Detection of Vibrio cholerae O1 in animal stools collected in rural areas of the Limpopo Province

Vibrio cholerae (V. cholerae), the causative agent of cholera, has been responsible for various outbreaks worldwide and may be associated with animal faeces. In an attempt to understand the occurrence of this organism in the environment, 230 faecal samples were collected from pigs, chickens, goats, donkeys, cows and pigeons in rural areas of the Limpopo Province. Bacterial DNA was extracted from the faecal samples using a guanidium thiocyanate-based method. The DNA was screened for the presence of the sodB, rfb, FlaE, 16S rRNA and ctxA genes associated with V. cholerae, V. cholerae O1, V. cholerae O139 using 2 multiplex polymerase chain reactions (m-PCR). The V. cholerae sodB gene was detected in 74 of the 230 samples tested. Detection rates for the faecal samples obtained from individual species were as follows: cows (55/74), chickens (8/74), goats (2/74), donkeys (4/74), pigs (3/74) and pigeons (2/74). V. cholerae O1 was detected in (17/74) cow and (3/74) chicken samples, of which (9/17) cow samples and (3/3) chicken samples tested positive for toxigenic V. cholerae O1. The presence of this organism in faecal samples, taken close to water sources used by the villagers, raises the possibility that the causative V. cholerae O1 strain of the most recent outbreak in South Africa was present in the area 6 months prior to the outbreak.Keywords: Vibrio cholerae, PCR, animal faeces, cholera toxi

Modulation of galactic protons in the heliosphere during the unusual solar minimum of 2006 to 2009

The last solar minimum activity period, and the consequent minimum modulation conditions for cosmic rays, was unusual. The highest levels of galactic protons were recorded at Earth in late 2009 in contrast to expectations. Proton spectra observed for 2006 to 2009 from the PAMELA cosmic ray detector on-board the Resurs-DK1 satellite are presented together with the solutions of a comprehensive numerical model for the solar modulation of cosmic rays. The model is used to determine what mechanisms were mainly responsible for the modulation of protons during this period, and why the observed spectrum for 2009 was the highest ever recorded. From mid-2006 until December 2009 we find that the spectra became significantly softer because increasingly more low energy protons had reached Earth. To simulate this effect, the rigidity dependence of the diffusion coefficients had to decrease significantly below ~3 GeV. The modulation minimum period of 2009 can thus be described as relatively more "diffusion dominated" than previous solar minima. However, we illustrate that drifts still had played a significant role but that the observable modulation effects were not as well correlated with the waviness of the heliospheric current sheet as before. Protons still experienced global gradient and curvature drifts as the heliospheric magnetic field had decreased significantly until the end of 2009, in contrast to the moderate decreases observed during previous minimum periods. We conclude that all modulation processes contributed to the observed increases in the proton spectra for this period, exhibiting an intriguing interplay of these major mechanisms

Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds

Commercialization ofBrassica napus.L (oilseed rape) meal as protein diet is gaining moreattention due to its well-balanced amino acid and protein contents. Phytic acid (PA) is a majorsource of phosphorus in plants but is considered as anti-nutritive for monogastric animalsincluding humans due to its adverse effects on essential mineral absorption. The undigested PAcauses eutrophication, which potentially threatens aquatic life. PA accounts to 2-5% in matureseeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes.Breeding polyploids for recessive traits is challenging as gene functions are encoded by severalparalogs. Gene redundancy often requires to knock out several gene copies to study theirunderlying effects. Therefore, we adopted CRISPR-Cas9 mutagenesis to knock out threefunctional paralogs ofBnITPK. We obtained low PA mutants with an increase of free phosphorusin the canola grade spring cultivar Haydn. These mutants could mark an important milestone inrapeseed breeding with an increase in protein value and no adverse effects on oil contents

A rapid and low-cost DNA extraction method for isolating Escherichia coli DNA from animal stools

The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA from goat, chicken, pig, cow and human stool samples. Two versions of the lysis buffer, with and without α-casein, were tested to alleviate PCR inhibition associated with DNA isolated from stool samples. Results obtained show that, this method using the lysis buffer containing α-casein, produces PCR ready DNA at a fraction of the cost of commercial DNA extraction kits.Key words: DNA extraction, Escherichia coli, polymerase chain reaction amplification (PCR), stool samples

Optimisation of methods for the collection and detection of bacterial pathogens from diarrhoeal human faecal samples using a novel stool collection kit

