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Clinical Veterinary Microbiology
Comparison of Cultivation and PCR-Hybridization for Detection ofSalmonella in Porcine Fecal and Water Samples
Ingrid Feder, Jerome C. Nietfeld, John Galland, Teresa Yeary, Jan M. Sargeant, Richard Oberst, Mark L. Tamplin, John B. Luchansky
Ingrid Feder
Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, 1 and Department of Diagnostic Medicine/Pathobiology 2 and Food Animal Health and Management Center, 3 College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506
Jerome C. Nietfeld
Department of Diagnostic Medicine/Pathobiology 2 and
John Galland
Food Animal Health and Management Center, 3 College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506
Teresa Yeary
Jan M. Sargeant
Richard Oberst
Mark L. Tamplin
Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, 1 and
John B. Luchansky
DOI: 10.1128/JCM.39.7.2477-2484.2001
A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37°C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42°C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37°C followed by overnight enrichment in RV10 at 42°C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42°C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viableSalmonella bacteria. Presumptive Salmonellaisolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invAgene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement betweenSalmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, whileSalmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detectingSalmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.
Samonella bacteria shed in the feces of asymptomatic swine are important sources of environmental and carcass contamination (3, 19, 29, 30). They can be shed from swine at levels of less than 10 CFU/g of feces (19) and remain viable in soil for more than a year (28). Detection of Salmonella enterica in fecal or water samples can be limited by low numbers of the bacterium (e.g., less than 10 CFU/g), thus necessitating the use of an enrichment step. Enrichment and preenrichment broths can dilute inhibitory compounds produced by competing bacteria in the sample, as well as aid in recovery of injured, stressed, or lag-phase bacterial cells (6,21). Generally, isolation of Salmonella from samples, such as feces, that contain approximately >107 aerobic bacterial cells/g requires a selective enrichment medium that permits the growth of Salmonella while inhibiting the growth of other aerobic bacteria (21, 24). However, some selective enrichment media also inhibit the growth of Salmonellabacteria that are stressed or damaged (4, 6).
Although bacteriological assays have historically been the method of choice for the recovery of Salmonella from feces and environmental samples, PCR has become an important technique for more-rapid detection of pathogens in feces and environmental samples when an isolate is not required (5, 7, 37, 38). Like bacteriological assays, PCR often requires enrichment of fecal samples to increase Salmonella numbers and to aid in the dilution of compounds that may interfere with the PCR (5, 7, 22, 23, 37, 38,40, 42).
Discrepancies between detection of Salmonella nucleic acid sequences in feces by the PCR and recovery of the bacterium on synthetic media have been noted in favor of the PCR; however, one comparative study did not use the same enrichment broth for comparing the two approaches, bacteriological cultivation and PCR, and some studies did not include a preenrichment step in the cultivation procedure (5, 7, 38). Also, the PCR primers selected can lead to inaccurate results. False-positive results arise from mispriming of nucleic acid sequences that are similar to target DNA, particularly when samples contain DNA from ingested material, fecal flora, and/or mammalian cells (31). For instance, there is over 90% homology between the genomes ofSalmonella and Escherichia coli(34), and this can cause considerable mispriming and lead to false-positive amplicons. Misprimed amplicons similar in length to the amplicon of interest are also difficult to distinguish on agarose gels but can be identified by Southern hybridization with internal nucleotide probes (31, 38, 43).
An improved understanding of the agreement between the bacteriological recovery of Salmonella, the current "gold standard" to which all other Salmonella detection tests are compared, and the PCR-based detection of Salmonella is warranted to determine if the latter could be used as a definitive or a presumptive test. As a presumptive test, the PCR could be used to determine samples from which a representative isolate should be obtained for further characterization. For this use, results from the PCR must be known within 18 to 24 h so that an isolate can be obtained before appreciable cell death occurs in the retained sample or in the primary enrichment broth (32). As a definitive test, the PCR, followed by Southern hybridization for the confirmation of amplicons, could be used to determine which samples contain SalmonellaDNA.
The purpose of this research was to compare cultivation- and PCR-based methods for the detection of Salmonella in porcine fecal and water samples from swine farms. The null hypothesis was that no difference would be measured between these methods for detectingSalmonella.
Sample collection and bacterial isolation.Samples were enriched by one of three methods: (i) overnight preenrichment in tryptic soy broth (TSB; Difco Laboratories Inc., Detroit, Mich.) at 37°C and overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10; Difco) at 42°C (TSB-RV10 enrichment method), (ii) overnight preenrichment in a laboratory-prepared medium previously designated 3MC broth (2, 20) at 37°C and overnight enrichment in RV10 at 42°C (3MC-RV10 enrichment method), and (iii) direct overnight enrichment in RV10 at 42°C (with no secondary enrichment) (RV10 enrichment method). All samples were collected within 100 mi of the laboratory. The samples were placed directly into 10 ml of TSB, 3MC broth, or RV10 and stored on ice in a cooler until processed later the same day.
In the first trial, 92 swabs of fresh swine feces, consisting of rectally inserted, cotton-tipped applicators or cotton-tipped applicators that had been inserted into fresh swine feces on the floor, collected from the nursery and farrowing barns at three swine farms (farm 1, 4 March 1998, n = 29 ; farm 2, 10 March 1998, n = 22 ; and farm 3, 6 April 1998, n = 41 ) were placed into 10 ml of TSB and preenriched overnight at 37°C (Fig. 1). One hundred microliters of each sample was then transferred into 10 ml of RV10 and enriched overnight at 42°C (Fig. 1). In the second trial, 24 water samples (1 ml), collected from bowl waterers or mud holes from farm 3 on the same day that the fecal samples were collected, were placed into 10 ml of 3MC broth and preenriched overnight at 37°C (Fig. 1). One hundred microliters of each sample was then transferred into 10 ml of RV10 and enriched overnight at 42°C (Fig. 1). In the third trial, 34 swabs of fresh swine feces collected from the pen floors of a swine finishing facility at a fourth farm (13 April 1998) were placed directly into 10 ml of RV10 (Fig. 1) and incubated overnight at 42°C. After enrichment in RV10, all 150 samples were subcultured onto modified semisolid Rappaport-Vassiliadis (Difco) agar plates (Fig. 1) by drop (50-μl) inoculation (11) and incubated for 24 h at 42°C. Samples displaying motile growth around the initial drop of the inoculum were subcultured onto Hektoen Enteric (Difco) agar plates for the isolation of single colonies and incubated at 37°C for 24 h (Fig. 2). Bacterial colonies with typicalSalmonella morphology (i.e., clear with black centers for most Salmonella serovars or clear without black centers forS. enterica serovar Choleraesuis) were tested by slide agglutination with Salmonella antigen group A-I, Vi polyvalent antiserum (Difco). In addition, Salmonellaisolates that tested positive using the polyvalent antiserum were forwarded to the National Veterinary Service Laboratory (Ames, Iowa) for further authentication and identification to species level using antisera specific for O type and H type (i.e., univalent antiserum).
Flow diagram of the methods used to isolate or detectSalmonella in fecal or water samples from swine feces. ∗, after DNA extraction, 10 μl of sample DNA was used per PCR. MSRV, modified semisolid Rappaport-Vassiliadis agar; HE, Hektoen Enteric agar.
Comparison of the time to Salmonelladetection via Southern hybridization with that by serotyping of a single colony isolate. MSRV, modified semisolid Rappaport-Vassiliadis agar; HE, Hektoen Enteric agar; TSA, tryptic soy agar.
DNA extraction and purification.Samples for DNA extraction and subsequent PCR amplification were retrieved directly from the broths after the initial enrichment. The DNA was extracted as described previously by Lou et al. (23) with some modifications. The 10-ml overnight samples were vortexed for 2 min, and particulate matter was allowed to settle for 10 min at room temperature. A 1.5-ml portion of the resultant supernatant was transferred to a microcentrifuge tube and centrifuged at room temperature for 2 min at 2,800 × g . One milliliter of this supernatant was removed to a second microcentrifuge tube and centrifuged at room temperature for 15 to 20 min at 12,000 × g . The supernatant was discarded, and the pellet was resuspended in 1 ml of sterile high-pressure liquid chromatography-grade H2O (Fisher Scientific, St. Louis, Mo.) and centrifuged at room temperature for 5 min at 12,000 × g . This wash step was repeated, and the pellet was resuspended in 500 μl of sterile H2O, boiled for 5 min, and then centrifuged at room temperature for 5 min at 12,000 × g . Eighty microliters of sample supernatant and 20 μl of a 5× stop solution (2% sodium dodecyl sulfate, 25% glycerol, 0.05% bromophenol blue, and 0.05% xylene cyanol) were added to 60 μl of sterile H2O and incubated for 15 to 20 min at 70°C. Ninety microliters of this supernatant-stop mixture was added to a Sepharose CL-6B gel filtration spin column and centrifuged at 700 × g for 2 min at room temperature. If blue dye was noted in the resultant filtrate, centrifugation was repeated using a fresh spin dialysis column. The resultant filtrate was used for the PCR.
