 kms, engine size 8, automatic transmission, seats 5, doors 4,front wheel drive, petrol engine.
It was bought in Sydney NSW 2143
paid cash for the car, $5250.
There are many Holden Commodores circulating around the streets, making their way to their second and third owners. Good things to look out for if you're buying or selling a Holden Commodore is paint deterioration or signs of peeling in the clear coat. Cars that have had more regular polishing will have a much better chance of a lasting coat. While Holden changed their servicing periods to 15,000kms, engines do much better with oil changes at 10,000kms so check the service history and see how the individual car has been taken care of. If you want to make sure the automatic transmission is still in good nic, go for a test drive and listen for strange sounds, shaking, or stickiness on shifting.
The beloved Holden Commodore 2010 range comes in either a Sedan or a Wagon with several different variations in each category. Today, the average 2010 Holden Commodore sells from anywhere between $6,000 and $19,000 if it's in extremely good condition, with low miles, sold through a platform. Prices will vary outside of this range depending on kilometres driven, wear and tear, extra features, and the servicing history.
-Berlina: The Berlina comes in Dual Fuel and International variants and has the option of 6.0L pulp, 3.6L LPG, and 3.0L ULP engines with six or 4-speed automatic transmissions. The used Berlinas start off at about $4,500 for private sales.
-International: The International has a 3.0L ULP engine with a 6-speed auto transmission and also comes in a D/Fuel variant with a 3.6L LPG engine with a 4-speed auto trans. These start around $6,900 AUD.
-Omega: The standard Omega variation comes with the 3.0L ULP engine and 6-speed auto transmission. It also comes as a D/Fuel, D/Fuel 60th anniversary, and 60th Anniversary variations, all with a 3.6L LPG engine and a 4-speed automatic transmission. These start around $5,800 AUD.
-SS: The SS has a 6.0L PULP engine and a 6-speed transmission in either auto or manual. They start selling at around $16,00 AUD.
-SS-V: These come as the standard SS-V, the redline edition, or the 60th anniversary edition and all three have the same engine and transmission options as the SS. These start around $14,900 for the SS-V, $12,000 for the 60th anniversary, and $17,900 for the redline.
-SS-V SE: This variant has the same options as the SS and starts selling in private, used sales, at about $18,000.
-SV6: This variant comes with a 3.6L ULP engine and either a manual or automatic 6-speed transmission.
-Berlina: The Berlina also has an international variant and both have a 3.0L ULP engine with 6-speed automatic transmission. They start selling around $4,500 in used, private sales.
-International: This variation has the same engine and transmission as the Berlina and starts used sales around $6,500 AUD.
-Omega: The Omega also has the standard engine and transmission setup and starts selling around $5,500. It also has a 60th anniversary edition that has a 3.6L ULP, 4-speed auto trans.
-SS:This comes with the 6.0L PULP engine with either a manual or automatic 6-speed transmission. Sales starts around $14,000.
-SS-V: This has the same options as the SS but also comes in redline edition.
-SS-V SE: Same as above.
-SV6: This only comes with a 3.6L ULP with 6-speed automatic transmission.
Not buying or selling a Holden Commodore? Check out our other buying and selling guides over here.
Sold in: Sydney
2012 Nissan Dualis – are you selling
2012 Nissan Dualis – are you selling
Condition report – The car has no spare key, sunroof, leather or Sat Nav. chipped paint across the bonnet and drivers door . It needs a detail inside and out.
Odometer shows – 122918 kms, engine size 5, Manual transmission, seats 5, doors 5, FWD, Petrol engine.
It was bought in Sydney 2761
Paid $4,750 cash for the car
The Dualis was created, and named, for serving as a family-friendly hatchback as well as it's dual purpose as a small sized SUV four-wheel-drive. This unique vehicle mash-up was essentially the first of it's own category here in Australia and ranked up some healthy sales in each of it's sections.
While it has more ground clearance than most hatchbacks and handles off-roading relatively well, it doesn't quite have the height of typical four-wheel drive. Interestingly enough, most of the actual sales made in Australia were for two-wheel-drives, indicative of the intention for most buyers. That said, some do opt for the 4WD to cross more rugged terrain in the Aussie bush. If you're into that, allow for a good few days to get parts imported if something goes wrong. As long as the issue isn't with the standard fit safety equipment, most mechanics should be able to do a sufficient job at repairing a Dualis. Otherwise, there are dealerships in many areas of the country as Nissan has been driving the Aussie roads for over half a century.
While this particular Nissan was made in a UK factory as opposed to the original Japanese factories, the quality appears to be on par and still decently reliable, compared to other Nissan models.
The 2012 Nissan Dualis is fitted with a 2.0 litre 4 cylinder engine with option for a 6-speed manual transmission or Continuously Variable Automatic transmission that has an option for manually operated ratios.
When dealing with a used vehicle, whether buying or selling a Nissan Dualis, the transmission and gear changing is one of the first things to look for. Any excessive noise, roughness, or stickiness on shifting should be inspected by someone experienced in transmission issues.