Bacterial pathogens such as Escherichia coli, Salmonella-, Shigella- and Vibrio species are known to be common causative agents for diarrhoeal disease in humans. This study aimed to develop a culture-independent PCR assay for the detection of bacterial pathogens present in human faecal samples collected using a less intrusive faecal collection technique, the Bio-wipe kit. A multiplex-PCR (m-PCR) was optimised targeting the E. coli mdh gene, the Salmonella IpaB gene, the Vibrio sodB gene, and the Ial and IpaH genes present in entero-invasive E. coli and Shigella spp. The influence of the DNA extraction method, and sensitivity and specificity of the m-PCR and the Bio-wipe storage conditions on the detection of the bacterial pathogens was investigated. A guanidium thiocyanate DNA extraction method used with laboratory-prepared spin columns could successfully extract DNA from 93% of the samples analysed. The m-PCR could successfully identify and differentiate between the various pathogens tested and was specific for the selected pathogens. Faecal matter was successfully recovered from used Bio-wipes and the bacterial DNA could be detected from these samples at concentrations of 10 cfu. Bacterial DNA could be recovered from the Bio-wipes 5 to 10 d after use when the Bio-wipes were stored at 30°C and 14 d after usage when stored at ambient temperature. Thus the Bio-wipe kit, along with the m-PCR, can be used for collection and detection of bacterial pathogens during outbreaks and in rural settings.Keywords: Bio-wipe kit, bacterial pathogens, faecal matter, PCR, DNA extractio

Comparison of capsule-mixed versus hand-mixed glass ionomer cements Part II: Porosity

Glass ionomer restorative cements (GIC) are routinely used in dental practice. During mixing, air incorporation may lead to higher porosity with subsequent weakening of the cement. The degree of porosity will determine whether capsule-mixed or hand-mixed GIC are mechanically stronger for clinical use. To compare the porosity of four commercially available dental glass ionomer cements, supplied in both hand mix and capsule-mix formulations, by evaluating number of voids (%), total volume of voids (mm3 ) and volume percentage of voids (%). Eighty samples were manufactured from hand-mixed GIC: Riva Self Cure; Fuji IX GP ; Ketac Universal, Ketac Molar Easymix, and equivalent capsule-mixed GIC: Riva Self Cure; Fuji IX GP ; Ketac Universal Aplicap and Ketac Molar Aplicap. Micro-CT scanning was used to evaluate porosity. The number of voids (mm3 ), total volume of voids (mm3 ) and the volume percentage of voids (%) were calculated

A rapid and low-cost DNA extraction method for isolating Escherichia coli DNA from animal stools

The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA from goat, chicken, pig, cow and human stool samples. Two versions of the lysis buffer, with and without _-casein, were tested to alleviate PCR inhibition associated with DNA isolated from stool samples. Results obtained show that, this method using the lysis buffer containing _-casein, produces PCR ready DNA at a fraction of the cost of commercial DNA extraction kits & copy; 2011 Academic Journals

The genetic relatedness of E. coli associated with post- collection drinking water contamination in rural households

Rural households are often dependent on rivers, springs, boreholes or standpipes some distance from their homes for their daily water requirements. Water for drinking and domestic use is consequently stored in containers in-house which are prone to post-collection contamination. The objective of the study was to determine the most likely origin or place of introduction of E. coli associated with post-collection contamination in rural households, by assessing the degree of genetic relatedness of E. coli present in the stored water and other environmental samples. E. coli isolates were obtained using either mFC agar with confirmation of indole production (44 isolates) or Colilert®-18 (52 isolates). Amplified fragment length polymorphism (AFLP) fingerprinting was applied to determine the genetic relatedness of E. coli isolated from in-house storage containers, drinking cups, hand-swab samples, cattle dung and from the source water (spring water). DNA fingerprints of E. coli produced a number of clusters (>85% similarity scores calculated with the cosine coefficient). Identical E. coli genetic patterns were observed at closely linked points within the domestic pathway of water handling, such as between hand-swab and drinking-cup samples, between storage container and source isolates, and between drinking cups, source water and storage containers. The results indicated that AFLP fingerprinting could be applied to determine the genetic relatedness of E. coli isolated from closely linked points within the domestic pathway of water use within a household. However, the high genetic diversity observed for E. coli bacteria isolated from the difffferent water and environmental samples tested in this study, hampered the identification of post collection points of contamination.Keywords: typing, fingerprinting, amplified fragment length polymorphism, E. coli, water quality, genetic relatedness, AFL

Preoperative B-type natriuretic peptides in patients undergoing noncardiac surgery: a cumulative meta-analysis

Background: A plethora of studies have shown elevated preoperative natriuretic peptide measurements to predict postoperative mortality and adverse cardiac events.Objectives: The current study aimed to demonstrate this overwhelming association and to show that further studies of this nature are unwarranted.Methods: A cumulative meta-analysis of 28 studies was conducted where the primary outcomes of  mortality and adverse cardiac events were associated with elevated preoperative natriuretic peptides.Results: Cumulative meta-analysis demonstrated an odds ratio trending to a constant of 5.66, with a marked narrowing in the 95% confidence interval.Conclusions: Further studies aiming only to demonstrate an association between a preoperative natriuretic peptide threshold and the risk of postoperative adverse cardiac events are not justified. Future investigation should focus on the clinical implications of these data and the application of these findings with regard to further investigation, optimisation and appropriate adaptation of perioperative management.Keywords: BNP, major adverse cardiac event, myocardial injury, natriuretic peptides, non-cardiac surgery, NT-proBNP, outcome