Sepharose CL-6B spin columns were prepared essentially as described by Maniatis et al. (25). Sepharose CL-6B (Sigma, St. Louis, Mo.) was washed three times with equal amounts of 10 mM Tris-Cl–1 mM EDTA (TE) buffer (pH 8.0). Buffer was removed for a final concentration of 60% Sepharose CL-6B. Autoclaved 60% Sepharose CL-6B in TE buffer (800 μl) was added to a 1-ml tuberculin syringe packed with glass wool to about 1 cm. The spin column was centrifuged at 700 × g for 2 min to remove excess TE buffer. Additional 60% Sepharose CL-6B was added, and centrifugation was repeated until the Sepharose CL-6B reached a final height of the 1-ml mark on the tuberculin syringe.
PCR.To optimize the efficacy of target DNA amplification, thawed reagents were held on ice during setup for the PCR. All reactions included a hot start technique (31) in HOTSTART reaction tubes (HOTSTART Micro 50; Molecular Bio-Products, Inc., San Diego, Calif.). All PCR assays were performed using 10 μl of template and 90 μl of the PCR mixture. Templates were heated to 94°C for 5 min before addition of PCR core reagents. Final concentrations of PCR reagents were 1.8 mM MgCl2; 1× PCR buffer II at 10% of the total volume; 200 μM (each) deoxynucleoside triphosphate; 2.5 U of Taq DNA polymerase (Gene Amp core reagents; Perkin-Elmer Corporation, Foster City, Calif.) per 100 μl; and 0.3 μMSalmonella oligonucleotide primers (37, 38) (Gibco BRL, Life Technologies, Rockville, Md.). The DNA templates were amplified by use of one denaturation cycle at 94°C for 2 min; 5 amplification cycles at 94°C for 60 s, 52°C for 30 s, and 72°C for 30 s; 40 cycles at 94°C for 30 s, 52°C for 15 s, and 72°C for 30 s, followed by an extension cycle at 72°C for 15 min; and a hold cycle at 4°C.
S. enterica serovar Choleraesuis DNA extracted by the Sepharose CL-6B method from RV10, 3MC broth, and TSB was used to determine optimal MgCl2 concentrations for PCR amplification. Negative controls, uninoculated TSB, 3MC broth, and RV10 samples and E. coli grown in TSB, were subjected to Sepharose purification. Positive controls consisted of Sepharose purifications from a pure culture of S. enterica serovar Choleraesuis grown overnight separately in TSB, RV10, and 3MC broth seeded with a swab of swine feces.
Detection of amplified products.Twelve-microliter aliquots of a PCR sample and 3 μl of sample application buffer (0.25% bromophenol blue, 40% [wt/vol] sucrose in water) were analyzed using ethidium bromide (EtBr)-stained agarose gels. To confirm amplification of PCR products detected on agarose gels as Salmonella, Southern hybridization (36) was conducted using a previously described digoxigenin-labeled, internal, oligonucleotide probe corresponding to nucleotides 106 to 125 of the Salmonella invA gene (37, 38). Nylon membranes (MSI, Westboro, Mass.) were prehybridized followed by hybridization with the probe as specified by Boehringer Mannheim (Indianapolis, Ind.). The DIG Easy Hyb buffer (Boehringer Mannheim) was used for the prehybridization and hybridization steps. Using the DIG Wash and Block buffer set (Boehringer Mannheim), probe that hybridized to the amplifiedSalmonella nucleotide segment was detected using the antidigoxigenin-alkaline phosphatase antibody (α-DIG-Alkaline Phosphatase-Fab fragments; Boehringer Mannheim), a chemiluminescent substrate for alkaline phosphatase (CSPPD; Boehringer Mannheim), and radiographic film (medical X-ray film; Fuji, Tokyo, Japan). The nylon membrane was exposed to radiographic film for 2 h. The PCR amplicons observed as bands on EtBr-stained agarose gels were compared to the corresponding bands on the radiographic film.
Data analysis.For the purposes of this study, samples that were positive by either cultivation (i.e., bacterial isolation with serological confirmation) or PCR (i.e., the PCR with confirmation using Southern hybridization) were considered positive, even if the samples were negative using either one of these detection methods alone. This assumption, using the combinatorial results of the PCR and cultivation to identify Salmonella present in a sample, enabled us to estimate the false negatives for each test. For example, a false negative would be defined as a sample that tested negative by cultivation and positive by the PCR and vice versa. Since the true-negative status of the samples was not known, specificity could not be reported. For this reason, we used the term "relative specificity" to approximate specificity. The relative specificity and sensitivity (26) of the PCR were determined by comparison to bacteriological results. For statistical analysis, a 2 × 2 comparison table was constructed to show the association ofSalmonella-positive results between the two detection strategies. The three different enrichment methods used were not compared because different samples were used for each enrichment scheme. The relative specificity and the sensitivity of PCR-hybridization and EtBr-stained agarose gels were calculated with the total number of positive samples as the standard by using a 2 × 2 table as described previously (26), and the differences between the two methods were compared.
Agreement between the cultivation- and PCR-based methods for detection of Salmonella was evaluated by use of the kappa statistic (17, 27, 35). The kappa statistic measures agreement between two tests that is beyond chance, with chance being a value of zero and with 1.00 being complete agreement, not by chance (8). Agreement between tests on positive samples is given as much weight by the kappa statistic as agreement between tests on negative samples. Kappa values were summarized as unacceptable (<0.3), acceptable (0.3 to ≤0.5), good (0.5 to 0.7), and excellent (>0.7) (27).
Detection of Salmonella by either cultivation- or PCR-based methods.Of the 150 samples tested in this study, 65 (43%) were positive for Salmonella by either cultivation or PCR-hybridization (data not shown). No Salmonella bacteria were detected on farm 1 or farm 2 by either approach alone (Table1). Salmonella bacteria were detected in 61% (25 of 41) of the fecal samples from farm 3 (Table 1). On this farm, Salmonella bacteria were also detected in 63% (15 of 24) of the water samples (Table 1). Detection ofSalmonella in fecal samples from the finishing farm (i.e., farm 4) was 74% (25 of 34) (Table 1). Of the 65Salmonella-positive samples from all of the farms tested, 47 (72%) were positive by cultivation, 52 (80%) were positive by PCR-hybridization, and 51 (79%) were positive by visualization of the PCR amplicons on EtBr-stained agarose gels (data not shown). Therefore, there were 18 false negatives with respect to cultivation, 13 false negatives with respect to the PCR-hybridization detection, and 14 false negatives with respect to EtBr-stained agarose gels. Only 34 (52%) of the 65 positive samples were detected by both cultivation and PCR-hybridization (data not shown).
Detection of Salmonella using cultivation, PCR-hybridization, and a combination of cultivation and PCR-hybridization following enrichment using three different media
In the present study, five Salmonella serovars were detected from two of the four clinically normal swine herds tested (data not shown). At a nursery-farrowing farm, S. enterica serovar Choleraesuis biotype kunzendorf, S. enterica serovar Anatum,S. enterica serovar Derby, and S. entericaserovar Enteritidis were isolated from feces, whereas serovar Anatum and serovar Derby were isolated from water samples. At the finishing farm, S. enterica serovar Choleraesuis, S. enterica serovar Enteritidis, and S. enterica serovar Bredeney were isolated from fecal samples.