While the Dualis is marketed as somewhat of an off-road vehicle, the 2012 and later versions, especially two-wheel-drives, aren't really designed for too much wear and tear. If the exterior looks pretty dinged up, you'll want to make sure the car has a regular service history and proper detailing.
Check out one of our Nissan Buying & Selling Guides here.
Sold in: Sydney
2013 suzuki alto -sell my car
2013 suzuki alto –sell my car
Condition report – The car has no sunroof, leather, Sat Nav or hub caps. A small dent in the passengers side door. It needs a full detail inside and out.
Odometer reads – 70,000 kms, engine size 4, Automatic transmission, seats 4, doors 5, front wheel drive, petrol engine.
It was bought in Sydney 2213
paid cash for the car
We buy all makes and models throughout Central Coast, 2002 onwards, under 180,000 kms. No roadworthy certificate required.
Are You Selling is Central Coast's leading car buying service
Type in "sell my car Central Coast" and you" find hundreds of listings with different information and costs for selling a used car in the area. While a private online sale is one option for selling a car, it can quickly become stressful when buyers consistently don't show up or take you half-way through the selling process only to change their mind. At areyouselling.com.au we have spent over 35 years in the car business creating a simple process that takes the hassle out of selling your car so you don't have to waste precious time and energy. We have local teams of professionals all over the country who are ready to make the whole experience a breeze. Get started today and you'll have cash for you car in no time at all, you might even enjoy the process!
If you're thinking of selling your car in the big smoke instead, head to our Sydney page and get started there!
Selling a car is easy with our simple 3-step process:
- Fill out a brief sell my car enquiry form, it only takes a second.
- We'll contact you to arrange an inspection at your location in the Central Coast, and one of our vehicle experts will put together a free car valuation.
- We give cash for cars within 24 hours of setting the price!
Why should I use areyouselling.com.au to sell my car in the Central Coast?
- We'll buy makes and models of all kinds, not just the most popular cars!
- We don't require a roadworthy check so you don't have to waste time and money.
- You get a free car valuation from professionals.
- Cash for cars, in three simple steps.
We love cars and we love the Central Coast! Here are our favourite tips for navigating the roads:
There are so many reasons to drive to the Central Coast with long sandy beaches, wetlands, and meetings of rivers with the ocean. When driving in a new area, it's always good to brush up on road rules to keep yourself and other drivers safe!
Many rules are the same throughout Australia, but there are definitely some discrepancies state to state. In most cities, the speed limit is 50km/hr in urban and residential areas, down to 40 in school zones, and up to 100 or 110km/hr on the highway. In NSW, there are a few rules that are good to know about as they tend to come with some decent fines. For example, eating behind the wheel can get you a ticket of over $400 if you get caught, and will cost you three demerit points! Yikes. Even parking your own car and forgetting to lock it will cost you $112 if you stray more than three metres away.
A rather obscure but courteous fine is the $187 you'll pay if you splash people waiting for the bus on a wet day. It's probably a good idea not to do that even if no cops are around though. Honking unnecessarily can also cost you over $300, so make sure to save it for warnings and when needed to stay safe.
If you're unsure on the road rules and you're driving in the Central Coast, make sure you brush up and avoid the fines!
To obtain a quick sell my car quote find your closest city.
Sell My Car Melbourne, Sell My Car Sydney, Sell My Car Adelaide, Sell My Car Brisbane, Sell My Car Hobart, Sell My Car Darwin, Sell My Car Canberra
We guarantee payment before collection & within 24 hours
We guarantee the highest price
We come to you, Australia-wide
We are Australia's largest buyer, operating in 6 states
We have thousands of happy customers!
Speak to real people and get real prices
Refer a friend who sells their car to us and receive a $100 Coles or Myer gift card.
Moving overseas? No luck selling privately? Need to sell your vehicle quickly?
Save yourself the time and hassle by selling your car to Are You Selling. We offer guaranteed cash for cars that are post-2002 and have travelled less than 180,000km.
We specialize in buying all car brands including Ford, Holden, Nissan and Toyota plus all other makes and models, including all luxury vehicles such as Audi, BMW and Mercedes Benz.
With more than 35 years' industry experience, you can trust the experts at Are You Selling to get you the best money for your car whilst ensuring a fast, easy and safe transaction.
Simply contact us to sell your car; we'll take care of the rest.
Enquire with us today!
Variety – the Children's Charity is a national not-for-profit organisation that is committed to empowering Australian children who are sick, disadvantaged or have special needs to live, laugh and learn.
By giving practical equipment, programs and experiences, Variety helps children in need to overcome whatever obstacles they face so they can live life to the fullest.


