Rapid detection of Streptococcus uberis in raw milk by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) method to detect Streptococcus uberis in raw milk was developed and evaluated. Three genes (sodA, pauA, cpn60) were assessed for their suitability as targets in LAMP. The analytical sensitivity was 120, 120, and 12 fg per assay for the sodA, pauA, and cpn60 assays, respectively, with a detectable signal within 8 min for the highest concentration (12ng/assay) and ~60 min for the lowest concentrations. The LAMP assays correctly identified 7 Strep. uberis strains among a set of 83 mastitis pathogens. To enable DNA isolation from raw milk, a new method was used in which a pretreatment with a cocktail of lysing enzymes was performed before an established procedure. This method resulted in an analytical sensitivity of 48 cfu/assay for the sodA LAMP assay using raw milk spiked with Strep. uberis, corresponding to 2.4×10(4) cfu/mL milk. For raw milk samples from cows experimentally infected with Strep. uberis, results of enumeration were largely reflected by results of LAMP. Evaluation of the sodA LAMP assay with 100 raw milk field samples, of which 50 were Strep. uberis culture-negative and 50 Strep. uberis culture-positive, showed that the assay had a diagnostic sensitivity of 96.0% and a diagnostic specificity of 96.0%. In conclusion, the described LAMP assay may offer a simple alternative for convenient and sensitive detection of S. uberis in raw milk, provided a compatible rapid DNA isolation procedure is available.

Increased fat and polyunsaturated fatty acid content in sow gestation diet has no effect on gene expression in progeny during the first 7 days of life.

The 'developmental origins of health and disease' hypothesis proposes not only that we are what we eat, but also that we could be what our parents ate. Here, we aimed to improve health and performance of young piglets via maternal diets based on the hypothesis that maternal nutritional interventions change metabolic programming in piglets, reflected by differential gene expression early in life. Therefore, sows were fed either a regular diet, based on barley, wheat and wheat by-products, sugar beet pulp, palm oil and oilseed meal, or a high-fat (HF) diet consisting of the regular diet supplemented with an additional amount of 3.5% soybean oil and 1% fish oil at the expense of palm oil and wheat. Performance results, physiological parameters and gene expression in liver of piglets and blood of piglets and sows at day 7 after farrowing from both diet groups were compared. The HF diet tended to enhance growth rate of the offspring in the first week of life. No significant differences in gene expression in liver tissue and blood could be detected between the two groups, neither with whole-genome microarray analysis, nor with gene specific qPCR analysis. In this study, the feeding of a high-fat diet with increased amounts of polyunsaturated fatty acid (PUFA) to gestating sows under practical farm settings did not induce significant changes in gene expression in sows and offspring.

The Actim Partus test to predict pre-term birth in asymptomatic high-risk women.

The Actim Partus test has been shown to be a useful predictor of pre-term birth in symptomatic women, but limited research has been carried out in high-risk asymptomatic women. This is a pilot study to evaluate the use of this test as a direct comparator with the fetal fibronectin test. All asymptomatic high-risk women attending a pre-term surveillance clinic over a 9-month period, took an Actim Partus and fetal fibronectin test, between 23(+0)-24(+6) weeks' gestation. A total of 45 women were eligible. The positive and negative predictive values of the Actim Partus test for delivery at ≤ 37 weeks' gestation were 0% and 70%, respectively, compared with the fetal fibronectin test, with values of 67% and 79%, respectively. It was concluded that the Actim Partus test did not perform well as a predictor of pre-term birth in high-risk asymptomatic women.

Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis.

Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.

Survival patterns of Streptococcus suis serotypes 1 and 14 in porcine blood indicate cross-reactive bactericidal antibodies in naturally infected pigs.

Streptococcus suis serotype (cps) 1 and cps14 have been detected in association with severe diseases such as meningitis and polyarthritis in pigs. Though these two cps are very similar, only cps14 is an important zoonotic agent in Asia and only cps1 is described to be associated with diseases in suckling piglets rather than weaning piglets. The main objective of this study was to assess restriction of survival of cps14 and cps1 in porcine blood by IgG and IgM putatively cross-reacting with these two cps. Furthermore, we differentiate recent European cps1/14 strains by agglutination, cpsK sequencing, MLST and virulence-associated gene profiling. Our data confirmed cps1 of clonal complex 1 as an important pathotype causing polyarthritis in suckling piglets in Europe. The experimental design included also bactericidal assays with blood samples drawn at different ages of piglets naturally infected with different S. suis cps types including cps1 but not cps14. We report survival of a cps1 and a cps14 strain (both of sequence type 1) in blood of suckling piglets with high levels of maternal IgG binding to the bacterial surface. In contrast, killing of cps1 and cps14 was recorded in older piglets due to an increase of IgM as demonstrated by specific cleavage of IgM. Heterologous absorption of antibodies with cps1 or cps14 is sufficient to significantly increase the survival of the other cps. In conclusion, IgM elicited by natural S. suis infection is crucial for killing of S. suis cps1 and cps14 in older weaning piglets and has most likely the potential to cross-react between cps1 and cps14.