Comparison of detection methods.Of the 65 positiveSalmonella samples, only 52% (34 of 65) were detected by both cultivation and PCR-hybridization; overall agreement (kappa) between both methods was 53% (data not shown). For the TSB-RV10 enrichment method, Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples. Likewise, for the 3MC-RV10 enrichment method,Salmonella bacteria were detected by both methods in 73% (11 of 25) of the positive samples. However, for the RV10 enrichment method, Salmonella bacteria were detected by both methods in only 24% (6 of 25) of the positive samples. Therefore, when a TSB preenrichment followed by enrichment in RV10 was used, cultivation failed to detect 3% (3 of 92) of the positive samples while PCR-hybridization failed to detect Salmonella in 5% (5 of 92) of the positive samples (Table 2). In contrast, when a preenrichment step was not performed and samples were directly enriched into RV10, there was a higher rate of false-negative reactions as cultivation failed to detect 41% (14 of 34) of the positive samples (Table 2). The agreement (kappa) between cultivation and PCR-hybridization was 76% for samples that were preenriched (data not shown). Agreement (kappa) between cultivation and PCR-hybridization was higher for the TSB-RV10 (71%) and 3MC-RV10 (63%) methods than for the RV10 enrichment method (6%) (data not shown). Compared to cultivation, PCR-hybridization displayed 77% sensitivity and 96% relative specificity with respect to the TSB-RV10 enrichment method, 79% sensitivity and 90% relative specificity with respect to the 3MC-RV10 enrichment method, and 55% sensitivity and 39% relative specificity with respect to the RV10 enrichment method. Also, in comparison to bacterial isolation and biochemical identification of presumptive Salmonella isolates, PCR-hybridization detected Salmonella 3 days earlier than if a preenrichment was used and 2 days earlier if preenrichment was not performed (Fig. 2).
A 2 × 2 comparison of Salmonelladetection by cultivation and PCR-hybridization versus the combined results of cultivation and PCR-hybridization
Comparison between agarose gel electrophoresis and Southern hybridization for the detection of PCR amplicons.Nine amplicons, three from each of the three enrichment methods, were visualized on EtBr-stained agarose gels but did not hybridize to theSalmonella-specific oligonucleotide probe (Table3). Therefore, the corresponding false-positive rates for detection of Salmonellaamplicons on agarose gels for each type of enrichment were 3, 13, and 9% for the TSB-RV10, 3MC-RV10, and RV10 enrichment methods, respectively (Table 3). Southern hybridization detected only one false-negative sample compared to EtBr-stained agarose gel electrophoresis detection of the PCR amplicons, as one band was seen on a radiograph of a Southern hybridization that was not seen on the corresponding EtBr-stained agarose gel when the RV10 enrichment method was used (Table 3).
A 2 × 2 comparison of Salmonelladetection by PCR and EtBr-stained agarose gels with that by PCR-hybridization
Neither cultivation nor PCR-hybridization alone detected all of the 65 Salmonella-positive samples from swine farms. However, the results from this study indicate that the PCR is equivalent to cultivation for delineating positive samples, particularly when a preenrichment step is used. Additionally, our results and those of others (38, 43) establish the importance of nucleic acid probes to evaluate the PCR amplicons visualized on EtBr-stained gels. With respect to gel electrophoresis and the visualization of EtBr-stained gels, Southern hybridization improved both the sensitivity and relative specificity of the PCR. While we had only one false positive (0.67%; 1 of 150), two other studies, one using feces and one using blood as the biological samples, have shown higher false-positive rates (38, 43). More specifically, 13 of 21 (54%) fecal samples tested from a single colony of beagles had PCR amplicons that could be detected on an EtBr-stained agarose gel but not on the corresponding radiographic film after Southern hybridization. The higher false-positive rate with respect to feces was most likely due to the cross-reaction of the PCR primers with a commensal bacterium or other DNA within the fecal specimens of the beagle colony tested. As there is no way of determining background flora, or other background DNA, within a sample containing DNA that may cross-react, it is imperative to perform hybridization. For this reason, Southern hybridization was used to confirm presumptiveSalmonella positives from PCR-based assays just as serotyping was used to confirm presumptive Salmonella from cultivation-based strategies. While the PCR-hybridization approach was equivalent to the bacteriology-based technique, the former only confirms the isolate at the genus level and does not delineateSalmonella species or serovar. For epidemiological purposes, the species or serovar of Salmonella may be of interest, particularly on swine farms where Salmonella prevalence is high and where more than one Salmonella species or serovar can be isolated (1, 9, 29, 33). In this regard, univalent O typing can be done within 1 day, whereas confirmation ofSalmonella isolates by serotyping could take an additional 1 to 2 weeks depending on the difficulty of flagellar typing (i.e., H typing).
S. enterica serovar Choleraesuis biotype kunzendorf, the most common cause of salmonellosis in swine (16, 41), and serovar Choleraesuis, a rare serovar in swine in the United States (16), generally have been isolated only from clinically ill swine (14, 37, 41). Our ability to detect serovar Choleraesuis, an H2S-negative biotype, was aided by using modified semisolid Rappaport-Vassiliadis agar, a medium previously described for the enhanced recovery of Salmonella from human feces (13, 18). Detection by this medium is based on motility and not on H2S production as determined on Hektoen Enteric or XLT4 agar (11, 12, 13, 18). In contrast to our study, the predominant Salmonella serotypes recovered from swine feces in other studies included S. enterica serovar Heidelberg, S. enterica serovar Mbandaka, S. enterica serovar Typhimurium, S. enterica serovar Worthington, and S. enterica serovar Tennessee (1, 9,10, 33).
At the farms where Salmonella was detected by either cultivation or the PCR technique, the detection ofSalmonella ranged between 61% (25 of 41) and 74% (25 of 34). Previously, field trials using PCR-based and culture-based methods for the detection of Salmonella from nonsymptomatic pigs have not been reported. Detection of Salmonella from swine intestinal swabs was 40% (two of five) using either bacteriology-or PCR-based methods when intestinal specimens from five dead pigs suspected of having salmonellosis were tested (37). As another example, detection of Salmonella was about 8% (6 of 79) and 9% (7 of 79) using bacteriology-and PCR-based methods, respectively, when intestinal specimens from 79 pigs with diarrhea were tested (14). In a study of clinically normal swine, the detection of Salmonella in feces averaged 25% from the feces from 24 of 29 farms tested (10). In additional studies of clinically normal swine, the detection ofSalmonella in feces was 37% (186 of 504) in feces from one farm (33) and ranged between 3 and 22% for seven other farms tested (9). Frequencies this high could challenge the Salmonella reduction practices at slaughter and could present an appreciable risk to public health.
RV10 was used as an enrichment medium for all samples in this study as Rappaport-Vassiliadis broths are optimal for isolatingSalmonella from swine feces (1, 15, 39). The RV10 enrichment method (i.e., no preenrichment) did not perform as well as the other two methods that used a preenrichment step and subsequent RV10 enrichment. While the RV10 enrichment method may not be an ideal method for the isolation of some Salmonella species from feces, Salmonella could be detected by the PCR method after enrichment in RV10. As reported by Stone et al. (37), a Rappaport-Vassiliadis enrichment broth inhibited the detection ofSalmonella by the PCR, whereas in the present study RV10 was not inhibitory to PCR amplification of DNA. Within the RV10 enrichment group, cultivation detected only 44% (11 of 25) of the positive samples even though detection of Salmonella in fecal samples at this finishing farm was 74% (25 of 34). Our ability to detectSalmonella using the PCR was independent of the enrichment medium and was most likely due to the DNA extraction technique used.
Unfortunately, the experimental design of this study did not permit comparisons among the different enrichment techniques, so we do not know if the addition of a preenrichment step would have improved the isolation of Salmonella in the RV10 enrichment group. Since the three enrichment methods used in this study were not directly compared, we also do not know if the enrichment protocol used for a sample affected the recovery of specific Salmonellaserotypes. However, some media may be better than others for the recovery of Salmonella. Further studies to compare the efficacies of selective preenrichment, nonselective preenrichment, and no preenrichment using the same sample are warranted.
The kappa statistic was used to evaluate the agreement of results between bacteriology-and PCR-based methods. When a preenrichment was used (i.e., TSB or 3MC broth), agreement between bacteriology-and PCR-based methods was higher than the agreement between bacteriology-and PCR-based methods from the RV10 enrichment method. According to the kappa statistic, the PCR-hybridization approach was as effective as cultivation for detecting Salmonella for the TSB-RV10 and 3MC-RV10 enrichment groups. These results indicate that enrichments in TSB-RV10 and 3MC-RV10 were equivalent to the PCR-hybridization procedure, while enrichment in RV10 was not optimal. In addition, the PCR-based detection may have outperformed cultivation with respect to the RV10 enrichment group due to a high number of nonviable Salmonella bacteria. It can be argued that bacterial cells in field samples need a resuscitating step, as they can be damaged. These damaged cells may not grow or may die in selective media. Likewise, samples containing only dead cells that are diluted into a synthetic medium must have a final concentration of 4 × 102 cells/ml to have one cell/2.5 μl, the volume used in the PCR. Therefore, it is highly unlikely that the PCR-hybridization method detected dead cells in this study.