text:
Homecoming 2017
September 15, 2017
Niobrara Cougars held Homecoming the week of September 11, 2017. This year's theme is "Surf Up." The spirit days were as follows:
Friday – Blue, Black, and Silver Day
Coronation was held on Monday, September 11, following the JV Football game at Verdigre. Freshmen attendants were Emily Parks and Gavin Chohon. Sophomore attendants were Summer Key and Ken Justo. Junior attendants were Logan Reynolds and Erika Kitto. The Queen Candidates were: Jeanene Henry, Kendra Hrbek, Isabel Rife, and Genesis Saul. King Candidates were Ricky Bickerstaff, Dakota Crosley, Kobe Keo, and Christian Swalley. Crownbearers were Madisyn Swanson, daughter of Scott and Pam Swanson, and Zachary Bolling, son of Chad and Susan Bolling. Ashley Guenther and Brady Pike, the 2016 Queen and King crowned the new King and Queen.
The 2017 Homecoming King was Dakota Crosley, son of Kyle and Lynda Crosley, and the 2017 Homecoming Queen was Jeanene Henry, daughter of Rob Henry and Teena Denny.
Students had a lot of fun dressing up for the different spirit days. In the Elementary, the 3rd grade was the winner on the spirit award and the 2nd grade was runner-up.
In the Middle School, Kennedi Reynolds and Chase Pritchett won the spirit awards.
Kalyn Nielsen and Adam DeKay won the spirit awards for the high school.
Mrs. Jenny Motacek was the staff member with the most spirit.
On Friday, all students were treated to grilled hamburgers for lunch and it was a gorgeous day to eat outside. The pep rally was held in the afternoon. Many fun games and competitions were played.
The week came to an end with the Homecoming dance on Friday evening at Verdgire.
Click on all the Bold, Underlined words to go see more pictures.