World Health Organisation multicentre randomised trial of supplementation with vitamins C and E among pregnant women at high risk for pre-eclampsia in populations of low nutritional status from developing countries.

OBJECTIVE: To determine if vitamin C and E supplementation in high-risk pregnant women with low nutritional status reduces pre-eclampsia. DESIGN: Multicentred, randomised, controlled, double-blinded trial. SETTING: Antenatal care clinics and Hospitals in four countries. POPULATION: Pregnant women between 14 and 22 weeks' gestation. METHOD: Randomised women received 1000 mg vitamin C and 400 iu of vitamin E or placebo daily until delivery. MAIN OUTCOME MEASURES: Pre-eclampsia, low birthweight, small for gestational age and perinatal death. RESULTS: Six hundred and eighty-seven women were randomised to the vitamin group and 678 to the placebo group. Groups had similar gestational ages (18.1; SD 2.4 weeks), socio-economic, clinical and demographical characteristics and blood pressure at trial entry. Risk factors for eligibility were similar, except for multiple pregnancies: placebo group (14.7%), vitamins group (11.8%). Previous pre-eclampsia, or its complications, was the most common risk factor at entry (vitamins 41.6%, placebo 41.3%). Treatment compliance was 87% in the two groups and loss to follow-up was low (vitamins 2.0%, placebo 1.3%). Supplementation was not associated with a reduction of pre-eclampsia (RR: 1.0; 95% CI: 0.9-1.3), eclampsia (RR: 1.5; 95% CI: 0.3-8.9), gestational hypertension (RR: 1.2; 95% CI: 0.9-1.7), nor any other maternal outcome. Low birthweight (RR: 0.9; 95% CI: 0.8-1.1), small for gestational age (RR: 0.9; 95% CI: 0.8-1.1) and perinatal deaths (RR: 0.8; 95% CI: 0.6-1.2) were also unaffected. CONCLUSION: Vitamins C and E at the doses used did not prevent pre-eclampsia in these high-risk women.

Supplementation of piglets with nutrient-dense complex milk replacer improves intestinal development and microbial fermentation.

Weaning of piglets causes stress due to environmental, behavioral, and nutritional stressors and can lead to postweaning diarrhea and impaired gut development. The diet changes experienced during weaning require extensive adaptation of the digestive system. A well-developed piglet that had creep-feed experience before weaning performs better after weaning. In the current study, the effect of providing sow-fed piglets with a supplemental nutrient-dense complex milk replacer (NDM) on gut development and growth performance was studied. Litters of sows with similar parities (3.6 ± 0.8) and similar numbers of live born piglets (13.5 ± 0.3) were assigned to 1 of 2 groups: 1 group of piglets had ad libitum access to NDM from Day 2 through 21 after birth, whereas the other group was used as controls. Nutrient-dense complex milk replacer-fed piglets were shown to be significantly heavier after 21 d of supplementation compared with the control piglets. At Day 21, 3 piglets from each litter were euthanized for morphological and functional analyses of the intestinal tract. The small intestines of NDM-fed piglets had significantly higher weights (g) as well as significantly higher relative weight:length ratios (g//cm) compared with the small intestines of control piglets ( < 0.05). Morphometric analysis demonstrated that villi length and numbers of goblet cells did not differ between groups. However, NDM-fed piglets had deeper crypts ( < 0.001) and an increased expression of the cell-proliferation marker proliferating cell nuclear antigen in crypts ( < 0.05), suggesting higher cell-proliferation rates. The gene encoding IGF-1 showed a tendency to higher gene expression in the jejunum from NDM-fed piglets ( = 0.07) compared with the jejunum from control piglets, suggesting that IGF-1 might be involved in the regulation of cell proliferation and intestinal growth. Finally, as a result of dietary fiber in NDM, piglets showed significantly increased concentrations of metabolic fermentation products. This suggests differences in metabolic activity in the colon between treatment groups. In conclusion, providing sow-fed piglets with NDM before weaning stimulates intestinal proliferation, leading to increased circular growth. Nutrient-dense complex milk replacer supplementation might, therefore, help piglets through the transition period at weaning by increased BW and increased capacity for uptake of nutrients.

Clinical accuracy of inflationary oscillometry in pregnancy and pre-eclampsia: Omron-MIT Elite.