While used in this study to compare PCR- and bacteriology-based assays, the kappa statistic has some disadvantages. The kappa statistic measures agreement of negative results as well as positive results. As such, a high kappa value, such as 90%, could indicate that the compared tests are equivalent in missing a positive sample. Since the samples used in this study were from farms, we do not know if both bacteriology-and PCR-based assays missed anySalmonella-positive samples. Another weakness of the kappa statistic pertains to the cutoff value that should be used to distinguish between poor and good agreement. The kappa statistic cutoff values are strictly arbitrary and vary by source (8, 17, 27,35). In spite of these weaknesses, the kappa statistic was used because the presence or absence of Salmonella in the samples was unknown, so a measure of agreement, not accuracy, between the two tests was required. When using the kappa statistic to evaluate the agreement between the two methods, the researcher must determine what is acceptable, particularly in the range between 0.3 and 0.7, as the scientist may reasonably find 0.3, as well as a higher value such as 0.60, to be unacceptable.
Salmonellosis in swine is an economic concern to the swine producer and a human health risk, and a reliable and rapid technique forSalmonella detection would be useful. Historically, the PCR has been used as a confirmatory test following bacterial isolation. We provide evidence here that the PCR could be used in combination with cultivation to improve Salmonella detection. For the synthetic medium combinations used in this study, the PCR worked as well or better at delineating positive samples. When an isolate is required, the PCR could be used as a presumptive or screening test. The organism could then be cultivated from the enrichment broth used for the PCR or from the original sample. However, the observation that the PCR detected only 72% (34 of 47) of those samples which cultivation identified as positive indicates that additional improvements are warranted before PCR replaces cultivation as the gold standard for detection of Salmonella from swine.
This research was supported by the Kansas Agricultural Experiment Station through its funding of the NC-62 Committee "Enteric Diseases of Swine and Cattle: Prevention, Control, and Food Safety," the Department of Diagnostic Medicine and Pathobiology, and the Recombinant DNA Laboratory of the Food Animal Health and Management Center, Kansas State University (KSU), Manhattan.
We thank Linda Cox (KSU) for technical support, George Stewart (KSU) and Doreene Hyatt (KSU) for reviewing early drafts of the manuscript, and Eileen Schofield (KSU) and James Smith (USDA, Agricultural Research Center, Eastern Regional Research Center) for editing the manuscript. We also thank the National Veterinary Diagnostic Laboratory for the confirmation and identification to species and serovar level of theSalmonella isolates.
Received 23 January 2001.
Returned for modification 4 March 2001.
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Journal of Clinical Microbiology Jul 2001, 39 (7) 2477-2484; DOI: 10.1128/JCM.39.7.2477-2484.2001
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A quality vacuum cleaner is a substantial purchase for most consumers. Even the highest quality vacuum cleaner will need repairs at some point in its life time. One of the more common repairs for aging vacuums is cord replacement. Vacuum power cords are stretched, twisted and dragged all around the house and eventually the cord will start to look like a bit of a fire hazard. When the cord begins to look frayed or ragged it's time for a replacement. You can make an appointment to bring your machine to a local vacuum repair shop or attempt fixing it yourself.
Step 1. Repairing a vacuum cleaner power cord is a relatively simple fix and if you're fairly handy you should be able to repair the cleaner yourself. Every vacuum is different and you should consult your manual and warranty before starting this project.
Start by removing the old frayed cord. The power cord is attached to the vacuum motor in the interior of the unit. The wires need to be detached from the motor in order for the power cord to come free from the vacuum.
Step 2. To access the vacuum's motor, the machine needs to be partially disassembled. If your vacuum has a brush system you'll need to remove the roller and belt to access the motor. Remove the bottom plate and pull the brush roll and belt out of the vacuum. Next you'll need to remove all the fasteners holding the motor housing. Finally you'll need to detach the handle from the motor by unscrewing the fasteners holding it in place. Be careful with the exposed wires after the motor has been removed from the rest of the vacuum. Before you detach the power cord, make note of its orientation- this will be useful when installing the new cord. Finally, remove the cord.
Step 3. Installing the new cord begins with taking parts from your old cord. Transfer all retaining parts from the old cord to your new cord. It's all downhill from here. Twist the wires together and orient them as they were before you began the repair. Everything should look exactly the way it was before interfering.
Step 4. Reassemble the vacuum cleaner by reversing your disassemble process. Replacing old, worn cords is necessary to keep your home safe. Mangled cords could cause a fire and damage your machine. Cord replacement is fairly simple and you should be able to do this repair at home saving you the time and money of visiting a vacuum repair shop.
If your vacuum cleaner isn't working properly, then you might be tempted to buy a new one, and throw the broken one away. Before you do buy a new one, you might want to fix your existing vacuum cleaner.
Here's why you should fix it instead.
Although you may have never taken a vacuum apart before, you'll be reassured to know that most problems are easy to resolve, and so you don't need to be an engineer, electrician or mechanic to fix yours.
Parts for the major brands are easy to get hold of, so there's a good chance that you can get the parts you need for your brand and model of cleaner.
The majority of problems won't take won't take long to fix, so you won't be without your vacuum cleaner for very long.
As well as not being able to clean your home, there is the inconvenience of being without it. You'll have to send your cleaner to an authorized dealer, or take it to a local repair center. You've then got to wait until they get round to looking at your vacuum, and then you need to be in when they send it back. If that sounds like hard work to you, then you'll definitely want to have a go at fixing yours yourself.
Fixing your own vacuum cleaner is much cheaper than buying a new vacuum cleaner, or having it repaired professionally. As well as the cost of parts, there is also the cost of labor, which is likely to be quite high.
By repairing your own, there is much less hassle involved, and there is no need to research a new vacuum cleaner. You won't need to find out whether your model is still being made, or more about the features and benefits of newer, or more expensive models.
Instead of taking your vacuum cleaner to the tip and buying a new one, by repairing it, you're actually doing your bit for the environment too. There's less landfill, and less natural resources are required too.
You'll also have the satisfaction of a job well done, and the skills and knowledge to tackle other problems that might arise with your vacuum cleaner, or perhaps other domestic items.
Carrying our repairs on your vacuum are cost effective, and will mean that your cleaner will be able to provide many more years of trusty reliable service. You might only need to replace the filters, or you might have to change the motor or the power cord, but you'll be saving money, time and the environment, all at the same time.


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Pagcor's 1H income up 2.5 pct on higher revenues
Jul 20, 2016 Newsdesk Latest News, Philippines, Top of the deck
Casino regulator-cum-operator the Philippine Amusement and Gaming Corp (Pagcor) on Tuesday reported net income of PHP2.45 billion (US$52.2 million) for the first six months of 2016, up by 2.5 percent from the prior-year period.
Income from gaming operations stood at PHP26.08 billion in the first six months of 2016, up 19.6 percent from a year earlier. In the three months to June 30, income from gaming operations reached PHP15.0 billion, a 35.4 percent sequential improvement compared to the first quarter.
Pagcor said it was 29.4 percent ahead of its own PHP20.16-billion target for income from gaming operations in the six-month period.
Total income for the first half of 2016 rose 8.6 percent year-on-year to PHP26.92 billion, Pagcor reported. For the second quarter alone, total revenue was PHP15.37 billion, up 33.1 percent from PHP11.55 billion in the first three months of the year.
In its latest report, Pagcor said that total operating expenses for the six months to June 30 increased by 3.7 percent year-on-year to PHP7.04 billion.
The state-run company, an operator of publicly-owned casinos as well as the regulator for the country's entire casino industry, contributed a total of PHP17.43 billion to state funds in the first half of the year. That figure included PHP12.36 billion directly transferred to the Bureau of the Treasury. Pagcor is required by law to pass at least 50 percent of its annual gross earnings to the government's treasury bureau.