text:
Love. Light. Tribal. Perspective.
Tag: Warrior
Coffee with an Indian | EP_11: Comity of the Sun | Podcast
We get our power from the Sun. We get our power from the Sun. -Chief Good Day ——- If you love Coffee with an Indian… * Try Coffee with an Indian_BLACK: the Extended / Raw / R-Rated version of each
Which Gender Has The Spiritual Skin-In-The-Game?
Mother Nature? A coincidence, I don't think so! I don't give a damn what anyone says, I've witnessed women hold-down sacred space with more devotion and tact and with more strength than all the men in that circle, all the way
Divine Protection? What's That?
"Fear will kill you": As my Grandma Helen used to say, "don't live in fear—fear will kill you". This concept was something I would hear for most of my childhood. So believe me when I say, it has taken me


text:
I'm a huge bath and body junkie–I always love trying out new shower gels and lotions. So when iFabbo offered up this 8-piece set of travel-size shower gels and lotions from Italian brand Perlier, I jumped at the chance to try a new brand. Read on for my thoughts on the four different scents!
The product: Perlier 8-piece Mini Set, $19.95; available through HSN. Use the promo code "ifabbo5" to save $5 off your Perlier purchase!
The claim: Moisture-rich scented products leave skin silky-soft. Made with natural ingredients like olive oil and organic honey. Not tested on animals.
I tried it: I went through these scents multiple times to get a good impression of them, and my most surprising find was that the shower gels and lotions smelled a bit different from each other–that is to say, one might be too strong and the other just right. So I'll go through the shower gels first, and the lotions separately.
The full eight piece set comes in a clear zippered TSA-style bag (not pictured). I plan to reuse this during travel, as it's just the perfect size for toting beauty essentials. The shower gels come in 1.6-oz. tubes and have a nice middling consistency, neither too thick nor too runny. These would be awesome for tucking in a travel tote. I kind of wish the lids had a flip top rather than screwing on, but that's a minor gripe; I just think it would have been easier to handle in the shower.
The shower gels have fairly mild scents. The White Almond turned out to be my least favorite; this scent just reminds me of hotel showers, for some reason! The Olivarium smells mildly of olive oil, but not so much as to be overpowering. The Honey is mild and calming, while the Shea Butter & Citrus was probably my favorite; I have a soft spot for citrus bath products and this one doesn't have that artificial orange smell of some bath gels.
Now, on to the lotions. The 1-oz. tubes don't offer much if you're going on vacation for multiple days but I would say there is enough product to cover the body at least 2-3 times. The consistency is fairly thin, with the White Almond a bit more like a hand lotion and the Shea Butter & Citrus akin to a hand cream. These also have a screw-on cap which makes it easier to squeeze out the last drops of lotion.
The White Almond was again a miss, not for formula's sake but only because I've never been a huge fan of this particular scent. The Olivarium, surprisingly, was a huge miss; the scent was far too strong, especially compared to the shower gel. It was actually difficult for me to finish the tube, tiny as it was! The Honey was nice, but my favorite was the Shea Butter & Citrus, which was rich enough to use as a hand cream/cuticle cream. I used this on my legs while testing out a round of tanning products to ensure my skin stayed soft and hydrated.
My verdict: Overall, I think the concept is cool, the fragrances just weren't quite my cup of tea. For myself, I'm not sure I would repurchase the full size of these products, as they are a bit on the expensive side and are not as natural as they seem to appear–the bath gels do contain sulfates, which I prefer to avoid. As a gift set, this would be nice for someone whose fragrance preferences you just don't know–with four to choose from, you've got better odds of finding a winner!


text:
Edited by Caitlin Cesa + Rachel Jones
Meet three artists whose local roots influence the subject matter, materials, and process for their greatest works.
Jason Sauer is a classically trained painter living in the Garfield neighborhood. From large outdoor murals to small Flemish-inspired impressionistic paintings on metal, Sauer can create art from anything. He frequently uses the distinctly American demolition derby as a high-art form. By reclaiming discarded automobiles and transforming them, he conceives performance art that lets fate and happenstance create a sculpture. These sculptures are then set in a calm, sterile museum environment as a record of the brutality of human emotional release. Sauer also works with locally sourced objects, using his art to create a dialogue within his community. pittsburghartcar.com.
Best known for oil paintings, which are couched in observational realism, William DeBernardi's artwork involves figure-based imagery concerning common human activity and behavior, often within the context of seemingly everyday environments. A Professor Emeritus of Painting and Drawing at Carlow University, DeBernardi has exhibited in various regional, national, and international galleries, universities, and museums. DeBernardi's recent exhibitions include "An American Festival" at the Penn State University New Kensington Gallery and "People, Places, and Things" at the Southern Alleghenies Museum of Art. williamdebernardi.com.
Kim Fox credits her aesthetic, subject matter, and choice of materials to her childhood in rural Western PA. Fox, who earned a Bachelor of Fine Arts in Printmaking at the College of Charleston, recently started exploring regional arts and crafts with a more rural bent, creating her own patchworks out of vintage tins and salvaged wood. Her tin quilts have been shown in "New Order: Collage Now" at the Future Tenant Gallery and at SPACE Gallery in Pittsburgh; in Providence, RI, in the "Memory Quilt Collage" exhibit; and in "Beyond the Bed Covers" at A.I.R. Gallery in Brooklyn, NY. Fox is also an itinerant art teacher in regional schools, as well as a designer and illustrator. workerbird.com.
Buy Amoxil online