OBJECTIVE: To evaluate the accuracy of the Omron MIT Elite in pregnancy and pre-eclampsia according to the British Hypertension Society protocol (BHS). DESIGN: Prospective observational study. SETTING: Antenatal clinics and wards at St. Thomas' Hospital (London, UK). POPULATION: Forty-five pregnant women including 15 with pre-eclampsia. METHODS: Nine sequential same arm blood pressure (BP) measurements were taken from each woman by trained observers, alternating between mercury sphygmomanometry and the test device. MAIN OUTCOME MEASURES: Grading criteria of the BHS protocol (A/B grade=pass; C/D=fail). RESULTS: The Omron MIT Elite achieved a grade A/A in both pregnancy and pre-eclampsia. The mean difference (SD) between the mercury standard and the device in pregnancy was -1.1 (5.2)mmHg and 1.5 (4.8)mmHg for systolic and diastolic BP respectively compared to 0.2 (5.3)mmHg and 2.2 (5.5)mmHg in pre-eclampsia. CONCLUSION: The Omron MIT Elite can be recommended for use in pregnancy and pre-eclampsia according to the BHS protocol. To date, this is the most accurate automated BP device validated in pre-eclampsia.

Development and validation of a blinded hybrid device according to the European Hypertension Society protocol: Nissei DM-3000.

Both automated and auscultatory blood pressure (BP) devices have their strengths and accuracy limitations. Hybrid devices, such as the Nissei DM-3000, are mercury free and provide both automated and auscultatory measurement modes. The aim of this study was to validate all measurement modes of the Nissei DM-3000 device according to the European Society of Hypertension (ESH) protocol, as well as to develop and validate a 'blinded' auscultatory measurement mode. Different measurement modes were developed and evaluated in separate studies. Nine sequential same-arm BP measurements were taken alternating between simultaneous mercury sphygmomanometer readings and the device. The latter seven measurements were analysed according to the requirements of the ESH protocol. All measurement modes of the device passed the ESH protocol. The blinded mode achieved the best results with a mean difference+/-s.d. of -0.1+/-2.6 and 0.04+/-2.4 mm Hg for systolic BP (SBP) and diastolic BP (DBP), respectively. The most accurate auscultatory measurement results were obtained with a deflation rate of 2.5 mm Hg s(-1) achieving a mean difference+/-s.d. of -0.6+/-4.4 (for SBP) and -1.4+/-2.8 mm Hg (for DBP). The automated mode achieved a mean difference+/-s.d. of -0.8+/-6.0 (SBP) and 0.8+/-4.8 mm Hg (DBP). The Nissei DM-3000 device is a suitable replacement for the mercury sphygmomanometer.

Calibration accuracy of hospital-based non-invasive blood pressure measuring devices.

Accurate blood pressure (BP) measurement is dependent on a trained observer using validated and properly maintained equipment. BP devices should be checked regularly to ensure that their calibration remains within the European Standard specification of +/-3 mm Hg. This study assessed the air leakage rates and calibration accuracy of BP devices in use at a large teaching hospital, using a calibrated electronic pressure gauge as reference. Air leakage rates were recorded over 1 min and static pressures were recorded at 250/200/150/100/50/0 mm Hg for computer download and analysis. A total of 127 devices were assessed (18 mercury, 62 aneroid and 47 automated). In total, 22 different models of devices were available, of which 11 were automated and only 4 had published evidence of a validation using a recognized protocol (British Hypertension Society, Association for the Advancement of Medical Instrumentation or International Protocol). Only 3% (n=4) of devices had an air leakage rate within 4 mm Hg per min and 25% (n=32) of devices failed to meet the European calibration standard of +/-3 mm Hg. Respective failure rates were 6% (1/18) for mercury, 31% (19/62) for aneroid and 26% (12/47) for automated devices. Inaccurate BP measurement of only 3 mm Hg can have detrimental effects in the patient. This study shows a quarter of devices currently in use at a large teaching hospital to have an unacceptable calibration error. Regular maintenance and calibration checks are vital in ensuring that BP is measured as accurately as possible.

Selection of recombinant antibodies specific for pathogenic Streptococcus suis by subtractive phage display.

A semisynthetic antibody phage display library was used to select recombinant antibodies directed against surface components of a pathogenic strain of Streptococcus suis serotype 2 and against extracellular factor (EF), a protein known to be exclusively associated with pathogenic S. suis serotype 2 strains. Three distinct monoclonal phage antibodies directed against conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage antibodies were isolated that recognized EF. To isolate antibody fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this procedure, only one distinct phage antibody was found, and it was shown to be directed against EF. This demonstrates the selectivity of the applied procedure and confirms that EF is indeed differentially expressed by pathogenic and nonpathogenic strains. It also shows that EF is a very dominant antigen in phage antibody selections.