The Philippines Commission on Audit said in a recent report that Pagcor underpaid by PHP15.40-billion – over a five-year period – the central government's 50 percent share of the earnings generated by the casino industry regulator. Pagcor argues that "it is not liable for the alleged under remittance … as it has correctly remitted the national government share".
Pagcor posted net income of PHP3.78 billion in 2015, an increase of 16.2 percent from 2014, according to official data.
Pagcor says inter-agency work has...
Pagcor 2022 income up 66pct y-o-y, tops...

redpajama_set_name: RedPajamaCommonCrawl


Investment-related articles that generate the most hype frequently focus on speculative highfliers. While there's always a time and place for that, especially if you're in your twenties, a well-rounded portfolio should include well-capitalized industry stalwarts. These so-called billion-dollar stocks provide an extra level of confidence in uncertain times.
And uncertain is exactly what we're witnessing in the markets today. Primarily, our new foreign policy under President Donald Trump has left many experts scratching their heads. Last month, Trump made history when he met with North Korean dictator Kim Jong Un. At the same time, the White House adopted an aggressive stance on oil-producer Iran.
Naturally, these events have weighed on benchmark indices. After flirting with a recovery, the Dow Jones is back in red ink against this year's opener. It's difficult to determine when the markets will regain its footing. While some sectors, such as crude oil, have bounced sharply from their doldrums, other sectors, including consumer staples and industrials, have noticeably stalled.
Buying billion-dollar stocks at this juncture is simply a smart strategy in light of unusual market dynamics. Here are ten heavily capitalized companies to consider for the second-half of this year.
President Trump lives a charmed life. From the get-go, outrageous controversies impacted his election campaign. But as we all know, consequences did not come to "Teflon Don." Later, as POTUS, Trump rattled foreign-policy experts with his unorthodox directness. Yet if current trends continue, Trump could potentially craft peace in the Korean peninsula.
Cynically speaking, peace is great for humanity, but not so much for billion-dollar stocks like Northrop Grumman Corporation (NYSE:NOC). Not too take anything away from the defense company's innovative technologies, but NOC thrives under the threat of warfare. For the military-industrial complex, North Korea might as well be a giant cash register.
So I understand the initial reason why NOC stock slipped. Last month, shares dropped 6% in the markets. On a year-to-date basis, NOC is just 1% above parity. But I believe the selloff is overdone, presenting opportunities for shrewd investors.
Just look at Northrop Grumman's financials, which are a head above several billion-dollar stocks in the defense sector. NOC has a fairly stable balance sheet, highlighted by an improving cash-to-debt ratio. On the income statement, Northrop features above-average profitability margins. Finally, year-to-year, NOC has consistent free cash flow.
Thanks to high-profile projects such as the F-35 Lightning II fighter jet, Lockheed Martin Corporation (NYSE:LMT) may be the most recognized among defense-related billion-dollar stocks. That said, Lockheed Martin's market performance is rather disappointing. On a YTD basis, LMT stock is down nearly 6%.
Essentially, the entire loss can be pinned on June's volatility, where LMT dropped 6.8%. Of course, this was the month where President Trump and Kim first met face-to-face during an unprecedented summit.
As with Northrop Grumman, Lockheed thrives under conflict. After all, what's the point of developing the world's best fighter jet if you have no need for it?
Also, Lockheed's management team isn't doing LMT stock any favors. In recent years, the defense contractor has racked up substantial long-term debt, with only a modest rise in cash. Also, shares appear overvalued relative to the defense industry.
But I think traders overestimate Trump's ability to negotiate peace with North Korea. It's tempting to believe that he has the Midas touch given his prior, shocking successes. And no, we shouldn't doubt our President to pull off another miracle.
But this is North Korea that we're talking about. We're still a long ways away from putting our guard down, which is why I'm bullish on LMT eventually recovering.
Technically, Sturm Ruger & Company Inc (NYSE:RGR) just misses the cut-off to be included in my list of billion-dollar stocks. However, if RGR pulls together a few good sessions, it could easily reach and exceed the minimum threshold. Plus, gun companies offer an overlooked bull case.
For both moral and performance reasons, mainstream analysts usually shun RGR stock, which I think is a mistake. According to a 2016 John Hopkins study, "medical errors may kill more people than lower respiratory diseases like emphysema and bronchitis do." That, according to CNN, would mean "death by doctor" is a far worse killer than firearms-related deaths.
We can argue all day about the statistics. My point is, wayward doctors are dangerous, yet no one threatens to ban all hospitals.
Moreover, people who avoid RGR for moral or political reasons are likewise avoiding potential profitability. One of the biggest developments in the firearms sector is the Concealed Carry Reciprocity Act. If fully passed, the bill enables concealed-carry permit seekers an easier road to acceptance. Long story short, the weakest state towards gun control – namely Florida – will become the standard for every other state.
This means Californians, which have long been denied such permits, can apply for them with reasonable assurances of acceptance. That bodes very well for RGR, which boasts a wide range of concealed-carry specific firearms.
Despite the many foreign-policy controversies that Trump generated, he has one economic ace up his sleeve: a robust labor market. Specifically, the civilian unemployment rate has trended down throughout his administration. Sure, critics argue that former President Obama set the foundation for employment opportunities. Still, Trump did his part by implementing his pro-business agenda.
Most importantly, businesses feel confidence towards the current administration in spite of its obvious flaws. That's why I think investors should consider staffing agency Robert Half International Inc. (NYSE:RHI). Set aside all the politics: it's clear that companies are in hiring mode. Even I'm receiving dozens of email solicitations from RHI businesses, and I don't expect them to stop.
Furthermore, the job offers aren't the usual high-volume, low-wage fares. Instead, several of the solicitations are frankly tempting. Admittedly, my story is anecdotal. But the declining unemployment and the currently positive consumer sentiment are very much real. Save for a catastrophic event, you may expect the labor market to at least hold steady, which is positive for RHI stock.
On a technical note, RHI is down more than 6% since mid-June, providing a decent discount.
Medical care and health services is becoming an obscenity. In 2016, healthcare costs hit $3.3 trillion, or $10,348 per person, according to Centers for Medicare & Medicaid Services. This year, Reuters reported that healthcare experts project a 5.3% increase in medical-related costs.
No wonder why Tenet Healthcare Corp (NYSE:THC) sports a $3.5 billion market capitalization. But the real story is how quickly it got to that level. THC is one of the hidden gems among billion-dollar stocks, gaining over 126% YTD. That said, its incredibly lucrative industry could continue to support THC move higher for the rest of 2018.
Tenet is a major player in the hospital and outpatient business. At present, the company owns 68 hospitals and 470 outpatient centers across 47 states. It also employs 32,000 active physicians, and 33,000 nurses.
The one drawback to THC stock is that its financials aren't as great compared to its peers. For instance, Tenet carries substantial debt relative to its cash position. Also, its three-year revenue growth is middling compared to the medical-care industry.
However, the counterargument is that America is aging rapidly due to the Baby-boomer demographic. As a longer-term investment, THC still has plenty of life left.
At some point, Amazon.com, Inc. (NASDAQ:AMZN) will no longer be eligible for a list of billion-dollar stocks. Instead, it will join Apple Inc. (NASDAQ:AAPL) as the elite few companies who have hit $1 trillion in market cap. For now, both companies must settle for their multi-billionaire status, with Apple clearly in the lead.
But if I'm being completely honest, I like AMZN to hit the $2 trillion threshold first. I'll freely admit that this point is pure conjecture. But in Apple's case, their business depends on selling their products and services. With Amazon.com, you get everything and the kitchen sink.
It's possible, considering the wide array of Amazon products infiltrating everyday life on a seemingly monthly basis. You can practically live your entire life within the Amazon ecosystem.
I don't disagree with Brumley. Back when Apple was on the verge of making history, I entertained the idea that Amazon could overtake it; I just didn't have the intestinal fortitude to write it down for publication.
But the bottom line is that AMZN is an automatic investment, a pure no-brainer. Consider that last year, we we're having heated debates about where shares go after hitting $1,000. Now, the conversation is about going for $2,000. Over the next several years, don't be surprised to see bitcoin-like prices.
Although it's a risky idea, I like Capital One Financial Corp. (NYSE:COF) due to the current economic situation. As I previously mentioned, the labor market is very strong, highlighted by multi-year low unemployment rates. In addition, consumer sentiment is strong, indicating that American families are willing to splurge a little.
That's a net positive for credit-card issuer COF, which represents a paradigm-shift from several years earlier. This is also the reason why I'm willing to set aside some weaknesses in Capital One's financials, such as high debt, and declining annual margins.