text:
InteractiveFull MotionTiltFixed
OmniClassic SeriesOmniSelect SeriesOmniElite Series
Safe & CertifiedCF TechnologyVESA Mount InterfacesEnvironmentally Responsible
Press Releases
Award Winning ProductsIn the News
OmniMount's professional-grade ceiling mounts provide single and back-to-back TV mounting options. With versatility and flexibility built right in, these TV mounts are ideal for any ceiling application.
Understanding the importance of compatibility with all projector sizes and brands, OmniMount projector mounts are designed as universal mounting solutions.
Find compatible mounts for your TV or projector.
U.S. Contact
Saint Paul, MN, U.S.A.
E-mail: email@example.com
EMEA Contact
Amersfoort, Netherlands
E-mail: firstname.lastname@example.com
Using only numbers (in/lb or mm/kg), please enter the width & height
of your flat panel's mounting holes followed by the panel's weight (without stand).


text:
|For the week of May 30, 2017 – Vol. 15, Issue 21
>> Market Update
QUOTATION OF THE WEEK… "April is the cruelest month…" –from "The Waste Land" by T.S. Eliot, British poet, playwright and critic
INFO THAT HITS US WHERE WE LIVE … The Nobel prize winning laureate probably didn't have the housing market in mind when he penned his famous line. But his description of April might be apt for housing now, as both new and existing home sales dipped last month. New homes delivered the cruelest headline number, down 11.4%. But that was because revisions to March pushed new home sales up, to their fastest pace yet in the recovery. And at a 569,000 unit annual rate, April new home sales are still high compared to 2016. Plus, the median sales price dropped, indicating builders are more sensitive to affordability.
Existing home sales saw less of a drop in April, slipping just 2.3%. And, hey, they still hit a 5.57 million unit annual rate, up from a year ago. This monthly dip was also set up by fabulous numbers the prior month, when March existing home sales came in at their fastest pace in more than a decade. Nationally, supply remains tight, but inventories increased. Demand stayed strong, with properties typically on the market just 29 days, the shortest time period in the last six years. Freddie Mac's chief economist opined, "With home sales, housing starts and home values up, 2017 is shaping up to be the best year for housing in over a decade."
BUSINESS TIP OF THE WEEK… Look for new leads, but don't forget to cultivate the clients and prospects you already know. Introduce them to new ideas; ask them to refer you to friends, relatives and colleagues.
>> Review of Last Week
RAISING THE BAR… The mood on Wall Street went super positive once again. At the end of the week, the broadly-based S&P 500 and the tech-heavy Nasdaq both reached fresh all-time highs, while the blue-chip Dow finished just 0.2% below its all-time record close. FOMC Minutes from the last Fed meeting revealed "it would soon be appropriate" to tighten monetary policy again, meaning hike rates in June. This shows that the central bank has confidence in the economic outlook, attributing slower Q1 growth to transitory factors. Friday's revised Q1 GDP had the economy growing at a better than expected 1.2% annual rate.
The week ended with the Dow UP 1.3%, to 21080; the S&P 500 UP 1.4%, to 2416; and the Nasdaq UP 2.1%, to 6210.
Bonds largely ended the week with modest gains in spite of the upwardly revised Q1 GDP number. The 30YR FNMA 4.0% bond we watch finished the week UP .01, at $105.45. In Freddie Mac's Primary Mortgage Market Survey for the week ending May 25, national average 30-year fixed mortgage rates sank to a new low for the year. Remember, mortgage rates can be extremely volatile, so check with your mortgage professional for up-to-the-minute information.
DID YOU KNOW?… Realtor.com reports that of the top 26 college degrees that would enable graduates to buy a home in four years, 22 were in engineering. Petroleum Engineering led, with a starting salary that could get the graduate a home in just 2.6 years .
>> This Week's Forecast
PENDING HOME SALES, CONSUMER SPENDING, INFLATION REBOUND; MANUFACTURING, JOBS GROW… It's nice to see Pending Home Sales forecast back in positive territory in April. Likewise Personal Spending and the Core PCE Prices inflation measure are also expected to rebound. Manufacturing is predicted to stay in growth territory, according to the Midwest Chicago PMI and the national ISM Index. May Nonfarm Payrolls are forecast to come in just under 200,000, with Hourly Earnings continuing to rise, a good thing for housing.
U.S. financial markets were closed yesterday, May 29, in observance of Memorial Day.
>> The Week's Economic Indicator Calendar
Weaker than expected economic data tends to send bond prices up and interest rates down, while positive data points to lower bond prices and rising loan rates.
Economic Calendar for the Week of May 29 – Jun 2
>> Federal Reserve Watch
Forecasting Federal Reserve policy changes in coming months… The probability for a rate hike in June is now solidly above 80%, but rates are expected to stay untouched through the summer. Note: In the lower chart, an 84% probability of change is only a 16% likelihood the rate will stay the same.
Current Fed Funds Rate: 0.75%-1.0%
Probability of change from current policy:
| Ron Schulz
Senior Loan Officer
13140 Coit Rd # 502
Dallas, TX 75240
Office: 214-346-5279
Mobile: 214-794-4014
Fax: 972-284-0715


text:
Angel Baubles (set of 8)
A set of 8 wooden or perspex laser cut and engraved decorations. The angels are decorated with an intricate engraved lacey motif. The angels in the small set measure approximately 40 x 50mm, the larger set average 80 x 100mm. Cut from 3mm wood or perspex and threaded on to satin ribbon. All wooden products are finished and sealed with a clear matt varnish.


text:
WoodlandsMangiare > Vegetariani ...
Sei il proprietario di questa EcoAzienda?
Conferma le EcoCompetenze 0
Condividi la tua opinione su quest'EcoAzienda
Woodlands has evolved from serving mainly south Indian dishes to the diverse Pan Indian vegetarian destination it is today. We are proud to cater to vegetarian, vegan, gluten free and Jain (no onion or garlic) diners from London and all over the world. Mr Sood's son, Rajat an ex banker and his wife Pooja, a Chartered Accountant are now at the helm of the company.
Non sono presenti certificazioni


text:
Is that Alisia Dragoon complete? If so them I'm interested.
Man you have a lot of PSP games.
I must have missed that. I didn't see any PSP games listed.
A Black Falcon: no, computer games and video games are NOT the same thing. Video games are on consoles, computer games are on PC. The two kinds of games are different, and have significantly different design styles, distribution methods, and game genre selections. Computer gaming and console (video) gaming are NOT the same thing."
Is Phantasy Star complete? In the photo the manual is missing.
How much for Shining in the darkness?
I'm interested in that Midnight Resistance.
Sega Dude
Visit my Instagram @RetroMegabit or RetroMegabit.com
Hey all, I'm back! Staying at home all day has allowed me to get my shit together and get it up on here. So massive bump!
There are currently 1 users browsing this thread. (0 members and 1 guests)


text:
Types of Prints
Print Only
Professionally printed on Fuji Deep Matte paper to provide a deep matte non-reflective surface that feels velvety soft.
Need the photo mounted and framed? Click here to learn more about the framing process.
Fine Art Canvas Wraps are printed directly onto museum-quality canvas material using high-quality archival inks. Lustre Laminate is applied to each piece to protect the surface of the print for generations to come. Minimum frame depth of 1 1/2"
Acrylic Prints are beautiful photo prints face-mounted to plexiglass with polished edges. Each print is printed on LuxJet Metallic paper a mounted to a firm DiBond backing. To make hanging an easier task each Acrylic piece of art comes standard with a French Cleat to ensure your art will be displayed with beauty and confidence.


text:
Mum & Dad horror trailer online
Mum & Dad, the British horror from writer and director Steven Sheil, has arrived online and it's looking pretty unnerving. While it doesn't give away too much story or much horror, it does certainly make you feel very uncomfortable and give you the feeling that some seriously bad things are about to happen.
The film is apparently very good, very scary and a cracker for the horror fans, and the trailer is right here.
The story for Mum & Dad, according to the official blurb, goes something like this:
"Mum and Dad, and their 'adopted' children, Birdie & Elbie, work at the airport. The family live off whatever they scavenge from cargo holds, offices and hotels - including a steady stream of transient workers who populate the airport's soulless hub. When Lena, a young Polish office cleaner, is befriended by Birdie, she gets drawn into a nightmarish world of torture, murder and perversity. Imprisoned in a suburban House of Horrors and designated a 'Mummy's Girl', Lena's only options appear to be to become part of the family - and join them in their insanity - or die."
Oh which option would you choose? The story sounds pretty interesting, and when you see the trailer after knowing that then you get a bit more from it.
I think the characters and set-up could be a little clearer in the trailer, but some of the shots and the line deliveries look really good. See what you think, one for the horror fans for sure.