A stronger labor market will likely translate to higher sales. In fact, we're already seeing that. In its latest first-quarter earnings report, COF generated $6.74 billion in revenues, up 5.3% against the year-ago level. Right now, COF stock is down more than 6%, indicating that traders are still focused on the company's weaknesses, and not its potential.
That suits me just fine because Capital One is best suited for smaller, everyday purchases, which is the kind of purchasing that is readily available now. What Americans, especially younger ones, are not doing are major acquisitions, such as home ownership.
It's a cynical argument, but COF has an ideal situation: an economy that's strong, but not too strong.
I'll say it right now. I don't know anybody who gets excited about investing in Sherwin-Williams Co (NYSE:SHW). Buying SHW is perhaps the investment equivalent of watching paint dry. But herein lies the company's longer-term potential.
For those who were lucky to have bought real estate prior to the market going bonkers, companies like Sherwin-Williams provide an easy means to boost home values. Additionally, their website offers multiple instructional videos and tutorials for individuals looking to upgrade their home's appearance.
More importantly, the boom in real-estate prices has had a positive impact towards Sherwin-Williams' financials. In Q1, SHW brought in nearly $4 billion in revenue, up a staggering 44% from the year-ago quarter.
As The Motley Fool contributor Joe Tenebruso points out, Sherwin-Williams has a significant advantage over rival Home Depot Inc (NYSE:HD) because the former makes its own products. With Amazon and the e-commerce industry disrupting traditional sectors, SHW can use its brand-power as a protective moat.
Finally, SHW stock is a secular play. If you need to paint or renovate your home, you can't put it off forever.
President Trump is enamored with his own direct style towards foreign policy. Inexplicably, his no-nonsense, shoot-first, ask-questions-later approach has paid off dividends. Yet a conspicuous weakness towards political directness is that it locks you in. Flexibility is a virtue, while inflexibility is a liability due to the lack of available options.
I think Trump and the American public will find this out the hard way. We're playing hardball with the Chinese, while we're deliberately throwing at the batter's head with Iran. This dynamic is a substantial headwind towards crude oil supplies, which in turn is bullish for Exxon Mobil Corporation (NYSE:XOM).
While speculative oil companies are much more exciting than the majors, Exxon Mobil's stability shouldn't be overlooked. Since the energy market crashed in 2014, management has focused on cutting waste and unnecessary expenditures. As such, it's a leaner, meaner version of itself, and is well-prepared to handle a boost in oil prices.
And that's exactly what we'll probably get. Iran is the world's third-biggest oil producer, and we'll lose access to that enviable channel. Knowing this, XOM stock will rise sooner or later.
I hate to admit it, but gold prices have been disappointingly poor. I'm noticing a pattern, which is that the yellow metal starts off promisingly, only to bitterly fail later. Last month was particularly volatile for gold, sending it down well below the critical $1,300 threshold.
But despite this disappointment, I don't think we've heard the last from the precious metals sector. It is a risky play, though, so only consider Barrick Gold Corp (USA) (NYSE:ABX) if you've got an iron stomach. If you do, I think you'll end up being pleasantly surprised with ABX stock.
In some respects, I understand why gold, and gold stocks aren't meeting expectations this year. As with defense stocks, fostering peace with hostile nations does away with the "fear factor." Buying gold bullion to thrive in a post-apocalyptic world is pointless if the country threatening us with that scenario is no longer considered a threat.
But I don't trust North Korea, and I'm also leery about Trump's Middle Eastern policy. If something sounds too good to be true, it usually is. We're not done with the fear trade, so I'm still bullish on gold stocks like ABX. It may be a loser now, but I'm almost certain the gold market will turn around.
Article printed from InvestorPlace Media, https://investorplace.com/2018/07/10-billion-dollar-stocks-for-q3-and-beyond/.


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Series of Explosions Rock Kabul
February 25, 2010 / 10:48 PM / AP
A series of explosions rocked an area near a hotel in the heart of the Afghan capital Friday. Witnesses said suicide bombers were to blame and that gunmen opened fire at the site. Two police were killed and one injured, according to authorities.
Abdul Ghafor Sayedzada, a top investigator for the Kabul police, confirmed the blasts, which woke up residents near the Kabul City Center, a nine-story shopping area that includes the Safi Landmark Hotel.
Najibullah, a 25-year-old hotel worker, said he ran out of the hotel in his underwear when he heard the first explosion. He said he saw two suicide bombers on the site.
"I saw foreigners were crying and shouting," said Najibullah, whose face and hands were covered in blood. "It was a very bad situation inside. God helped me; otherwise I would be dead. I saw one suicide bomber blowing himself up on the first floor of the hotel."
There was no official confirmation about the target of the explosions, but an official from the Interior Ministry said one suicide bomber detonated an explosive near the shopping area. The official, who spoke on condition of anonymity because an investigation was still under way, said more than one suicide bomber might have been involved.
The blasts occurred on Friday, the first day of the Afghan weekend when fewer people are on the streets.
A building was on fire in front of the four-star hotel, which is close to most government offices. A reporter for The Associated Press said gunmen opened fire at the site. Shattered glass covered the streets. A large plume of black smoke was seen rising from the area. Afghan police, armed with Kalashnikov rifles, moved in taking cover in the doorways of shops.
It was the first attack in the Afghan capital since Jan. 18, when teams of suicide bombers and gunmen targeted government buildings, leaving 12 dead, including seven attackers.
First published on February 25, 2010 / 10:48 PM


During the Pacific Alliance's third roadshow on September 1-8, nine businesspeople from Peru, six from Chile, nine from Colombia and eight from Mexico met with more than 300 Chinese tour operators in Beijing, Shanghai, Guangzhou and Hong Kong (around 80 per city) with the goal of bringing more Chinese tourists to the Alliance's member countries.
"The Alliance allows us to work together to offer tourists from China the chance to step out and discover the history, food and overwhelming natural beauty of our countries in a single trip, giving them a fuller picture of the unforgettable experiences our destinations offer. Peru welcomes them with open arms, ready to exceed the expectations of even the most discerning traveler," said Marisol Acosta, director of Tourism Promotion at PROMPERÚ.
In the roadshow's third iteration, the Alliance's four tourism boards (SERNATUR, ProColombia, the Tourism Board of Mexico – MTB – and PROMPERÚ) were able to explain to attendees the ins and outs of this integration mechanism and the tourist attractions of each participating country.
In terms of tourism, the Pacific Alliance economic agreement has allowed the agencies promoting this industry to implement work plans designed to create new business opportunities for companies through macro-roundtables, international trade shows and roadshows.
These events include the 2nd Pacific Alliance Macro-Roundtable in Mexico (2015), which was attended by 16 Chinese wholesale agencies, the 3rd Macro-Roundtable in Peru (2016), with the participation of 20 Chinese companies, and the 4th Macro-Roundtable in Chile, which also had wholesale agencies from that destination. This last event led to around 2534 business meetings.
Last year, 23 tour operators from the Pacific Alliance promoted their destinations at the second roadshow held in Beijing, Shanghai, Guangzhou and Hong Kong. At this event, 287 Chinese businesspeople were trained and more than 1300 meetings were held, with potential deals worth more than US$6 million.
It should be pointed out that in 2016, China became the world's foremost outbound tourism market with 135 million tourists, according to the World Tourism Organization. The growing number of tourists flocking to Pacific Alliance countries was one reason it reached this milestone. 2016 saw a 34% jump up from the previous year, to a total of 136,220 Chinese tourists. Most Chinese travelers seek experiences related to nature or culture, so this market is increasingly interested in the countries of the trade bloc.


"Fake news has it that we must sacrifice the NEA and the NEH for better stewardship of our national wealth and interests. It is claimed that these agencies are wasteful and unnecessary. But in fact, they are among the most important investments our society can make. By their means, we increase our chances to understand who we are, and thus to envision how we might better proceed. Their conceptual roots are in the alphabet agencies of the Great Depression when a staggering economic crisis called for a political shift in the way that the United States cared for its population, in some ways not unlike the present moment.
A few hours after his inauguration, Franklin Roosevelt swore in his entire cabinet en masse, so as to hit the ground running. In the next 105 days, in the depths of the emergency, in un-ending special session, Congress created and passed the Emergency Banking Act, the Agricultural Adjustment Act, the Federal Emergency Relief Administration, the Civilian Conservation Corps, the Works Progress Administration, the Truth-in-Securities Act, the Glass-Steagall Act and the National Industrial Recovery Act. The new president was able to persuade millions that they had "nothing to fear, but fear itself." In all, there were 15 major bills. It was a vigorous and astonishing three and a half months.
The contrast with the current 100-day count-down could not be more stark. From the shocking disarray of presidential appointments to the ugly attempt of Congress to take healthcare away from millions of Americans, to the amplification of racism, Islamophobia, anti-Semitism and xenophobia, to the spate of executive orders designed precisely to undo whatever could be undone of what remains of the New Deal's legislation and spirit, Trump's first 100 days are the Bizarro version of the earlier period.
The two-endowment agencies each receive about $148 million a year now. The budget for public broadcasting, currently $445 million, has been more consistent over the years. Together they still account for only $741 million, or much less than one-tenth of 1 percent of the United States' annual federal spending, an amount supporters say is too small to make a difference.
Proponents of preserving endowment grants are increasingly speaking of them in terms that many Republicans can love – as investments that spur job creation; as public-private partnerships that award grants that are matched by private donations; and as programs that help returning veterans or people who live in rural communities.
But these defenses, though factual and earnest, and maybe even necessary, are themselves a species of "fake news." To our adversaries, no matter how small we make ourselves, it will never be small enough. However little we claim to cost, we will still be too expensive, and however much we claim to multiply value, we will be worth too little. The premise is fake. We are aligning ourselves with the disrespect of our adversaries while attempting to gain their regard. We are fighting with one hand tied behind our backs while being shoved into a corner. It is hurtful to see.
The real news makes a much larger claim: that the Federal government benefits from the CPB, the IMLS and the NEA and the NEH not because they save money but because they support another economy: that of the nation itself. This larger expenditure is in the currency of self-recognition. For calculations about the NEA and the NEH, neither the "E" (money) nor the "A" and the "H" (arts and humanities) are as important as the "N" (national). The reason to fight for the NEH and the NEA is not because we need them in order to have arts and humanities, which we will have in any case, but because we need them to know how to imagine ourselves as a nation. As in Betsy deVos's hands the voucher system systematically aims to destroy the public schools, in Trump's hands the destruction of the NEH and the NEA aims to voucherize the public itself. But the public cannot be supported by special interests and wealthy individuals because those are specific and exceptional rather than representative actors.
During the Great Depression, under the direction of a brilliant young economist named Roy Stryker, Roosevelt's Farm Security Administration sent more than a score of American's most accomplished documentary photographers out into the field to take photographs that would show Americans to one another as "deserving" of government aid. This was done, if you will, under the sign of "fake news," that is to say, the economic argument that New Deal programs of social support were saving far more than they cost. In point of fact, the enemies of the administration did not allow most of the programs to reach their full potential. No matter what the factual ratio of cost to benefit, state supported welfare was seen as creeping socialism and as something to be beaten back.
But the real news is what happened anyway. Walker Evans, Dorothea Lange, Russell Lee, Gordon Parks, Margaret Bourke-White, John Vachon, John Delano, Arthur Rothstein, Esther Bubbly, Marion Post Walcott, and many others took the opportunity to work extravagantly beyond bounds to make a collective portrait of the spirit of the American people that is unsurpassed, one of our greatest national treasures.
I am Principal Investigator of the NEH-supported Photogrammar Project, codirected by Lauren Tilton and Taylor B. Arnold. Photogrammar has made an interactive, geospatial map of the more than 170,000 photographs produced between 1935-1945 by the Farm Security Administration and its successor, the Office of War Information. Because Photogrammar makes this enormous archive of images so easily searchable, it is possible to see at scale the extraordinary extra expenditure these artists laid out in the work that they did, work that had no need to be as fine as they made it. Most of the images were circulated in newsprint-quality reproductions or in government reports. Much less effort would have sufficed. And yet, the government got more.
My friend and colleague, Laura Wexler and I had the opportunity to present the DOCC at the EduTech Horizons workshop held at the National University of Singapore for members of the International Association of Research Universities (IARU) of which Laura's school, Yale, is a member. We were in friendly, interesting, and interested territory even as we presented the project to technologists who weren't necessarily feminists, and to a truly international crowd with representatives from a significant number of continents and disciplines. Given that internationalization and feminist education are both core values of FemTechNet, it was gratifying to see the enthusiasm in this diverse audience.
I knew we were at home when in his opening address, Professor Lakshminarayanan Samavedham from NUS' Centre for Teaching and Learning reminded us to think beyond efficiency towards effectiveness in digitally-enhanced education, explaining that by this he meant experiences that were built to be engaging, personalized and authentic, just like the DOCC … (Professor John Traxler, from England's University of Wolverhampton, a specialist on mobile computing and education, suggested we all stop using the term "technologically-enhanced" and instead dub those efforts not up to speed on technology as "technologically-deficient learning.") Given this start, Laura and I felt fully supported to share the passionate, active, distributed, technofeminist, co-production of knowledge at the heart and daily practice of the DOCC.
We felt like we had more to add to the conversation when Lynne O'Brien, Associate Vice-Provost of Digital and Online Education Initiatives at Duke, warned against digital efforts that simply replicated older forms and formats of teaching, concluding that "the faculty of the future needs to know more about learning." This seemed a both prescient and somehow curious call, given that the powerhouse Universities assembled at the meeting are best known for (and have joined together) because of their awesome capacities at Research. Dr. Abelardo Pardo Sanchez from the University of Sydney, a powerful researcher in his own right, had a great deal to teach us about how the quickly developing field of learning analytics might be of use not just for higher education writ large (built as it is from big data) and its developing cultures of tabulation but also for understanding, optimizing, and enhancing instructors' knowledge about what happens in their own classrooms.
FemTechNet understands that technologies are complex systems with divergent values and cultural assumptions. We work to expand critical literacies about the social and political implications of these systems.
FemTechNet is cyberfeminist praxis: we recognize digital and other technologies can both subvert and reinscribe oppressive relations of power and we work to make these complex relations of power transparent.
FemTechNet is hard at work creating better tools.
FemTechNet has no observers, only participants.
Accountability is a feminist technology.
Collaboration is a feminist technology.
Collectivity is a feminist technology.
Care is a feminist technology.
because, of course, we have long known we had to perform our feminist praxis in sites in and out of the academy, in multiple formats and to different audiences. And now we might all agree that a new part of our feminist digital pedagogy is also to divvy up the temporal spectrum, and each take some responsibility to hold down the short or medium and even, yes, long form, making sure we are present in the immediate, gratifying flows of Twitter as well as guaranteeing that we are lying safe for the long run on paper in a library.
Crank (or should I say crunk) it back a day, and move the (my) body to Rutgers, and similar conversations were happening, under the same title, only in a different room, and to a similar but unique crowd (online and off: see Adeline Koh's Storify version).
Now, you might ask, why two conferences, two cities, three days? What is this telling us about this metronome and its unique piano-home? A conference, as you all know, is a kind of medium speed but fully-placed venture: long talks, all day in one room, some need for a coffee and pee break, but the sustaining, necessary gratification of f2f: we must be present to each other … sometimes.
As was true just a year or two ago, when the fembot collective and the femtechnet one found ourselves forming in distinct places, for varied (feminist, digital) ends, but at the same time, and then worked together to divvy up some of that HUGE map-of-affective-labor, this current synchronicity marks a pulse we can all be nourished and energized by across our differences. Rutgers and Michigan held these sister conferences because they want to up their digital games. That's because over just the past few years a large enough number of us have organized in a lot of places, temporalities, and forms, so as to create visibility, community, and output, so as to make it crystal clear what was always true: that there's a new and old game in many time-frames and in a world of places; miss it to your own loss.
While teaching at Peking University, she asked her students the verboden: would they share their family photos with her. It seems within the intellectual context she produced they were happy to oblige: exposing the usually hidden shape, class, possessions and other details of their private lives. However, Wexler learned that her students could only share photo albums of their own lives, as their parent's (and grandparent's) albums had been expunged, for fear of retaliation regarding the politics of personal life during the cultural revolution.
She shared her findings about the "staging of inaccessibility" in places like contemporary China where forgetting is what sustains people (in part, Wexler thinks, because of the absence of a human right's discourse to create a framework for remembering). She learned that continuous telling, and continuity between telling, is often the function of our (ever growing and easily sharable archive of) home images of self and family.
It was painful and perplexing to think about the gaps between (visuality of) Chinese generations (also being discussed this week around different generations of Germans ), but I've been thinking about such gaps myself (ones that actually occur outside of systems of the state sanctioning of ongoing forgetting and within a society where human rights discourse prevails), in relation to gaps across feminist generations, as well as my father's forced forgetting and remembering of his own Holocaust trauma.
Along this vein, it was truly interesting to learn that while (after twenty-five years!), neither of remembered the other's face, the other's voice, intellect, and shared feminist ethos were as vivid as they had been in the 1980s, photos to record or no.


Traits in a woman are quickly noticed – and honestly, that has nothing to do with beauty (happens more often than you know of girls!). Her personality, attitude, body language and the right mixture of maturity & modesty make a girl spot-on.
And that's it. There you're, not only for men, but women are intimidated by you too. You know why? Because that slightly differentiable quality in you makes you win any challenges using the charm you're gifted with. Or, sometimes situations help you train! But there are only a few who, understand true beauty right from the inside. My shout out for those who think looks matter the most – here's changing your opinion with the abundances women are being created with.
A person is immediately judged by the way she expresses her ideologies. Remember first impressions matter. Largely. An assessment could be formed within seconds and if you succeed giving out the right vibes with a right amount of confidence, there's no stopping you. So when entering into a meeting room for a big business, or to influence a client, or even more casual – walking into a first date, be sure to dazzle the world with the pinch of confidence in such a way that they feel comfortable around you – ready to be hypnotized by whatever you're going to say next.
In this case, the manoeuvre here is that you don't have to pretend something that you are not. A beautiful woman with an ample of sense of humor can kill men because guess what, she is not a typical woman who only is aligned to talk about beauty, fairness, and chores. No! She is more than that and this particular trait is kind of hard to not notice. A woman who exudes, cheers a party up and takes care of the fun element is surely refreshing for a man.
Tips: Studies say that women fall for men who keep them happy, comfort them with humor. The same theory goes for men too. There's no faking in any sense so laugh out loud.
No men like to be outsmarted but when they come closer to a woman who is equivalently smart and witty, that's the affection never goes off the circle. What makes a woman ingenious? The way she speaks, the way she carries herself, the way she never acclaims or interacts with others without knowing the facts, the way she knows her stuff, speaks when needs to, keeps the right friends closer, and when she is around, nothing, I REPEAT, nothing can go wrong. This doesn't mean women have to steal a march on, but with your keen sharpness, you're inevitable.
There's no bigger failure for a woman (no matter how beautiful she is) if she doesn't possess any passion in life. People around you in general look for genuinely, passion that you follow up and how badly you're involved with it. This is one thing in a woman that is infectious and can clean-bold any person. Whether you're passionate about art or the things and people you love — show that you dream, you inspire, and the key things that drive you to become better selves. What's even sexier is woman passionately talking about the ambitions that she wouldn't let go off until dawn. But only talking could be unheard till you turn those things into reality.
Like it or not, if a woman is a selfless deed, men would never let her go off the site. Knowing that she is humble and cares for the surrounding, she believes in making a difference in the lives – is one thing that drives them toward you. Seeing you don the hat of a determined, definite personality, they will adore your strength and independence. Nothing is more empowering for a man than being affectionate to a woman who survives and helps other survive.
So next time if you dress up to kill, make sure to mushroom the ultimate traits in you that could enable the license for you to go from Good to Wow!


Savannah area small business owners are invited to attend a speed-mentoring conference on Thursday, March 8 from 8:30 a.m. to 12:30 p.m. at the Savannah Morning News Auditorium, 1375 Chatham Pwy.
The conference is an opportunity for small business owners to learn about a variety of topics, ask questions and network with executives who have decades of experience in the small business field.
Among the topics that will be covered are finding the money, the power of social media, finding your niche, when to outsource, HR headaches, cash flow-business lifeblood, pricing for profit, understanding financial reports, preparing a budget, deciding which product or service to keep and creating ads that sell.
Participants will meet with mentors for 25 minutes on each subject. Members of the Savannah chapter of SCORE and the University of Georgia Small Business Development Center, along with top executives from area businesses, will be on hand to facilitate the speed-mentoring process.
The event will also include lunch and a presentation by Timothy Mescon, president of Columbus State University.
Mescon, who served as dean at the Michael J. Coles College of Business at Kennesaw State University in Atlanta prior to becoming Columbus State's fourth president in 2008, is the author of more than 200 articles and cases and has co-authored four books, including Entrepreneurship: Venture Initiation, Management and Development.
He has written extensively about retail banking, bank marketing and innovation and creativity in the financial services industry. He was the featured speaker at the AMEX/SCORE Speed Mentoring Conference in Atlanta.
"This is an excellent opportunity for members of the Savannah small business community to interact with and learn from high-level business professionals," said Marjorie Young, president of the Savannah chapter of SCORE.
Registration is $19.95 apiece, $9.95 for additional parties from the same organization and includes the mentoring sessions, lunch and Mescon's presentation. Participants will also have the opportunity to schedule a follow-up appointment.
The Savannah Morning News and Comcast are the event's media sponsors.


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Nicotine concentrate in the form of salts (salicylate) which are naturally occurring in the tobaco plant. This property enables an increased absorption rate. Generally for smaller pod or Juul type non-sub ohm devices. It is imperative to take special care as the higher milligrams will be much 'smoother' on the throat with little to no aftertaste. Clarity is an amber hue.
Comes in a white, UV light-impact resistant HDPE container. Further dilution and process may be required. Remember to store refrigerated or in a cool place away from heat, which will assist in retaining freshness.
Note: Nicotine salts will cause a colour change. This is completely normal due to its complexity and is in no way a reflection of freshness or quality.


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//Unless required by applicable law or agreed to in writing, software
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//WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
//See the License for the specific language governing permissions and
//limitations under the License.
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// Code generated by Alibaba Cloud SDK Code Generator.
// Changes may cause incorrect behavior and will be lost if the code is regenerated.

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redpajama_set_name: RedPajamaGithub


text:
GM Modular is all geared up to unveil "Showroom on the wheels" at Acetech Hyderabad 2023!
Maharashtra 24x7 > Business > GM Modular is all geared up to unveil "Showroom on the wheels" at Acetech Hyderabad 2023!
GM's most luxurious showroom on wheels is now in Hyderabad!
Hyderabad (Telangana) [India], January 23: GM Modular is all set to roll out its trailblazing and one-of-a-kind initiative 'Showroom on wheels' at Hyderabad Acetech from 20th to 22nd of January 2023. This showroom on wheels is a state-of-the-art bus fully equipped with the latest innovations like designer switches, decor lighting, home automation solutions and more from GM. The bus will be at Acetech Hyderabad and will be a good opportunity for everyone to experience the cream of the crop products in an unique way. This bus is already turning heads wherever it goes.
Acetech is a coveted trade fair in Asia, well renowned for hosting a wide spectrum of allied events. It offers revolutionary avenues of networking opportunities for all professionals involved in various phases of architecture, construction, and design. During the Mumbai edition in November 2022, GM unveiled the first look of this unique concept, 'showroom on the wheels.' The bus is designed by Mr. Dilip Chhabria, the acclaimed automobile designer in India.
Speaking about it, Jayanth Jain, GM's MD and CEO said, "Over the years GM has added a varied range of home electrical products to its portfolio and we always look for opportunities for our consumers to experience them all at one place. Today we are a one-stop-solution for all your electrical needs. Right from wires and pipes to switches and home automation solutions, we have it all. Acetech is a perfect platform for us to introduce 'the most luxurious showroom on wheels in the world' – A state-of-the art bus designed by the best auto designer in India Mr. Dilip Chhabria. With this bus, we will be able to showcase our high quality GM products to every corner of the country.The bus is one of its kind with an extremely futuristic design and equipped with all the latest gadgets.
Experience the world's most luxurious showroom on wheels at Acetech Hyderabad.
Jan 23, 2023 Leave a Comment on GM Modular is all geared up to unveil "Showroom on the wheels" at Acetech Hyderabad 2023!Business
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Where do I download add ons?
Where do I download Add ons? There is a listing on the download page but no link.
We pulled that download while we are working on getting those functions working with the latest version posted.
We will repost them soon.